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  1. 1. 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: ABSTRACTOvarian follicular dynamics in the laboratory rat has been reviewed. Major physiological andbiochemical events during follicular growth and maturation as well as during atresiaincluding apoptosis have been described based on literature survey. Natural factors likegenetic make up and hormones and artificial factors like hormone specific antibodies areknown to influence this follicular dynamics in both directions. Anti gonadotropic effects ofProlactin in other rat models has been briefly reviewed. The advantages of using the presentmodel have been pointed out. Keywords: Ovary, Atresia, Follicles, Prolactin, GonadotropinsINTRODUCTIONProlactin was discovered in 1928 and is groups. That raised the third mystery i.e.found in all vertebrates including humans. extensive microhetrogeneity in structure andThe name ’prolactin’ is derived from its hence its relevance to physiology. Theestablished role, in female mammals, in mechanism of action of prolactin has beenmammopoiesis. That raised the first mystery studied extensively which gave raise toregarding its role in human male and in non fourth mystery as to why it does not followmammalian vertebrates. More than 300 the classical second messenger model ineffects have been produced by injecting it signaling pathways. As mentioned earlierinto animals of all phylogenic groups. That prolactin has effects in all vertebrate groupsraised the second mystery i.e. absence of and these effects can be grouped underany reliable bioassay for prolactin till today. seven categories. These are effects on waterProlactin has been purified and and salt balance seen dramatically in fish,characterized from a number of vertebrate on growth and morphogenesis seen in aIJBPAS, February, 2013, 2(2)
  2. 2. Murlidhar K et al Research Articleamphibians, on metabolism, on reaches maturity. Atresia is a type ofimmunoregulation, on skin, on behavior and Programmed cell death or Apoptosis.lastly but not the least important on Apoptosis was first discovered by Carl Vogtreproduction and lactation. In spite of in 1842. The morphological characteristic ofexhibiting multiple physiological effects on apoptosis includes cell shrinkage, plasmaa variety of tissues like brain (behavior), membrane blebbing and apoptotic bodygonads and mammary tissues, accessory sex formation. Atretic follicles undergo severalorgans like ventral prostate, cells of immune changes which include retraction ofsystem like phagocytes and lymphocytes etc granulosa cells and initiation of granulosano disease whose origin can be ascribed to cell apoptosis. After most of the granulosamutations in Prolactin or prolactin receptor cells are lost, more severe changes occur asgenes has yet been discovered. This leads to atresia progresses, including segmentationthe fifth mystery i.e. there is no known of the oocytes and cytoplasmicclinical model of prolactin deficiency. vacuolization. Most follicles that leave theHyperprolactinemia due to tumors of resting stage and begin to grow do notpituitary lactotrophs is the only known mature fully but instead undergo atresiapathological condition. Long term hyper- during the developmental process. Moreprolactinemia can lead to amenorrhea in than 99.9% of the ovarian follicles presentwomen, loss of libido in men and infertility at birth never reach ovulation in human andin both. The interaction of prolactin with this figure is 77% for the mouse. Thegonadotropins leading to antagonism can number of follicles developing to theoccur at the pituitary level where it is known preovulatory stage is thus far fewer than theto inhibit the action of GnRH [1] and at the number undergoing atresia. Follicles cangonadal level. Models to study this become atretic at any stage of development.antagonistic interaction are not available yet. Successful follicle development depends onOvarian follicular dynamics including the presence of survival factors that promotegrowth and atresia can be judiciously follicle growth and also protect cells fromstudied to investigate this antagonism apoptosis. These include factors producedbetween gonadotropins and prolactin. within the ovary as well as theMammalian females are born with definite gonadotropins Luteinizing hormone (LH)number of follicles in the ovary. With the and Follicle stimulating hormone (FSH). Inpassing period of time this follicular pool the absence of survival factors, endogenousdecreases and undergoes atresia [2, 3]. apoptosis pathways within the follicleFollicular atresia is degeneration and become activated and lead to follicularresorption of an ovarian follicle before it atresia.. Several molecules that regulate bIJBPAS, February, 2013, 2(2)
