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Endocrinology Vol. 142, No. 6 2184-2193
Copyright © 2001 by The Endocrine Society

ARTICLES

Perspective: The Ovarian Follicle—A Perspective in 20011

JoAnne S. Richards, Ph.D.

Department of Molecular and Cellular Biology Baylor College of Medicine Houston, Texas 77030

Address all correspondence and requests for reprints to: JoAnne S. Richards, Ph.D., Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: joanner{at}bcm.tmc.edu


   Introduction
Top
 Introduction
Organizers of the ovarian...
 FSH: An external driver...
 LH: an external switch...
 When local organizational...
 Summary
 References
 
In the past century, tremendous progress has been made in our understanding of the endocrine regulation of fertility. The identification and purification of peptide and steroid hormones and our knowledge of their actions in endocrine cells has led to major advances in contraceptive development, in vitro fertilization, and endocrine therapy. The advances in molecular biology have led to our understanding of how hormones and growth factors regulate the expression of specific genes in endocrine cells. Today, the disruption and deletion of selected genes in mice is becoming routine and null mutations for many proteins expressed in and regulating endocrine cell function have been generated. These mutant mice have brought molecular reproductive endocrinologists full circle. We have returned to ablation-replacement bioassays used by the early endocrinologists, albeit in a much more sophisticated way and with more elaborate and specific mechanisms. The new age endocrinology and our knowledge of endocrine cell function will expand even more explosively now that the cloning of the mouse and human genomes nears completion. In the next century, new hormones, new receptors, new signaling pathways for old receptors, and new transcription factors will no doubt be identified and our understanding of the complex pathways regulating endocrine cells should be markedly enhanced. The goal of this minireview is to highlight some recent advances in our knowledge of how hormones and growth factors impact the formation and growth as well as the termination of the ovarian follicle. Of particular relevance is the importance of the microenvironments that control oocyte-cumulus cell functions and granulosa-theca cell functions within the follicle and how hormones, such as FSH and LH, impact these microenvironments. Highlighting what we know about the ovarian follicle in 2001 may help us set goals for 2100. Hopefully, basic science and clinical discoveries in the next century will unravel the endocrine, molecular, and cellular bases of premature ovarian failure, polycystic ovarian syndrome, and ovarian cancer.


   Organizers of the ovarian follicle and its microenvironments
Top
 Introduction
 Organizers of the ovarian...
FSH: An external driver...
 LH: an external switch...
 When local organizational...
 Summary
 References
 
The formation of primordial follicles depends on the presence of the female germ cells, the oocytes, and their interactions with surrounding somatic cells, the pregranulosa cells. Our current knowledge of the genes and factors that control gonadogenesis and oocyte migration to the genital ridge is increasing but still remains relatively limited (1, 2). Members of the transforming growth factor-ß (TGFß) family appear to be key regulators of ovarian organization and development at several stages. Bmp4 and Bmp8b, which are produced by the extraembryonic ectoderm are essential for the generation of primordial germ cells (PMGs) (3) (Figs. 1 and 2). Once germ cells are formed, gonadal development is regulated by the expression of specific transcription factors in the somatic cells of the indifferent gonad (Fig. 1Go). Among these factors are Lhx1, Lhx9, WT1, Wnt 4, and the orphan nuclear transcription factor steroidogenic factor-1 (SF-1) (1, 4, 5) (Figs. 1Go and 2Go). Although originally cloned and identified as a transcription factor that regulates steroid hydroxylase genes, it is clear that SF-1 has other functions. Mice null for SF-1 have no gonads, no adrenal and altered pituitary and hypothalamic function, corresponding to sites of its expression in somatic cells of these tissues (5, 6). SF-1 has now been shown to regulate transcription of many genes including, Müllerian-inhibiting substance (MIS) (7, 8), LHß (9) and the FSH receptor (10) to name a few. Expression of SF-1 and the FSH receptor are also mediated by the E-box binding transcription factor upstream stimulatory factor (USF) (11, 12, 13), which also regulates such diverse genes as RIIß the regulatory subunit of type II cAMP-dependent protein kinase and COX-2 cyclo-oxygenase-2 (14). In addition, transcription of SF-1 and MIS, as well as other gonadal genes, is regulated by members of the GATA family of transcription factors of which GATA-4 and GATA-6 are expressed in granulosa cells of developing follicles (15, 16).



