SENESCENT ALTERATIONS OF LH AND
TESTOSTERONE REGULATION AND
HYPOTHAlAMIC CATECHOLAMINES

IN THE MALE RAT

esis for the Degree 0? H. S.
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AMA ELALABFA IAALELR

1975

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ABSTRACT

SENESCENT ALTERATIONS OF LH AND TESTOSTERONE
REGULATION AND HYPOTHALAMIC
CATECHOLAMINES IN
THE MALE RAT

BY

Anna Elizabeth Miller

The effects of advanced age on the reproductive
control system was studied in male rats by examining
changes in serum testosterone and LH concentrations and
alterations which occur in the response to administration
of Luteinizing Hormone Releasing Hormone (LHRH), Human
Chorionic Gonadotropin (HCG), and L-dopa, and in hypothala-
mic catecholamine concentrations. Young adult (3-6 mo) and
aged (20-30 mo) male Long-Evans rats were used in these
studies.

The effect of HCG on serum testosterone was studied
in two trials. In the first experiment, 23 young and 27
aged male rats were assigned to one of three groups which
received jugular vein injections of 0.5 ml of physiological
saline or 0.5 ml of saline containing 1 or 5 IU of HCG.
Testosterone was measured by radioimmunoassay in serial
blood samples taken by orbital sinus puncture before injec-

tion and at 15, 30, and 60 minutes after treatment.

Anna Elizabeth Miller

Resting serum testosterone concentrations were found to be
significantly lower in aged male rats than in young male
rats. In the saline treated groups, testosterone remained
stable throughout the sampling period. After treatment with
HCG, serum testosterone was steadily increased in both age
groups over the sampling period. This increase in testos-
terone was approximately twice as great in young as in aged
males, and the 5 IU dose of HCG stimulated a greater
increase in testosterone than did 1 IU in both young and
aged groups.

In the second trial, groups of 32 young and 31 aged
ma1e rats were assigned to one of 4 groups and received
intravenous injections of 0.5 ml of physiological saline
or 0.5 m1 of saline containing 1, 5, or 20 IU of HCG.
Serial blood samples were taken from each rat prior to and
at 45, 90, and 150 minutes after the HCG treatment. Serum
testosterone was increased in both young and aged rats
following all 3 doses of HCG. The increases after each
HCG treatment were smaller in aged rats compared to young
rats. The increase in serum testosterone concentrations
following HCG injection was sustained throughout the
sampling period in both age groups.

In a second experiment, 17 aged and 24 young male
rats received 3 intravenous injections of 500 ng LHRH at
75-minute intervals. Serum LH was measured by radioimmuno-
assay in serial blood samples taken before each LHRH

injection and 15 minutes following each drug treatment.

Anna Elizabeth Miller

Young males were found to have higher LH concentrations and
higher serum LH concetrations 15 minutes after the first
LHRH injection than aged male rats. However, serum LH
levels were similar in both age groups before and after the
second and third LHRH injections.

In a third experiment, groups of 8 aged and 8
young male rats received intravenous injections of 500 ng
of LHRH. Serum testosterone was measured in serial blood
samples taken before and 15, 30, and 60 minutes after LHRH
injection and was found to be progressively increased
following LHRH injections in the young group while not being
significantly increased over saline injected controls in
the aged group.

A fourth experiment examined hypotha1amic catecho-
lamine content of young and aged male rats. Groups of 16
aged and 16 young male rats were decapitated and their
hypothalami collected and weighed. Hypothalamic norepine-
phrine and dopamine were determined from hypotha1amic
extracts by microflourescence after alumina absorption.
Hypothalamic d0pamine and norepinephrine content were both
found to be about twice as great in young as in the aged
rats.

In a fifth experiment, groups of 19 aged and 30
young male rats were given an intravenous injection of
500 ng LHRH prior to and shortly after 10 days of treatment
with L-dOpa. Serum LH was measured in blood samples taken

before and at 15 and 45 minutes after LHRH injection.

Anna Elizabeth Miller

Although the LHRH reSponse prior to L-dOpa treatment
showed that serum LH was higher 15 minutes after LHRH
injection in the young than in the aged rats, serum LH 15
minutes after LHRH treatment at the end of the L-dOpa
injection regime was similar for both ages.

These data suggest that with age, functional
deterioration of gonadotropin control occurs at the level
of the gonad, pituitary, and at the hypothalamus. Although
aged male rats have lower serum concentrations of LH and
testosterone than young males, the responsiveness of the
testes and pituitary to LH and LHRH stimulation indicates
that these tissues are capable of maintaining higher levels
of secretion. These data are interpreted to indicate that
the primary dysfunction in the gonadal control system in
the aged male rat occurs in the hypothalamus or other

neural regulatory tissues.

SENESCENT ALTERATIONS OF LH AND TESTOSTERONE
REGULATION AND HYPOTHALAMIC
CATECHOLAMINES IN

THE MALE RAT

BY

Anna Elizabeth Miller

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Physiology

1976

ACKNOWLEDGMENTS

I would like to express my sincere appreciation to
Dr. Gail D. Riegle, my major advisor, for his patient
assistance and inspiring example as both a scientist and
a person.

I am also grateful to the other members of my
guidance committee, Dr. Harold Hafs and Dr. Edward Convey
for their interest and advice in the final stages of my
masters program.

I would also like to express my appreciation to
Dr. E. M. Bogdanove at the Medical College of Virginia who
kindled my interest in endocrinology and showed me how
exciting scientific research can be.

The friendship and helpfulness of Sandra M. Wood
and Donald W. McKay have been of immeasurable importance
to me, along with the rest of the Endocrine Research Unit
staff.'

I must also thank my parents, Loentine V. Goff and
the late John B. Beltz for giving me the financial Oppor-
tunities and the emotional support to be all I can be.

And I must certainly thank my husband, Michael E.
Miller, for his concern and understanding, and for always

providing the confidence I lack.

ii

TABLE OF CONTENTS

LIST OF FIGURES . . . . . . . . .
INTRODUCTION . . . . . . ‘ . . . .
LITERATURE REVIEW . . . . . . . .

Gonadotropin Control. . . . . . .
Aging. . . . . . . . . . . .

Introduction. . . . . . . . .
Female--General Considerations. . .
Female-~Gonadal Function. . . . .
Female--Pituitary Function . . . .
Female--Hypothalamic Function . . .
Male--General Considerations . . .
Male--Gonadal Function . . . . .
Male--Pituitary Function. . . . .
Male--Hypothalamic Function. . . .

METHODS. . . . . . . . . . . .

Experimental Animals. . . . . . .
Blood Collection . . . . . . . .
Hormone and Drug Treatment. . . . .
Hypothalami Collection and Preparation.
Catecholamine Assay . . . . . . .
Radioimmunoassay for Testosterone . .
Radioimunnoassay for LH. . . . . .

EXPERIMENTAL . . . . . . . . . .

Experiment 1. The Effects of HCG on Serum

Testosterone. . . . . . . .
Experiment 2. The Effects of Multiple
Injections on Serum LH . . . . .

iii

LHRH

Page

31
42

Page

Experiment 3. The Effects of LHRH Injections

on Serum Testosterone. . . . . . . . . . 46
Experiment 4. Hypothalamic Norepinephrine
and DOpamine Content . . . . . . . . . 50

Experiment 5. The Effects of Chronic L-dopa
Treatment on Pituitary ReSponsiveness to

LHRH . O O O O C O O O O O O O O I 54
DISCUSSION. 0 O O O O O C O O 0 O O I 0 59
LIST OF REFERENCES . . . . . . . . . . . . 65

iv

Figure

1.

LIST OF FIGURES

Effects of intravenous administration of

0.5 ml saline on serum testosterone con-
centrations in young and aged male rats.

Effects of intravenous administration of l
or 5 IU HCG on serum testosterone con-
centrations in young and aged male rats.

Effects of intravenous administration of l,
5, or 20 IU HCG on serum testosterone
concentrations in young and aged rats .

Effects of three conservative injections of
500 ng LHRH on serum LH concentrations in
young and aged male rats. . . . . .

Effects of intravenous administration of
500 ng LHRH on serum testosterone concen-
trations in young and aged male rats. .

Hypothalamic catecholamine content in young
and aged male rats. . . . . . . .

Effects of intravenous administration of
500 ng LHRH on serum concentrations in
young and aged male rats prior to and
following 10 days of L-dOpa treatment .

