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llllllllllilllllfl'

l mama?
Michigan Sta te
i University

This is to certify that the
thesis entitled
Involvement of Ovarian Follicles in
Prostaglandin F20: Induced Luteal
Regression in Cattle

presented by
Trudy Lynn Hughes

has been accepted towards fulfillment
of the requirements for

Master's degree in Animal Science

 

f

ajor professor

Roy Fogwell

Date May 92 1985

 

0—7639 MSUi: an ‘m'mnfiw ‘ ' '1, '“H, ', Institun'on

'W.“-".

 

 

MSU

 

 

RETURNING MATERIALS:
Place in book drop to-
remove this checkout from

 

 

 

LIBRARIES .
w your record. FINES_ W11]
' be charged if book is

returned after the date
fl stamped below.
NM
1 1+ 1

 

 

INVOLVEMENT OF OVARIAN FOLLICLES IN
PROSTAGLANDIN F20 INDUCED LUTEAL

REGRESSION IN CATTLE

BY

Trudy Lynn Hughes

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1985

~3igtleifilo‘tv

 

ACKNOWLEDGEMENTS

I wish to extend my sincere gratitude to my major
professor, Dr. Roy Fogwell. Not only did Dr. Fogwell
challenge me to complete difficult tasks and to develop
myself as a scientist, he has given me the tools necessary
by providing materials, technical assistance, time and an
inspirational role model. I would also like to thank the
other members of my graduate committee for their time and
guidance, Dr. R. W. Dukelow and Dr. J. J. Ireland who
graciously took over in the absence of Dr. WhIL. Smith and
Dr. E. M. Convey.

A hearty thanks to the past and present members of the
Reproductive Physiology group for their time consuming
assistance. ‘When the schedule was tight, I could always
depend on the generous and pleasant aid of Jerry Hartung,
Jim Kesner, Mark Ledoux and Alex Villa-Godoy. In addition,
I would like to thank Kathy Koudele, Kwanyee Leung, and
Sharon Tonetta for their encouraging friendship and
tolerance.

Finally, I wish to thank my parents for their support,

advice and enthusiasm.

ii

TABLE OF

LIST OF FIGURES . . . .

ABBREVIATIONS . . . . .

INTRODUCTION . . . . .

REVIEW OF LITERATURE .

CONTENTS

Development and Maintenance of

/

Mechanism of Action of LE on

Corpora

Production of Progesterone‘

Causes of Luteolysis in Non-Pregnant Animals

Luteotropic Factors

Availability of LH .

Dynamics of Luteal Receptors for

0

During the Estrous Cycle .

Steroidogenesis and Accumulation

of cAMP

Luteolytic Factors

Evidence Suggesting PGan is

the Luteolytic Factor
Mechanism of Action of PGan in
Luteolysis

Hormonal Control of PGan Secretion

Role of Oxytocin in Luteal Regression
Evidence for a Direct Luteolytic

Action of Estradiol—l78 on Luteal

Cells

Luteolytic Effects of Estradiol-17B.
During the Estrous Cycle .

Intraluteal Effects of Estradiol- 178
and Other Follicular Products

iii

Page

vi

b

\Immmm

10

10
12
l6

18
20
21

MODEL OF INITIATION OF LUTEAL
EXPERIMENTAL OBJECTIVES . .

MATERIALS AND METHODS . . . .

RESULTS 0 O O O O O O O O O 0

DISCUSSION 0 O O O O O O O 0

SUMMARY AND CONCLUSIONS . . .

LIST OF REFERENCES . . . . .

REGRESSION AND

iv

 

Page

23

26

31

39

44

45

Figure

1.

LIST OF FIGURES
Page

Concentrations of estradiol-l7B in serum of
heifers. During surgery on day 9 postestrus,
ovarian follicles were destroyed (hatched

bars) or not destroyed (open bars). Data

are means i SEM. Only animals receiving

saline on day 14 are reported for day 15.

Numbers within brackets represent numbers

of heifers sampled . . . . . . . . . . . . . 33

Characteristics of pulsatile secretion of

LH in serum of heifers. During surgery on

day 9 postestrus, ovarian follicles were
destroyed (hatched bars), or not destroyed

«open bars). Animals included for day 15
received an injection of saline on day 14.

Data are means 1 SEM. Only animals receiving
saline on day 14 are reported for day 15.

Numbers within brackets represent number of
heifers sampled . . . . . . . . . . . . . . . 35

Concentrations of progesterone in serum of
heifers. During surgery on day 9
postestrus, ovarian follicles were destroyed
(x-Irrad), or not destroyed (Control). On
day 14, heifers were injected with Saline
(open circles, dashed lines), or PGFZa
(closed circles, solid linesL. Days of
surgery and injection are indicated by
arrows. Data are daily means i SEM. Num-
bers within brackets represent number of
heifers sampled . .. . . .. . . .. . .. . 38

cAMP

hCG
ng

Pg
PGan
PGFM

SEM

ABBREVIATIONS

adenosine 3',5'-cyclic monophosphate

gram(s)

hour(s)

human chorionic gonadotrophin

nanogram(s)

picogram(s)

prostaglandin F20

13,14-dihydro-15 keto PGF, a PGan metabolite

standard error of the mean

vi

ABSTRACT
INVOLVEMENT OF OVARIAN FOLLICLES IN

PROSTAGLANDIN F a INDUCED LUTEAL
REGRESSIO IN CATTLE

BY
Trudy Lynn Hughes

Factors potentially'involved:h1initiating luteolysis
in cattle were inspected. To investigate changes in luteo-
tropic support, frequency and amplitude of pulses of
luteinizing hormone were characterized on days 8, 13 and 15
postestrus. Interactions between products from ovarian
follicles and prostaglandin F2 on (PGFZOL) on luteal regression
were also tested. Changes in serum concentrations of pro-
gesterone were monitored after an injection of saline or
PGFZQ on day 14 to heifers whose ovarian follicles had been
destroyed or had undergone sham destruction on day 9
postestrus.

Amplitude of pulses of luteinizing hormone was greater
on day 15, whereas neither basal concentrations nor fre-
quency of pulses changed with time. Thus, there was no
reduction in availability of luteotropic support by day 15.
PGFZG was significantly less efficacious in causing luteo-
lysis in animals whose follicles had been destroyed than in
control animals. Products of ovarian follicles interact
with PGFZG to heighten responsiveness of corpora lutea to

luteolytic factors.

 

INTRODUCTION

Luteinizing hormone (LH) is the primary luteotropin in
cows as presence of LH is required for development and
maintenance of bovine corpora lutea (Hansel et al., 1973;
Hoffman et al., 1974). Reduced availability of circulating
LH achieved by immuninactivation results in early luteal
regression in cattle (Snook et al., 1969; Hoffman et al.,
1974). Mean concentrations of LH do not decrease near the
time functional luteal regression begins as demonstrated by
decreasing concentrations of progesterone in peripheral
plasma (Spicer et al., 1981; Villa-Godoy et al., 1985).
However, it is not known whether the pulsatile pattern of
release of LH is altered during this period (Rahe et al.,
1980; Walters et al., 1984). Though maintenance of de-
veloped corpora lutea was not investigated, McNeilly et a1.
(1984) demonstrated pulsatile secretion of LH is required
for the formation of functional corpora lutea in ewes. In
addition, in cows and primates there is a synchronous rela-
tionship between the pulsatile release of gonadotropins and
the pulsatile release of progesterone from mature corpora
lutea which suggests a functional relationship might exist
between the two during diestrus (Walters et al., 1984; Healy

et al., 1983). Thus a reduction in the frequency or

2
amplitude of pulses of LH during late diestrus could be
responsible for the decline in luteal function.

