INTERACTION BETWEEN PROLACTIN AND GONADOTROPINS

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

a

IJBPAS, February, 2013, 2(2)

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: [email protected]

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

found in all vertebrates including humans.

The name ’prolactin’ is derived from its

established role, in female mammals, in

mammopoiesis. That raised the first mystery

regarding its role in human male and in non

mammalian vertebrates. More than 300

effects have been produced by injecting it

into animals of all phylogenic groups. That

raised the second mystery i.e. absence of

any reliable bioassay for prolactin till today.

Prolactin has been purified and

characterized from a number of vertebrate

groups. That raised the third mystery i.e.

extensive microhetrogeneity in structure and

hence its relevance to physiology. The

mechanism of action of prolactin has been

studied extensively which gave raise to

fourth mystery as to why it does not follow

the classical second messenger model in

signaling pathways. As mentioned earlier

prolactin has effects in all vertebrate groups

and these effects can be grouped under

seven categories. These are effects on water

and salt balance seen dramatically in fish,

on growth and morphogenesis seen in

Murlidhar K et al Research Article

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amphibians, on metabolism, on

immunoregulation, on skin, on behavior and

lastly but not the least important on

reproduction and lactation. In spite of

exhibiting multiple physiological effects on

a variety of tissues like brain (behavior),

gonads and mammary tissues, accessory sex

organs like ventral prostate, cells of immune

system like phagocytes and lymphocytes etc

no disease whose origin can be ascribed to

mutations in Prolactin or prolactin receptor

genes has yet been discovered. This leads to

the fifth mystery i.e. there is no known

clinical model of prolactin deficiency.

Hyperprolactinemia due to tumors of

pituitary lactotrophs is the only known

pathological condition. Long term hyper-

prolactinemia can lead to amenorrhea in

women, loss of libido in men and infertility

in both. The interaction of prolactin with

gonadotropins leading to antagonism can

occur at the pituitary level where it is known

to inhibit the action of GnRH [1] and at the

gonadal level. Models to study this

antagonistic interaction are not available yet.

Ovarian follicular dynamics including

growth and atresia can be judiciously

studied to investigate this antagonism

between gonadotropins and prolactin.

Mammalian females are born with definite

number of follicles in the ovary. With the

passing period of time this follicular pool

decreases and undergoes atresia [2, 3].

Follicular atresia is degeneration and

resorption of an ovarian follicle before it

reaches maturity. Atresia is a type of

Programmed cell death or Apoptosis.

Apoptosis was first discovered by Carl Vogt

in 1842. The morphological characteristic of

apoptosis includes cell shrinkage, plasma

membrane blebbing and apoptotic body

formation. Atretic follicles undergo several

changes which include retraction of

granulosa cells and initiation of granulosa

cell apoptosis. After most of the granulosa

cells are lost, more severe changes occur as

atresia progresses, including segmentation

of the oocytes and cytoplasmic

vacuolization. Most follicles that leave the

resting stage and begin to grow do not

mature fully but instead undergo atresia

during the developmental process. More

than 99.9% of the ovarian follicles present

at birth never reach ovulation in human and

this figure is 77% for the mouse. The

number of follicles developing to the

preovulatory stage is thus far fewer than the

number undergoing atresia. Follicles can

become atretic at any stage of development.

Successful follicle development depends on

the presence of survival factors that promote

follicle growth and also protect cells from

apoptosis. These include factors produced

within the ovary as well as the

gonadotropins Luteinizing hormone (LH)

and Follicle stimulating hormone (FSH). In

the absence of survival factors, endogenous

apoptosis pathways within the follicle

become activated and lead to follicular

atresia.. Several molecules that regulate


c


apoptosis are summarized here. Two of the

experimental models to understand the

process of atresia are by performing

hypophysectomy or by immunoneutraliztion

method. Low level of gonadotropins

especially Follicle stimulating hormone

(FSH) and low level of Estradiol is the

major inducer of apoptosis in primary and

small antral follicles. It has been shown that

androgens are atretic in action and estradiol

is anti-atretic. Role of prolactin in

macrophage invasion in ovarian atresia is

also known. There are various apoptotic

markers studied in rat such as Cathepsin-d

(lysosomal enzyme) which increases during

atresia.

