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THESIS

This is to certify that the
dissertation entitled

REPRODUCTIVE CONTROL SYSTEMS IN THE AGING RAT

presented by

Anna E. Miller

has been accepted towards fulﬁllment
of the requirements for

Ph.D. degree in Physiology

(J

V w Major professor

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REPRODUCTIVE CONTROL SYSTEMS IN THE AGING RAT

BY

Anna E. Miller

A THESIS

Submitted to
Michigan State University

Department of Physiology

ABSTRACT
REPRODUCTIVE CONTROL SYSTEMS IN THE AGING RAT

BY

Anna E. Miller

Nine separate experiments were performed in order to
examine the effects of age on the hypothalamic-pituitary—
gonadal unit in male and female Long-Evans rats. Aged male
rats demonstrate lower serum testosterone and LH concentra-
tions and a reduced response to acute stimulation by HCG or
LHRH as compared to young adult male rats. However, chronic
Leydig cell stimulation by HCG results in similar increases
in serum testosterone in young and aged. male rats and
restores testicular responsiveness to acute gonadotropin,
indicating that the testis of the aged male rat remains
capable of significant response. An in vitro study
suggests similar hypothalamic content of biologically active
LH-releasing hormone activity in young and aged groups. An
experiment to characterize serum testosterone concentrations
during different times of the day indicates the present of a
diurnal pattern of serum testosterone in young male rats

which was not present in aged male groups and also of small

episodical surges of pituitary LH and testicular testoster—
one secretion which show lower average concentrations and
less variability with increased age.

Experiments in the female rat support a hypothesis of
multiple factors affecting changes in the estrous cycle and
fertility in the aging female rat. The magnitude of the LH
and progesterone surge is progressively decreased with
increased age and this reduction presumably ultimately
results in increased estrous cycle irregularities and con-
tributes to the development of the constant estrous state.

A longitudinal breeding study' demonstrates decreased
fertility at nine to ten months of age in female rats, which
is prior to the loss of normal ovarian cyclicity. An in—
creased gestation length accompanied by longer maintenance
of elevated serum progesterone concentrations in aged com-
pared to young rats is also demonstrated.

Although hypothalamic LH-releasing content in vitro is
not found to be decreased with age in the female rat,
multiple LHRH injections could increase serum LH in aged
rats to concentrations which were similar to those found
following similar hormone treatment in young groups. A
hypothesis of altered neurotransmitter function in the
hypothalamus contributing to decreased LH-releasing hormone

secretion is discussed.

ACKNOWLEDGMENTS

I wish to express my thanks to Dr. Gail Riegle for pro—
viding me with the opportunity and the support to undertake
these studies. It was indeed an honor and a pleasure to
have been his student. I also wish to thank those who have
served on my guidance committee, Dr. Jerry Scott, Dr. Ed
Convey, Dr. Tom Emerson, Dr. Joseph Meites, Dr. Lyn Clemens,
Dr. Keith Demarest and Dr. Bdb Ringer, for their advice and
encouragement. Appreciation is also expressed to my col—
leagues, especially Donald McKay and Sandra Wood, for their

many hours of assistance.

TABLE OF CONTENTS

ListOf Tables 000............OOIOOOOOOOOOOOOOOO

List Of Figures 0....O.........OOOOOOOOOOOOOOOOO

Introduction
Aging and Reproduction .....................

Effect of Age on Reproductive
Control Systems in the Male ..............

Effect of Age on Reproductive
Control Systems in the Female ............

Experimental
Introduction ...............................
Materials and Methods
Experimental Animals ..................
Blood Collection ......................

Radioimmunoassay for Testosterone
and Progesterone .....OOOOOOOIOOOOOOO

Radioimmunoassay of LH ................

Experiment 1: The Effect of Age on Reproductive Control

Systems in the Male Rat

A. Responsiveness of the Aged Male Rat
Testis to Chronic HCG Stimulation ...

Material 8 and MethOdS O O O O O O O O O O I O I O 0
Re SUI ts O O O O O I O I O O O O O O I O O O O O O O I O O O I O O
DiSCUSSion . C C C O O O O O O O O O O O O I O O O O O O O O O

B. Hypothalamic LH-Releasing Activity in
the Aged Male Rat ...................

17

3O

3O

31

32

35

37
39
39

43

45

C.

Experiment 2:

mterials and MethOdS ......OOOOOOOOOIOCOIOO

Results ......

DiSCUSSion ......OOOOOOOOOOOOOOOOOO......OOO

Temporal Patterns of Serum LH and Testosterone
and Pituitary and Testicular Responsiveness

to LHRH in the Aged Male Rat ...............

Materials and Methods .....................

Temporal Patterns of Serum Testosterone

and LH ......OOOOOOOOOOOOOOIOO0.000....

LHRH Stimulation of LH and Testosterone

secretion ......OOOIOOOOOOOOOOO00......

Results ........O.........OOOOOOOOOOOOOOOOO

DiscuSSion 00.0.0.0.........OOOOIOOOOOOOOOO

Mechanisms in the Female Rat

A.

B.

C.

Hypothalamic LH-Releasing Activity in

Intact and Ovariectomized Rats .......

Materials and Methods ................

Resu1ts ....0.0.0.000...00.00.000.000.

DiscuSSion 0.0.0.0000.........OOOOOOOOOO

The Effect of Age on Reproductive Control

Serum LH Following Multiple LHRH Injections in
Aging Female Rats ......OOIOOOOOOOOOOOOO

Materials and Methods ..............
Results 0.0.0.........OOOOOOOOOOOOOO

DiSCUSSion .........OOOOOOOOOOOOOOOO

The Effect of Age on Reproduction in
Repeatedly Mated Rats ..............

Materials and Methods ..............
Resu1ts 0.0.0.0.........OOOOOOOOO...

DiscuSSionO......OOOOOOOOOOOOOOOOOO

Page
46

48

50

55
56

56

57
.58

67

72
73
74

77

78
80
81

85

89
91

92

97

D. Temporal Changes in Serum Progesterone in
Aging Female Rats .......................... 104

Materials and Methods ...................... 105
Results . ........ ........................... 107
Discussion ................................. 113
E. Serum Progesterone During Pregnancy and
Pseudo—pregnancy and Gestation Length in

the Aging Rat coo-oooooooooooooooooooooooooo 121

Material 8 and MethOds O O O O O O O O O O O O O O O O O O O O O O 1 2 3
Resu1ts ....OOOOOOOOOOOOO......OOOOOOOOOI... 125
DiSCUSSion ......OOOOOOOOOOOOOOI...0.0.0.... 133
F. Endocrine Factors Associated with the

Development of the Constant Estrous State

in Aging Female Rats ....................... 139
Materials and MethOds 0 O O O O O O O O O O O O O O O O O O O O O 142
Resu1ts I O O O O OOOOOOOOOOOOOOO O 0 O O O O O O O O 0 O O O O O 144

DiscuSSj-on 00.......0.........OOOOOOOOOOOOOO 150

Summary and Conclusions

The Reproductive Control System of the
Aging Male Rat 00......00.0.00...0.00.00.00.00... 161

The Reproductive Control System of the
Aging Female Rat ................. ......... ...... 165

LiStOf References 0.00.00...O.......OOOOOOOOOOOOOOOOOO 173

10.

11.

LIST OF TABLES

Serum LH and Testosterone Concentration in Intact
and Gonadectomized YOung and Aged Rats ............

Effect of Hypothalamic Extracts on LH Release from
Incubated Rat Pituitaries .........................

Group Means and Range of Individual Serum
Luteinizing Hormone and Testosterone from Serial
B100d samples in male mtg ....IOOOOOOOOOOOOOOOOOOO

Individual Rat Mean and Range of Serum Testosterone
(ng/ml) from Serial Blood Samples in Male Rats ....

Serum LH from Serial Blood Samples in Aging Male
Rats Experimentz0......0............OOIOOOOOOOI.O

Serum LH Concentrations in Intact and Gonadecto-
mized Ybung and Aged Female Rats ..................

Effect of Hypothalamic Extracts on LH Release from
Incubated mt PitUitaries ......OOIOOIOOOIOOOO0....

The effect of Age on Initiation of Repeated
Pregnancies on Reproduction in the Rat ............

Effect of Age on Pregnancy in Cycling
Female Rats ......OOOOCOO......OOOOOOOOOOO0.0.0....

Evidence for Ovulation in Aged Long-Evans Rats ....

The Effect of Mating on Serum LH in Aging Constant
Estrous Rats ......OIOOOOOOO......OOOOOOOOOOIOOOOOI

51

61

62

64

74

75

98

126

145

146

10.

11.

LIST OF FIGURES

The Effect of 7 Days of HCG Treatment on Serum
Testosterone in Ybung and Aged Male Rats ........

The Effect of Injection of 0.2 and 2.0 IU of
HCG on Serum Testosterone in Young and Aged
Male Rats 00.00.000.000.000000000000000000.00....

The Effects of 0.25, 0.5 and 1.0 Young and Aged
Hypothalamic Equivalents from Intact and
Gonadectomized Male Rats on LH Release from
Incubated Pituitary Halves ......................

Serum Testosterone from Serial Blood Samples
Collected at 2 Hour Intervals in YOung,
Middle-Aged and Aged Male Rats ..................

Serum Testosterone from Serial Blood Samples
Collected at 38 Hour Intervals in YOung, Middle-
Aged and Aged Male Rats .........................

The Effect of Injection of 1 mg/g bw LHRH on
Serum LH and Testosterone in YOung, Middle-Aged
and Aged Male Rats 0....O.....OOOOOOOOOOOOOOOOOOO

The Effect of Injection of 5 mg/g bw LHRH on
Serum LH and Testosterone in YOung, Middle-Aged
and Aged Male Rats 0....O......IOOOOOOOOOOOOOO...

The Effects of 0.25, 0.5 and 1.0 Young and Aged
Hypothalamic Equivalents from Intact and
Gonadectomized Female Rats on LH Release from
Incubated Pituitary Halves ......................

The Effect of Serial Intravenous LHRH Injections
on Serum LH Concentration in Young Diestrous
and Aged Pesistent Diestrous Female Rats ........

The Effect of Serial Intravenous LRH Injections
on Serum LH Concentration in Ybung Estrous and
Aged Constant Estrous Female Rats ...............

The Effect of Age and Repeated Pregnancies

on Fecundity ......OOOOOOOOOOII00......0.0.00.0...

42

‘52

59

6O

65

66

76

82

84

93

The Effect of Age and Repeated Pregnancies on
Litter Size and Average Newborn Pup Weight ....... 95

The Effect of Age and Repeated Pregnancies on
the Proestrous LH Surge .......................... 96

The Effect of Age on Serum Progesterone at
Proestrous, Estrous, and Diestrus Day 2 Stages
of the Ovarian Cycle ............................. 108

Serum Progesterone Concentrations During the
Proestrus Hormone Surge in Young and Aged
Female Rats ....OOOOOOOOOOI......OOOOOOOOOOOOOOOOO 109

Serum LH Concentrations During the Proestrous
Homone surge ..................OOOOOOOOOOOOOOO... 111

Temporal Changes in Serum Progesterone in YOung

and Aged Female Rats at Proestrous, Estrous,

and Diestrus Day 2 Stages of Their

Ovarian Cycles ................................... 112

Serum Progesterone Concentrations in Noncycling
Constant Estrous, Pseudopregnant, and Constant
Diestrous Aged Female Rats ....................... 114

Serum Progesterone Concentrations from Groups of
Aging Female Rats on Days 1 and 6 After Mating ... 127

Serum Progesterone Concentrations from Groups
of Aging Female Rats on Days 11 and 16
After Mating OOOOOOIOOOOOIOOOO......OOOOOOOO...... 128

Serum Progesterone Concentrations from Young
and Aged Female Rats on Days 1, 2, 3, 4 and 5
After Mating 00.00.000.000.......OIOOOOOOOOOOOOOOO 13o

Serum Progesterone Concentrations from Young and
Aged Female Rats on Days 19, 20, 21, and 22
After Mating 0.0.0.0....O.......OOOOOOOOOOOOOOOOOO 132

The Effect of 2 Hour Stress on Serum Progesterone
in Aged Constant Estrous Rats .................... 147

Serum Progesterone Concentrations from Stress
Interrupted Constant Estrous Female Rats and

Similarly Aged Normally Cycling Female Rats

on Diestrus Day 1 ................................ 149

Proestrus Serum Progesterone in Cycling vs.
Constant Estrous Rats After Stress ............... 151

26.

27.

Proestrus Serum LH in Cycling vs. Constant
Estrous Rats After Stress ... ...... ............... 152

The Effect of Injection of 0, 0.5 or 2 mg. of
Progesterone on Proestrous Serum LH in Aged
Cycling Female Rats .............................. 153

INTRODUCTION

Aging is believed to affect the function of all physio-
logical systems. Recognized aging effects on body systems
include anatomical degeneration, reduced functional reserve,
reduced responsiveness to control inputs and loss of syn—
chrony between and within physiological systems. A substan-
tial amount of information about the biology of aging has
been collected in recent years. However, answering the
fundamental questions concerning specific molecular causes
of aging changes in body function and whether the effects of
aging can be reversed remains the challenge.

As major regulators of body function which are recog-
nized to influence all aspects of physiology, the endocrine
systems have always fascinated gerontologists. Early inves-
tigators often thought that aging of the endocrine systems
could be directly linked to the effects of aging on all body
systems. Aging changes in the endocrine control systems
that regulate reproduction are particularly well documented.
In fact, many early investigators postulated that age deter-
ioration of gonadal function was the primary cause of bio-
logical aging.

Major advances in our understanding of the biochemistry,

physiology and pharmacology of reproductive control systems

2

have been made in recent years. Significant age-related
alterations in resting hormone concentrations, and decreases
in the ability of components of reproductive control systems
to respond to control input have been identified in several
species. Current biochemical-physiological techniques offer
promise for clinical identification and treatment of age-
related alterations in reproductive function.

The primary goals of most studies of the biology of
aging are twofold: to develop a better understanding of the
etiology of age effects on human function; and to identify
potential clinical procedures to treat or reverse adverse
aging effects on function. The effects of aging on human
reproductive function are particularly dramatic. 1m: other
known biological change with age is as finite or as irre-
versible as the menopausal changes which occur in women. A
significant problem for scientists attempting to relate
their work to aging problems of human significance concerns
appropriate selection of animal models. To date, no animal
species 'has been identified that Shows identical aging
effects on reproductive function as found in humans.

The laboratory rat has been extensively utilized as a
model of mammalian aging. In addition, the rat has been a
primary model for basic studies of endocrine and reproduc-
tive control systems. Although the effects of age on repro-
duction in the rat are somewhat different from that occur-
ring in humans, particularly related to menopause, I believe

that my studies and experiments reported from other

3
laboratories indicate that the rat can be an appropriate
model for the study of premenopausal aging in women and as a
model for the decline in reproductive function in aging men.

Agingiand Reproduction

 

Although the effects of aging on reproduction received
extensive study by early biologists, modern scientists and
clinicians have not given sufficient attention to the effect
of age on human sexuality and reproductive control systems.
When dealing with the elderly, current clinicians are much
more likely to concentrate their thinking in terms of age
effects on hearing, sight, arthritis and senility than on
age effects on human sexuality. Behavioral scientists have
likewise been concerned with the effectiveness of health
care delivery systems, nursing homes, and pension and social
security systems, also at the expense of a better under-
standing of sexuality in the aged (Butler, 1978). Schneider
(1978) pointed out societal attitudes toward the elderly
tend to identify them as relatively sexless. Butler (1978)
has summarized our societal expectations toward sexual
stereotypes in the aged. Men over the age of 50 are typic-
ally considered sexually impaired. waever, Butler points
out that most aging men do not have physiological bases for
failure of sexual performance and indicates a high success
rate of treatment of sexual impotence in aged men.

The societal attitude toward aging women in our society
is even more troublesome than with men. Butler describes

the sexual metamorphosis of women from desirable, sexy young

4

things, to sexually interesting mature women which at about
the age of 50 are expected to decline into sexual oblivion.
In addition, women are often victimized by the myths of the
menopause. These myths include the onset of mental altera-
tions including depression, defeminization and the loss of
sexual desire. These attitudes toward the sexuality of
older men and women are carried into our institutions. The
fact that no provision for sexual privacy normally exists in
most homes for the aged, hospitals or other forms of public
institutions for the aged may be a reflection of these
attitudes.

Schneider (1978) has hypothesized that our failure to
give attention to reproductive function in the aged is
related to a mistaken belief that pathologies of these
organs and tissues do not have life threatening potential in
the elderly When compared to cardiovascular, renal, pulmon-
ary or other physiological systems. However, Schneider
pointed out that pathological involvement of the reproduc-
tive system such as prostatic or breast carcinomas is one of
the leading causes of morbidity and mortality in the older
patient.

Aging changes in reproduction and reproductive endocrine
control systems have been extensively studied in the past
few years. A wealth of new information and understanding
regarding the anatomical and physiological parameters that
are associated with aging effects in reproductive control

mechanisms have been obtained.

5

Although the loss of reproductive function with in-

creased age is of concern for many animal breeders, the
effect of age on human reproduction has not been as much of
a concern in our society in the past because most men and
women had completed their desired procreation before fertil-
ity was significantly affected by aging. In recent years,
however, fertility problems have become of increasing in-
terest and concern, largely because of the growing trend
toward postponing motherhood until the thirties and even
forties. Thus, the desired childbearing years are increas-
ing coinciding with the premenopausal years. Recent statis-
tics (Schwartz and Mayaux, 1982) indicate that fertility
declines significantly in women after age 30. However,
despite growing medical knowledge of reproductive mechan-
isms, the precise reasons for this decrease in fertility
cannot be readily explained. Increasing age can certainly
be related to increasing incidence of uterine disease, tubal
obstruction and ovulatory disorders, but aging processes
affecting the hypothalamic-pituitary-ovarian axis are also
implicated. Thus, although the changes in reproductive
function occurring with the menopause in women are dramatic,
it must be remembered that aging effects on reproduction are
continuous and important changes in reproductive control
systems occur throughout the biological lifespans of humans
and in all mammals.

Major advances have recently been made in our under-

standing of the relationship of the hypothalamus—pituitary

6

regulation of the gonadotropin secretion and the mechanisms
of gonadal gametogenesis and steroidogenesis in the aging
mammal. It has become clear that there are significant
differences in the effect of age on parameters of the repro-
ductive control systems among various species. :n: is my
objective in this thesis to summarize current thought con—
cerning the mechanisms of aging effects on the reproductive
control system and to specifically consider the results of
my experiments concerning the effect of aging on reproduc-
tion in the rat in the context of our understanding of aging
effects on reproduction in other mammals, particularly
humans.

Effect of Age on Reproductive
Control Systems in the Male

 

 

The most consistent effect of aging on reproduction in
males is the progressive decrease in sexual interest, libido
and sexual activity from maturity into old age (Bishop,
1970). Although aged males of all mammalian species have
decreased sexual function, individual males have been shown
to be fertile at advanced age (Bishop, 1970). The most
complete studies of the effect of age on male sexuality and
reproduction have been done in men and were first reported
by Kinsey et a1. (1948). Additional studies of the effect
of age on sexual activities in men have been made by Newman
and Nichols (1960) and Martin (1975). These workers have
reported a progressive decrease in orgasmic frequency from
age 35 onward. The numbers of sexual encounters decrease

with increasing age in men until by age 65 to 79 a majority

7

of men in their study reported less than 20 sexual encoun-
ters per year with considerable numbers of men reporting no
sexual activity. Similar observations have been made with
domestic mammals. Fraser (1968) and Rowson (1959) reported
sexual apathy and impotence in older, domestic, male mammals
which cause problems in breeding programs. In summary,
Martin (1975) pointed out that a large number of variables,
including environmental, psychological and physiological may
be involved in determining the level of human male sexual
fuction. Certainly all these variables affect reproductive
function during aging. It is the intent of this reviewer to
focus on the physiological variables.

The anatomy of the testes is significantly affected by
aging. Bishop (1970) points out the inconsistencies between
measures of sexual activity and anatomical evidence of
testicular degeneration and decreased spermatogenesis which
occur in males. Some males show little sexual interest with
near normal spermatogenesis, some continue sexual activity
with little» or no spermatogenesis and others lose ‘both
sexual interest and spermatogenesis with increasing age.

Degenerative changes in the testes of aging men include
seminiferous tubule fibrosis, decreased spermatogensis and
thickening of the basement membrane of the seminiferous
tubule (Nelson and Heller, 1945: Engle, 1952; and Balze, et
al., 1954). Decreased interstitial cell size and number
often accompanies seminiferous tubule degeneration in aging

males. Similar degenerative testicular changes occur in the

8

aging bull (McEntee, 1958). MacLeod and Gold (1953) found
increased sperm counts and ejaculate volumes and decreased
sperm motility with age in men ranging from 25 to 50 years
of age. Natoli, et a1. (1972), studied seminal character-
istics in men ranging from 45 to 91 years of age. Although
these workers found a decrease in the numbers of normal
sperm per ejaculate with aging, they found no age effect on
the total number of sperm in each ejaculate. These studies
suggest that decreased ejaculatory frequency tends t1) com-
pensate for the decrease in spermatogenesis in older men.
In addition, these studies imply age-related alterations in
the normalcy and fertility of sperm produced in aging semi-
niferous tubules.

Testosterone is recognized to be essential for the
maintenance of the male reproductive system and the stimu—
lation of sexual behavior. Many of the previously mentioned
alterations in sexual activity and spermatogenesis in aged
males are consistent with a hypothesis of decreased testos-
terone synthesis in the aged. Several investigators have
measured blood testosterone in aging men. Although the
early work of Hollander and Hollander (1958) showed de—
creased testosterone concentrates in aged human testicular
venous blood, several subsequent early studies failed to
find age—related alterations in serum testosterone concen-
trations in blood samples collected from adolescence to old
age (Coppage and Cooner, 1965; Kent and Acone, 1966; Gandy

and Peterson, 1968). 131 more recent years several

9

investigators have completed major studies of large numbers
of men indicating decreased blood testosterone concentra-
tions with increasing age (Baker, et al., 1976; Greenblatt,
et al., 1976; Mazzi, et al., 1974; Rubens, et al., 1974;
Stearns, et al., 1974; Vermeulen, 1976). Vermeulen (1976)
found a progressive decrease in plasma testosterone concen-
trations from 50 years of age. This decrease in total blood
testosterone was accompanied by an increase in plasma tes-
tosterone binding capacity which resulted in a major de—
crease in free testosterone from 10.6 ng/lOO ml in 20 to 50
year old men to only 3.6 ng/100 ml in men over age 65.

The effect of age on hormone receptors is another vari-
able which can affect endocrine control systems. .Altera-
tions in tissue responsiveness to hormone regulation, in—
cluding androgen stimulation of reproductive accessory
tissue, is a common alteration that can occur with biolog-
ical aging. Decreased androgen receptors in the aging male
rat have been reported in the liver (Roy, et al., 1974) in
the prostate (Shain, et al., 1973) and in the hypothalalmus,
cerebral cortex, pituitary and testis (Chauknyiska and
Vassileva—Popova, 1977).

Relatively less is known about the effect of age on
testicular endocrine function in other species. A study by
Eleftheriou and Lucas (1974) found no changes in plasma
testosterone in two strains of aging mice. In addition,
Nelson, et al. (1975) failed to show decreased plasma tes-

tosterone concentration in C57BL/6J male mice at 28 months

10
of age. On the other hand Leathem an Albrecht (1974) found
sharply reduced testicular As-Bﬂ hydroxysteriod dehydro-
genase activity, suggesting decreased testosterone secretion
in Long-Evans male rats at age 18 and 24 months of age.

This suggestion of decreased serum testosterone in aging
male Long-Evans rats was confirmed by my M.S. thesis which
concerned blood testosterone concentrations and testicular
responsiveness to human choronic gonadotropin (HCG) in the
aging male rat (Miller, 1976). My study showed sharply
decreased resting serum testosterone in 22 to 30 month old
male rats compared to 4-month old controls. Separate groups
of young and aged males received intravenous injections of
0, 1, 5, or 20 IU of HCG. Serum testosterone was measured
from serial blood samples collected at 45, 90, and 150
minutes after HCG injection. Serum testosterone was in-
creased in both young and aged groups following all HCG
treatments. However, the increase was smaller in the aged
compared to the young rat. The result of our initial exper-
iment did not identify whether the decrease in basal testos-
terone and testicular responsiveness to HCG was due to
interstitial cell pathologies in aged rats or an age-related
failure in testicular stimulation.

Several groups have measured the effects of HCG treat—
ment in aged men. Although Mazzi, et a1. (1974) found lower
basal blood testosterone in the older man, they reported
similar percentage increases in testosterone after HCG

treatment. 0n the other hand, Rubens, et a1. (1974) found

11
both lower blood testosterone and reduced testicular re-
sponsiveness to HCG stimulation in aged compared to young
men.

Changes in endocrine control systems of aged males could
also involve steroids other than testosterone. Pirke and
Doerr (1975) and Rubens, et al. (1974) have reported in-
creased free and total plasma estradiol and estrone in aged
men. The increase in estrogen is thought to be responsible
for the increase in testosterone binding globulin reported
in the blood of aged men (Vermeulen, 1976) and the resultant
decrease in free testosterone in these men.

