Rat Ovarian Follicular Dynamics: A Model to Study Prolactin and Gonadotropins Interaction

IJBPAS, February, 2013, 2(2): X-X
ISSN: 2277–4998

RAT OVARIAN FOLLICULAR DYNAMICS: A MODEL TO STUDY
INTERACTION BETWEEN PROLACTIN AND GONADOTROPINS

AGGARWAL N, KUMARI N AND MURALIDHAR K*
Hormone Research Laboratory, Department of Zoology, University of Delhi, Delhi-
110007
*Corresponding Author: E Mail: kambadur@hotmail.com
ABSTRACT
Ovarian follicular dynamics in the laboratory rat has been reviewed. Major physiological and
biochemical events during follicular growth and maturation as well as during atresia
including apoptosis have been described based on literature survey. Natural factors like
genetic make up and hormones and artificial factors like hormone specific antibodies are
known to influence this follicular dynamics in both directions. Anti gonadotropic effects of
Prolactin in other rat models has been briefly reviewed. The advantages of using the present
model have been pointed out.
Keywords: Ovary, Atresia, Follicles, Prolactin, Gonadotropins
INTRODUCTION
Prolactin was discovered in 1928 and is groups. That raised the third mystery i.e.
found in all vertebrates including humans. extensive microhetrogeneity in structure and
The name ’prolactin’ is derived from its hence its relevance to physiology. The
established role, in female mammals, in mechanism of action of prolactin has been
mammopoiesis. That raised the first mystery studied extensively which gave raise to
regarding its role in human male and in non fourth mystery as to why it does not follow
mammalian vertebrates. More than 300 the classical second messenger model in
effects have been produced by injecting it signaling pathways. As mentioned earlier
into animals of all phylogenic groups. That prolactin has effects in all vertebrate groups
raised the second mystery i.e. absence of and these effects can be grouped under
any reliable bioassay for prolactin till today. seven categories. These are effects on water
Prolactin has been purified and and salt balance seen dramatically in fish,
characterized from a number of vertebrate on growth and morphogenesis seen in

a
IJBPAS, February, 2013, 2(2)

Murlidhar K et al Research Article

amphibians, on metabolism, on reaches maturity. Atresia is a type of
immunoregulation, on skin, on behavior and Programmed cell death or Apoptosis.
lastly but not the least important on Apoptosis was first discovered by Carl Vogt
reproduction and lactation. In spite of in 1842. The morphological characteristic of
exhibiting multiple physiological effects on apoptosis includes cell shrinkage, plasma
a variety of tissues like brain (behavior), membrane blebbing and apoptotic body
gonads and mammary tissues, accessory sex formation. Atretic follicles undergo several
organs like ventral prostate, cells of immune changes which include retraction of
system like phagocytes and lymphocytes etc granulosa cells and initiation of granulosa
no disease whose origin can be ascribed to cell apoptosis. After most of the granulosa
mutations in Prolactin or prolactin receptor cells are lost, more severe changes occur as
genes has yet been discovered. This leads to atresia progresses, including segmentation
the fifth mystery i.e. there is no known of the oocytes and cytoplasmic
clinical model of prolactin deficiency. vacuolization. Most follicles that leave the
Hyperprolactinemia due to tumors of resting stage and begin to grow do not
pituitary lactotrophs is the only known mature fully but instead undergo atresia
pathological condition. Long term hyper- during the developmental process. More
prolactinemia can lead to amenorrhea in than 99.9% of the ovarian follicles present
women, loss of libido in men and infertility at birth never reach ovulation in human and
in both. The interaction of prolactin with this figure is 77% for the mouse. The
gonadotropins leading to antagonism can number of follicles developing to the
occur at the pituitary level where it is known preovulatory stage is thus far fewer than the
to inhibit the action of GnRH [1] and at the number undergoing atresia. Follicles can
gonadal level. Models to study this become atretic at any stage of development.
antagonistic interaction are not available yet. Successful follicle development depends on
Ovarian follicular dynamics including the presence of survival factors that promote
growth and atresia can be judiciously follicle growth and also protect cells from
studied to investigate this antagonism apoptosis. These include factors produced
between gonadotropins and prolactin. within the ovary as well as the
Mammalian females are born with definite gonadotropins Luteinizing hormone (LH)
number of follicles in the ovary. With the and Follicle stimulating hormone (FSH). In
passing period of time this follicular pool the absence of survival factors, endogenous
decreases and undergoes atresia [2, 3]. apoptosis pathways within the follicle
Follicular atresia is degeneration and become activated and lead to follicular
resorption of an ovarian follicle before it atresia.. Several molecules that regulate