The ovaries are covered by a sheet of

squamous or cuboidal epithelium, the

germinal or serous epithelium which rests

on the basement membrane. Beneath the

serous layer is a layer of dense connective

tissue termed as tunica albuginea. On the

edge of the ovary, the hilum is attached to

the broad ligament by the mesovarium. The

ovary is organized into two principal

regions the medulla and a peripheral part

called the cortex. Embedded in the stroma

of the cortex are the stages of follicles and

reflects different stages of growth and

development [4]. The majority of mammals

restrict oogonial development (development

of primordial germ cells PGC) to prenatal

development or to shortly after birth [4]. In

most mammals before or soon after birth,

PGC’s (Primordial germ cells) are

transformed into primary oocytes

characterized by long prolonged meiotic

prophase and surrounded by a squamous

layers of pregranulosa cells. These

primordial follicles constitute the resting

pool of non-growing follicles, which

progressively depleted during the

reproductive life span. Primordial follicles

proceed by growing into primary follicles in

which oocyte is surrounded by cuboidal

granulosa cells. Later stages then by a series

of mitotic divisions in granulosa cell layer,

unilayered primary follicles are converted

into multilayered preantral stage designated

as secondary follicle. A secondary follicle

becomes invested with thecal cells with time

and comprises of full grown oocytes

surrounded by zona pellucida. With the

appearance of an antral cavity, the

secondary follicle is converted into a

tertiary follicle. The number of granulosa

cell is very high at this stage and acquires a

large antrum –fluid filled cavity. This large

antral follicle has now become Graafian

follicle and is ready to ovulate. Those

follicles which do not ovulate degenerate by

atresia. Following the ovulation of large

dominant follicle, the follicle wall collapses

to transform into corpus luteum. Eventually

corpus luteum degenerates and is present as

a white scar of dense connective tissue, the

corpus albicans. Gonadotropins control the

growth and differentiation of the steroid

hormone secreting cells of the ovary [5].


d


Estrogens are synthesized in granulosa cells.

LH drives thecal synthesis of androgens and

androgens are subsequently aromatized to

estrogens in adjacent granulosa cells. In

granulosa cells FSH stimulates transcription

of the gene encoding aromatase which

aromatizes androgens into estrogens. The

word estrus is a Latin adaptation of the

Greek word oistrus. This term was first used

by Heape to describe “special period of

sexual desire of the female”. Heape further

described different stages of the cycle as it

applies to mammals during breeding season.

He used the term anestrous to describe the

non breeding season or period of rest in

female mammal when the ovaries and

accessory reproductive organs are relatively

quiescent and attempts of mating by male

are resisted. Heape also used the prefixes

pro-, di-, met- along with the suffix –estrus

to describe the stages of the cycle between

the periods of estrus during the sexual

season. The first part of the cycle he termed

proestrus which last for 12-14 hours. The

next period is estrus period at which female

is receptive to male the length of this period

is 25-27 hours. In the absence of coitus,

estrus is succeeded by a short phase called

metestrus phase which lasts for 6-7 hours.

The following period diestrus is of variable

duration in different species of 55-57 hours.

During this time, ovarian secretions prepare

the reproductive tract for the receipt of

ovum, newly fertilized shortly after mating

on estrus. If fertilization has not taken place

the, mammal returns to proestrus and the

cycle begins anew. The laboratory rat is a

nonseasonal, spontaneous ovulating,

polyestrous animal. Ovulation occurs every

4-5 days throughout the year.

Hypophysectomy is the removal of

hypophysis or pituitary gland. When the

procedure is performed before sexual

maturity, the reproductive tract remains

undeveloped and non-functional. There is

also a general lack of growth. If performed

after sexual maturity, there will be a loss of

reproductive function along with atrophy of

gonads and accessory reproductive

structures. In rats after hypophysectomy it is

observed that the number of healthy follicles

decreases considerably. Female rats

hypophysectomized at 28 days of age were

killed at various time intervals and healthy

follicles were classified as primary follicles

with two or more granulosa cell layer and

vesicular follicle. By 10thday and 38thday

after hypophysectomy, the average number

of primary follicles in ovary was 102 and 20

respectively [6] compared to 213 on the day

after operation, similarly vesicular follicles

at the same time intervals were 99 and 3

compared to 160 on day 1. Dependency of

preantral follicles on gonadotropin support

is reflected with experimental evidences and

observations where long term injection of

gonadotropin–releasing hormone (GnRH)

agonist greatly increases the percentage of

follicles smaller than 35μm, while

decreasing the percentage9 of follicles


e


greater than 35μm from 14% in controls to

1% in the treated groups [7]. Also follicular

growth in rats hypophysectomized on day

22 and injected 10days later with [³H]

thymidine shows labeling of granulosa cells

in small follicles in intact animals.