If the gonadal control system remains functional with
age, the decrease in testicular secretion and serum testos-
terone concentration should be detected by hypothalamic-
hyp0physea1 negative feedback systems and result in
increased pituitary gonadotropin secretion. This relation-
ship between testicular endocrine fuction and gonadotropin
secretion in aging males has received considerable atten-
tion. Early studies of this control system in aging men
showed that the increase in urinary gonadotropins with
increasing age was much smaller' than. the ‘postmenopausal
increase in gonadotropins in women (Pedersen-Bjergaard and
Jonnesen, 1948). Recent studies by Baker, et a1. (1976),
Rubens, et a1. (1974) and Stearns, et a1. (1974) have demon-
strated increased circulating concentrations of luteinizing
hormone (LH) and follicle stimulating hormone (FSH) in aging

men. The increase in FSH reported in these studies was

12

consistently greater than that reported for LH. This in-
crease in gonadotopin secretion by aged men suggest the
pituitary is responsive to the decrease in testosterone in
these subjects. H0wever, it is not clear why the increase
in gonadatropin secretion is not of sufficient magnitude to
effectively restore testicular secretion of testosterone to
the concentrations of this hormone that are present in young
men.

In contrast to the findings in aged men there is no
evidence for increased LH secretion in the aged rat. Pre-
vious studies in our laboratory and by others have demon-
strated decreased serum LH concentrations in the aged male
rat (Riegle, et al., 1976; Riegle and Meites, 1976; Meites,
et al., 1978). In contrast to the data from men, these
studies indicate that the aged male rat does not increase LH
secretion in response to the decrease in testosterone,
suggesting a neuroendocrine imbalance in the rat rather than
the primary testicular alteration believed to contribute to
decreased testosterone concentrations in men.

The responsiveness of the pituitary to 'hypothalamic
stimulation has been tested in aged men and rats. Studies
of pituitary responsiveness to Lﬂ-releasing hormone (LHRH)
in aged men have consistently showed increased LH secretion.
Mazzi, et a1. (1974) found similar increases in LH after
LHRH in young and aged men over 70 years. Rubens, et al.
(1974) and Hashimoto (1973) reported a relatively reduced

responsiveness of aged men to LHRH. However, this reduced

13

responsiveness reflects high pretreatment LH concentrations
since both groups had similar maximal serum LH concentra-
tions after LHRH were similar in young and aged men. 0n the
other hand, Haug, et a1. (1974) and Snyder, et a1. (1975)
reported decreased LH responsiveness to LHRH in older men in
terms of both magnitude of the increase in blood LH after
LHRH stimulation and the maximum concentration of LH
attained. Snyder, et al. (1975) also found decreases in the
increase in plasma FSH following LHRH stimulation in the
older men. These data support a hypothesis of altered
neuroendocrine function as well as Leydig cell failure in
aged men.

My M.S. thesis showed that the pituitary of aged male
rats is capable of increasing serum LH concentrations after
LHRH stimulation. The magnitude of the increase in LH
secretion after a single LHRH injection was smaller in aged
compared to young male rats (Riegle and Meites, 1976).
However, we found similar serum LH in young and aged male
rats following multiple LHRH injections. This finding was
supported by our report of decreased pituitary LH content
and reduced LH release after LHRH treatment in pituitary
incubates from aged compared to young male rats. (Riegle,
et al., 1976).

Reported aging changes in the gonadal control system of
male mice are not similar to what we found in the rat.
Finch (1978) reported similar serum LH concentrations in 10

and 28 month old male C57BL/6J mice. In addition, this

14
study showed similar increases in testosterone secretion
from young and aged mice testis slices, incubated with
variable concentrations of LH. This study suggests no
functional impairment of the male reproductive control sys-
tem with aging in this strain of mice.

In recent years there has been increasing recognition in
the role of the hypothalamus in the regulation of reproduc-
tion and its possible role in the effect of age on reproduc—
tive function in the male. It is believed that the hypo—
thalamus synthesizes LHRH primarily in the preoptic region.
The gonadotropin releasing hormone is transported to the
median eminence where, upon stimulation, the pituitary
stimulation factor is released into the hypothalamic-
hypophyseal portal vascular system. A large number of
experiments suggest that the secretion of hypothalamic
hormones is regulated by the neurotransmitters of the cen-
tral system. IFuxe and Hokfelt (1969) demonstrated the
presence of dopamine containing neurons in the external
layer of the median eminence by histochemical techniques.
These workers also reported that dopamine release from these
neurons was correlated with pituitary gonadotropin release.
In recent years numerous laboratories have attempted to
establish the role of specific hypothalamic neurotransmit-
ters with the control of hypothalamic gonadotropin releasing
hormone secretion, often with conflicting evidence and
conclusions. Kamberi, et al. (1970) showed that dopamine

could stimulate FSH release from pituitaries incubated with

15

hypothalamic fragments, and Schneider and McCann (1970)
found third ventricle injection of catecholamines stimulated
pituitary gonadotropin and inhibited prolactin secretion.
Results of other experiments have suggested that hypothal-
amic gonadotropin stimulating hormone secretion may be
specifically controlled by the activity of norepinephrine
(Sawyer, et al., 1974; Cocchi, et al., 1974). Although
Fernstrom. an.‘Wurtman (1977) summarized. the evidence for
neurotransmitter regulation of LHRH secretion as inconclu-
sive, many other investigators believe that neurotransmitter
function is directly involved with the regulation of hypo-
thalamic function.

Several experimenters working on the effect of aging on
endocrine function have postulated significant age related
alterations in hypothalamic sensitivity to control input.
Dilman (1976) has suggested decreased hypothalamic sensitiv~
ity to multiple hormone inputs. Aschheim (1976) hypothe-
sized increased hypothalamic sensitivity to estrogens in
aged rats. A series of experiments from our laboratory
(Riegle and Miller, 1978), and from Dr. Meites' group
(Meites, et al., 1978) suggest altered hypothalamic respon-
siveness to testosterone feedback related to castration,
stress and the catecholamine precursor L-Dopa.

The decrease in hypothalamic responsiveness to control
inputs is consistent with the hypothesis of decreased hypo-
thalamic neurotransmitter function in aged rats. In a

preliminary experiment included in my M.S. thesis, we found

     

16

decreased. whole ‘hypothalamic dopamine and norepinephrine
content in 24-26 month old compared to 4 month old male rats
(Miller, et al., 1976). Although this reduction in content
does not pmove there are functional deficiencies in these
neurotransmitters at specific hypothalamic neurons involved
in gonadotropin control, these findings are consistent with
the hypothesis of involvement of neurotransmitters with the
reduction of hypothalamic-pituitary function in the aged
rat. Our report of reduced catecholamines content in aged
male rats has been confirmed and expanded by the report of
Simpkins, et a1. (1977) showing decreased hypothalamic
content and turnover' of' dopamine and norepinephrine and
increased serotonin turnover in old compared to young male
rats and our recent report of"hypothalamic and. median
eminence function in aged rats (Riegle, et a1. 1979). It is
assumed that these changes in hypothalamic neurotransmitter
function are related to the age—related decrease in hypo-
thalamic responsiveness to changes in testosterone negative
feedback in the aged male rat.

In summary, aging has been reported to affect reproduc-
tive function in males of all mammalian species studied. On
the other hand, the specific effects of biological aging
varies markedly among the species studied. Decreased sperms
atogenesis and fertility seems characteristic of all
species. Aging men have been reported to have alterations
in both testosterone secretion and hypothalamic responsive-

ness to control input. On the other hand, the aged male rat

17
has decreases in both testosterone and LH which seem revers—
ible with LH and LHRH stimulation which suggests that age
alterations in hypothalamic responsiveness may be the pri—
mary lesion in the reproductive control system in this
species.

Effect of Age on Reproductive
Control Systems in the Female

 

 

The most fundamental effect of aging on the mammalian
female reproductive control system is the irreversible
decline in oocytes which begins during fetal and neonatal
life and continues through the reproductive lifespan of the
individual (Jones, 1970). The best known and most widely
documented consequence of aging on female reproductive
control systems is the loss of ovarian function which occurs
in women. Although the number of normal oocytes is ex-
hausted at, or shortly after, menopause in women (Jones,
1970), other species experience reproductive failure with
substantial numbers of oocytes remaining in the ovaries
(Talbert, 1978). In many instances, estrous cycles, and at
least in some instances, ovulation, can be induced by hor-
mone or drug therapy in aging females that have experienced
loss of ovarian cycles or infertility (Finch, 1978). How-
ever, decreased fertility precedes ovarian cycle loss in all
species which have been studied to date. These findings
suggest that the loss of oocytes is not the primary factor
involved in the failure to reproduce in these species.

The effect of age on reproductive function has been most

extensively studied in tﬂua laboratory rodent, particularly

18

in the rat. Finch (1978) has proposed two phases of repro-
ductive senescence in aging female rodents. The first
phase, which is characterized by the loss of fertility in
animals which retain apparently normal ovarian cycles,
begins before the rat reaches the middle of its normal
lifespan. The changes in reproductive control systems in
the rat during this interval appear to be similar in many
respects to the premenopausal decline of fertility in women.
The second phase begins later in the rat's lifespan and is
characterized by alterations in the patterns of the rat
estrous cycle.

Aschheim (1976) has extensively studied the effect of
aging on estrous cycles in the rat. The first change in
vaginal cytological patterns in the rat is the development
of increased incidence of cornified cells in vaginal lavages
which leads to the development of constant estrous states.
At more advanced ages, increased numbers of the aging female
rats develop vaginal cytological patterns characteristic of
repetitive pseudopregnancies or they may eventually enter a
noncyclic, diestrous state. A large number of studies have
shown that the loss of ovarian function in the aged rat is
not accompanied by the large increase in serum gonadotropin
(Riegle and Miller, 1978; Meites, et al., 1978; Finch, 1978)
which is characteristic of postmenopausal women (Heller and
Heller, 1939).

A great deal of attention has recently been directed

toward the identification of specific lesions occurring in

19

the aging hypothalamic-pituitary-gonadal control system of
the laboratory rodent. Many studies have shown decreased
litter size prior to decreased ovulatory rates in a variety
of species (Adams, 1970; Fekete, 1946: Harman and Talbert,
1970). Age—related alterations in reproduction Which result
in decreased fertilization and inhibition of the development
of the preimplanted egg has been proposed. The normalcy of
oocytes from aged mammalian females has been studied by
transplanting fertilized eggs from old donors into the uteri
of young recipients. Although an initial study of ova
transplanted from aged to young female hamsters by Blaha
(1964) indicated decreased fertility from the eggs from aged
hamsters compared to eggs transported from young donors to
young recipient controls, subsequent studies by Gosden
(1974), Jones (1970) and Talbert and Krohn (1966) utilizing
somewhat different postovulatory transplant times have shown
similar viability of aged compared to young donor ova trans-
planted to young recipient mice and rabbits. In addition,
these same studies show sharply decreased survival of eggs
transplanted from young donors to aged recipients compared
to transplants between young donors to young recipients.

These data indicate that the decrease in fertility of
aged mammalian females occurs much earlier than any known
alteration in ovarian reproductive potential and implicate a
deterioration in the ability of the reproductive tract,
presumably the uterus, to sustain normal gestation with

increased age. Age-related alterations in uterine function

20

have been implicated in the decrease in fertility of both
premenopausal women and in mid-aged rodent females. Failure
of the uterus to sustain pregnancy could involve both ana-
tomical changes in reproductive tract tissue which impair
their ability to sustain the conceptus and age changes in
endocrine stimulation of uterine function. Blaha (1967) and
Holinka and Finch (1977) have reported decreased decidual
tissue development after uterine endometrial stimulation in
aged hamsters and mice. Since this decrease in decidualiza-
tion in aged females could reflect declines in either the
magnitude of hormone stimulation or endometrial responsive-
ness, several investigators have considered the effect of
age on ovarian steroid secretion.

Both direct and indirect estimates have been made on the
effect of aging (n1 luteal progesterone secretion, Fekete
(1946), Green (1957), and Harman and Talbert (1970) have
reported reduced numbers of luteal cells in the ovaries of
postovulatory aging mice. In addition, attempts to increase
blood progesterone concentration in aged mice by surgical
ovarian implants from young donors (Blaha, 1970) or proges-
terone injections (Gosden, 1975) have resulted in increased
numbers of fertilized eggs undergoing implantation. On the
other hand, Spilman, et al. (1972) found similar plasma
progesterone concentrations during pregnancy in young and
aged rabbits and Larson, et al. (1973) showed no effect of
postovulatory progesterone injection on the fertility of

aged rabbits. The effect of age on progesterone secretion

21
and the biological activity of progesterone is of funda-
mental importance and warrants additional consideration in
the search for understanding the effect of aging on repro-
ductive control systems.

The influence of progesterone in pregnancy could be
influenced by age-related alterations in uterine responsive-
ness to the hormone. For instance, normal cytosol receptor
function is essential for progesterone function. 5e11, et
a1. (1972) found induction of progesterone receptors in
estrogen treated ovariectomized rat. Since estrogen and
progesterone receptors can be influenced by the presence of
these hormones, a preexisting deficiency in either of these
hormones could result in impairment of uterine responsive-
ness to the steroids. Additional experiments need to be
conducted to clarify these important areas.

Information is currently available concerning aging
effects on estrogen secretion in laboratory rodents (Lu, et
al., 1979; Steger, et al., 1979; Page and Butcher, 1982).
Our preliminary data indicate reduced serum estradiol con-
centrations in aged constant estrous rats (45 pg/ml) com-
pared to young proestrous (122 pg/ml) or estrous (93 pg/ml)
rats (Miller and Riegle, unpublished). Age related altera—
tions in estrogen secretion could be important in many of
the changes occurring in reproduction in rodent species and
warrant further experimentation.

Age alterations in estrogen receptor function could also

affect the biological activities associated with the

22
hormone. Peng and Peng, (1973) reported decreased estradiol
uptake in aged rat hypothalami and pituitaries compared to
young controls. Estimates of estradiol receptor concentra-
tion indicate progressively reduced receptor control in the
hypothalamus and cerebral cortex (Haji, et al., 1981;
Kanungo, et al., 1975) but not in the pituitary (Haji, et
al., 1981). Estradiol receptor content is also progres-
sively reduced in the uterus of the aged rat (Hsueh, et al.,
1979) with the concentration at 30 months of age only about
15% of that at 4 months of age.

Although there has been considerably more data collected
related to estrogen and progesterone function in aging women
than in rodent species, most of these studies have concerned
changes in the menopausal rather than the premenopausal
years. Talbert (1978) points out that as women approach
menopause, failure of ovulation and corpus luteum formation
becomes an increasingly common occurrence. Preliminary
studies on premenopausal women (Sherman and Kbrenmen, 1975)
suggest that blood estradiol levels are reduced by about 50%
throughout their ovarian cycle. There is also evidence that
corpora lutea formed in these premenopausal women may be
anatomically abnormal and may secrete less progesterone.
The decrease in progesterone in these women is hypothesed to
result in shortened menstrual cycles or early abortion of
the fetus if pregnancy has occurred (Collett, 1954; Novak,

23

Mean values for pregnandiol of 24 hour urine samples
(Klapper and Wilson, 1962), luteal progesterone secretion
(Sherman, et al., 1976), and plasma 17-dehydroxy-
progesterone (Abraham, et al., 1969) have all been reported
to 'be decreased in postmenopausal women. The work of
Greenblatt, et a1. (1976) and others has also established
that. ovarian estrogen secretion, particularly estradiol,
decreases in the menopausal woman. Poortman, et a1. (1973)
have shown that peripheral metabolism of androgens, includ-
ing those of ovarian origin, is a major source of estrogen
in postmenopausal women. These workers showed that ovarian
vein concentrations of estrogens in postmenopausal women
were only about one third that found in premenopausal women
while androgen synthesis is increased to about twice the
level of premenOpausal ovarian secretion. In summary, there
are major endocrinological alterations occurring with the
menopause. Most of the studies of this system have con-
cerned the loss of ovarian gamete and endocrine function.
There have been only minimal attempts to consider specific
age-related lesions which may occur in the hypothalamic-
pituitary-gonadal control system in the premenopausal woman.

Our laboratory and others have proposed an alteration in
hypothalamic-pituitary unit sensitivity to steroid feedback
in the aging mammal. HOwever, the sharp increase in post-
menopausal FSH and LH secretion reported by Odell and
Swerdloff (1968) and others and their demonstrated ability

to induce ovulatory-like gonadotropin surges using steroid

24

treatments in postmenopausal women challenges this theory
when it is applied to women. Another proposed mechanism to
explain the «decline in fertility‘ in premenopausal again
women was the proposed exhaustion of normal oocytes in the
aging ovary. However, the previously mentioned fertility of
transplanted eggs from aged rodents and the description of
anatomically normal primordial follicles in non-ovulatory
menopausal women (Costoff and Mahesh, 1975) challenges this
theory. These findings offer partial support to Longcope's
(1974) hypothesis of an age-related change in ovarian sensi-
tivity to gonadotropin in the menopausal years. Ovarian
factors associated with this apparent insensitivity to
gonadotropins, including differences in gonadotropin re-
ceptors in the human ovary, remain to be identified.

Pituitary gonadotropin secretion is sharply increased in
postmenopausal women. FSH and LH concentrations were re-
ported to increase and to lose their typical cyclicity
during the menstrual cycles of the early climacteric
(Adamopoulos, et al., 1971; Yahia, et al., 1964). Everitt
(1976) hypothesized that increased gonadotropin secretion in
premenopausal women could contribute to the loss of oocytes
in the menopausal ovary. HOwever, a premenopausal increase
in gonadotropin secretion has not been uniformly reported.
Kohler, et al. (1968) have not shown increased gonadotropin
secretion in women until after the climacteric. The in-
crease in gonadotropin secretion in postmenopausal women

appears to occur as a direct result of reduced negative

25
feedback associated with alteration in ovarian estradiol and
progesterone secretion since Odell and Swerdloff (1968)
showed that restoration of these hormones reduced the post-
menopausal increase in gonadotropin secretion.

Although aged female rodents lose their reproductive
capacity long before they reach their maximal longevity,
there is minimal evidence for increased gonadotropin secre-
tion. associated. with. this infertilityu ‘Wilkes, et a1.
(1979) reported increased serum FSH at 10 a.m. in proestrus
in 12 month old rats with regular estrous cycles compared to
6 month old controls. Although Clemens and Meites (1971),
Wilkes, et a1. (1978), and Lu, et a1. (1979) found modest
increases in serum FSH in older acyclic rats, the magnitude
of this increase does not approach the FSH concentrations
characteristic of postmenopausal women. Aschheim (1976) has
reported the presence of deficiency cells in ovarian inter-
stitial tissue whose anatomical appearance could be restored
by LH injections. This suggestion of inadequate gonadotro—
pin secretion in aged female rats is supported by direct
measurements of serum LH concentrations in aged groups.
Several laboratories have shown that the aged female rat
maintains increased serum prolactin but reduced LH concen-
trations (Huang, et al., 1976; Shaar, et al., 1975: Watkins,
et al., 1976). In addition, Shaar, et al. (1976) showed
smaller increases in serum LH following ovariectomy in aged
compared to young female rats. These data could reflect

decreased pituitary' capacity for gonadotropin secretion.

26

Although Watkins, et a1. (1976) reported decreased pituitary
responsiveness in terms of increased serum LH after a single
LHRH injection in aged compared to young female rats, the
increase in LH stimulated by LHRH indicates that the aging
rat is capable of sustaining greater pituitary gonadotropin
secretion than normally occurs. This Observation of similar
pituitary responsiveness to LHRH in young and aged rats is
similar to the reports of Tsai and Yen (1971) and Wentz, et
a1. (1975), showing no difference in pituitary response to
LHRH stimulation in pre and postmenopausal women.

Many investigators believe the hypothalamus is a primary
site of age-related alterations in female reproductive
control systems in mammalian species. Changes in hypothala—
mic responsiveness to control systems input have been re-
ported in aging females by several laboratories (Dilman,
1976; Meites, et al., 1978; Riegle and Miller, 1978). Aging
effects on the ability of hypothalamic neurons to synthesize
LHRH could contribute to the decrease in gonadotropin secre-
tion. However, in a preliminary study, we found no age
differences in 'biological LHRH activity in 'hypothalamic
extracts from young and aged rats (Riegle, et a1. 1976).
These data were confirmed by the experiments of Steger, et
a1. (1979) who found similar immunoassayable LHRH in hypo-
thalamic extracts from. young and aged rats. These (data
suggest that age alterations in the ability of the rat

hypothalamus to release LHRH, rather than aging effects on

27
LHRH synthesis, contribute to the decrease in pituitary LH
secretion.

Many researchers have shown that the release of ,hypo-
thalamic hormones, including LHRH, into the hypothalamic-
hyp0physea1 portal vessels in the median eminence can be
influenced by alterations in hypothalamic neurotransmitters.
In particular, Fuxe and Hokfelt (1969) and Sawyer, et a1.
(1974) and others have shown an association with hypothal-
amic dopamine and norepinephrine function with LHRH secre-
tion. The decrease in hypothalamic dopamine and norepi—
nephrine content and turnover previously described in the
male rat suggests that loss of catecholamine function could
contribute to the decrease in hypothalamic LHRH release and
the loss of fertility in the female rat. This hypothesis
was at least in part challenged by the report of Wilkes, et
al. (1979) indicating increased median eminence norepineph-
rine content on the morning of proestrus in regularly cycl-
ing 12 month compared to 6 month old controls. In recent
experiments, our laboratory has extended study to the aged
female rat and have found decreased dopamine content and
turnover in the hypothalamus and median eminence of aged
pseudopregnant-like or constant estrous rats compared to
young controls with normal estrous cycles (Riegle, et al.,
1979; Demarest, et al., 1982).

In addition, there is considerable indirect evidence
which supports the hypothesis of decreased catecholamine

function as a primary cause of reproductive failure in aging

28

rats. Riegle and Meites (1976) and Watkins, et al. (1976)
showed decreased pituitary responsiveness of L-Dopa inhibi—
tion of prolactin secretion in aged male and female rats
compared to young controls. As mentioned previously, one of
the first alterations in the ovarian cycle of the aging rat
is the development of the constant estrous state. Clemens
and Meites (1971) and Wilson (1974) showed resumption of
estrous cycles and ovulation in aged constant estrous rats
receiving controlled electrical stimulation of the preoptic
region of the hypothalamus. Finch (1978) has summarized a
large number of studies which have shown that a variety of
experimental treatments, including progesterone, ACTH,
L—Dopa, lergotrile, iproniazid or epinephrine injections or
stress treatments, were capable of apparent reinitiation of
estrous cycles in aging constant estrous rats. Although
some of these treatments may reinitiate estrous cycles by
affecting hypothalamic neurotransmitter and LHRH secretion,
direct evidence of effects of these agents on hypothalamic
monoamines is lacking. Several of these treatments, includ-
ing progesterone, ACTH and epinephrine injections or stress,
could act by increasing progesterone secretion which, in
turn could be involved in the regulation of gonadotropin
secretion by the hypothalamic-pituitary unit.

In summary, current experimental evidence suggests more
than a single mechanism involved with the effects of aging
on female reproductive control systems. Aging rats and the

menopausal woman are reported to have «decreased ovarian

29
steroid secretions. The primary decrease in ovarian steroid
secretion results in sharply increased gonadotropin secre-
tion in postmenopausal women. Although the relationship
between ovarian steroid and gonadotropin secretion in pre-
menopausal women is not clear, current experimental data in
the aging rat suggest that decreased steroid secretion may
be associated with reduced pituitary gonadotropin secretion.
Similarly, there is minimal evidence for hypothalamic in—
volvement with decreased fertility in aging women whereas
decreased sensitivity of hypothalamic gonadotropin control
mechanisms to control input seems to be unquestionably a
significant alteration of the reproductive control system of

the aging female rat.

EXPER IMENTAL

Introduction

The focus of my graduate research has been to identify
age-related alterations in reproductive control systems of
the laboratory rat. The data collected in the Ph.D. re-
search have been organized and presented in eight separate
manuscripts. Seven of these papers are already published.
The other one is currently undergoing editorial review. The
remaining portion of this thesis is organized to reflect
these research publications. My approach to this presen-
tation is to first describe materials and procedures which
are common to all of the experiments, then to summarize my
findings related specifically to the aging male rat, and
finally to present my findings concerning age effects of the

female rat reproductive control system.

Materials and Methods

Experimental Animals

 

Rats used in these studies were of the Long-Evans
strain. All of the rats utilized were bred and raised in
our colony from genetic stock obtained from Blue Spruce
Farms, Altamont, New York and Charles River Breeders,

Wilmington, Massachusetts. All rats were housed in the

30

31

Endocrine Research Unit's rat colony under controlled light
(12 hour light cycle) and temperature (22 1 Co) and were
allowed free access to Wayne Lab-Blox (Allied Mills,
Chicago, Illinois) and tap water. Only rats which were
maintaining stable body weights and were free of obvious
tumors or disease were used in these studies. Animals known
to be sick or diseased were removed from the colony and
destroyed. None of the experimental animals received anti—
biotic or other drug therapies to combat respiratory disease
or other maladies.