Page

34

37

39

44

48

52

57

INTRODUCTION

While man has historically been fascinated with the
changes that accompany advanced age and has time and again
been haunted with hopes of reversing or retarding this
apparently inevitable process, the state of experimental
gerontology is still at the descriptive stage, a relative
infant in the bio-medical research field.

The progression of life from conception through
growth, develOpment, physiological aging and the onset of
diseases leading to death, while primarily under genetic
control, clearly involves physiological control systems.
Gerontologists have long been fascinated by the possibility
of a direct relationship between aging and changes in
hormonal function. Many investigators have related changes
in endocrine control system function to aging. Reduction
of reproductive function with increasing age has been a
universal observation in mammalian species. A large amount
of recent experimental work has shown that reproduction is
regulated by complex control systems involving the central
nervous system, the anterior pituitary gland and the gonads.
Most hormones secreted by these tissues have been shown to

be influenced by the aging phenomenon. Alterations in

certain response thresholds and changes in the function of
the components of these control systems also appear to be
involved with the loss of reproductive function. It is
hoped that a better understanding of the effects of aging
on the endocrine neurophysiological control mechanisms will
be applicable to other neurophysiological control systems
and increase our knowledge of the biology of aging.

The research described here was done in an effort
to characterize the major sources of the alterations
occurring with age in the reproductive control system of
the male rat. The rat was selected as a model primarily
because of its short life span and the relative ease in
acquiring modest numbers of senescent animals in a short
time. The reproductive control system was regarded as an
apprOpriate model for an aging study since it has measurable
impairments occurring with age and the normal neuroendocrine
control system has been quite thoroughly established in

this species.

LITERATURE REVIEW

GonadotrOpin Control

 

The literature published in recent decades con-
cerning reproductive control mechanisms is voluminous and
frequently conflicting. No attempt will be made here to
comprehensively review this topic. The purpose of this
review is to summarize recent findings and the relation-
ships relevant to our study of the control of male repro-
ductive function with age. Many recently published reviews
have given more exhaustive and comprehensive consideration
to the control of reproduction (Greep and Astwood, 1973,
1974, 1975).

Testicular activity has been clearly shown to be
controlled by the pituitary gonadotropins follicle stimu-
lating hormone (FSH) and luteinizing hormone (LH). It was
initially thought that FSH was primarily concerned with
the regulation of spermatogenesis, while LH was the primary
regulator of Leydig cell growth and androgen secretion
(Greep et al., 1936; Greep and Fevold, 1937). The finding
that the administration of testosterone alone is sufficient
to maintain spermatogenesis in hypophysectomized animals

(Nelson and Merckel, 1938) somewhat complicated this theory.

Also, a linear relationship between the capacity of the
Leydig cells to produce testosterone and the serum levels of
gonadotropins has not been demonstrated. It is now thought
that LH directly controls the secretion of androgens by the
testes and, along with FSH, then indirectly influences
spermatogenesis through its control over steroidogenesis.

Pituitary gonadotropins have also been shown to
control growth of the testes. Steinberger and Steinberger
(1972) have shown that at puberty in rats, increases in
plasma levels of LH and FSH are associated with dramatic
increases in testicular weight which largely reflects
growth of the seminiferous tubules. These workers also
found that in neonatal rats estrogen administration suffi-
cient to block gonadotropins also blocks growth of the
seminiferous tubules. However, it has also been shown
that several androgens, especially testosterone, can stimu-
late growth of the seminiferous tubules without the pres-
ence of gonadotrOpins, although the degree of stimulation
is significantly less than that seen with gonadotrOpins
(Steinberger and Steinberger, 1972). The growth of the
testes appears to be a complex process, involving several
different tissues, and it appears that the hormonal control
may be similarly complicated, involving both the gonado-
tropins and testosterone.

It is now accepted that the anterior pituitary
secretion of gonadotrOpins is largely controlled by the

hypothalamus. In male rats, median eminence lesions

result in atrophy of the testes and accessory organs, with
a dramatic decrease in plasma FSH and LH, and an increase
in prolactin secretion (DeVoe et al., 1965). The existence
of specific releasing factors that control pituitary secre-
tions is also well established. Highly purified LH-
releasing hormone (LHRH) is active in nanogram doses to

release LH in_vivo and in vitro (Fawcett and McCann, 1971).

 

Luteinizing Hormone Releasing Hormone (LHRH) has been found
to act directly on the pituitary gland to release LH, since
it is active (a) when injected into the peripheral circula-
tion in animals with lesions in the median eminence that
eliminate neural control of the gland, (b) when micro-
injected into the interstitial tissue of the pituitary,

(c) when perfused directly into a hypophysial portal

vessel, or (d) when added to pituitary incubates in vitro

 

(McCann, 1970).

Moore and Price (1932) were perhaps the first to
recognize that secretion of gonadotrOpins by the pituitary
is normally held in check by the feedback action of gonadal
steroids. When rats are orchidectomized, both plasma and
pituitary LH show a rapid and prolonged rise. Kalra et al.
(1971) demonstrated that administration of gonadal steroids
in castrates inhibits gonadotrOpin secretion. Single
injections of low doses of testosterone are capable of
rapidly lowering plasma LH.

Several workers have implanted minute amounts of

steroids into either the hypothalamus or the anterior

pituitary in an effort to localize their site of action in
modifying gonadotrOpin release (Rose and Nelson, 1957;
Davidson and Sawyer, 1961; Lisk, 1962; Bogdanove, 1963;
Chowers and McCann, 1965). Evidence indicates a direct
effect of steroids on the anterior pituitary, along with a
hypotha1amic site of feedback such that the hypothalamic
content of LHRH is lowered. Furthermore, alterations in
the level of hypotha1amic releasing factors have been shown
to follow castration (Meites, 1970).

Several studies have shown that hypotha1amic control
of gonadotropin secretion by the anterior pituitary involves
the monoamine containing neurons of the hypothalamus as
well as other neural mechanisms whose actions may be inde-
pendent of or directly associated with the effects of the
catecholamines.{ Ungerstedt (1971) has shown that catecho-
laminergic tracts enter the hypothalamus and innervate many
anatomical regions. Lesion and stimulation studies have
shown these catecholamine tracts to be involved in hypo-
thalamic control of anterior pituitary secretions. Fuxe
(1969) has proposed that dopaminergic tracts terminating
in the region of the primary plexus of the hypothalamic-
pituitary portal blood system control the secretion of
releasing factors into the portal blood.

Direct evidence of catecholaminergic regulation of
hormone release has been provided by studies correlating
catecholamine administration with hormone secretion.

Dopamine injection into the third ventricle of estrogen

primed rats resulted in increased LHRH in portal blood
(Kamberi et al., 1969), and in systemic blood (Schneider
and McCann, 1970a), and increased serum LH in rats
(Schneider and McCann, 1970b; Kamberi et al., 1970).
Systemic injection of L-dopa, the precursor for dopamine,
has been shown to increase serum LH concentrations in female
rats (Watkins et al., 1975). However, these data do not
indicate the mechanism by which L—dOpa stimulates an
increase in LH. The recent studies of Sawyer et al. (1974)
and Cocchi et al. (1974) suggest that the release of hypo-
tha1amic LHRH is stimulated by norepinephrine. These
reports would suggest that the systemically-injected L-dOpa
was converted to norepinephrine for biological activity.
Following castration, Wurtman et a1. (1969) found that
norepinephrine synthesis is increased and Donoso et a1.
(1967) reported increased hypotha1amic norepinephrine
content.

On the other hand, not all investigators agree with
the forementioned data. Fuxe and Hokfelt (1969) concluded
that catecholamines inhibit gonadotrOpin secretion and did
not detect norepinephrine changes following castration.

The molecular mechanism of catecholamine regulation of the
release of hypothalamic factors regulating gonadotrOpins
and comprehension of how multiple stimulatory and inhibitory

inputs may effect this system remain to be identified.

Aging

Introduction

 

The physiological control system mechanisms
governing reproduction have received considerable attention
in recent years, particularly regarding the location,
specificity, sensitivity, and mechanisms of action of the
reproductive control system components. While interest in
the effects of aging on reproductive control systems is
increasing, insight into the causes and origins of funda-
mental alterations and deterioration of reproductive
function associated with advanced age is still minimal.
Much of the work done in this field continues to be
oriented toward descriptive and quantitative data con-
cerning the effects of age on reproduction, with the
causative factors and mechanisms yet to be elucidated.