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apoptosis are summarized here. Two of the transformed into primary oocytes
experimental models to understand the characterized by long prolonged meiotic
process of atresia are by performing prophase and surrounded by a squamous
hypophysectomy or by immunoneutraliztion layers of pregranulosa cells. These
method. Low level of gonadotropins primordial follicles constitute the resting
especially Follicle stimulating hormone pool of non-growing follicles, which
(FSH) and low level of Estradiol is the progressively depleted during the
major inducer of apoptosis in primary and reproductive life span. Primordial follicles
small antral follicles. It has been shown that proceed by growing into primary follicles in
androgens are atretic in action and estradiol which oocyte is surrounded by cuboidal
is anti-atretic. Role of prolactin in granulosa cells. Later stages then by a series
macrophage invasion in ovarian atresia is of mitotic divisions in granulosa cell layer,
also known. There are various apoptotic unilayered primary follicles are converted
markers studied in rat such as Cathepsin-d into multilayered preantral stage designated
(lysosomal enzyme) which increases during as secondary follicle. A secondary follicle
atresia. becomes invested with thecal cells with time
The ovaries are covered by a sheet of and comprises of full grown oocytes
squamous or cuboidal epithelium, the surrounded by zona pellucida. With the
germinal or serous epithelium which rests appearance of an antral cavity, the
on the basement membrane. Beneath the secondary follicle is converted into a
serous layer is a layer of dense connective tertiary follicle. The number of granulosa
tissue termed as tunica albuginea. On the cell is very high at this stage and acquires a
edge of the ovary, the hilum is attached to large antrum –fluid filled cavity. This large
the broad ligament by the mesovarium. The antral follicle has now become Graafian
ovary is organized into two principal follicle and is ready to ovulate. Those
regions the medulla and a peripheral part follicles which do not ovulate degenerate by
called the cortex. Embedded in the stroma atresia. Following the ovulation of large
of the cortex are the stages of follicles and dominant follicle, the follicle wall collapses
reflects different stages of growth and to transform into corpus luteum. Eventually
development [4]. The majority of mammals corpus luteum degenerates and is present as
restrict oogonial development (development a white scar of dense connective tissue, the
of primordial germ cells PGC) to prenatal corpus albicans. Gonadotropins control the
development or to shortly after birth [4]. In growth and differentiation of the steroid
most mammals before or soon after birth, hormone secreting cells of the ovary [5].
PGC’s (Primordial germ cells) are

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Estrogens are synthesized in granulosa cells. the, mammal returns to proestrus and the
LH drives thecal synthesis of androgens and cycle begins anew. The laboratory rat is a
androgens are subsequently aromatized to nonseasonal, spontaneous ovulating,
estrogens in adjacent granulosa cells. In polyestrous animal. Ovulation occurs every
granulosa cells FSH stimulates transcription 4-5 days throughout the year.
of the gene encoding aromatase which Hypophysectomy is the removal of
aromatizes androgens into estrogens. The hypophysis or pituitary gland. When the
word estrus is a Latin adaptation of the procedure is performed before sexual
Greek word oistrus. This term was first used maturity, the reproductive tract remains
by Heape to describe “special period of undeveloped and non-functional. There is
sexual desire of the female”. Heape further also a general lack of growth. If performed
described different stages of the cycle as it after sexual maturity, there will be a loss of
applies to mammals during breeding season. reproductive function along with atrophy of
He used the term anestrous to describe the gonads and accessory reproductive
non breeding season or period of rest in structures. In rats after hypophysectomy it is
female mammal when the ovaries and observed that the number of healthy follicles
accessory reproductive organs are relatively decreases considerably. Female rats
quiescent and attempts of mating by male hypophysectomized at 28 days of age were
are resisted. Heape also used the prefixes killed at various time intervals and healthy
pro-, di-, met- along with the suffix –estrus follicles were classified as primary follicles
to describe the stages of the cycle between with two or more granulosa cell layer and
the periods of estrus during the sexual vesicular follicle. By 10thday and 38thday
season. The first part of the cycle he termed after hypophysectomy, the average number
proestrus which last for 12-14 hours. The of primary follicles in ovary was 102 and 20
next period is estrus period at which female respectively [6] compared to 213 on the day
is receptive to male the length of this period after operation, similarly vesicular follicles
is 25-27 hours. In the absence of coitus, at the same time intervals were 99 and 3
estrus is succeeded by a short phase called compared to 160 on day 1. Dependency of
metestrus phase which lasts for 6-7 hours. preantral follicles on gonadotropin support
The following period diestrus is of variable is reflected with experimental evidences and
duration in different species of 55-57 hours. observations where long term injection of
During this time, ovarian secretions prepare gonadotropin–releasing hormone (GnRH)
the reproductive tract for the receipt of agonist greatly increases the percentage of
ovum, newly fertilized shortly after mating follicles smaller than 35μm, while
on estrus. If fertilization has not taken place decreasing the percentage9 of follicles

