[Frontiers in Bioscience S3, 680-697, January 1, 2011]

Apoptosis in ovary

Xuan Jin1 , Li-Juan Xiao 2, Xue-Sen Zhang 2, Yi-Xun Liu2

1The First Affiliated Hospital of Nanchang University, Nanchang Jiangxi 330006, 2State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academyof Sciences, Beijing 100101, China


1. Abstract
2. Introduction
3. Apoptosis during follicular development
3.1. Follicular development
3.2. Follicular atresia
4. Hormonal regulation of ovarian apotosis
4.1. Primordial follicles
4.2. Preantral follicles
4.3. Early antral follicles
4.4. Preovulatory follicles
4.5. Periovulatory follicles
5. Apoptosis in corpus luteum
6. Signal pathway of factors in regulating apoptosis in ovary
6.1. Intracellular molecular mechanism of ovarian cell death
7. Conclussion and expetive
8. Acknologement
9. References


Folliculogenesis is a complex process involving dramatic morphological and functional changes in granulosa and theca cells. This process is sequential and dictated specifically by tightly regulated response to endocrine hormones and intra-ovarian regulators. In mammalian ovaries, only a few number of presented follicles in a fetal ovary can reach ovulatory status during follicular development; more than 99% of the follicles in the ovary undergo a degenerative process known as "atresia" induced by apotosis. It is characterized by distinct biochemical and morphological changes such as DNA fragmentation, plasma membrane blebbing and cell volume shrinkage. Apoptosis in ovary is regulated by a number of endocrine, locally produced intracellular mediators in a stage-specific and time-dependent manner. New knowledge of hormones and cell factors which regulate granulosa cell or oocyte apoptosis and their possible signaling pathways underlying intracellular events has made important contributions in advancing our understanding mechanism of follicular atresia.


Apoptosis, also named programmed cell death, is a necessary process in a living organism to maintain proper development, and eliminate cell damage or excess (1, 2). In embryogenesis, for example, the fetus to form fingers and toes is needed by removing the tissue between them. It is also necessary for body to destroy cells that threatening integrity of organism (3). Over a century ago, there was no "term" for "apoptosis" or "programmed cell death". The earliest description of the physiological cell death recognized as distinct from pathological tissue destruction was derived from morphological evaluations (2). Later, a series of criteria were set up to identify the programmed cell death (4).

Apoptosis is an active form of programmed cell death, which is characterized by distinct biochemical and morphological changes, such as DNA fragmentation, plasma membrane blebbing and cell volume shrinkage. The characteristic structural and molecular events of apoptosis distinguish from necrosis, which is a group of cells die at same time (4). Apoptosis always occurs in a single cell surrounded by viable cells. In multicellular organisms physiologically removing cells by apoptosis is a mandatory process in maintenance of homeostasis of individual. Apoptosis occurs in embryonegesis, embryo development and in various adult tissues, including reproductive tracts.

Generally it is thought there are two pathways for regulation of cell apoptosis: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway (5, 6). In death receptor pathway, apoptosis is triggered by the binding of cell death receptors (Fas, TNFR etc.) located in the plasmic membrane with their complementary death activator (FasL, TNF etc.). The binding of death receptor and activator induces receptor accumulation and formation of a death-inducing signal complex (DISC). This complex recruits and activates a number of apoptotic executors, such as procaspase-8 (7-10). Activated caspases-8 also can cleave Bid, increasing its pro-death activity and translating it to mitochondria, where it causes the mitochondrial cytochrome c release. Both of these two pathways occur in gonads and uterus.

Apoptosis is an essential physiological process, by which tissues function normally. A balance of cell proliferation, differentiation and apoptosis plays an important role in a healthy organ, and any imbalance of the processes can lead to organ dysfunction and developmental abnormalities. Apoptosis in ovary is regulated by a number of endocrine, locally produced mediators. However, the signaling pathways underlying the intracellular events induced by these regulators are not clear. This chapter was focused on cell apoptosis in ovary by reviewing available data published in the recent literature, including our recent publications in the fields. The review would also address new knowledge of hormones and cell factors as well as their possible signaling pathways underlying the intracellular events, which regulate somatic and germ cell apoptosis in the gonad.


3.1 Follicular development

During embryogenesis, primordial germ cells migrate from yolk sac through dorsal mesentery of hindgut to genital ridge, and the somatic cells derive from mesenchyme of genital ridge. Both of germ cells and somatic cells proliferate till each germ cell is enclosed by one layer somatic cells, named follicular epithelial cells,to form primordial follicles. After mitosis occurred in somatic cells, the germ cells undergo first meiotic division, called primary oocytes. The primary oocytes become arrested in the diplotene stage of meiosis, until the primordial follicles start to grow and finally reach ovulatory stage.

Folliculogenesis is a complex process involving dramatic morphological and functional changes in granulosa and theca cells. This process is sequential and dictated by specifically, tightly regulated response to endocrine hormones and intraovarian regulators. They control follicular development by determining which of the growing follicles continue to develop and differentiate, and which become atretic. Mammalian ovaries contain thousands of thousand primordial follicles that are the only source of gametes during the entire reproductive life. However, there is study provides evidence that challenges the validity of the belief, the results showed bone marrow and peripheral blood as potential sources of female germ cells, that could sustain oocyte production in adulthood (11, 12). Primordial follicle consists of an oocyte surrounded by a single layer of flattened pre-granulosa cells. The primordial follicles may survive more than 50 years in woman ovary. Once a group of primordial follicles begin to grow, they will develop and differentiate either into dominant follicle (s) and ovulate, or undergo atresia at various stages of development. During onset of primordial follicle growth, flattened pre-granulosa cells become cuboidal and begin to proliferate. The enclosed oocyte begins to grow at the same time (13, 14). It is interesting to note why and how some primordial follicles are capable of starting to grow while their neighbor sisters remain quiescent. The signal (s) for selection of primordial follicle growth is not clearly known.

Growth of granulosa cells in the follicle is a key process in initiation and development of primordial follicle. The early growth stage of primary follicles (with mono-layer cuboidal granulosa cells) and secondary follicles (with stratified granulosa cells but without antrum) is characterized by a dramatic increase in proliferation of granulosa cells, identified in the rapid increase in number and size. Subsequently, granulosa cells separate from each other resulting in formation of follicular antrum, which is called antral follicle. Meanwhile, meiosis restarts in secondary oocyte, germinal vesicle (GV) disappearance, called GV breakdown (GVBD), and the first polar body divides. At last, the selected follicle burst, and the oocyte is ovulated, the rest of follicular cells further differentiate into a new endocrine organ, called corpus luteum (15).

3.2 Follicular atresia

In newly formed embryonic ovary, the germ cells leave mitotic cycle, and begin with meiotic divisin, the meiotic division of the oocytes become arrested in the first prophase (16). During both mitosis and meiosis, large numbers of germ cells are culled from the ovary for as yet unknown reasons, resulting in less than one-third of the total number of potential germ cells being endowed in the ovary within primordial follicles shortly after birth (17, 18). In human fetal ovaries, the maximum number of germ cells observed in 5 month of pregnancy is about 6.8�106. At birth, the number of germ cells in the primordial follicles has decreased markedly to less than 20% of the maximum number, due to apoptosis of germ cells occurring before formation of ovarian follicles (19). Detailed analyses of germ cell degeneration in rodent and human fetal ovary suggest that there are several discrete waves of germ cell loss occurring, such as attrition of dividing oogonia, degeneration of pachytene stage oocytes, and loss of diplotene stage oocytes (17-19).

Although male mammals generally retain germline stem cells for spermatogenesis in testis throughout their adult life, oocyte production in most of female mammals is believed to cease before birth (16, 18, 20-22). The central concept of reproductive biology "basic biological doctrine that during the life of the individual there neither is nor can be any increase in the number of primary oocytes beyond those originally laid down when the ovary was formed" (23). But the belief of the concept has been challenged by Tilly et al, based on the data of rates of oocyte degeneration and clearance, as well as the chemical 9,10-dimethylbenz (α) anthracene (DMBA) induced synchronization of atresia in mouse ovary. They have demonstrated that in addition to existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary, the transgenic female mouse bone marrow may be also a potential source of germ cells to sustain oocyte production in adulthood (11, 12). However there is no evidence to determine whether those oocytes can fertilize normally and develop into viable offspring. Most ovarian follicles (>99.9%) present at birth never reach ovulatory status, but undergo atresia at various time point along this extended developmental pathway.

Apoptotic process follows a particular pattern during different phases: in fetal ovaries, most of apoptotic activity was detected in germ cells; while in adult quiescent cortical follicles apoptosis was occurred originating from both oocyte and granulosa cells. It has been demonstrated that it is the oocyte which initiates the apoptotic process and induces the follicular atresia. The process always begins from the oocyte and then extends to the surrounding follicular cells leading to the growing follicle atresia. (24). Thus, it seems that the apoptotic signals can communicate from a single cell to all over the other cell types inside the follicle. Finally, the whole follicular structure has become atretic, while the surrounding stromal cells remain viable.