While this report is concerned only with the
effects occurring in the male with advanced age, a majority
of the aging studies to date concern females, and thus the
particulars of reproductive control system alterations with
age are more thoroughly known in females. Since many of
the components of the male reproductive control system have
equivalent counterparts in the female which are often
ultimately found to Operate in a similar manner, the
effects of aging on both the male and female reproductive

control systems will be reviewed here.

Female--General Considerations

 

Decreased reproductive output has long been recog-
nized as a consequence of advanced age in mammalian species.
Several studies have shwon a decrease in litter size of
aging laboratory rodents (Blaha, 1964; Adams, 1970;
Thorneycroft and Soderwall, 1969a). Rugh and Wohlfromm
(1967) reported a decrease in average litter size in mice
from 9.5 at 3-5 mo to 7.6 at 10—12 mo of age.

Cyclic patterns of vaginal cytology in laboratory
rodents are also altered with age (Mandl and Shelton, 1958;
Ingram, 1959; Clemens and Meites, 1971; Peng and Huang,
1972). Aschheim (1976) has been characterizing these
changes in the rat for the past several years. Although
some aged rats remain cyclic throughout their lifespan,
Aschheim reports 2 changes in the estrous cycles of rats
which become evident in his colony at about 12 to 15 mo of
age. Beyond 10 months of age increased percentages of his
rats show either constant estrous or repetitive pseudo-
pregnant vaginal smears. Aschheim reported that rats
showing constant estrous vaginal smears predominate in the
second years of rat life (12 to 14 mo) with the repetitive
pseudOpregnant state becoming dominant in the third year
(24 to 36 mo).

Ingram (1959) also reported a change in the sexual
behavior of aged rats with age, i.e., sexual acceptance of
the male was less strongly associated with vaginal

estrous-type cornification.

10

Female-~Gonadal Function

 

There are many possible explanations for the
alterations that occur in the outward signs of reproductive
activity with age. Several hypotheses have been suggested
to explain the effect of aging on litter size. The most
characteristic effect of aging on the ovary is the decline
in the number of oocytes (Krohn, 1967). Although the
number of oocytes are exhausted soon after menOpause in
women (Jones, 1970), other species experience reproductive
failure with substantial numbers of oocytes remaining in
the ovaries. In most rodents the rate of loss of oocytes
is linear with increasing age and rarely reaches zero
before death. Studies by Adams (1970) showed that litter
size decreases more rapidly than ovulation rate with
increasing age, and Mandl and Shelton (1959) reported that
in rats, reduced fertility precedes depletion of
ovarian oocytes. Thorneycroft and Soderwall (1969a) found
a 7 fold increase in preimplantation deaths and a 2 fold
increase in postnidation resorption in aged hamsters as
another explanation for the decrease in litter size with
age.

Several structural changes in the ovary have been
reported to be associated with aging. Takacs and Verzar
(1968) reported increased ovarian collagen content with
age. Thorneycroft and Soderwall (1969b) found that senes-
cent hamsters had fewer ovarian follicles than young female

hamsters, while Aschheim (1976) reported that the number of

11

eggs ovulated are normal in aged rats which retain normal
estrous cycles. Harmon and Tolbert (1967) reported a
higher incidence of degenerate-looking corpora lutea in the
ovaries of old pregnant mice and concluded that there was

a reduction in luteal function. In addition, the produc-
tion of corpora lutea decreases before there is any
evidence of failure by the follicles to respond to FSH
stimulation (Jones and Krohn, 1961). This observation
coupled with the appearance of "deficiency" cells in ovarian
interstitial tissue of aging rats (Wolfe, 1943) suggested
that the ability of the pituitary to supply ovarian stimu-
lation may be impaired with increasing age.

Ovarian steroid production in the human female
changes dramatically during menOpause in spite of increases
in gonadotropic hormone secretion. Pincus et a1. (1954)
reported a progressive decrease in total urinary estrogens
in women between the ages of 40 and 60 years. The relative
decline in estradiol is greater in this interval than the
decreases in estrone or estriol (Procope, 1969). Mattingly
and Huang (1969) found increased ovarian production of
androgens during the interval of declining estrogen secre-
tion in women. In addition, Dilman (1976) has shown an
increased production of noncalssical phenolsteroids
in the aging female. Adamapoulos et al. (1971) has
reported decreased pregnandiol excretion in women
approaching menOpause, which suggests that ovarian proges-

terone secretion may also be effected by aging. The effect

12

of aging on ovarian sex steroid secretion in laboratory
rodent species remains almost totally unknown. Most
estimates of aging effects on rat ovarian steroid secretion
have been derived indirectly, using vaginal cytology as an
index of sex steroid secretion.

Alterations in vaginal cyclicity with age have also
been correlated with ovulatory and luteinizing activity of
the ovary. Although the ovaries of constant estrous aged
rats are anovulatory, atrophic and contain no corpora lutea
(Aschheim, 1976; Clemens and Meites, 1971), aged repetitive
pseudOpregnant rats show normal ovulation at intervals of
12 to 30 days. The presence of freshly formed corpora
lutea and the ability of the uterus to form deciduoma after
endometrical trauma suggest that aged pseudOpregnant rats

are secreting considerable ovarian progesterone.

Female--Pituitary Function

 

Pituitary gonadotrOpin secretion is markedly
altered in aged females. Everitt (1976) points out evi-
dence for overstimulation of the ovary by excessive
pituitary gonadotrOpin secretion in aged women that could
lead to premature exhaustion of the ovary. However,
Baranov et al. (1972) found that the excretion of estrogen,
pregnandiol, and gonadotropins was not altered in women
until after the first missed menstrual period. Llewellyn-
Jones (1971) proposed that the decrease in the FSH:LH ratio

found with advanced age may be responsible for the changes

13

in follicular development and sex steroid secretion associ-
ated with menopause.

In contrast, there is no evidence for increased LH
secretion in aged rats. Aschheim (1976) cited the existence
of ovarian deficiency cells in the interstitial tissue
beginning at 13 months of age as evidence of inadequate LH
stimulation. Aschheim also found that LH injection or
pituitary implants would make the deficiency cell undetec-
table in the aged rats, and injections of purified LH in
aged constant estrous rats will result in the resumption of
normal estrous cycles. This suggestion of inadequate LH
stimulation of aged rat gonads is supported by reports from
our laboratory in both male and female rats (Shaar et al.,
1975; Riegle and Meites, 1976). These studies have demon—
strated that serum LH is diminished and serum prolactin is
elevated in aged rats. Accordingly, Pecile et a1. (1966)
has reported prOportionally higher numbers of acidophils
present in aged rat pituitaries than in those of young rats.

Several workers have shown that pituitaries of
young rats can maintain ovarian function in ovaries trans-
planted from aged donors to young recipients (Peng and
Huang, 1972; Aschheim, 1976). Similarly, Pecile et a1.
(1966) found that gonadal function in young hypOphysecto-
mized female rats, as assessed by vaginal estrous cyclicity
and by uterine and ovarian weight, could be restored by
transplanting pituitary tissue from young donor rats.

Transplantation from older donors did not restore

l4

gonadotropic function. However, Peng and Huang (1972) also
showed that pituitaries from aged donors transplanted under
the median eminence region of hyp0physectomized young adults
could sometimes restore estrous cycling and fertility.

The data summarized thus far indicate important
age differences in gonadal steroid and pituitary gonado-
trOpin secretion in aged women and rats. These observations
support the view that the ovary is not the primary site of
malfunction in reproductive control systems in the aging
rat. Although functional alterations at the hypOphysial
level are implicated, they may not be totally responsible
for senescent impairment of reproduction.

Pituitary gonadotrOpin content and pituitary
responsiveness to hypotha1amic stimulation is also effected
by aging. Clemens and Meites (1971) found increased
prolactin content in aged constant estrous female rats.
Although Matsyama (1966) reported increased gonadotrOpin
content in 12 mo old constant estrous rat pituitaries,
Clemens and Meites (1971) have reported decreased pituitary
LH content. Watkins et a1. (1975) and Riegle and Meites
(1976) recently reported lower serum LH concentrations in
aged rats in response to acute LHRH injections than in

similarly treated young rats.