Blood Collection

 

Most of the experiments included in this thesis involved
serial blood collections from individual rats. Our standard
procedure for blood collection involved orbital sinus punc-
ture using a capillary tube under light ether anesthesia.
After sinus puncture, venous pressure causes blood to flow
through the capillary tube and into 12 x 75 mm disposable
culture tubes for collection. In recent years we and others
(Euker, et al., 1975; and Ruisseau, et al., 1978) have shown
that acute stress can affect blood hormone concentrations.
In our experiments we attempted to minimize nonspecific
stress effects on blood hormone concentrations by standard-
ization of rat handling techniques associated with blood
collection. Rats were removed from their cages and placed
in ether-saturated desiccator jars. The rats were then
quickly transported to a surgery room where blood samples

were collected. The volume of each blood sample collected

32
ranged from 0.5 to 1.5 m1 depending on the number of serial
blood collections included in the experimental design and
the amount of serum needed for the hormone analyses.

Blood samples were allowed to clot at room temperature
from 30 to 60 minutes and then refrigerated overnight.
Serum was separated by centrifugation, decanted into clean
10 x 75 mm disposable culture tubes and was stored at -20°C
until used for steroidal or protein hormone radioimmuno-
assays.

RadioimmunoassayAfor Testosterone
and Progesterone

 

 

The radioimmunoassay procedure for testosterone deter—
mination was a modification of the technique validated by
Mongkonpunya, et a1. (1975) and that for progesterone a
modification of the progesterone radioimmunoassay outlined
and validated for the Niswender antibody by Gibori, et al.
(1977).

A single serum sample large enough to furnish duplicates
of two serum volumes for final assay (i.e., if we wished to
assay duplicates of 25 and 100 ul of serum, we would initi-
ally extract at least 250 ul of serum) was decanted into a
glass stoppered 15 m1 extraction tube. Three m1 of N-hexane
was added to each tube, the tubes were securely stoppered
and vigorously agitated for two minutes. After this extrac-
tion, the tubes were stored at -20°C for about thirty
minutes in order to freeze the aqueous phase. The hexane
was then decanted into clean 12 x 75 mm culture tubes. The

extract was then taken to dryness in a water bath evaporator

33

by passing a slow stream of air over the solvent with the
tubes maintained at 42 i 2°C. The extraction procedure was
then repeated for each serum sample with the solvent from
the second extraction added to the dried culture tube that
contained the first extract residue and then dried down
again. Extraction efficiency was estimated by the addition
of tritiated testosterone or progesterone (about 3000 dpm)
to serum samples subjected to the same extraction procedure.
The solvent from these recovery extractions was decanted
into scintillation vials and the radioactivity compared to
similar amounts of tritiated hormone pipetted directly into
counting vials. Aliquots of pooled rat serum were included
in each assay' to determine interassay' variability» In
addition, solvent blanks were included in each steroid
radioimmunoassay.

At the end of the extraction procedure a known volume of
phosphate buffered saline was added to each culture tube to
solubilize the extraction residue. After addition of the
saline, the contents of the tubes were mixed by vortexing
for ten seconds and the tubes were covered and stored over-
night at 30°C. The next day duplicates of two serum equiva-
lents were pipetted from the extract—containing culture
tubes and transferred to clean 12 x 75 mm culture tubes.
The content of these tubes was then adjusted to 200 ul total
volume by the addition of appropriate amounts of phosphate

buffered saline.

34

All tubes then received 200 ul of phosphate buffered
saline which contained progesterone or testosterone anti-
body. The antibodies used were Dr. Niswender's # 666 anti-
testosterone and # 337 anti-progesterone (Dr. G.D.
Niswender, Colorado State University, Fort Collins,
Colorado). The concentration of antibody was adjusted to
optimize assay conditions for binding and sensitivity (anti-
progesterone use used at 1:2000 and anti-testosterone at
1:15000 dilutions). After addition of the antibody, the
content of the tubes was stirred 'by' vortexing for ten
seconds and the tubes were stored at room temperature for
thirty minutes to allow equilibration. At this time 100 ul
of phosphate buffered saline containing about 20,000 dpm of
chromatographically purified H3 -1,2,6,7 Testosterone or H3
-1,2,6,7 Progesterone was added to each tube. The content
of eadh tube was again mixed by vortexing for ten seconds
and the tubes were stored overnight at 3°C to allow equi-
libration of the antibody-antigen complex.

To separate free from antibody bound steroid, 1 ml of
cold 25% polyethylene glycol 4000 (Fisher Scientific, Fair
Lawn, New Jersey) was added to each tube. The content of
each tube was mixed by vortexing and the tubes were placed
in an ice bath for a thirty minute equilibration. At the
end of the equilibration, the antibody bound and free hor-
mone were separated by centrifugal separation of the poly-
ethylene glycol, which binds the free hormone, in a refrig-

erated centrifuge.

35

After centrifugation, a 1.0 m1 aliquot of the super-
natant fluid of each tube was decanted into mini liquid
scintillation vials into which 5 ml of aqueous counting
scintillant. (Amersham.IOorporation, Arlington. Heights,
Illinois) was added. The content of the scintillation vials
was mixed, allowed to equilibrate in light shielded boxes
and counted on a liquid scintillation counter (Searle Ana-
lytical Delta 300 Liquid Scintillation System).

The counts from the unknown serum extracts were arith—
metically corrected for hormone loss during the extraction
procedures and for the solvent 'blanks. Hormone content
(ng/ml) of’ the unknown serum. samples was determined 'by
comparison. of the counts of the unknown with standard
curves which were constructed as the log of hormone concen-
tration from three sets of assay tubes which contained known
amounts of progesterone or testosterone. Separate sets of
standard curves were prepared for each centrifuge run.

Radioimmunoassaygof LH

 

The basic radioimmunoassay procedure for LH determina-
tion was that described in the NIAMDD kits (Dr. A. F.
Parlow, University of California, Los Angeles). At least
two serum volumes in duplicate (from 10 to 200 ul, depending
on the expected serum LH content) were pipetted into 12 x 75
mm disposable culture tubes. Sufficient phosphate buffered
saline, containing 0.1% gelatin, was added to bring the
total volume to 400 ul. One hundred ul of NIAMDD anti-rat

LH, diluted to 1:10,000 in 3% normal rabbit serum in

36

phosphate buffered saline was added to each tube. The
content of each tube was mixed by vortexing for ten seconds
and the tubes were stored overnight at 3°C. The next day,
100 ul of iodinated rat LH diluted to a concentration of
about 30,000 dpm was added to each tube. The iodinated LH
was generously supplied by Dr. Meites. The content of each
tube was mixed by vortexing for 10 seconds and the tubes
were stored overnight at 3°C. On the following day, the
antibody—antigen complex was precipitated by the addition of
100 ul of a second antibody produced by specific immuni-
zation of sheep against rabbit gamma globulin. The second
antibody was produced in our laboratory and titrated for
maximal assay efficiency (usually from 1:60 to 1:80 dilu-
tions). The content of each tube was again mixed by vor-
texing and the tubes were returned to the cold (3°C) for
three more days of equilibration. On the third day, 3 m1 of
cold phosphate buffered saline was added to each assay tube.
The antibody-antigen precipitate was concentrated by centri-
fugation at 2800 rpm (5,500 g) in a refrigerated centrifuge
(Sorvall, RC-5). At the end of the centrifugation, the
supernatant fluid containing the free hormone fraction was
poured off and discarded. Each assay tube was counted in
our automatic (gamma. counting system (Searle Model 1197,
Automatic Gamma System).

Unknown serum LH concentrations were determied by com-
parison of the dpm of the precipitates of each assay tube

with standard curves constructed as dpm as a function of the

37
log dose of standard LH (NIAMDD rat LH RP-l). Samples of
pooled rat serum were run in each assay to allow estimation
of interassay variation in LH determination. Individual
assay variables in terms of nonspecific binding and total
antibody binding were determined for each LH assay. The
nonspecific binding tubes contained only the diluent for the
LH antibody in place of the antibody containing diluted
rabbit serum. The total antibody binding tubes were equiva-
lent to zero hormone standards. The counting time for each
assay was calculated as that interval required to record
10,000 disintegrations from the total count tubes.
Experiment 1: The Effect of Age on

Reproductive Control Mechanisms
in the Male Rat

A. Responsiveness of the Aged
Male Rat Testis to Chronic HCG
Stimulation

 

 

 

The studies included in my Master's thesis (Miller,
1976) showed sharply reduced basal serum testosterone con-
centrations in aged compared to young male rats. In addi—
tion, these studies showed that the aged rat had smaller
increases in testosterone following intravenous injection of
1, 5 or 20 IU of HCG than was found in young male rats.

These data were of interest in that they seemed similar
to the reported effects of age on testicular function in
men. Although Coppage and Cooner (1965) and Gandy and
Peterson (1968) found similar blood testosterone concentra-
tions in the human male from adolescence to old age, most

recent studies have shown sharply reduced blood testosterone

38

in aged human males (Kirscher and Coffman, 1968; Persky, et
al., 1971: Longcope, 1973; Vermeulen, 1976: Baker, et al.,
1976). In addition, Longcope (1973) reported a smaller
increase in blood testosterone after HCG stimulation in aged
compared to young' men and ‘Vermeulen (1976) reported an
increase in plasma testosterone binding protein capacity in
aged men which, coupled with the decreased total testoster-
one concentrations, resulted in a dramatic reduction in free
testosterone in the older men.

The similarity in our initial data concerning the effect
of age on Leydig cell function in the rat and data collected
from older men suggested that the rat may be a particularly
useful model to study age effects on reproductive function
in men. However, it is recognized that an acute injection
of tropic hormone may not demonstrate the secretory poten-
tial of an endocrine tissue, particularly one that has not
recently received normal stimulation. consistent with our
findings in rats, Rubens, et al. (1974) found that a group
of 65 to 90 year old men treated with 1500 IU of HCG per day
for three days had much smaller increases in plasma testos-
terone than a similarly treated group of men 20 to 50 years
of age. These data were interpreted as further evidence of
an inherent age decline in Leydig cell function in these
men. However, these types of acute experiments fall short
of elucidating whether the aged testis can be stimulated to
greater secretion under more chronic conditions. The

following experiment was designed to consider the effect of

39
chronic HCG stimulation on Leydig cell function in aged male
rats compared to similarly treated young male groups.
Materials and Methods

Separate groups of 16 young (5 months) and aged (22 to
26 months) male rats received subcutaneous injections of
physioloigical saline containing 0 or 5 IU of HCG/100 gm bw
(Ayerst Laboratories, New Ybrk) for seven consecutive days.
Testosterone was measured in blood samples collected before
the treatment was started and 24 hours after the final HCG
injection.

The effect of the chronic HCG treatment regime on an
acute Leydig cell response was measured by determining serum
testosterone (concentration. in serial blood samples taken
before and at one, two and three hours after intravenous
injection of 0.2, or 2.0 IU of HCG/100 gm bw. This test of
acute responsiveness after chronic HCG stimulation was
started 24 hours after the final daily subcutaneous treat-
ment.

Differences within and between age groups were tested by
multivariant analysis of variance and analysis of variance
for repeated measurements. Only differences with a proba-
bility of error of less than 0.05 were considered as signif—
icant.

Results

The effect of seven consecutive days of treatment with 5

ID of HCG/100 gm bw on basal serum testosterone are shown in

Figure 1. Serum testosterone (concentrations in 'the

40

30

nag/in!”
0 0|

d
”I

Se ru mi Testosterone
o

 

 

 

 

A c c ace
vouuc. AGED

BEFORE HCG AFTER HCG

Figure 1. The Effect of 7 Days of HCG Treatment on Serum
Testosterone in Young and Aged Male Rats. Seven days of
daily subcutaneous injection of 0.5. ml. of saline con-
taining 5 ID of HCG/100 gm bw or 0.5 ml of saline alone were
given to young (n = 32) male rats. Testosterone is
expressed as group means (ng/ml serum) with the indicated
standard error of the means from blood samples collected
under light ether anesthesia before HCG injections and 24
hours after the last HCG treatments (n = 16 in control and
HCG treated groups).

 

41

pretreatment blood sample were greater in young than in aged
rats (P<0.01). The difference between young and aged groups
receiving the saline vehicle control injections was sus-
tained throughout the treatment regime. On the other hand,
both groups of rats receiving the chronic HCG treatment had
similar large increases in testosterone concentration 24
hours after the final HCG injection (P<0.01). Serum testos-
terone concentrations were not different in young and aged
rats following the HCG injections.

Figure 2 illustrates the effect of intravenous HCG
injection on serum testosterone concentrations in young and
aged groups which had received 0 to 5 IU of HCG/100 gm bw
for seven days previous to this experiment. Although the
0.2 IU/100 gm bw injection of HCG stimulated increased tes-
tosterone concentrations in both the young and aged control
groups, this level of acute HCG stimulation did not cause a
further increase in serum testosterone above the elevated
initial testosterone levels in either age group which had
received the chronic HCG pretreatment. Intravenous injec-
tion of 2.0 IU of HCG/100 gm bw stimulated increased serum
testosterone in young and aged HCG ‘treated and control
groups (P<0.05). The increase in testosterone following 2.0
IU of HCG injections was greater in the young control than
in the aged group which was not pretreated with chronic HCG.
However, this level of HCG stimulation resulted in similar
stimulation. of serum testosterone concentration in 'both

young and aged groups which had received HCG pretreatment.

42

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43

Discussion

The results of this experiment suggest that the decrease
in serum testosterone found in the aged male Long-Evans rat
is not due to an impairment of Leydig cell steroidogenesis
or to decreased sensitivity to gonadotropin stimulation.
Although the aged male rat has sharply decreased basal serum
testosterone and reduced responsiveness to acute testicular
stimulation (Miller, 1976), the increase in blood testoster-
one concentrations following either acute or chronic gonado-
tropin stimulation suggests the Leydig cells of the aged
male rat are capable of sustaining much greater testosterone
secretion than normally occurs. The similarity of testi-
cular response to seven days of HCG treatment indicates that
there may be no significant decline in steroidogenic capac-
ity of the Leydig cells. These results are consistent with
the report of Leathem and Albright (1974) thatlls—gg- hydro-
xysteroid dehydrogenase activity of the aged male rat testis
could be restored by similar HCG treatments as were employed
in this study and other recent reports (Steger, et al.,
1979: Geisthovel, et al., 1981; Kaler and Neaves, 1981)
showing no decrease in LH binding by aged male rat Leydig
cells. In addition, Kaler and Neaves (1981) found that
diminished testosterone in aged male rats could not be
attributed to functional or numerical deficits in the Leydig
cell population. These data suggest that the primary factor
involved with decreased testosterone secretion of the aged

male rat is reduced gonadotropin stimulation of the testis.

44

Several recent studies have shown decreased serum LH in
the aged male rat (Riegle and Meites, 1976; Bruni, et al.,
1977; Gray, 1978: Pirke, et al., 1979). If the gonadal
control system of the aged male rat was functioning nor-
mally, we would expect that the decrease in testosterone
negative feedback would stimulate the hypothalamic-pituitary
unit to increase LH secretion. Although the aged male rat
has somewhat smaller increases in serum. LH following a
single LHRH injection. than young' male rats (Riegle and
Meites, 1976; Bruni, et al., 1977), the increase in ILH
secretion is sufficient to stimulate increased Leydig cell
steroidogenesis (Miller, 1976) and to maintain normal serum
testosterone concentrations. These data suggest that age
changes in neuroendocrine control of hypothalamic LHRH may
be a major factor in the reduction in pituitary and tes-
ticular function in the male rat.

Earlier studies have found decreased neuroendocrine
responsiveness to acute stress stimulation of LH secretion
(Riegle and Meites, 1976) in aged compared to young male
rats. These experiments suggest that a primary effect of
age on hypothalamic regulation of gonadotropin secretion
involves a change in its responsiveness to regulatory input.
Considerable recent evidence suggests that changes in hypo-
thalamic neurotransmitters, particularly involving catechol-
amine function, are associated with the regulation of LHRH
secretion. My Master's thesis indicated decreased hypo-

thalamic content of both dopamine and norepinephrine in aged

45
male rats. This observation has been confirmed by Simpkins,
et a1. (1977), Riegle, et al. (1979) and Demerest, et a1.
(1980) who also found decreased catecholamine turnover in
the hypothalamic and median eminences of aged male rats.

In summary, these data indicate that the decrease in
blood testosterone of the aged male rat is not due to an
alteration in steroidogenic capacity of the testicular
Leydig cell. Our results suggest that reduced testosterone
secretion occurs secondarily to an age-related reduction in
LH secretion which, in turn, may be associated with an
alteration in the control of hypothalamic secretion of LHRH._

B. Hypothalamic LH-Releasing
Activity in the Aged Male Rat

 

Several previous studies from our laboratory have shown
decreased hypothalamic—pituitary responsiveness to gonadec-
tomy (Shaar, et al., 1975), L-dopa (Riegle and .Meites,
1976), and stress (Riegle and Meites, 1976). The increase
in serum LH following LHRH injection (Riegle and Meites,
1976; Bruni, et al., 1977) suggests that the pituitary can
sustain sufficient LH secretion to support testicular ster-
oidogenesis and implicates failure of 'hypothalamic LHRH
secretion as a major lesion in the gonadal control system of
the aged male rat. An age-related alteration in the capac-
ity of hypothalamic peptidergic neurons to synthesize and
store LHRH could contribute to this alteration in neuro-
endocrine function. The fbllowing experiment was designed

as a preliminary attempt to determine the effect of age on

46
hypothalamic content of LH-stimulating activity in intact
and orchidectomized male rats.
Materials and Methods

Young adult (3 and 5 months) aged (22 to 26 months)
intact and gonadectomized male rats were used in these
studies. The gonadectomized rats had been surgically pre-
pared eight weeks prior to the estimation of hypothalamic
LH-releasing activity.

The procedures for handling hypothalamic tissue and the
pituitary incubations were modifications of the techniques
of Shaar and Clemens (1974). Groups of fifteen or sixteen
young and aged rats were decapitated as rapidly as possible
after removing them from their cages. Trunk blood was
collected at decapitation for subsequent. measurement of
serum LH and testosterone. The skull was quickly opened,
the hypothalamus exposed, and the hypothalamic island was
dissected out and pooled by groups in tissue homogenizers
containing 0.1 ml of cold 0.4 N perchloric acid for each
hypothalamus included in the group. The weight of the
hypothalamic tissue removed ranged from 17 or 22 mg and was
not different between intact and gonadectomized, young or
aged groups. After homogenization, the homogenate was
transferred to a centrifuge tube and the homogenizer was
rinsed with an additional 0.1 m1 of cold 0.4 N perchloric
acid per hypothalamus extracted. The homogenate from each
group was centrifuged at 20,000 x g for 30 minutes at 3°C.

The supernatant was decanted and brought to a pH of 7.25 by

47
the addition of cold 1 N NaOH. After neutralization, the
volume of the hypothalamic extract was adjusted to 0.25 ml
for each hypothalamus in each group.

Hypothalamic LH-releasing activity was measured by
adding 0.25, 0.5 or 1.0 hypothalamic equivalents to young
male rat paired hemisectioned pituitary incubates. The
pituitaries were incubated at 37.5°C in medium 199 (Difco,
Detroit, Michigan) whidh was maintained at pH 7.25 to 7.35

by the addition of 0.165 gm of NaHCO /100 ml of culture

2
medium and constant gasing with 95% O2 -5%/CO2 in a Dubnoff
incubator at 60 cycles/min. Anterior pituitary halves were,
incubated in 2 ml of medium 199 in glass 12 x 75 mm culture
tubes. After a one hour preincubation, the culture medium
was decanted and discarded and 2 ml of fresh culture medium
was added. The control pituitary half received only medium
199. The paired treated pituitary half received either
0.25, 0.5, or 1.0 hypothalamic equivalent in the 2 ml of
medium 199. Average culture medium osmolarity ranges were
from 305 to 330, 325 to 350, 355 to 380 and from 385 to 430,
mOsm/L for the control tubes, and the tubes containing 0.25,
0.5 and 1.0 hypothalamic equivalents, respectively (5120
Vapor Pressure Osmometer, Wescor, Inc., Logan, Utah). At
the end of a fOur hour incubation, the culture medimm was
decanted, diluted with phosphate buffered physiological
saline, and frozen until the LH assays could be completed.
These data were analyzed using the "t" test for paired

observations to compare the response of the treated

48

pituitary halves with their corresponding controls. Differ—
ences between groups treated with hypothalamic extracts were
tested by multivariant analysis of variance.
Results

The effect of age on serum testosterone and LH from the
trunk blood samples is shown in Table 1. Both LH and tes-
tosterone were higher in intact young male rats than in aged
groups (P<0.01) in both age groups. However, the young
gonadectomized rats maintained higher serum LH than did the

aged gonadectomized group. (P<0.05).

hEouoeoenom umuwe exem3 unmwmn

enema mnonm me oemmmnmxm mnonmumoumee one an Enumme

 

 

Sam M.
m.nm H 0.0mm m.H u H.e «H.o H em.n mm emnmm mans news
9
4 ed... M H63 mé H 83 mmé H $.m S m .m 39: 9:6»
nomquOuomoenoo evennH uoeunH .oz AOEV med macho
eaﬁE\mnv ma Enumm A~E\mnv
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muem owed one mnno» oeNﬂEouomoenow one
uoeunH nﬂ nOwueuunoonou enoneumOpmeB one an Enuem

a mqmde

50

The responsiveness of incubated pituitaries to hypo-
thalamic extracts is shown in Table 2 and is plotted as
percent increases in LH release from hypothalamic extract
treated compared to pituitary halves in Figure 3. Hypo-
thalamic extract addition to the incubation medium increased
LH release in all groups (P<0.05). Age differences in the
effect of hypothalamic extracts on LH release were not
significant in either intact or gonadectomized groups.
Although the intact male group appeared to have greater
hypothalamic LH-releasing activity than was found in the
castrate group, the experimental design required separate
incubation on separate days for each group which makes
direct comparison of response between intact and orchidec-
tomized male groups unreliable.

Discussion

The decrease in serum testosterone in aged male rats and
reduced serum LH in intact and gonadectomized aged male rats
are in agreement with previous reports from our laboratory
(Shaar, et al., 1975,: Watkins, et al., 1975; Miller and
Riegle, 1978a) and with other more recent reports (Geist-
hovel, et al., 1981; Gray, 1978; Pirke et al., 1979).

These data indicate that the aged male rat hypothalamic—
pituitary unit is less responsive to alterations in steroid—
al feedback than the young male rat. We have previously
reported that the aged rat pituitary can increase LH secre-
tion after LHRH injection (Riegle and Meites, 1976). If the

aged male rat hypothalamus was functionally intact, its

51

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.mme mo 08 m on m wuo3 muen mnnowm

H mnemE enoum me oemmeumxm emeeﬂmu mu
.mma EnwooE nan

meanenwnnwm wen eHeE OE m oenOMuommﬂEmﬁ omnwem nuﬂ3 omuennonﬂ mne3 euoenuxm owEeHenpomeme

Sam

 

 

 

5mm H omoa omH H OMB oma H mmoa mm H oah oo.H m
mma H mhha mva H Hooa «ma H omma Nwa H HooH om.o m
maez
Nam H mama ¢NH H hem Hog H eom mm H moo mm.o m oeNﬂEonomoenoo
055 H heme Hmm H mmha mmm H hump aha H Noma oo.H m
«we H Hume mmm H omwa owma H mums ohm H Ohha om.o m mHez uoeunH
oeuemue Houunoo oeuemne Honunoo .m.m n macho
oommd omnsow

 

naxneuﬂnuam mE\mnv emeeaem ma

 

eeowueuanuﬂm pea omuennonH Eoum
emeoaem ma no muueuuxm oaseaenuomhm mo uommmm

N mqm<9

Figure 3. The Effects of 0.25, 0.5 and 1.0 Young and Aged
Hypothalamic Equivalents from Intact and Gonadectomized Male
Rats on LH Release from Incubated Pituitary Halves. YOung
male pituitary halves were incubated 4 hours in medium 199.
LH release is plotted as the average % increase of 8 hypo-
thalamic extract-treated pituitary halves compared to their
paired control pituitary halves.

52

U1 RELEASING ACTIVITY OF YOUNG AND AGED INTACT MALE
HYPOTHALAMIC EXTRACTS

eeeeeeeee eoe

.
......0.0.0.I.O.I.'.' .. .U. .

I ... D

e e .

e e e e e e e e e
I
.e.e.e.e.e.0.0.0

. .
0.0.0.0.I.e.e.e

0

e
C
e
e
e
:e

.

.

X Increase In LI-I Release
§

0 e e v
0.0.0....
0.. 0

I

.e
.0
.

 

 

 

 

 

 

 

 

 

        
    

      

                 
   

 

' v A
0.50
Hypothelemlc EquIveIenIe
LH RELEASING ACTIVITY OF YOUNG AND AGED CASTRATED MALE
HYPOTHALAMIC EXTRACT s
m
s we
a:
.a
5 mm
8
e
2 .
o ,. .
5 3'
.1 ‘°‘ 5::
m :-° '-°-. .

 

 

 

 

 

 

 

Hypothelemlc EquIveIente

Figure 3.