It is interesting that although in the growing follicles, follicular atresia is mainly induced by granulosa cells, there species-specific difference in the apoptotic process was observed. The initiation area of granulosa cell apoptosis is different among species, it is therefore suggested that different local mechanisms to regulate apoptotic process may be present (25-27). For example, at the earliest stage of follicular atresia, the apoptotic granulosa cells are randomly scattered in ovaries of rodents; while in bovine ovary, apoptotic granulosa cells are located on outer surface of follicular wall; and in porcine ovary, apoptotic granulosa cells are located on inner surface of granulosa layer. Moreover, during early to middle stages of follicular atresia, there is no apoptotic cells observed in the theca external layer, although detachment and degeneration of granulosa layer, fragmentation of basement membrane, and the apoptotic endocrine cells in theca internal layer were observed.


Physiological death of a cell is usually under control of multiple extracellular factors, and the balance of survival and apoptotic signals determines a cell fate. Follicular cell apoptosis is regulated by multiple hormones, including pituitary hormones as well as local growth factors, cytokines, and steroids (28, 29). Diverse hormones and growth factors can act as survival factors to inhibit apoptosis, or as apoptotic factors to induce cell demise, such as Fas ligand, tumor necrosis factor (TNF)-α, interleukin-6, and gonadotropin-releasing hormone (GnRH) (30-35), through endocrine, paracrine and autocrine mechanisms. The factors that regulate apoptosis in diverse tissues appear to be tissue-specific. In ovary, the action of these factors is dependent on the stage of follicular development as shown in figure 1.

4.1 Primordial follicles

In primordial follicles, oocyte apoptosis is likely responsible for follicular degeneration. FSH is important for follicular development, however, FSH is unlikely to exert a direct action on primordial follicles because FSH receptors have not yet developed at this stage (36, 37). In fact, follicles do not express functional FSH receptors until the secondary stage of follicular development (36-38). Stem cell factor (SCF) is important for survival of primordial follicles in fetal as well as postnatal ovaries by preventing oocytes from apoptosis (39, 40). Another oocyte-derived factor which is important for survival of small follicles is growth and differentiation factor 9 (GDF-9). In mice lacking GDF-9 follicles do not develop beyond the primary or early secondary stage (41). Just recently we have demonstrated that androgen receptor (AR) and Foxo3a was expressed in 2 day-old mouse oocyte, treatment of the cultured ovaries with testosterone induced the ovarian Foxo3a phosphorylation and primordial follicular growth and development via PI3-K/Akt signal pathway. In contrast, the oocyte GDF9 was down-regulated by the androgen at late stage of culture via the same signal pathway. Therefore we suggested that intra-ovarian hyperandrogenism might be the main culprit for excessively growing follicle by inducing the Foxo3a exclusion from oocyte nucleoli and for follicular arrest by directly down-regulating GDF9 expression.

4.2 Preantral follicles

It has been reported that FSH is important for development of preantral follicles in vivo, and FSH can also enhance expression of steroidogenic enzymes (43, 44). Decrease of circulating gonadotrophins through hypophysectomy (45) or blockade of luteinizing hormone/follicle stimulating hormone (LH/FSH) surge (46) lead to massive atresia of pre-ovulatory follicles on the day of pro-oestrus. The hormonal regulation of apoptosis in granulosa cells during follicular atresia appears very complex, and probably involves a classic cell crosstalk of oocyte - granulosa cells - theca-interstitial cells, as well as interaction among the cells at different stage of follicular development (47-49). For example, when the preantral follicles isolated from 12-14 day old rats were cultured in serum-free medium, a spontaneous onset of apoptosis was observed in the granulosa cells, similar to that in preovulatory follicles. FSH, as well as its downstream mediator cAMP could not inhibit apoptosis of the granulosa cells in the cultured preantral follicles (50). This finding suggests that the action of gonadotrophins on granulosa cell apoptosis may be mediated via other gonadotrophin - sensitive cells, such as cells in follicles at a later stage of development, rather than directly on the preantral follicles. Besides gonadotrophins, locally produced survival factors may also play important roles in regulation of follicular atresia. Sex steroids have been demonstrated to be essential important ovarian factors for follicle development (51). In human, ovine or porcine ovaries estradiol production by atretic follicles was lower, while androgen production was higher (52-54). However, in rat and hamster ovaries, the production of both estrogen and androgen decreased in atretic follicles (46, 55-57). In general, changes in steroidogenesis can be observed prior to morphological signs of atresia (46, 55, 58). Liu et al have demonstrated that endogenously-produced estrogen in the developing follicles synergistically with FSH can enhance the aromatase activity and differentiation of LH receptors,and is essential for follicular differentiation and dominate follicle formation. Decrease in endogenously estrogen in the follicle may lead to its atresia (59, 60).

In-situ analysis of DNA fragmentation on histological sections of ovaries has demonstrated that apoptosis induced by estrogen withdrawal in hypophysectomized rats is confined to the granulosa cells in early antral and pre-antral follicles, but no increase in DNA breakdown in primordial and primary follicles was demonstrated (51). ERαβ double knock-out mice are infertile because of follicular arrest (61, 62). Nevertheless, early follicular growth and development can also occur in these mice, even though mature Graafian follicles do not form.

In contrast to estrogen, which inhibits granulosa cell apoptosis, androgen promotes the cell apoptosis (28). In vivo, treatment with androgen causes a dose- and time- dependent decrease in ovarian weight (63, 64) and an increase in morphological signs of atresia in estrogen - treated hypophysectomized rats (64).

Other locally produced growth factors, including keratinocyte growth factor (KGF), fibroblast growth factor (bFGF) are also important for survival of preantral follicles. KGF, a member of FGF family which produced by thecal cells and receptors of that are present in granulosa cells, suppresses apoptosis in cultured rat preantral follicles (65). Similar effect of FGF on apoptosis has also observed in cultured preantral follicles (65).

4.3 Early antral follicles

In human and rodent, the early antral stage of follicle development is the most critical stage. At this stage a functional FSH receptor is expressed in the granulosa cells, and follicle survival becomes mainly dependent on FSH stimulation (13). FSH is able to suppress apoptosis by up to 60%, but its action was partially reversed by insulin growth factor (IGF) binding protein IGFBP-3, suggesting that some of the physiological effects of FSH may be mediated by IGF (66). Local production of IGF-I plays an important intra-ovarian role in augmentation of gonadotrophin stimulation of follicle differentiation. (67, 68). IGF and its binding protein (IGFBP) are important for oocyte maturity, and granulosa cells differentiation during follicle development. Gene expression for IGF-binding protein in granulosa cells definitely differed between normal women and women with polycystic ovary syndrome (69-72). IGFBP-4 and -5 are produced by rat granulosa cells (73, 74). FSH treatment increases IGF-I production (75, 76), but decreases IGFBP secretion in ovaries (77). High concentrations of IGFBP have been detected in atretic human follicles of both normal and polycystic ovarian syndrome patients (78, 79). In situ mRNA analysis has further demonstrated presence of IGFBP in atretic,but not in healthy follicles (74). IGF-I, as well as FSH prevent spontaneous onset of apoptosis in cultured follicles (47), however, they can not prevent apoptosis in isolated granulosa cells, in spite of presence of their receptors on the granulosa cells (80), indicating that theca cells may be important for mediating the suppressive effect of IGF-I and gonadotrophins on apoptosis.

The mechanisms controlling the follicular growth involve the interaction between local growth factors which are expressed throughout development and extra-follicular factors. A large number of follicular growth factors, such as member of bone morphogenetic family, BMP-15, epidermal growth factor (EGF) and growth differentiation factor-9 (GDF-9) control the initiation of follicular growth and early preantral development. During antral follicle development, the oocyte secretes factors that stimulate granulosa cell proliferation and differentiation, modulate apoptosis and suppress progesterone production, thereby preventing premature luteinisation (81, 82). In the development competence of in vitro-matured (IVM) cumulus oocyte complexes (COCs), epidermal growth factor (EGF) has been proved functionally mimicked the action of FSH and could completely replace FSH for nuclear maturation, specific inhibition of EGF receptor (EGFR) inhibited both EGF- and FSH-induced meiotic resumption (83-87). Besides of EGF, growth hormone (GH), IGFs and IGFBPs also play an important role in preantral follicle growth through their binding with GH receptor, which are located both in the oocyte and follicular somatic tissues. In vitro studies and knockout experiments shows GH stimulates the development of small antral follicles to gonadotrophin-dependent stages, as well as maturation of oocytes. In antral follicles, IGFs stimulate granulose cell proliferation and steroidogenesis in most mammals (88, 89).

4.4 Preovulatory follicles

At the preovulatory stage, both granulosa and theca cells in the follicle express LH receptors and are able to respond to impending LH surge. It has been reported that FSH and LH both suppressed the degree of apoptosis in isolated preovulatory rat follicles (47). At this stage, interaction of granulosa cells and theca cells produces the highest estrogen in the follicle, as shown in Figure 2, that may be important for preventing the selected dominate follicle atresia and going to ovulation (90).