Female--Hypotha1amic Function

 

Many investigators now believe that a primary site

of age related alteration in the reproductive control system

15

is the hypothalamus. Dilman (1976) suggests that aging of
the hypothalamus reduces its sensitivity to estrogen feed-
back, possibly due to impaired neurotransmitter synthesis
(Frolkis, 1966). This theory is supported by reports of
reduced inhibition of ACTH secretion by glucocorticoids in
aged rats (Riegle and Hess, 1972; Riegle, 1973). Shaar

et a1. (1975) also demonstrated a smaller increase in serum
LH after castration in aged rats as compared to young rats
and a diminished response to negative feedback by adminis-
tered gonadal steroids. However, Odell and Swerdloff
(1968) and Wise et a1. (1973) found similar hypotha1amic-
pituitary sensitivity to estrogen feedback in both pre- and
post-menOpausal women.

Alterations in hypotha1amic function have not been
correlated with anatomical changes of the hypothalamus with
age. Andrew (1956) showed no cellular destruction in the
supraoptic and paraventricular nuclei in the senile human
hypothalamus. Finch (1973) also reported that brain weight
and cellularity do not vary with age.

Several studies suggest that differences in the
concentration of biOgenic amines and the ability of the
hypothalamus to secrete its releasing hormones is of great
significance in the effect of aging on reproductive control
systems. Finch (1973) found that catabolism of norepine-
phrine and dOpamine is decreased in aged mice. Clemens and
Meites (1971) reported elevated hypotha1amic follice

stimulating hormone releasing factor (FRF) activity in

16

aged constant estrous rats compared to that of young ones
on the day of estrous.

Attempts have been made to experimentally alter
hypotha1amic control mechanisms. Systemic progesterone
treatment of several days duration to aged rats in constant
estrus is capable of inducing ovulation in association with
at least one apparently normal estrous cycle (Clemens
et al., 1969). Clemens et al. (1969) also demonstrated that
direct electrical stimulation of the hypotha1amic preoptic
area of old constant estrous rats can cause ovulation.
Their report further showed that prolonged systemic epine-
phrine administration could induce ovulation in old rats,
the effect possibly mediated through alteration of certain
central nervous system neuroendocrine functions. Similarly,
chronic administration of L-dOpa or iproniazid, drugs
assumed to increase hypotha1amic catecholamine availability,
restored regular cycling patterns in old constant estrous
rats (Quadri et al., 1973). Watkins et a1. (1975) reported
a decreased response to acute hypotha1amic stimulation with
L-dopa in aged rats as compared to young rats. It was
found that the resulting increase in LH and decrease in
prolactin was less pronounced in aged rats.

Riegle and Meites (1975) demonstrated that young
rats respond to acute stress with increased serum LH, while
aged rats show no such increase with stress, suggesting
some alterations in the CNS mediated hypotha1amic-pituitary

mechanism with age.

l7

Male--General Considerations

 

While the male reproductive system is often regarded
as less complex than that of females, it also seems that
investigators have a more incomplete understanding of the
details concerning male reproductive control mechanisms,
and particularly the alterations occurring in them with
age. In male reproduction, advanced aging is characterized
by the progressive decrease in spermatogenesis, hormonal

secretion, secondary sex characteristics, and libido.

Male--Gonadal Function

 

The testes of senescent rats have been found to be
significantly smaller than those of young adults (Peng
et al., 1973) and similar findings have been reported in
men (Stearns et al., 1974). A reduction in size and
activity of the seminiferous tubules has also been
reported in aged males(Adams, 1972). Although sperm
production has been found to decrease, abundant sperm have
been found in the testis and epididymis of sexually
inactive aged males (Bishop, 1970; Peng et al., 1973).
This observation that the loss of gametogenesis is not the
primary lesion associated with decreasing fertility in
aging males is in agreement with data from the female. The
most common degenerative change in the testis with age is
fibrosis in the seminiferous tubules. The numbers and
histological appearance of Leydig cells of the testis have

also been found to decline with advancing age (Albert,

18

1961, Peng et al., 1973). On the other hand, Lynch and
Scott (1950) reported increased structural evidence for
activity of sertoli cells associated with atrOphied Leydig
cells in the aged human testis. Here it was postulated that
increased sertoli cell secretions of estrogens could
inhibit LH secretion and contribute to the atrophy of the
Leydig cells. Peng et a1. (1973) also reported decreased
seminal vescicle weight in aged rats.

There appears to be significant species differences
in hormonal changes occurring with age. Eleftheriou and
Lucas (1974) reported no decrease in serum testosterone
concentrations in aging mice. Reports generally agree that
plasma testosterone decreases in men over 70 years of age
(Vermeulen et al., 1972; Persky et al., 1971; Rubens et al.,
1974; Stearns et al., 1974), although Vermeulen et a1.
(1972) observed changes in testosterone to be highly vari-
able. Ghanadian et a1. (1975) found serum testosterone in
aged male rats decreased as compared to young adults.

There have also been some reports of decreased
testicular ability to respond to acute stimulation. Long-
scope (1973) showed that testosterone increased more in
young than in aged men after administration of HCG.
Similarly Rubens et a1. (1974) reported decreased Leydig
cell response to HCG in aged men, and suggested that the
decreased testosterone secretion with age has a primary

testicular origin.

19

Male--Pituitary Function

 

In support of the theory of primary testicular
degeneration in men, Stearns et a1. (1974) reported that
mean LH concentrations rise after 40 yr of age, with
gonadotropin concentrations being inversely related to
testicular size. Stearns et a1. (1974) hypothesize that in
men there is a primary decline in testicular function
beginning at 45-50 yr of age, with a resulting increase in
pituitary LH secretion. However, alterations in the
reproductive system of male rats appear to be somewhat
different. Riegle and Meites (1976) reported decreased
serum LH in the aged male rat as compared to the young
adult. Several reports have also shown decreased pituitary
LH with age (Debeljuk et al., 1972; Peng et al., 1973;
Riegle et al., 1976) in the rat. Debeljuk et a1. (1972)
has also demonstrated that the pituitary response to acute
LHRH stimulation is decreased in aged male rats. These
reports indicate that testicular degeneration is not the
primary source of dysfunction in the reproductive control

system in the rat.

Male--Hypothalamic Function

 

There are conflicting reports concerning changes
occurring in the hypotha1amic sensitivity to negative feed-
back with age. Peng et a1. (1973) reported a larger
increase in plasma LH after castration in aged males than

in young male rats. Shaar et a1. (1975) found the opposite

20

result, accompanied by an increase in pituitary sensitivity
to testosterone negative feedback in aged male rats.

In summary, observations indicate that reproductive
control mechanisms are altered in aging mammals. It
appears, however, that the specific changes are not coinci-
dent in humans and in the rat. Data from men and women
imply primary degeneration of the gonad in both sexes may
result in the increased serum LH concentrations demonstrated
with advancing age. The rat, on the other hand, shows no
such increase in pituitary secretion and other sources of
degeneration besides the gonad may be implicated. There is
considerable evidence which suggests age alterations in the
function of the ovary, pituitary, and hypothalamus in the
female rat. To date, much less is known concerning aging
effects of gonadal control systems in the male. The experi-
ments to be presented were intended to further characterize
the alterations occurring in the reproductive control system
of the male rat with age and to try to localize the primary

site of these alterations.

METHODS

Experimental Animals

 

Young adult and aged male Long Evans rats (Blue
Spruce Farms, Altamont, New York) were used in these
studies. Rats included in the young group were three to
six months of age, while those classified as aged ranged
from twenty to thirty months. The studies included in
this report have considered the effects of age on these
two age groups only, with no consideration of intermediate
ages. Rats to be used in aged groups were obtained either
as retired breeders or surplus rats raised in our colony.
All rats were housed in the Endocrine Research Unit's rat
colony under conditions of controlled light (12 hr light
cycle) and temperature (21°-22°C) and given free access
to Wayne Lab-Blox (Allied Mills, Chicago, Ill.) and water.
Mean body weights for young and aged rats were 436 and
502 grams respectively. In some studies, rats were sub-
jected to more than one experimental treatment. In these
instances, all rats were allowed a period of recovery of
at least three weeks following previous experimental use to
assure adequate hematocrit and blood volume recovery before

they were considered suitable for further experimentation.

21

22

Blood Collection

 

Rats were removed from their cages and transported
to a surgery room before experimentation. All blood samples
were taken under light ether anesthesia by orbital sinus
puncture using heparinized capillary tubes. The volume of
each blood sample ranged from 1 to 1.5 ml. Pretreatment
blood samples were obtained within 30 to 60 seconds after
the rats were first disturbed. This method of blood
sampling has been shown to keep alterations of hormone
concentrations due to stress effects at a minimum (Euker
et al., 1975).