  3. 3. Murlidhar K et al Research Articleapoptosis are summarized here. Two of the transformed into primary oocytesexperimental models to understand the characterized by long prolonged meioticprocess of atresia are by performing prophase and surrounded by a squamoushypophysectomy or by immunoneutraliztion layers of pregranulosa cells. Thesemethod. Low level of gonadotropins primordial follicles constitute the restingespecially Follicle stimulating hormone pool of non-growing follicles, which(FSH) and low level of Estradiol is the progressively depleted during themajor inducer of apoptosis in primary and reproductive life span. Primordial folliclessmall antral follicles. It has been shown that proceed by growing into primary follicles inandrogens are atretic in action and estradiol which oocyte is surrounded by cuboidalis anti-atretic. Role of prolactin in granulosa cells. Later stages then by a seriesmacrophage invasion in ovarian atresia is of mitotic divisions in granulosa cell layer,also known. There are various apoptotic unilayered primary follicles are convertedmarkers studied in rat such as Cathepsin-d into multilayered preantral stage designated(lysosomal enzyme) which increases during as secondary follicle. A secondary follicleatresia. becomes invested with thecal cells with timeThe ovaries are covered by a sheet of and comprises of full grown oocytessquamous or cuboidal epithelium, the surrounded by zona pellucida. With thegerminal or serous epithelium which rests appearance of an antral cavity, theon the basement membrane. Beneath the secondary follicle is converted into aserous layer is a layer of dense connective tertiary follicle. The number of granulosatissue termed as tunica albuginea. On the cell is very high at this stage and acquires aedge of the ovary, the hilum is attached to large antrum –fluid filled cavity. This largethe broad ligament by the mesovarium. The antral follicle has now become Graafianovary is organized into two principal follicle and is ready to ovulate. Thoseregions the medulla and a peripheral part follicles which do not ovulate degenerate bycalled the cortex. Embedded in the stroma atresia. Following the ovulation of largeof the cortex are the stages of follicles and dominant follicle, the follicle wall collapsesreflects different stages of growth and to transform into corpus luteum. Eventuallydevelopment [4]. The majority of mammals corpus luteum degenerates and is present asrestrict oogonial development (development a white scar of dense connective tissue, theof primordial germ cells PGC) to prenatal corpus albicans. Gonadotropins control thedevelopment or to shortly after birth [4]. In growth and differentiation of the steroidmost mammals before or soon after birth, hormone secreting cells of the ovary [5].PGC’s (Primordial germ cells) are cIJBPAS, February, 2013, 2(2)
  4. 4. Murlidhar K et al Research ArticleEstrogens are synthesized in granulosa cells. the, mammal returns to proestrus and theLH drives thecal synthesis of androgens and cycle begins anew. The laboratory rat is aandrogens are subsequently aromatized to nonseasonal, spontaneous ovulating,estrogens in adjacent granulosa cells. In polyestrous animal. Ovulation occurs everygranulosa cells FSH stimulates transcription 4-5 days throughout the year.of the gene encoding aromatase which Hypophysectomy is the removal ofaromatizes androgens into estrogens. The hypophysis or pituitary gland. When theword estrus is a Latin adaptation of the procedure is performed before sexualGreek word oistrus. This term was first used maturity, the reproductive tract remainsby Heape to describe “special period of undeveloped and non-functional. There issexual desire of the female”. Heape further also a general lack of growth. If performeddescribed different stages of the cycle as it after sexual maturity, there will be a loss ofapplies to mammals during breeding season. reproductive function along with atrophy ofHe used the term anestrous to describe the gonads and accessory reproductivenon breeding season or period of rest in structures. In rats after hypophysectomy it isfemale mammal when the ovaries and observed that the number of healthy folliclesaccessory reproductive organs are relatively decreases considerably. Female ratsquiescent and attempts of mating by male hypophysectomized at 28 days of age wereare resisted. Heape also used the prefixes killed at various time intervals and healthypro-, di-, met- along with the suffix –estrus follicles were classified as primary folliclesto describe the stages of the cycle between with two or more granulosa cell layer andthe periods of estrus during the sexual vesicular follicle. By 10thday and 38thdayseason. The first part of the cycle he termed after hypophysectomy, the average numberproestrus which last for 12-14 hours. The of primary follicles in ovary was 102 and 20next period is estrus period at which female respectively [6] compared to 213 on the dayis receptive to male the length of this period after operation, similarly vesicular folliclesis 25-27 hours. In the absence of coitus, at the same time intervals were 99 and 3estrus is succeeded by a short phase called compared to 160 on day 1. Dependency ofmetestrus phase which lasts for 6-7 hours. preantral follicles on gonadotropin supportThe following period diestrus is of variable is reflected with experimental evidences andduration in different species of 55-57 hours. observations where long term injection ofDuring this time, ovarian secretions prepare gonadotropin–releasing hormone (GnRH)the reproductive tract for the receipt of agonist greatly increases the percentage ofovum, newly fertilized shortly after mating follicles smaller than 35μm, whileon estrus. If fertilization has not taken place decreasing the percentage9 of follicles dIJBPAS, February, 2013, 2(2)