53

release of LII-stimulating substances should be increased
when serum testosterone concentrations are reduced. Al-
though hypothalamic secretion of LH-releasing substances in
aged intact male rats was not sufficient to maintain concen-
trations of blood LH and testosterone which are similar to
that found in young adult male rats, further reduction of
serum steroid concentration by gonadectomy results in
sharply increased LH secretion. The magnitude of the in-
crease in serum LH concentration was smaller in aged than in
the young rats. However, the proportion of LH increase
between the young and aged groups is roughly similar when
pre- and post-gonadectomy hormone concentrations are com—
pared. Although these data suggest some reduced ability of
aged male rat hypothalamic-pituitary units to sustain LH
after gonadectomy, the control system is responsive to
sustaining higher serum LH concentrations than normally
found in the aged rat. The similarity of LH-releasing
activity of hypothalamic extracts between young and aged
groups supports this hypothesis that the aged rats are
capable of stimulating greater pituitary LH secretion than
was measured both before castration when serum testosterone
and LH concentrations were both low and after gonadectomy
when pituitary LH secretion was sharply stimulated.

This experiment implies a significant age reduction in
the responsiveness of the hypothalamus leading to decreased
secretion of factors which regulate pituitary gonadotropin

secretion. Several alterations in neuroendocrine function

54

could be associated with aging changes in hypothalamic
responsiveness. The possibility of age-related changes in
neurotransmitter regulation of LHRH secretion is receiving a
great deal of current attention. The ability of major
alterations of ‘hypothalamic biogenic amines function to
affect hypothalamic release of pituitary regulating sub-
stances has been established. The hypothalamus contains
large amounts of dopamine and norepinephrine (Palkovits, et
al., 1974). Alterations in the activity of these catechol-
amines has been associated with gonadotropin control mecha-
nisms (Fernstrom and Wurtman, 1977). We and others have
reported decreased ‘hypothalamic content and turnover. of
catecholamines in aged male rats (Miller, et al., 1976;
Simpkins, et al., 1977). Although absolute proof of the
relationship between hypothalamic catecholamine function and
the effect of aging on gonadal control mechanisms is not
firmly established, the decrease in hypothalamic catechola-
mine function is consistent with the reduced gonadotropin
secretion found in this experiment.

In summary, these data indicate no significant change in
hypothalamic LH-releasing activity in the intact or orchid-
ectomized aged male rat. This experiment confirms the
hypothesis of significant alteration in the responsiveness
of the 'hypothalamus toI control input. in the aged rat.
Although the hypothalamus contains sufficient LII-releasing
activity to stimulate higher levels of pituitary and gonadal

endocrine function, aging effects on the neuroendocrine

55
control system are hypothesized to result in reduced hypo-
thalamic hormone secretion.
C. Temporal Patterns of Serum.LH
and Testosterone and Pituitary and

Testicular Responsiveness to LHRH
in the Aged Male Rat

 

 

Recent studies have shown decreased serum testosterone
concentrations in aged men (Kirschner and Coffman, 1968;
Baker, et al., 1976). One study showed that the decreased
plasma testosterone after age fifty in men was accompanied
by an increase in plasma testosterone binding capacity which
resulted 511:3 sharply reduced free testosterone concentra-
tion (Vermeulen, 1976). The decreased serum testosterone in
aged men is accompanied by increased serum concentrations of
FSH and LH (Baker, et al., 1976; Vermeulen, 1976). Although
the increase in gonadotropin secretions are believed to
partially compensate for decreased Leydig cell function, it
has been shown that the Leydig cells of aged men have re-
serve capacity for still further testosterone secretion
(Rubens, et al., 1974) and that pituitaries of aged men can
maintain similar serum LH concentrations following LHRH
injection as pituitaries from young men (Hashimoto, et al.,
1973; Rubens, et al., 1974). These data indicate probable
aging effects in Leydig cell function. and.‘hypothalamic—
pituitary unit responsiveness to negative feedback in aging
men.

Although the aging male rat is also characterized by
decreased serum testosterone concentrations (Miller and

Riegle, 1978a; Pirke, et al., 1978; Steger, et al., 1979),

56

the aged male rat has decreased serum LH concentrations
(Shaar, et al., 1975: Riegle and Meites, 1976: Gray, 1978;
Geisthovel, et al., 1981) rather than the increased blood
gonadotropins found in aged men. Both Leydig cell secretion
of testosterone and pituitary secretion of LH in aged male
rats can be stimulated to serum concentrations similar to
those after similar treatment in young males if aged male
rats receive chronic HCG or LHRH treatments (Miller and
Riegle, 1978a; Miller and Riegle, 1978b). These findings
suggest significant age effects of hypothalamic-pituitary LH
secretory control mechanisms in the aging rat.

It is accepted that testosterone secretion occurs in
episodical bursts reflecting patterns of LH secretion,
rather than in continuous, sustained secretion. Previous
studies of aged male rat testicular function have usually
measured serum testosterone concentrations at only one
sampling interval. The objective of the present study was
to determine the effect of aging on patterns of testosterone
secretion in the male rat by sequentially measuring serum
testosterone and LH in aging rats through a 24 hour period.
In addition, we determined the patterns of pituitary and
testicular responsiveness of LHRH injection in young, medium
and aged male rats.

Materials and Methods

Temporalypatterns of serum testosterone and LH. Two

experimental procedures were used to determine daily

patterns of LH and testosterone secretion. In the first

57
experiment, groups of ten young (4 months), medium (13
months) and aged (20 months) male rats were subjected to
serial blood collections every two hours for 24 hours be-
ginning at 5 a.m. In the second experiment the interval
between blood collections was extended to ndnimize effects
of hypovolemia and anesthesia—blood collection stress on
hormone control mechanisms. In this study, groups of 14
young (4 months), medium (13 months) and aged (22 months)
male rats were subjected to serial blood collections at 38
hour intervals beginning at 1 p.m. This bleeding schedule
also produced a more random sequence of blood sampling times’
(1 p.m., 3 a.m., 5 p.m., 7 a.m., et cetera.)

LHRH stimulation of LH and testosterone secretion

 

Groups of 20 young (3 months), medium (12 and 13 months)
and aged (22—26 months) male rats received jugular vein
injections of 1 or 5 ng of synthetic LHRH (Lot 628059,
Calbiochem, San Diego, California)/gm bw. Blood samples
were collected from all rats just before the LHRH injec-
tions. After the hormone treatment (0 time) the rats in
each age group receiving each level of LHRH stimulation were
randomly divided into two subgroups (10 rats/subgroup).
Serial blood samples were collected from one subgroup 15 and
90 minutes after LHRH stimulation. Blood samples from the
second subgroup were taken at 45 and 150 minutes after the
intravenous injection. Serum LH and testosterone concentra-

tions were measured from each blood sample.

58

Differences between and within age groups were tested by
multivariant analysis of variance and analysis of variance
for repeated measurements by use of the program BALANOVA
supplied by the Michigan State University Computer Labora-
tory. Only differences with a probability of error of less
than 0.05 were considered as significant.
Results

Temporal changes in serum testosterone are illustrated
as group means in Figures 4 and 5. Individual and groups
means and ranges of testosterone for the twelve sampling
intervals are included in Tables 3 and 4. In the first
experiment, in which rats were serially sampled 12 times in
23 24 hour period (Figure 4), average testosterone concen—
trations in the first blood samples were higher in young
than in aged male rats, with the medium aged males having
intermediate hormone concentrations. Serial blood sampling
reduced serum testosterone concentration (P<0.05) in all
three age groups. Average testosterone values were not
different between the age groups by the third sampling
interval (9 a.m.). Serum testosterone concentrations were
decreased proportionally greater by this experimental pro-

cedure in the young compared to the aged rats.

59

Serum Testosterone ng/ml

\/\.

ﬁxk/ \- A .
..A

um Tom Dem 11am 1pm 3PM 590‘" 7PM “Pm “PM 1am 31m

   

Thne

Figure 4. Serum Testosterone from Serial Blood Samples
Collected at 2 Hour Intervals in Young, Middle-Aged and Aged
Male Rats. Blood samples were collected under light ether

anesthesia beginning at 5 a.m. Testosterone is expressed as
the group means (ng/ml serum, N = 10)

60

 

nglml

/-
' 13 mo

 

Serum Testosterone

 

Inn 3” 59'“ 19m 9'.“ ﬂpun 1am 3am Gem Tun Dem 1am
TIme

Figure 5. Serum Testosterone from Serial Blood Samples
Collected at 38 Hour Intervals in Young, Middle-Aged and
Aged Male Rats. Blood samples were collected under light
ether anesthesia. Testosterone is expressed as the group
means (ng/ml serum, N = 10/group).

61

Table 3

Group Means and Range of Individual
Serum Luteinizing Hormone and Testosterone from
Serial Blood Samples in Male Rats

 

 

 

 

 

Testosterone Luteinizing hormone
Age N M Range M Range
Young 14 4.09 i .26 .67-17.70 36.6 i 2.8 4.0-3oo.o'
Medium 14 1.91 i .07 .58- 7.21 18.6 i 1.6 2.0-208.0

Aged 14 1.26

l+

e08 041- 4e10 11e3 e9 2.0- 5800

|+

 

aTwelve blood samples were collected from each rat at 38-hour
intervals. Young rats were 2 months, medium—aged 13 months, and
aged 22 months of age. Testosterone and luteinizing hormone
reported as mean : SEM ng/ml of serum. Range reported as ng/ml
of serum within the 168 blood samples for each age group.

62

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N enm3 even mnnow .mae>ueunw Mooglmm ue wen ﬁoee Bonn oeuoeaaoo mne3 moamﬁem oooame

 

 

 

 

 

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om.NIom. Nm.H m ¢O.mlmh. hN.N o mm.m Iv¢.H 0H.¢ m
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mH.NImm. oH.H a NN.vah. oo.N N ON.oaloo.H no.m H
mmnem 2 new owned 2 pem mmnem z uem
em me em 3:633: muse»

 

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Eoum A~E\mnv wnouwumonmma Enuwm m0 mmnem one new: Hem Hesoﬁ>wonH

.¢ OHQeB

63

In the second experiment with the extended blood collec—
tion schedule (Figure 5, Tables 3, 4, and 5), average tes—
tosterone and LH concentrations of the young group were
higher than the medium or aged groups of rats (P<0.05). The
younger rats also showed more fluctuation between average
hormone concentrations (Figure 5) than did either the medium
or the aged groups. A diurnal pattern of testosterone
concentration with reduced serum testosterone in the early
evening hours was present in the young and medium aged rats
but absent in the aged group. The aged group did not have
significant differences in testosterone concentration at any
of the blood sampling intervals.

Serum concentrations of LH and testosterone following
intravenous LHRH injection are plotted in Figures 6 and 7.
The young groups had higher pretreatment LH concentration
than the mediwm or aged groups (P<0.01). Pretreatment LH
was also higher in medium than in aged groups. The 1 ng
LHRH/gm bw treatment (Figure 6) stimulated increased serum
LH concentration in all age groups at both the 15 and 45
minutes post-injection blood sampling intervals. Serum tes—
tosterone concentrations were higher in the young group than
in either the medium or aged group of rats receiving the 1
ng LHRH treatment at all blood collection times (P<0.05).
Although this level of LHRH treatment stimulated significant
increases in LH secretion in all age groups, only the young
male rats had increased testosterone which was present in

the 45 minutes post-injection blood sample.

in Aging

64

TABLE 5

Experiment 2

Serum LH from Segial Blood Samplesa
Male Rats

 

------------------ LH (ng/ml)c

 

Sampling

Time Young Medium Aged

1 p.m. 115.0 3 17.5 32.7 i 6.7 24.0 i 3.3
3 p.m. 15.1 i 2.6 11.8 i 1.6 7.4 i 1.5
5 p.m. 32.6 i 7.3 16.2 i 2.9 11.6 i 2.6
7 p.m. 69.6 ’5 13.6 62.1 i 20.6 24.2 i 6.4
9 p.m. 22.5 3 4.7 9.5 3 1.8 9.7 i 1.5
11 p.m. 28.5 i 3.5 11.3 i 2.2 7.2 i 1.0
1 a.m. 20.5 i 3.6 12.9 i 2.3 7.5 i 1.2
3 a.m. 32.1 i 4.2 15.4 i 2.4 13.2 i 4.3
5 a.m. 20.9 i 5.6 13.0 i 2.5 8.9 3 2.6
7 a.m. 34.2 i 4.7 9.1 i 1.3 5.4 i 0.6
9 a.m. 24.0 i 3.7 20.1 i 3.3 9.5 i 1.6
11 a.m. 24.4 i 4.9 9.6 i 1.3 7.1 i 1.1

 

Young rats were 2 mo, medium 13 mo, and aged 22 mo of age.
cLH expressed as group means (n = 14

) i SEM

:Blood samples were collected from each rat at 38 hour intervals.

65

senuu LH AFTER 1 n9 LHRH/gm bw

 

 

 

6 1s 4‘6 0'0 150

HInutes After LHRH

85mm teetosnnone AFTER 1 a. LHRH/gm bw

 

 

 

 

 

"1
‘0' , muno
~
0
g N I ﬁ.
.‘ *m. e
A"/ -..—
.—. 0‘0
' ' ' 130
o 6 1s 46 no

Ilnutes Aﬂer LHRH

Figure 6. The Effect of Injection of 1 ng/g bw LHRH on Serum
LH and Testosterone in Young, Middle-Aged and Aged Male
Rats. Blood samples were collected under light ether anes-

thesia before and at 15, 45, 90 and 150 min. after intra-
venous injection of LHRH in all three groups (N

- 10/group).

Figure 7. The Effect of Injection of S mg/g bw LHRH on Serum
LH and Testosterone in Young, Middle-Aged and Aged Male
Rats. Blood samples were collected under light ether anes—
thesia before and at 15, 45, 90, and 150 min. after intra-
venous injection of LHRH in all three groups (N = 10/group).

66

seen» [.01 AFTER 5 ng LHRIVgrn hw

one
venue
eom stone:
E .6...
o
s
I
...1

I .

 

 

71 1'6 4'5 0'0 150

NInutee After LHRH

SERUM TESTOSTERONE AFTER 5 "I LHRH cm In:
28‘

15‘

myml

10‘

 

 

 

 

 

 

 

0 1'3 45 90 150

IInutee After LHRH

Figure 7.

67

The pretreatment serum LH and testosterone concentra-
tions were again greater in the young than in the aged
groups receiving the 5 ng/gm bw LHRH treatment (Figure 7).
This LHRH treatment markedly stimulated serum LH concen-
trations in all age groups (P<0.01). This increase in LH
was also uniformly greater than that stimulated by 1 ng/gm
bw (P<0.01) and was sustained through the 90 ndnutes post-
injection blood sampling interval for all groups and through
150 minutes for the two older groups. Differences in LH
concentration between groups were significant before LHRH
injection, ‘between. the young and aged groups at the 15‘
minute interval and between the young and aged groups 150
minutes after LHRH treatment.
Discussion

The results of these experiments confirm and extend our
previous findings and hypotheses concerning age effects on
gonadal control systems in the male rat (Miller and Riegle,
1978a). These data show that young male rats maintain
consistently higher serum testosterone and LH concentrations
than do mid—aged or aged male rats. The young male group
also had much more variability among sampling time means and
a greater range of hormone concentration among serial blood
samples collected from individual rats. This variability
was progressively decreased in the mid-aged and in the aged
group. If the gonadal control system remained functionally
intact, the decrease in testosterone in the aged male rat

should stimulate increased gonadotropin secretion due to the

68

loss of negative feedback (Davidson, 1969). However, serum
LH concentrations were consistently decreased in the aged
rat. These findings are in agreement with our previous
reports of reduced serum LH concentrations (Riegle and
Meites, 1976) and smaller increases in serum LH following
gonadectomy in aged compared to young male rats (Shaar, et
al., 1975). On the other hand, several gonadal control
system components of the aged male rat remain responsive to
stimulation. We have shown that the reduced serum testos-
terone of the aged male rat could be stimulated to concen-
trations similar to young rats by several days of HCG treats
ment (Miller and Riegle 1980a), the low serum LH of the aged
male rat can be stimulated to concentrations similar to that
found in young groups following serial LHRH treatment
(Miller and Riegle, 1978b) and hypothalamic content of LH
stimulating activity was similar in young and aged rats
(Miller and Riegle, 1978c).

These findings offer strong support to the hypothesis of
significant age-related change in the responsiveness of the
neuroendocrine mechanisms which regulate pituitary gonado-
tropin secretion. Although hypothalamic neurons contain
biologically active LHRH, their responsiveness to a variety
of factors which stimulate LH release, including acute
stress stimulation of LH release (Riegle and Meites, 1976),
reduced testosterone negative feedback after castration
(Shaar, et al., 1975), and LH release after L-dopa treat-

ments (Riegle and Meites, 1976) are reduced in the aged male

69

rat. An early* study' on the responsiveness of the
hypothalamic-pituitary unit to negative feedback inhibition
by testosterone after gonadectomy suggested increased sensi-
tivity to testosterone inhibition in aged compared to young
orchidectomized rats (Shaar, et al., 1975). This finding
was confirmed by a similar study in young and old orchidec-
tomized rats which received testosterone from silastic
capsule implants (Pirke, et al., 1978). These observations
of apparent increased neuroendocrine sensitivity to a nega-
tive feedback inhibitor are contrary to the developing
evidence of significant reductions in specific neurotrans-
mitter function (Carlsson, 1978; Finch, 1978; McGear and
McGear, 1978) and reduced hypothalamic testosterone receptor
concentrations (Chouknviska and Vassileva-Popova, 1977) and
warrant further consideration.

Although acute stress has been shown to increase serum
LH in young male rats (Euker, et al., 1975), the results of
our first attempt to study temporal hormone changes clearly
indicate that the combined stress of serial anesthesia,
handling, and blood collection result in sharply reduced
serum testosterone concentrations. The decrease in testos-
terone occurs in association with suppression of LH secre-
tion. Although the initial serum LH concentrations from the
rats subjected to serial blood sampling at two hour inter-
vals were similar to those measured in the second temporal
experiment with the 38 hour sampling intervals, LH concen—

trations in the first experiment were sharply reduced by the

70
third blood sample (5 p.m.) and remained at near baseline
assay sensitivity for the remaining sampling times in all
groups.

Although precise mechanisms regulating secretory activ-
ity of LHRH synthesizing neurons in the brain are unknown,
direct and indirect evidence implicates neurotransmitter
involvement within the hypothalamus and in other brain
regions (Fernstrom and Wurtman, 1977). Decreased hypothal-
amic and median eminence content and turnover of dopamine
and norepinephrine and increased serotonin turnover occur—
ring with increased age have been reported by several lab—
oratories (Miller, et al., 1976: Simpkins, et al., 1977;
Finch, 1978). It is widely presumed that alterations in
these or other yet unidentified neurotransmitters affect
LHRH neuron synthesis and secretion.

Factors which regulate the episodical bursts of LH
secretion in the young male rat are also unknown. Our blood
sampling intervals were much too infrequent to show close
associations with LH release followed by stimulation of tes-
tosterone secretion. However, our data do show consistent
progressive decreases in the range of LH concentrations
measured in individual blood samples and in the overall
average LH values measured from the young compared to the
middle, compared to the aged groups (LH concentrations from
all blood samples averaged 36.6, 18.6 and 11.3 ng/ml serum
from the young, middle, and aged groups respectively: in-

dividual serum samples ranged from 4 to 300 ng/ml in the

71
young, from 2 to 208 ng/ml in the middle, and from 2 to 58
ng/ml in the aged group).

The increase in serum LH following LHRH treatments
indicate that the aged male rat maintains a high degree of
pituitary responsiveness to hypothalamic gonadotropin stimu—
latory activity. These data are in agreement with our
previously reported findings (Miller and Riegle, 1978b) and
suggest that although the serum LH concentrations in the 15
minutes post—injection blood sample of the aged group is
statistically less than that from young males, this differ-
ence probably reflects chronically low levels of pituitary
stimulation and is not considered to be of major biological
significance. The higher serum LH concentrations in the
medium and aged groups compared to the young rats at 150
minutes is in agreement. with. our earlier work in aged
females (Miller and Riegle, 1978b) and suggest age-related
differences in the duration of pituitary LH secretion or in
the metabolism and removal of LH from the circulation.

These data indicate decreased Leydig cell responsiveness
to LH stimulation in the medium and aged groups. However,
our earlier experiments (Miller and Riegle, 1978a) indicated
that the decrease in testicular responsiveness to acute
stimulation was due to consistently low levels of LH stimu-
lation in the aged rat, rather than actual biological dif-
ferences in responsiveness to the product of pituitary
secretion as measured by radioimmunoassay. These findings

together now suggest that a functional impairment of normal

72
neuroendocrine responsiveness to gonadotropin control input
in the aged male rat results in decreased pituitary LH
secretion and markedly reduced testicular responsiveness to
13L The decreased testicular responsiveness results in a
consistently low level of testosterone secretion without the
episodical bursts of secretion which are characteristic of
younger males.
Experiment 2: The Effect of Age on
Reproductive Control Mechanisms
in the Female Rat
A. Hypothalamic LH-Releasing

Activity in Intact and Ovari-
ectomized Rats

 

 

The effect. of age on gonadotropin secretion in the
female rat is not as marked as for the aged male whidh was
described in the first section of this thesis. The effect
of age on blood LH concentration appears to be related to
the reproductive state of the old rat. Previous work in our
laboratory indicated reduced LH concentration in aged
pseudopregnant and noncyclic constant diestrous rats com-
pared to young rats in the diestrous phase of their estrous

cycle (Watkins, et al., 1975). (M1 the other hand, aging

constant estrous rats maintain higher serum LH concentra-
tions than that measured in young rats at estrous or dies-
trous stages of their ovarian cycle. However, the aged
constant estrous rats apparently secrete insufficient LH for
full follicular maturation and ovulation. Additionally,
aged rats had smaller increases in serum LH following

ovariectomy than young rats (Shaar, et al., 1975). These

73

experiments indicate that at least in some situations of
gonadotropin stimulation, aged female rats secrete less LH
than do young rats. In addition, Clemens and Meites (1971)
and Lu, et al., (1979) found elevated serum FSH concentra-
tions in aged constant estrous rats. These reports suggest
that the effect of age of FSH control mechanisms in the rat
may be different than age effects on LH secretion.

It is hypothesized that failure of adequate LH secretion
during the proestrus surge may be a factor contributing to
the decrease in fertility of the aging female rat. The
ability of hypothalamic neurons to synthesize and store LH-
releasing hormone is one factor which could affect hypo-
thalamic stimulation of pituitary LH secretion. The follow-
ing study was undertaken to measure LH-releasing activity of
hypothalamic extracts from young and aged intact and ovari-
ectomized female rats.

Materials and Methods

Young adult (3 and 5 months) and aged (22 to 26 months)
intact and gonadectomized female rats (n = 15 and 16/group)
were used in this experiment. The gonadectomized groups were
surgically prepared eight weeks prior to the estimation of
hypothalamic LH-releasing activity. The methods and proce-
dures used for collection of trunk blood, for preparation of
the hypothalamic extracts and the incubation of the extracts
with paired male pituitary halves was identical to that
described in section l-B for the experiment with male rat

hypothalamic extracts.

74

Results

Average serum LH concentrations for the experimental
groups are shown in Table 6. Serum LH was higher in the
young intact than in the aged group (P<0.05). 111 concen-
tration was increased after ovariectomy in both age groups
(P<0.01). Although the increase in LH following gonadectomy
was nearly proportional in both groups, the magnitude of the

increase was greater in the young group.

TABLE 6

Serum LH Concentrations in Intact and
Gonadectomized Young and Aged Female Rats

 

 

Age Serum LH (ng/ml)a b
Group (Mo) No. Intact Gonadectomized
Young Female 3-5 mo 31 47.4 + 6.7 407.1 + 48.1
Aged Female 22-26 mo 32 21.8 + 6.4 139.3 + 11.6

 

aSerum LH expressed as group means 1 SEM

bEight weeks after gonadectomy

The responsiveness of incubated rat pituitaries to
hypothalamic extracts is shown in Table 7 and is plotted as
percent increases in LH secretion over control pituitary
half LH secretion in Figure 8. Addition of increased hypo—
thalamic extract stimulated increased LH release from incu-

bated pituitaries for all groups. The effect of age on

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Figure 8. The Effects of 0.25, 0.5 and 1.0 Young and Aged
Hypothalamic Equivalents from Intact and Gonadectomized
Female Rats on LH Release from Incubated Pituitary Halves.
Young male pituitary halves were incubated 4 hrs. in medium
199. LH release is plotted as the average percent increase
of 8 hypothalamic extract-treated pituitary halves compared
to their paired control pituitary halves.

76

LR RELEASING ACTIVITY OF YOUNG AND AGED INTACT FEMALE
HYPOTHALAMIC EXTRACTS

X Increese In LH Releeee

 

V 0.25 A V 0.50 A Y 1.0 A

HypothelemIc Equivalents

LN RELEASING ACTIVITY OF YOUNG AND AGED CASTRATED FEMALE
HYPOTHALAMIC EXTRACTS

X Increeee In L" Beleeee

      

A Y A Y
Y 0.25 0.50 1.0 A

Hypothelemlc EqulveIente

Figure 8.

77
hypothalamic LH-releasing activity content was not signifi—
cant for either the intact or gonadectomized groups.
Discussion

These data confirm the previous experiment on the aged
male rat and indicate that the aged rat hypothalamus con—
,tains substances (presumably LHRH) capable of stimulating LH
secretion. These results reaffirm the hypothesis that aged
rat hypothalami have sufficient LHRH synthesizing capacity
to stimulate greater pituitary gonadotropin in both intact
and ovariectomized female rats and emphasize the importance
of age changes in neuroendocrine responsiveness to gonado-
tropin control input as a major alteration of the reproduc-
tive control mechanisms of the aging rat.