Exogenous administration of estradiol-17(30r prosta-
glandin an.(PGFZCO causes functional luteolysis in cows and
ewes. Uterine synthesis and release of PGF2<xis stimulated
by increasing concentrations of estradiol-178 in the uterine
blood supply (Barcikowski et al., 1974). Concentrations of
estradiol-17B in serum increase during late diestrus before
luteal function begins to decline in sheep and cows
(Barcikowski et al., 1974; Fogwell et al., 1985). Thus,
estradiol-178 may trigger an increase in uterine synthesis
and release of PGan which then in turn results in luteo-
lysis. Indeed, luteal lifespan in heifers is extended fol-
lowing destruction of ovarian follicles (Fogwell et al.,
1985; Villa-Godoy et a1», 1985) which are the primary
sources of estradiol-17B in cattle (Ireland and Roche,
1983a; Ireland et al., 1984). However, in addition to the
positive effect of estradiol-17B on uterine secretion of
PGan, results from experiments with ewes (Gengenbach et
al., 1977) and cows (Hixon et al., 1983) indicate that an
additional role for estradiol-17E3in luteal regression
exists. There is evidence that estradiol-178 and PGFZG
synergize during luteal regression since a combination of
exogenous estradiol and PGan is a more efficacious luteo-
lytic treatment than either hormone administered alone
(Gengenbach et al., 1977; Hixon et al., 1983). However, it

is not known if ovarian follicles, or their products

3
influence the ability of PGFZG to cause luteal regression in
cows.

This experiment was designed to address the following
two questions. Firstly, does the pattern of pulsatile
secretion of LH change before the spontaneous decline of
luteal function occurs? Secondly, does destruction of
ovarian follicles alter the efficacy of exogenous PGFZG to

cause luteal regression in heifers?

REVIEW OF LITERATURE

WWW

- Two or three days before estrus, the ovulatory follicle
of a cow is the largest follicle on either ovary (Dufour et
al., 1972). As luteinization of the ovulatory follicle
occurs during the\periovulatory period of several species,
the major end product of steroidogenesis within this folli-
cle shifts from estradiol-178 to progesterone (Channing,
1980; Murdoch and Dunn, 1982; Ireland and Roche, 1983b). In
cows, hyperplasia of the corpus luteum continues until day 9
(day 0 = estrus; Donaldson and Hansel, 1965). Furthermore,
changes in the type of cells comprising the corpus luteum
also occur. Luteal cells may be divided into two steroido-
genic types based on cell size and origin. Granulosa cells
give rise to large luteal cells while thecal cells develop
into small luteal cells, which in turn differentiate into
large luteal cells as the corpus luteum ages (Donaldson and
Hansel, 1965; Fritz et al., 1981; Alila et al., 1983). The
importance of this differentiation to luteal function will
be discussed in following sections.

Presence of LH is required for the functional and

morphological differentiation of follicular cells to luteal

cells as well as being required for the maintenance of

 

5

bovine luteal cells in zitrg (Gospodarowicz and
Gospodarowicz, 1972, 1975). Similarly, primate corpora
lutea in yiyg require LH (Hutchinson et al., 1984). Reduced
availability of circulating LH causes decreased weights of
corpora lutea and early luteal regression in cows (Snook et
al., 1969; Hoffman et al., 1974). DH is the primary luteo-
tropin in the cow (Hansel et al., 1973; Hoffman et al.,
1974) though other factors such as PGIZ (Milvae and Hansel,
1980), PGE2 (Godkin et al., 1977), insulin (Veldhuis et al.,
1984), and increased blood flow to the corpus luteum
(Niswender et al., 1976; Wise et al., 1976) have luteotropic
capabilities.

nechani§m_gf_agtign_cf_LH

9W

LH exerts luteotropic effects by a cascade of events:
1) LH binds to receptors for LH which are present in the
luteal cell membrane (Rao et al., 1979; Hwang et al., 1983),
2) membrane bound LH activates the adenylate cyclase system,
3) adenylate cyclase catalyzes intracellular production of
cAMP and, 4) cAMP acts at several control points in steroid-
ogenesis to stimulate production of progesterone (Marsh,
1976; Williams et al., 1978; Darbon et al., 1980). Oxytocin
is also present in high concentrations within the bovine
corpus luteum (Wathes and Swann, 1982; Wathes et al., 1984)
and though it is secreted concomitantly with progesterone
from the ovary during late diestrus (Flint and Sheldrick,

1983; Walters et al., 1984) no role for LH in control of

 

6
ovarian production or release of oxytocin has been demon-
strated.

Since LH is necessary for the development and mainte-
nance of corpora lutea in cows, it may be hypothesized that
control of luteal regression may depend upon several vari-
ables which influence 1uteotropic support. These variables
include: 1) amount of LH which is available to the corpus
luteum, defined hereafter as concentration of LH in serum,
2) number and affinity of receptors for LH in luteal tissue,
3) ability of the LH receptor complex to stimulate activity
of the adenylate cyclase system, 4) ability of adenylate
cyclase to increase production of cAMP, 5) effectiveness of
cAMP to stimulate various control points in steroidogenesis,
6) interference by luteolytic factors at any of the previ-
ously mentioned control points. The objective of this re-
view is to discuss the importance of the aforementioned
variables to spontaneous luteolysis. A general model which
summarizes and integrates this information will be presented
in conclusion of this section and will introduce the objec-

tives of my thesis research.

LQLQQLLQEIQ_E§QLQL§

Ayailability_gfi_gfi. Low basal concentrations of LH are
maintained throughout diestrus in the cow (Spicer et al.,
1981; Villa-Godoy et al., 1985). However, Rahe et a1.

(1981), and Walters et a1. (1984) found changes in the

 

 

 

 

7
pattern of pulsatile secretion of LH occurs between early
and mid-diestrus. From day 3 to day 11 postestrus, fre-
quency of pulses of LH decreases while amplitude of these
pulses increases. Changes in secretory pattern of LH during
diestrus are largely due to alterations in the concentra-
tions of progesterone and estradiol—178 found in serum
(Goodman and Karsch, 1980; Goodman et al., 1981), but other
non-steroidal and uncharacterized follicular products may
also have effects (Barraclough et al., 1979; Cummins et al.,
1983). Matton et a1. (1981) and Ireland et a1. (1979) found
both follicular inventories and concentrations of steroids
within follicles are altered between days 11 to 17 of the
estrous cycle, and it is probable these changes are re-
flected in serum concentrations of ovarian products to which
the hypothalamus and pituitary are exposed. Thus, there is
reason to believe that a change in the pulsatile pattern of
release of LH occurs during late diestrus before a decline
in secretion of progesterone from the bovine corpus luteum
occurs. While mean daily concentration of LH does not
change near the time luteal function declines, the pulsatile
pattern of secretion may be altered and contribute to fac-
tors causing 1utea1 regression in non—pregnant cows and

heifers.

