Human Reproduction Update Advance Access originally published online on February 10, 2005
Human Reproduction Update 2005 11(2):162-178; doi:10.1093/humupd/dmi001
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Apoptosis in the ovary: molecular mechanisms
Department of Pathology, Assiut University Hospitals, Assiut, Egypt
Email:mrh17{at}swissinfo.org
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Abstract |
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Cell death was first described in rabbit ovaries (Graaffian follicles), the phenomenon being called ‘chromatolysis’ rather than apoptosis. In humans, the ovarian endowment of primordial follicles is established during fetal life. Apoptotic cell death depletes this endowment by at least two-thirds before birth, executed with the help of several players and pathways conserved from worms to humans. To date, apoptosis has been reported to be involved in oogenesis, folliculogenesis, oocyte loss/selection and atresia. Several pro-survival and pro-apoptotic molecules are involved in ovarian apoptosis with the delicate balance between them being the determinant for the final destiny of the follicular cells. This review critically analyses the current knowledge about the biological roles of these molecules and their relevance to the dynamics of follicle development. It also presents the existing literature and assesses the gaps in our knowledge.
Key words: apoptosis / death / ovary
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Introduction |
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The term apoptosis (of Greek origin) means dropping off, as in leaves from a tree. Apoptosis is a genetically determined and biologically functional mode of cell death. The widespread existence of apoptosis among vertebrates and invertebrates suggests conservation of its molecular components. Few organs, if any, provide such a paradigm for apoptosis as the ovary. This is due to the cyclicity of ovarian development. In the ovary, the mechanisms underlying decisions of life and death involve cross-dialogue between pro-apoptotic and pro-survival molecules. Morphologically, apoptosis is found in ovarian follicles throughout fetal and adult life. During fetal life, apoptosis is localized to the oocytes, whereas in adult life, it is detected in granulosa cells of secondary and antral follicles. Hypotheses of mechanisms underlying this apoptotic loss include: (i) ‘quality control’ to eliminate meiotic anomalies; (ii) a deficit in survival factors; and (iii) a ‘self-sacrifice’. This review surveys current knowledge about: (i) the discovery, morphology and detection of apoptosis; (ii) the dynamics of follicle development; (iii) the roles of the four major arms of apoptosis/survival pathways in the folliculogenesis, oocyte loss/selection and atresia [B cell/lymphoma-2 family (Bcl-2), tumour necrosis factor (TNF), caspases, transforming growth factor (TGF)-ß and other molecules]; (iv) the contributions of the other molecules; and (v) the existing gaps in our knowledge about these issues.
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Historical aspects |
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The studies of the Australian pathologist John Kerr, on liver atrophy, provided the first clue to the existence of apoptosis. He noticed that individual hepatocytes (outside the zone of necrosis) evolved into small rounded cytoplasmic masses, with some containing pyknotic chromatin. Further histochemical and ultrastructural analysis revealed membrane-bound cellular fragments containing well-preserved organelles. Kerr called this phenomenon ‘shrinkage necrosis’, and in a seminal article in the British Journal of Cancer he proposed the term ‘apoptosis’ for this type of cell death (Kerr et al., 1972
; Hussein et al., 2003a). Over two decades, Kerr and Wyllie have defined apoptosis as a distinct entity of cell death in terms of morphology, biochemistry and incidence (Kerr et al., 1972; Wyllie, 1993). |
Morphology and detection of apoptosis |
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In tissues, the ultrastructural features of apoptosis include: (i) condensation of the nuclear chromatin into sharply circumscribed masses; (ii) convolutions of the nuclear and cellular outlines; (iii) fragmentation and budding of the cell and the production of the membrane-bound apoptotic bodies; and (iv) phagocytosis of the apoptotic bodies by the macrophages. The phagocytosed bodies are finally reduced to unrecognizable residues. The histological features of apoptosis include cytoplasmic vacuolization, chromatin condensation as well as the appearance of eosinophilic apoptotic bodies as round cytoplasmic masses or as masses of pyknotic chromatin surrounded by a narrow rim of the cytoplasm (Figure 1). In culture, the features of apoptosis include cell shrinkage, surface convolutions, formation of protuberances with buddings and subsequent formation of the apoptotic bodies. Biochemically, apoptosis is characterized by rapid nuclear DNA cleavage which occurs in two stages. Initially, cleavage occurs rapidly, mostly by topoisomerase II, into 200–300 kilobase pair fragments. Then cleavage of the double-stranded internucleosomal DNA by DNAse I/DNAse II results in the formation of oligonucleosome-sized fragments. The morphological evaluation remains the standard tool for detection of apoptosis. Other techniques include: (i) DNA agarose gel electrophoresis with the formation of DNA ladder; (ii) flow cytometry; and (iii) terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling and in situ end labelling (Wyllie, 1993
).
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During human oogenesis, the ultrastructural changes of apoptosis involve the two main phases of the meiotic process: an earlier one concerning the oogonia and oocytes in the preleptotene stage, and a later one that mainly concerns the oocytes in the pachytene stage (De Pol et al., 1998
). In atretic oocytes, nucleolar segregation, cytoplasmic or nuclear condensation, apoptotic body formation and chromatin margination along the nuclear membrane never occur. Instead, early morphological changes in atretic oocytes include retraction of granulosa cell and oocyte-derived microvilli as well as the condensation of mitochondria with the loss of cristae. These changes coincide with initiation of granulosa cell apoptosis. After loss of most of the granulosa cells, more severe changes occur, including segmentation of the oocyte and cytoplasmic vacuolization (Devine et al., 2000). |
Genetic design of apoptosis |
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Two general mechanisms are operative in apoptosis: one mechanism is triggered by the binding of death molecules to cell surface receptors (death receptor-mediated events), while the other is generated by signals arising within the cell (mitochondria-mediated events) (Hussein et al., 2003a
). Death receptor-mediated apoptotic events
These events are initiated by the binding of the death receptors [Fas, tumour necrosis factor receptor (TNFR), interferon (IFN) and TNF-related apoptosis-inducing Ligand (TRAIL) receptors] to their ligands. This binding initiates ligation of the receptors and transmission of the apoptotic signals through death domains (DD), death effector domains and caspase recruitment domains (CARD). The death domains are found in cytoplasmic proteins including Fas-associated protein with death domain (FADD), TNF receptor-associated protein with death domain (TRADD), and receptor interacting protein (RIP) and in transmembrane proteins, including TNF-R1, TRAIL-R1/DR4 and TRAIL-R2/DR5 (Hussein et al., 2003a) (Figure 2). The CARD mediates the activation of adaptor proteins and procaspases, leading to caspase activation and apoptosis. These pathways are regulated by Flip (FLICE inhibitory protein), which prevents initiator caspases and the inhibitor of apoptosis (IAP) (Hussein et al., 2003a).