A. growing body of experimental data implicate age-
related changes in catecholaminergic neurotransmitters with
the regulation of LHRH secretion from hypothalamic pepti-
dergic neurons. There remains considerable controversy
concerning the relative importance of stimulatory and/or
inhibitory activity of specific neurotransmitter substances
related to LHRH control (Fernstrom and Wurtman, 1977).
Negro-Vilar, et a1. (1979) have shown that _i__n vitro hypo—
thalamic LHRH secretion is influenced by both dopamine and

norepinephrine and normal 'hypothalamic function. of ‘both

neurotransmitters is essential for optimal secretory pro-
cesses. The reported decreases in hypothalamic catecholamine
content and turnover in aged rats (Demerest, et al., 1982)

are consistent with the hypothesis that impairment of

78
catecholamine function results in reduced LHRH secretion.
However, it must be emphasized that the relationship between
the changes in hypothalamic catecholamine function and aging
alterations in reproductive control mechanisms will require
a great deal more experimentation before the hypothesis can
be regarded as dogma.

B. Serum.LH Following Multiple
LHRH Injections in Aging Female Rats

 

 

Although the precise neuroendocrine mechanisms involved
with age-related alterations in reproduction remain unre-
solved, current information from various species suggests
that age may affect several components of the hypothalamic-
hypophyseal-gonadal control system.

Although the effect of age on ovarian endocrine function
is not as dramatic as that measured for testosterone in the
old male rat, there is ample evidence of alterations occur-
ring in the female with increased age. Huang and Meites
(1975), Aschheim (1976), and others have shown that normal
ovarian cyclicity is replaced by periods of constant estrous
and repetitive pseudopregnancies. The effects of age on
gonadal function and the reduction in gonadal steroid pro-
duction with age in men and women is accompanied by in-
creased blood gonadotropin concentrations (Adamopoulos, et
al., 1971; Baker, et al., 1976; Lazarus and Eastman, 1976).
On the other hand, there is no evidence for similar in-
creases in gonadotropin release in aged female rats. Riegle
and Meites (1976) reported decreased serum LH levels in aged

male rats. In female rats the effect of age on serum LH

79

levels is variable and related to the variable (ovarian
states of the aged rat. Although aged constant estrous rats
have higher serum LH concentrations than do young cycling
female rats in estrous or diestrous stages of their ovarian
cycles, the LH level in aged constant estrous rats is non-
cyclic and is much less than that found in young rats at
proestrus (Watkins, et al., 1975). This study also showed
that aged rats with repetitive pseudopregnancies or aged
anestrous rats have serum LH levels which are lower than LH
levels in young rats at diestrus. In addition, several
studies have shown that ovaries from young rats grafted into
aged female rats resume the endocrine state of the recipient
before the transplant (Aschheim, 1976).

An age associated decrease in pituitary responsiveness
to LHRH could contribute to the decrease in LH secretion,
affecting gonadal control and fertility in aged rats. Pre-
vious experiments from our laboratory have shown smaller
increases in serum LH following single intravenous LH re-
leasing hormone (LHRH) injections in aged compared to young
rats of 'both sexes (Watkins, et al., 1975; Riegle and
Meites, 1976). The responsiveness to acute LHRH injection
in aged rats indicates that their pituitaries are capable of
secreting greater amounts of LH than they normally maintain.
Hewever, suppressed endocrine function leads to a functional
disuse atrophy in most endocrine systems. The long-term
reduced LH secretory activity which is characteristic of the

aged rat could at least partially account for the reported

80

decrease in pituitary responsiveness to acute LHRH in aged
compared to young rats. The current study was designed to
consider the effects of more sustained LHRH stimulation on
serum LH concentrations in aging female laboratory rats in
order to ascertain whether or not this reduction in secre-
tion is indeed due to a primary dysfunction of the pitui-
tary.
Materials and Methods

Young adult animals used in these studies were four and
five months of age and aged groups ranged from 24 to 28
months of age. The aged females were multiparous rats
obtained as retired breeders at nine months of age (Blue
Spruce Farms, Altamont, New York). Experimental groups
included aged females with either constant estrous or per-
sistent diestrous (repetitive pseudopregnant) vaginal
cytology and young females at estrous or diestrus day two of
their ovarian cycles. Old rats were considered to be con-
stant estrus or persistent diestrus if they had at least
eight consecutive days of cornified or leucocytic vaginal

smears, respectively.

Experiments were begun at 10:00 a.m. to minimize any
unrecognized diurnal variation in pituitary responsiveness.
A pretreatment blood sample was taken by orbital sinus
puncture under light ether anesthesia. All experimental
groups (12 rats/group) received three serial intravenous
injections of LHRH. Initially, 500 ng of LHRH (Eli Lilly,

Inc., Indianapolis, Indiana) was injected into the exposed

81
jugular vein. Serial blood samples averaging about 1 ml
were taken from each rat 15, 75, 90, 150 and 165 minutes
after the first LHRH injection. Similar intravenous LHRH
treatments (500 ng) were also administered at the 75 and 150
minutes blood sampling intervals.

The statistical analysis of the data included analysis
of variance of the pretreatment LH concentrations and multi-
variant analysis of variance to determine interaction be-
tween age groups, treatments, and treatment responses.
Results

Figure 9 compares serum LH levels in young, day two
diestrous and aged pseudopregnant (persistent. diestrous)
female rats. Serum LH concentration in the samples taken
prior to LHRH administration (0, 75, and 150 minutes) were
different in the aged compared to the young groups (P<0.05).
LH was higher in the young group than the aged group at 0
time (16.3 vs. 8.6 ng/ml). However, in the 150 minute
sample, LH levels in the aged group far exceeded that of the
young rats (141.0 vs. 35.7 ng/ml). Serum LH concentration
was increased after each LHRH injection in both age groups
(P<.0001). The increase in serum LH 15 minutes following
each injection was greater in both the young and aged groups
with each successive LHRH injection (P<.01). Although the
increase in serum LH following the first LHRH injection was
greater (P<.05) in the young than the aged group (112 vs. 64
ng/ml), the apparent differences between pretreatment and

the 15 minute post injection LH values in the young and aged

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83
groups following the second and third LHRH injections were
not statistically different.

The effects of multiple LHRH injections on serum LH
concentrations in young estrous and aged constant estrous
female rats are illustrated in Figure 10. LH levels in the
blood samples taken from the aged group before each LHRH
injection (0, 75 and 150 minutes), increased with each
sampling interval (P<.01). Mean LH concentrations increased
from 13.5 to 79.1 to 207.9 ng/ml from the 0 minute to the 75
minutes to the 150 minutes sampling interval. Serum LH was
increased after each LHRH injection. in ‘both. age groups
(P<.0001). The increase in LH in the 15 minutes sample was
greater for the young than the aged groups (P<.01). Al—
though the increases in serum LH concentrations in the young
group were similar after the second and third LHRH injec-
tions to the response after the first injection, the aged
group had greater increases in LH after the second and third
LHRH injections (the increase in serum LH after the first
injection was 163 ng/ml: after the second, 474 ng/ml; and
after the third, 479 ng/ml). In addition, the increases in
LH were similar after the second and third LHRH injections

in the young and aged groups.

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Discussion

These data suggest substantial differences in young and
aged rat pituitary response to multiple LHRH stimulations.
Levels of serum LH measured after the initial LHRH injection
were uniformly lower in aged compared to young experimental
groups. This finding is in agreement with our previous
reports in aged rats of both sexes (Riegle and Meites, 1976;
Watkins, et al., 1975). However, the results of this study
show that age-related differences in serum LH are much less
prominent following multiple LHRH injections than after an
acute stimulation, in agreement with the report of Wise and
Ratner (1980) indicating similar LHRH responsiveness in 8-12
month compared to 3-4 month old female rats. The marked
increase in serum LH following the second and third LHRH
treatments in the aged female rats and the young diestrus
female group is similar to the accepted self-priming effects
of LHRH previously shown to be involved in the proestrus LH
surge. It is presumed that the increased responsiveness to
LHRH involves changes in gonadal steroids which can affect
pituitary responsiveness to LHRH, the self-priming effects
of LHRH on pituitary LH secretion which results in increased
LH synthesis, or changes in the secretory processes within
pituitary gonadotrophs resulting in more rapid LH secretion
response. The marked increase in serum LH levels in the
second and third preinjection blood samples of the aged

female groups suggests either a more sustained LH release

86
following LHRH or more likely, a slower rate of removal of
the hormone from the circulation in these animals.

Although this study indicates that the aged rat can
sustain substantial serum LH levels under experimental
conditions of high LHRH stimulation for limited time
periods, there is ample evidence that in fact, the secretory
activity of the pituitary of the aged rat is significantly
reduced. We and others have consistently found low basal
serum LH levels in aged male rats and in aged female rats in
various reproductive states. Van der Schoot (1976) and
Cooper, et. a1. (1980) reported reduced serum LH levels
during the proestrous surge in 10-12 month old cycling rats
compared to 3-5 month old cycling rats which we confirmed in
section 2D of this thesis. These low blood gonadotropin
concentrations 'have ‘been implicated in the decrease in
reproductive performance of aged rats of both sexes by
several laboratories.

We and others have hypothesized that the most funda-
mental age-related alteration in the reproductive control
system occurs in the neural regulation of hypothalamic
hormone release. In the previous section of this thesis, we
showed that hypothalami of aged rats contain sufficient
gonadotropin releasing activity to stimulate LH release from
incubated rat pituitaries. Studies by Shaar, et al. (1975)
and Huang, et a1. (1976) demonstrate smaller increases in
serum LH and FSH following orchidectomy and ovariectomy in

aged male and female rats compared to young control groups.

87
These findings indicate that although the reproductive
control system in aged rats does respond to decreased nega—
tive feedback following gonadectomy, the response is less
than that which occurs in the young adult rat. These data
suggest that decreased responsiveness of the LHRH-release
mechanism, rather than loss of LHRH content or pituitary
responsiveness to LHRH, is the major contributor to age
effects in this control system.

The constant estrous state in aged female rats seems to
be involved with failure of hypothalamic mechanisms re-
sponsible for the proestrous hormone surge. Crighton and
Schneider (1969) showed that the preoptic area of the rat.
hypothalamus contains gonadotropin releasing hormone.
Electrical stimulation of this region can cause ovulation in
rats (Everett and Quinne, 1966) and lesions of the preoptic
area have been shown to reduce gonadotropin releasing hor-
mone content in this region (Mess, 1969) and cause persis-
tent vaginal cornification in young female rats (Crighton
and Schneider, 1969). In addition, the preoptic area has
been reported to be the site of the stimulatory effect of
estrogen on gonadotropin release. Clemens et a1. (1969)
showed that electrical stimulation of the preoptic area will
stimulate ovulation in constant estrous rats. Aschheim
(1976) concluded that aged constant estrous rats have suf-
ficient serum LH levels to stimulate minimal follicular
development and estrogen secretion. Although the hypo-

thalamic mechanisms have apparently become less responsive

88

to endogenous estrogen or other hormone stimulation of
gonadotropin release. The results of the current study show
that aged rats can sustain high levels of serum LH if the
hypothalamic regulation of LH release is circumvented by the
injection of LHRH, which reinforces the concept that the
ability of the hypothalamus to secrete gonadotropin releas-
ing factors is impaired in the aged rat.

Although the precise mechanisms involved in the deter-
ioration of hypothalamic function in the aged rat are not
understood, a substantial amount of experimental data sug-
gests that hypothalamic catecholamines may be involved.
Hypothalamic catecholamines have been shown to influence.
anterior pituitary secretions presumably either by affecting
the hypothalamic pituitary-regulating hormones' secretory
mechanisms or by the catecholamines acting directly on the
pituitary, as has been demonstrated for control of prolac-
tin. Increased hypothalamic catecholamine function has been
implicated in the release of pituitary LH and the inhibition
of pituitary prolactin release (Sawyer, 1975). The decrease
in serum LH and increase in serum prolactin that we have
found in the aged rat is consistent with a hypothesis of
decreased hypothalamic catecholamine function. This hypo-
thesis is supported by reports of decreased hypothalamic
catecholamine content and neuronal catecholamine turnover
rates in aged rats and mice (Riegle and Miller, 1978:

Meites, et al., 1978; Finch, 1978). On the other hand,

Wilkes, et a1. (1979) found increased median eminence

89
norepinephrine content in 12 month old rats with regular
estrous cycles compared to six month old controls. A great
deal more experimentation is required to understand the
molecular basis for changes in hypothalamic catecholamine
function in aging rats and to understand how changes in
hypothalamic catecholamines may be related to alterations in
hypothalamic responsiveness to the multiple stimulatory and
inhibitory inputs it receives.

C. The Effect of Age on Reproduction
in Repeatedly Mated Female Rats

 

In the past few years we and others have studied the
effects of increasing age on several parameters of the,
reproductive control system of the laboratory rat. The
aging female rat is characterized by loss of regular ovarian
cycles (Huang and Meites, 1975; Aschheim, 1976; Lu et al.,

1979). Although some rats remain cyclic throughout their

lifespan, these authors report increased incidence of con-
stant estrus and repetitive pseudopregnant states in the
vaginal cytologies of aged rats. It has also been demon-
strated that aged constant estrous rats will return to
regular estrous cycles following stimulation of the hypo—
thalamus directly or with presumably centrally acting drugs
or hormones (Finch, 1978; Lehman, et al., 1978; Meites, et
al. 1978). Previous work from our laboratory and others has
shown decreased serum LH and increased serum prolactin in
both male an female rats of increasing age (Watkins, et al.,
1975; Riegle and Meites, 1976; Takahashi, et al., 1980).

The previous section of this thesis showed that the

90
decreased serum LH in aged female groups could be restored
with. chronic administration: of LHRH (Miller and Riegle,
1978). These data have been interpreted as evidence for
age-related alterations in hypothalamic sensitivityto
control inputs (Aschheim, 1976; Meites, et al., 1978; Riegle
and Miller, 1978). Although our bioassay study showed that
the hypothalamus of the aged rat contains similar LH-
releasing activity as that of young rats (Miller and Riegle,
1978), the aged hypothalamus apparently releases less of
this factor than young rats since the hypothalamic-pituitary
unit of the aged rat appears to be less responsive to physi-
ological changes in sex steriod concentrations in various
reproductive states. In addition, the hypothalamic-
pituitary unit of the aged rat is less responsive to
castration-induced. decreased gonadal steroids (Shaar, et
al., 1975; Gray and Wexler, 1980) or systemic L—dopa injec-
tions (Riegle and Meites, 1976).

These experiments have been interpreted to indicate that
neuroendocrine alterations in the aging rat may be assoc—
iated with alterations of the estrous cycle normalcy in the
female rat. Although neuroendocrine mechanisms undoubtedly
contribute to the loss of reproductive function in the aged
rat, there are other factors which also contribute. The
most characteristic effect of aging on mammalian ovarian
function is the decline in the number of oocytes remaining
in the ovary with increasing age (Talbert, 1978). Aging

laboratory rodents show decreased litter size (Asdell, 1941;

91
Blaha, 1964). Although the decrease in oocyte numbers may
ultimately contribute to the decreased reproduction in aged
mammals, Adams (1970) showed that litter size decreased
prior to a decrease in ovulation rate. These observations
suggest increased post-ovulatory interruption of reproduc-
tion in the aged mammal.

Arvay (1976) reported that multiple pregnancies accel-
erated certain indices of biological aging in the female rat
during the reproductive period, but contributed to overall
longevity. Similarly, Gonzalez-Lima (1981) found that
pregnancy and lactation retarded the subsequent onset of
sterility. The study described here represented our initial
attempt to study age effects on reproductive control systems
during the interval when the rat loses its ability to repro-
duce. The experiment was undertaken to determine the inter-
action between age and repeated pregnancies on longevity,
the maintenance of normal ovarian cyclicity and the ability
of the laboratory rat to successfully reproduce.

Materials and Methods

Twenty-four two month old female rats were randomly
assigned to two groups of twelve each. Both groups of rats
sustained serial pregnancies, beginning at two months of age
for the first group and at nine months of age for the second
group. To accomplish this, rats with proestrus vaginal
smears were individually placed in cages containing two
reproductively experienced male rats. Male rats used in

this study ranged from four to ten months of age. We have

92

not found decreased fertility in male rats over this age
frame in our colony. Successful mating was ascertained by
the presence of sperm in the vaginal smear on the morning of
estrus. Mated rats were placed in individual cages for
littering. The number of pups born, number of live pups and
litter weights at birth and on days 7, 14, 21 and 24 were
recorded. At 24 days the litters were weaned and the
mother's vaginal smears were taken until proestrus was
detected and the rat was again mated for the next pregnancy.
This procedure was continued from two months of age through
nine gestational cycles when, at seventeen months of age,
only one rat littered.

At 4 p.m. on the day of proestrus a single blood sample
was taken by orbital sinus puncture under light ether anes—
thesia. Serum was collected from this blood sample for
subsequent LH radioimmunoassay.

Results

The percentage of rats successfully mated and the per-
centages of rats littering are plotted as a function of age
and number of pregnancies in Figure 11. All of the rats
showed normal ovarian cyclicity and were successfully mated
through six months of age. The percentage of rats cycling
and mated ranged from 73% to 83% between 7 1/2 and 14 1/2
months of age. Sixty percent of the rats were still cycling
and were mated at seventeen months of age when the experi—

ment was discontinued.

93

EFFECT OF AGE ON REPRODUCTION

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Fecundity. The percentage of rats with normal

ovarian

cycles which were successfully mated and the percentages of
both total and mated rats producing litters are plotted as a

function of rat age and litter number.

94

The percentage of rats delivering litters (Figure 11)
are plotted both as the proportion of successfully mated
rats and as the percentage of all rats included at the
various stages of the study (twelve rats for litters
sequence numbers 1-4, twenty-four rats beginning at litter
five). The reduction in successful pregnancies with in-
creased age was much more dramatic than the previously
described loss of ovarian cyclicity in this study. The
numbers of rats which delivered normal litters fell pro-
gressively from eleven of twelve rats at four months of age
to only one of twenty—two in seventeen month old rats.

The effect of age and repeated pregnancy on litter size)
and average pup weight are shown in Figure 12. Neither age
nor number of previous pregnancies affected the numbers or
weight of the pups born. The range of average litter size
was from 7 to 10.3 pups/litter and mean pup weight at birth
ranged from 5 to 7 gms.

Mean serum LH concentrations of rats exhibiting an LH
surge at 4 p.m. on proestrus are plotted as a function of
age and litter number in Figure 13. Serum LH concentration
was not affected by age or serial pregnancy number. All
rats did not show a proestrus LH surge in the 4 p.m. blood
sample. Only serum LH concentrations greater than 50 ng/ml
were considered indicative of an LH surge and were included
in the calculation of mean LH surge levels. The numbers
above each column indicate the fraction of the normally

cycling-mated rats with 4 p.m. LH surges. It should be

Figure 12. The Effect of Age and Repeated Pregnancies on
Litter Size and Average Newborn Pup Weight. Group means are
plotted with indicated standard errors of the means. The
numbers above each column indicate the number of litters

born at each interval.

 

 

 

 

 

 

 

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12. EFFECT OF AGE ON LITTER SIZE
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96

EFFECT OF AGE ON PROESTROUS LH SURGE

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Figure 13. The Effect of Age and Repeated Pregnancies on the

Proestrous LH Surge. Serum LH concentrations from 4 PM
proestrous blood samples are plotted as group means (ng/ml
serum) with indicated standard errors of the means. The

numbers above each column indicate the proportion of rats
with detectable LH surges (serum LH greater than 50 ng/ml at
each breeding interval).

97
noted that these fragmentary data are not totally represen-
tative of the characteristics of the proestrus LH surge.
There was no correlation between rats with demonstrable 4
p.m. LH surges and successfully mated rats which delivered
normal litters.

The age when serial pregnancies were started did not
affect any of the parameters of reproduction included in
this study (Table 8). The age when serial pregnancies were
initiated also had no effect on rat longevity. Eleven rats
from each group were alive at eighteen months of age. Six
rats from group one and seven rats from group two were still
in the colony at twenty-four months of age and three rats.
from each group were still alive at thirty months of age.
Discussion

These data indicate that there is a sharp, continuous
reduction in the ability of aging Long-Evans rats to suc-
cessfully reproduce and that the age when repeated preg—
nancies was initiated did not influence any of the param-
eters of reproduction in the aging rat which were included
in this study. The aging female rat exhibits a gradual
increase in estrous cycle irregularities including the
development of persistent vaginal cornification, pseudo-
pregnancies of varying lengths, or diestrous states (Huang
and Meites, 1975; Aschheim, 1976; Lu, et al., 1979). In
previous experiments we and others have shown increased

serum prolactin and a smaller increase in LH following

ovariectomy in aged compared to young adult female rats

98

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99

(Riegle and Miller, 1978). Our studies (Miller and Riegle,
1978a; 1978b; 1978c) and the work of other investigators
(Meites, et al., 1978; Finch, 1978; Gray and Wexler, 1980)
suggest that aging changes in hypothalamic function contri-
bute to the loss of normal estrous cycles in the rat.
However, most of the changes in ovarian cyclicity and the
previously documented neuroendocrine changes in reproductive
control systems of the aging rat occur after the end of the
rat's normal fertile lifespan (Jones, 1970). The results of
this study confirm that aging effects on neuroendocrine
mechanisms of reproductive control systems reduce the pro-
portion of aging rats that are cycling and mated and contri-I
bute to the loss of reproduction. However, the sharper
reduction in percentage of rats littering compared to per—
centages of rats with normal estrous cycles indicate that
other post-ovulatory factors are major contributors to the
loss of reproduction in the aged rat.

Van der Schoot (1976) reported decreased proestrous LH
secretion in twelve month old rats. Cooper, et al., (1980)
demonstrated that the LH surge may be more variable, lower
and appear later in ten month old cycling rats. In a fol-
lowing section of this thesis we have also shown reduction
in the magnitude of the proestrous LH surge in 12-16 month
old compared to 4 month old control rats. YOung rats in our
colony show peak proestrous LH concentrations from 3 to 7
p.m. The similarity of LH concentrations with increasing

age in this experiment was unexpected. However, our data

100

also indicate significant numbers of rats did not have a
proestrous LH surge at the 4 p.m. sampling interval. Al-
though the measurement of serum LH from a single blood
sample does not preclude the possibility of aging effects on
the timing of the LH surge and ovulation, the results of
this limited experiment do not support the hypothesis that
changes in the neuroendocrine control of the proestrous LH
surge contribute to the loss of reproductive function.

The studies of Talbert (1978) and Mandl and Shelton
(1959) suggest that normal ovulation was occurring in the
cycling, mated rats used in this study. This hypothesis is
supported by the report of Harman and Talbert (1970) which.
showed no difference in the number of eggs ovulated per
ovarian cycle in mice from four to thirteen months of age.
The hypothesis of normal ovulation rate occurring at the age
when fertility is lost is also supported by our data pre-
sented in section 2 F of this thesis. In addition, studies
by Talbert and Krohn (1966), and Gosden (1974) showed no
difference in the survival of ova from aged mice compared to
that of ova from young mice when both were transplanted to
young multiparous uterine hosts. On the other hand, ova
collected from young donors which were transplanted into
aged recipients showed much smaller rates of survival,
indicating that the aged uterus was less capable of sus-
taining normal pregnancies. Although these experiments
clearly implicate uterine relationships to infertility in

aging rats, additional experimentation will be required

101
before conclusions can be made concerning the cytogenetic
normalcy of ova from aging rats and possible roles of aging
ova in the decline of reproductive function.

Several investigators have suggested that the decrease
in post-ovulatory reproductive capacity of the aging mammal
could be due to alterations in endocrine support of uterine
function. In a review of corpora luteal function in aging
rodents, Talbert (1978) concluded that although the evidence
was contradictory, most studies do not suggest that the
decrease in reproduction in aged mammals could be contrib-
uted to by decreased progesterone secretion. This conclu—
sion is supported by our recent experiments reported in
section 2 E of this thesis which show no difference in serum
progesterone in young compared to aged rats made pregnant or
pseudopregnant by mating. The ability of the uterus to
sustain pregnancy could also be affected by estrogen func-
tion.

Less is known about the effect of aging on estrogen
secretion and its role in the decline in reproduction in
mammalian species. Estrogen is markedly decreased in post-
menopausal women and Sherman. and. Korenmen. (1975) showed
nearly a 50% reduction in estradiol during the premenopausal
years in women. In the middle aged female rat, Lu, et al.,
(1979) report serum levels of estradiol (17 i l to 18 i 2
pg/ml) which were significantly lower than young rats during
diestrus day two or proestrus. Serum levels of estradiol in

aged rats that were repetitive pseudopregnant or constant

102
diestrus were also relatively low. However, Page and
Butcher (1982) found no significant difference in the serum
estradiol concentrations found in young (3 to 6 month) and
middle aged (12 month) normally cycling rats.