Endogenous IGF-1 also partially mediates suppression of apoptosis by gonadotrophins. LH receptor stimulation results in an increase in IGF-1 mRNA content in cultured preovulatory follicles, while IGFBP-3 results in a dose-dependent decrease in the apoptosis suppressive effect by LH receptor stimulation (47). Besides of IGF-1, cytokine IL-1β mediates part of apoptosis suppressive effect of gonadotrophins in rats, while IL-1β receptor antagonist partially decreases effect of gonadotrophins (91). Insulin is another survival factor for cultured rat preovulatory follicles. Although insulin has no effect on isolated preovulatory rat granulosa cells, it can decrease sensitivity of cultured follicles to apoptosis, suggesting involvement of other ovarian cells (80). EGF, bFGF, and GH suppress apoptosis effectively in follicles at preovulatory stage.

4.5 Periovulatory follicles

Follicles that survive to periovulatory stage are dependent on endogenous LH surge. After LH surge, follicles are less susceptible to atresia than those are at earlier stages (92). Inhibition of LH surge by hypophysectomy or pentobarbital treatment causes follicles to degenerate (93, 46). The suppression of LH to apoptosis is partly mediated by endogenous production of pituitary adenylate cyclase-activating polypeptide (PACAP) (94).

Shortly after LH surge, expression of nuclear progesterone receptor is induced in both rat and human granulosa cells, which coincides with apoptosis suppressive effects of progesterone. In rat, expression of progesterone receptors is transient, while in human, it is prolonged (95, 96). Progesterone functions as a regulator of apoptosis via its nuclear receptor at periovulatory follicles (92). Mice lacking both isoforms of the progesterone receptor (A and B) are anovulatory, indicating that progesterone has a direct effect in ovary (97, 98). Progesterone regulates expression of the genes, such as PACAP, and its receptor PAC1 (99, 100). In immature and preovulatory stage (before LH surge), progesterone attributes an apoptosis-inhibiting effect via a GABA receptor-like receptor, but that seems not related with apoptosis (92).


The corpus luteum (CL) is developed by extensive cellular reorganization and neovascularization of remnants of evacuated follicle following ovulation. CL is a transient endocrine organ that secretes progesterone to support early pregnancy. If implantation is unsuccessful, luteolysis is initiated. In both rodent and primate, development of CL is a rapid process with very high cellular turnover (101-103). A CL is usually developed within hours in rat and mouse, and within days in monkey and human. A mature CL receives the greatest blood supply per unit tissue in the whole body (103). However, if the implantation is unsuccessful, the functional phase of the CL is terminated and luteolysis is initiated. Associated with these repetitive cycles of luteal development and regression is an extensive connective tissue remodeling and extracellular matrix degradation (104, 105).

It has been reported that CL function is regulated by various bioactive substances, such as gonadotropins, steroids, and growth factors. CL regression can be initiated by the release of PGF2α from uterus. PGF2α inhibits the gonadotropin-stimulated CL progesterone production, after the initial decrease in the steroidogenesis, more chronic effects of PGF2α take place, including loss of gonadotropin receptors and disruption of the cytoskeleton, and preventing progesterone secretion, eventually followed by morphological changes in the steroidogenic cells, and loss in both size and weight of CL (106). Another important factor initiating CL regression is prolactin secretion in the estrous cycles (107). Using a chemical drug to block prolactin secretion, corpora lutea are increased by weight (108). Furthermore, chemical blockade of the proestrous prolactin surge diminishes apoptosis in the regressing corpora lutea (109).

Luteal regression is a complex process that involves two phases. The first phase, named functional regression which means the functional ability of corpus luteum to sustain pregnancy lost at this stage, is defined as termination of secretion of appreciable quantities of progesterone (110) and occurs during the 4- to 5-day estrous cycle. The second phase, called structural luteolysis, is defined as the complete morphological regression of corpus luteum mainly by cell apoptosis. A great decrease in weight and size occurs at this stage. The latter process is executed long after the initial decline in progesterone secretion and corpus lutea may remain in the ovary throughout several estrous cycles before their complete dissolution (111, 112). The main mechanism involved in reduction in size and weight of corpus luteum is removal of luteal cells by apoptosis and subsequent phagocytosis (113, 114).

Our serious experiments in monkey, rat and mouse have demonstrated involvement of matrix remodeling proteases in the processes of tissue remodeling during CL formation and luteolysis. Matrix remodeling proteases includes plasminogen activator (PA) (115, 116) and matrix metalloproteinase (117-123) system. Our experiment results suggest that coordinated expression of tissue type plasminogen activator (tPA) and its inhibitor type-1 (PAI-1) in corpus luteum at late stage of CL development in primate, rat and mouse induces CL regression, leading to luteal cell apoptosis (115, 116, 124). In addition to the PA system, recent evidence suggests that the luteal tissue remodeling is also regulated by MMP/TIMP system (117). The TIMPs have been reported to stimulate cell growth, impact angiogenesis, and induce cell apoptosis (125-127). Recently we demonstrated that coordinated expression of MMP-2, -14 and TIMP-1, -3 may have a potential role in the CL formation and the function, while the interaction of MMP-2, -9, -14 and TIMP-1, -2, -3 might also play a role in CL regression at the late stage of CL development in the primate (127).

At early and late stages of CL development an extensive tissue remodel and cell apoptosis occur. Using VEGF and its receptors as well as StAR as the marker molecules of CL function, we have designed experiments to look at the possible effect of cytokines and RU486 on CL regression and apoptosis (128-133). As compared to the control, a single administration of RU486 significantly increased VEGF expression in the CL during early pregnancy in monkey. However, twice administration of RU486 significantly declined the single-RU486-induced VEGF expression (p=0.005), the mechanism, however is not known.

IFN-g can inhibit progesterone production of luteal cells (128, 129, 132, 133) and induce luteal cell apoptosis in varies species including human (134, 135). After treatment of monkey with IFN-g , as compared to the control, the sharp endothelial cells in the CL altered into round. However, unlike TNF-a treatment, the VEGF levels did not increase after administration of IFN-g . These results suggest that endothelial cells might be going to degenerate, IFN-g may induce angiolysis in the primate CL even at the early state of CL development. Thus, IFN-g could promote luteal degression by inducing the cell apoptosis in the primate.

Evidence in recent years has shown that caspase-3 exerts an important role in luteal cell apoptosis of bovine and cattle (136, 137). It has also reported that activation of protein kinase C (PKC) signal pathway and cAMP accumulation could protect bovine luteal cells from apoptosis by suppressing caspase-3 mRNA expression (138).


Selection of apoptosis or survival of granulosa cells and oocyte is a critical process in determining the fate of follicular development. In mammals, at least 60 different proteins and signaling molecules have been identified as constituents of intracellular framework that governs apoptosis. Although a number of endocrine and paracrine factors have been shown as survival or apoptosis-inducing factors in these cells in vivo or in vitro (139, 140), their molecular mechanisms and the underlying intracellular events are not completed illuminated. Several intracellular signaling pathways have been linked directly to promoting granulosa cell or oocyte survival, including pathways such as gonadotrophin- and vasoactive intestinal peptide (VIP)-induced cAMP formation (48, 141), mitogen-activated protein kinase (MAPK) (142), and phosphoinositol-3-kinase-Akt (143, 144). PI3K/Akt pathways play an important role in mediating anti-apoptotic action of SCF in oocytes of primordial follicles. Jin et al (39) demonstrated that the anti-apoptotic effect of SCF on oocytes was significantly inhibited by the PI3K inhibitor (Figure 3). Moreover, PI3K inhibitor could also revere the effect of SCF on the expression of Bcl-xL and Bax (Figure 4). MAPKs activation is a key event in many cellular processes, including proliferation, differentiation, and apoptosis (145). There are three main classes of MAPK: Erks, c-Jun amino-terminal kinases (JNKs), and P38 proteins (146-148). Erks are important mediator of many factors, such as FSH (149), SCF (150). Inhibition of Erks activity with PD98059 distinctly reduces FSH-induced DNA synthesis in immortalized granulosa cells and over-expressing a recombinant novel growth factor type 1 receptor for FSH. Study on granulosa cells isolated from equine chorionic gonadotropin-primed immature rats revealed that activities of Erks,MEK kinase and Raf-1 were reduced with a concomitant decrease in phosphorylation level of the proapoptotic factor, Bad, prior to onset of granulosa cells apoptosis (142). Another important signal molecule stimulated by FSH is cyclic AMP (cAMP), signaling via cAMP enhances resistance of hen granulosa cells to apoptosis. Blocking cellular phosphodiesterase activity in forskolin-stimulated primary granulosa cells by isobutylmethylxanthine, which maintains high level of intracellular cAMP, led to further enhancement of cell death (141, 48).

It has been reported that the regulated phosphorylation of Tyr residues is a major control mechanism for the processes as diverse as cell survival, proliferation, differentiation, and metabolism. The opposing activities of protein tyrosine kinases (PTKs) and PTPs accurately regulate protein phosphor-Tyr (pTyr) levels (151, 152). Several reports indicated that PTKs play important roles in regulating intracellular events of granulosa cells after stimulation with various factors (47, 66, 80, 153). For instance, EGF, IGF/insulin and bFGF prevent spontaneous onset of apoptosis in cultured granulosa cells by activating their respective tyrosine kinase receptors (47, 80). IL-1β acts through cytoplasmic PTKs, called Janus kinases (JAKs), that is an effective survival factor for preovulatory follicles in vitro (66). Other signal molecules, such as Ca2+, protein kinase C, heat-shock proteins may also involve in transduction of factors regulating the apoptosis in ovary.