Since long there has been questions related

to how such large number of follicles

undergo degeneration while only one

dominant follicle is selected to mature and

ovulate, and what are the factors regulating

cell death of such a large number of follicles

since birth and continues throughout the

reproductive life span. There are evidences

from rat models which can tell us that there

is a direct endocrine hormonal control of

this process with which large number of

follicles undergo atresia or programmed cell

death. Granulosa cell undergoes apoptosis

during follicular atresia. The degeneration

of granulosa cells as atresia advances has

characteristics of apoptotic cell death [8].

Atresia can be induced by first

administering immature rats with PMSG

(pregnant mare’s serum gonadotropin) and

then withdrawing it, PMSG shows both

biological and binding activity of both FSH

when injected in heterologous species and

activity of both FSH and LH in same

species [9].

FSH induces granulosa cell divisions in large

Antral follicles and in intact rats. Decreased

level of FSH causes decreased granulosa cell

division and appearance of pyknotic nuclei

[10]. Falling levels of gonadotropins on the

morning of estrus initiates atresia of large

follicles that take place on metestrus. FSH

(follicle stimulating hormone) levels goes

down in evening of estrus, so FSH levels

were augmented by prolonging the surge of

FSH by giving single injection of PMSG

(0.025IU/gm body weight) during peri-

ovulatory period at 8h on estrus an increase

in higher number of healthy follicles were

observed at 12h of metestrus by a decrease

in large atretic follicles in ovaries. This

simply signifies that the atresia is triggered

by decline in FSH concentration during the

morning of estrus due to which normally

large atretic follicles are observed at

metestrus. This is due to decline in

concentration of FSH during the morning of

estrus and can be prevented by prolonging

the surge of FSH with administration PMSG

[11]. Similarly other factors like ratio of

estrogen to progesterone, [12], androgens

[13] obviously influenced by the levels of

steroidogenic enzymes [14] also affect

follicular growth and atresia.. Histochemical

and biochemical alterations in granulosa

cells often precede definite morphologic

change sin atretic granulosa sells. These

include an increase in lysosomal enzymes

such as acid phosphatase, amino peptidase

[15]. A model system to study the

biochemical mechanism of follicular atresia

in rats [16] was characterized using

histological and biochemical studies. PMSG

and PMSG antiserum was used to induce

the follicular growth and atresia of


f


preovulatory follicles. Ovarian histology

during these PMSG and PMSG - treatment

periods was recorded under a light

microscope. An analysis of lysosomal

enzyme Cathepsin-D activity of granulosa

cells from similarly treated ovaries showed

that there was a reduction in Cathepsin-D

activity during the histologically observable

follicular growth and there was an increase

in Cathepsin-D activity during atresia.. Pre

ovulatory follicular atresia was studied

using pregnant mare serum gonadotropin

(PMSG)-primed rats (15 IU/rat) which were

deprived of hormonal support either by

allowing the metabolic clearance of the

PMSG or by injecting a specific PMSG

antiserum (PMSG a/s). Atresia was

monitored by an increase in lysosomal

Cathepsin-D activity and a decrease in the

receptor activity of the granulosa cells

isolated from the preovulatory follicles. It

was shown that the increase in lysosomal

activity the decrease in receptor activity

seen at 96 h after PMSG (or PMSG plus

PMSG a/s) could be arrested both by follicle

stimulating hormone (FSH) and Luteinizing

hormone (LH). Injection of cyanoketone or

Clomiphene citrate together with FSH/LH

prevented this 'rescue' suggesting a role for

estrogens in the regulation of atresia.

Although the administration of estradiol-17

beta (20 micrograms/rat) together with

PMSG a/s could show a 'rescue effect' in

terms of reduction in Cathepsin-D activity,

the gonadotropin receptor activities of these

granulosa cells were not restored. The

injection of dihydrotestosterone (DHT) to

48 h PMSG-primed rats induced atresia as

noted by an increase in Cathepsin-D activity.

However, the exogenous administration of

FSH along with DHT prevented this atretic

effect suggesting that DHT is not having a

direct effect on atresia. Determination of

androgen: estrogen content of the granulosa

cells and an analysis of the individual

profile of androgen and estrogen revealed

that the increase in Cathepsin-D activity

could be correlated only with the decrease

in granulosa cell estrogen content. This

along with the observation that granulosa

cells showed a loss of estrogen synthesis

well before the increase in Cathepsin-D

activity strongly points out that the lack of

estrogen rather than an increase in androgen

is the principle factor responsible for the

atresia of preovulatory follicles in the rat.