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Mitochondria-mediated apoptotic events
The death signals cause the pro-apoptotic Bcl-2 proteins (especially Bax) to allow cytochrome c to leak out of the mitochondria. The released cytochrome c and apoptotic protease-activating factor 1 (Apaf-1) bind to caspase 9, which then activates the caspase cascade, leading to cell death. Also, the induction of the pro-apoptotic BH3-only domain proteins [Bid, Bad, Noxa and p53-up-regulated modulator of apoptosis (PUMA)] can relay the signals to the mitochondria. Once induced, these proteins can facilitate the assembly of pro-apoptotic Bax and Bak into the pores in the outer mitochondrial membrane (Hussein et al., 2003a,b). The process involves changes in mitochondrial permeability and release of various factors involved in apoptosis, including cytochrome c and apoptosis-inducing factor (Figures 2, 3 and 5 and Tables II and III).
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Caspase cascade and apoptosome assembly
Caspases include both ‘initiator’ caspases (8 and 9) and ‘effector’ caspases (3, 6 and 7). Once activated, these proteases cleave enzymes and proteins essential to the cell's viability. Caspases share in the formation of an ‘apoptosome’ complex, which is a multi-protein complex consisting of cytochrome c, Apaf-1, pro-caspase-9 and ATP. Its formation is initiated by the release of cytochrome c from mitochondria into the cytosol where it binds Apaf-1 and, in the presence of dATP/ATP, induces the oligomerization of Apaf-1. This complex recruits pro-caspase-9, which then undergoes autoactivation to promote recruitment and cleavage of caspase-3. Caspase-3 then cleaves its target substrates to effect the changes associated with apoptosis (Hussein et al., 2003b) (Figures 2, 3 and 5 and Tables II–IV).
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Dynamics of follicle development |
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Our understanding of oogenesis, folliculogenesis, oocyte loss, follicle selection and atresia is still incomplete. In humans, the ovarian reserve of follicles is established during fetal life, then it is gradually depleted during follicular development. At least two-thirds of the oocytes present in the reserve die by apoptosis before birth. Apoptosis is an essential component of ovarian function and development. Indeed, it is the mechanism that makes the female biological clock tick. During fetal life, apoptosis mainly involves the oocyte. Alternatively, it involves the granulosa cells of the growing follicle during the adult life. Hypothetically, mechanisms underlying the exhaustion of the ovarian reserve of follicles include: (i) ‘quality control’ leading to the elimination meiotic anomalies; (ii) a deficit in survival factors produced by somatic neighbouring cells; (iii) a ‘self-sacrifice’ or ‘altruistic death’ (Monniaux, 2002
) (Figure 2). Follicle development during fetal life
Shortly after the fourth week of gestation, primordial germ cells migrate from the yolk sac to the gonadal ridge and proliferate to a total of 
7 x 106 cells. In the second half of pregnancy, this number declines to 1 x 106 oocytes. Therefore, during early fetal life 7 x 106 oocytes are formed in the human ovary. This number is sharply reduced before birth through apoptotic cell death of the oocytes. Apoptosis is the highest between weeks 14 and 28, and then decreases thereafter towards term. Of note, the mitochondrial DNA is more liable to undergo mutations than genomic DNA and most of the mitochondria are inherited from the oocytes. Thus the quality of the mitochondria seems to have a crucial role in the life/death decision of the oocyte. In support of this idea, microinjection of mitochondria purified from healthy (non-apoptotic) granulosa cells into the oocyte decreases the possibility of apoptosis in these cells. After birth, most oocytes perish, and by puberty the ovary loses 75% of its follicles (Perez et al., 2000) (Figure 3).
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Follicle development during adult life
During adult life, a number of primordial follicles start growing during each menstrual cycle. Usually only one follicle will ovulate and the fate of the rest of the follicles is atresia through apoptotic cell death. Ultimately, only 450 follicles will ovulate during a woman's reproductive life. Between puberty and menopause, 250 000 follicles are destined for atresia. During a typical menstrual cycle, 1000 follicles begin growing, with only one or two being destined for ovulation. After ovulation, the dominant follicle forms the corpus luteum. The latter is responsible for the production of progesterone and maintenance of the endometrium during early pregnancy. Apoptosis is also responsible for corpus luteum regression, i.e. luteolysis (Tilly et al., 1991, 1992; Tilly, 1996, 1997; Morita and Tilly, 1999; Vaskivuo and Tapanainen, 2003) (Figure 3).
Folliculogenesis
Folliculogenesis is the development of the ovarian follicles. It commences with the recruitment of the primordial follicles and ends with either ovulation or death by atresia. The follicles include primordial (resting follicles), primary (follicles activated for development or atresia), secondary (large-sized follicles) and tertiary follicles (Fortune et al., 2000; Suh et al., 2002). The process of follicular development and survival depends on autocrine and paracrine signalling involving growth factors from granulosa cells, theca cells, stromal–interstitial cells, and the oocytes. These factors include several molecules such as bone morphogenetic protein (BMP)-4, Bcl-2, Kit, FGF, NOBOX, NTS/Trkb, survivin, XIAP, GDF-9, NAIP, AHR, GATA-4, SCF, integrin, gonadotrophins and TRAIL (Tables II and III). In particular, the growth factor bone morphogenetic protein-4 (BMP-4) and its receptor (BMPR-IB) are critical for follicular development: BMP-4-treated ovaries had a higher proportion of developing primary follicles and fewer arrested primordial follicles than did untreated controls, indicating that BMP-4 promotes primordial follicle development and the primordial–primary follicle transition (Gougeon, 1996; Nilsson and Skinner, 2003) (Table I).
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Negative and positive selection of ovarian follicles
During follicle recruitment, an increase in the level of circulating FSH allows some antral follicles to escape apoptotic demise. Among this cohort, a leading follicle dominates by secreting more estrogen and inhibin to suppress FSH release. This action negatively selects the remaining follicles in the cohort, leading to their ultimate loss (negative selection). At the same time, the increased production of local growth factors allows positive selection of the dominant follicle and eventual ovulation (positive selection) (McGee and Hsueh, 2000).