  5. 5. Murlidhar K et al Research Articlegreater than 35μm from 14% in controls to morning of estrus initiates atresia of large1% in the treated groups [7]. Also follicular follicles that take place on metestrus. FSHgrowth in rats hypophysectomized on day (follicle stimulating hormone) levels goes22 and injected 10days later with [³H] down in evening of estrus, so FSH levelsthymidine shows labeling of granulosa cells were augmented by prolonging the surge ofin small follicles in intact animals. FSH by giving single injection of PMSGSince 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 increaseundergo degeneration while only one in higher number of healthy follicles weredominant follicle is selected to mature and observed at 12h of metestrus by a decreaseovulate, and what are the factors regulating in large atretic follicles in ovaries. Thiscell death of such a large number of follicles simply signifies that the atresia is triggeredsince birth and continues throughout the by decline in FSH concentration during thereproductive life span. There are evidences morning of estrus due to which normallyfrom rat models which can tell us that there large atretic follicles are observed atis a direct endocrine hormonal control of metestrus. This is due to decline inthis process with which large number of concentration of FSH during the morning offollicles undergo atresia or programmed cell estrus and can be prevented by prolongingdeath. Granulosa cell undergoes apoptosis the surge of FSH with administration PMSGduring follicular atresia. The degeneration [11]. Similarly other factors like ratio ofof granulosa cells as atresia advances has estrogen to progesterone, [12], androgenscharacteristics of apoptotic cell death [8]. [13] obviously influenced by the levels ofAtresia can be induced by first steroidogenic enzymes [14] also affectadministering immature rats with PMSG follicular growth and atresia.. Histochemical(pregnant mare’s serum gonadotropin) and and biochemical alterations in granulosathen withdrawing it, PMSG shows both cells often precede definite morphologicbiological and binding activity of both FSH change sin atretic granulosa sells. Thesewhen injected in heterologous species and include an increase in lysosomal enzymesactivity of both FSH and LH in same such as acid phosphatase, amino peptidasespecies [9]. [15]. A model system to study theFSH induces granulosa cell divisions in large biochemical mechanism of follicular atresiaAntral follicles and in intact rats. Decreased in rats [16] was characterized usinglevel of FSH causes decreased granulosa cell histological and biochemical studies. PMSGdivision 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 eIJBPAS, February, 2013, 2(2)
  6. 6. Murlidhar K et al Research Articlepreovulatory follicles. Ovarian histology granulosa cells were not restored. Theduring these PMSG and PMSG - treatment injection of dihydrotestosterone (DHT) toperiods was recorded under a light 48 h PMSG-primed rats induced atresia asmicroscope. An analysis of lysosomal noted by an increase in Cathepsin-D activity.enzyme Cathepsin-D activity of granulosa However, the exogenous administration ofcells from similarly treated ovaries showed FSH along with DHT prevented this atreticthat there was a reduction in Cathepsin-D effect suggesting that DHT is not having aactivity during the histologically observable direct effect on atresia. Determination offollicular growth and there was an increase androgen: estrogen content of the granulosain Cathepsin-D activity during atresia.. Pre cells and an analysis of the individualovulatory follicular atresia was studied profile of androgen and estrogen revealedusing 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 decreasedeprived of hormonal support either by in granulosa cell estrogen content. Thisallowing the metabolic clearance of the along with the observation that granulosaPMSG or by injecting a specific PMSG cells showed a loss of estrogen synthesisantiserum (PMSG a/s). Atresia was well before the increase in Cathepsin-Dmonitored by an increase in lysosomal activity strongly points out that the lack ofCathepsin-D activity and a decrease in the estrogen rather than an increase in androgenreceptor activity of the granulosa cells is the principle factor responsible for theisolated from the preovulatory follicles. It atresia of preovulatory