Using DNA 3'-terminal labeling, immunohistochemistry, in situ hybridization we comparatively examined the correlation expression of inhibin, LH receptor in granulosa cells, and the tPA activity in oocytes at the same section of the follicle. High level of tPA mRNA in oocytes was detected at early stages of follicular development, but tPA protein activity in the oocyte was not detected until the onset of meiosis maturation at late stage of follicular development triggered by the LH surge. The high level expression of inhibin in GC observed in the early stage follicles may play an essential role in preventing the tPA mRNA translation into its protein in the oocytes. Once inhibin expression decreases in the GC of developing follicles at early stage, the increasing tPA protein activity in the oocyte may induce certain morphological changes in the oocyte similar to GVBD, leading to the oocyte apoptosis and the follicle atresia in the under-developed follicles. Based on this finding, we have proposed a mechanism of follicular atresia originating from oocyte apoptosis (Figure 5) (154, 155)

6.1 Intracellular molecular mechanism of ovarian cell death

Although there are many different hormonal signals to regulate apoptosis in ovary, the intracellular cascade of events appears to share common features (Figure 6.). Bcl-2 system is important in regulation of ovarian cell apoptosis in vivo. Bcl-2 is a proto-oncogene, which encodes a membrane-anchored intracellular protein to prevent apoptosis induced by various stimuli (156, 157). Expression of Bcl-2 has been detected in ovary of many species (158, 159). In the transgenic mice over-expression of Bcl-2 was detected in the ovary, while the follicular cell apoptosis was suppressed, and followed by enhancing folliculogenesis and an increased incidence of benign ovarian teratoma development, indicating that Bcl-2 associated regulatory system is operating in the ovary (160). In contrast, ablation of functional Bcl-2 through targeted disruption of the gene (gene 'knock-out') leads to significantly fewer oocytes and primordial follicles in the postnatal ovary (161). Another member of Bcl-2 gene family is Bax, a death-susceptibility gene. The protein of Bax was originally identified via its ability to non-covalently interact with Bcl-2 in cells (162). This interaction is thought to blunt Bcl-2 bioactivity and thus serve as proapoptotic member. With oocyte in-vitro maturation experiment, Bcl-2 mRNA expression is significantly higher in cumulus-oocyte-complexes (COC) cells associated with mature oocytes than those associated with immature oocytes, and levels of Bax expression appear to be positively correlated with apoptosis in each of these cell lineages (163-165). Knudson and his colleagues (166) noted "a marked accumulation of unusual atretic follicles" containing "numerous atrophic granulosa cells that presumably failed to undergo apoptosis". Primordial oocytes within the ovaries of Bax null mice were completely resistant to apoptosis induced by exposure to a widely used chemotherapeutic drug in vivo (167). Similarly, granulosa cells within degenerating follicles of Bax-deficient mice also appear to be resistant to induction of apoptosis (166). A significant defect in primordial and primary follicle atresia rates was detected in Bax-deficient female mice, leading to a marked reduction in the incidence of postnatal oocyte death. Moreover, in aged Bax mutant females, defect in oocyte death leads to a dramatic prolongation of ovarian life span (168). These results support a fundamental role for Bax in mediating apoptosis in both oocyte and granulosa cells.

In addition to Bcl-2 and Bax, several other members of Bcl-2 gene family have been found expressed in ovary and play important role on oocyte survival (169, 170, 164), such as Bad, which acts as an important pro-apoptotic ligand by bridging upstream signaling proteins, 14-3-3 and P11, to the channel-forming anti-apoptotic Bcl-2 family proteins (171). In the ovary, Bad plays an important role in mediating communication from different upstream signal transduction pathways to the Bcl-2 regulated apoptotic decision step. Gonadotropins and other upstream survival factors, such as IGF-1 and insulin, activate Akt/PKB kinase to phosphorylate Bad to allow binding of 14-3-3 proteins, leading to dampening of Bad-induced cell killing (171-174). Bad phosphorylation has been suggested to be an important mechanism by which upstream survival factors suppress apoptosis.


Apoptosis in ovary is a complex, but a regulated process, it plays important roles in reproduction under various physiological conditions. Dysregulation of cell apoptosis in the reproductive tract causes infertility.and reproductive diseases.

Apoptosis often begins before birth, and continuously throughout reproductive life. Balance of cell proliferation and apoptosis plays an important role in a healthy organ, any imbalance of these two processes can lead to organ dysfunction and developmental abnormalities. To better understand mechanism of cell apoptosis can help to find ways to prevent its inappropriate occurrence and to improve reproductive health and give more helpful insight on treatment of reproductive diseases.


This work and the related publications were supported by the Major Research Plan (2006CB0F1002), the"973" project (2006CB504001, 2007CB947502), the CAS Innovation Project (KSCA2-YW-R-55,072AC41101) and the National Nature Science Foundation of China (No: 30618005, 30230190, 30600311,31071018).as well as the WHO/Rockefeller Foundation.

The author would greatly thank Drs. Fei Gao, Peng Wei, Zhao-Yuan Hu, C-X Guo, Xin-Chon Zhou, Xiao-Min Mu, Kui Liu, Qing Feng, Xin-Lei Chen, Hong-Juan Gao, Xiao-Ben Han, Yin-Chuan Li, Ru-Jin Zou, Fu-Qing Yu, Je Ma, Qin-Qin Song, Fu-Hua Xu, Guo-Qing Fu for their contributions to the review data in the Lab.


1. G. Majno and I. Joris: Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol, 146, 3-15 (1995)
PMid:7856735    PMCid:1870771

2. R.A. Lockshin and C.M. Williams: Programmed cell death. I. Cytology of degeneration in the intersegmental muscles of the Pernyi silkmoth. J Insect Physiol, 11, 123-133 (1965)

3. J.W. Saunders: Death in embryonic systems. Science, 154, 604-612 (1966)

4. N.K. Kuan and E. J. Passaro: Apoptosis: programmed cell death. Arch Surg, 133, 773-775 (1998)

5. J.M. Adams and S. Cory: The bcl-2 protein family: arbiters of cell survival. Science, 281, 1322-1326 (1998)

6. J.C. Reed: Mechanisms of apoptosis. Am J Pathol, 157,1415-1430 (2000)
PMid:11073801    PMCid:1885741

7. A. Agic, S. Djalali, K. Diedrich and D. Hornung: Apoptosis in endometriosis. Gynecol Obstet Invest, 68 (4),217-223 (2009)

8. A.P. Sinha Hikim, Y. Lue, M. Diaz-Romero, P.H. Yen, C. Wang and R.S. Swerdloff: Deciphering the pathways of germ cell apoptosis in the testis. J Steroid Biochem Mol Biol, 85,175-182 (2003)

9. M.E. Guicciardi and G.J. Gores: Life and death by death receptors. FASEB J, 23 (6), 1625-1637 (2009)
PMid:19141537    PMCid:2698650

10. X. Luo, I. Budihardjo, H. Zou, C. Slaughter and X. Wang: BID, a BCL-2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell, 94, 481-490 (1998)

11. J. Johnson, J. Canning, T. Kaneko, J.K. Pru and J.L. Tilly: Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature, 428,145-150 (2004)

12. J. Johnson, J. Bagley, M. Skaznik-Wikiel, et al: Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell, 122, 303-315 (2005)

13. A.N. Hirshfield: Development of follicles in the mammalian ovary. Int Rev Cytol, 124, 43-101 (1991)

14. R. Braw-Tal: The initiation of follicle growth: the oocyte or the somatic cells? Mol Cell Endocrinol, 187 (1-2), 11-18 (2002).