The aforementioned observations have led most investi—
gators to conclude that age effects on uterine structure and
fuction are major contributors to age-related decreases in
fertility. Biggers, Finn and McLaren (1962) concluded that
increased deposition of collagen in the aging mouse uterus
contributed to the loss of reproductive capacity. In the
aged rat, the uterine connective tissue stroma enlarges to
comprise a large part of the myometrium (Soriero, 1978)..
Finn, et a1. (1963) suggested that the high collagen content
of the aging mouse uterus may impair fertility by decreasing
uterine vascularity, which in turn could result in decreased
nutritional support of the conceptus. Although Buradk, et
a1. (1941) reported that the rate of collagen deposition in
the aging rat uterus is slower in fertile breeders than in
nulliparous females of comparable age, the results of the
present study suggest that the loss of reproductive capacity
is not affected by the numbers of pregnancies experienced in
the reproductive lifespan of the rat.

Another factor which could contribute to the loss of
reproductive function in the aged rat is failure of normal
implantation. Maibenco and Krehbiel (1973) found reduced
uterine response to hormones in aged ovariectomized rats and

increased leucocytic infiltration and infections in

103

naturally’ mated eighteen. month old rats. Shapiro and
Talbert (1974) reported. decreased. decidualization in ‘the
aged mouse uterus. Soriero (1978) concluded that the aged
female has decreased uterine tissue response and delayed
implantation. Although the design of this study prevented
detection of the stage of reproductive failure, it is
assumed that the loss of function occurred in the early
stages of pre- and post- implantation development since no
aborted fetuses were detected in the study.

The similarity of average litter size and pup weight
in litters born of aged compared to young females in this
study suggests an "all or none" type of interference with-
reproduction. Rather than producing litters of smaller size
or impairing the growth of fetuses, age effects on reproduc-
tion in the Long—Evans rat seem to be manifested in total
loss of reproductive function. This observation is consis-
tent with our observation of similar "all or none" effects
of stress on post-ovulatory reproduction in this strain of
rats (Euker and Riegle, 1973). Our findings conflict with
the reports of Asdell, et a1. (1974) and Ingram, Mandl, and
Zuckerman (1958). These studies showed similar age-related
decreases in numbers of rats littering as found in our work.
However, they report decreased average litter size in the
aged rat. These findings suggest significant strain dif-
ference in this variable in the aging rat or that the rela-

tively small numbers of older rats producing litters in this

104

study may not be representative of a larger population of
aging rats.

D. Temporal Changes in Serum

Progesterone in Aging Female Rats

Aging changes in the reproductive control system are

well documented in lnemmalian females. During the past
several years, we and others have considered the effect of
aging on the 'hypothalamic-pituitary-ovarian axis in the
laboratory rat. Although the postmenopausal human ovary
does not contain sufficient number of normal oocytes to
maintain reproductive function (Novak, 1970), the aged rat
ovary' has ‘been shown to retain. considerable .numbers of.
oocytes throughout the lifespan of the rat (Mandl and
Shelton, 1959). Since the rat loses normal reproductive
capacity at 9-12 months of age (Miller, et al., 1979) the
decline in oocyte numbers does not appear to be the primary
factor involved with the failure to reproduce.

The aging female rat is characterized by loss of regular
ovarian cycles (Huang and Meites, 1975; Aschheim, 1976; Lu,
et al., 1979) with increased incidence of constant estrous
and repetitive pseudopregnant patterns of vaginal cytology.
The aged constant estrous rat will return to regular ovarian
cycles following several types of hypothalamic stimulation
(Meites, et al., 1978). Although we previously showed that
hypothalamic extracts from the aged rat have similar LHRH
activity as those from young rats (Miller and Riegle, 1978),

the hypothalamic-pituitary unit of the aged rat was less

105
responsive to castration-induced decreases in gonadal ster-
oids (Shaar, et al., 1975). These data have been inter-
preted to indicate decreased hypothalamic sensitivity to
control inputs with age (Meites, et al., 1978; Riegle and
Miller, 1978).

Secretion of gonadotropin by the hypothalamic-
hypophyseal unit is influenced by steroid hormones. The
negative feedback relationship between gonadal steroids and
gonadotropin secretion is well established. However, in-
creased gonadotropin secretions in response to LHRH stimula-
tion have been induced by controlled increases in both
estrogen and progesterone in intact proestrous and gonadec-i
tomized rats (Vilchez-Martinez, et al., 1974; Aiyer, et al.,
1976). We and others have consistently shown decreased
serum gonadotropin concentrations in intact and gonadectom-
ized aged female rats which could be related to reductions

in sex steroid availability. The purpose of the present

Study was to measure temporal changes in serum progesterone
in young and aged cycling rats and in aged constant estrous,
Pseudopregnant and diestrus female rats in order to begin to
identify changes in the pattern of sex steroid secretion
Occurring with age which may be related to alterations in

Ovarian cycles and decreased fertility.

Ma terials and Methods
The first experiment was designed to measure serum

Progesterone concentration at 1600 hours in aging rats

106
during proestrous, estrous and diestrous day two stages of
their ovarian cycle. Rats included in this study were 3,
12, 18 or 24 months of age (n = 12/group). Only rats with
at least two previous regular ovarian cycles were used in
the study. Repetitive blood samplings were scheduled at
least five days apart.

The second experiment considered temporal serum proges-
terone and LH changes during the proestrus hormone surge.
Serial blood samples were taken from groups (n = 12/group)
of young (3 mo.) and aged (12 - 24 mo.) regularly cycling
proestrous rats at 1400, 1800, 2200 and 0200 hours.

Temporal serum progesterone concentrations were also.
measured in the third experiment. Groups of twenty young (3
mo.) and twenty aged (12 — 24 mo.) regularly cycling rats at
proestrous, estrous and diestrous day two stages of their
ovarian cycles were randomly divided into two groups. In an
effort to minimize blood sampling effects on neuroendocrine
control mechanisms, serial blood samples were collected from
one subgroup at 0400, 1200, and 2000 hours and from the
other subgroup at 0800, 1600 and 2400 hours.

In addition, temporal serum progesterone was measured in
groups of aged constant estrous, pseudopregnant and dies-
tJ’-"<>us rats. Rats were considered to be constant estrous if
t-1'1ey had at least eight consecutive days of cornified vagi-
rial smears. The pseudopregnant group consisted of rats
having between eight and twelve consecutive days of leuco-

thic vaginal smears which followed one or two days of

107

cornified vaginal cytology. The diestrous rats had at least
twenty-four days of consecutive leucocytic vaginal smears
without cornification. Thus, groups of twenty twelve month
old and twenty twenty-four month old constant estrous,
sixteen twenty-four month old pseudopregnant, and sixteen
twenty-four month old diestrous rats were also divided into
subgroups and serially bled at either 0400, 1200, and 2000
‘hours or at 0800, 1600, and 2400 hours.

Differences within and between age groups were tested by
multivariant analysis of variance and analysis of variance
for repeated measurements. Only differences with a proba-

bility of error of less than 0.05 were considered signifi—

cant .

Results

Figure 14 illustrates serum progesterone concentration
in 3, 12, 18, and 24 month old rats at 1600 hours on pro-
estrous, estrous, and diestrous day two stages of their
Ovarian cycles. Mean progesterone concentrations were
higher in the three month old group than in any of the aged
groups at all three stages of the ovarian cycles. Serum
Progesterone concentrations were not different among any of
the three higher aged groups at this sampling time.

We next considered the effect of age on the increase in
Serum progesterone which accompanies the proestrous hormone
8Llarge (Figure 15). Serum progesterone was increased on the

afternoon of proestrus in both age groups. The magnitude of

108

”T Proestrus Estrue Dlestrue

Progesterone nglml

 

 

 

 

 

 

 

 

   

a 12 u a a
AGE. MONTHS

Figure 14. The Effect of Age on Serum Progesterone at Pro—
estrous, Estrous, and Diestrus Day 2 Stages of the Ovarian
Cycle. Progesterone is expressed as the group mean (ng/ml
serum) with the indicated standard errors of the means from
blood samples collected under light ether anesthesia at 1600
h in 3, 12 and 18, and 24 mo. old rats (N = 12/group).

109

PROESTRUS

 

Progestetone nglml

 

 

 

 

 

 

 

 

 

TIME

Figure 15. Serum Progesterone Concentrations During the Pro-
estrus Hormone Surge in Young and Aged Female Rats. Proges—
terone is expressed as the group mean (ng/ml serum) with the
indicated standard errors of the means from blood samples
collected under light ether anesthesia at 1400, 1800, 2200,
and 0200 h in 3 and 12 to 24 mo. old rats (n = 12/group).

110

the increase and the proportional increase over basal pro-
gesterone levels were both greater in the young than in the
aged group. The sustained increase in progesterone at 0200
hours also indicates a longer duration of elevated proges-
terone in young compared to aged proestrous rats. Serum LH
concentration from these young and aged groups during the
proestrous hormone surge are shown in Figure 16. The effect
of age on LH concentrations was similar to that for proges-
terone. Although both age groups had increased LH during
proestrus, the increase was greater in the young than in the
aged group.

Temporal changes in serum progesterone concentration ati
four hour intervals from proestrous, estrous, and diestrous
day two young and aged rats are shown in Figure 17. The
changes in progesterone during proestrous are similar to the
data from the second experiment. Serum progesterone was
similar between aged groups at 0400, 0800, and 1200 hours.
The increase in progesterone at 1600, 2000, and 2400 hours
was much greater in the young compared to the aged group.
The higher progesterone concentrations in the young compared
to aged groups was sustained through the estrous stage of
the ovarian cycle. Mean serum progesterone levels were of
greater magnitude in the young than in the aged group during
estrous at all sampling intervals except at 2000 hours.
Additionally the young group showed a mid-day increase in
progesterone which was not detected in the aged group. Both

age groups showed a surge in serum progesterone on diestrous

lll

PROESTROUS, CYCLING RATS

1500

m 3 m0! “3‘2

12'16 m0, ":12

 

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LH nulml

 

 

 

 

 

 

 

 

 

2 pm 6 pm
TIME

Figure 16. Serum LH Concentrations

During the Proestrous
Hormone Surge .

LH is expressed as the group mean (ng/ml
serum) with the indicated standard errors of the means from

serial blood samples collected under light ether anesthesia.

Figure 17. Temporal Changes in Serum Progesterone in Young
and Aged Female Rats at Proestrous, Estrous, and Diestrus
Day 2 Stages of Their Ovarian Cycles. Progesterone is
expressed as the group mean (ng/ml serum, N = 10) with the
indicated standard errors of the means from serial blood
samples collected under light ether anesthesia at 0400,
0800, 1200, 1600, 2000 and 2400 h (N = 10/group).

112

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113
day two which was of greater magnitude and of longer dura-
tion in the young group.

The patterns of serum pmogesterone concentration found
in both young and aged cycling rats were not found in any of
the noncycling aged groups (Figure 18). Serum progesterone
concentrations were uniformly low in both twelve and twenty-
four month old constant estrous groups. Mean progesterone
concentrations were higher in aged repetitive pseudopregnant
and constant diestrous rats than progesterone levels during
estrous or diestrous day two in young or aged cycling rats.
However, progesterone levels were not significantly differ—
ent between aged (twenty-four month) rats classified as
repetitive pseudopregnant or during the leucocytic period of
those designated constant diestrus by vaginal cytological

characteristics.

Discussion

The results of these experiments indicate substantial
alterations in serum progesterone with age in cycling rats.
The aged cycling rat had lower progesterone concentrations
at most stages of the estrous cycle which were sampled in
this study. The aged groups had smaller increases in serum
progesterone and shorter duration of increased progesterone
concentrations during both the proestrous and diestrous day
two hormone surges. This decrease in serum progesterone
could contribute to age-related decreases in reproduction in

the rat and could be associated with the decrease in LH

114

CONSTANT ESTRUS

 

 

 

 

 

 

 

  

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E
3 ESE 24 mo
e
C
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o
2
n. Run-1': r—‘Fﬂ I‘M-bl: l ' I .--- "
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Figure 18. Serum Progesterone Concentrations in Noncycling
Constant Estrous, Pseudopregnant, and Constant Diestrous
Aged Female Rats. Progesterone is expressed as the group
means (ng/ml serum) with the indicated standard errors of
the means from serial blood samples collected under light
ether anesthesia at 0400, 0800, 1200, 1600, 2000, and 2400 h
from 12 and 24 mo. old constant estrous (N = 10) and 24

month old pseudopregnant and constant diestrous (N = 8)
rats.

115
secretion found in these studies and commonly associated
with aging in the rat.

It is agreed that the rat and most other rodent species
retain considerable numbers of oocytes long after they fail
to successfully reproduce (Mandl and Shelton, 1959; Talbert,
1978). However, the number of follicles that develop to the
vesicular stage is somewhat related to the numbers of pri-
mordial follicles remaining in the ovary (Erickson, 1966)
which can limit the numbers of follicles maturing and form-
ing potential ovulatory oocytes (Talbert, 1976). Although
reproductive failure does not occur in the rat because of a
lack of oocytes, defective oocytes or changes in ovulatory-
conditions could contribute to the inability of the aged
female to reproduce. The development of more large non—
ovulatory follicles has been hypothesized to be responsible
for the secretion of additional estrogen which could be
related to alterations occurring with age in the development
of the reproductive tract and the control of gonadotropin
secretion (Foote, 1967).

The origin of the proestrous increase in progesterone
and its relationship to the proestrous gonadotropin surge
has received considerable attention. Several studies have
shown an adrenocortical role in the increased progesterone
secretion during proestrus (Mann and Barraclough, 1973a;
Shaikh and Shaikh, 1975). Increased adrenocortical pro-

gesterone secretion has been shown to occur before the

proestrous gonadotropin surge (Shaikh and Shaikh, 1975).

116
The increase in serum progesterone, on the morning of pro-
estrous, has been shown to facilitate gonadotropin secretion
and induce earlier mating behavior and ovulation in the rat
(Nequin, et al. 1975) and to cause LH release in Nembutol
blocked rats (Kobayashi, et a1. 1973). It is also clear
that increased estradiol concentrations can augment pitui-
tary responsiveness to LHRH (Vilchez-Martinez, et a1. 1974).
It has recently been reported that increased blood estra-
diol, presumably of ovarian origin can increase the duration
of the adrenal progesterone secretion during proestrus in
the rat (Campbell, et a1. 1977). On the other hand, this
study also showed a stress-induced increase in adrenal.
estradiol secretion in proestrous but not in metestrus

cycling rats suggesting that the proestrous hormone milieu

can have specific effects on adrenocortical steroidogenesis.

On the other hand, adrenalectomized rats have normal
estrous cycles and ovulate (Feder, et al. 1971). Decreased
progesterone in intrauterine device-bearing hamsters caused
a delay in LH secretion (Saksena and Shaikh, 1974) and the
critical period for LH release was extended in adrenalectomr
ized rats (Feder, et al. 1971). Interruption of the pro—
estrous LH surge with Nembutol also affects the increase in
progesterone during proestrus, indicating that the ovulatory
LH surge increases ovarian progesterone secretion during and
after the peak of gonadotropin release (Everett, 1967). It
has been proposed that the primary role of adrenocortical

progesterone secretion is to facilitate LH secretion and

117
estrous behavior at specific time intervals (Mann and
Barraclough, 1973b).

These findings suggest several possible variables in
steroid—gonadotropin interaction that could affect repro-
duction in the aging rat. The smaller increase in serum
progesterone concentration during diestrous day two and
proestrus could reflect an age—related decrease in adrenal
or ovarian progesterone secretion with age. Additionally,
the decrease in both magnitude and duration of the proges-
terone increase could be due to interactions with undetected
changes in adrenocortical or ovarian estrogen secretion in
the aged rats. The reported effects of decreased proges-i
terone secretion on gonadotropin control systems are con-
sistent with the decrease in basal serum LH levels and the
reduced proestrous LH surge in the aged rats from this
study. This reduction in serum LH is in agreement with
previous reports of smaller increases in LH secretion
following acute LHRH injections or gonadectomy (Meites, et
al. 1978; Riegle and Miller, 1978; Gray and Weler, 1980) in
the aged compared to the young female rat.

It is not clear whether the reduced progesterone con-
centrations found on diestrous day two are characteristic of
post-coital progesterone secretion. There is considerable
evidence of decreased uterine capacity to sustain pregnancy
in aging rodents. An increase in degenerate-appearing
corpora lutea has been associated with decreased numbers of

implantation sites in mice (Harmon and Talbert, 1970) and

118
the pregnancy rate of mice has been improved by progesterone
injections on days 2-9 of pregnancy (Gosden, 1975). How-
ever, no decrease in blood progesterone was found in aged
pregnant rabbits (Spilman, et al. 1972) and we recently
reported no differences in serum progesterone between young
and aged rats on days one through six and on days eleven and
sixteen of pregnancy (Riegle and Miller, 1978; Section 2 E

of this thesis).

These findings suggest that if differences in proges-
terone availability contribute to loss of fertility in the
aging rat, the effects are related to the control of gonado-
tropin secretion which stimulates ovulation or involve the
ability of the uterus to respond to progesterone. This
latter hypothesis could reflect differences in progesterone
receptors. In addition, estradiol has been shown to induce
uterine progesterone receptors (Feil, et al. 1972). There-
fore, a pre-existing estrogen deficiency could markedly
alter uterine responsiveness to circulatory progesterone.

With increasing age, rat estrous cycles become irregular
and increasing proportions of aging female rats develop a
constant cornified vaginal cytology, commonly referred to as
the constant estrous state (Aschheim, 1976). A large
variety of treatments, including the use of certain drugs,
hormones and stress, have been shown capable of restoring
vaginal cytologic changes characteristic of ovarian cycles
in these rats (Finch, 1978; Lehman, et a1. 1978). Although

it is agreed that aged constant estrous rats are

119

anovulatory, the alterations in the neural, pituitary,
gonadal mechanisms which account for this endocrine state
remain unclear. Our results confirm the previous hypothesis
that progesterone secretion was reduced in the senile con-
stant estrous rat (Weisz and Lloyd, 1965). Several investi—
gators have shown an increase in the incidence of ovulatory
ovarian cycles in constant estrous rats following proges-
terone injections (Everett, 1940; Meites, et. al. 1978).
However, at this time it is not clear whether the effective-
ness of progesterone is due to increased hypothalamic LHRH
secretion, increased pituitary responsiveness to LHRH,
progesterone effects on hypothalamic monoamine availability-
or other neuroendocrine mechanisms.

The similarity of serum progesterone concentrations
which we found in repetitive pseudopregnant and constant
diestrous rats was not expected. The ovarian cycles of
aged pseudopregnant rats have previously been shown to be
ovulatory followed ‘by development of functional corpora
lutea (Huang and Meites, 1975; Lu, et al., 1979). Although

the presence of corpora lutea was not ascertained in this

study, it is presumed that the pseudopregnant state in these
rats was due to luteal maintenance of high serum proges-
terone concentrations. Our data do not support the hypo-
thesis that the aged rats classified as pseudopregnant by
vaginal cytologic criteria. consistentlyr ovulate and. form

functional corpora lutea. We found serum progesterone

concentrations in these rats to be highly variable. Average

120

progesterone concentrations for the group classified as
pseudopregnant was considerably less than the 40-100 ng/ml
concentrations we and others (Feder, et al. 1971) have
reported in induced pseudopregnancy in younger aged groups.
Although some of these rats had high progesterone concen-
trations typical of pseudopregnancy in younger rats, others
had low progesterone which could suggest failure of ovula-
tion and luteinization. Resolution of this discrepancy will
require further measures of progesterone secretion coupled
with anatomical observation of ovarian tissues or establish-
ment of the magnitude or the nocturnal-diurnal pattern of
prolactin secretion in these rats classified. as ;pseudo-
pregnant on the basis of vaginal cytological changes.

On the other hand, constant diestrous rats do not show
evidence of estrous cycles or ovulation by changes in
vaginal cytology. It has been assumed that these rats would
have minimal serum progesterone concentrations. However,
our data suggests significant progesterone secretion, pre-
sumably from adrenocortical or nonluteal ovarian sources.
Our findings also suggest that vaginal cytological data may
not always clearly indicate the functional state of the
hypothalamic hypophyseal-ovarian—axis.

In summary, these experiments demonstrate that signifi-
cant decreases in serum progesterone concentration occur
during the estrous cycle of aging female rats. In addition,
progesterone concentrations were reduced in aged constant

estrous rats and generally elevated in experimental groups

121
identified as pseudopregnant or constant diestrous. These
data suggest that alterations in progesterone secretion
could contribute to the decline in gonadotropin secretion,
ovulation and fertility in aging rats which have sustained
their ovarian cyclicity and decreased progesterone secretion
could be associated with the induction of aberrant ovarian
cycles and the absence of ovarian cycles in aging female
rats. These experiments do not indicate whether the primary
alteration between sex steroid secretion and gonadotropin
control mechanisms involve failure of gonadal and adreno-
cortical steroidogenic tissues to secrete adequate hormone
which facilitates gonadotropin release or a failure of.

gonadotropin stimulation of progesterone secretion.

E. Serum Progesterone During
Pregnancy and Pseudopregnancy and
GEStation Length in the Aging Rat

 

Decreased reproductive function is well documented in
aging laboratory rats and other mammalian species (Arvay,
1976 and Talbert, 1978). During the past few years we and
others have considered the effect of aging on the
hypothalamic-pituitary-gonadal control system in the labor-
atory rat. Although postmenopausal women are characterized
by substantial increases in serum gonadotropins (Odell and
Swerdloff, 1968) which occurs as a consequence of reduced
ovarian steroid secretion, the aging rat has reduced serum
LH and increased prolactin concentrations when compared to

young rats with normal reproductive function (Riegle and

122

Miller, 1978). The aged rat has reduced serum LH concen-
trations following a single LHRH stimulation (Riegle and
Meites, 1976; Watkins, et al., 1975) and smaller LH in—
creases after gonadectomy (Shaar, et al., 1975; Gray and
Wexler, 1980). However, we found similar LH concentrations
in young and aged rats following multiple LHRH injections
(Miller and Riegle, 1978, Section 2 B of this thesis) which
is in agreement with an experiment studying pituitary re-
sponsiveness to LHRH in aging C57BL/56 mice (Finch, et al.,
1977). These data suggest that the aged female rat retains
sufficient pituitary function to stimulate ovarian function
and that alterations in hypothalamic gonadotropin control.
systems could contribute to changes in ovarian cycles and
the decreased fertility characteristic of this species.

To date, most studies of aging effects on laboratory
rodent female reproductive control systems have considered
changes in very aged (after 24 months of age or older)
animals with obvious alterations in ovarian cycles. How-
ever, rodents characteristically begin to show a reduced
ability to reproduce at a much earlier age, when most are
still exhibiting regular estrous cycles (Thung, et al.,
1956). Several studies have shown that reproductive failure
first occurs in rodents while their ovaries contain con-
siderable oocytes (Mandl and Shelton, 1959), and they are
capable of producing normal numbers of oocytes at ovulation
(Harman and Talbert, 1970). Oocyte transfer experiments

have shown that eggs from old donors are capable of normal

123
development when transplanted into young recipients (Finch,
1978) suggesting that uterine failure contribues to the
decline in fertility. Morphological studies (Harman and
Talbert, 1970) have suggested that an increase in
degenerate-appearing corpora lutea may be associated with
implantation failure in aged mice. In addition, some in-
vestigators have found improved reproduction in aging mice

receiving progesterone treatments (Gosden, 1975). These

studies suggest that decreased progesterone secretion could
contribute to the failure of the uterus to sustain pregnancy
in aged mammals.

The purpose of the present study was to consider the.
effect of increasing age on serum progesterone concentra-
tions in mated normally cycling female rats and in aged

constant estrous rats made pseudopregnant by mating.

Material 5 and Methods

The reproductive status of the rats were established by
daily vaginal lavage. Only rats with at least two consecu-
tive four or five day ovarian cycles were considered to be
regularly cycling. Rats maintaining at least eight days of
c(Dnsecutive cornified vaginal smears were considered to be

in the constant estrous state. Rats to be mated were indi-

Viciually placed in a cage of two males (four to eight months
of age) in the late afternoon on the day of proestrus or

designated day of constant estrus. Successful mating was

124
determined by the presence of sperm in the vaginal lavage
performed the following morning.

The first experiment considered the effect of pregnancy
and pseudopregnancy on serum progesterone in separate groups
of cycling rats at 4, 7, 9, ll, 13, 15, 20 and 22 months of
age and groups of constant estrous rats at 11, 15, 20 and 22
months of age. Serum progesterone was measured in serial
blood samples taken between 2 and 4 p.m. on days 1, 6, 11,
and 16 after mating. All rats were maintained in the colony
for the entire gestation period. The number of pups born
and parturition time was recorded for each rat successfully
completing gestation. Most young rats in our colony deliv—
ered their litters in the late afternoon of the twenty-
second day of gestation. In this study 6 p.m. was desig-
nated as the end of the twenty-second day of gestation.
Pregnant rats were checked for the presence of pups at
twelve hour intervals to estimate parturition time.