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greater than 35μm from 14% in controls to morning of estrus initiates atresia of large
1% in the treated groups [7]. Also follicular follicles that take place on metestrus. FSH
growth in rats hypophysectomized on day (follicle stimulating hormone) levels goes
22 and injected 10days later with [³H] down in evening of estrus, so FSH levels
thymidine shows labeling of granulosa cells were augmented by prolonging the surge of
in small follicles in intact animals. FSH by giving single injection of PMSG
Since long there has been questions related (0.025IU/gm body weight) during peri-
to how such large number of follicles ovulatory period at 8h on estrus an increase
undergo degeneration while only one in higher number of healthy follicles were
dominant follicle is selected to mature and observed at 12h of metestrus by a decrease
ovulate, and what are the factors regulating in large atretic follicles in ovaries. This
cell death of such a large number of follicles simply signifies that the atresia is triggered
since birth and continues throughout the by decline in FSH concentration during the
reproductive life span. There are evidences morning of estrus due to which normally
from rat models which can tell us that there large atretic follicles are observed at
is a direct endocrine hormonal control of metestrus. This is due to decline in
this process with which large number of concentration of FSH during the morning of
follicles undergo atresia or programmed cell estrus and can be prevented by prolonging
death. Granulosa cell undergoes apoptosis the surge of FSH with administration PMSG
during follicular atresia. The degeneration [11]. Similarly other factors like ratio of
of granulosa cells as atresia advances has estrogen to progesterone, [12], androgens
characteristics of apoptotic cell death [8]. [13] obviously influenced by the levels of
Atresia can be induced by first steroidogenic enzymes [14] also affect
administering immature rats with PMSG follicular growth and atresia.. Histochemical
(pregnant mare’s serum gonadotropin) and and biochemical alterations in granulosa
then withdrawing it, PMSG shows both cells often precede definite morphologic
biological and binding activity of both FSH change sin atretic granulosa sells. These
when injected in heterologous species and include an increase in lysosomal enzymes
activity of both FSH and LH in same such as acid phosphatase, amino peptidase
species [9]. [15]. A model system to study the
FSH induces granulosa cell divisions in large biochemical mechanism of follicular atresia
Antral follicles and in intact rats. Decreased in rats [16] was characterized using
level of FSH causes decreased granulosa cell histological and biochemical studies. PMSG
division and appearance of pyknotic nuclei and PMSG antiserum was used to induce
[10]. Falling levels of gonadotropins on the the follicular growth and atresia of