follicles in the rat.was shown that the increase in lysosomal Prolactin receptors have been identified onactivity the decrease in receptor activity T and B lymphocyte cells as well as onseen at 96 h after PMSG (or PMSG plus macrophages [17]. [18] studied effects ofPMSG a/s) could be arrested both by follicle prolactin on immune challenge withstimulating hormone (FSH) and Luteinizing lipopolysaccharides (LPS) on thehormone (LH). Injection of cyanoketone or macrophage invasion into follicular andClomiphene citrate together with FSH/LH luteal compartment and occurrence ofprevented this rescue suggesting a role for apoptosis/atresia due to this macrophageestrogens in the regulation of atresia. invasion. Wistar rats were injected withAlthough the administration of estradiol-17 ovine prolactin or LPS or vehicle salinebeta (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 soterms of reduction in Cathepsin-D activity, that ovaries can be obtained on proestrusthe gonadotropin receptor activities of these day1. From each female rat one ovary is fIJBPAS, February, 2013, 2(2)
  7. 7. Murlidhar K et al Research Articlefixed for histological studies and another for and do not show overt signs of atresia. Theimmunohistochemistry studies. In estrous up regulation of anti-apoptotic and survivalrats the total (antral plus preovulatory) % of factors is hypothesized to account for theatretic follicle increased from 41% in accumulation of small antral folliclescontrol to 45%in Prolactin treated to 56% in whereas the lack of developmentalLPS treated rat. Apoptotic cells were progression is explained by the abnormalobserved in granulosa cell layer and rarely endocrine environment in PCOS patients,in thecal cell layer and inversely notably FSH suppression. PCOS is thus amacrophages were observed in thecal cells unique pathology deviating significantlyand less than 5% in granulosa cells.. The from the normally concomitant occurrenceoverall mean number of macrophages per of developmental arrest and atresia of thefollicle increased after treatment and more follicle. Current lines of evidence supportsignificantly higher in LPS treated animals. the role of a survival/apoptotic balance asPre treatment with either of LPS or prolactin one of likely multiple mechanismsshowed reduced progesterone response to explaining PCOS symptoms persistence ofFSH in vitro, in comparison to controls the follicles.ovaries. The same effect was not seen with In a normal animal undergoing natural agingforkskolin (an adenylate cyclase stimulator) process, follicular growth and maturation isinduced progesterone secretion, which must asynchronous. The superovulated pseudobe due to disrupted ovarian cyclicity caused pregnant immature rat, which is used as aby LPS and prolactin treatment. The model to perform bioassay for Luteinizingreduced steroidogenic response to FSH hormone, the animals receive injections ofmight be the cause of increased number of PMSG to initiate new but synchronizedatretic follicles following LPS and prolactin wave of follicular growth [19]. This ‘Parlowtreatment. rat’ also would be a good model. Indeed aThe follicular atresia is also pertinent to the better model, to investigate not onlypathogenesis of PCOS. Many defects arise underlying mechanisms including signalingin PCOS patients, primarily chronic pathways but also to study interactionanovulation and hyperandrogenism. The among factors which influence this processprimary defect is at the level of the ovary, in vivo. Hence the putative interactionparticularly at the level of folliculogenesis between Prolactin and gonadotropins can bewherein the ovary contains many small investigated using such models. Forantral follicles (at least more than 10 example we have already demonstrated thatfollicles between 2-8 mm in diameter). Prolactin can antagonize FSH action in thisThese follicles are arrested in development model [20] but also that a peptide, derived gIJBPAS, February, 2013, 2(2)
  8. 8. Murlidhar K et al Research Articlefrom the internal amino acid sequence of after sexual maturity, there will be a loss ofbuffalo pituitary prolactin, can increase reproductive function along with atrophy ofascorbic content of ovaries [19]. gonads and accessory reproductiveFig: Concentrations of various hormones structures. In rats after hypophysectomy it isin peripheral plasma obtained at 2hour observed that the number of healthy folliclesintervals throughout each day of 4-day decreases considerably.estrous cycle of the rat. Taken from: [4] Female Rats hypophysectomized at 28daysPhysiology of Reproduction 2006 The first of age were killed at various time intervalspart of the cycle he termed proestrus which and healthy follicles were classified aslast for 12-14 hours. The next period is primary follicles with two or moreestrus 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 afterIn the absence of coitus estrus is succeed by hypophysectomy, the average number ofshort phase called metestrus phase which primary