Follicular atresia
With the increasing serum FSH concentrations at the start of the menstrual cycle, one follicle produces increasing amounts of estrogen and thus progresses towards maturity at mid-cycle. As a feedback mechanism, FSH secretion falls, and therefore the remaining follicles undergo apoptosis and become atretic (Hughes and Gorospe, 1991; Tilly et al., 1991). Although the exact signals, receptors and intracellular signalling pathways leading to apoptosis within granulosa cells are unclear, it is likely that: (i) multiple molecules are involved (such as Fas, caspases, TNF, TVB, Par-4, p53, prohibitin, c-Myc, IFN, endothelins); (ii) these molecules include both survival (such as gonadotrophins, insulin-like growth factor-1, interleukin-1ß, epidermal growth factor, basic fibroblast growth factor, TGF-
, bcl-2, bcl-xlong) and atretogenic factors (TGF-ß, interleukin-6, androgens, reactive oxygen species, bax, Fas antigens, p53, TNF and caspases) (Driancourt et al., 1998; Tilly, 2001; Jiang et al., 2003); and (iii) the outcome depends upon a delicate balance between these molecules (Tables I–IV).
Luteolysis
The phenomenon of luteolysis, or regression of corpus luteum, terminates the female reproductive cycle in humans. Luteolysis is both a functional and morphological process. The former is characterized by an initial decline of progesterone secretion. Alternatively, the morphological luteolysis entails alterations in the cellular structure of corpus luteum and its gradual involution in the ovary into a small scar composed of connective tissue (corpus albicans). The latter persists in the ovary, often for several weeks (McCracken et al., 1999). A wide variety of molecules has been proposed as mediators of structural luteolysis such as TNF, Fas/Fas ligand, caspase-3, Bax, prohibitin, BMP ligands (BMP) and receptors (Table I). The involvement of these molecules is supported by two observations. First, the temporal relationships between apoptosis and luteolysis. Second, luteolysis is associated with altered expression of some molecules such as Bcl-2/Bax ratio and TNF in corpus luteum (Sugino et al., 2000).
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Molecular mechanisms involved in the dynamics of ovarian development |
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 TOPAbstractIntroductionHistorical aspectsMorphology and detection of...Genetic design of apoptosisDynamics of follicle development Molecular mechanisms involved in... Conclusions and future...References |
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The ovarian dynamics are orchestrated by plethora of molecular mechanisms. The latter are mediated by several pro-apoptotic and pro-survival molecules. Some of these molecules are involved in the process of: (i) atresia such as Bcl-2 family members, TNF and caspases; (ii) follicle selection/loss such as Bcl-2, Bax, FSH, inhibin, Fas ligand, caspases; and (iii) luteolysis such as Fas/Fas ligand, caspase-3, Bax, prohibitin, BMP ligands and receptors (Table I). The following sections examine these molecules.
Bcl-2 family
The B cell/lymphoma-2 family (Bcl-2) of proteins includes both inhibitors (Bcl2, Mcl-1 and Bcl-XL) and promoters (Bax, Bcl-2-associated X protein) of apoptosis. This family contributes to the checkpoints between cell surface and internal death signals, formation of apoptosome and activation of the caspase cascade (Sato et al., 1994).
Profiles of Bcl-2 family members
The Bcl-2 gene is a pro-survival molecule identified through its involvement in B-cell lymphomas. It is a membrane-associated protein that resides in the nuclear envelope and mitochondria. Its molecular properties are summarized in Tables I and II. It exerts its pro-survival functions by: (i) modulating the mitochondrial release of cytochrome c and the interaction of Apaf-1 with caspase-9; (ii) binding to Bax; and (iii) blocking apoptosis induced by c-Myc (Luo et al., 1997) (Figures 1 and 2). Bax is another pro-apoptotic Bcl-2 homologue that resides either in the cytoplasm or in the cell membrane and can therefore antagonize the protective role of Bcl-2. Its molecular properties are summarized in Tables I and II. Induced myeloid leukaemia cell differentiation protein (Mcl-1) has sequence similarity to Bcl2, and its pro-survival actions are mediated either through heterodimerization with Bax or reduction of c-Myc-induced apoptosis. Alternatively, Bcl-XL (long isoform) has stronger pro-survival effects than Bcl-2 itself. The expression of this protein protects cells from entering p53-mediated apoptosis (Boyd et al., 1995) (Figures 2 and 3).
Bcl-2 and ovarian apoptosis
Bcl-2 (pro-survival), Bax (pro-apoptotic) and c-Myc are expressed in granulosa cells of both fetal and adult ovaries, suggesting their possible role in atresia (Nandedkar and Dharma, 2001). Bcl-2 is found mainly in the developing follicles while Bax is seen mainly in the atretic follicles (Van Nassauw et al., 1999).
Bcl-2 protein expression is found in all components of the human fetal ovaries (19–33 gastational weeks) to overcome extensive apoptotic activity (Abir et al., 2002). This expression is related to gonadotrophin levels where higher levels of gonadotrophins increase the expression of bcl-2 and decrease the expression of Bax (Sugino et al., 2000). Also, whereas Bcl-2/Bax ratio regulates apoptosis of the primordial follicle under SCF (survival molecule), Bax-mediated apoptosis in oocytes is mostly due to intrinsic meiotic checkpoints (Felici et al., 1999).
The role of Bcl-2 in ovarian apoptosis is supported by several experimental findings including: (i) decreased numbers of follicles in bcl-2-deficient mice (Ratts et al., 1995); (ii) excessive expression of bcl-2 leads to decreased follicular apoptosis and atresia (Hsu et al., 1996; Morita and Tilly, 1999); (iii) bax-deficient mice have abnormal follicles with an excessive number of granulosa cells (Perez et al., 1999); and (iv) Bax expression is high in the atretic follicles as compared to the healthy ones (Kugu et al., 1998). Other Bcl-2 family members involved in ovarian apoptosis include, Mcl-1, Bok (Bcl-2-related ovarian killer), BOD (Bcl-2-related ovarian death agonist) and BAD (Bcl2 antagonist of cell death, pro-apoptotic). The activity of the pro-apoptotic ligand BAD is regulated by upstream follicle survival factors. In contrast, Mcl-1 and Bok regulate cytochrome c release, and, together with the recently discovered Diva/Boo, control downstream Apaf-1 homologues and caspases (Tilly et al., 1995; Hsu et al., 1998).