e


preovulatory follicles. Ovarian histology granulosa cells were not restored. The
during these PMSG and PMSG - treatment injection of dihydrotestosterone (DHT) to
periods was recorded under a light 48 h PMSG-primed rats induced atresia as
microscope. An analysis of lysosomal noted by an increase in Cathepsin-D activity.
enzyme Cathepsin-D activity of granulosa However, the exogenous administration of
cells from similarly treated ovaries showed FSH along with DHT prevented this atretic
that there was a reduction in Cathepsin-D effect suggesting that DHT is not having a
activity during the histologically observable direct effect on atresia. Determination of
follicular growth and there was an increase androgen: estrogen content of the granulosa
in Cathepsin-D activity during atresia.. Pre cells and an analysis of the individual
ovulatory follicular atresia was studied profile of androgen and estrogen revealed
using pregnant mare serum gonadotropin that the increase in Cathepsin-D activity
(PMSG)-primed rats (15 IU/rat) which were could be correlated only with the decrease
deprived of hormonal support either by in granulosa cell estrogen content. This
allowing the metabolic clearance of the along with the observation that granulosa
PMSG or by injecting a specific PMSG cells showed a loss of estrogen synthesis
antiserum (PMSG a/s). Atresia was well before the increase in Cathepsin-D
monitored by an increase in lysosomal activity strongly points out that the lack of
Cathepsin-D activity and a decrease in the estrogen rather than an increase in androgen
receptor activity of the granulosa cells is the principle factor responsible for the
isolated from the preovulatory follicles. It atresia of preovulatory follicles in the rat.
was shown that the increase in lysosomal Prolactin receptors have been identified on
activity the decrease in receptor activity T and B lymphocyte cells as well as on
seen at 96 h after PMSG (or PMSG plus macrophages [17]. [18] studied effects of
PMSG a/s) could be arrested both by follicle prolactin on immune challenge with
stimulating hormone (FSH) and Luteinizing lipopolysaccharides (LPS) on the
hormone (LH). Injection of cyanoketone or macrophage invasion into follicular and
Clomiphene citrate together with FSH/LH luteal compartment and occurrence of
prevented this 'rescue' suggesting a role for apoptosis/atresia due to this macrophage
estrogens in the regulation of atresia. invasion. Wistar rats were injected with
Although the administration of estradiol-17 ovine prolactin or LPS or vehicle saline
beta (20 micrograms/rat) together with 3days before the experimentation.
PMSG a/s could show a 'rescue effect' in Treatment is initiated on day of estrus so
terms of reduction in Cathepsin-D activity, that ovaries can be obtained on proestrus
the gonadotropin receptor activities of these day1. From each female rat one ovary is

f


fixed for histological studies and another for and do not show overt signs of atresia. The
immunohistochemistry studies. In estrous up regulation of anti-apoptotic and survival
rats the total (antral plus preovulatory) % of factors is hypothesized to account for the
atretic follicle increased from 41% in accumulation of small antral follicles
control to 45%in Prolactin treated to 56% in whereas the lack of developmental
LPS treated rat. Apoptotic cells were progression is explained by the abnormal
observed in granulosa cell layer and rarely endocrine environment in PCOS patients,
in thecal cell layer and inversely notably FSH suppression. PCOS is thus a
macrophages were observed in thecal cells unique pathology deviating significantly
and less than 5% in granulosa cells.. The from the normally concomitant occurrence
overall mean number of macrophages per of developmental arrest and atresia of the
follicle increased after treatment and more follicle. Current lines of evidence support
significantly higher in LPS treated animals. the role of a survival/apoptotic balance as
Pre treatment with either of LPS or prolactin one of likely multiple mechanisms
showed reduced progesterone response to explaining PCOS symptoms persistence of
FSH in vitro, in comparison to controls the follicles.
ovaries. The same effect was not seen with In a normal animal undergoing natural aging
forkskolin (an adenylate cyclase stimulator) process, follicular growth and maturation is
induced progesterone secretion, which must asynchronous. The superovulated pseudo
be due to disrupted ovarian cyclicity caused pregnant immature rat, which is used as a
by LPS and prolactin treatment. The model to perform bioassay for Luteinizing
reduced steroidogenic response to FSH hormone, the animals receive injections of
might be the cause of increased number of PMSG to initiate new but synchronized
atretic follicles following LPS and prolactin wave of follicular growth [19]. This ‘Parlow
treatment. rat’ also would be a good model. Indeed a
The follicular atresia is also pertinent to the better model, to investigate not only
pathogenesis of PCOS. Many defects arise underlying mechanisms including signaling
in PCOS patients, primarily chronic pathways but also to study interaction
anovulation and hyperandrogenism. The among factors which influence this process
primary defect is at the level of the ovary, in vivo. Hence the putative interaction
particularly at the level of folliculogenesis between Prolactin and gonadotropins can be
wherein the ovary contains many small investigated using such models. For
antral follicles (at least more than 10 example we have already demonstrated that
follicles between 2-8 mm in diameter). Prolactin can antagonize FSH action in this
These follicles are arrested in development model [20] but also that a peptide, derived