Blood samples were allowed to clot at room tempera-
ture for at least 30 min and then refrigerated overnight.
Serum was then separated by centrifugation, decanted, and
stored at 20°C until the day of radioimmunoassay for LH or

testosterone.

Hormone and Drug Treatment

 

Several experiments were performed involving
treatment with synthetic luteinizing hormone releasing
hormone (LHRH). Each rat was under light ether anesthesia
when given 500 ng LHRH (Eli Lily Co., Indianapolis, Ind.)
in 0.5 m1 of physiological saline into an exposed jugular
vein. In other experiments, human chorionic gonadotropin
(HCG) at doses ranging from 1 to 20 10 was administered

intraveneously in 0.5 ml of saline. When L-dopa was given,

23

15 mg dosages of the drug suspended in 0.5 m1 of saline
were given twice daily (W8 am and W5 pm) for 10 consecutive

days either subcutaneously or intraperitoneally.

Hypothalami Collection and Preparation

 

The procedure for handling hypotha1amic tissue was
a modification of that of Shaar and Clemens (1974). Sixteen
young and 16 aged male rats were decapitated as rapidly as
possible after removal from their cages. Hypothalamic
tissue (3 x 3 x 2 mm) was then quickly collected from each
rat, weighed, and placed in a tissue homogenizer containing
0.2 ml cold 0.4 N perchloric acid. The tissue was manually
homogenized, the homogenate decanted, and the homOgenizer
rinsed with another 0.2 ml of cold 0.4 N perchloric acid.
The combined homogenate for each hypothalamus was centri-
fuged at 20,000X g for 30 min at 3°C. The supernatant was
then decanted into a clean tube containing 0.1 ml of 190
EDTA (Sigma Chemical Co., St. Louis, Mo.) and stored at

-20°C until assayed.

Catecholamine Assay

 

Hypothalamic catecholamine assays were conducted
in Dr. James Clemens' laboratory at the Eli Lily Company in
Indianapolis, Indiana, under the technical direction of
Dr. Carl Shaar. Norepinephrine and dopamine were measured
by a modification of the microflourescent technique of
Laverty and Taylor (1968). Eight hypotha1amic extracts

from each age group were used for dopamine quantification,

24

with the remaining 8 extracts used for measurement of
norepinephrine. Each individual frozen preparation was
thawed, transferred to a beaker and 2 ml of l M sodium
acetate buffer and 1 ml of 1% disodium EDTa were added;
then 240 mg of heat activated, neutral, grade 1 alumina
were added and the mixture swirled for 5 minutes. After
mixing, the supernatant was decanted and discarded. The
alumina with the adsorbed catecholamines was then

washed into a micro column containing 140 mg of alumina.
The column was washed 3 times with triple distilled water,
after which catecholamines were eluted into evaporating
tubes with 5 m1 of .2 N acetic acid. After evaporation
to dryness on an evapormix, the residue was resuspended in
.4 ml of triple distilled water and catecholamine deter-
minations were made by acid fluorescence using a system of
reverse blanks and an internal standard. Ten ul of phos-
phate buffer (PH 6.5), 10 pl of .02 N iodine and 50 ul of
alkaline sulfite solution were added to 100 pl of the
resuspended extract. After 5 minutes, 30 pl of glacial
acetic acid were added to develop the fluorescence.
Fluorescence of norepinephrine was read at excitation wave
length of 392 mu and emission of 490 mu. After 40 min

of heating at 100°C, dopamine fluorescence was measured at
320 m excitation and 380 m emission. Analysis of these
data was performed using Student's t-test and a probability

less than 0.05 was considered significant.

25

Radioimmunoassay for Testosterone

 

The radioimmunoassay procedure for testosterone
determination was that described and validated by Mongkon-
punya et al. in 1975.

Duplicate aliquots (100 pl) from the unknown serum
samples to be assayed were dispensed into 16 x 100 mm
disposable culture tubes (Scientific Products, McGaw Park,
111.). In order to account for losses occurring during the
extraction procedure, 3000 dpm of 3H-l,2 testosterone (New
England Nuclear, Boston, Mass.) was added to a third group
of a representative number of tubes (10-20 per assay) con-
taining a standard serum (100 pl). All tubes then received
2 m1 of benzene: hexane (1:2), were vortexed for 30 seconds
each, and were stored at -20°C for at least 1 hr in order
to freeze the aqueous phase. The organic solvent from the
tubes containing unknown serum samples were then decanted
into 12 x 75 mm disposable culture tubes, and those with
3H- testosterone were decanted into scintillation vials.

Culture tubes containing various known amounts of
purified testosterone (Sigma Chemical Co.) were pipetted
from a stock solution having a concentration of 10 ng/ml.
At least three sets of standards containing 0.0, 0.02,
0.05, 0.10, 0.25, 0.50, 0.75, 1.0, 1.5, and 2.0 ng were
included in each assay to serve as reference standards.

All serum extracts and testosterone standards were
dried by air. Testosterone antibody (Niswender antiserum

to testosterone - 3 - oxime - bovine serum albumin, #666)

26

was diluted 1:3000 in 0.1% gelation in 0.1 M phosphate
buffered saline and 200 In of this solution was added to
each tube. Tubes were vortexed briefly and allowed to
incubate at room temperature for 30 min. At this time
approximately 30,000 dpm 3H-1,2,6,7-testosterone (New
England Nuclear, Boston, Mass.) diluted in 200 pl gel PBS
was added to each tube, vortexed, and refrigerated at 4°C
for 24 hr to allow the complexing reaction between antigen
and antibody to attain equilibrium.

To separate free from bound testosterone, a dextran-
coated charcoal solution was made with 0.025 gm of dextran
150 and 0.25 gm of carbon decolorizing neutral Norit sus-
pended in 100 ml distilled water, and 0.5 ml of this
solution was added to each tube. Each tube was then vor-
texed briefly, chilled in an ice bath for 10 min and
centrifuged in a refrigerated centrifuge at 2500 x g for
10 min. A 0.5 ml aliquot of the supernatant fluid of each
tube was then diluted with 5 ml of liquid scintillation
cocktail (Research Products International Corp., Elk Grove
Village, Ill.) in scintillation vials for quantification of
radioactivity in a liquid scintillation spectrometer
(Nuclear Chicago, Des Plaines, Ill.). Vials made to check
extraction recovery also received 5 ml of scintillation
fluid and were counted. For comparison among assays,
standard rat serum and blank extraction tubes are also

assayed with each set of unknown serum samples.

27

All tubes were counted for four minutes. Standard
curves were drawn on semilogarithmic paper correlating CPM
with the log of the reference standard doses. Values for
all unknown serum samples derived from the standard curves
were then transformed into ng/ml concentration units.
Duplicate values were averaged and statistical analysis was
performed using Student's t-test. A critical alpha

probability value of 0.05 was selected for these analyses.

Radioimmunoassay for LH

 

The radioimmunoassay procedure for LH determination
was that described and validated by Monroe et al. in 1968
and routinely used in the laboratory of Dr. J. Meites at
Michigan State University.

Radio-iodination of purified rat LH (LER 1056 LH)

with 125

I was performed at Dr. Meites' facilities. Labeled
hormone was eluted through a l x 15 cm Bio-Gel P60 column
and then diluted to a concentration of approximately 30,000
counts per minute (CPM) as counted by an automatic well
counter (Nuclear-Chicago, model 1085L; Des Plaines, Ill.)
using 0.1% gelation phosphate buffered saline (PBS). Anti-
ovine antiserum (supplied by Dr. Niswender, Fort Collins,
Colo.) was made by immunization of rabbits with purified
hormone and was diluted to a concentration of l:28,000 for
use in the assay. The antigen-antibody complex was pre-

cipitated using a second antiserum resulting from specific

immunization of sheep against rabbit gamma globulin. This

28

ovine-rabbit gamma globulin serum was diluted to a working
concentration which from various bleedings ranged from 1:35
to 1:60.