Tumor necrosis factor family
The tumor necrosis factors family members are mostly type II transmembrane proteins. The members involved in ovarian development and atresia include TNF-, Fas/Fas-Ligand (FasL), TRAIL, a TNF decoy receptor homologue (Wada et al., 2003) (Figures 2 and 3).
Molecular profiles of TNF family members
TNF interacts with two cell surface receptors, denoted TNFR1 and TNFR2, resulting in receptor aggregation and recruitment of signalling proteins. While TNFR2 encourages proliferation, TNFR1 induces cytotoxic effects. TNFR1 exerts its effects through the death domain in its cytoplasmic region, which interacts with intracellular transducers, resulting in complex aggregation and the activation of molecules such as caspases, which lead to cell death. A second death pathway is initiated by the TNF-associated proteins known as TRAF. TRAF serve as adapter proteins that recruit downstream molecules which ultimately result in the activation of nuclear factor kappa B (NF
B) by degradation of IKB (Hsu et al., 1996; Malinin et al., 1997) (Figures 1 and 2).
The Fas/APO-1 receptor is a type-I transmembrane protein of the TNF family located on the cell surface. When Fas ligand binds to this receptor, apoptosis occurs in sensitive cells both in vitro and in vivo. Studies suggest that death induced by this ligand and TNF involves interactions with a family of cysteine proteases related to the ICE family of proteins (interleukin 1ß-converting enzyme) (Fulda et al., 1997; Deveraux et al., 1999). TRAIL, a cell surface molecule, is a member of the TNF family. Although the in vivo role of the TRAIL/TRAIL receptor system is not fully determined, antibodies that neutralize TRAIL can inhibit IFN-ß-induced apoptosis. Also, the induction of TRAIL/Apo2L by IFN-ß is responsible for its greater ability to induce apoptosis. After IFN precipitates the synthesis of TRAIL, the latter activates the TRAIL death receptors TRAIL-R1 and TRAIL-R2, possibly forming a heterotrimeric TRAIL receptor complex, which then activates multiple apoptotic cascades in the cell. TRAIL-induced apoptosis is dependent on the caspase cascade and can be completely inhibited by caspase inhibitors. The expression or lack of expression of certain apoptotic inhibitors such as FLIP (FLICE-inhibitory protein) may determine sensitivity to TRAIL-mediated apoptosis. Ironically, one of the inhibitory signals which protects both normal tissue from apoptosis results from the activation of the transcription factor NFB by both TRAIL binding to the decoy/inhibitory receptor DcR2 and to the death receptors TRAIL-R1 and TRAIL-R2. This pathway may result in increased resistance to TRAIL at low concentrations, causing the induction of apoptosis only at high molecular concentrations, and is the topic of ongoing investigations (Nguyen et al., 2000; Chawla-Sarkar et al., 2001; Franco et al., 2001).
TNFR-associated factors
TNFR-associated factors (TRAF) are cytoplasmic adapter proteins that function as signalling intermediates and play important roles in cellular apoptosis and survival. Although little is known about their exact mechanisms, they seem to mediate the formation of larger signalling complexes. These complexes contain several adapter proteins such as TRADD and FADD, kinases including NIK (NFB-inducing kinase) and JNK (c-Jun N-terminal kinase), and proteases of the caspase family (Arch and Thompson, 1998) (Figures 1 and 2). TNF-associated factor TRAF-2 is a member of the TRAF family associated with other signal effector molecules, such as germinal centre kinase (GCK) (Ivanov et al., 2000), a molecule which appears to be involved in TNF-mediated activation of the SAPK (JNK) (Pombo et al., 1995). Recruitment of signal molecules, such as the protein kinase NIK, by TRAF-2 results in the activation of the pro-survival molecule NFB and increased cell survival (Malinin et al., 1997) (Figures 2 and 3).
The biological roles of TNF family members depend upon their mechanisms of action. For instance, binding of FasL with its receptor results in recruitment of the death domains, other intracellular transducers, resulting in the activation of caspase cascades and apoptosis (Bakker et al., 1999; Raisova et al., 2000). In contrast, TNF- can act as both pro-survival and pro-apoptotic molecule, depending on the receptor subtype activated. It can promote granulosa cell survival by increasing XIAP and FLIP expression via the IB–NFB pathway. Alternatively, its pro-apoptotic action is mediated either through the activation of caspase cascade (Jiang et al., 2003) or by reducing BcL-2 protein levels. TRAIL is a new member of the TNF family. The binding of TRAIL Ligand to cell surface receptors containing death domains induces apoptosis. Interestingly, TRAIL induces apoptosis only in tumour cells, not in normal cells. Normal cells may be protected from TRAIL-induced apoptosis by the presence of TRAIL decoy receptors, which compete with the death receptors for binding of TRAIL Ligand (Ashkenazi and Dixit, 1999). The molecular profiles of these molecules are summarized in Table III.
TNF family and ovarian apoptosis
The members of the TNF receptor family, TNF-, Fas (and their ligands) and TRAIL have critical roles in the ovarian atresia. Their roles are reflected by their expression not only in the apoptotic granulosa cells of healthy and atretic antral follicles but also in the embryonic gonad (Driancourt et al., 1998; Kim et al., 1999). These roles are supported by the following experimental observations: (i) the higher contents of FasL mRNA in granulosa and theca cells from atretic as compared with healthy follicles (Porter, 1997); (ii) the ability of Fas to induce granulosa cell apoptosis (Hakuno et al., 1996); (iii) the expression of multiple death domains related to TNF proteins in hen granulosa cells (Bridgham and Johnson, 2001); (iv) TRAIL and its receptors are expressed in the ovarian follicles (Bobe and Goetz, 2001); and (v) the expression of TRAIL-decoy receptor-1 is markedly reduced in the granulosa cells of atretic follicles (Wada et al., 2003). The expression of these proteins is closely linked with gonadotrophin levels. In this regard, gonadotrophin deprivation (as serum withdrawal) or treatment are associated with expression or disappearance of FasL/Fas and p53 (in the granulosa cells) respectively (Kim et al., 1999; Hu et al., 2001). Of note, TVB is an avian death domain-containing receptor belonging to the TNF receptor family. It represents the fourth death domain-containing receptor. It is expressed within hen granulosa cells with higher levels in the atretic than the healthy follicles (Bridgham and Johnson, 2002) (Figures 2 and 3).