15. N. Manabe, Y. Goto, F. Matsuda-Minehata, N. Inoue, A. Maeda, K. Sakamaki and T. Miyano: Regulation mechanism of selective atresia in porcine follicles: regulation of granulose cell apoptosis during atresia. Journal of Reproduction and Development, 50, 493-514 (2004)

16. H. Peters: Migration of gonocytes into the mammalian gonad and their differentiation. Proceedings of the Royal Society of London (B), 259,91-101 (1970)

17. H.M. Beaumont and A.M. Mandl: A quantitative and cytological study of oogonia and oocytes in the foetal and neonatal rat. Proceedings of the Royal Society of London (B), 155, 557-579 (1961)

18. K. Borum: Oogenesis in the mouse: a study of the meiotic prophase. Experimental Cell Research,24, 495-507 (1961)

19. T.G. Baker: A quantitative and cytological study of germ cells in human ovaries. Proceedings of the Royal Society of London-Biological Sciences, 158, 417-433 (1963)

20. S. Zuckerman: The number of oocytes in the mature ovary, Recent Prog Horm Res, 6, 63-108 (1951)

21. A. McLaren: Meiosis and differentiation of mouse germ cells. Symp Soc Exp Biol, 38, 7-23 (1984)

22. L.D. Anderson and A.N. Hirshfield: An overview of follicular development in the ovary: from embryo to the fertilized ovum in vitro. Md Med J, 41, 614-620 (1992)

23. R. Pearl and W.E. Schoppe: Studies on the physiology of reproduction in the domestic fowl. J Exp Zool, 34,101-118 (1921)

24. C. Tingen, A.Kim and T.K. Woodruff: The primordial pool of follicles and nest breakdown in mammalian ovaries. Mol Hum Reprod, 15 (12), 795-803 (2009)

25. J.L. Black and B.H. Erickson: Oogenesis and ovarian development in the prenatal pig. Anat Rec, 161, 45-55 (1968)

26. N. Manabe, Y. Kimura, A. Myoumoto, H. Matsushita, C. Tajima, M. Sugimoto and H. Miyamoto: Role of granulosa cell apoptosis in ovarian follicle atresia. In: Yamada T, Hashimoto Y (eds), Apoptosis: Its Roles and Mechanism Tokyo. Academic Societies Japan, 97-111 (1998)

27. N. Manabe, F. Matsuda-Minehata, Y. Goto, A. Maeda, Y. Cheng, S. Nakagawa, N. Inoue, K. Wongpanit, H. Jin, H. Gonda and J. Li: Role of cell death ligand and receptor system on regulation of follicular atresia in pig ovaries. Reprod Domest Anim, 2, 268-272 (2008)

28. M.G. Hunter and F.Paradis: Intra-follicular regulatory mechanisms in the porcine ovary. Soc Reprod Suppl, 66, 149-164 (2009)

29. Y.L. Miao, K. Kikuchi, Q.Y. Sun and H. Schatten: Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility. Hum Reprod Update, 15 (5),573-585 (2009)

30. T.A. Ferguson and T.S. Griffith: The role of Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) in the ocular immune response. Chem Immunol Allergy, 92, 140-154 (2007)

31. N. Manabe, Y. Goto, F. Metasuda-Minehata, N. Inoue, A. Maeda, K. Sakamaki and T. Miyano: Regulation mechanism of selective atresia in porcine follicles: regulation of grannulosa cell apoptosis during atresia. J Reprod Dev, 50 (5), 493-514 (2004)

32. D. Crespo, E. Bonnet, N. Roher, S.A. Mackenzie, A. Krasnow, F.W. Goetz, J. Bobe and J.V. Planas: Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mchanism of trout. Reprod Biol Endocrinol, 12,8,34 (2010)

33. A. Korzekwa, S. Murakami, I. Woclawak-Potocka, M.M. Bah, K. Okuda and D.J. Skarzynski: The influence of tumor necrosis factor alpha (TNF) on the secretory function of bovine corpus luteum: TNF and its receptors expression during the estrous cycle. Reprod Biol, 8 (3), 245-262 (2008)

34. A. Maeda, Y. Goto, F. Metsuda-Minehata, Y. Cheng, N. Inoun and N. Manabe: Changes in expression of interleukin-6 receptors in granulosa cells during follicular atresia in pig ovaries. J Reprod Dev, 53 (4), 727-736 (2007)

35. C. Metallinou, B. Asimakopoulos, A. Schroer and N. Nikolettos: Gonadotropin-releasing hormone in the ovary. Reprod Sci, 14 (8), 737-749 (2007)

36. E. Clelland and C. Peng: Endocrine/paracrine control of zebrafish ovarian development. Mol Cell Endocrinol, 312 (1-2), 42-52 (2009)

37. M. Mihm and A.C. Evans: Mechanisms for dominant follicle selection in monovulatory species: a comparison of morphological, endocrine and intraovarian events in cows, mares and woman. Reprod Domest Anim, 43 Suppl 2, 48-56 (2008)

38. H.Z. Liu, F.H. Xu and Y.X. Liu: Effect of EGF on initiation of primordial follicle growth in ovary of newborn rat. Sci Chin, 43 (5), 8-16 (2000)

39. X. Jin, C.S. Han, F.Q. Yu, P. Wei, Z.Y. Hu and Y.X. Liu: Anti-apoptotic action of Stem Cell Factor on oocytes in primordial follicles and its signal transduction. Mol Reprod Dev, 70 (1), 82-90 (2005)

40. X. Jin, C.S. Han, X.S. Zhang, F.Q. Yu, S.H. Guo, Z.Y. Hu and Y.X. Liu: Stem cell factor modulates the expression of steroidogenesis related proteins and FSHR during ovarian follicular development. Front Biosci, 10, 1573-1580 (2005)

41. J. Dong, D.F. Albertini, K. Nishimori, T.R. Kumar, N. Lu and M.M. Matzuk: Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature, 383, 531-535 (1996)

42. J.L. Yang, C.P. Zhang, L. Li, L. Huang, S.Y. Ji, C.L. Lu, C.H. Fan, H. Cai, Y. Ren, Z.Y. Hu, F. Gao and Y.X. Liu: Testosterone induces redistribution of forkhead Box-3a and down-regulation of growth and differentiation factor 9 (GDF9) mRNA expression at early stage of mouse folliculogenesis. Endocrinology, 151 (2), 774-782 (2010)

43. L. Dunkel, J.L. Tilly, T. Shikone, K. Nishimori and A.J. Hsueh: Follicle-stimulating hormone receptor expression in the rat ovary: increases during prepubertal development and regulation by the opposing actions of transforming growth factors beta and alpha. Biol Reprod, 50, 940-948 (1994)

44. A.S. Rannikki, F.P. Zhang and I.T. Huhtaniemi: Ontogeny of follicle-stimulating hormone receptor gene expression in the rat testis and ovary. Mol Cell Endocrinol, 107, 199-208 (1995)

45. D.L. Ingram: The effect of hypophysectomy on the number of oocytes in the adult albino rat. J Endocrinol, 9, 307-311 (1953)

46. R.H. Braw and A. Tsafriri: Follicles explanted from pentobarbitone-treated rats provide a model for atresia. J Peprod Fertil, 59, 259-265 (1980)

47. S.Y. Chun, H. Billig, J.L. Tilly, I. Furuta, A. Tsafriri and A.J. Hsueh: Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor-I. Endocrinology, 135, 1845-1853 (1994)

48. J.A. Flaws, A. DeSanti, K.I. Tilly, R.O. Javid, K. Kugu, A.L. Johnson, A.N. Hirshfield and J.L. Tilly: Vasoactive intestinal peptidemediated suppression of apoptosis in the ovary: poteintial mechanisms of action and evidence of a conserved anti-atretogenic role through evolution. Endocrinology, 136, 4351-4359 (1995a)

49. J.L. Tilly, J.A. Flaws, A. DeSanti, K. Kugu, J.S. Rubin and A.N. Hirshfield: Role of intrafollicular growth factors in maturation and atresia of rat ovarian follicles. Biol Reprod (Supplement 1), 52, 159 (1995b)

50. E. McGee, N. Spears, S. Minami, S.Y. Hsu, S.Y. Chun, H. Billig and A.J. Hsueh: Preantral ovarian follicles in serum-free culture: suppression of apoptosis after activation of the cyclic guanosine 3' 5'-monophosphate pathway and stimulation of growth and differentiation by follicle-stimulating hormone. Endocrinology, 138, 2417-2424 (1997a)

51. D.L. Ingram: The effect of oestrogen on the atresia of ovarian follicles. J Endocrinology, 19, 123-125 (1959)

52. R.S. Carson, J.K. Findlay, I.J. Clarke and H.F. Burger: Estradiol, testosterone, and androstenedione in ovine follicular fluid during growth and atresia of ovarian follicles. Biol Reprod, 24, 105-113 (1981)

53. W.S. Maxson, A.F. Haney and D.W. Schomberg: Steroidogenesis in porcine atretic follicles: loss of aromatase activity in isolated granulosa and theca. Biol Reprod, 33, 495-501 (1985)

54. R.M. Moor, H.M. Dott and D.G. Cran: Macroscopic identification and steroidogenic function of atretic follicles in sheep. J Endocrinol, 77, 309-318 (1978)

55. J.T.J. Uilenbroek, P.J.A. Woutersen and P. van der Schoot: Atresia of preovulatory follicles: gonadotropin binding and steroidogenic activity. Biology of Reproduction, 23, 219-229 (1980)

56. R.H. Braw, S. Bar-Ami and A. Tsafriri: Effect of hypophysectomy on atresia of rat preovulatory follicles. Biol Reprod, 25, 989-996 (1981)

57. P.F. Terranova: Steroidogenesis in experimentally induced atretic follicles of the hamster: a shift from estradiol to progesterone synthesis. Endocrinology, 108, 1855-1890 (1981)

58. P.D. Jolly, D.J. Tisdall, D.A. Heath, S. Lun and S. McNatty: Apoptosis in bovine granulosa cells in relation to steroid synthesis, cyclic adenosine 3'5'-monophosphate response to follicle-stimulating hormone and luteinzing hormone, and follicle atresia. Biol Reprod, 51, 934-944 (1994)

59. Y.X. Liu and A.J.W. Hsueh: Autocrine role of endogenously-produced estrogen in the enhancement of aromatase activity, progesterone production and LH receptor in cultured rat granulosa cells. Chinese J. Physiol. Sci, 1 (2), 1-9 (1986)

