By postnatal d 2, meiotically arrested oocytes associate with somatic cells to form primordial follicles, the first stage of folliculogenesis (4). Although the signals that trigger formation of primordial follicles and the eventual recruitment of a dormant primordial follicle into the growing pool are still unknown, the genes critical for these processes are being identified, and their functions are being analyzed (Table 4A). The subsequent period of preantral follicle growth, consisting of granulosa cell proliferation and oocyte growth, is relatively slow, and only a few mitotic figures are observed in granulosa cells at this stage (38). Initiation of follicle growth in the mouse is not restricted to sexual maturity, but in fact begins within the first wk postnatally. Since there is no evidence for a reserve pool of larger follicles, it appears that once a follicle enters the growing pool, it is normally committed to a program of growth and differentiation, culminating in either apoptotic death of the granulosa cells (atresia) or ovulation of the mature oocyte (39).
Several proteins are known to be expressed by mouse primordial follicles and follicles during the initiation period. The oocyte of the newly recruited follicle begins to secrete its unique extracellular glycoprotein matrix, called the zona pellucida (ZP). ZP formation at this early stage of folliculogenesis suggests that it may be important for oocyte-granulosa cell coupling, or may play a role in continued follicle development (37). One of the genes involved in regulating the expression of all the three mouse ZP genes is Figa (Factor In the Germline a). Figa is a basic helix-loop-helix transcription factor that is first expressed in oocytes prior to primordial germ cell formation, and is also expressed in oocytes of primary and later follicles (40). Although Figa (with a heterodimeric partner) binds to an E-Box in the ZP-1, ZP-2, and ZP-3 genes to regulate transcription of these genes, Figa must also function prior to this point, because mice without Figa have a normal number of oocytes at birth, but subsequently fail to form primordial follicles and demonstrate dramatic apoptosis of these oocytes over the next few days (41). Thus, Figa not only regulates expression of the ZP genes, but may also induce the expression of an oocyte gene involved in "recruitment" of pregranulosa cells to surround the oocytes and form primordial follicles.
To study the function of the ZP, all three of the ZP genes have been mutated in mice. Interestingly, mice deficient in any of the three major components of the ZP (ZP1, ZP2, or ZP3) show no defects at any stage of folliculogenesis (41-44). ZPl-knockout mice are subfertile, with litter sizes 50% of wild-type controls, whereas ZP2- and ZP3-knockout mice are absolutely infertile. The reasons for these differences in fertility are secondary to the differences in the formation of the ZP in these knockouts. ZP2-knockout and ZP3-knockout mice fail to form a ZP; this absence of a zona leads to failure of fertilization and progression to the two-cell stage. In contrast, ZP1 knockout mice have a thin ZP, and the integrity of this ZP is compromised; these structural defects have resulted in a 80% reduction in the number of two-cell embryos recovered after superovulation. Thus, ZP absence (in the case of ZP2- and ZP3-knockouts) or alteration (in the case of ZP1 knockout), decreases ovulation and fertilization in vivo, confirming an important predicted role of the ZP in female reproduction.
Apoptotic cell death occurs in oocytes and granulosa cells of both primordial follicles and growing follicles. Members of the Bcl2-related protein family play either positive or negative roles in regulating apoptosis (45,46) (see Chapter 6). Bcl2 and BclxL protect against apoptosis, while Bax, which can heterodimerize with Bcl2 and BclxL, counters their protective effect and promotes cell death when overexpressed. Bax is expressed in granulosa cells and oocytes, and plays a critical role in regulating ovarian-cell death; 6-wk old Bax-deficient mice have three times more primordial follicles than controls and one-half the number of atretic primordial follicles. This difference in the rate of follicu-lar-pool depletion results in the presence of growing, functional follicles at 640 d of age. Despite the presence of growing follicles, no corpora lutea or pregnancies have been seen in these very old mice. Ovulation could be induced by injection of exogenous gonadotropins, indicating that reproductive senescence is caused by a combination of follicular depletion and pituitary dysfunction. Thus, Bax inactivation produces a surplus of nonatretic follicles, extends the function of the ovary into advanced chronological age, and may provide additional insight into the molecular basis of oocyte depletion associated with menopause in humans.
Growth of preantral follicles, corresponding to type 3b to type 5b follicles in the mouse, is gonadotropin-independent, and is regulated primarily by intraovarian and intrafollicular mechanisms. In the FSHp knockout mouse (47) or in the hypogonadal (hpg) mouse, in which a naturally occurring mutation in the gonadotropin-releasing-hormone-gene markedly reduces the synthesis of both FSH and LH from the pituitary (48), preantral follicle growth proceeds normally, confirming the gonadotropin-inde-pendence of this stage of development. However, several mouse models have clearly demonstrated that signaling from the granulosa cells to the oocyte, as well as from the oocyte to the granulosa cells, is necessary for preantral follicle growth and does not require extragonadal input (Table 4B). For example, intrafollicular signaling of kit ligand from granulosa cells to c-kit on the oocyte is critical for preantral follicle development. All mouse oocytes express c-kit, whereas granulosa cells of one-layered growing follicles, preantral follicles, and the outer (mural) layers of preovulatory follicles express kit ligand (49,50). Two hypomorphic kit ligand alleles at the Sl locus, Slt and Slpanda, permit prenatal ovarian development (in contrast to the other alleles mentioned earlier) and initiation of follicular growth, but result in follicular arrest before the two-layer follicle stage (51-53). In Slpanda homozygous mutant ovaries, kit ligand expression is virtually absent (53), because of a large paracentric inversion located 115 kb 5' of the kit ligand coding sequences (54). In addition, blocking antibodies to the c-kit receptor administered to mice during the first 2 wk after birth inhibit ovarian follicular development beyond the one-layer primary-follicle stage (55), further supporting the essential role of kit ligand/c-kit signaling at this stage of follicular development.