Caspases
Caspases are are family of highly conserved cysteine proteases that mediate the course of apoptotic cell suicide. Caspases are the main effector molecules in ovarian apoptosis. They are activated in two ways in the granulosa cells: (i) cell surface receptors; and (ii) members of the Bcl-2 family of proteins. Experimental evidence supports the expression of caspase-1, caspase-3, DNA fragmentation factor, IL1-ß converting enzyme (ICE), CPP32 and Apaf-1 in human granulosa cells (Flaws et al., 1995; Boone and Tsang, 1998; Van Nassauw et al., 1999; Fenwick and Hurst, 2002). In the ovary, caspase-3 is expressed in luteal and thecal cells of healthy corpus luteum as well as in the granulosa cells of atretic follicles. It is absent in granulosa cells of healthy follicles. This expression is regulated by gonadotrophin and may be altered as part of the apoptotic process in the granulosa cells (Boone and Tsang, 1998) (Figures 2 and 3).
Protease-apoptosis activating factor-1
Protease-apoptosis activating factor-1 (Apaf-1) is a putative homologue of the Caenorhabditis elegans gene, ced-4, that contributes to the induction of apoptosis by binding to cytochrome c and caspase 9. The resulting complex aggregates in the cytoplasm (Song et al., 1999). Caspase-9 then activates the caspase cascade, leading to cell death (Figures 1 and 2). In the ovary, Apaf-1 is abundant in granulosa cells of early antral follicles, whereas in vivo gonadotrophin priming completely suppresses Apaf-1 expression and granulosa cell apoptosis (Deveraux and Reed, 1999). The inhibitors of apoptosis (IAP) families are a new class of apoptosis inhibitors that function by inhibiting the action of caspases (Deveraux and Reed, 1999). In the ovary, increased X-linked inhibitor of apoptosis (XIAP) expression and suppressed follicular apoptosis are important determinants in the regulation of follicular development by FSH. The FSH-induced XIAP expression is mediated via the NFB pathway through activation of phosphatidylinositol 3-kinase rather than the classical IB kinase (Li et al., 1998; Wang et al., 2002). NFB is a transcription factor which, when activated, inhibits apoptosis triggered by diverse stimuli. Transcription factors from this family are activated by TNF, essentially counteracting the effects of the latter. NFB can be activated both through the death receptors for TRAIL, TRAIL-R1 and TRAIL-R2 and through one of the decoy receptors for this ligand, TRAIL-R4. NFB then modulates the expression of several other inhibitory proteins, such as A1, a Bcl-2 homologue and additional molecules that inhibit the action of caspases and therefore seems to play a critical role in ovarian apoptosis (Degli-Esposti et al., 1997a,b; Deveraux and Reed, 1999) (Figures 2 and 4).
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p53 pathway
p53 is a stress response gene that encodes a 53 kDa oncosuppressive nuclear protein with a Mr of 53 000. The p53 protein exists as a tetramer that accumulates in the cytoplasm during the G1 (Gap1) phase and migrates to the nucleus at the start of the S (synthesis) phase.
Molecular profile of p53
This protein has up to seven different functional properties, can activate 20 different promoters, repress 26 different promoters and enhancers, and can interact with >35 cellular and viral proteins. Also, p53 is a synaptic point where upstream and downstream cross-talk dictates the final destiny of the cell (Haapajarvi et al., 1999). The components of the upstream pathway have not been identified and seem most likely to include protein kinases that phosphorylate p53 protein. The downstream p53 ‘effector’ genes include mdm2 (murine of double minute 2) GADD45 (growth arrest and DNA damage inducible protein), and p21Cip1/Waf1 (wild-type p53-activated fragment 1) genes (Haapajarvi et al., 1999). The molecular properties of p53 are summarized in Figure 4 and Table III.
p53 and ovarian apoptosis
The expression of p53 protein in the apoptotic granulosa cells of atretic follicles suggests its possible role in atresia (Kim et al., 1999). This role is supported by several experimental leads: (i) inhibition of p53 expression is associated with a marked reduction in the number of apoptotic granulosa cells and atretic follicles (Tilly et al., 1995); (ii) overexpression of p53 can induce apoptosis in cAMP-stimulated cells. The underlying mechanisms of this role include: (i) p53 modulation of the bcl-2 and bax gene transcriptional activity; and (ii) p53/cAMP interactions through alteration of p53/bax ratio and inhibition of clusterin gene (an apoptosis-associated protein) (Tilly et al., 1995; Amsterdam et al., 1996). In this regard, cAMP deprivation or treatments are associated with either inhibition or enhancement respectively of p53-induced apoptosis (Zwain and Amato, 2001).
Transforming growth factor-ß family
TGF-ß has recently generated much interest from evidence of its critical role in determining the follicle fate. TGF family members (TGF-ß) are multifunctional cytokines that control cellular proliferation, differentiation and interaction with the extracellular matrix. TGF-ß is synthesized as a prohormone that is cleaved in the secretory pathway into an amino- and a carboxy-terminal fragment. Its members include TGF-ß, activin, inhibin, BMP and growth differentiation factor 9 (GDF-9), the Nodals, anti-Müllarian hormone, decapentaplegic and many other structurally related proteins. Generally, they function through Smad to modify the expression of specific sets of target genes. Ski and SnoN proteins are Smad co-repressors. The former was originally discovered as the product of a retroviral oncogene (v-ski) that causes transformation in chick embryo fibroblasts. Its cellular counterpart c-Ski and the related SnoN protein are Smad 3- and Smad 4-interacting proteins (Miyazono, 2000; Piek and Roberts, 2001).