Prolactin receptors have been identified on

T and B lymphocyte cells as well as on

macrophages [17]. [18] studied effects of

prolactin on immune challenge with

lipopolysaccharides (LPS) on the

macrophage invasion into follicular and

luteal compartment and occurrence of

apoptosis/atresia due to this macrophage

invasion. Wistar rats were injected with

ovine prolactin or LPS or vehicle saline

3days before the experimentation.

Treatment is initiated on day of estrus so

that ovaries can be obtained on proestrus

day1. From each female rat one ovary is


g


fixed for histological studies and another for

immunohistochemistry studies. In estrous

rats the total (antral plus preovulatory) % of

atretic follicle increased from 41% in

control to 45%in Prolactin treated to 56% in

LPS treated rat. Apoptotic cells were

observed in granulosa cell layer and rarely

in thecal cell layer and inversely

macrophages were observed in thecal cells

and less than 5% in granulosa cells.. The

overall mean number of macrophages per

follicle increased after treatment and more

significantly higher in LPS treated animals.

Pre treatment with either of LPS or prolactin

showed reduced progesterone response to

FSH in vitro, in comparison to controls

ovaries. The same effect was not seen with

forkskolin (an adenylate cyclase stimulator)

induced progesterone secretion, which must

be due to disrupted ovarian cyclicity caused

by LPS and prolactin treatment. The

reduced steroidogenic response to FSH

might be the cause of increased number of

atretic follicles following LPS and prolactin

treatment.

The follicular atresia is also pertinent to the

pathogenesis of PCOS. Many defects arise

in PCOS patients, primarily chronic

anovulation and hyperandrogenism. The

primary defect is at the level of the ovary,

particularly at the level of folliculogenesis

wherein the ovary contains many small

antral follicles (at least more than 10

follicles between 2-8 mm in diameter).

These follicles are arrested in development

and do not show overt signs of atresia. The

up regulation of anti-apoptotic and survival

factors is hypothesized to account for the

accumulation of small antral follicles

whereas the lack of developmental

progression is explained by the abnormal

endocrine environment in PCOS patients,

notably FSH suppression. PCOS is thus a

unique pathology deviating significantly

from the normally concomitant occurrence

of developmental arrest and atresia of the

follicle. Current lines of evidence support

the role of a survival/apoptotic balance as

one of likely multiple mechanisms

explaining PCOS symptoms persistence of

the follicles.

In a normal animal undergoing natural aging

process, follicular growth and maturation is

asynchronous. The superovulated pseudo

pregnant immature rat, which is used as a

model to perform bioassay for Luteinizing

hormone, the animals receive injections of

PMSG to initiate new but synchronized

wave of follicular growth [19]. This ‘Parlow

rat’ also would be a good model. Indeed a

better model, to investigate not only

underlying mechanisms including signaling

pathways but also to study interaction

among factors which influence this process

in vivo. Hence the putative interaction

between Prolactin and gonadotropins can be

investigated using such models. For

example we have already demonstrated that

Prolactin can antagonize FSH action in this

model [20] but also that a peptide, derived


h


from the internal amino acid sequence of

buffalo pituitary prolactin, can increase

ascorbic content of ovaries [19].

Fig: Concentrations of various hormones

in peripheral plasma obtained at 2hour

intervals throughout each day of 4-day

estrous cycle of the rat. Taken from: [4]

Physiology of Reproduction 2006 The first

part of the cycle he termed proestrus which

last for 12-14 hours. The next period is

estrus period at which female is receptive to

male the length of this period is 25-27 hours.

In the absence of coitus estrus is succeed by

short phase called metestrus phase which

lasts for 6-7 hours. The following period

diestrus is of variable duration in different

species of 55-57 hours. During this time,

ovarian secretions prepare the reproductive

tract for the receipt of ovum, newly

fertilised shortly after mating on estrus. If

fertilisation has not taken place the,

mammal returns to proestrus and the cycle

begins anew. The laboratory rat is a

nonseasonal, spontaneous ovulating,

polyestrous animal. Ovulation occurs every

4-5days throughout the year.

Effect of Hypophyesctomy on Rat

Ovarian Follicular Development And

Atresia

Hypophysectomy is the removal of

hypophysis or pituitary gland. When the

procedure is performed before sexual

maturity, the reproductive tract remains

undeveloped and non-functional. There is

also a general lack of growth. If performed

after sexual maturity, there will be a loss of

reproductive function along with atrophy of

gonads and accessory reproductive

structures. In rats after hypophysectomy it is

observed that the number of healthy follicles

decreases considerably.