P .11! 0 -._ R ‘0 o o H D IO 0‘ 0-
Cycle. Affinity of luteal receptors for LH remains constant
throughout diestrus and initiation of luteal regression

(Diekman et al., 1978; Rao et al., 1979). However,

 

8
concentration of receptors for LH in corpora lutea decreases
concomitantly with luteal functions (Diekman et al., 1978;
Spicer et al., 1981). In addition, there is a decrease in
binding capacity of corpora lutea for LH during mid-diestrus
which precedes any significant decline in concentrations of
progesterone in serum (Spicer et al., 1981). One factor
which reduces the concentration of receptors for LH during
diestrus is theldifferentiation of small luteal cells into
large cells as the corpus luteum ages (Fitz et al., 1981;
Fitz and Sawyer, 1982; Alila, 1983). Fitz et al.\(l982)
found the number of binding sites for LH are 10 fold greater
in small luteal cells than in large cells. Thus, as the
proportion of large luteal cells increases, the capacity of
corpora lutea to bind LH declines and corpora lutea could
become less responsive to stimulation by LH (Ursely and
Leymarie, 1979; Koos and Hansel, 1980; Fitz et al., 1982;
Hoyer et al., 1984). Basal secretion of progesterone from
bovine luteal cells in yitrg_does decline between corpora
lutea collected on day 10 postestrus and those collected on
day 15 postestrus even though plasma progesterone levels
were maintained during this interval. However, it is not
clear if the ability to respond to LH is involved in this
diminished ability to secrete progesterone. LH stimulated
production of progesterone from corpora lutea also declined
between day 10 and 15 postestrus through the percent of

progesterone produced following LH treatment in comparison

9
to basal production of progesterone was maintained (Milvae
and Hansel, 1983).

If reduced availability of LH to the luteal cell, which
is caused either by depressed concentrations of LH in serum
or by a decline in binding capacity of luteal tissue for LH,
is involved in luteal regression an increase in concentra-
tions of LH in serum would extend luteal function. In fact,
injections of human chorionic gonadotrophin in cows (hCG;
Wiltbank et al., 1961) and infusions of LH in ewes (Karsch
et al., 1970) during diestrus prolong the lifespan of
corpora lutea. However, these treatments do not block
luteolysis from eventually taking place indicating that
variables in addition to availability of LH are involved in

luteal regression.

WWW Increased

proportion of large luteal cells during late diestrus (Fitz
et al., 1981; Fitz and Sawyer, 1982) may cause a decrease in
responsiveness to luteotropic support for reasons in addi-
tion to the fact that large cells contain fewer receptors
for LH (Fitz et al., 1982). LH does not stimulate accumula-
tion of cAMP in large luteal cells, nor can CAMP increase
secretion of progesterone from large cells as it does from
small cells (Fitz et al., 1982; Hoyer et al., 1984). Pro-
gesterone secretion is thus negatively modulated during late
diestrus by decreased binding capacity of luteal cells for

LH, decreased proportion of cells which are responsive to

 

10
LH, decreased production of LH stimulated production of

cAMP, and decreased responsiveness of luteal cells to cAMP.

MW
Eyidgngg Suggesting Pngct i§ 1mg Lgtgglytig Fagtgz.

Hansel et a1. (1973) and Inskeep (1973) summarized numerous
studies that determined uterine production of PGFZG is
involved in luteal regression in cows and ewes. The bovine
corpus luteum is another source of prostaglandins including
those with luteotropic, PGI2, and luteolytic, PGFZG, effects
(Milvae and Hansel, 1983). Blockade of synthesis of
endogenous prostaglandins achieved by injecting indomethacin
(Smith and Lands, 1971) in ewes and heifers prevents normal
luteal regression (Lewis and Warren, 1977). Additionally,
in liLLQ addition of indomethacin to luteal tissue of ewes
(Evard et al., 1978) and cows (Pate and Condon, 1984) in—
creases the ability of LH to stimulate synthesis of pro-
gesterone. Immunogenic inactivation of endogenous PGan
(Scaramuzzi and Baird, 1976; Fairclough et al., 1981) or
removal of uterine produced PGan by hysterectomy (Brunner
et al., 1969; Bolt and Hawk, 1975) prolongs the lifespan of
corpora lutea in cows and ewes. Therefore, both ovarian and
uterine production of PGcmxhave implicated roles in
luteolysis. Short term treatment (1 h or less) of luteal
cells with PGan 13 11:19 (Hixon et al., 1983; Heath et al.,
1983) and injection of PGFZa in 1119 (Hixon and Hansel,
1974; Heath et al., 1983; Schallenberger et al., 1984)

results in an immediate increase in secretion of

 

ll
progesterone. This effect is probably due to massive exocy—
tosis of progesterone and oxytocin containing granules which
are found in both large and small luteal cells (Quirk et
al., 1979; Sawyer et al., 1979; Heath et al., 1983). Fol-
lowing this period of increased secretion luteal concentra-
tions of progesterone (Heath et al., 1983) and serum concen-
trations of progesterone (Hixon and Hansel, 1974;
Schallenberger et al., 1984) decline rapidly. This sequence
of events indicates that the ultimate action of PGFZG is
luteolytic and not luteotropic.

Large luteal cells contain more receptors for PGde
(Fitz et al., 1982) and are more responsive to PGFZG than
are small luteal cells (Heath et al., 1983). Rao et a1.
(1979) reported a progressive increase in number and
affinity of receptors for PGFZG in bovine corpora lutea
during the estrous cycle. These findings are consistent
with the concept that the proportion of large luteal cells
increases during diestrus and that this change could be
involved in luteal regression. Furthermore, a lower propor-
tion of large luteal cells during early diestrus and luteal
development could in part explain the failure of exogenous
PGan administered before day 4 postestrus to cause luteoly-
sis (Saumande and Chupin, 1981; Battista et al., 1984).
Since large cells are less responsive to LH than small
cells, it is not surprising that an inverse relationship
exists between ability of bovine luteal cells to produce

progesterone and to bind PGan (Henderson and McNatty,

12
1977). In addition to the intraluteal changes occurring
during diestrus which increase the ability of corpora lutea
to respond to PGan, distinct elevations of PGFZa in serum
occur as luteal functions declines (McCracken, 1980; Auletta
et al., 1984; Fogwell et al., 1985). Therefore, during late
diestrus PGan becomes more available to luteal cells as
concentrations of PGFZG in serum increase and ability of
luteal cells to bind PGan peaks. These PGan related
events are coincident with the decreased ability of luteal
cells to respond to LH and the two factors in combination

may result in luteal regression.

Mechanism of Action of PGFZG in Luteolysis. PGan acts
at several control points in luteal cell function to exert
its luteolytic effects. Decreased luteal function after
treatment with PGFZG occurs prior to a decrease in concen-
tration or affinity of receptors for LH in luteal tissue
(Grinwich et al., 1976; Thomas et al., 1978; Spicer et al.,
1981). Thus, subsequent points in the LH stimulated release
of progesterone are important. In yitrg, PGFZa inhibits LH
stimulated adenylate cyclase activity and subsequent produc-
tion of cAMP by luteal tissue of rats (Grinwich et al.,
1976; Thomas et al., 1978). However, when CAMP is supplied
to luteal cells of rats by addition of dibutryl cAMP to
culture media (Jordan, 1981) or by increasing intracellular
levels of cAMP by treating bovine luteal cells with cholera
toxin or forskolin (Pate and Condon, 1984), production of

progesterone continues to be inhibited by PGan. Therefore,

 

l3
inhibitory effects of PGan on LH stimulated production of
progesterone occur at control points in steroidogenesis both
before and after accumulation of cAMP.