TGF-ß signalling entails two basic steps: receptor activation and Smad activation. There are three mammalian isoforms for TGF-ß (TGF-ß1, 2 and 3), which represent secreted ligands. These ligands initiate downstream signalling events by activation of transmembrane receptors with cytoplasmic serine–threonine kinase domains. TGF-ß interact with the TGF-ß type II receptor (TGF-ßRII), with subsequent recruitment of a TGF-ß type I receptor into a heterotetrameric receptor complex. The ligand-binding type II receptor kinase is constitutively active and activates type I receptors. This phosphorylation event results in the activation of the type I receptor kinase and downstream signalling. The ligand assembles a receptor complex that phosphorylates Smad. Upon phosphorylation, ligand/receptor-specific Smad translocates to the nucleus, interacts with a specific partner protein and recruits the other transcriptional agents (Miyazono, 2000; Piek and Roberts, 2001; Wieser, 2001). All the biological effects of TGF-ß are Smad-dependent (Miyazono, 2000). In the ovary, the TGF-ß ligands, receptors and mediators (Smad) are present at distinct points throughout folliculogenesis, suggesting discrete roles for each of these ligands during follicle maturation (Bristol and Woodruff, 2004) (Tables I–IV and Figure 5).
Inhibin and activin
Inhibin- subunit protein is expressed in all follicle stages. This expression increases in intensity within the mural granulosa cells in large antral follicles. The inhibin ßA and ßB subunit proteins, in addition to the activin type I (ActRIB) and activin type II receptor (ActRIIB), are expressed by the granulosa cells of primordial and primary follicle. Additionally, inhibin ßA subunit is expressed in the theca cells of the secondary to large antral follicles (Gougeon, 1994; Hsueh et al., 1996; Billiar et al., 2003; Bristol and Woodruff, 2004).
TGF-ß ligands
TGF-ß1 is strongly expressed in the corpus luteum and oocytes. It is weakly expressed in the theca cells of the large healthy and atretic follicles. It is absent in the granulosa cells of small and medium-sized follicles. Alternatively, TGF-ß2 and 3 as well as TGF-ß type II receptor are expressed in the oocytes and granulosa cells of the antral follicles (Christopher, 2000; Bristol and Woodruff, 2004).
Growth differentiation factor-9
Growth differentiation factor-9 (GDF-9) is a secreted member of TGF-ß. It is expressed at high levels in the oocytes and its deficiency blocks folliculogenesis. Its absence results in the loss of theca cell layer markers (17-hydroxylase, LH receptor and c-kit, the receptor for kit ligand) around GDF-9-deficient follicles. Therefore, in the absence of GDF-9, the follicles cannot generate a signal to recruit theca cell precursors to surround the follicle. Also, the primary follicles of GDF-9-deficient mice have up-regulation of kit ligand and inhibin . These secreted growth factors (c-kit/inhibin) are regulated in a paracrine fashion by GDF-9 (Elvin et al., 1999).
Bone morphogenetic protein family
The BMP family is formed of several ligands (BMP-2, -3, -3b, -4, -6, -7, -15) and receptors (BMPR-IA, -IB, -II). The altered expression of the BMP genes affects folliculogenesis (recruitment, selection, atresia), ovulation and luteogenesis (luteinization, luteolysis). In this regard, BMB mRNA are expressed in a cell-specific manner in the oocyte, granulosa, theca interstitial, theca externa, and corpus luteum. Also, BMP undergoes dynamic changes during follicular and corpus luteum morphogenesis (Erickson and Shimasaki, 2003; Erickson et al., 2004).
Smad
The TGF-ß mediators (Smad 2 and 4) are expressed in the granulosa cell cytoplasm of all follicles. Smad 3 is detected in the granulosa cell nucleus, the oocyte, and the theca cell nucleus of all follicles. Smad 3 plays critical roles in the regulation of: (i) ovarian follicle growth; and (ii) the expression of Bax and Bcl-2. The critical role of Smad is suggested by the reduction of fertility in Smad 3-deficient mice, as compared with wild-type animals (Tomic et al., 2002; Symonds et al., 2003).
Inhibitors of apoptosis family
The inhibitors of apoptosis family (IAP), including both survivin and livin, comprise a new class of apoptosis inhibitors that functions by inhibiting the action of caspases. Survivin is a unique member of the IAP family as it contains a single baculovirus IAP repeat domain combined with a COOH-terminal -helix coiled-coil domain instead of the more common zinc-binding RING finger. Because of this combination, survivin can regulate both cell proliferation and apoptosis. Survivin is a pro-survival molecule that enhances the progression through the cell cycle. It localizes to the mitotic spindle and interacts with several pro-apoptotic caspases. In the ovary, high levels of survivin expression during follicle development occur in mitotically active granulosa cells from undifferentiated, pre-hierarchal follicles. In these cells, survivin acts as a bifunctional protein associated with regulation of the cell cycle and the inhibition of apoptosis (Johnson and Bridgham, 2002). Moreover, in Xenopus laevis, survivin mRNA is present in the earliest stages of Xenopus oocytes and it accumulates during oogenesis. The decrease in survivin mRNA is associated with both the slowing of the cell cycle and the onset of apoptosis (Murphy et al., 2002).
X-linked inhibitor of apoptosis protein (XIAP) is a member of a family of intracellular pro-survival proteins. It is the most potent caspase inhibitor encoded in the genome. In the ovary, XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth. Also, XIAP is an important intracellular modulator of the TNF- death-signalling pathway in granulosa cells. Its expression is regulated by TNF- via a NFB-mediated mechanism (Xiao et al., 2001). Moreover, XIAP expression in the granulosa cells is regulated by gonadotrophins during follicular development. The mechanisms by which XIAP suppresses granulosa cell apoptosis involve the phosphatidylinositol 3-kinase (PI 3-K) survival pathway. Gonadotrophin treatment increases granulosa cell XIAP and phospho-Akt protein contents and suppresses apoptosis. In addition, gonadotrophin withdrawal can induce granulosa cell apoptosis. The increased apoptosis is accompanied by marked decreases in XIAP expression and phosphorylation of Akt protein (Li et al., 1998; Asselin et al., 2001; Xiao et al., 2001; Sauerwald et al., 2002; Wang et al., 2002, 2003; Johnson et al., 2004).
Stem cell factor ligand and c-KIT receptors
Kit and Mgf gene encode KIT receptor and ligand respectively. KIT–KIT ligand interaction has pro-survival effects in the oocytes, primordial, primary and antral follicles. In the fetal gonad, KIT–KIT ligand interaction has cytoprotective effects on the oocytes. In postnatal ovaries, this interaction contributes to the initiation of the folliculogenesis, controls oocyte growth and theca cell differentiation. It protects the preantral follicles from apoptosis. In the antral follicles, it modulates the ability of the oocytes to undergo cytoplasmic maturation and enhances thecal androgen output (Driancourt et al., 1998, 2000).