follicles in ovary was 102 and 20lasts for 6-7 hours. The following period respectively [6] compared to 213 on the daydiestrus is of variable duration in different after operation, similarly vesicular folliclesspecies of 55-57 hours. During this time, at the same time intervals were 99 and 3ovarian secretions prepare the reproductive compared to 160 on day 1. Dependency oftract for the receipt of ovum, newly preantral follicles on gonadotropin supportfertilised shortly after mating on estrus. If is reflected with experimental evidences andfertilisation has not taken place the, observations where long term injection ofmammal returns to proestrus and the cycle gonadotropin–releasing hormone(GnRH)begins anew. The laboratory rat is a agonist greatly increases the percentage ofnonseasonal, spontaneous ovulating, follicles smaller than 35μm, whilepolyestrous animal. Ovulation occurs every decreasing the percentage9 of follicles4-5days throughout the year. greater than 35μm from 14% in controls toEffect of Hypophyesctomy on Rat 1% in the treated groups [5]. Also follicularOvarian Follicular Development And growth in rats hypophysectomized on dayAtresia 22 and injected 10days later with [³H]Hypophysectomy is the removal of thymidine shows labelling of granulosa cellshypophysis or pituitary gland. When the in small follicles in intact animals.procedure is performed before sexual Factors Affecting Follicular Atresiamaturity, the reproductive tract remains Since long there has been questions relatedundeveloped and non-functional. There is to how such large number of folliclesalso a general lack of growth. If performed undergo degeneration while only one hIJBPAS, February, 2013, 2(2)
  9. 9. Murlidhar K et al Research Articledominant follicle is selected to mature and ovulatory period at 8h on estrus an increaseovulate, and what are the factors regulating in higher number of healthy follicles werecell death of such a large number of follicles observed at 12h of metestrus by a decreasesince birth and continues throughout the in large atretic follicles in ovaries. Thisreproductive life span. There are evidences simply signifies that the atresia is triggeredfrom rat models which can tell us that there by decline in FSH concentration during theis a direct endocrine hormonal control of morning of estrus due to which normallythis process with which large number of large atretic follicles are observed atfollicles undergo atresia or programmed cell metestrus. This is due to decline indeath. Granulosa cell undergoes apoptosis concentration of FSH during the morning ofduring follicular atresia. The degeneration estrus and can be prevented by prolongingof granulosa cells as atresia advances has the surge of FSH with administration PMSG.characteristics of apoptotic cell death [8]. B. Ratio of Estrogen AndAtresia can be induced by first Progesteroneadministering immature rats with PMSG An evidence for a potential mechanism(pregnant mare’s serum gonadotropin) and underlying follicular atresia The imbalancethen withdrawing it, PMSG shows both of estrogen and progesterone levelsbiological 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 byspecies [9]. performing gel electrophoresis of genome of A. Affect of Gonadotrophins granulosa cells from experimental andFSH induces granulosa cell divisions in control, a DNA fragmentation ladder waslarge Antral follicles and in intact rats. observed specific to apoptosis on day 4 afterDecreased level of FSH causes decreased PMSG injection and on the same day serumgranulosa cell division and appearance of levels of estrogen were 4 fold higher thanpyknotic nuclei [10]. Falling levels of controls and progesterone levels weregonadotropins on the morning of estrus elevated up to 7-8 fold on day 4 and day5initiates atresia of large follicles that take then from controls, showing imbalance inplace on metestrus. FSH (follicle estrogen: progesterone levels. In ratsstimulating hormone) levels goes down in Changes in Estrogen level is one of theevening 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 inby giving single injection of PMSG estrogen level just after the LH surge.(0.025IU/gm body weight) during peri- Estrogen treatment prevents follicular iIJBPAS, February, 2013, 2(2)
  10. 10. Murlidhar K et al Research Articleatresia 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 muchthat PMSG injection given to immature rats much higher in DHT treated rats. Numberdecreases the atresia of large antral follicles inside each bar represents the number ofand also a sharp decline in pyknotic index is animals from which a single ovary wasbeen stated [7]. It is possible that FSH and examined. From [13] Since the ovaries ofPMSG exert their antiatretic action through PMSG primed hypophysectomized ratsthe stimulation of follicular estrogen would be expected to contain considerableproduction. 