Female Rats hypophysectomized at 28days

of age were killed at various time intervals

and healthy follicles were classified as

primary follicles with two or more

granulosa cell layer and vesicular follicle.

By 10thday and 38thday after

hypophysectomy, the average number of

primary follicles in ovary was 102 and 20

respectively [6] compared to 213 on the day

after operation, similarly vesicular follicles

at the same time intervals were 99 and 3

compared to 160 on day 1. Dependency of

preantral follicles on gonadotropin support

is reflected with experimental evidences and

observations where long term injection of

gonadotropin–releasing hormone(GnRH)

agonist greatly increases the percentage of

follicles smaller than 35μm, while

decreasing the percentage9 of follicles

greater than 35μm from 14% in controls to

1% in the treated groups [5]. Also follicular

growth in rats hypophysectomized on day

22 and injected 10days later with [³H]

thymidine shows labelling of granulosa cells

in small follicles in intact animals.

Factors Affecting Follicular Atresia

Since long there has been questions related

to how such large number of follicles

undergo degeneration while only one


i


dominant follicle is selected to mature and

ovulate, and what are the factors regulating

cell death of such a large number of follicles

since birth and continues throughout the

reproductive life span. There are evidences

from rat models which can tell us that there

is a direct endocrine hormonal control of

this process with which large number of

follicles undergo atresia or programmed cell

death. Granulosa cell undergoes apoptosis

during follicular atresia. The degeneration

of granulosa cells as atresia advances has

characteristics of apoptotic cell death [8].

Atresia can be induced by first

administering immature rats with PMSG

(pregnant mare’s serum gonadotropin) and

then withdrawing it, PMSG shows both

biological and binding activity of both FSH

when injected in heterologous species and

activity of both FSH and LH in same

species [9].

A. Affect of Gonadotrophins

FSH induces granulosa cell divisions in

large Antral follicles and in intact rats.

Decreased level of FSH causes decreased

granulosa cell division and appearance of

pyknotic nuclei [10]. Falling levels of

gonadotropins on the morning of estrus

initiates atresia of large follicles that take

place on metestrus. FSH (follicle

stimulating hormone) levels goes down in

evening of etsrus, so FSH levels were

augmented by prolonging the surge of FSH

by giving single injection of PMSG

(0.025IU/gm body weight) during peri-

ovulatory period at 8h on estrus an increase

in higher number of healthy follicles were

observed at 12h of metestrus by a decrease

in large atretic follicles in ovaries. This

simply signifies that the atresia is triggered

by decline in FSH concentration during the

morning of estrus due to which normally

large atretic follicles are observed at

metestrus. This is due to decline in

concentration of FSH during the morning of

estrus and can be prevented by prolonging

the surge of FSH with administration PMSG.

B. Ratio of Estrogen And

Progesterone

An evidence for a potential mechanism

underlying follicular atresia The imbalance

of estrogen and progesterone levels

attributes to granulosa cell apoptosis [12].

15 IU PMSG was injected in immature rats.

Granulosa cell apoptosis was observed by

performing gel electrophoresis of genome of

granulosa cells from experimental and

control, a DNA fragmentation ladder was

observed specific to apoptosis on day 4 after

PMSG injection and on the same day serum

levels of estrogen were 4 fold higher than

controls and progesterone levels were

elevated up to 7-8 fold on day 4 and day5

then from controls, showing imbalance in

estrogen: progesterone levels. In rats

Changes in Estrogen level is one of the

reason why small follicles undergo atresia.

It is observed that there is sharp decline in

estrogen level just after the LH surge.

Estrogen treatment prevents follicular


j


atresia in hypophysectomized immature rats

[21]. Through various studies about effect

of PMSG and follicular atresia it is shown

that PMSG injection given to immature rats

decreases the atresia of large antral follicles

and also a sharp decline in pyknotic index is

been stated [7]. It is possible that FSH and

PMSG exert their antiatretic action through

the stimulation of follicular estrogen

production.

C. Affect Of Androgen On Follicular

Atresia

[13] studied the effect of androgen on

follicular growth and found that androgens

posses atretic effects on ovaries. Immature

cycling rats were given PMSG to initiate

follicular growth and 54hrs later hCG

(human chorionic gonadotropin) was given

to induce ovulation. Then to see the effect

of non-aromatizable androgen 5α-

dihydrotestosterone (DHT), to a second set,

PMSG primed hypophysectomized rats

were given increasing doses of DHT prior to

sacrifice or hCG treatment. Along with this,

to see the possible antiatretic affects of

estradiol in the system other groups of

PMSG primed rats were given increasing

doses of estradiol alone or in combination

with DHT. To serve as controls adult rats

were sacrificed at the morning of estrus.