In order for bovine luteal cells to remain functional
in yitro they must remain in a morphological configuration
resembling epithelial cells. Presence of LH is required to
maintain this configuration while PGan inhibits this con—
figuration (Gospodarowicz and Gospodarowicz, 1975). Thus
morphological differentiation of luteal cells is one area of
luteal cell function that PGan influences. Also, PGFZa may
affect viability of luteal cells. One of the earliest
events in luteal regression is increased lysosomal formation
and lysosomal enzyme activity which lead to autophagocytosis
and degeneration of luteal cells (McClellan et al., 1977L
Lysosomes may be a site of interaction between luteolytic
and luteotropic hormones as binding sites for PGFZa and LH
are present in lysosomal membranes of bovine corpora lutea
(Mitra and Rao, 1978). Thus, PGan may interfere with
luteal function by affecting the morphology and viability of
luteal tissue in addition to direct effects on LH stimulated
steroidogenesis. Additionally, PGFZa reduces the amount of
blood flow to the ovary (Niswender et al., 1976). Thus PGan
may impair luteal function by diminishing the amount of
luteotropic agents and required metabolites delivered to the

corpus luteum.

Hormonal Control of PGan Secretion. Increased

synthesis (Huslig et al., 1979) and secretion (Lewis et al.,

14
1977; McCracken, 1980; Auletta et al., 1984; Fogwell et al.,
1985) of PGan from the uterus during late diestrus is
controlled by a complex interaction between progesterone,
estradiol and oxytocin. Progesterone administered on days 0
to 3 postestrus results in premature luteal regression
(Battista et al., 1984). The mechanism by which pro-
gesterone produces this effect may be by lowering mean
concentration of LH in serum (Battista et al., 1984). How-
ever, an interaction with PGan is implied as hysterectomy
(Moor et al., 1966; Woody et al., 1968) or treatment with
indomethacin (Lewis et al., 1977a) prevents progesterone
induced luteolysis. To this end, it has.been demonstrated
that administration of progesterone to ewes increases
uterine content of PGan (Wilson et al., 1972) and hastens
the occurrence of the first peak of PGan prior to luteal
regression (Ottobre et al., 1980). Additionally, adminis—
tration of 100 mg/day of progesterone before day 3
postestrus followed by exogenous PGFZG does not shorten the
length of estrous cycles (11.7 days) more than administra-
tion of progesterone alone (13.2 days; Battista et al.,
1984). In contrast, exogenous progesterone decreases con—
tent and concentration of PGan in endometrial tissue of
ovariectomized ewes (Wilson et al., 1972) and concentration
of PGde in plasma remains low (Ford et al., 1975) or is
decreased (Fairclough et al., 1983). Therefore, Ford et a1.

(1975) suggest the stimulatory effect of progesterone on

 

15
uterine secretion of PGan is indirect and acts to prime the
uterus to respond to estradiol-178.

Injections of estradiol into corpora lutea of monkeys
cause increased concentrations of luteal PGan (Auletta et
al., 1978). Similarly, infusions of estradiol-17B into
uterine arterial blood of ewes results in increased uterine
synthesis and release of PGFZa (Barcikowski et al., 1974).
These effects of estradiol on uterine secretion of PGFZa are
influenced by presence of progesterone however. Injections
of estradiol-178 in intact ewes on days 9 and 10 postestrus,
increase uterine secretion of PGan but the same dose of
estradiol—178 is ineffective on days 4 and 5 unless the
animals receive injections of progesterone on days 1 through
5 (Ford et al., 1975). Given alone, 10 mg of progesterone
on days 1 through 5 did not alter secretion of uterine PGan
(Ford et al., 1975). Likewise, injections of estradiol-178
cause early luteal regression in intact animals but injec-
tions of estradiol on day 5 or 6 were only luteolytic if
preceded by injections of progesterone on days 1 through 4
(Warren et al., 1973).

The scenario of progesterone priming followed by
increased serum concentrations of estradiol—178 to cause
uterine release of PGan fits well with the profiles of
concentrations of these hormones in blood during diestrus.
During this time, secretion of progesterone begins to
decline as secretion of estradiol—178 and PGan increases

(Barcikowski et al., 1974; Fogwell et al., 1985).

16
Alterations in numbers of endometrial binding sites for
progesterone and estradiol-178 occur during the cycle in
parallel to the changes occurring in concentrations of these
steroids in serum (Kimbal and Hansel, 1974; Zelenski et al.,
1982). On days 13 to 14 postestrus, concentrations of
progesterone declines and peaks of estradiol-178 become
associated with peaks of PGF20‘ and the amplitude of peaks of
PGan increase with time (Barcikowski et al., 1974). A
decline in progesterone may facilitate the stimulatory ac—
tion of estradiol-178 on the uterus as progesterone dimin-
ishes translocation of the estradiol/receptor complex and
retention of this complex within the nucleus (Okulicz et
al., 1981; Smanik et al., 1982). Indeed, presence of
exogenous progestogens during late diestrus in ewes de-
creases the amplitude of pluses of the prostaglandin F
metabolite 13, l4-dihydro-15-keto-PGF (PGFM; Fairclough et
al., 1983). Within 18 h following removal of progestogen
impregnated vaginal sponges from cows, a marked increase in

PGFM was detected in plasma (Smith et al., 1979L

Role of Qxytocin in Luteal Regreeeion. In addition to
the ovarian steroids previously discussed, a role for oxy—
tocin in luteolysis has been suggested (Hansel and Wagner,
1960; Armstrong and Hansel, 1959; Sheldrick et al., 1980).
However, luteolysis induced by oxytocin may be due to oxy-
tocin stimulated release of PGFZG from the uterus rather

than a direct effect of oxytocin on luteal tissue since

 

l7
hysterectomy completely blocks oxytocin induced luteolysis
(Armstrong and Hansel, 1959; Brunner et al., 1969).

Oxytocin stimulates synthesis and release of PGFZG from
endometrial tissue (Roberts et al., 1976) and increases
plasma concentrations of PGFM in ewes (Fairclough et al.,
1984). Effectiveness of oxytocin to increase release of
PGFZG or its metabolite during late diestrus (Roberts et
al., 1976; Fairclough et al., 1984) is correlated with
increased concentrations of estradiol—178 in plasma
(Barcikowski et al., 1974). In ovariectomized ewes infusion
of estradiol-173 for 6 h increases oxytocin induced PGan
release from the uterus. Infusion of progesterone for 2 to
6 days blocks the stimulatory interaction between estradiol-
178 and oxytocin on PGde release. However, following 10
days of infusion of progesterone, estradiol augmented oxy-
tocin induced release of PGan was increased 50 to 100 fold
above the release induced without previous exposure to pro—
gesterone (McCracken, 1980). McCracken (1980) suggests
estradiol-17B and progesterone alter uterine responsiveness
to oxytocin through inducing increased receptors for oxyto-
cin in the endometrium.