g


from the internal amino acid sequence of after sexual maturity, there will be a loss of
buffalo pituitary prolactin, can increase reproductive function along with atrophy of
ascorbic content of ovaries [19]. gonads and accessory reproductive
Fig: Concentrations of various hormones structures. In rats after hypophysectomy it is
in peripheral plasma obtained at 2hour observed that the number of healthy follicles
intervals throughout each day of 4-day decreases considerably.
estrous cycle of the rat. Taken from: [4] Female Rats hypophysectomized at 28days
Physiology of Reproduction 2006 The first of age were killed at various time intervals
part of the cycle he termed proestrus which and healthy follicles were classified as
last for 12-14 hours. The next period is primary follicles with two or more
estrus period at which female is receptive to granulosa cell layer and vesicular follicle.
male the length of this period is 25-27 hours. By 10thday and 38thday after
In the absence of coitus estrus is succeed by hypophysectomy, the average number of
short phase called metestrus phase which primary follicles in ovary was 102 and 20
lasts for 6-7 hours. The following period respectively [6] compared to 213 on the day
diestrus is of variable duration in different after operation, similarly vesicular follicles
species of 55-57 hours. During this time, at the same time intervals were 99 and 3
ovarian secretions prepare the reproductive compared to 160 on day 1. Dependency of
tract for the receipt of ovum, newly preantral follicles on gonadotropin support
fertilised shortly after mating on estrus. If is reflected with experimental evidences and
fertilisation has not taken place the, observations where long term injection of
mammal returns to proestrus and the cycle gonadotropin–releasing hormone(GnRH)
begins anew. The laboratory rat is a agonist greatly increases the percentage of
nonseasonal, spontaneous ovulating, follicles smaller than 35μm, while
polyestrous animal. Ovulation occurs every decreasing the percentage9 of follicles
4-5days throughout the year. greater than 35μm from 14% in controls to
Effect of Hypophyesctomy on Rat 1% in the treated groups [5]. Also follicular
Ovarian Follicular Development And growth in rats hypophysectomized on day
Atresia 22 and injected 10days later with [³H]
Hypophysectomy is the removal of thymidine shows labelling of granulosa cells
hypophysis or pituitary gland. When the in small follicles in intact animals.
procedure is performed before sexual Factors Affecting Follicular Atresia
maturity, the reproductive tract remains Since long there has been questions related
undeveloped and non-functional. There is to how such large number of follicles
also a general lack of growth. If performed undergo degeneration while only one

h


dominant follicle is selected to mature and ovulatory period at 8h on estrus an increase
ovulate, and what are the factors regulating in higher number of healthy follicles were
cell death of such a large number of follicles observed at 12h of metestrus by a decrease
since birth and continues throughout the in large atretic follicles in ovaries. This
reproductive life span. There are evidences simply signifies that the atresia is triggered
from rat models which can tell us that there by decline in FSH concentration during the
is a direct endocrine hormonal control of morning of estrus due to which normally
this process with which large number of large atretic follicles are observed at
follicles undergo atresia or programmed cell metestrus. This is due to decline in
death. Granulosa cell undergoes apoptosis concentration of FSH during the morning of
during follicular atresia. The degeneration estrus and can be prevented by prolonging
of granulosa cells as atresia advances has the surge of FSH with administration PMSG.
characteristics of apoptotic cell death [8]. B. Ratio of Estrogen And
Atresia can be induced by first Progesterone
administering immature rats with PMSG An evidence for a potential mechanism
(pregnant mare’s serum gonadotropin) and underlying follicular atresia The imbalance
then withdrawing it, PMSG shows both of estrogen and progesterone levels
biological and binding activity of both FSH attributes to granulosa cell apoptosis [12].
when injected in heterologous species and 15 IU PMSG was injected in immature rats.
activity of both FSH and LH in same Granulosa cell apoptosis was observed by
species [9]. performing gel electrophoresis of genome of
A. Affect of Gonadotrophins granulosa cells from experimental and
FSH induces granulosa cell divisions in control, a DNA fragmentation ladder was
large Antral follicles and in intact rats. observed specific to apoptosis on day 4 after
Decreased level of FSH causes decreased PMSG injection and on the same day serum
granulosa cell division and appearance of levels of estrogen were 4 fold higher than
pyknotic nuclei [10]. Falling levels of controls and progesterone levels were
gonadotropins on the morning of estrus elevated up to 7-8 fold on day 4 and day5
initiates atresia of large follicles that take then from controls, showing imbalance in
place on metestrus. FSH (follicle estrogen: progesterone levels. In rats
stimulating hormone) levels goes down in Changes in Estrogen level is one of the
evening of etsrus, so FSH levels were reason why small follicles undergo atresia.
augmented by prolonging the surge of FSH It is observed that there is sharp decline in
by giving single injection of PMSG estrogen level just after the LH surge.
(0.025IU/gm body weight) during peri- Estrogen treatment prevents follicular