Result showed, DHT treatment significantly

reduced the numbers of primary follicles to

PMSG treated animals.

Fig: Effect of DHT on numbers of primary,

secondary, tertiary and atretic ovarian

follicles from PMSG primed

hypophysectomised induced female rats.

Note: number of atretic follicles is much

much higher in DHT treated rats. Number

inside each bar represents the number of

animals from which a single ovary was

examined. From [13] Since the ovaries of

PMSG primed hypophysectomized rats

would be expected to contain considerable

amounts of estradiol one of the actions of

DHT in causing atresia could be

interference with the action of estradiol.

When estradiol is injected along with DHT

the atresia inducing effects of DHT is

prevented. One of the prominent effect

shown by [13] on ovaries from PMSG/DHT

treated hypophysectomized rats given

estradiol was both reappearance of healthy

follicles and reduction in atresia when

compared to PMSG/DHT treated rats. So

estradiol rescues follicles from androgen

induced degeneration. Intra-ovarian

androgens may act to prevent the follicle

from completion of one or more steps in

maturational process by hindering in

estrogen action.

D. Changes In Steroidogenic

Enzymes Activity

Steroidogenic enzyme activity increases

following preovulatory surge of

gonadotropin and there is decreased

estrogen production leading to atresia [13].

It was found that activity of C17,20-lyase

and 20α –hydroxysteriod dehydrogenase

(20α-SDH)changes during atresia.


k


Preovulatory follicles at different time were

explanted after stimulation by hCG /LH and.

It was reported that there was gradual

decrease in lyase activity and increase in

20alpha-hydroxysteriod dehydrogenase after

3h incubation.

Fig: Lyase and 20α- SDH activity in rat

preovulatory follicles. The follicles were

isolated on morning of proestrus at 0hr, 3, 6,

9 after administration of 5IU of hCG [14].

Fig: Effects of hypophysectomy on

follicular lyase and 20α-SDH activity.

Follicles from the intact rats were explanted

on the morning of the day of proestrus at 0h,

6h, 12h, 24h after hypox [14]. Overall

steroidogenesis is stimulated within 4-6h of

LH/hCG and the sectretion of androgens

and estradiol is diminished after LH/ hCG

stimulation follicles in which atresia was

induced experimentally and steroidogenic

changes include decreased production of

follicular estrogen. After hypophysectomy ,

it resulted in atresia of preovulatory follicles

due to withdrawal of tonic levels of

gonadotropin and was accompanied by

marked decrease in lyase activity and

increase in 20α-SDH within 6h. The activity

was measured from homogenates of

Graafian follicle by evaluating conversion

of precursors to products. Follicles of most

species in late stages of atresia exhibit

germinal vesicle nreakdown. Then the

oocytes from atretic follicles are cultured ,

germinal vesicle breakdown increased, 235

of the oocyte fragmented. During atresia the

cumulus cells lose contact with the rat

oocyte (From Ingram : The Ovary 1962).

Enzyme Marker For Studying Follicular

Atresia In Rodents

Histochemical and biochemical alterations

in granulosa cells often precede definite

morphologic change sin atretic granulosa

sells. These include an increase in

lysosomal enzymes such as acid

phosphatase, aminopeptidase [15].

Cathepsin-D- Lysosomal Enzyme As a

Marker Of Follicular Atresia

A model system to study the biochemical

mechanism of follicular atresia in rats [16]

was characterized using histological and

biochemical studies. PMSG and PMSG

antiserum was used to induce the follicular

growth and atresia of preovulatory follicles.

Ovarian histology during these PMSG and

PMSG - treatment periods was recorded

under a light microscope. An analysis of

lysosomal enzyme cathepsin-D activity of

granulosa cells from similarly treated

ovaries showed that there was a reduction in

cathepsin-D activity during the

histologically observable follicular growth

and there was an increase in cathepsin-D

activity during atresia. The group [22]

further used this model system in tracing the

path of atresia. Preovulatory follicular

atresia was studied using pregnant mare

serum gonadotropin (PMSG)-primed rats

(15 IU/rat) which were deprived of

hormonal support either by allowing the

metabolic clearance of the PMSG or by


l


injecting a specific PMSG antiserum

(PMSG a/s). Atresia was monitored by an

increase in lysosomal cathepsin-D activity

and a decrease in the receptor activity of the

granulosa cells isolated from the

preovulatory follicles. It was shown that the

increase in lysosomal activity the decrease

in receptor activity seen at 96 h after PMSG

(or PMSG plus PMSG a/s) could be arrested

both by follicle stimulating hormone (FSH)