During mid-to—late diestrus there is an increased
number of receptors for oxytocin in the endometrium (Roberts
et al., 1976), increased concentration of oxytocin within
corpora lutea (Schams et al., 1984) and in peripheral blood
(Schams et al., 1980; Flint and Sheldrick, 1983; Walters et

al., 1984). Therefore, ability of oxytocin to cause

18
increased uterine release of PGcmxiS temporally correlated
with luteal regression. A cycle of positive feedback may be
generated during late diestrus between ovarian secretion of
oxytocin and uterine secretion of PGan. An injection of
PGan causes increased release of oxytocin from the ovary
(Flint and Sheldrick, 1982L Similarly, vaginal distention
causes early luteal regression (Hansel and Wagner, 1960) and
increased secretion of oxytocin (Roberts and Share, 1968)
possibly due to increased levels of PGan secreted following
vaginal stimulation (McCracken et al., 1980). Estradiol
enhances while progesterone blocks the ability of vaginal
distention to increase plasma concentrations of oxytocin

(Roberts and Share, 1969).

E ' ' D' Lu 'c A ' E d' -
lZfi on Loteel gelle. Destruction of ovarian follicles, the
primary source of circulating estradiol-178, results in
prolonged lifespan of corpora lutea in ewes (Karsch et al.,
1970) and cows (Fogwell et al., 1985; Villa-Godoy et al.,
1985). Following destruction of ovarian follicles, adminis-
tration of estradiol benzoate results in early luteal re-
gression in ewes (Hixon et al., 1975) as do injections of
estradiol in intact animals (Hansel et al., 1973; Cook et
al., 1974; Karsch and Sutton, 1976). Estradiol induced
luteal regression is not, however, entirely dependent upon
presence of the uterus. While hysterectomy completely
blocks the ability of exogenous oxytocin and progesterone to

cause luteal regression (Brunner et al., 1969), exogenous

l9

estrogen significantly depresses luteal content and plasma
concentrations of progesterone in hysterectomized cows and
ewes although estrogen's ability to cause luteal regression
is greatly diminished (Brunner et al., 1969; Gengenbach et
al., 1977). Also, destruction of ovarian follicles which
results in diminished levels of estradiol blocks PGde in-
duced luteal regression in ewes (Hixon et al., 1975). One
proposed mechanism of action for estradiol is a synergism
with PGFZG on corpora lutea, though a direct interaction in
cows has not yet been demonstrated in yitro (Hixon et al.,
1983). In contrast, results from studies conducted with
sheep support the hypothesis. Injection of low doses of
estradiol—178 and pGde in hysterectomized ewes whose fol-
licles had been destroyed resulted in complete luteal re—
gression in 3 of 4 ewes, while either treatment alone was
only marginally effective (Gengenbach et al., 1977L
Neither are the luteolytic effects of an injection of
estradiol due to the ability of estradiol-178 to depress
concentrations of LH in serum when concentrations of pro—
gesterone are high (Beck et al., 1976; Goodman et al., 1981;
Karsch et al., 1980). After ovarian follicles were de—
stroyed, injection of estradiol in ewes results in an in-
crease in basal concentration of LH before concentrations of
progesterone are depressed (Hixon et al., 1975; Gengenbach
et al., 1977). In addition, infusion of LH does not block
estrogen induced luteolysis (Cook et al., 1974). Thus

estradiol-178's luteolytic effects are not entirely

20
dependent on the effects of estradiol-17B on the uterus and
concentrations of LH in serum. As will be discussed in a
following section, estradiol-17B can act directly on luteal

cells to affect their steroidogenic function.

Lt 'cE c E d'-78D'n E u
dele, Luteal cells of cows (Kimball and Hansel, 1974) and
ewes (Glass et al., 1984) contain binding proteins for
estradiol—178 indicating that the corpus luteum is a target
tissue for estradiol—173. Concentrations of cytosolic bind-
ing proteins for estradiol-178 increase between early and
mid-diestrus in corpora lutea of ewes (Sheridan et al.,
1975; Glass et al., 1984) and cows (Kimball and Hansel,
1974) before luteal function declines. Also, the concentra—
tion of binding proteins is highly correlated with concen-
trations of estradiol-17B found in the plasma of cows during
this time (Kimball and Hansel, 1974L Additionally, large
steroidogenic cells contain 3.5 fold higher concentration of
cytosolic estradiol-17B receptors than do small luteal cells
collected from ewes on day 10 postestrus (Glass et al.,
1984). The combination of increased availability of
estradiol-17B in serum (Barcikowski et al., 1974; Fogwell et
al., 1985) and increased concentration of binding proteins
for estradiol—17B in luteal cells during late diestrus, in
part, explains why the luteolytic effectiveness of exogenous
estradiol is increased between early to late diestrus

(Warren et al., 1973).

 

21

I E E '-7Bd
Folliooler_groooot§, Estradiol-17B depresses the ability of
LH to stimulate synthesis and release of progesterone (Moody
and Hansel, 1971; Williams and Marsh, 1973) from both large
and small bovine luteal cells (Ursely and Leymarie, 1979).
Luteolytic actions of estradiol—178 occur in part after LH
activation of adenylate cyclase since estradiol-178 does not
significantly affect cAMP accumulation in LH stimulated
luteal cells, and treatment of luteal cells with dibutyrl
CAMP or cholera toxin only partially overcomes the inhibi—
tory effects of estradiol—17B (Williams and Marsh, 1978).
Conversion of pregnenolone to progesterone is catalyzed by 38
-hydroxysteroid dehydrogenase. The activity of this enzyme
is inhibited by estradiol—178 (Akbar et al., 1972; Caffrey
et al., 1979) and thus represents one site in the steroido—
genic pathway influenced by estradiol—178.

Antagonistic interaction between cAMP and estradiol-17B
may also influence luteal cell function. Bodwin et a1.
(1981) demonstrated that inverse relationships exist between
cAMP in the cytosol and nuclear uptake of the estrogen/
receptor complex and between cytosolic estradiol-178 and
nuclear binding of cAMP in mammary tissue of rats. As
nuclear translocation of the receptor is critical for
steroid hormone action (Yamamoto and Alberts, 1976), I
suggest that decreased ability of corpora lutea to respond
toIJias the luteal tissue ages either due to<iiminished

number of receptors for LH or alterations in proportion of

 

 

 

 

22
small to large luteal cells, would result in diminished
accumulation of cAMP within luteal cells. Decreased concen-
trations of cAMP would increase the ability of corpora lutea
to respond to estradiol-178. In addition to the effects of
estradiol~178, follicular fluid diminishes adenylate cyclase
activity and cAMP accumulation in gonadotropin stimulated
ovarian tissue (Amsterdam et al., 1979; Ledwitz-Rigby, 1980)
and results in depressed production of progesterone
(Shemesh, 1979). Karsch et al. (1970) observed that de-
struction of ovarian follicles decreases by four fold the
amount of exogenous LH that is required to maintain corpora
lutea in intact animals.