Duplicate aliquots from the unknown serum samples
to be assayed were dispensed into 12 x 75 mm disposable
culture tubes (Scientific Products, McGaw Park, 111.), and
then diluted to a volume of 0.5 ml with gel PBS. A volume
of 200 pl of the working rabbit antiserum (anti-LH) was
added to each tube and the tubes vortexed briefly. Equili-
bration of the complexing reaction between available
hormone antigen and antibody was accomplished during 24
hours of incubation at 4°C. At the end of the incubation,
100 pl of the labeled LH solution was added to each tube,
the tubes vortexed, and incubated for another 24 hours at
4°C, allowing the competitive reaction between the unknown
amount of endogenous hormone in the serum sample and the
radio-labeled hormone to equilibrate.

Following this incubation, 200 ul of the ovine anti-
rabbit gamma globulin antibody solution was added to each
tube. The tubes were then briefly vortexed and refrigerated
at 4°C for 72 hours to allow maximum antigen-antibody com-
plexing and precipitation. At the end of 72 hours 3 ml of
cold PBS were added to each tube and the tubes were centri-
fuged at 2200 RPMs for 30 min. The supernatant was poured
off and each tube placed in a plastic holding jacket for

counting in the automatic gamma well counter.

29

Culture tubes containing known amounts of purified
hormone were also included in the assay to serve as refer-
ence standards. Five identical samples of 11 different
doses ranging from 0.8 to 40.0 ng of purified NIH LH RP-l
was used for this purpose. Total count tubes, normal
rabbit serum tubes (NRS) and total antibody binding tubes
were also included in the assay procedure in order to
determine general binding characteristics of each assay.
Only radio-labeled hormone was added to the total count
tubes and these tubes were a reflection of the total effi-
ciency of count recovery. Normal rabbit serum (NRS) tubes
received 200 ul of a standard rabbit serum diluted in gel
PBS in place of hormone specific antibody, in order to
monitor non-specific binding activity. Total antibody
binding tubes were equivalent to "zero hormone" standards.
The counting time for all tubes in a given assay was deter-
mined by the time necessary for the "zero hormone" standards
to equal 10,000 CPM. Also, the non-specific activity
represented by the NRS tubes was subtracted from all sample
tubes by appropriate adjustment of the background setting
on the gamma counter.

Standard curves were drawn on 3-cyc1e semi-
logarithmic graph paper correlating CPM with the log of the
dose of reference standard hormone. These standards curves
showed 50% cold hormone binding at 8-15 ng. Quantitative
serum hormone data for all unknown serum samples derived

from the standard curves were then transformed into ng/ml

30

concentration units. Duplicate values were averaged and
statistical analysis was performed on the experimental data
using Student's t-test. A critical alpha probability of

less than 0.05 was considered significant.

EXPERIMENTAL

Experiment 1. The Effects of HCG on
Serum TEstosterone

 

 

Introduction:

Relatively little is known about testicular func-
tion in aged male mammals. Although aged men have been
reported to have lower blood concentrations of testos-
terone than young men (Persky et al., 1971; Vermeulen,
1976), other studies have shown this age-related decline
in blood testosterone levels in men to be variable
(Vermeulen et al., 1972). Other reports have suggested
that the metabolism of pregnenolone is slower in older men
17‘C20
hydroxylase activity (Axelrod, 1972). The decrease in blood

and that the aged human testis may have less C

testosterone of the human male is in conflict with a recent
report showing no change in plasma testosterone in aging
mice (Eleftheriou and Lucas, 1974). Although our laboratory
has reported decreased serum LH in aged male rats (Riegle
and Meites, 1976), little information is available concern-
ing blood testosterone concentrations or testicular respon-
siveness in the aged male rat. This study was undertaken to
compare serum testosterone levels and testicular responsive-

ness to gonadotropin stimulation in the aged male rat.

31

32

Procedures:

The effect of HCG on serum testosterone was studied
in two trials. In the first trial, 23 young and 27 aged
male rats were assigned to one of three treatment groups to
be given either 0.5 ml of physiological saline or 0.5 ml of
saline containing 1 IU or 5 IU of HCG via jugular injection.
Serial blood samples were taken from each rate before the
intravenous injections and at 15, 30, and 60 minutes after
injection.

In a second trial, groups of 32 young and 31 aged
male rats were assigned to one of 4 experimental groups
and were given either intravenous injections of 0.5 ml of
physiological saline or 0.5 ml of saline containing 1, 5,
or 20 IU of HCG. Serial blood samples were taken from
each rat before the intravenous injection and at 45, 90,

and 150 minutes after the HCG treatments.

Results:

Figure 1 illustrates serum testosterone concentra-
tions in the control groups of young and aged rats which
received only the saline injections in the first experi-
ment. Serum testosterone concentrations were significantly
lower in aged male rats than in young male rats before
treatment and throughout the sampling period. Testosterone
concentrations remained stable throughout the sampling

period in both groups.

Figure l.

33

Effects of intravenous administration of 0.5 ml
saline on serum testosterone concentrations in
young and aged male rats.

These data illustrate serum testosterone levels
in saline injected control rats. Serum testos-
terone concentration expressed in ng/ml appears
on the ordinate, with interval of time in min-
utes after injection represented on the
abscissa. Blood samples were taken prior to
injection of saline, and 15, 30, and 60 minutes
afterwards. The solid line represents the
means for 8 young rats and the dashed line
signifies those for 6 aged animals. Standard
errors are indicated with bars above and below
each mean.

 

34

 

 

 

 

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TIME (min)

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35

The response of young and aged male rats to l and
5 ID of HCG is plotted in Figure 2. Pretreatment concentra-
tions of serum testosterone were higher in young than in
the aged rats. After intravenous injection of l or 5 ID of
HCG, rats in both age groups had sharp, steady increases in
serum testosterone. This increase in testosterone, however,
was approximately twice as great in young as in aged males.
Also, both young and aged rats showed a greater increase
in serum testosterone following injection of 5 IU of HCG
than did the groups injected with l IU of HCG.

Figure 3 shows the results of the second trial
which considered the effects of HCG stimulation over an
extended sampling period. As in the first experiment,
serum testosterone concentrations in the control groups
which received only the saline injections were significantly
lower in the aged male rats than in young male rats. Serum
testosterone was increased in both young and aged rats
following all 3 doses of HCG. The increase in testosterone
was smaller in the aged rats compared to the young rats at
each dose and at all sampling intervals. The increase in
serum testosterone levels following HCG injection was sus-
tained throughout the sampling period in rats of both age
groups. The increase after 20 IU was not significantly
greater than after 5 ID except for the initial sample at

45 minutes in the aged group.

 

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Discussion:

These data indicate that there are substantial
alterations in testosterone secretion in aged male rats
both in terms of resting serum testosterone concentrations
and testicular responsiveness to acute gonadtrOpin stimula-
tion. The decreased testosterone concentrations in blood
samples from the control groups of aged rats are in general
agreement with data from aged human males (Persky et al.,
1971; Vermeulen, 1976), but are quite different from the
level of plasma testosterone reported in aged mice (Elef-
theriou and Lucas, 1974).

The increased serum testosterone concentrations in
both age groups following HCG stimulation in the initial
HCG experiment demonstrated that the testes of the aged male
is capable of responding to stimulation by gonadotropin.
However, this experiment did not indicate whether maximal
testicular steroid secretion had been stimulated or what the
time course of increased serum testosterone levels following
HCG administration was in either the young or the aged
groups. Since testosterone concentrations had not peaked
prior to 60 minutes, it was possible that given sufficient
time after HCG injections, aged rats would attain serum
testosterone concentrations similar to the levels achieved
following HCG injections in the young groups. However, in
the second trial, serum testosterone concentrations were
also higher in the young males than in aged male groups at

all sampling intervals. The longer experimental sampling

41

interval, and the increased dose of HCG used in the second
HCG experiment, suggests that 5 ID of HCG was sufficient to
show maximal testicular effect. The 20 IU of HCG injection
was more effective than the 5 IU HCG injection in stimula—
ting serum testosterone only at the 45-minute sampling
interval in the aged male group. Serum testosterone con-
centrations in the aged male 5 and 20 10 treatment groups
were similar at both the 90- and the 150-minute sampling
intervals. These data also suggest that the peak in serum
testosterone concentrations occurs between 45 and 60
minutes following HCG injection in both age groups and

that these elevated testosterone levels are sustained for

at least 150 minutes after the injection of gonadotrOpin.