Stem cell factor (SCF) is a growth factor that has profound effects on the proliferation, migration, differentiation and survival of numerous cell types, including those of the ovary. The proto-oncogene c-KIT is a transmembrane tyrosine kinase receptor activated by the binding of SCF ligand. SCF can partially reduce primordial germ cell apoptosis. C-Kit and SCF are expressed: (i) in oogonia of the primordial and primary follicles; and (ii) strongly in oocytes of preantral follicles and weakly in granulosa and thecal cells. The expression of c-kit/SCF in oocytes: (i) gradually decreases as the follicles develop; (ii) is specific in all developmental stages of ovarian follicles; (iii) decreases after the follicle starts to grow; and (iv) negatively regulates Fas-mediated apoptosis in vivo (Gougeon and Busso, 2000). Moreover, the addition of SCF to culture can down-regulate BAX expression and therefore significantly reduce apoptosis in the oocyte (Felici et al., 1999).
Granzyme B
Granzymes are exogenous serine proteinases that are released from cytoplasmic granules of cytotoxic lymphocytes (CTL) and NK cells. These granules contain beside granzymes other proteins including a pore-forming protein (Perforin). Upon binding of the CTL to a target cell, the contents of the granules are released in the intercellular space where perforine can ‘perforate’ the target cell membrane by forming transmembrane pores. Through these pores the granzymes can enter the cytosol of the target cell. Granzyme B activates the intracellular cascade of caspases resulting in the killing of the target cells. Granzyme B can cleave and activate pro-caspase-8, -3 and -7. In this regard, pro-caspase-3 appears to be a major physiological substrate of Granzyme B (Bladergroen et al., 2001; Amsterdam et al., 2003a,b). In the ovary, Granzyme B is expressed and activated in granulosa cells, therefore allowing the apoptotic signals to bypass mitochondria-mediated apoptosis. Thus this role can preserve the steroidogenic activity until complete cell destruction occurs. This apoptotic pathway can maintain the cyclicity of estradiol and progesterone release in the estrus/menstrual cycle even during the initial stage of apoptosis (Amsterdam et al., 2003a,b).
Prohibitin
Prohibitin is an evolutionarily conserved protein involved in the regulation of cell death and survival. It is localized in the mitochondrial membrane and thus its expression is related to the mitochondrial destabilization. Prohibitin protein is expressed in the oocytes, granulosa cells, corpus luteum, and atretic follicles. The increased levels of prohibitin correlate with the initial events of apoptosis. In this regard, the phosphorylation of prohibitin can be induced by estrogen and therefore it is involved in the regulation of granulosa cell proliferation and the ontogony of ovarian follicles. In atretic follicles, prohibitin is translocated from the cytoplasm to the nucleus and this event is associated with the atretic events (Thompson et al., 1999, 2001, 2004; Hu et al., 2001).
Integrins
Integrins are transmembrane molecules that link the cell to the cytoskeleton and can influence cell survival and death. In addition to their adhesion properties, integrins also participate in signal transduction, with loss of signalling resulting in apoptosis. Integrins are expressed on the surface of primordial follicles and can mediate their adhesive interactions with the extracellular matrix, somatic cells and neighbouring cells (De Felici, 2000). There is a possible association between granulosa cell apoptosis of tertiary follicles and integrin expression in ovarian follicles. In this regard, the expression of integrin-ß1 and -6 is: (i) strong in intact primordial/primary, secondary and tertiary follicles; (ii) weak in atretic tertiary follicles but not present in atretic primary or secondary follicles. Interestingly, apoptosis is only found in granulosa cells of tertiary follicles lacking the expression of integrins (Giebel et al., 1996, 1997; Giebel and Rune, 1997).
Endothelins
Endothelins (ET) are a family of vasoactive peptides involved in granulosa and luteal cell function. They include three isopeptides (ET-1, -2 and -3), with various biological effects including vasoconstriction, mitogenesis and steroidogenesis (Iwai et al., 1991). Endothelins are signal peptides that exert their biological roles through binding to two G-protein-coupled receptors with apparently opposite effects. ET-1 is up-regulated during luteal regression and may act as apoptosis inducer during luteolysis (Schams et al., 2003). The binding site for ET-1 is localized in the granulosa cells (Otani et al., 1996). In humans, granulosa–lutein cells are sites of ET reception and action. They inhibit cyclic AMP-dependent FSH-mediated function. The ET (A) receptor participates in this effect (Furger et al., 1995). ET can influence prostaglandin (PG) synthesis and release from luteal cells (Otani et al., 1996; Miceli et al., 2001).
Insulin growth factor and its receptors
Insulin growth factor and its receptors (IGF-I, IGF-IR) are expressed in the granulosa cells of healthy antral follicles (A.Li et al., 1999; Y.Z.Li et al., 1999). IGF-I is necessary for the completion of follicular development in mice. Analysis of IGF-I mRNA revealed an increase in its transcripts in healthy follicles with the transition from primary follicle to preantral and antral stages. By contrast, IGF-I transcripts were low in atretic follicles and absent in corpus luteum. Therefore, IGF-I plays an important role in stimulating and sustaining the proliferation of the granulosa cells. IGF binding protein-4 (IGFBP-4) mRNA expression in granulosa cells is restricted to apoptotic and atretic follicles, i.e. IGFBP-4 is an atretogenic candidate for ovarian follicles. IGFBP-5 transcript levels, on the other hand, are elevated in granulosa cells of healthy primary and secondary follicles but decreased in subsequent follicular stages and in atretic follicles. Conversely, IGFBP-2 mRNA is constitutively expressed in granulosa cells (Wandji et al., 1998).
Gonadotrophins
Gonadotrophin-mediated inhibition of apoptosis in ovarian granulosa cells is linked in part to changes in the expression of several cell death-related genes. In this regard, gonadotrophins can induce the expression of bcl-2, GATA-4, FLIP and XIAP (pro-survival molecules) and decrease the expression of Bax, Apaf-1, Fas/FasL and p53 (pro-apoptotic molecules) (Kim et al., 1999; Robles et al., 1999; Wang et al., 2002). GnRH-I and GnRH-II are expressed in granulosa and luteal cells (Leung et al., 2003). The loss of trophic hormonal support is translated into a reduction of the specific mitogen-activated protein kinase (MAPK) module along with a decrease in the level of the phosphorylated form of BAD. These events preceed the onset of apoptosis in the granulosa cells (Gebauer et al., 1999; Peter and Dhanasekaran, 2003).