and luteinizing hormone (LH). Injection of

cyanoketone or clomiphene citrate together

with FSH/LH prevented this 'rescue'

suggesting a role for estrogens in the

regulation of atresia. Although the

administration of estradiol-17 beta (20

micrograms/rat) together with PMSG a/s

could show a 'rescue effect' in terms of

reduction in cathepsin-D activity, the

gonadotropin receptor activities of these

granulosa cells were not restored. The

injection of dihydrotestosterone (DHT) to

48 h PMSG-primed rats induced atresia as

noted by an increase in cathepsin-D activity.

However, the exogenous administration of

FSH along with DHT prevented this atretic

effect suggesting that DHT is not having a

direct effect on atresia. Determination of

androgen: estrogen content of the granulosa

cells and an analysis of the individual

profile of androgen and estrogen revealed

that the increase in cathepsin-D activity

could be correlated only with the decrease

in granulosa cell estrogen content. This

along with the observation that granulosa

cells showed a loss of estrogen synthesis

well before the increase in cathepsin-D

activity strongly points out that the lack of

estrogen rather than an increase in androgen

is the principle factor responsible for the

atresia of preovulatory follicles in the rat.

Role of Prolactin in Increased Infiltration

of Macrophage In Follicles

Preovulatory surge of LH (luteinizing

hormone) causes preovulatory prolactin

surge which induces the expression of a

monocyte chemo-attractant protein in

corpora lutea after ovulation causing

invasion of macrophages. Prolactin

receptors have been identified on T and B

lymphocyte cells as well as on macrophages

[17]. [18] studied effects of prolactin on

immune challenge with lipopolysaccharides

(LPS) on the macrophage invasion into

follicular and luteal compartment and

occurrence of apoptosis/atresia due to this

macrophage invasion. Porton wistor rats

were injected with ovine prolactin or LPS or

vehicle saline 3days before the

experimentation. Treatment is initiated on

day of estrus so that ovaries can be obtained

on proestrus day1. From each female rat one

ovary is fixed for histological studies and

another for immunohistochemistry studies.

In oestrous rats the total (antral plus

preovulatory) % of atretic follicle increased

from 41% in control to 45%in Prolactin

treated to 56% in LPS treated rat. Apoptotic

cells were observed in granulosa cell layer

and rarely in thecal cell layer and inversely


m


macrophages were observed in the cal cells

and less than 5% in granulosa cells.

Fig: The percentage numbers of healthy (H)

and atretic (A) follicles observed in rats

treated with prolactin (PRL),

lipopolysaccharide (LPS) or vehicle

(control) for 3 days prior to expected oestrus

[18].

The overall mean number of macrophages

per follicle increased after treatment and

more significantly higher in LPS treated

animals. Pre treatment with either of LPS or

prolactin showed reduced progesterone

response to FSH in vitro dispersates in

comparison to controls ovaries. The same

effect was not seen with forkskolin (a

adenylate cyclase stimulator) induced

progesterone secretion, which must be due

to disrupted ovarian cyclicity caused by LPS

and prolactin treatment. The reduced

steroidogenic response to FSH might be the

cause of increased number of atretic

follicles in effect of LPS and prolactin

treatment.

Signal Transduction During Granulosa

Cell Apoptosi

Bcl-2 Family members including several

antiapoptotic (Bcl-2, Bcl-x),proapoptotic

multidomain (Bax, Bak, Mtd/Bok,

Boo/Diva) and BH-3 only ( Bad, Bim/Bod)