42

Experiment 2. The Effects of Multiple
LHRH InjectIOns on Sérum.LHI'

 

 

Introduction:

Several laboratories have hypothesized that signifi-
cant changes occur in hypotha1amic-pituitary control of
anterior pituitary hormone secretions during aging. Our
laboratory has recently reported that single LHRH injec-
tions stimulate smaller increases in serum LH in aged
compared to young adult rats of both sexes (Watkins et al.,
1975; Riegle and Meites, 1976). Since these aged rats had
lower pretreatment serum LH concentrations, it is possible
that they are receiving less endogenous hypotha1amic
releasing hormone stimulation than are young rats. This
experiment was designed to determine the effect of a longer
duration of LHRH stimulation on pituitary LH secretion in

young and aged rats.

Procedure:

Groups of 17 aged and 24 young males received 3
intravenous injections of 500 ng of LHRH at 75-minute
intervals. Serum LH was measured in serial blood samples
taken before each LHRH injection and 15 minutes following

each drug treatment.

Results:
The data in Figure 4 show that the young male rats
had higher pretreatment serum LH concentrations and higher

serum LH 15 minutes after the first LHRH injection. On the

Figure 4.

43

Effects of three consecutive injections of
500 ng LHRH on serum LH concentrations in
young and aged male rats.

These data illustrate the effects of three LHRH
injections given 75 minutes apart on serum LH
levels in groups of 24 young and 17 aged male
rats. Serum LH concentration expressed in
ng/ml appears on the ordinate, with interval
of time in minutes after the first injection
represented on the abscissa. Blood samples
were taken prior to and 15 minutes following
each LHRH injection. Solid line represents
means of young rats and the dashed line signi-
fies those of aged rats. Standard errors are
indicated by bars above and below each mean.

 

L H (ng./ml.)

3001

250'

200'

ISO:

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50‘

 

44

 

7? 9‘0 I53 E5
TIME (min)

45

other hand, serum LH concentrations were similar in rats of
both ages before and after the second and third LHRH

injections.

Discussion:

The lowered pretreatment serum LH concentrations
and reduced responsiveness following the first LHRH injec-
tion in the aged rats are in agreement with the report of
Riegle and Meites (1976). The overall similarity of serum
LH concentration between rats in the two aged groups in
this experiment clearly indicates that the pituitary of aged
male rat's pituitary is capable of a higher level of
activity than it normally maintains, at least for the
experimental interval tested. The ability of the pituitary
to increase LH secretion after orchidectomy in aged rats
(Peng et al., 1973; Shaar et al., 1975) also seems to indi-
cate that the ability of the hypotha1amic-pituitary control
system to increase LH secretion remains functional. These
data lend support to the hypothesis (Dilman, 1971) that the
hypotha1amic-pituitary control system becomes less sensi-
tive to feedback control in aged male rats. Thus, the
decrease in tonic function at the testicular and hypOphysial
level with age may be only secondarily due to a degenera-
tion of function at those sites in response to a primary
chronic decrease in the stimulatory activity of higher

centers, most likely the hypothalamus and CNS.

46

Experiment 3. The Effects of LHRH
Injections on Serum Testosterone

 

 

Introduction:

The previous experiments have shown that serum LH
concentrations are increased in aged male rats following
LHRH injections and serum testosterone concentrations are
increased after HCG stimulation. This study considered
the effect of LHRH treatment on serum testosterone concen-

trations.

Procedure:

Groups of 8 aged and 8 young male rats received
intravenous injection of 500 ng of LHRH. Serum testosterone
was measured in serial blood samples taken before and 15,

30, and 60 minutes after LHRH injection.

Results:

The data in Figure 5 show that the pretreatment
concentration of testosterone was higher in young than in
aged rats. Serum testosterone concentrations were progres-
sively increased following LHRH injections in young rats,
while serum testosterone in aged rats was not significantly

increased over saline injected control levels.

Discussion:
Although the previous experiments have shown that
the aged male rats' testes and pituitaries are capable of

responding to LHRH and HCG stimulation, serum testosterone

Figure 5.

47

Effects of intravenous administration of 500 ng
LHRH on serum testosterone concentrations in
young and aged male rats.

These data illustrate the effects of LHRH injec-
tion on serum testosterone concentrations in
groups of 8 young and 7 aged male rats. Serum
testosterone concentration expressed in ng/ml
appears on the ordinate, with interval of time
in minutes after injection represented on the
abscissa. Blood samples were taken prior to
injection of LHRH and 15, 30, and 60 minutes
afterwards. The solid line represents means
for young rats and the dashed line signifies
those for aged animals. Standard errors are
indicated by bars above and below each mean.

 

48

 

 

 

IO-I

:Eas uzomuhmohwm»

TIME (min)

49

did not increase in this experiment following LHRH. There
are several factors which could be involved with this dis-
crepancy. The increase in serum LH following LHRH may be
less than the LH activity associated with the HCG injec-
tions, or the testes may not respond to the LH endogenously
secreted by the aged rat pituitary. Both hypotheses will
require additional experimentation before either can be

applied to these data.

50

Experiment 4. Hypothalamic Norepinephrine
and Dopamine Content

 

 

Introduction:

The hypothalamus contains large amounts of nore-
pinephrine and dopamine (Palkovits et al., 1974) which have
been shown to be involved in control of release of
anterior pituitary regulatory-hypotha1amic hormones.
Experiments from our laboratory and others have suggested
fundamental changes in hypotha1amic control of endocrine
function with aging. It is conceivable that these changes
in hypotha1amic catecholamine content are related to the

effects of aging on this control tissue.

Procedure:

Groups of 16 young and 16 aged male rats were
decapitated as rapidly as possible after removing them from
their cages. Each hypothalamus was weighed and homogenized
as previously described. Hypothalamic norepinephrine and
dopamine was determined separately from 8 hypotha1amic

extracts from each age group.

Results:

DOpamine, and norepinephrine content in hypothalami
of young and aged male rats are shown in Figure 6. In both
instances, the hypothalamic content of catecholamines of
young rats was about twice that found in the aged group.
Average hypotha1amic dOpamine and norepinephrine content of

young rats was 32.5 i 9.3 mg per hypothalamus and

51

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47.6 i 10.7 mg per hypothalamus, resPectively. Hypothalamic
content of dopamine and norepinephrine in aged rats

averaged only 15.6 i 2.5 mg and 22.8 i 1.8 mg per hypotha1a-
mus, respectively. Hypothalamic weights averaged approxi-
mately 20 mg with no significant difference in size between

the two age groups.

Discussion:

The results of this study suggest that important
changes in hypotha1amic catecholamine function may accom-
pany aging. Although it is agreed that both adrenergic and
dopaminergic pathways are involved in regulation of
hypotha1amic endocrine secretions, the precise mechanism
by which these amines regulate hypotha1amic secretion
remains to be determined. Techniques such as those used
in this study measure only the catecholamine content of the
whole hypothalamus. Current concepts of the mechanism of
amine control of hypothalamic function suggest that differ—
ences in hypotha1amic catecholamine content in specific
areas of the hypothalamus and turnover in individual hypo-
tha1amic nuclei may be more important in understanding the
mechanisms of hypothalamic control (Palkovits et al., 1975).

Although the present data do not indicate differ-
ences in hypotha1amic function, in particular anatomical
regions, they indicate that there are major decreases in
catecholamine function in our aged male rats which is con-
sistent with our earlier reports of alterations in adreno-

cortical (Riegle, 1973) testicular and pituitary secretions.

54

Experiment 5. The Effects of Chronic
L-dOpa Treatment on Pituitary
ResponéIVeness to LHRH
Introduction:

We and others have hypothesized the changes in
hypotha1amic function may be involved in age-related altera-
tions in adrenocortical and gonadal function. Quadri et a1.
(1973) demonstrated that daily administration of epine-
phrine, iproniazid or L-dopa could cause the resumption of
cycling in aged constant estrous rats, suggesting that a
deficiency of hypothalamic catecholamines may be responsible
for the reduced ability of the pituitary to release LH and
an increased pituitary release of prolactin in aged rats.

In addition, Watkins et a1. (1975) found that a single
systemic injection of L-dOpa could increase serum LH con-
centrations and reduce serum prolactin concentrations in
both young and aged female rats. The present study con-
sidered the effects of chronic L-d0pa treatment on serum LH

following LHRH injection.