In summary, due to alterations in the type of luteal
cells present, the responsiveness of corpora lutea to LH may
decline while responsiveness to estradiol-178 may increase
during late diestrus. These alterations in binding capa-
city for LH and estradiol-17B in combination with increased
serum concentrations of estradiol-178 can result in the
initial decline in production and release of progesterone
from corpora lutea. Diminished serum concentrations of
progesterone in combination with high levels of oxytocin and
estradiol—17B and increased concentration of binding pro-
teins for estradiol—178 and oxytocin in the uterus, causes
increased uterine secretion of PGF2a and luteal regression

ensues .

MODEL OF INITIATION OF LUTEAL REGRESSION
AND EXPERIMENTAL OBJECTIVES

E D n
Lutegtreeis_zagtgrs

Luteal tissue contains in—
creased receptors for LH.
Steroidogenic cells are
primarily small in size
which in turn are the most
responsive cells to stimula-
tion by LH. Pulses of LH
are of low amplitude but
high frequency.

M

Luteal tissue is still quite
responsive to LH stimulation
but a decrease in specific
binding for LH occurs as the
proportion of steroidogenic
cells which are large
increase. Pulses of LH are
of high amplitude and low
frequency.

c L
Luteolyti£_2agtgrs

Concentrations of luteal
receptors for PGan and
estradiol-17B are low as are
concentrations of PGFZa and
oxytocin in blood. A tran-
sitory increase in estradiol
occurs. The uterus is unre—
sponsive to estradiol and
oxytocin stimulation and
contains a low concentration
of receptors for estradiol
and oxytocin.

C L m

Concentration of luteal re-
ceptors for PGFZa and
estradiol—178 are increased
but levels of these hormones
in blood are low. The
uterus has increased concen-
tration of receptors for
estradiol-17B and oxytocin
though high levels of pro-
gesterone blocks their
ability to stimulate release
of PGFZa.

I . . . R .
Degline_gf_Luteal_£ungthiBegins

L 'c F c

Proportion of steroidogenic
cells which are large peaks.
Large cells contain a lower
concentration of receptors
for LH and are less respon—
sive to LH stimulation for
accumulation of cAMP and
production of progesterone.
While basal secretion of LH
is unchanged, it is not
known if the pattern of
pulses is altered.

‘c F c

Increased proportion of
large luteal cells results
in increased concentration
of receptors for estradiol-
178 and PGan. Increased
concentrations of estradiol-
178 in peripheral plasma are
found due to increased se—
cretion of estradiol-178
from ovarian follicles. The
ability of estradiol-17B and
oxytocin to stimulate
uterine secretion of PGFZa
is increased. Whether or
not ovarian follicles of
cows are necessary for the
luteolytic effects of PGan
to be manifested is not
known.

Luteal_3egressien

Luteal receptors for LH
steadily decline. As secre-
tion of progesterone de-
clines basal concentrations
of LH in plasma increase.
Pulses of LH demonstrate
decreased amplitude and in-
creased frequency.

Previous exposure of the
uterus to high levels of
progesterone during the pre—
ceeding stages of luteal
function followed by de—
clining levels of proges-
terone in serum during this
stage allows estradiol-178
from ovarian follicles to
stimulate increased release
Of PGde from the uterus.
Increased PGF a secretion is
also due to e fects mediated
by oxytocin from the corpus
luteum which are augmented
by estradiol-178.

The corpus luteum is respon-
sive to PGF G and estradiol-
178 since i contains an
increased proportion of
large luteal cells. Ini—
tially PGF a causes in-
creased reIease of proges—
terone and oxytocin due to a
massive exocytosis of luteal

 

25

W (Continued)
Wis—Factors Waiters

granules. Meanwhile PGde
blocks LH induced cAMP
accumulation and production
of progesterone. Increased
secretion of oxytocin in
turn further stimulates
uterine secretion of PGF 0.
Long term exposure of lugeal
cells to PGFZG results in
loss of luteal receptors for
LH and autophagocytosis.
Experimental oojeotiyee
Parts of this model are well documented as described in
the Review of Literature. Other aspects are suppositional
and require further investigation. Of particular interest
to this investigation are those factors involved in the
stage I have termed "initiation of luteal regression." In
order to clarify factors that are involved with the initial
decline in function of the bovine corpus luteum the follow-
ing questions were posed:
(1) Is an alteration in the pattern of pulsatile release of
LH associated with initiation of luteal regression?

(2) Do follicular products affect response of corpora lutea

to exogenous PGFZG?

 

MATERIALS AND METHODS

After exhibiting two or more estrous cycles of 17 to 24
days, twenty Holstein heifers were assigned at random to a 2
x 2 factorial experiment. Main effects were: 1) destruc-
tion of ovarian follicles on day 9 (estrus = day 0), and 2)
injection of PGan on day 14 postestrus. Visible follicles
were destroyed in 10 heifers by electrocautery then ovaries
were x-irradiated (x-irrad) as described by Villa-Godoy et
a1. (1985). The remaining 10 heifers (control) experienced
similar surgical manipulations except no follicles were
cauterized and ovaries were not x-irradiated (Villa-Godoy et
al., 1985; Fogwell et al., 1985). On day 14 postestrus,
heifers received an intramuscular injection of 15 mg PGdel
(n=10) or 3 ml of 2 percent Tham2 buffered saline (Saline;
n=10). The dosage 0f PGan was selected as the minimal
effective level required to cause luteolysis in cows as
determined by Lauderdale (1979). Therefore, the four groups
(5 heifers/group) were: x-irrad PGanr x—irrad saline,

control PGFZa, and control Saline.

 

1Lutylase", the Upjohn Company.

2Tris (Hyroxy methyl) Aminomethane, Fisher Scientific Company.

26

 

 

27

To evaluate success of destruction of follicles twenty
days following estrus, eleven days after surgery, x—irrad
heifers were ovariectomized supravaginally. Corpora lutea
were isolated from ovarian stroma and weighed. The ovarian
stroma was then sliced into 1 to 2 mm sections and each
section was examined macroscopically for follicles ; 2 mm.
Only animals whose ovaries were free from visible follicular
development were accepted as x-irrad heifers.

From day 8 through day 20 postestrus, jugular venous
blood was collected from indwelling cannulas every 8 h to
determine concentrations of progesterone in serum and thus
monitor luteal function. To examine pulsatile release of
LH, blood was sampled every 15 min from 0800 to 2000 h on
days 8 and 13 postestrus from all animals and on day 15 from
heifers injected with saline. Day 15 was selected as the
last day of monitoring the pulsatile pattern of secretion of
LH based on the observation by Villa-Godoy et a1. (1985)
that a decline in luteal function in similarly treated
control heifers can be detected as early as day 16
postestrus. Since our first experimental objective was to
determine if a change in the pulsatile pattern of release of
LH occurred before luteal function declined sampling subse-
quent to day 15 was not conducted. For the same reason
animals receiving PGan on day 14 were not sampled on day 15
for LH. Samples of blood were allowed to clot at room
temperature before refrigeration at 4°C for 24 to 48 h.

After centrifugation, serum was decanted and stored at -20°C

28
until assayed for concentrations of LH (Convey et al.,
1976), estradiol-17B (Carruthers and Hafs, 1980), and pro-
gesterone (antibody against progesterone as validated by
Convey et al., 1977; assay as described by Louis et al.,
1973). Due to insufficient volume of serum in samples taken
every 8 h, assays for estradiol-17B were limited to pools of
samples collected every 15 min on days 8, 13 or 15 pos—
testrus. Coefficients of variation within and among assay
of serum pools were 3.9% and 11.0% for LH, 6.4% and 17.4%
for progesterone, and 11.5% and 9.8% for estradiol—17B.
Sensitivities of assays, defined as the first point on the
standard curve lower than the 95% confidence interval of the
buffer controls, were 0.075 ng for LH, 0.04 ng for proges-
terone, and 0.8 pg for estradiol-17B.