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 DHTfollicular growth and found that androgens the atresia inducing effects of DHT isposses atretic effects on ovaries. Immature prevented. One of the prominent effectcycling rats were given PMSG to initiate shown by [13] on ovaries from PMSG/DHTfollicular growth and 54hrs later hCG treated hypophysectomized rats given(human chorionic gonadotropin) was given estradiol was both reappearance of healthyto induce ovulation. Then to see the effect follicles and reduction in atresia whenof non-aromatizable androgen 5α- compared to PMSG/DHT treated rats. Sodihydrotestosterone (DHT), to a second set, estradiol rescues follicles from androgenPMSG primed hypophysectomized rats induced degeneration. Intra-ovarianwere given increasing doses of DHT prior to androgens may act to prevent the folliclesacrifice or hCG treatment. Along with this, from completion of one or more steps into see the possible antiatretic affects of maturational process by hindering inestradiol in the system other groups of estrogen action.PMSG primed rats were given increasing D. Changes In Steroidogenicdoses of estradiol alone or in combination Enzymes Activitywith DHT. To serve as controls adult rats Steroidogenic enzyme activity increaseswere sacrificed at the morning of estrus. following preovulatory surge ofResult showed, DHT treatment significantly gonadotropin and there is decreasedreduced the numbers of primary follicles to estrogen production leading to atresia [13].PMSG treated animals. It was found that activity of C17,20-lyaseFig: Effect of DHT on numbers of primary, and 20α –hydroxysteriod dehydrogenasesecondary, tertiary and atretic ovarian (20α-SDH)changes during atresia. jIJBPAS, February, 2013, 2(2)
  11. 11. Murlidhar K et al Research ArticlePreovulatory follicles at different time were cumulus cells lose contact with the ratexplanted after stimulation by hCG /LH and. oocyte (From Ingram : The Ovary 1962).It was reported that there was gradual Enzyme Marker For Studying Folliculardecrease in lyase activity and increase in Atresia In Rodents20alpha-hydroxysteriod dehydrogenase after Histochemical and biochemical alterations3h incubation. in granulosa cells often precede definiteFig: Lyase and 20α- SDH activity in rat morphologic change sin atretic granulosapreovulatory follicles. The follicles were sells. These include an increase inisolated on morning of proestrus at 0hr, 3, 6, lysosomal enzymes such as acid9 after administration of 5IU of hCG [14]. phosphatase, aminopeptidase [15].Fig: Effects of hypophysectomy on Cathepsin-D- Lysosomal Enzyme As afollicular lyase and 20α-SDH activity. Marker Of Follicular AtresiaFollicles from the intact rats were explanted A model system to study the biochemicalon 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 andsteroidogenesis is stimulated within 4-6h of biochemical studies. PMSG and PMSGLH/hCG and the sectretion of androgens antiserum was used to induce the follicularand estradiol is diminished after LH/ hCG growth and atresia of preovulatory follicles.stimulation follicles in which atresia was Ovarian histology during these PMSG andinduced experimentally and steroidogenic PMSG - treatment periods was recordedchanges include decreased production of under a light microscope. An analysis offollicular estrogen. After hypophysectomy , lysosomal enzyme cathepsin-D activity ofit resulted in atresia of preovulatory follicles granulosa cells from similarly treateddue to withdrawal of tonic levels of ovaries showed that there was a reduction ingonadotropin and was accompanied by cathepsin-D activity during themarked decrease in lyase activity and histologically observable follicular growthincrease in 20α-SDH within 6h. The activity and there was an increase in cathepsin-Dwas measured from homogenates of activity during atresia. The group [22]Graafian follicle by evaluating conversion further used this model system in tracing theof precursors to products. Follicles of most path of atresia. Preovulatory follicularspecies in late stages of atresia exhibit atresia was studied using pregnant maregerminal vesicle nreakdown. Then the serum gonadotropin (PMSG)-primed ratsoocytes from atretic follicles are cultured , (15 IU/rat) which were deprived ofgerminal vesicle breakdown increased, 235 hormonal support either by allowing theof the oocyte fragmented. During atresia the metabolic clearance of the PMSG or by kIJBPAS, February, 2013, 2(2)