i


atresia in hypophysectomized immature rats follicles from PMSG primed
[21]. Through various studies about effect hypophysectomised induced female rats.
of PMSG and follicular atresia it is shown Note: number of atretic follicles is much
that PMSG injection given to immature rats much higher in DHT treated rats. Number
decreases the atresia of large antral follicles inside each bar represents the number of
and also a sharp decline in pyknotic index is animals from which a single ovary was
been stated [7]. It is possible that FSH and examined. From [13] Since the ovaries of
PMSG exert their antiatretic action through PMSG primed hypophysectomized rats
the stimulation of follicular estrogen would be expected to contain considerable
production. amounts of estradiol one of the actions of
C. Affect Of Androgen On Follicular DHT in causing atresia could be
Atresia interference with the action of estradiol.
[13] studied the effect of androgen on When estradiol is injected along with DHT
follicular growth and found that androgens the atresia inducing effects of DHT is
posses atretic effects on ovaries. Immature prevented. One of the prominent effect
cycling rats were given PMSG to initiate shown by [13] on ovaries from PMSG/DHT
follicular growth and 54hrs later hCG treated hypophysectomized rats given
(human chorionic gonadotropin) was given estradiol was both reappearance of healthy
to induce ovulation. Then to see the effect follicles and reduction in atresia when
of non-aromatizable androgen 5α- compared to PMSG/DHT treated rats. So
dihydrotestosterone (DHT), to a second set, estradiol rescues follicles from androgen
PMSG primed hypophysectomized rats induced degeneration. Intra-ovarian
were given increasing doses of DHT prior to androgens may act to prevent the follicle
sacrifice or hCG treatment. Along with this, from completion of one or more steps in
to see the possible antiatretic affects of maturational process by hindering in
estradiol in the system other groups of estrogen action.
PMSG primed rats were given increasing D. Changes In Steroidogenic
doses of estradiol alone or in combination Enzymes Activity
with DHT. To serve as controls adult rats Steroidogenic enzyme activity increases
were sacrificed at the morning of estrus. following preovulatory surge of
Result showed, DHT treatment significantly gonadotropin and there is decreased
reduced the numbers of primary follicles to estrogen production leading to atresia [13].
PMSG treated animals. It was found that activity of C17,20-lyase
Fig: Effect of DHT on numbers of primary, and 20α –hydroxysteriod dehydrogenase
secondary, tertiary and atretic ovarian (20α-SDH)changes during atresia.

j


Preovulatory follicles at different time were cumulus cells lose contact with the rat
explanted after stimulation by hCG /LH and. oocyte (From Ingram : The Ovary 1962).
It was reported that there was gradual Enzyme Marker For Studying Follicular
decrease in lyase activity and increase in Atresia In Rodents
20alpha-hydroxysteriod dehydrogenase after Histochemical and biochemical alterations
3h incubation. in granulosa cells often precede definite
Fig: Lyase and 20α- SDH activity in rat morphologic change sin atretic granulosa
preovulatory follicles. The follicles were sells. These include an increase in
isolated on morning of proestrus at 0hr, 3, 6, lysosomal enzymes such as acid
9 after administration of 5IU of hCG [14]. phosphatase, aminopeptidase [15].
Fig: Effects of hypophysectomy on Cathepsin-D- Lysosomal Enzyme As a
follicular lyase and 20α-SDH activity. Marker Of Follicular Atresia
Follicles from the intact rats were explanted A model system to study the biochemical
on the morning of the day of proestrus at 0h, mechanism of follicular atresia in rats [16]
6h, 12h, 24h after hypox [14]. Overall was characterized using histological and
steroidogenesis is stimulated within 4-6h of biochemical studies. PMSG and PMSG
LH/hCG and the sectretion of androgens antiserum was used to induce the follicular
and estradiol is diminished after LH/ hCG growth and atresia of preovulatory follicles.
stimulation follicles in which atresia was Ovarian histology during these PMSG and
induced experimentally and steroidogenic PMSG - treatment periods was recorded
changes include decreased production of under a light microscope. An analysis of
follicular estrogen. After hypophysectomy , lysosomal enzyme cathepsin-D activity of
it resulted in atresia of preovulatory follicles granulosa cells from similarly treated
due to withdrawal of tonic levels of ovaries showed that there was a reduction in
gonadotropin and was accompanied by cathepsin-D activity during the
marked decrease in lyase activity and histologically observable follicular growth
increase in 20α-SDH within 6h. The activity and there was an increase in cathepsin-D
was measured from homogenates of activity during atresia. The group [22]
Graafian follicle by evaluating conversion further used this model system in tracing the
of precursors to products. Follicles of most path of atresia. Preovulatory follicular
species in late stages of atresia exhibit atresia was studied using pregnant mare
germinal vesicle nreakdown. Then the serum gonadotropin (PMSG)-primed rats
oocytes from atretic follicles are cultured , (15 IU/rat) which were deprived of
germinal vesicle breakdown increased, 235 hormonal support either by allowing the
of the oocyte fragmented. During atresia the metabolic clearance of the PMSG or by