The second experiment considered the effect of age on
serum progesterone concentration during early and late

gestation. In this study progesterone was measured in

serial blood samples taken from young (4 mo., n 14) and
aged (13 - 16 mo., n = 26) cycling rats at 4 p.m. on days 1,
2, 3, 4, and 5 of early pregnancy and at 8 a.m. on days 19,
20, 21, and 22 of pregnancy. Daily vaginal smears were
taken from all rats throughout their expected gestation

period. Five of the aged rats resumed estrous cycles be-

tween days fourteen and nineteen of their gestation period

125

and were not included in the rats serially bled in late
gestation. The number of pups born and parturition time was
recorded for each rat successfully completing gestation.
Results

The effects of increasing age~cn1 successful pregnancy,
mean litter size and average gestation length in groups of
regularly cycling rats ranging from four to twenty-two
months of age are illustrated in Table 9. The effect of age
on the proportion of rats producing litters was apparent by
eleven months of age. In this study, no successful repro-
duction was obtained in rats older than thirteen months.
Increased maternal age was also associated with increased'
gestation length. Average gestation intervals progressively
increased from 22.2 days in four month old rats to 23.7 days
in the thirteen month old females.

Changes in serum progesterone on days 1, 6, 11, and 16
of pregnancy and pseudopregnancy are plotted in Figures 19
and 20. The data are shown on histograms which include
progesterone concentrations in mated cycling rats which
delivered litters, mated cycling rats which did not deliver
litters (considered pseudopregnant), and aged mated constant
estrous rats made pseudopregnant by mating. Average serum
progesterone concentrations on day one of pregnancy ranged
from 1.8 to 9.7 ng/ml and were not different between cycling
rats ‘which littered, cycling rats that, did not. produce
litters and mated constant estrous rats. Average proges-

terone concentrations were markedly increased in all groups

 

126

---- ---: o m mm

 

IIII till 0 m ON
.11. ---: o «H ma
p.mm >.v m NH ma
m.mm ¢.m w ¢H HH
H.MN N.h OH ma m
m.m~ m.oH ma ¢H h
N.NN ®.HH AH NH w
Ammwpv coﬂumumow ouwm Hopped ooumuqu .02 tops: .02 AOEV om<

 

mumm mHmth mcﬂﬂohu CH hocmcmoum so mm¢ mo uoommm

m mam¢9

Figure 19. Serum Progesterone Concentrations from Groups of
Aging Female Rats on Days 1 and 6 After Mating. Each age
group is subdivided into rats with regular ovarian cycles
that produced litters (Cycling, Littered), those with
regular ovarian cycles that did not produce litters
(Cycling, Pseudopregnant), and mated constant estrous rats
(Constant estrous, Pseudopregnant). Serial blood samples
were collected between 1400 and 1600 h under light ether
anesthesia. Progesterone is expressed as group means (ng/ml
serum) with indicated standard errors of the means.

127

DAY 1
I ICycllng,thtered
I ICyollng,Peeudopreg.
on I IConetent Estrous.
Peeudopreg.
E
S
2 4o
2
2
8
C
e
3 so
:

 

 

 

:rgrymrrgr‘ammmmrﬂmm

O
AGE, MONTHS

menu”. thtered
I—ICycung, Peeudopreg.

meow-m Eetroue,

Peeudopreg.

W DAYS

 

-

E eo‘

\ ~ . x

O 9&5;

c . I.
x H»,
\\ \\
\\

 

Progeeterone
g
I.
ﬂ}-
I.
I
:‘I—
:F.

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, 1’ , ,v , .I‘.
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' ’1 M4 4 x 411‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4 7 e n n n 1: t3 1: 15 20 20 22 22
AGE. MONTHS

Figure 19.

Figure 20. Serum Progesterone Concentrations from Groups of
Aging Female Rats on Days 11 and 16 After Mating. Each age
group is subdivided into rats with regular ovarian cycles
that produced litters (Cycling, Littered), those with
regular ovarian cycles that did not produce litters
(Cycling, Pseudopregnant), and mated constant estrous rats
(Constant estrous, Pseudopregnant). Serial blood samples
were collected between 1400 and 1600 h under light ether
anesthesia. Progesterone is expressed as group means (ng/ml
serum) with indicated standard errors of the means.

 

 

 

 

 

 

 

 

 

 

 

128

I ICycIlng. thtered

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

00‘ DAY 11
E -_ ICycllng, Peeudopreg.
meant Eetroue. FL
Peeudopreg.
en
5 ea
2
. FL rL \ 1
§ .1. * 3*
s .. 1 ..L.
g #‘ +7 1%
1 55:5; r
20
f g
- 4 7 e 11 11 11 13 1: 1s 1s 20 20 22 22
AGE. NONTHS
I I6 cll . thtered
on 10 " “9
eo I ICycllng. Peeudopu'eg.
mConetent Eetroue.
J1 Poeudopreg.
ea

Progeeterone no! 1n!
5

 

 

 

 

 

 

 

 

 

 

.L

 

 

 

 

 

 

 

 

 

[‘1

 

Figure 20.

 

1a 1: 1s 15 2o 20 22 7 22
ACE , MONTHS

129
by day six of pregnancy or pseudopregnancy. Although there
were substantial variations between individual age group
means, progesterone concentrations were not consistently
different between littering-cycling females, nonlittering-

cycling rats and constant-estrous-pseudopregnant groups.

The similarity between groups and the absence of age
associated variations in serum progesterone was still
apparent in the data from day eleven after mating. No
differences were found in progesterone between littering
groups, cycling-pseudopregnant or constant-estrous-
pseudopregnant groups. The average progesterone concen-
tration on day eleven of pregnancy from the 43 rats that-
subsequently littered was 55.2 ng/ml. The 64 rats of the
cycling and constant estrous pseudopregnant groups at the
same interval after mating averaged 48.0 ng progesterone/ml.

By day sixteen after mating, average progesterone con—
centrations were higher in the groups producing litters than
in the pseudopregnant groups (P<.05). Average progesterone
concentrations were 60.8 ng/ml on day sixteen in the preg-

nant groups, but were decreased to only 25.7 ng/ml in the

cycling pseudopregnant group, and 24.0 ng/ml in the constant
estrous pseudopregnant group.

The second experiment considered the effect of age cu:
serum progesterone in early and late pregnancy. Average
serum progesterone concentration from regularly cycling

mated young and aged groups are shown as a function of the

days of early pregnancy in Figure 21. Serum progesterone

130

1001
I IYoung, emo, n.“ I
W [—1 _J_
Aged,13-1Bmo, n: 21

h

2f Him H,“ H

Days of Pregnancy

Progeeterone ng/ml
‘
o

 

 

 

 

 

 

 

 

 

 

Figure 21. Serum Progesterone Concentrations from Young and
Aged Female Rats on Days 1, 2, 3, 4 and 5 After Mating.
Serial blood samples were collected at 1600 h under light
ether anesthesia. Progesterone is expressed as group means
(ng/ml serum) with the indicated standard errors of the
means.

131

concentrations were progressively increased with developing
pregnancy in both aged groups. Average serum progesterone
concentrations were not different between the two age groups
during this interval. Although the mean progesterone con-
centration of the young group was somewhat higher than that
of the older group on day two, elevated progesterone on day
two was not related to successful pregnancy in the aged
rats. Progesterone concentrations averaged 27.2 ng/ml in
the twelve rats in the aged group that produced litters,
whiCh was not different from the average progesterone level
in the non-littering pseudopregnant rats from this group
(21.4 ng/ml).

Eleven of the mated aged rats from the second experiment
(Figure 22) did not produce litters but maintained consis-
tently elevated progesterone concentrations sufficient to
block resumption of regular ovarian cycles. Factors in-
volved with continued progesterone secretion (presumably of
luteal origin) in these rats were not identified in this
study. However, the duration of pseudopregnancy in this
group did exceed that normally induced by cervical stimula-
tion alone, inferring possible corpora lutea stimulation by
uterine factors, which suggests postnidation reproductive
failure in this group.

Changes in serum progesterone on days 19, 20, 21, and 22
of pregnancy are plotted in Figure 22. Average progesterone
concentrations were decreased in late pregnancy in both the

young group and the aged group which produced litters. The

132

Pnocssrsnons LATE-GESTATION I |V°“""”"'"° m“

I IAged. Uttered n:12

En: I Aged, No Litters M11

60

Progesterone rug/ml

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

1e 20 2‘
Dey ot Geetetlon

Figure 22. Serum Progesterone Concentrations from Young and
Aged Female Rats on Days 19, 20, 21 and 22 After Mating.
The aged group was subdivided into a group that produced

1 itters and another group that did not . Serial blood
sampl es were col 1 ected at 0800 h under 1 ight ether anes—
thesia . Progesterone is expressed as group means (ng/ml

serum) with indicated standard errors of the means.

133
reduction in progesterone on day twenty-two of pregnancy was
greater in the young than in the aged group whidh produced
litters. The aged group which did not produce litters had
similar progesterone concentrations at each blood sampling
interval. Average length of gestation was 22.1 days in the

young group and 22.9 in the aged group.

Discussion

These experiments suggest that alterations in proges—
terone availability to the uterus are not the primary reason
for reproductive failure in the aging rat. The maximal age
of successful reproduction in the rat in these studies was
sixteen months. We and others (Jones and Krohn, 1959:
Miller and Riegle, 1979) have shown that the failure to
reproduce precedes the termination of estrous cycles in
aging rats and mice. Alterations in oocyte viability in
aging mammals may be related to decreased fertility. How»
ever, several studies have suggested no differences in
oocyte survival when eggs from young and aged donors were
transplanted into young host uteri (Talbert and Krohn, 1961;
Mauer and Foote, 1971) and drastically reduced egg survival
when oocytes were transplanted from young donors to aged
uterine environments.

These findings suggest that failure of the aged uterus
to sustain gestation contributes to the decline in fertility
of aging female mammals. Experiments considering whether

this uterine failure is due to changes in hormonal support

134

or aging effects in uterine function have produced conflict—
ing data. Reduced decidualization responses have ‘been
reported from ovariectomized estrogen and pmogesterone
primed aging mice and hamsters compared to decidualization
in young adult control animals (Finn, 1966; Blaha, 1967).
The decline in fertility of aging mice has been associated
with morphological evidence of corpora lutea degeneration
(Harman and Talbert, 1970). Other experiments have shown
smaller corpora lutea of pregnancy in aged mice and hamsters
compared to young controls (Thorneycroft and Soderwall,
1969; Gosden, 1974).

The possibility of reduced luteal progesterone secre—l
tions contributing to loss of fertility in aging is sup-
ported by experiments showing increased implantation rates
in aging hamsters which have received young ovarian implants
beneath the kidney capsule (Blaha, 1970) and increased
survival of fetuses in mice receiving progesterone injec-
tions on days two through nine of pregnancy (Gosden, 1975).

The results of the present study do not support the
hypothesis of reduced corpora lutea progesterone secretion
during pregnancy in aged rats and suggest that the cycling
or constant estrous female rats of any age can form func-
tional corpora lutea. Similar serum progesterone concen-
trations were found from days one to eleven after mating in
aging rats with regular estrous cycles that did not produce
normal litters, aged constant estrous groups which did not

produce litters, and the younger age groups which produced

135
normal litters. In addition, these data indicate substan-
tial differences in serum progesterone from pseudopreg-
nancies induced by cervical stimulation in this study com-
pared to progesterone concentrations in similarly aged
females showing spontaneous vaginal cytology which has been
classified as persistent pseudopregnancy, as described in
the previous section of this thesis. In the rat, cervical
stimulation by mating on the evening of proestrous estab-
lishes two daily surges of prolactin secretion, the noc-
turnal surge from 3-5 a.m. and a diurnal surge from 4-7
p.m., which will support luteal secretions characteristic of
pregnancy or pseudopregnancy (Freeman and Neill, 1972; Smith-
and Neill, 1976). The results of this study indicate that
coital stimulation of constant estrous female rats induces
similar effects on endocrine control systems. The luteo-
tropic function of prolactin in the mated rat is augmented

by LH stimulation of luteal cells which is obligatory for

normal progesterone secretion between days seven and twelve
after mating and is replaced by placental luteotropic secre-
tions in the latter half of pregnancy (Morishige and
Rothchild, 1974).

The similarity of serum progesterone during the early
postcoital period in groups producing litters and those not
producing litters suggests that alterations in neuroendo-
crine factors related to pituitary hormone stimulation of

luteal tissue formation and function were not primary causes

for infertility in the aged groups. Reports from our

136

laboratory ‘have previously indicated decreased serum LH
concentration after ovariectomy in aged compared to young
female rats (Shaar, et al., 1975) and decreased sensitivity
of the hypothalamic-pituitary unit to L-dopa inhibition of
prolactin secretion in the aged female rat (Watkins, et al.,
1976). We and others have interpreted these and similar
data to indicate age-related alterations in hypothalamic-
pituitary sensitivity and responsiveness to control input in
the rat. Although we did not measure serum LB or prolactin
concentrations in these experiments, the results of this
study suggest that changes in hypothalamic-pituitary re-
sponsiveness to control input in the aging rat with regular)
ovarian cycles or in aging constant estrous rats are not
sufficient to interfere with pituitary stimulation of luteal
function. The decreased serum progesterone at postcoital
day sixteen in the groups which did not deliver litters
indicates that these rats did not have normal placental
luteotropic stimulation of progesterone secretion.

The design of this experiment does not allow determina-
tion of the stage of reproductive failure in the aging rat.
Failure of ovulation is not generally considered to be a
primary alteration of reproductive systems in aging rodents.
No change in post-ovulatory corpora lutea numbers have been
reported in aging mice (Harman and Talbert, 1970) or rabbits
(Adams, 1970). In a separate experiment we have assessed
the ovulation rate of twelve month old Long-Evans rats with

regular ovarian cycles by counting the numbers of eggs

137
recovered from the ballooned section of the fallopian tube
on the day of estrus. In our study, fifteen of eighteen
animals ovulated. The average number of eggs recovered from
ovulatory rats was 11.1 and was not different from three
month old rats. These data indicate that most rats are
continuing to ovulate at the age interval when fertility
rates are declining rapidly and support the hypothesis of
post-ovulatory fertility loss in aging rats. Coital cervi-
cal stimulation is believed to be sufficient to stimulate
prolactin secretion and maintain the nocturnal and diurnal

surges of prolactin secretion required for early luteal
function. The failure of normal uterine development results.
in a loss of corpora lutea support in the later stages of
pregnancy.

Average gestation length was increased about 1.5 days in
the aged rats in this study. This increase in gestation
length is in agreement with age effects on gestation lengths
in hamsters (Soderwall, 1960) and mice (Holinka, et al.,
1978). Increased gestation length could be associated with
reduced fertility in aging rats, since experimentally in-
duced prolonged gestation has been shown to cause fetal
mortality in mice (Kroc, et al., 1959) and rats (Moore,
1963). Serum progesterone concentrations in the present
study were similar on days 19, 20, and 21 of gestation in
young and aged rats which produced litters. Although aver-

age progesterone was decreased on day twenty-two of gesta-

tion in both age groups, the decline in progesterone was

138

less in the aged compared to the young group. This differ-
ence in the rate of decline of progesterone in late preg-
nancy in aging rats is in agreement with a report in aged
mice (Holinka, et al., 1978) and suggests that maintenance
of luteal progesterone secretion was related to the in-
creased length of gestation in the aged rat since a major
decrease in plasma progesterone is thought to be essential
for the onset of parturition in the rat.

Factors controlling reduction of luteal progesterone
secretion and the onset of parturition are poorly under-
stood. Changes in blood concentration or biological activ-
ity of a large number of hormones and uterine factors,-
including progesterone, estrogen, prostaglandins and fetal
glucocorticoids, have been implicated in controlling the
onset of parturition (Ryan, 1977). All these factors act at
least in part by decreasing serum progesterone concentra-
tions or decreasing the biological activity of progesterone.

Biological actions of hormones depend on target cell
receptor function as well as extracellular hormone concen-
trations. Estrogens have been shown to influence uterine
progesterone receptors (Leavitt, et. al., 1977). It is
possible that undetected alterations of estrogen secretion
are affecting progesterone function in late gestation in the
aging rat. The specific role of factors believed to in-
fluence the onset of parturition in the aging rat as well as
consideration of other prepartum gestational parameters such

as the time of implantation remains to be resolved.

139

In summary, these data indicate that the loss of fertil-
ity in the aged rat is not due to failure of luteal tissue
formation or progesterone secretion during early gestation.
Our experiments suggest anatomical failure of fertilization,
egg development or implantation or functional alterations in
the ability of the uterus to sustain pregnancy. In addi-
tion, we have shown increased gestation length in the aged
rat indicating age-related changes in the timing of endo-
crine changes regulating pregnancy in the rat. Identifica-
tion of the specific alterations in pregnancy control
systems which affect reproduction in the aged rat warrant

further consideration.

F. Endocrine Factors Associated
with the Development of the Constant
Estrous State in Aginngemale Rats

 

 

Decreased reproductive function with increased age is
especially well documented in the female laboratory rat and
inbred mouse (Arvay, 1976; Talbert, 1978; Finch, 1978). The
first effect of age on fertility in these species occurs at
a time when most females have normal, regular estrous cycles

(Thung, Boot and Muhlback, 1956), and substantial numbers of
oocytes in their ovaries (Mandl and Shelton, 1959) which
produce normal numbers of eggs at ovulation (Harman and
Talbert, 1970). Oocyte transfer experiments 'have shown
that eggs from aged hamster (Blaha, 1964), mouse (Gosden,
1974) and rabbit (Adams, 1970) donors have higher survival

rates when transferred to young compared to aged recipients.

140

These data have been interpreted to indicate significant
post-ovulatory decreases in the ability of aged females to
reproduce.

The aging female rodent also has significant changes in
the regulation of estrous cycles which is also thought to
contribute to the loss of fertility. Although some aging
rats retain essentially normal estrous cycles throughout
their lifespan, others show marked changes in ovarian cycles
beginning at about nine to ten months of age. At this age
increased numbers of rats show increased occurrence of
cornified vaginal epithelial cells typical of continuous
estrogen stimulation of this tissue (Huang and Meites, 1975;
Aschheim, 1976). This condition often persists for several
days or weeks and is commonly classified as constant
estrous. With increasing age, some rats show occasional
irregular 1-3 day intervals of cornified vaginal epithelium

and on the basis of vaginal lavage characteristics have been

classified as repetitive pseudopregnant. At more advanced
ages, increased proportions of rats show no evidence of
estrous cycles or periodic vaginal cornification and have
been classified as persistent diestrus.

Changes in reproductive control mechanisms associated
with the decline in fertility have received considerable
recent attention. Increased serum FSH concentrations have
been reported in ten to fourteen month old and twenty-four
month old constant estrous and pseudopregnant female rats

(Clemens and Meites, 1971; Wilkes, et al., 1978: Lu, et al.,

141

1979). On the other hand, no studies have shown increased
serum LH in the aged female rat. Previous experiments from
our laboratory (Shaar, et al., 1975; Watkins, et al., 1975)
and studies by other groups (Aschheim, 1976; Meites, et al.,
1978) indicate reduced serum LH and smaller increases in LH
secretion after gonadectomy or single LHRH injections in
aged compared to young female rats. On the other hand, aged
female rats receiving multiple LHRH treatments had increases
in serum LH which were similar to young rats receiving the
same treatment (Miller and Riegle, 1978) and aged rats can
be stimulated to ovulate (Meites, et al., 1978; Lehman, et
al., 1978).

To date, many of the experiments concerning age effects
on reproduction in the rat have considered very old (twenty-
four months) compared to young (two to four months) groups,
without giving major attention to intermediate ages when
fertility is first affected. One of the changes which
occurs at this early interval is the development of constant
estrous. Endocrine factors associated with the pattern of
vaginal cytology (8 or more consecutive days of cornified
epithelial cells) constant estrous state are not well under-
stood. The following experiments were designed to consider
changes in the reproductive control system associated with
the development and maintenance of this alteration in repro-

ductive function.

142
Materials and Methods

Rats used in these experiments ranged from eleven to
sixteen months of age. The reproductive status of the rats
was established by daily vaginal lavage. Only rats with at
least two previous consecutive four or five day ovarian
cycles were considered to be regularly cycling. Rats were
considered to be in the constant estrous state if they
showed at least eight consecutive days of cornified epi—
thelial cells in their daily vaginal lavage (the duration of
two normal estrous cycles).

The first experiment considered the effect of coital
stimulation of rats in the constant estrous state on LH
secretion and ovulation. In one study, a group of constant
estrous rats were individually placed in cages containing
two reproductively experienced six month old males at 7:30
a.m. Mating was verified by the presence of sperm in
vaginal lavages collected at noon. The ability of coital
stimulation to activate ovulatory mechanisms was determined
by the presence of oocytes in oviducts removed at 11 a.m. on
the following day. A second study was designed to consider
the possibility that the time sequence for ovulation could
be different in the constant estrous rat. In this study a
group of eighteen rats received coital stimulation as pre-
viously outlined. Evidence of ovulation in this group was
based on measurement of LH in blood samples collected at 3
and 5 p.m. on the day of mating and by the presence of

fetuses in the uterus on day eight following coital

143
stimulation. In addition to these experiments on rats in
the constant estrous state, ovulatory rate was determined by
oviductal egg collection at 11 a.m. on the day of estrus in
a group of eighteen, twelve month old rats with regular
estrous cycles.

Stress is one of the several factors which has been
employed to induce estrous cycles in constant estrous rats
(Finch, 1978). The basic experimental design utilized in
the remainder of this study was to interrupt constant es-
trous by subjecting rats to two hours of restraint stress
and then to measure serum progesterone and LH concentrations
during the following ovarian cycle ‘which. could lead to
ovulation and the formation of corpora lutea or the reinit-
iation of the constant estrous state.

The effect of the stress treatment on progesterone
secretion was determined by measuring progesterone in blood
samples taken before the stress was initiated and at the end
of the two hour restraint. In addition, progesterone con-
centrations were measured in blood samples collected at 4
p.m. on the first day of leucocytic cell-dominated vaginal
lavage following the stress and at 4 and 8 p.m. on the day
the stressed rats exhibited their first characteristic
proestrous ‘vaginal lavage following ‘the stress treatment
(the vaginal smear is dominated by' nucleated epithelial
cells). Similar blood samples were collected at the same

times on diestrous day one and on proestrous from similarly

aged rats which were maintaining regular estrous cycles.

144
The final experiment included in this study considered
the effect of progesterone treatment on the proestrous LH
surge. In this study separate groups of ten, fifteen month
old rats with normal estrous cycles received a subcutaneous

injection of 0, 0.5 or 2 mg of progesterone (Sigma Chemical

Company, St. Louis, Missouri) at 8 a.m. on proestrus. LH
was measured in blood samples collected at 5 and 8 p.m. on
proestrus.

Differences between groups was determined by analysis of
variance. Only differences with a probability of error of

less than 0.05 were considered significant.

Results

The effects of increasing age and reproductive state on
ovulation rates in these rats are illustated in Tables 10
and 11. Ovulation rates in 12 month old rats with normal
estrous cycles were similar to three month old controls.
Although the constant estrous rats all were successfully
mated, as determined by the presence of sperm in their

vaginal lavage, none of the rats exhibited normally bal-

looned oviducts or had oviductal oocytes on the day after
mating. In addition, only two of ten mated constant estrous
rats had implanted fetuses eight days after mating. The
mated rats showed a modest increase in sermm LH concentra-
tion at 3 and 5 p.m. after coital stimulation.

Figure 23 illustrates the effect of stress on serum

progesterone concentrations. The two hour stress treatment

145

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Effect of 2hr Stress on Serum Progesterone

In Aged Constant Estrous Hats

40

3O

1O

 

 

 

 

 

Before After
Stress Stress

Figure 23. The Effect of 2 Hour Stress on Serum Progesterone
in Aged Constant Estrous Rats. Serial blood samples were
collected under light ether anesthesia prior to and immed-
iately following the stress treatment. Progesterone .is

expressed as group means (ng/ml serum) with indicated
standard errors of the means.

148
increased serum progesterone from 8.0 ng/ml before the
stress to 34.2 ng/ml at the end of the treatment regime.
All 26 rats included in this group exhibited an interruption
in their estrous state. .After one additional post-stress
day of cornified cells in their vaginal lavage, the rats had
two to four days of leucocytic cells in their vaginal smear
which was followed by one day of nucleated cells typical of

proestrous and a resumption of the constant estrous state by
23 of the 26 rats. In summary, these rats had vaginal
cytological changes characteristic of a normal ovarian cycle
followed by presumed failure of ovulation and resumption of
the constant estrous state.

The character of the vaginal lavage following the stress
treatment suggests normal luteal function in these rats.
Average progesterone concentrations at 4 p.m. on apparent
diestrous day one from stress-interrupted constant estrous
rats and similarly aged normally cycling rats are plotted in
Figure 24. In this experiment, 44 of 47 rats receiving
stress treatments had their constant estrous state inter-
rupted. Progesterone averaged 44.0 ng/ml following the
stress treatment in these rats. Average progesterone con—
centration on apparent diestrous day one was only 4.8 ng/ml
which was less than diestrous day one progesterone in simi—
larly aged cycling rats (15.6 ng/ml) (P<0.01).

Serum progesterone and LH concentrations at 4 and 8 p.m.
on proestrous from stress interrupted constant estrous and

similarly aged, normally cycling rats are plotted in Figures

149

 

nglml

 

Progesterone

 

 

 

 

 

Conetent Eetrue Conetent Eetme Cycling
End of Streee Dleetrue 1 Dleetrue 1

Figure 24. Serum Progesterone Concentrations from Stress
Interrupted Constant Estrous Female Rats and Similarly Aged
Normally Cycling Female Rats on Diestrus Day 1. Blood
samples were collected under light ether anesthesia immed-
iately following the stress treatment and at 1600 h on
diestrus Day 1 in both the stress interrupted constant
estrous rats and the regularly cycling female rats of

similar age. Progesterone is expressed as group means
(ng/ml serum) with indicated standard errors of the means.