  12. 12. Murlidhar K et al Research Articleinjecting 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-Dincrease in lysosomal cathepsin-D activity activity strongly points out that the lack ofand a decrease in the receptor activity of the estrogen rather than an increase in androgengranulosa cells isolated from the is the principle factor responsible for thepreovulatory follicles. It was shown that the atresia of preovulatory follicles in the rat.increase in lysosomal activity the decrease Role of Prolactin in Increased Infiltrationin 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 (luteinizingboth by follicle stimulating hormone (FSH) hormone) causes preovulatory prolactinand luteinizing hormone (LH). Injection of surge which induces the expression of acyanoketone or clomiphene citrate together monocyte chemo-attractant protein inwith FSH/LH prevented this rescue corpora lutea after ovulation causingsuggesting a role for estrogens in the invasion of macrophages. Prolactinregulation of atresia. Although the receptors have been identified on T and Badministration of estradiol-17 beta (20 lymphocyte cells as well as on macrophagesmicrograms/rat) together with PMSG a/s [17]. [18] studied effects of prolactin oncould show a rescue effect in terms of immune challenge with lipopolysaccharidesreduction in cathepsin-D activity, the (LPS) on the macrophage invasion intogonadotropin receptor activities of these follicular and luteal compartment andgranulosa cells were not restored. The occurrence of apoptosis/atresia due to thisinjection of dihydrotestosterone (DHT) to macrophage invasion. Porton wistor rats48 h PMSG-primed rats induced atresia as were injected with ovine prolactin or LPS ornoted by an increase in cathepsin-D activity. vehicle saline 3days before theHowever, the exogenous administration of experimentation. Treatment is initiated onFSH along with DHT prevented this atretic day of estrus so that ovaries can be obtainedeffect suggesting that DHT is not having a on proestrus day1. From each female rat onedirect effect on atresia. Determination of ovary is fixed for histological studies andandrogen: estrogen content of the granulosa another for immunohistochemistry studies.cells and an analysis of the individual In oestrous rats the total (antral plusprofile of androgen and estrogen revealed preovulatory) % of atretic follicle increasedthat the increase in cathepsin-D activity from 41% in control to 45%in Prolactincould be correlated only with the decrease treated to 56% in LPS treated rat. Apoptoticin granulosa cell estrogen content. This cells were observed in granulosa cell layeralong with the observation that granulosa and rarely in thecal cell layer and inversely lIJBPAS, February, 2013, 2(2)
  13. 13. Murlidhar K et al Research Articlemacrophages were observed in the cal cells survival. These changes in gene expressionand less than 5% in granulosa cells. in granulosa cells correspond with shifts inFig: The percentage numbers of healthy (H) levels of Bax, Bcl-2, and Bcl-xlong proteinsand atretic (A) follicles observed in rats as well [23]. In support of these genetreated with prolactin (PRL), expression studies, inactivation of the baxlipopolysaccharide (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 undergoThe overall mean number of macrophages apoptosis as the follicles proceeds throughper follicle increased after treatment and atresia [24]. Bax functions a key modulatormore significantly higher in LPS treated of granulosa cell fate in diverse species ofanimals. Pre treatment with either of LPS or mammals. The BH-3 only family member,prolactin showed reduced progesterone Bad, is expressed in granulosa cells andresponse to FSH in vitro dispersates in theca interna cells of the rat ovary, and overcomparison to controls ovaries. The same expression of Bad in rat granulosa cellseffect was not seen with forkskolin (a induces apoptosis [25]. Caspases cysteineadenylate cyclase stimulator) induced aspartate-specific proteins are essential inprogesterone secretion, which must be due formation of apoptosome (apoptoticto disrupted ovarian cyclicity caused by LPS machinery) and require release ofand prolactin treatment. The reduced cytochrome-c from destabilizedsteroidogenic response to FSH might be the mitochondria an event triggered by thecause of increased number of atretic actions of proapoptotic Bcl-2 familyfollicles in effect of LPS and prolactin members. Processing and activation oftreatment. caspase-9 and effector caspase such asSignal Transduction During Granulosa caspase -3 have been reported [26] in ratCell Apoptosi ovary. Preliminary studies have shown thatBcl-2 Family members including several inactivation of caspase-9 gene in miceantiapoptotic (Bcl-2, Bcl-x),proapoptotic results in defective granulosa cell apoptosismultidomain (Bax, Bak, Mtd/Bok, and follicular atresia in vivo.Findings haveBoo/Diva) and BH-3 only ( Bad, Bim/Bod) established the existence of an inverseproteins are involved in determining correlation between caspase-3 expressiongranulosa cell fate. Gonadotropin mediated and apoptosis in granulosa cells of theinhibition of apoptosis in rat granulosa cells rodent [26, 27]. A central role for Caspase-3results in alteration in ratio of bax to bcl-2 in executing granulosa cell death has beenand bcl-xlong expression favouring cell shown by studies of caspase -3 deficient mIJBPAS, February, 2013, 2(2)