Procedure:

Groups of 19 aged and 30 young male rats received
an intravenous injection of LHRH. Serum LH concentrations
were measured in blood samples from all rats taken before
and at 15 and 45 minutes after hormone injection. These
rats were then randomly divided into subgroups. Aged male
rats received intraperitoneal injections of 0.5 ml of saline

or 15 mg of L-dOpa suspended in 0.5 ml of saline, twice

55

daily for ten days. The young rats were assigned to one of
2 experimental groups. The young control rats received

0.5 ml of saline alone, twice daily. The L-dOpa treated
group was subdivided with half of the group receiving 15 mg
of L-dopa suspended in saline by intraperitoneal injection
twice daily with the other treated group receiving a similar
L-dopa suspension subcutaneously twice daily for the lO-day
period. On the 11th day, 15 hours after the last L-dOpa
injection all the rats received a second LHRH injection.
Serum LH concentrations were measured in blood samples

before and at 15 and 45 minutes after LHRH injection.

Results:

Although the pretreatment LHRH response showed that
serum LH concentrations were higher 15 minutes after LHRH
injection in the young than in the aged rats, serum LH 15
minutes after LHRH treatment at the end of the L-dOpa
injection regime was similar for both ages (Figure 7).
Intraperitoneal L-dOpa injection did not affect serum LH
concentrations in either age groups. On the other hand,
serum LH concentrations in young rats receiving L-dOpa by
subcutaneous injection were lower 15 minutes following LHRH

than serum LH in either the control or intraperitoneal

injected groups.

Discussion:
Although acute administration of L-dopa by intra-

ventricular (Kamberi et 31.. 1970b and 1971a) or systemic

56

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injection (Watkins et al., 1975) has been shown to affect
pituitary release of LH and prolactin, interpretation of
these experiments suggest that chronic L-dOpa treatment
does not significantly affect the concentration of these
hormones in serum. Several factors could be involved with
the inconsistency. The forementioned studies considered
only the acute effects of this drug on serum hormone
levels. In addition, Watkins et a1. (1975) studied L-dOpa
effects in female rats. Riegle and Meites (1976) showed
that similar L-dopa treatments could acutely affect serum
prolactin, but not LH in male rats. On the other hand,
Quadri (1973) found that catecholamine injections similar
to those used in this study would reinitiate estrous

cycles in aged constant estrous rats. These experiments

do not indicate whether systemic L-dOpa injections effect
catecholamine function in the hypothalamus. The results

of this study, as well as the effect of acute L-dOpa
treatment, suggest that any effect of L-dopa on hypotha1amic
function is short-lived and indicates that chronic adminis-
tration of L-dopa may have little effect on neuroendocrine

control systems.

DISCUSSION

The data reported here indicates substantial
alterations in several components of reproductive control
systems in the male rat. There are, first of all, signifi-
cant changes in testosterone secretion in aged male rats,
both with respect to basal serum testosterone concentra-
tions and in testicular responsiveness to acute gonado-
trOpin stimulation. The decrease in resting testosterone
concentrations in the serum of aged male rats is consistent
with reports concerning senescent men (Persky et al., 1971;
Stearns et al., 1974; Vermeulen et al., 1972), while data
concerning the concentrations of plasma testosterone in
aged male mice has been quite different (Eleftheriou and
Lucas, 1974). The response of both young and aged rats to
the various doses of HCG given in the first experiment were
dose related and exhibited the same dynamics. However, the
amplitude of the testicular response was proportionally
less in the aged groups than in the young groups in all
cases. Explanation for this decrease in testicular func-
tion and reSponsiveness with age may involve a decrease in
the effectiveness of the systemic circulation carrying HCG

to the gonad, a decrease in the numbers or specificity of

59

60

testicular binding sites for HCG, a decreased activation of
the intracellular machinery to enhance testosterone synthe-
sis and secretion, and most probably a decrease in the
secretory capacity of the testes with age simply due to
decreased numbers of functioning Leydig cells. While
testicular function is somewhat impaired, it is to be empha-
sized that the testes of aged animals do respond to
gonadotrOpin stimulation, and the control system at the
level of the testis appears to remain intact.

Results from the second experiment verified an
earlier report from our laboratory (Riegle and Meites,
1976) that pituitaries of aged rats secrete less LH after
a single acute LHRH injection than that of young rats. The
inconsistency here lies in the logic that if the
hypotha1amic-pituitary mechanism were responding to
decreased negative feedback that should accompany low blood
testosterone levels, serum LH and pituitary responsiveness
to LHRH would be expected to be increased rather than
showing the decrease reported.

A second experiment also demonstrated that serum
LH concentrations after LHRH stimulation of a longer dura-
tion were not significantly different between the age
groups. Thus, pituitaries of aged rats are capable of
substantial secretory activity, certainly more than they
normally maintain. TOgether, the first two experiments
suggest that the decrease in serum testosterone and

decreased testicular response to acute HCG stimulation may

61

first reflect a long-term lack of normal LH stimulation of
the testes. This would account for the reported atrOphy of
Leydig cells in testes of aged males (Albert, 1961; Peng

et al., 1973).

These experiments also indicate that the decrease
in tonic function of the aged rat testis is not due to a
degeneration of function originating at the level of the
pituitary since it clearly retains a considerable capacity
to respond. Instead, these data seem to support the
hypothesis that the sensitivity of hypotha1amic-pituitary
control system to feedback control is altered with age
(Dilman, 1971), resulting in a chronic decrease in the
stimulatory activity of the CNS in general or more specifi-
cally the hypothalamus. The decrease in the stimulatory
activity of these higher centers would then be the causative
factor in the decrease in tonic functions at the testicular
and hypophysial level.

This theory, while it does account for the decrease
in the ability of the aged testis to function and respond
to stimulation, it does not completely explain the virtual
lack of testicular response to LHRH stimulation in aged
males in the third experiment. It is possible that the
complete lack of testicular response to LHRH may be due to
an additive effect of decreased responsiveness at both the
pituitary and testicular levels, resulting in a complete
flattening of the response curve. However, analysis of

acute HCG and LHRH experiments together might also suggest

62

the possibility that while the LHRH injections do stimulate
secretion of adequate amounts of LH by the aged male rat
pituitary, perhaps this endogenous LH secreted by the aged
rat is an anahormone, with impaired biological activity,
thus also explaining the subsequent lack of testicular
response.

A great deal of experimental evidence suggests
that hypotha1amic releasing hormone secretion can be influ-
enced by hypotha1amic catecholamine function. The ability
of the hypotha1amic neurons to secrete releasing hormones
could be related to changes occurring with age in the
hypothalamic response to stimulatory or inhibitory input
and the availability of neurotransmitters which have been
shown to influence hypotha1amic endocrine function. Inter-
pretation of data presented herein suggests that important
changes in hypotha1amic catecholamine function may accompany
old age. Although the methods used can recognize only
gross changes in hypotha1amic catecholamine content and
cannot distinguish between individual nuclei, they do show
that there are major decreases in catecholamine function in
aged male rats which are consistent with the alterations in
pituitary and testicular secretions. Perhaps the cause of
the decline in function and responsiveness of the testes
and pituitary originates with the decrease in hypotha1amic
catecholamine content. In any case, these preliminary
results would suggest that aging differences in the ability

of the hypothalamus to secrete its releasing hormone is of

63

great signifiCance in the effect of aging on reproductive
control systems and that hypotha1amic catecholamine con-
centrations play an integral part. Clearly, much more
experimentation in this area is necessary before the effects
of aging on the hypothalamus and the resulting impact on

the endocrine system as a whole is understood.

Recent studies suggest that systemically injected
L-dOpa may be converted to hypotha1amic catecholamines with
biological activity. Previous experiments have shown that
acute treatment with L-dOpa will result in a transient
decline in serum prolactin and an increase in serum LH
(Watkins et al., 1975) in young rats, and similar but less
dramatic changes following acute L-dopa injection in aged
rats. These reports, coupled with the present data con-
cerning decreased hypotha1amic catecholamines with age,
stimulated an attempt to examine the effects of L-dOpa
treatment of a longer duration. However, in this study
chronic treatment with L-dOpa did not significantly change
pituitary responsiveness to LHRH in the aged males as
was anticipated.

Simultaneous assessment of all experiments presented
generally suggest that with age, functional deterioration
of gonadotropin control occurs at the level of the gonad,
the pituitary and at the hypothalamus, with primary dys-
function occurring in the hypothalamus. The testes and
pituitary of the aged rat may function in response to a

chronic lack of stimulation originating at the hypothalamus,

64

which has become less sensitive to feedback control than
young rats. More experimentation is necessary to understand
more specifically the nature and degree of hypothalamic

alterations.

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