150

25 and 26. Average progesterone concentrations were higher
at both 4 and 8 p.m. (P<0.01) in the cycling compared to the
constant estrous stress-interrupted group. Average proges-
terone from the cycling group was 24.2 ng/ml at 4 p.m. and
58.3 ng/ml at 8 p.m. compared to 7.2 ng/ml at 4 p.m. and
15.7 ng/ml at 8 p.m. from the constant estrous group. LH
concentrations were similarly ‘higher in the same blood
samples collected from cycling rats compared to the constant
estrous group (P<0.01). Sermn LH averaged 377 ng/ml at 4
p.m. and 579 ng/ml at 8 p.m. in the cycling group compared
to 107 ng/ml at 4 p.m. and 166 ng/ml at 8 p.m. in the con-
stant estrous group (Figure 26).

The effect of progesterone injection on the proestrous
LH surge is plotted in Figure 27. Both 0.5 and 2.0 mg of
progesterone injected at 8 a.m. increased serum LH concen-
trations at 5 p.m. (P<0.01) and the 2.0 ng treatment in-
creased LH at the 8 p.m. blood sampling interval (P<0.05).
Discussion

The results from. these experiments indicate that
inadequate proestrous LH secretion may be associated with
the failure to ovulate and the initiation of the constant
estrous state in aging female rats. Since ovulation can be
stimulated in aging rats by gonadotropin injection, and
estrous cycles, with presumed ovulation, can be induced by
treatment of constant estrous rats with a wide variety of
hormones, drugs or treatments known or presumed to stimulate

hypothalamic-pituitary release of gonadotropins in the aging

151

Proestrous Serum Progesterone In Cycling
vs Constant Estrous Rats After Stress

_I_ [—1 Cycling

m Constant Estrous

GO

   

Progesterone n9! ml

20

 

 

W

 

 

4pm 8pm

Figure 25. Proestrous Serum Progesterone in Cycling vs.
Constant Estrous Rats After Stress” IBlood samples 'were
collected under light ether anesthesia at 1600 h and at 2000
h on the day of proestrus which occurred following the
stress treatment in the constant estrous rats and on the day
of proestrus in normally cycling female rats of similar age.

Progesterone is expressed as group means (ng/ml serum) with
indicated standard errors of the means.

152

AGED, PROESTROUS RATS

 

 

 

 

 

 

   

I ICycllng, n:12
Constant
Estrous, n:22

400
E
u
c
I:

-‘ 200

0

4pm 8pm

Figure 26. Proestrous Serum LH in Cycling vs. Constant
Estrous Rats After Stress. Blood samples were collected
under light ether anesthesia at 1600 h and at 2000 h on the
day of proestrus which occurred following the stress treat-
ment in the constant estrous rats and on the day of proes-
trus in normally cycling female rats of similar age. LH is
expressed as group means (ng/ml serum) with indicated
standard errors of the means.

153

Effect of Proestrous Progesterone on LH Secretion

Age : 15 mo
n: 10 I

1200‘
1000

800‘

LH nglrnl
a1
8

b
O
O

200‘

1

75pm 8pm 5pm 8pm 5pm 8pm
0 Prog. 0.5 mg Prog. 2.0 mg Prog.

 

 

 

 

 

 

 

 

Figure 27. The Effect of Injection of O, 0.5 or 2 mg. of
Progesterone on Proestrous Serum LH in Aged Cycling Female
Rats. Injection of progesterone was performed at 0800 h and
blood samples were collected at 1700 h and 2000 h on the day
of proestrus. L3 is expressed as group means (ng/ml serum)
with indicated standard errors of the means.

154
rat (Finch, 1978; Lehman, et al., 1978; Meites, et al.,
1978), failure of adequate gonadotrOpin stimulation of
follicular maturation and ovulation is believed to be in-
volved with the agewrelated loss of normal ovarian cycles

and reproduction function.

Although decreased LH has been consistently reported in
aged male rats (Riegle and Meites, 1976; Bruni, et al.,
1977; Riegle and Miller, 1978), data from aged female rats
are more variable and are related to the reproductive state
of the aged female. A report from our laboratory reported
decreased serum LH in aged rats with pseudopregnant. or
persistent diestrous vaginal cytological states compared to)
young rats in the diestrous stage of their estrous cycle

(Watkins, et al., 1975). On the other hand, we found aged

constant estrous rats have higher LH concentrations than
found in young diestrous or estrous groups but less LH than
present during the proestrous hormone surge. Other reports
have found unchanged LH concentrations in both middle—aged
and aged female rats (Wilkes, et al., 1978; Lu, et al.,
1979) and a temporary delay in the onset of the proestrus
gonadotropin surge (Cooper, et al., 1980; Wise, 1982).
Serum FSH concentrations have been reported to be increased
in aging constant estrous and pseudopregnant rats (Clemens
and Meites, 1971; Wilkes, et al., 1978; Lu, et al., 1979)
and during the proestrous surge in 12 month old rats with

regular estrous cycles (Wilkes, et al., 1979). These data

155

offer strong evidence that gonadotropin secretion is not
uniformly depressed in aged female rats.

We have reported a smaller proestrous LH surge in cyclic
12 to 16 month old compared to three month old young rats
(Section 2 D of this thesis). In that study, peak LH con-
centration measured in the aged group at 6 p.m. (310 ng/ml)
was less than one-quarter that of the young rats. However,
the ovulation data from the current experiment implies that
the reduced proestrous LH surge in aging cycling rats is
sufficient for follicular maturation and normal ovulation.
Other important variables that could affect gonadotropin
stimulation of the ovary include possible age differences in'
the biological activity of the pituitary hormones measured
in peripheral blood samples and age-related decreases in
gonadal responsiveness to gonadotropins. Age-related alter-
ations in the biological activity of blood TSH and insulin
have been reported (Klug, et al., 1978), however this has
not been shown to be true of LH in rats (Parkening, et al.,
1982). There are several reports of no change in LH binding
to the Leydig cells of aged male rats (Steger, et al., 1979;
Geisthovel, et al., 1981; Kaler and Neavens, 1981) and
similar data on female rats indicates that there is also no
significant difference in LH binding or aromatase activity
in granulosa cells (Erickson, et a1. 1979).

Our data support the concept of a gradual reduction in

the magnitude of the proestrous LH surge in aging rats which

is ‘hypothesized to lead to the development. of’ constant

156

estrous. Since the aged rat pituitary retains a high capac-
ity for LH secretion following LHRH stimulation (Miller and
Riegle, 1978; Meites, et al., 1978; Tang and Tang, 1981), it
is less likely that an increasing inability to secrete LH,
inherent in aging pituitary gonadotrophs, contribute signif-
icantly to this reduced LH secretion. These findings are
supportive of the hypothesis that age effects on the neuro-
endocrine regulation of hypothalamic secretion of LHRH is
the primary factor contributing to the loss of estrous
cycles and fertility.

Available evidence concerning the regulation of the
proestrous gonadotropin surge suggests that controlled'
increases in the secretion of both estradiol and proges-
terone during proestrus play prominent roles in this pro-
cess. Increased estradiol secretion has been shown to
augment pituitary responsiveness to LHRH (Vilchez—Martinez,
et al., 1974) and progesterone has been one of multiple
factors associated with reinitiation of estrous cycles in
aged animals. In the previous section of this thesis, we
reported smaller increases in the proestrous increase in
serum progesterone in aging compared to young female rats.
The origin of the proestrous increase in progesterone and
its relationship to the proestrous gonadotropin surge has
received considerable attention. Several studies have shown
an adrenocortical role in the increased progesterone secre-
tion at proestrous (Mann and Barraclough, 1973; Shaikh and

Shaikh, 1975). The increase in serum progesterone occurs

157
before the gonadotropin surge (Shaikh and Shaikh, 1975).
Progesterone has been shown to facilitate gonadotropin
secretion and induce earlier mating behavior and ovulation
in the rat (Neguin, et al., 1975) and to stimulate LH re-
lease in pentobarbital-blocked rats (Kobayaski, et al.,
1973). It has also been shown that increased blood estra-
diol, presumably of ovarian origin, can increase the dura-
tion of proestrous adrenal progesterone secretion (Campbell,
et al., 1977). These findings suggest that decreased
estrogen secretion during proestrous or the recognized

decreases in estrogen uptake and receptor numbers could be

associated to the age-related reduction in the proestrous LH.
surge (Peng and Peng, 1973; Kanungo, et al., 1975). In
addition, Campbell, et al., showed a stress-reduced increase
in adrenal estradiol secretion in proestrous but not in
metestrous cycling rats, suggesting that the proestrous
hormone milieu can have specific effects on adrenocortical
steroidogenesis. These experiments indicate that the
decrease in serum LH during the proestrous hormone surge in
stress-interrupted constant estrous rats could be related to
the decrease in progesterone. This concept is supported by
the increase in proestrous LH secretion we found in aged
cycling rats injected with progesterone on the morning of
proestrous.

On the other hand, adrenalectomized rats have normal

estrous cycles and cwolate (Feder, et al., 1971) and in-

terruption of the proestrous LH surge with pentobarbital

158
also decreases the increase in serum progesterone during
proestrous, indicating that the ovulatory LH surge affects
ovarian progesterone secretion during and after the release
of gonadotropins (Everett, 1967). These reports suggest
that reduced serum progesterone during proestrous in aging
rats can contribute to the smaller LH release but indicate
that the reduction in progesterone may not be the only or
the primary factor involved in the age-related alteration in
proestrous gonadotropin release.

The increase in progesterone in the post-stress blood
sample presumably occurs due to stress-induced ACTH stimu-
lation of the adrenal gland. The magnitude of this increase.
in progesterone suggests normal adrenocortical secretory
response and implicates age-related alteration in gonado-
tropin stimulation of ovarian or adrenal progesterone secre-
tion or altered ovarian responsiveness to gonadotropin as
primary factors involved with the reduced proestrous pro-
gesterone secretion in the aged rat.

A wide variety of treatments have been reported to be
capable of reinitiating estrous cycles in aging rats (Finch,
1978). In many of these studies evidence for the reinitia-
tion of ovarian cycles is based on cytological characteris-
tics of vaginal lavages rather than measured stimulation of
gonadotropin release or by verified ovulation. The results
from this study clearly indicate that these indirect cri-
teria are inadequate. We found that although rats receiving

stress treatments have apparently normal post-stress estrous

159

cycles, there is no evidence of normal luteal progesterone
secretion, proestrous surges of LH and progesterone are
sharply reduced, and a majority of animals resume their
constant estrous state without ovulating or developing
functional luteal tissue. :n: is presumed that several of
the factors associated with the restoration of estrous
cycles, in aged rats, including progesterone, ACTH, epi-
nephrine or various other stressors, could act similarly.

We found similar responses in mated constant estrous
rats. Although average serum LH concentrations at 3 and 5
p.m. in the mated rats were increased (7 and 10 hours after
initiation of copulatory behavior) over that in non—mated
constant estrous rats, these LH concentrations are lower
than average LH measured in similarly aged proestrous cycl-
ing rats and, together with the failure to recover eggs from
the fallopian tube, suggest that the increased LH secretion
induced by coital stimulation was insufficient to stimulate
ovulation.

In the past few years, several laboratories have shown
significant age-related alterations in hypothalamic neuro-
transmitter content and turnover in the aging rodent. These
studies have shown decreased norepinephrine content and
turnover in the hypothalamus (Miller, et al., 1976;
Simpkins, et al., 1977), reduced hypothalamic and median
eminence dopamine content and turnover (Finch, 1973; Miller,
et al., 1976: Simpkins, et al., 1977; Riegle, et al., 1979)

and increased hypothalamic serotonin turnover :hi aged

160

compared to young groups. It has been proposed that these
neurotransmitters regulate LHRH secretion from the pepti—
dergic hypothalamic neurons. Our current understanding of
neurotransmitter content of LHRH release indicates that
these agents may be involved in the age-related alterations
in the reproductive control system.

In summary, these experiments indicate that the loss of
estrous cycles in the aging rat involves multiple compo-
nents. Although the stress interrupted constant estrous
aging rat shows vaginal cytological changes which are
typical of the normal rat estrous cycle, these rats show
decreased proestrous progesterone and LH secretion and.
failure of normal ovulation and luteinization. Our pre-
liminary studies suggest that the proestrous release of LH
can be increased in the aging rat by progesterone adminis-
tration. These findings suggest a neuroendocrine alteration
with increasing age which reduces hypothalamic LHRH stimula-
tion of pituitary gonadotropin secretion. The decrease in
proestrous progesterone secretion, presumably' of ovarian
origin, is hypothesized to be one of the factors involved in
the reduction of hypothalamic stimulation of pituitary
gonadotropin secretion. Other neuroendocrine factors in-
cluding age-related decreases in hypothalamic and median
eminence catecholamine function are believed to contribute

to the loss of estrous cycles and fertility.

SUMMARY AND CONCLUSIONS

The Reproductive Control System of the Aginngale Rat

 

The data reported here indicates major alterations in

the endocrine reproductive control system of the aging male

rat.

1.

Specifically, these studies:

affirm our previous experiments showing a major
reduction in serum testosterone in aging male Long—
Evans rats.

indicate that the Leydig cells of the aged male rat
remain responsive to gonadotropin stimulation.

show that chronic Leydig cell stimulation by HCG
administration stimulates similar increases in
serum testosterone in young and aged male rats and
restores testicular responsiveness to acute gonado—
tropin stimulation.

suggest similar hypothalamic content of biologic—
ally active LH-releasing hormone activity in young
and aged, intact and gonadectomized male rats.
indicate the presence of a diurnal pattern of serum
testosterone concentrations in young male rats
which was not present in aged male groups.

imply that the aged male rat has small episodical

surges of pituitary LH and testicular testosterone

161

162

secretion which result in reduced average serum LH
and testosterone concentrations and less variabil—
ity in individual blood sample content of LH and
testosterone in older male rats.

7. ShOW’ that although the aged rat pituitary and
testes respond to LHRH stimulation, the magnitude
of the increase in LH and testosterone is reduced
in the older animals.

The aged male rat is recognized to have smaller testes,
reduced rates of spermatogenesis, reduced male accessory
gland size, decreased sexual libido and decreased fertility.
In addition, the aged male rat shows reduced skeletal muscle.
mass, less physical vigor, reduced aggression and progres-
sive thinning of his hair coat and skin. Although some of
the aforementioned changes in structure and function of the
aged male rat are undoubtedly associated with specific age
effects on these tissues, all of' these alterations (are
consistent with the biological effects expected to accompany
the decrease in testosterone concentrations reported in this
thesis. This progressive reduction in the biological avail—
ability and activity of testosterone with increasing age
undoubtedly contributes to the deterioration of function in
a variety of physiological systems with age.

Although our results clearly indicate reduced testicular
testosterone secretion with increasing age in the Long-Evans
rat, it is also apparent that the reduction in serum testos-

terone concentration occurs secondarily to a chronic

163

reduction in pituitary LH secretion rather than an inability
of testicular Leydig cell to respond to gonadotropin stimu-
lation. Our experiments confirm the hypothesis that the
pituitary and testis are capable of sustaining much higher
levels of endocrine function than that which normally occurs
in the aging male rat.

In addition, these experiments suggest that hypothalamic
peptidergic neuronal content of LH—releasing activity is not
reduced with increased age or increased rate of peptide
hormone synthesis and secretion which occurs following
gonadectomy. These data support our previous hypothesis of
significant age-related alterations in the responsiveness of’
hypothalamic neurons to control input in the male rat. A
large number of recent experiments have shown that signifi-
cant alteration in hypothalamic and median eminence content
and turnover of neurotransmitters, particularly of norepine—
phrine and dopamine, can profoundly affect gonadotropin
secretion. These findings have been interpreted to indicate
that age alterations in neurotransmitter function in speci-
fic regions of the hypothalamus and median eminence could
contribute to the alteration in hypothalamic stimulation of
pituitary secretion which occurs with increased age. This
hypothesis has been strengthened by the results of our
experiments and the work of several other laboratories which
show reduced hypothalamic and median eminence content and
turnover of dopamine and norepinephrine with increasing age

in the male rat. Although the association between changes

164

in neurotransmitters and hypothalamic endocrine function
remains an attractive hypothesis, it needs to be emphasized
that this relationship should not yet be accepted as dogma.
A large amount of additional experimentation must be com-
pleted to establish a quantitative relationShip 'between
these two variables.

There are similarities in the effect of age on repro-
ductive control systems in the male rat and men which
suggest that the rat can be a useful model for studing many
aspects of the effects of age on the reproductive control
mechanism in men. Both species commonly encounter decreased
peripheral testosterone activities with increased age.
Although, there are differences in endocrine reproductive
control system responsiveness to the decreased testosterone
between rats and. men, current. data indicates that. both
species are capable of sustaining sufficient pituitary
gonadotropin and testicular secretions to restore testos-
terone concentrations in the aged to that found in younger
males. These data indicate significant age—related alter-
ations in neuroendocrine control mechanisms related to
gonadal control. As indicated above, current hypotheses
relate these age-related changes in reproductive control
systems to alterations in neuronal fuction, particularly to
changes involving catecholaminergic neuronal systems. The
similarities between control system components between
species suggest that a better understanding of age effects

on basic neuroendocrine control of hypothalamic secretions

165

which regulate pituitary function in the rat should contri-

bute to our understanding of fundamental age effects on

other neuronal systems in humans.

The Reproductive Control System of the Aging Female Rat

These experiments also indicate significant alterations

in the endocrine regulatory systems pertaining to repro-

duction in the aging female rat. Specifically, the results

of these studies:

I.

affirm decreased fertility of female rats at nine
to ten months of age.

confirm evidence that the loss of fertility occurs
at an earlier age than the loss of normal ovarian
cyclicity in the aging female rat.

indicate that total litter loss with increasing age
is more characteristic of the decline in fertility
than decreased litter size.

suggest that hypothalamic content of LEI-releasing
activity is not different in young and aged intact
and gonadectomized female rats.

show that serial LHRH injections can increase serum
LH in aged female rats to concentrations similar to
that measured in young rats with normal estrous
cycles.

suggest age-related differences in the metabolism
and excretion of LH from the blood of aged rats.
establiSh the concept of reduced sermn concentra-

tions of progesterone and LH during the proestrous

10.

11.

12.

13.

166

hormone surge in aging female rats.

show that reduced serum progesterone concentrations
are also characteristic of aged compared to young
rats during estrous and diestrous stages of their
estrous cycles.

affirm low serum progesterone concentrations in
aging constant estrous rats.

indicate that vaginal cytology alone is not an
adequate index of progesterone secretion in aged
rats cytologically classified as constant diestrous
or repetitive pseudopregnant.

establish that average early pregnancy serum pro-
gesterone concentrations are similar in aging fe-
male rats with normal estrous cycles compared to
aged rats with constant estrous vaginal cytology
which were made pseudopregnant by coital stimula-
tion.

show similar serum progesterone concentrations
during early pregnancy among aging rats which
produced normal litters compared to rats which did
not produce litters.

show that aged pregnant and pseudopregnant rats
which did not produce normal litters have reduced
serum, progesterone concentrations ‘by';post-coital
day 16, indicating failure of early pregnancy or
suggesting age-related alterations in uterine-

placenta factors affecting luteal tissue secretion.

14.

15.

16.

17.

18.

19.

20.

167

indicate increased gestation length in aging female
rats associated with. longer' maintenance of in-
creased serum progesterone concentrations in aged
compared to young groups.

affirm normal ovulation rates in 10 to 16 month old
female rats with regular estrous cycles.

suggest that stress can interrupt the constant
estrous state of aging rats by stimulating proges-
terone secretion, presumably of adrenal origin.
indicate that constant estrous rats whose persis-
tent vaginal cornification is interrupted by stress
do not have normal diestrous luteal progesterone-
concentrations as similarly aged, regularly cycling
rats during diestrus.

show that constant estrous rats whose persistent
vaginal cornification is interrupted by stress have
smaller increases in serum LH and progesterone
during the post-stress proestrous ‘hormone surge
compared to similar aged regularly cycling rats at
proestrus.

establish the return to the constant estrous state
in the majority' of untreated stress-interrupted
constant estrous aging rats.

suggest that failure of progesterone secretion
during proestrus in aging rats contributes to the
development of the constant estrous reproductive

state.

168

The results of these experiments indicate multifaceted
components contribute to the loss of fertility in the aging
female rat. The early loss of fertility beginning at about
nine to ten months of age in rats with regular estrous
cycles seems independent of known changes in pituitary or
neuroendocrine control. Although we showed that 10 to 16
month old rats with regular ovarian cycles have a somehwat
smaller proestrous LH surge than young rats with regular
estrous cycles, they secrete sufficient gonadotropin to
stimulate normal ovulation, luteinization, and luteal pro-
gesterone secretion. These data imply increased postovula-
tory loss of reproduction in these rats.

Several experiments from a variety of laboratories have
shown that eggs collected from oviducts of aged doner rats
can develop normally in young recipient reproductive tracts.
These findings indicate that age-related abnormalities in
ova or age changes in their ability to be fertilized do not
contribute to the loss of fertility. In addition, aged
female rat corpora lutea maintain serum progesterone concen-
trations during early pregnancy which were similar to that
measured in normal young rats. These data imply normal
control of corpora lutea secretion during early pregnancy
in the aging rat and suggest age—related alterations in
uterine function contribute to the failure to reproduce.

There are also multiple possibilities for age-related
alterations in uterine function. Most experiments suggest

some alteration in embryo-placenta development or function

169

during the first half of gestation in aged animals since
normal corpora lutea regulation is lost during the last half
of pregnancy, when corpora lutea stimulation is dependent on
uterine stimulatory factors. Implantation or placentation
mechanisms could be affected in the aging rat. There could
also be age-related changes in tissue responsiveness to
hormones. In addition, there may be differences in decidua
formation or uterine blood flow which are necessary for
maintaining normal pregnancy.

My experiments also support a hypothesis of multiple
factors affecting the changes in the estrous cycle in the
aging female rat. We found that the magnitude of the LH-
surge is progressively decreased with increased age. It is
hypothesized that this reduction in proestrous LH secretion
ultimately results in failure of follicular' maturation,
ovulation and follicle luteinization, and to the development
of increased estrous cycle irregularities which are believed
to progressively lead to the development of the constant
estrous reproductive state. The results of my studies
indicate that the decrease in the magnitude of the proes-
trous LH surge may involve two factors. First there may be
reduced LH-releasing hormone secretion from the hypothalamus
and secondly, reduced adrenal or ovarian progesterone secre-
tion may result in either decreased hypothalamic-pituitary
unit responsiveness to proestrous controlling input or

decreased pituitary responsiveness to LHRH.

170

Our experiments and studies by others indicate that
stress-induced increases in progesterone secretion or direct
progesterone injections can restore regular ovarian cycles
based on cytological changes in vaginal lavages, and in some
instances this increase in progesterone is sufficient to
induce ovulation and luteinization in aging rats with con-
stant estrous alterations in their estrous cycles. This
relationship is strengthened by our finding of increased LH
secretion during the proestrous hormone surge in aged rats
following progesterone injection.

Although hypothalamic LH-releasing hormone content is
not decreased with increasing age in the female rat, the.
changes in reproductive function which accompany aging are
consistent with an alternate hypothesis of decreased LH-
releasing hormone secretion. In support of this hypothesis,
we showed that multiple LHRH injections could increase serum
LH in aged rats to concentrations which were similar to
those found following similar hormone treatment in young
groups. The hypothesis of decreased LH-releasing hormone
secretion is commonly associated with evidence for the
alteration of hypothalamic neurotransmitter function in aged
rats. The fundamental premise of this hypothesis is that
age-associated deterioration of hypothalamic catecholamin-
ergic function results in impairment of peptidergic neuronal
secretion of LH—releasing hormone which in turn leads to
reduced pituitary gonadotropin secretion and failure of

ovarian control mechanisms. However, there are obvious

171

problems associated with this interpretation. Additional
experiments involving study of the effect of restoration of
catecholamine function in aging animals, the effects of
premature reduction in specific neurotransmitter function,
and the interaction of catecholamine regulation of LHRH
secretion with peripheral steroid hormone effects on LHRH
secretion and pituitary responsiveness are necessary to
understand these relationships.

In summary, there are several factors which appear to
influence the usefulness of the rat as a model of aging
effects on the reproductive control systems of women. The
most obvious difference between the species is the lack of a,
clear menopausal state in the aging rat. Although the aging
female rat has a modest increase in blood FSH concentra-
tions, there is no evidence for an increase in LH secretion
with age-related decreases in ovarian gametogenic and ster-
oidogenic function. On the other hand, there is ample
evidence indicating increased post-ovulatory loss of fertil-
ity in the aging rat and in premenopausal women. These
findings suggest the mid-aged rat could be a very useful
model to study premenopausal changes in reproductive func-
tion in women. In addition, both the rat and human show
alterations in central nervous system function with in-
creased age. Better understanding of fundamental age
effects on neuronal function in the rat should be of major
significance for our understanding of and the development of

effective prevention or treatment schemes related to the

172

deterioration of central nervous system function which

occurs in aging humans.

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