Samples with undetectable concentrations of LH were
assigned a value equivalent to assay sensitivity. Pulses,
amplitude, frequency and baseline LH were determined using
the following criteria. A pulse of LH was defined as an
increase in concentration of LH which exceeds another value
within the preceding 30 min by twice the within assay
standard deviation (2 x SD = 0.96 ng). Amplitude of peaks
equals the difference between maximal value reached during a
pulse and the preceding nadir. Frequency of pulses refers
to the mean number of pulses experienced per animal during
the 12 h sampling period. Baseline was defined as the mean
of values equal to interpulse nadirs + sensitivity of the

assay.

 

29

Concentrations of estradiol-17B, progesterone and
baseline LH as well as frequency and amplitude of pulses of
LH were analyzed using error terms generated from linear
regression analyses for split plot designs (Alvey et al.,
1980). Specific contrasts among data on LH were examined
with the Bonferroni t test (Gill, 1978), while data on
estradiol-178 were contrasted with Scheffe's test (Gill,
1978).

To reduce heterogeneity of variance, daily mean concen-
trations of progesterone were calculated and used in the
analysis of variance for treatment effects. Progesterone
data were divided into two periods. Period 1 included day 8
to day 13 and was used to examine differences between surgi-
cal groups before injection of PGFZG or saline on day 14-
Period 2 included samples collected between the time of
injection and day 20. Dunnet's t test was used to determine
when a significant decrease in concentration of progesterone
occurred following injection of PGFZa or saline. Concentra—
tions of progesterone before surgery or before injection
were used as covariates in analyses of variance for Periods
l and 2 respectively. Weight of corpora lutea were con—
trasted using Welch's approximate t test (Gill, 1978).
During Period 2 concentrations of progesterone which fell
and remained below 2 ng/ml for two or more consecutive
samples was used as a limit to monitor impaired luteal
function in this study rather than1.ng/ml which is fre-

quently used. This was a conservative measure designed to

 

30
increase sensitivity to detect diminished luteal function in

individual animals.

 

RESULTS

Before injection of PGan on day 14 postestrus, one
control heifer had begun spontaneous luteal regression on
day 13 postestrus as assessed by a 70% reduction in concen-
tration of progesterone (11.5 mg/ml to 3.8 ng/ml), increased
concentrations of estradiol-17B (7.9 pg/ml), and increased
frequency of pulses of LH (6 pulses/5 hours). Data from
this heifer were excluded from all analyses.

No ovaries from x-irrad heifers contained follicles Z
2 mm on day 20 postestrus. Thus, electrocautery of fol—
licles and x—irradiation of ovaries destroyed existing fol-
licles and prevented growth of follicles. Furthermore,
concentrations of estradiol-178 in these heifers tended (P <
0.1) to be lower on day 15 (1.9 t 0.4 pg/ml) than on day 8
(4.0 i 0.5 pg/ml). In contrast, concentrations of
estradiol—17B in control heifers did not differ between day
8 and 15 (Figure 1). ,

Basal concentrations of LH did not differ among days
sampled or between surgical groups. Yet on days 13 and 15,
frequency (p < 0.05) and amplitude (p < .01) of pulses of LH
were greater than on day 8 in x-irrad heifers (Figure 2).

In control animals, frequency of pulses did not vary across

days though amplitude of pulses of LH was increased

31

 

 

Figure l.

 

32

Concentrations of estradiol-178 in serum of
heifers. During surgery on day 9 postestrus,
ovarian follicles were destroyed (hatched bars),
or not destroyed (open bars). Data are means i
SEM. Only animals receiving saline on day 14 are
reported for day 15. Numbers within brackets
represent number of heifers sampled.

* Concentrations of estradiol—17B in heifers
whose follicles were destroyed were less on day
15 than on day 8 (P < 0.1).

 

 

E * t—h\\\\\

 

 

A
U)

 

E th\\\\\\\\\\

 

Et-L

 

fl\\\\\\\\\\\\

 

seL

 

 

.L .2 (L 1.
(Iw/bd) ‘Iomvalsa

l5

l3
DAYS POSTESTRUS

 

Figure 2.

34

Characteristics of pulsatile secretion of LH in
serum of heifers.a During surgery on day 9
postestrus, ovarian follicles were destroyed
(hatched bars), or not destroyed (open bars).
Animals included for day 15 received an injection
of saline on day 14. Data are mean 1 SEM. Only
animals receiving saline on day 14 are reported
for day 15. Numbers within brackets represent
number of heifers sampled.

a See Materials_and_fletheds for criteria de-
fining pulses of LH.

* Greater than the comparable value for day 8 (P
< 0.05).

** Greater than the comparable value for day 8 (P
< 0.01).

LH
(ng/ml)

(ng/ml)

Pulses/I2h

 

35

Figure 2

BASELINE

(1o)
(9) (1o) (9) (5) (5)

ma as as

PULSE AMPLITUDE

Wang

PULSE FREQUENCY a“,

—|\)

— h)(fl h

—N0‘-¥>U|

r112 Fla

DAYS POSTESTRUS

 

 

36
(p < 0.01) on day 15 in control heifers and did not differ
from amplitude of pulses found in x-irrad heifers on day 15.
Thus availability of LH in serum, if defined as either
increased basal concentration of LH without change of pulsa-
tile secretion or constant basal concentration of LH with
increased frequency and/or amplitude of pulses of LH, was
increased between day 8 and 15 postestrus in both control
and x-irrad heifers.

As expected, there were no differences in profiles of
concentrations of progesterone in serum between control and
x-irrad heifers from day 8 to day 13 postestrus (Figure 3).
From day 14 through day 20, concentrations of progesterone
were above 2 ng/ml in all heifers injected with saline.
However, all control heifers injected with PGan demon-
strated concentrations of progesterone in serum below 2
ng/ml within 32 h after injection. In contrast, in x-irrad
heifers injected with PGan mean concentration of pro-
gesterone did not decline between injection of PGde and day
20 postestrus and weights of corpora lutea (5.4 t 1.1 gm)
did not differ from x-irrad heifers injected with saline
(5.0 i 0.1 gm). However, by day 20 postestrus three of five
x-irrad heifers injected with PGFZG had concentrations of

progesterone in serum fall and remain below 2 ng/ml.

 

 

Figure 3.

37

Concentrations of progesterone in serum of
heifers. During surgery on day 9 postestrus
ovarian follicles were destroyed (X-IRRAD), or
not destroyed (CONTROL). On day 14 postestrus,
heifers were injected with Tham buffered saline
(open circles) or PGFZG (closed circles). Days
of surgery and time of injection (T0) are indi-
cated by arrows. Data are daily means i SEM.
Numbers within brackets represent numbers of
heifers sampled.