  14. 14. Murlidhar K et al Research Articlemice, in which the mutant females were follicles between 2-8 mm in diameter).shown to possess numerous aberrant atretic These follicles are arrested in developmentfollicle containing granulosa cells that failed and do not show overt signs of atresia. Theto undergo apoptosis [28]. upregulation of anti-apoptotic and survivalImplication of Atresia In Ovary factors are hypothesized to account for theUnderstanding the basic biology of accumulation of small antral folliclesapoptosis and its control in ovarian whereas the lack of developmentalfollicular development has opened avenues progression is explained by the abnormalto explore with respect to clinical endocrine environment in PCOS patients,applications of these data. Although the vast notably FSH suppression. PCOS is thus amajority of these studies are still in the unique pathology deviating significantlydevelopment stages for improving human from the normally concomitant occurrencehealth and fertility. of developmental arrest and atresia of theFig: Flowchart showing Possible fields of follicle. Current lines of evidence supportstudy in understanding Follicular atresia. the role of a survival/apoptotic balance asPremature ovarian failure is defined as one of likely multiple mechanismsamenorrhea with hypo-oestrogenism and explaining PCOS symptoms persistence ofelevated gonadotrophins occurring before the follicles .the age of 40 years. In theory, ovarian CONCLUSIONfailure may occur because of a decreased The major help in studying follicular atresiapool of primordial follicles, because ovarian is provided by model systems developed forapoptosis is increased or accelerated or understanding events occurring during thebecause the follicle maturation is interrupted process in rodents. With the ongoingbefore the preovulatory stage. The research over event of follicular atresia rolemechanisms inducing premature ovarian of various factors affecting follicular atresiafailure have been described in a few number like low level of gonadotropins , ratio ofof cases [29]. estrogen to progesterone, androgen effectsThe follicular atresia is also pertinent to the in rodent models have helped inpathogenesis of PCOS. Many defects arise understanding temporal events occurringin PCOS patients , primarily chronic during atresia. Once a platform is set withanovulation and hyperandrogenism. The studies and experiments on rat modelprimary defect is at the level of the ovary, system then further studies proceeds withparticularly at the level of folliculogenesis trials and experimentation on humans canwherein 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 nIJBPAS, February, 2013, 2(2)
  15. 15. Murlidhar K et al Research Articlemodel suppressing the rate at which the Work reported here was supported by fundsimmature follicle pool is lost from the from Delhi University under the Doctoralovaries via atresia would result in a R&D support scheme.corresponding lengthening the ovarian life REFERENCESspan. In Bax deficient mice showed reduced [1] Muralidhar K, Rhoda Maneckjeerate of primordial and primary follicle and Moudgal NR, Inhibition of inatresia. With further studies and continuos vivo pituitary release of lutropin byresearch in this field researchers are trying prolactin in lactating rats,to find out various biomarkers for the event Endocrinology, 100, 1977, 1137-of atresia so that it can be traced early in 1142.ovarian follicles, like cathepsin-D, its [2] Krarup, Pedersen T, and Faber M,activity which increases in atretic follicles. Regulation of oocytes growth inIt can also be used for further studies as a mouse ovary, Nature, 224, 1969-biomarker for event of atresia in ovary. 188.Various molecular studies upon genes [3] Mandal AM and Shelton M, Ainvolved in apoptosis or programmed cell quantitative study of oocytes indeath of ovarian follicles are studied with young and old nulliparousknockout mice and mutant mice. These laboratory rats, J. Endocrinol., 18,studies in rat model system had majorly 1959, 444-450.helped in improving assisted reproductive [4] Knobil and Neill’s, Textbooktechnologies (where number of follicles are Physiology of Reproduction, 3rdneeded to make it successful by inducing Ed., 2006, Elsevier, Boston.superovulation), to postpone menopause, [5] Bokser L, Srkalovic G, Szepeshaziand to prevent ovarian damage premature K, and Schally AV, Recovery ofovarian failure and infertility in patients. pituitary- gonadal function in maleAfter knowing the whole chain of and female rats after prolongedbiochemical events occurring during administration of a potentfollicular atresia, it becomes easier for antagonist of luteinizing hormone-medical treatment of patients. Examples releasing hormone(SB-75),cited here paysoff in terms of basic Neuroendocrinology, 54, 1991,understanding of ovarian function and the 136-145.development of therapies to combat ovarian [6] Lane CE and Greep RO, Thefailure and infertility. follicular apparatus of the ovary hypophysectomized immature ratACKNOWLEDGEMENTS and the effects of hypophyseal oIJBPAS, February, 2013, 2(2)
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  19. 19. Murlidhar K et al Research Article FIGURE 1 sIJBPAS, February, 2013, 2(2)