60. K. Bruce, Y.X. Liu, X.C. Jia and A.J.W. Hsueh: Autocrine role of estrogen in augmentation of luteinizing hormone receptor formation in cultured rat granulosa cel1s. Biol. Reprod, 32, 1038-1050 (1985)

61. J.F. Couse and K.S. Korach: Estrogen receptor null mice: what have we learned and where will they lead us? Endocrine Reviews, 20, 358-417 (1999)

62. S. Dupont, A. Krust, A. Gansmuller, A. Dierich, P. Chambon and M. Mark: Effect of single and compound knockouts of estrogen receptors α (ERα) and (ERβ) on mouse reproductive phenotypes. Development, 127, 4277-4291 (2000)

63. R.W. Payne, A.A. Hellbaum and J.N. Owens: The effect of androgen on the ovaries and uterus of the estrogen treated hypophysectomized immature rat. Endocrinology, 59, 306-316 (1956)

64. S.G. Hillier and G.T. Ross: Effects of testosterone on ovarian weight, follicular morphology and intraovarian progesterone concentration in estrogen-primed hypophysectomized immature female rats. Biol Reprod, 20, 261-268 (1979)

65. E.A. McGee, S.Y. Chun, S. Lai, Y. He and A.J.W. Hsueh: Keratinocyte growth factor promotes the survival, growth, and differentiation of preantral ovarian follicles. Fertil Steril, 71, 732-738 (1999)

66. S.Y. Chun, K.M. Eisenhauer, S. Minami, H. Billig, E. Perlas and A.J. Hsueh: Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor. Endocrinology, 137, 1447-1456 (1996)

67. A.M. Mani, M.A. Fenwick, Z. Cheng, M.K. Sharma, D. Singh and D.C. Wathes: IGF1 induces up-regulation of steroidogenic and apoptotic regulatory genes via activation of phosphatidylinositol-dependent kinase/AKT in bovine granulosa cells. Reproduction, 139 (1), 139-151 (2010)

68. S. Furukuma, T. Onuma, P. Swanson, Q. Luo, N. Koide, H. Okada, A. Urano and H. Ando: Stimulatory effects of insulin-like growth factor 1 on expression of gonadotropin subunit genes and release of follicle-stimulating hormone and luteinizing hormone in masu salmon pituitary cells early in gametogenesis. Zoolog Sci, 25 (1), 88-98 (2008)

69. H. Kwon, D.H. Choi, J.H. Bae, J.H. Kim and Y.S. Kim: mRNA expression pattern of insulin-like growth factor components of granulose cells and cumulus cells in women with and without polycystic ovary syndrome according to oocyte maturity. Fertil Steril, May, (2010)

70. J. Brannian, K. Eyster, B.A. Mueller, M.G. Bietz and K. Hansen: Differential gene expression in human granulose cells from recombinant FSH versus human menopausal gonadotropin ovarian stimulation protocols. Reprod Biol Endocrinol, Mar, (2010)

71. F. Rey, F.M. Rodriguez, N.R. Salvetti, M.M. Palomar, C.G. Barbeito, N.S. Alfaro and H.H. Ortega: Insulin-like growth factor-II and insulin-like growth factor-binding proteins in bovine cystic ovarian disease. J Comp Pathol, 142 (2-3), 193-204 (2010)

72. K. Ozerkan, G. Uncu and M. Tufekci: Insulin-like growth factor-1 and insulin-like growth factor-binding protein-1 in patients with polycystic ovary syndrome during clomiphene citrate therapy. Int J Gynaecol Obstet, 108 (1), 71-72 (2010)

73. G.F. Erickson, A. Nakatani, N. Ling and S. Shimasaki: Localization of insulin-like growth factor-binding protein-5 messenger ribonucleic acid in rat ovaries during the estrous cycle. Endocrinology, 130, 1867-1878 (1992a)

74. G.F. Erickson, A. Nakatani, N. Ling and S. Shimasaki: Cyclic changes in insulin-like growth factor-binding protein-4 messenger ribonucleic acid in the rat ovary. Endocrinology, 130, 625-636 (1992b)

75. C.J. Hsu and J.M. Hammond: Gonadotropins and estradiol stimulate immunoreactive insulin-like growth factor-I production by porcine granulosa cells in vitro. Endocrinology, 120, 198-207 (1987)

76. E.R. Hernandez, C.T.J. Roberts, D. LeRoith and E.Y. Adashi: Rat ovarian insulin-like growth factor I (IGF-I) gene expression is granulosa cell-selective: 5'-untranslated mRNA variant representation and hormonal regulation. Endocrinology, 125, 572-574 (1989)

77. Adashi, E.Y., Resnick, C.E., Hernandez, E.R., Hurwitz, A. & Rosenfeld, R.G. (1990). Follicle-stimulating hormone inhibits the constitutive release of insulin-like growth factor binding proteins by cultured rat ovarian granulosa cells. Endocrinology, 126, 1305-1307.

78. N.A. Cataldo and L.C. Giudice: Follicular fluid IGF binding protein profiles in polycystic ovary syndrome. J Clin Endocrinol Metab, 74, 695-697 (1992a)

79. N.A. Cataldo and L.C. Giudice: IGF binding protein profiles in human ovarian follicular fluid correlate with follicular functional status. J Clin Endocrinol Metab, 74, 821-829 (1992b)

80. J.L. Tilly, H. Billig, K.I. Kowalski and A.J.W. Hsueh: Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol, 6, 1942-1950 (1992b)

81. M.G. Hunter and F. Paradis: Intra-follicular regulatory mechanisms in the porcine ovary. Soc Reprod Fertil Suppl, 66, 149-164 (2009)

82. M. Hsieh, A.M. Zamah and M. Conti: Epidermal growth factor-like growth factors in the follicular fluid: role in oocyte development and maturation. Semin Reprod Med, 27 (1), 52-61 (2009)

83. S.J. Uhm, M.K. Gupta, J.H. Yang, H.J. Chung, T.S. Min and H.T. Lee: Epidermal growth factor can be used in lieu of follicle-stimulating hormone for nuclear matulation of porcine oocytes in vitro. Theriogenology, 73 (8), 1024-1036 (2010)

84. J.K. Nyholt de Prada, Y.S. Lee, K.E. latham, C.L. Chaffin and C.A. VandeVoort: Role for cumulus cell-produced EGF-like ligands during primate oocyte maturation in vitro. Am J Physiol Endocrinol Metab, 296 (5), E1049-1058 (2009)
PMid:19276391    PMCid:2681310

85. O. Onagbesan, V. Bruggeman and E. Decuypere: Intra-ovarian growth factors regulating ovarian function in avian species: a review. Anim Reprod Sci, 111 (2-4), 121-140 (2009)

86. H. Fujinaga, M. Yamoto, T. Shikone and R. Nakano: FSH and LH upregulate epidermal growth factor receptors in rat granulosa cells. J Endocrinol, 140, 171-177 (1994)

87. A.M. Luciano, A. Pappalardo, C. Ray and J.J. Peluso: Epidermal growth factor inhibits large granulosa cell apoptosis by stimulating progesterone synthesis and regulating the distribution of intracellular free calcium. Biol Reprod, 51, 646-654 (1994)

88. R.S. Brogan, S. Mix, M. Puttabyatappa, C.A. VandeVoort and C.L. Chaffin: Expression of the insulin-like growth factor and insulin systems in the luteinizing macaque ovarian follicle. Fertil Steril, 93 (5), 1421-1429 (2010)

89. J.R. Silva, J.R. Figueiredo and R. Van den Hurk: Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis. Theriogenology, 71 (8), 1193-1208 (2009)

90. Y.X. Liu and A.J.W. Hsueh: Synergism between granulosa and theca-interstitial cells in estrogen biosynthesis by gonadotropin-treated rat ovaries : Studies on the two-cel1, two-gonadotropin hypothesis using steroid antisera. Biol Reprod, 35, 27-36 (1986)

91. S.Y. Chun, K.M. Eisenhauer, M. Kubo and A.J. Hsueh: Interleukin-1 beta suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. Endocrinology, 136, 3120-3127 (1995)

92. E.C. Svensson, E. Markstrom, M. Andersson and H. Billig: Progesterone receptor-mediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gondadotropin. Biol Reprod, 63, 1457-1464 (2000)

93. G.B. Talbert, R.K. Meyer and W.H. McShan: Effect of hypophysectomy at the beginning of proestrus on maturing follicles in the ovary of the rat. Endocrinology, 49, 687-694 (1951)

94. J. Lee, H.J. Park, H.S. Choi, H.B. Kwon, A. Arimura, B.J. Lee, W.S. Choi and S.Y. Chun: Gonadotropin stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) messenger ribonucleic acid in the rat ovary and the role of PACAP as a follicle survival factor. Endocrinology, 140, 818-826 (1999)

95. U. Natra and J.S. Richards: Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. Endocrinology, 133, 761-769 (1993)

96. M.F. Press and G.L. Greene: Localization of progesterone receptor with monoclonal antibodies to the human progestin receptor. Endocrinology, 122, 1165-1175 (1988)