k


injecting a specific PMSG antiserum cells showed a loss of estrogen synthesis
(PMSG a/s). Atresia was monitored by an well before the increase in cathepsin-D
increase in lysosomal cathepsin-D activity activity strongly points out that the lack of
and a decrease in the receptor activity of the estrogen rather than an increase in androgen
granulosa cells isolated from the is the principle factor responsible for the
preovulatory follicles. It was shown that the atresia of preovulatory follicles in the rat.
increase in lysosomal activity the decrease Role of Prolactin in Increased Infiltration
in receptor activity seen at 96 h after PMSG of Macrophage In Follicles
(or PMSG plus PMSG a/s) could be arrested Preovulatory surge of LH (luteinizing
both by follicle stimulating hormone (FSH) hormone) causes preovulatory prolactin
and luteinizing hormone (LH). Injection of surge which induces the expression of a
cyanoketone or clomiphene citrate together monocyte chemo-attractant protein in
with FSH/LH prevented this 'rescue' corpora lutea after ovulation causing
suggesting a role for estrogens in the invasion of macrophages. Prolactin
regulation of atresia. Although the receptors have been identified on T and B
administration of estradiol-17 beta (20 lymphocyte cells as well as on macrophages
micrograms/rat) together with PMSG a/s [17]. [18] studied effects of prolactin on
could show a 'rescue effect' in terms of immune challenge with lipopolysaccharides
reduction in cathepsin-D activity, the (LPS) on the macrophage invasion into
gonadotropin receptor activities of these follicular and luteal compartment and
granulosa cells were not restored. The occurrence of apoptosis/atresia due to this
injection of dihydrotestosterone (DHT) to macrophage invasion. Porton wistor rats
48 h PMSG-primed rats induced atresia as were injected with ovine prolactin or LPS or
noted by an increase in cathepsin-D activity. vehicle saline 3days before the
However, the exogenous administration of experimentation. Treatment is initiated on
FSH along with DHT prevented this atretic day of estrus so that ovaries can be obtained
effect suggesting that DHT is not having a on proestrus day1. From each female rat one
direct effect on atresia. Determination of ovary is fixed for histological studies and
androgen: estrogen content of the granulosa another for immunohistochemistry studies.
cells and an analysis of the individual In oestrous rats the total (antral plus
profile of androgen and estrogen revealed preovulatory) % of atretic follicle increased
that the increase in cathepsin-D activity from 41% in control to 45%in Prolactin
could be correlated only with the decrease treated to 56% in LPS treated rat. Apoptotic
in granulosa cell estrogen content. This cells were observed in granulosa cell layer
along with the observation that granulosa and rarely in thecal cell layer and inversely