PROGESTERONE(ngAnU

38

FIgure 3

Surgery Injection X-IRRAD

 

 

2..
I I I I I I I I I I I I I I
\I ¢ CONTROL
I2-
15—41 A
.. 46 I 5’ \ /
s- I
6—.
4_
2—
Ifi I I I Ifi I I *I I I

 

 

u I
8 9IOII I2 I3TOI4I5I6 I7|8 I920
DAYS POSTESTRUS

8- I IEPSL\€;_5\
Fir/WINE:
4-

(5)

(4)

 

DISCUSSION

Stable levels of progesterone continued through day 20
in saline injected control heifers indicating luteal
regression had not yet occurred in this group. In
experiments with animals of the same breed and approximate
age and using the same surgical procedures, Villa-Godoy et
a1. (1985) reported control animals experienced luteal
regression between 17 and 22 days postestrus. Thus, failure
to detect luteal regression by day 20 in saline injected
control heifers in this study is apparently due to random
variation in the length of estrous cycles (Bartol et al.,
1981) rather than to surgical manipulations.

In heifers, passive immunization against LH reduces
weights of corpora lutea (Snook et al., 1969) and decreases
concentrations of progesterone in serum (Hoffman et al.,
1974). Injection of hCG or LH in heifers (Wiltbank et al.,
1961; Brunner et al., 1969) and infusion of LH in ewes
(Karsch et al., 1970; Karsch et al., 1971) prolongs luteal
function. Decreased baseline concentrations, frequency or
amplitude of pulses would decrease luteotropic support and
could initiate luteal regression. However, we did not
detect any changes in the pulsatile secretory pattern for LH

on the days sampled which would lower availability of

39

 

 

40
concentrations of LH in serum to corpora lutea. On the
contrary, amplitude of pulses of LH increased 86% between
days 13 and 15 postestrus. Increased amplitude of pulses is
surprising since concentrations of progesterone and
estradiol-178, which are thought to be the primary factors
involved in altering the episodic pattern of release of LH
(Goodman and Karsch, 1980; Karsch et al., 1980; Beck et al.,
1976; Hausler and Malven, 1976), did not change in control
heifers during these days. Since luteal regression was not
detected during the sampling period of this study it is
still unclear if the pulsatile secretion of LH changes
immediately before function luteal regression begins
however.

Reduced concentrations of estradiol-178 in serum (Beck
et al., 1976; Goodman et al., 1981) and the possible
reduction in other non—identified follicular products
(Cummins et al., 1983; Barraclough et al., 1979) in x—irrad
heifers possibly accounts for the increased frequency of
pulses of LH. Destruction of ovarian follicles cause
increased lifespan of corpora lutea in heifers (Fogwell et
al., 1985; Villa-Godoy et al., 1985) because luteolytic
factors are attenuated and potentially because the avail-
ability of LH in serum is increased. Additionally, in—
creased secretion of LH may explain the maintenance of
corpora lutea in x-irrad heifers treated with PGan in this
study. This is unlikely however, since a 10 fold increase

in concentration of LH cannot block luteal regression in-

 

41
duced by a 25 mg injection of PGan in cows (Gonzalez-Mencio
et al., 1977), nor will exogenous LH prolong lifespan of
corpora lutea in cows or ewes indefinitely (Wiltbank et al.,
1961; Karsch et al., 1971). Therefore, failure of PGan to
induce luteal regression in x—irrad heifers is most likely
due to absence of follicular products and thus their effects
on luteal tissue rather than increased availability of LH in
serum.

As diestrus advances, corpora lutea are more responsive
to the luteolytic effects of estradiol-17B (Warren et al.,
1973) and PGan (Inskeep et al., 1973; Battista et al.,
1984) suggesting that the increase in plasma concentrations
of both hormones in late diestrus (Fogwell et al., 1985;
Barcikowski et al., 1974) may be involved in normal luteal
regression. On day 14 postestrus, 15 mg PGF2a caused a
rapid decline in serum concentrations of progesterone in
control heifers but not in x-irrad heifers. Thus, it is
evident that follicular products alter responsiveness of
bovine corpora lutea to exogenous PGan as was observed
previously in ewes (Gengenbach et al., 1977). Ovarian
follicles are important in normal luteal regression in
heifers because: 1) estradiol—178 stimulates uterine
secretion of PGFZa (Barcikowski et al., 1974), 2)
estradiol-178 potentiates and may be required for maximal
luteolytic effectiveness of PGan, and 3) estradiol-17B has
direct luteolytic actions on luteal function. Indeed

estradiol-178 depresses LH stimulated synthesis and release

42
of progesterone from bovine luteal cells (Williams and
Marsh, 1978; Ursely and Leymarie, 1979) past the point of LH
induced cAMP accumulation (Williams and Marsh, 1978).
Activity of 3B—hydroxysteroid dehydrogenase which converts
pregnenolone to progesterone is one point in the
steroidogenic pathway that estradiol-178 inhibits (Akbar et
al., 1972; Caffrey et al., 1979). In addition, non—
steroidal components of follicular fluid reduce luteal
function in primates (Stouffer et al., 1983), and depress
basal and LH induced secretion of progesterone in luteinized
bovine follicles (Shemesh et al., 1979) possibly by inhibit-
ing LH-sensitive adenylate cyclase activity (Amsterdam et
al., 1979; Ledwitz-Rigby, 1980). Attempts to demonstrate an
interaction between estradiol—17B and PGFZa directly on
luteal tissue in xitro have proven unsuccessful to date
(Hixon et al., 1983).

In summary, though no changes in the concentrations of
progesterone or estradiol-178 in serum were noted the ampli-
tude of pluses of LH increased while frequency of pulses and
baseline concentrations of LH remained the same in control
heifers. Therefore, there was no reduction of luteotropic
support by day 15 postestrus. Additionally, removal of
ovarian follicles resulted in increased amplitude and fre—
quency of pulses of LH prior to any detectable changes in
serum concentrations of progesterone and estradiol—17B.
Increased luteotropic support is not, however, believed to

to be the reason that destruction of ovarian follicles

 

43
results in the lengthening of the lifespan of corpora lutea
of cows. It is suggested that products of ovarian fol-
licles, such as estradiol-17B, act directly on corpora lutea
to affect the luteolytic efficacy of exogenous PGan and are
thus required for spontaneous regression of bovine corpora

lutea.

 

 

SUMMARY AND CONCLUSIONS

The study presented examines two factors that could be
involved in initiation of luteal regression. Based on
results from this study it is suggested that availability of
LH in serum is not diminished between mid-to-late diestrus
though it is unknown if a change in the pulsatile release of
LH is altered immediately before luteal function declines.
However, as suggested in the model, ability to respond to
luteotropic support may decline during this time and thus
corpora lutea require large increases in LH in serum to
offset this declined responsiveness.

The presence of ovarian follicles clearly facilitates
the ability of exogenous PGan to cause luteal regression.
This suggests products of ovarian follicles negatively
affect luteal function in ways other than the ability of
estradiol-178 to stimulate increased synthesis and release
of PGFZG from either the uterus or ovary. This luteolytic
action of ovarian follicular products is possibly directly
on luteal cells and interferes with LH induced stimulation
of progesterone secretion. Estradiol-178 and other follicu-
lar products may synergize with the luteolytic effects of
PGFZa on luteal cell function at the same or different sites
of LH stimulated steroidogenesis but this synergism directly
on luteal tissue remains to be elucidated.

44

 

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