97. J.P. Lydon, F.J. DeMayo, C.R. Funk, et al: Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev, 9, 2266-2278 (1995)

98. J.P. Lydon, F.J. DeMayo, O.M. Conneely and B.W. O'Malley: Reproductive phenotypes of the progesterone receptor null mutant mouse. J Steroid Biochem Mol Biol, 56, 67-77 (1996)

99. C. Ko, Y.H. In and O.K. Park-Sarge: Role of progesterone receptor activarion in pituitary adenylate cyclase activating polypeptide gene expression in rat ovary. Endocrinology, 140, 5185-5194 (1999)

100. C. Ko and O.K. Park-Sarge: Progesterone receptor activation mediates LH-induced type-1 pituitary adenylate cyclase activating polypeptide receptor (PAC (1)) gene expression in rat granulosa cells. Biochem Biophys Res Commun, 277, 270-279 (2000)

101. T.M. Hazzard and R.L. Stouffer: Angiogenesis in ovarian follicular and luteal development. Baillieres Best Pract Res Clin Obstet Gynaecol, 14, 883-900 (2000)

102. L.P. Reynolds, A.T. Grazul-Bilska and D.A. Redmer: Angiogenesis in the corpus luteum. Endocrine, 12, 1-9 (2000)

103. L.P. Reynolds and D.A. Redmer: Growth and development of corpus. luteum J Reprod Fertil Suppl, 54, 181-191 (1999)

104. I. Rothchild: The regulation of the mammalian corpus luteum. Recent Prog Horm Res, 37, 183-298 (1981)

105. G.D. Niswender and T.M. Nett: The corpus luteum and its control in infraprimate species, in The Physiology of Reproduction (eds. Knobil, E., Neill, J.), New York: Raven Press, 781-816 (2000)

106. J.L. Pate and P. Landis Keyes: Immune cells in the corpus luteum: friends or foes. Reproduction, 122, 665-676 (2001)

107. J.M. Bowen and P. Landis Keyes: Repeated exposure to prolactin is required to induce luteal regression in the hypophysectomized rat. Biol Reprod, 63, 1179-1184 (2000)

108. W. Wuttke and J. Meites: Luteolytic role of prolactin during the estrous cycle of the rat. Proc Soc Exp Biol Med, 137, 988-991 (1971)

109. S. Matsuyama, K.T. Chang, H. Kanuka, M. Ohnishi, A. Ikeda, M. Nishihara and M. Takahashi: Occurrence of deoxyribonucleic acid fragmentation during prolactin-induced structural luteolysis in cycling rats. Biol Reprod, 54, 1245-1251 (1996)

110. P.V. Malven: Hypophysial regulation of luteolysis in the rat. In: McKerns KW (ed.), The Gonads. New York: Appleton-Century-Crofts, 367-382 (1969)

111. K. Guo, V. Wolf, A.M. Dharmarajan, Z. Feng, W. Bielke, S. Saurer and R. Friis: Apoptosis-associated gene expression in the corpus luteum of the rat. Biol Reprod, 58, 739-746 (1998)

112. J.A. Long and H.M. Evans: The estrous cycle of the rat and its associated phenomena. Mem Univ Calif, 6, 1-148 (1922)

113. J.M. Bowen, R. Towns, J.S. Warren and P.L. Keyes: Luteal regression in the normally cycling rat: apoptosis, monocyte chemoattractant protein-1, and inflammatory cell involvement. Biol Reprod, 60, 740-746 (1999)

114. C.M. Telleria, A.A. Goyeneche, J.C. Cavicchia, A.O. Stati and R.P. Deis: Apoptosis indeced by antigestagen RU486 in rat corpus luteum of pregnancy. Endocrine, 15, 147-155 (2001)

115. K. Liu, A. Brandstrom, Y.X. Liu, T. Ny and G. Selstam: Coordinated expression of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 during corpus luteum formation and luteolysis in the adult pseudopregnant rats. Endocrinology, 137, 2126-2132 (1996)

116. K. Liu, Y.X. Liu, Z.Y. Hu, Y.J. Zou, Y.J. Chen, X.M. Mu and T. Ny: Temporal expression of urokinase type plasminogen activator, plasminogen activator inhibitor type-1 in rhesus monkey corpus luteum during the luteal maintenance and regression. Mol Cell Endocrinol,133, 109-116 (1997)

117. K. Liu, J.I. Olofsson, P. Wahlberh and T. Ny: Distinct expression of gelatinase A (matrix metalloproteinase (MMP)-2), collagenase-3 (MMP-13), membrane type MMP 1 (MMP-14), and tissue inhibitor of MMPs Type 1 mediated by physiological signals during formation and regression of the rat corpus luteum. Endocrinology, 140, 5330-5338 (1999)

118. W.C. Duncan: The human corpus luteum: remodelling during luteolysis and maternal recognition of pregnancy. Rev Reprod, 5, 12-17 (2000)

119. T.E.J. Curry and K. Osteen: Cyclic changes in the matrix metalloproteinase system in the ovary and uterus. Biol Reprod, 64,1285-1296 (2001)

120. K.A. Young, B. Tumlinson and R.L. Stouffer: ADAMTS/METH-1 and TIMP-3 expression in the primate corpus luteum: divergent patterns and stage-dependent regulation during the natural menstrual cycle. Mol Hum Reprod, 10, 559-565 (2004)

121. K.A. Young and R.L. Stouffer: Gonadotropin and steroid regulation of matrix metalloproteinases and their endogenous tissue inhibitors in the developed corpus luteum of rhesus monkey during menstrual cycle. Biol Reprod, 70, 244-252 (2004)

122. M.F. Smith, E.W. McIntush, W.A. Ricke, F.N. Kojima and G.W. Smith: Regulation of ovarian extracellular matrix remodelling by metalloproteinases and their tissue inhibitors: effects on follicular development, ovulation and luteal function. J Reprod Fertil Suppl, 54, 367-38 (1999)

123. Q. Feng, K. Liu, R.J. Zou and Y.X. Liu: The possible involvement of tissue type plasminogen activator in luteolysis of rhesus monkey. Human Reproduction, 8, 1640-1644 (1993)

124. M. Bond, G. Murphy, M.R. Bennett, A. Amour, V. Kna�uper and A.C. Newby: Localization of the death domain of tissue inhibitor of metalloproteinase-3 to the N terminus. Metalloproteinase inhibition is associated with proapoptotic activity. J Biol Chem, 275, 41358-41363 (2000)

125. M. Bond, G. Murphy, M.R. Bennett, A.C. Newby and A.H. Baker: Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent type II apoptotic pathway. J Biol Chem, 277, 13787-13795 (2002)

126. F. Mannello and G. Gazzanelli: Tissue inhibitors of metalloproteinases and programmed cell death: conundrums, controversies and potential implications. Apoptosis, 6, 479-482, 23-25 (2001)

127. X.L. Chen, H.J. Gao, F. Gao, P. Wei, Z.Y. Hu and Y.X. Liu: Temporal and spatial exepression of MMP-2,-9,-14 and their inhibitors TIMP-1, -2, -3 in the corpus luteum of the cycling rhesus monkey. Science in China Ser C, Life Science,48 (6), 1-9 (2005)

128. H.J. Gao, X.L. Chen, Z.H. Zhang, X.X. Song, Z.Y. Hu and Y.X. Liu: IFN-gamma and TNF-alpha inhibit expression of TGF-beta, its receptors tbetar-I and tbetar-II in the corpus luteum of PMSG/hCG treated thesus monkey. Front Biosci, 10, 2496-2503 (2005)

129. X.L. Chen, H.J. Gao, P. Wei, Z.H. Zhang and Y.X. Liu: Expression of apoptosis-related genes Fas/FasL, Bax/BcL-2 and caspase-3 in rat corpus luteum during luteal regression. Science in China (C), 46 (3), 273-285 (2003)

130. X.L. Chen, H.J. Gao, P. Wei, X.X. Song, Z.Y. Hu and Y.X. Liu: Regulatory effect of TGF-b1, TbR-II and StAR in corpus luteum of pregnant rhesus monkey. Acta Pharmacol. Sinica, 24 (5), 435-441 (2003)

131. Y.J. Chen, Q. Feng and Y.X. Liu: Expression of the Steroidogenic acute regulatory protein and LH receptor and their regulation by TNF-a in rat corpus lutea. Biol. Reprod, 60, 419-427 (1999)

132. Y.J. Chen, Q. Feng and Y.X. Liu: Expression of steroidogenic acute regulatory protein (StAR) and its regulation by TNF-a in rat corpus lutea Recent Progress in Molecular.Comparative Endocrinology P63-70 eds by H.B. Kwon, JMP Joss and SI Shii, Kwanggju, Republic of Korea (1999)

133. J. Suter, I.R. Hendry, L. Ndjountche, K. Obholz, J.K. Pru, J.S. Davis and B.R. Rueda: Mediators of interferon gamma-initiated signaling in bovine luteal cells. Biol Reprod, 64, 1481-1486 (2001)

134. S.M. Quirk, R.M. Harman, S.C. Huber and R.C. Cowan: Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis. Biol Reprod, 63, 49-56 (2000)