Growth-differentiation factor-9 (GDF-9 or Gdf9), a novel, oocyte-expressed member of the transforming growth factor p (TGF-P) superfamily of secreted growth factors, is also necessary for early preantral follicle growth. Gdf9 is first expressed by oocytes of type 3a follicles, and its expression persists in the oocyte through ovulation (56-59). We have generated a GDF-9-deficient mouse model by deleting exon 2, which encodes the entire GDF-9 mature region (60). Heterozygotes of both sexes and males homozygous for the deletion are fertile, but homozygous mutant females are completely infertile. Although follicular recruitment and initiation of growth is grossly normal, no follicles with two or more symmetric or concentric layers of granulosa cells are evident, indicating that folliculogenesis is blocked at the type 3b (primary) follicle stage in these mice (60). This defect in follicular development is not rescued by treatment of the mice with exogenous gonadotropins. In addition, kit ligand and a inhibin mRNA are elevated in these one-layer primary follicles (58). The increased signaling of kit ligand through its receptor, c-kit, in the oocyte, is a probable cause of the increased oocyte size in the Gdf9 knockout ovary (61), further supporting the importance of modulation of kit ligand/c-kit signaling in the postnatal ovary. This finding suggests that GDF-9 is a direct negative regulator of this signaling pathway.
At the early stages of oogenesis in the Gdf9 knockout ovary, the oocytes appear to be fairly normal. However, the absence of GDF-9 signaling, and potentially the resultant increase in kit ligand signaling, eventually leads to defects in oocyte meiotic competence and abnormal germinal vesicle breakdown, and spontaneous parthenogenetic activation of the oocytes, in addition to the increased rate of growth of the oocyte. At the electron-microscopic level, the GDF-9-deficient oocytes have several unusual features, including Golgi complexes composed of single lamellae instead of stacks and a decreased number of cortical granules. Additionally, cell-cell contacts between the oocyte and granulosa cells are unusual in that oocyte microvilli are clustered next to abnormal, large processes from surrounding somatic cells. These follicle cells subsequently invade the perivi-
telline space, and are associated with a loss of oocyte viability (60,61). The combined oocyte and granulosa cell abnormalities lead to eventual death of the oocyte, resulting in a follicular nest with granulosa cells surrounding a collapsed ZP remnant. The cells of the majority of these follicular nests are steroidogenic; these cells appear vacuolated because of the large number of lipid droplets, have an increased number of mitochondria (60), and express P450 side-chain cleavage, P450 aromatase, LH receptor, and a inhibin mRNA (58). Thus, although these nests of cells often resemble small corpora lutea, these follicles express both luteal (i.e., p450 side-chain cleavage and LH receptor) and nonluteal (i.e., p450 aromatase and a inhibin) markers, suggesting that the early loss of the oocyte (and possibly the absence of GDF-9) alters the differentiation program of these granulosa cells.
In the periovulatory follicle, the oocyte secretes important growth factors, which stimulate the synthesis of hyaluronic acid necessary for cumulus expansion and repress the synthesis of LH receptor and urokinase plasminogen activator (uPA) (59). Because of the early block in the growth of the Gdf9 knockout ovary, and based on the continued expression of GDF-9 beyond ovulation, we have studied the biological actions of GDF-9 in the periovulatory period, using pregnant mare serum gonadotropin (PMSG)-induced mouse granulosa cells cultured with recombinant mouse and human GDF-9 protein. Recombinant mouse GDF-9 induces hyaluronan synthase 2 (Has2), cyclooxygenase 2 (Cox2), and steroidogenic acute regulator protein (StAR) mRNA synthesis, and suppresses urokinase plasminogen activator and luteinizing hormone receptor (LHR) mRNA synthesis (57). In addition, GDF-9 stimulates in vitro cumulus expansion of oocytecto-mized cumulus cell-oocyte complexes (i.e., complexes in which the oocyte has been microsurgically removed) (57). Thus, GDF-9 is essential for granulosa-cell growth and function at early stages, and is also the oocyte-secreted factor responsible for modulating the expression of a number of cumulus cell genes critical during the periovulatory period.
The theca layer forms when the follicle achieves two layers of granulosa cells, and provides a source of aromatizable androgen to the adjacent granulosa cells, which is crucial for follicular estrogen production by enzymatic conversion (62). Theca cells differentiate from mesenchymal or stromal precursors adjacent to developing follicles. Theca-interstitial cell culture experiments in vitro show that rat preantral follicles with 2-5 layers of granulosa cells (but not one-layer or antral follicles) secrete a factor in the absence of gonadotropins that induces theca layer differentiation, including expression of cytochrome P450 17a-hydroxylase-C17-20 lyase (63). In GDF-9-deficient mice, a theca layer fails to form, despite the presence of increased FSH and LH (58,60). However, an identifiable theca layer is formed around the multilayer preantral follicles in the FSH-deficient ovary model (47). Taken together, these data support the presence of a paracrine, inductive signal secreted from preantral follicles with two or more layers of granulosa cells, which is necessary for theca layer development. GDF-9 is possibly an important direct or indirect regulator of these theca cell "recruitment/differentiation" factors.
Primordial and small preantral follicles (type 3a and 3b) represent a minute fraction of total ovarian cells, making it difficult to isolate genes and proteins involved in the earliest stages of follicular development. In addition, these stages have generally not proven amenable to extraovarian manipulation. However, development of these mouse models, in which folliculogenesis is arrested at an early stage, have defined some key factors involved in initiation and early preantral follicle growth.
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