Newborn ovary homeobox-encoding gene
The newborn ovary homeobox-encoding gene (Nobox) is an oocyte-specific homeobox gene expressed during folliculogenesis. The gene spans 14 kb and is encoded by eight exons. The Nobox gene maps to chromosome 6 in the mouse. In humans, a portion of the human gene encoding a Nobox homologue resides on chromosome 7q35. Nobox is preferentially expressed in primordial and growing oocytes at high levels. The lack of Nobox accelerates postnatal oocyte loss and abolishes the transition from primordial to growing follicles. This lack is associated with replacement of the ovarian follicles by fibrous tissue and down-regulation of GDF9 (Suzumori et al., 2002; Rajkovic et al., 2004).
Neurotrophins, brain-derived neurotrophic factor and TrkB kinase receptors
Neurotrophin (NT) signalling via TrkB kinase receptors is critical for folliculogenesis. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF) represent TrkB ligands. They are expressed in the mouse ovary both in the oocytes and granulosa cells. Full-length kinase domain-containing TrkB receptors are expressed in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes. In mice lacking TrkB or NT-4 or BDNF isoforms, there are: (i) a stage-selective deficiency in folliculogenesis that nullifies the ability of follicles to grow beyond the primary stage; (ii) reduced proliferation of granulosa cells; and (iii) failure of folliculogenesis in their ovaries grafted under the kidney capsule with massive oocyte death (Paredes et al., 2004).
Aryl hydrocarbon receptor
The aryl hydrocarbon receptor (AhR) is a member of a family of ligand-activated transcriptional regulators. It regulates the expression of 15 genes, including enzymes, growth factors and other transcription factors. AhR protein is expressed in the oocytes and granulosa cells of follicles at all stages of development. AhR is activated by polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons. Once bound by ligand, the AhR interacts with the AhR nuclear translocator protein to form the aryl hydrocarbon receptor complex. In the ovary, PAH exposure can cause destruction of oocytes within immature follicles, suggesting that AhR can mediate cell death signalling in the female germ line. Therefore, AhR can regulate the size of the oocyte reserve by affecting germ cell death during female oogenesis (Robles et al., 2000).
Neuronal apoptosis inhibitory protein
Neuronal apoptosis inhibitory protein (NAIP) is involved in ovarian folliculogenesis as it can prevent granulosa cell death. NAIP is induced by gonadotrophin, which is known to inhibit apoptosis in ovarian follicles. In mouse ovaries, NAIP mRNA expression is seen in the granulosa cells of developing follicles from the primary stage to the Graafian stages, whereas it is absent in the atretic follicles. Suppression of ovarian NAIP expression with antisense oligonucleotides can evoke a decrease in the number of morphologically normal ovulated oocytes, implying an indirect involvement of NAIP in germ cell development by enhancing the survival of granulosa cells (Matsumoto et al., 1999).
Prostate apoptosis response 4
Prostate apoptosis response 4 (par-4) encodes a transcription factor, Par-4, with a leucine zipper domain. Par-4 protein is widely expressed in various adult cell types. It is functionally required for apoptosis. Induction of Par-4 in cultured cells is found exclusively during apoptosis. Par-4 is expressed in apoptotic granulosa cells of atretic ovarian follicles. The widespread expression of Par-4 underscores the physiological importance of the protein (Boghaert et al., 1997).
Prostaglandin F2- receptor
The prostaglandin F2- receptor (PGF2-R) is found in three ovarian cell subpopulations: (i) cells of corpus luteum; (ii) thecal cells surrounding secondary and mature follicles; and (iii) primary and secondary interstitial cells. The PGF2-R is absent in the granulosa cells of primary, secondary mature follicles, the oocyte, and corpus luteum. During luteolysis, cells undergoing apoptosis stained for the presence of PGF2-R (Orlicky et al., 1992).
GATA-4
GATA-4 is a member of zinc finger transcription factors that regulates cell proliferation. During fetal development, GATA-4 mRNA and protein are localized to the granulosa cells, with expression being highest in the youngest ovaries and decreasing somewhat towards term. Also, this expression is localized to granulosa cells of primary and antral follicles. This expression is stimulated by exogenous gonadotrophins in cultured gonadal cell lines. Moreover, down-regulation of GATA-4 expression is associated with follicular atresia in adult mouse ovaries. Therefore, GATA-4 serves as a pro-survival molecule in the ovary (Heikinheimo et al., 1997; Viger et al., 1998; Laitinen et al., 2000; Vaskivuo et al., 2001).
Cellular-Myelocytomatosis oncogene
Cellular-Myelocytomatosis (c-Myc) oncogene is a nuclear phosphoprotein that can contribute to both cell proliferation and apoptosis. Interestingly, apoptosis has been observed in cells with up-regulated as well as down-regulated levels of c-Myc protein. c-Myc exerts its functions by interaction with other molecules such as Bcl-2, BAX and TGF-. In the human ovary, the expression of c-Myc may play a role in the molecular mechanisms of cell proliferation and apoptosis. In this regard, c-Myc expression is observed in the primordial, preantral and atretic follicles. Also, with the increase in the size of the follicles, c-Myc protein expression decreases in the oocytes and increases in the granulosa and theca cells (Li et al., 1998; Nandedkar and Dharma, 2001).
Interferons
Interferons are cytokines that have antiviral and antiproliferative functions, and include IFN- and IFN-ß (Type I Interferons) and IFN-
(a Type II interferon). These molecules exert their apoptotic functions by sensitizing the cells to apoptotic factors and inducing genes and proteins involved in several major apoptotic pathways. In the ovary: (i) Fas can inhibit the induction of apoptosis in luteal cells; (ii) INF- down-regulates production of Fas and therefore allows apoptosis to occur in these cells (Komatsu et al., 2003); and (iii) co-culturing INF--pretreated granulosa cells with zona-free oocytes can induce granulosa cell apoptosis (Hakuno et al., 1996).
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Conclusions and future directions |
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Today, a current literature search using ‘apoptosis’ and ‘ovary’ as key words reveals >1269 articles, and interest in apoptosis research shows no signs of abating. Apoptosis, a genetically regulated cell suicide, is an energy-requiring process that can be observed throughout the whole of the animal and plant kingdoms. It is morphologically distinct from the degenerate process of necrosis. Apoptosis permits the safe disposal of cells at t