135. K. Okuda, A. Korzekwa, M. Shibaya, S. Murakami, R. Nishimura, M. Tsubouchi, I. Woclawek-Potocka and D.J. Skarzynski: Progesterone is a suppressor of apoptosis in bovine luteal cells. Biol Reprod, 71, 2065-2071 (2004)

136. S.F. Carambula, T. Matikainen, M.P. Lynch, R.A. Flavell, P.B. Goncalves, J.L. Tilly, B.R. Rueda: Caspase-3 is a pivotal mediator of apoptosis during regression of the ovarian corpus luteum. Endocrinology, 143, 1495-1501 (2002)

137. Y. Tatsukawa, A. Bowloaksono, R. Nishimura, J. Komiyama, T.J. Acosta and O. Kiyoshi: Possible roles of intracellular cyclic AMP, protein kinase C and calcium ion in the apoptotic signaling pathway in bovine luteal cells. J Reprod Dev, published on line May 8 (2006)

138. A. Amsterdam and N. Selvaraj: Control of differentiation, transformation, and apoptosis in granulosa cells by oncogenes, oncoviruses, and tumor suppressor genes. Endocrinology Review, 18, 435-461 (1997)

139. E.A. McGee and A.J. Hsueh: Initial and cyclic recruitment of ovarian follicles. Endocrinology Review, 21, 200-214 (2000)

140. A.L. Johnson, J.T. Bridgham and T. Jensen: Bcl-X (LONG) protein expression and phosphorylation in granuloda cells. Endocrinology, 140, 4521-4529 (1999)

141. G. Gebauer, A.T. Peter, D. Onesime and N. Dhanasekaran: Apoptosis of ovarian granulosa cells: correlation with the reduced activity of ERK-signaling module. J Cell Biochem, 75, 547-554 (1999)

142. E. Asselin, Y. Wang and B.K. Tsang: X-linked inhibitor of apoptosis protein activates the phosphatidylinositol 3-kinase/Akt pathway in rat graunlosa cells during follicular development. Endocrinology, 142, 2451-2457 (2001)

143. A.L. Johnson, J.T. Bridgham and J.A. Swenson: Activation of the Akt/protein kinase B signaling pathway is associated with granulosa cell survival. Biol Reprod, 64, 1566-1574 (2001)

144. R.J. Davis: The mitogen-activated protein kinase signal transduction pathway. J Biol Chem, 268, 14553-14556 (1993)

145. T. Hunter: Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell, 80 (2), 225-236 (1995)

146. E.K. Kim and E.J. Choi: Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta, 1802 (4), 396-405 (2010)

147. B. Kaminska, A. Gozdz, M. Zawadzka, A. Ellert-Miklaszewska and M. Lipko: MAPK signal transduction underlying brain inflammation and gliosis as therapeutic target. Anat Rec, 292 (2), 1902-1913 (2009)

148. M.A. Bogoyevitch, K.R. Ngoei, T.T. Zhao, Y.Y. Yeap and D.C. Ng: c-Jun N-terminal kinase (JNK) signaling: recent advances and challenges. Biochim Biophys Acta, 1804 (3), 463-475 (2010)

149. X. Jin, C.S. Han, X.S. Zhang, J.X. Yuan, Z.Y. Hu and Y.X. Liu: Signal transduction of stem cell factor in primordial follicles development promotion. Mol Cell Endocrinol, 229 (1-2), 3-10 (2005)

150. K.Z. Guyton, Y. Liu, M. Gorospe, Q. Xu and N.J. Holbrook: Activation of mitogen-activated protein kinase by H2O2: Role in cell survival following oxidant injury. J Biol Chem, 271, 4138-4142 (1996)
PMid:9657968    PMCid:1219585

151. X. Wang, J.L. Martindale, Y. Liu and N.J. Holbrook: The cellular response to oxidative stress: influences of mitogen-activated protein kinase signaling pathways on cell survival. Biochem J, 333, 291-300 (1998)

152. G.A. Dissen, C. Romero, A.N. Hirshfield and S.R. Ojeda: Nerve growth factor is required for early follicular development in the mammalian ovary. Endocrinology, 142, 2078-2086 (2001)

153. J.L. Yan, Q. Feng, H.Z. Liu, et al: Expression of tPA, LH receptor and inhibin a, BA subunits during follicular atresia in rat. Science in China C, 42, 583-590 (2000)

154. X. Jin and Y.X. Liu: Follicular growth, differentiation and atresia. Chinese Science Bulletin, 48, 1786-1790 (2003)

155. J.C. Reed: Bcl-2 and the regulation of programmed cell death. J Cell Biol, 124, 1-6 (1994)

156. J.E. Chipuk, T. Moldoveanu, F. Llambi, M.J. Parsons and D.R. Green: The BCL-2 family reunion. Mol Cell, 37 (3), 299-310 (2010)

157. V. Ganesan and M. Colombine: Regulation of ceramide channels by Bcl-2 family proteins. FEBS Lett, 584 (10), 2128-2134 (2010)

158. W.G. McCluggage: Immunohistochemistry as a diagnostic aid in cervical pathology. Pathology, 39 (1), 97-111 (2007)

159. F. Matsuda-Minehata, N. Inoue, Y. Goto and N. Manabe: The regulation of ovarian granulose cell death by pro- and anti-apoptotic molecules. J Reprod Dev, 52 (6), 695-705 (2006)

160. V.S. Ratts, J.A. Flaws, R. Kolp, C.M. Sorenson and J.L. Tilly: Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology, 136, 3665-3668 (1995)

161. Oltvai, Z.N., Milliman, C.L. & Kosmeyer, S.J. (1993). Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74, 609-619.

162. M.D. Felici, A.D. Carlo, M. Pesce, S. Iona, M.G. Farrace, M. Piacentini: Bcl-2 and Bax regulation of apoptosis in germ cells during prenatal oogenesis in the mouse embryo. Cell Death Differ, 6, 908-915 (1999)

163. M. Filali, N. Frydman, M.P. Belot, L. Hesters, F. Gaudin, G. Tachdjian, D. Emilie, R. Frydman and V. Machelon: Oocyte in-vitro maturation: BCL2 Mrna content in cumulus cells reflects oocyte competency. Reprod Biomed Online, 19 suppl 4, 4309 (2009)

164. V. Anchamparuthy, R. Pearson and F. Gwazdauskas: Expression pattern of apoptotic genes in vitrified-thawed bovine oocytes. Reprod Domest Anim, Oct, (2009)

165. Y. Guillemin, P. Lalle, G. Gillet, J.F. Guerin, S. Hamamah and A. Aouacheria: Oocytes and early embryos selectively express the survival factor BCL2L10. J Mol Med, 87 (9), 923-940 (2009)

166. G.L. Perez, C.M. Knudson, L. Leykin, S.J. Korsmeyer and J.L. Tilly: Apoptosis-associated signaling pathways are required for chemotherapy-mediated female germ cell destruction. Nat Med, 3, 1228-1332 (1997)

167. G.I. Perez, R. Robles, C.M. Knudson, J.A. Flaws, S.J. Korsmeyer and J.L. Tilly: Prolongation of ovarian lifespan into advanced chronological age by Bax-deficiency. Nat Genet, 21, 200-203 (1999)
PMid:20169201    PMCid:2820548

168. S. Krajewski, M. Krajewska, A. Shabaik, H.G. Wang, S. Irie, L. Fong and J.C. Reed: Immunohistochemical analysis of in vivo patterns of Bcl-x expression. Cancer Research, 54, 5501-5507 (1994b)

169. L.L. Kujjo, T. Laine, R.J. Pereira, W. Kagawa, H. Kurumizaka, S. Yokoyama and G.I. Perez: Enhancing survival of mouse oocytes following chemotherapy or aging by targeting Bax and Rad51. PLoS One, 5 (2), 9204 (2010)

170. J.K. Pru, T. Kaneko-Tarui, A. Jurisicova, A. Kashiwagi, K. Selesniemi and J.L. Tilly: Induction of proapoptotic gene expression and recruitment of p53 herald ovarian follicle loss caused by polycyclic aromatic hydrocarbons. Reprod Sci, 16 (4), 347-356 (2009)

171. L. Del Peso, M. Gonzalez-Garcia, C. Page, R. Herrera and G. Nunez: Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science, 278, 687-689 (1997)

172. T.F. Gajewski and C.B. Thompson: Apoptosis meets signal transduction: elimination of a BAD influence. Cell, 87, 589-592 (1996)

173. J. Zha, H. Harada, E. Yang, J. Jockel and S.J. Korsmeyer: Serine phosphorylation of death agonist BAD in response to survival factor results in binding 14-3-3 not BCL-X (L). Cell, 87, 619-628 (1996)

174. S. Hu, S.J. Snipas, C. Vincenz, G. Salvesen and V.M. Dixit: Caspase-14 is a novel developmentally regulated protease. J Biol Chemistry, 273, 29648-29653 (1998)

Key Words: Apoptosis, Signal Pathway, Ovary, Follicle, Review

Send correspondence to: Yi-Xun Liu, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China, Tel: 86-10-64807038, Fax: 86-1064807583, E-mail:Liuyx@ioz.ac.cn