proteins are involved in determining

granulosa cell fate. Gonadotropin mediated

inhibition of apoptosis in rat granulosa cells

results in alteration in ratio of bax to bcl-2

and bcl-xlong expression favouring cell

survival. These changes in gene expression

in granulosa cells correspond with shifts in

levels of Bax, Bcl-2, and Bcl-xlong proteins

as well [23]. In support of these gene

expression studies, inactivation of the bax

gene in mice results in the accumulation of

abnormal follicles in the ovary, possessing

atrophic granulosa cells that fail to undergo

apoptosis as the follicles proceeds through

atresia [24]. Bax functions a key modulator

of granulosa cell fate in diverse species of

mammals. The BH-3 only family member,

Bad, is expressed in granulosa cells and

theca interna cells of the rat ovary, and over

expression of Bad in rat granulosa cells

induces apoptosis [25]. Caspases cysteine

aspartate-specific proteins are essential in

formation of apoptosome (apoptotic

machinery) and require release of

cytochrome-c from destabilized

mitochondria an event triggered by the

actions of proapoptotic Bcl-2 family

members. Processing and activation of

caspase-9 and effector caspase such as

caspase -3 have been reported [26] in rat

ovary. Preliminary studies have shown that

inactivation of caspase-9 gene in mice

results in defective granulosa cell apoptosis

and follicular atresia in vivo.Findings have

established the existence of an inverse

correlation between caspase-3 expression

and apoptosis in granulosa cells of the

rodent [26, 27]. A central role for Caspase-3

in executing granulosa cell death has been

shown by studies of caspase -3 deficient


n


mice, in which the mutant females were

shown to possess numerous aberrant atretic

follicle containing granulosa cells that failed

to undergo apoptosis [28].

Implication of Atresia In Ovary

Understanding the basic biology of

apoptosis and its control in ovarian

follicular development has opened avenues

to explore with respect to clinical

applications of these data. Although the vast

majority of these studies are still in the

development stages for improving human

health and fertility.

Fig: Flowchart showing Possible fields of

study in understanding Follicular atresia.

Premature ovarian failure is defined as

amenorrhea with hypo-oestrogenism and

elevated gonadotrophins occurring before

the age of 40 years. In theory, ovarian

failure may occur because of a decreased

pool of primordial follicles, because ovarian

apoptosis is increased or accelerated or

because the follicle maturation is interrupted

before the preovulatory stage. The

mechanisms inducing premature ovarian

failure have been described in a few number

of cases [29].

The follicular atresia is also pertinent to the

pathogenesis of PCOS. Many defects arise

in PCOS patients , primarily chronic

anovulation and hyperandrogenism. The

primary defect is at the level of the ovary,

particularly at the level of folliculogenesis

wherein the ovary contains many small

antral follicles (at least more than) 10

follicles between 2-8 mm in diameter).

These follicles are arrested in development

and do not show overt signs of atresia. The

upregulation of anti-apoptotic and survival

factors are hypothesized to account for the

accumulation of small antral follicles

whereas the lack of developmental

progression is explained by the abnormal

endocrine environment in PCOS patients,

notably FSH suppression. PCOS is thus a

unique pathology deviating significantly

from the normally concomitant occurrence

of developmental arrest and atresia of the

follicle. Current lines of evidence support

the role of a survival/apoptotic balance as

one of likely multiple mechanisms

explaining PCOS symptoms persistence of

the follicles .

CONCLUSION

The major help in studying follicular atresia

is provided by model systems developed for

understanding events occurring during the

process in rodents. With the ongoing

research over event of follicular atresia role

of various factors affecting follicular atresia

like low level of gonadotropins , ratio of

estrogen to progesterone, androgen effects

in rodent models have helped in

understanding temporal events occurring

during atresia. Once a platform is set with

studies and experiments on rat model

system then further studies proceeds with

trials and experimentation on humans can

begin. In one of the studies Bax-

proapoptotic protein is eliminated in mice


o


model suppressing the rate at which the

immature follicle pool is lost from the

ovaries via atresia would result in a

corresponding lengthening the ovarian life

span. In Bax deficient mice showed reduced

rate of primordial and primary follicle

atresia. With further studies and continuos

research in this field researchers are trying

to find out various biomarkers for the event

of atresia so that it can be traced early in

ovarian follicles, like cathepsin-D, its

activity which increases in atretic follicles.

It can also be used for further studies as a

biomarker for event of atresia in ovary.

Various molecular studies upon genes

involved in apoptosis or programmed cell

death of ovarian follicles are studied with

knockout mice and mutant mice. These

studies in rat model system had majorly

helped in improving assisted reproductive

technologies (where number of follicles are

needed to make it successful by inducing

superovulation), to postpone menopause,

and to prevent ovarian damage premature

ovarian failure and infertility in patients.

After knowing the whole chain of

biochemical events occurring during

follicular atresia, it becomes easier for

medical treatment of patients. Examples

cited here paysoff in terms of basic

understanding of ovarian function and the

development of therapies to combat ovarian

failure and infertility.

ACKNOWLEDGEMENTS

Work reported here was supported by funds

from Delhi University under the Doctoral

R&D support scheme.

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FIGURE 1

Education

INTERACTION BETWEEN PROLACTIN AND GONADOTROPINS