l


macrophages were observed in the cal cells survival. These changes in gene expression
and less than 5% in granulosa cells. in granulosa cells correspond with shifts in
Fig: The percentage numbers of healthy (H) levels of Bax, Bcl-2, and Bcl-xlong proteins
and atretic (A) follicles observed in rats as well [23]. In support of these gene
treated with prolactin (PRL), expression studies, inactivation of the bax
lipopolysaccharide (LPS) or vehicle gene in mice results in the accumulation of
(control) for 3 days prior to expected oestrus abnormal follicles in the ovary, possessing
[18]. atrophic granulosa cells that fail to undergo
The overall mean number of macrophages apoptosis as the follicles proceeds through
per follicle increased after treatment and atresia [24]. Bax functions a key modulator
more significantly higher in LPS treated of granulosa cell fate in diverse species of
animals. Pre treatment with either of LPS or mammals. The BH-3 only family member,
prolactin showed reduced progesterone Bad, is expressed in granulosa cells and
response to FSH in vitro dispersates in theca interna cells of the rat ovary, and over
comparison to controls ovaries. The same expression of Bad in rat granulosa cells
effect was not seen with forkskolin (a induces apoptosis [25]. Caspases cysteine
adenylate cyclase stimulator) induced aspartate-specific proteins are essential in
progesterone secretion, which must be due formation of apoptosome (apoptotic
to disrupted ovarian cyclicity caused by LPS machinery) and require release of
and prolactin treatment. The reduced cytochrome-c from destabilized
steroidogenic response to FSH might be the mitochondria an event triggered by the
cause of increased number of atretic actions of proapoptotic Bcl-2 family
follicles in effect of LPS and prolactin members. Processing and activation of
treatment. caspase-9 and effector caspase such as
Signal Transduction During Granulosa caspase -3 have been reported [26] in rat
Cell Apoptosi ovary. Preliminary studies have shown that
Bcl-2 Family members including several inactivation of caspase-9 gene in mice
antiapoptotic (Bcl-2, Bcl-x),proapoptotic results in defective granulosa cell apoptosis
multidomain (Bax, Bak, Mtd/Bok, and follicular atresia in vivo.Findings have
Boo/Diva) and BH-3 only ( Bad, Bim/Bod) established the existence of an inverse
proteins are involved in determining correlation between caspase-3 expression
granulosa cell fate. Gonadotropin mediated and apoptosis in granulosa cells of the
inhibition of apoptosis in rat granulosa cells rodent [26, 27]. A central role for Caspase-3
results in alteration in ratio of bax to bcl-2 in executing granulosa cell death has been
and bcl-xlong expression favouring cell shown by studies of caspase -3 deficient

m


mice, in which the mutant females were follicles between 2-8 mm in diameter).
shown to possess numerous aberrant atretic These follicles are arrested in development
follicle containing granulosa cells that failed and do not show overt signs of atresia. The
to undergo apoptosis [28]. upregulation of anti-apoptotic and survival
Implication of Atresia In Ovary factors are hypothesized to account for the
Understanding the basic biology of accumulation of small antral follicles
apoptosis and its control in ovarian whereas the lack of developmental
follicular development has opened avenues progression is explained by the abnormal
to explore with respect to clinical endocrine environment in PCOS patients,
applications of these data. Although the vast notably FSH suppression. PCOS is thus a
majority of these studies are still in the unique pathology deviating significantly
development stages for improving human from the normally concomitant occurrence
health and fertility. of developmental arrest and atresia of the
Fig: Flowchart showing Possible fields of follicle. Current lines of evidence support
study in understanding Follicular atresia. the role of a survival/apoptotic balance as
Premature ovarian failure is defined as one of likely multiple mechanisms
amenorrhea with hypo-oestrogenism and explaining PCOS symptoms persistence of
elevated gonadotrophins occurring before the follicles .
the age of 40 years. In theory, ovarian CONCLUSION
failure may occur because of a decreased The major help in studying follicular atresia
pool of primordial follicles, because ovarian is provided by model systems developed for
apoptosis is increased or accelerated or understanding events occurring during the
because the follicle maturation is interrupted process in rodents. With the ongoing
before the preovulatory stage. The research over event of follicular atresia role
mechanisms inducing premature ovarian of various factors affecting follicular atresia
failure have been described in a few number like low level of gonadotropins , ratio of
of cases [29]. estrogen to progesterone, androgen effects
The follicular atresia is also pertinent to the in rodent models have helped in
pathogenesis of PCOS. Many defects arise understanding temporal events occurring
in PCOS patients , primarily chronic during atresia. Once a platform is set with
anovulation and hyperandrogenism. The studies and experiments on rat model
primary defect is at the level of the ovary, system then further studies proceeds with
particularly at the level of folliculogenesis trials and experimentation on humans can
wherein the ovary contains many small begin. In one of the studies Bax-
antral follicles (at least more than) 10 proapoptotic protein is eliminated in mice

n


model suppressing the rate at which the Work reported here was supported by funds
immature follicle pool is lost from the from Delhi University under the Doctoral
ovaries via atresia would result in a R&D support scheme.
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Rat Ovarian Follicular Dynamics: A Model to Study Prolactin and Gonadotropins Interaction

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