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  		Figure 1. Schematic of expression and interactions of local organizers and external impact factors that control ovarian follicular development. Follicular development is dependent on sequential changes in local organizing factors (listed vertically for each stage of follicular development), cell-cell interactions as well as on external impact factors such as the pituitary gonadotropins FSH and LH. Formation of primordial germ cells (PGCs) occurs as a consequence of signals (Bmp4 and Bmp8) from the extraembryonic ectoderm (EEE) as well as cell-cell interactions via gap junctions, connexin 43. Somatic cells of the embryonic gonad determine gonadal sex and viability of the oocytes. Some factors involved in this process include Kit ligand KL, SF-1, Wilms tumor suppressor 1 (WT1), and Wnt 4 as well as connexin 43. After gonadogenesis, oocytes within primordial follicles express various factors that help initiate follicle growth. Among these are Fig{alpha}, a transcription factor obligatory for expression of zona pellucida proteins (ZP1,2,3), connexin 37, GCNF, Dazla, c-kit receptor. At a slightly later stage of development, oocytes control another critical event, the formation of the basal lamina and the appearance of theca cell layer, the latter of which is a target of GDF-9, a TGFß family member (indicated by the heavy red vertical arrow) (also see Fig. 2Go). The oocyte and the surrounding cumulus cells form one microenvironment in the follicle. The growth of these small follicles is regulated by other factors from granulosa cells such as MIS, SF1b (NR5A2), WT1, GATA-4, and Wnt4. As follicles continue to grow, additional growth supporting factors begin to appear. These include IGF-1, ER{alpha}/ß subtypes, FSH receptor, and cyclin D2. Once the follicle reaches a critical size, a consequence of granulosa cell proliferation, the granulosa cells attached to the basal lamina become distanced from the oocyte-cumulus complex and establish a second microenvironment in the follicle. At this time, the follicle becomes dependent on FSH and external factors to maintain these outer cells by interactions with the local factors such as IGF-1 (Fig. 3Go), whereas the oocyte-cumulus complex continues to reside as a special microenvironment. Ultimately, granulosa cells of preovulatory follicles begin to differentiate and express genes such as aromatase/CYP19, LH receptor and activin, inhibin, and Sgk. Theca cells differentiate to produce androgens via P45017{alpha}. These changes increase steroidogenesis and ultimately lead to the positive feedback of estradiol to trigger the LH surge. Following the impact of the LH surge, genes associated with ovulation (both cumulus expansion and follicle rupture) and luteinization are induced (Fig. 3).

 


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  		Figure 2. A schematic of the spatial and temporal expression of local organizing factors in the transition of primordial follicles to primary follicles. The generation of primordial germ cells is dependent on the expression of the organizers Bmp4 and Bmp8b coming from somatic, extraembryonic ectoderm. Gonadogenesis is dependent on the migration of the germ cells to the gonadal ridge and the expression of specific factors in the indifferent cells of the presumptive gonad. Primary follicles are established by the appearance of the Fig{alpha}-dependent transcription of zona pellucida proteins (ZP1,2,3), an extracellular matrix that defines and organizes the granulosa cells around the oocyte. The next layer of complexity involves the formation of the basal lamina, another extracellular matrix upon which the theca cells are organized, a process controlled in part by the oocyte-derived TGFß molecule, GDF-9. The potential sites of action of GDF-9 are indicated by the heavy yellow arrows and are discussed in the text. At this time the granulosa cells also express MIS, FSH-receptor and IGF-1/IGF-1 receptor, and cyclin D2. In response to the activation of these molecules granulosa cells proliferate, albeit slowly.

 
Recently, another orphan nuclear receptor within the NR5A subfamily of nuclear receptors NR5A2 (also known as liver receptor homologous protein-1) (17) has been identified and is expressed in equine and rodent ovarian tissues, specifically granulosa cells of growing follicles (18). NR5A2 (referred to herein as SF-1b) has high homology to SF-1 (17, 18). This raises interesting questions about the cell-specific expression and functional activities of these two highly-related but distinct receptors. It will be essential now to disrupt/delete the NR5A2/SF-1b gene and determine its role in the ovary (and other endocrine tissues?) as well as to perform tissue-specific knockouts of each SF-1 receptor, thereby bypassing the developmental defects in ovarian formation. Although other orphan receptors are expressed at high levels in the adult ovary, their specific functions in follicular cell differentiation are not entirely clear. For example, female mice null for DAX-1 appear normal (4, 19) despite the more severe phenotype in humans (4). Mice null for COUP-TFII are embryonic lethal at day E9.5 precluding an analysis of its function in ovarian tissues (20). Although COUP-TFII is a repressor of SF-1 on transcription of some genes including {alpha}2 macroglobulin in the rat ovary (21), whether its specific deletion in the ovary would alter the transcriptional activation of genes regulated by SF-1 and other steroid receptors is unclear. Lastly, specific coactivators of SF-1 in addition to CBP/p300 need to be identified and characterized.

The oocyte also plays a critical role in the formation and growth of ovarian follicles. Recent studies have identified a basic helix-loop-helix transcription factor that is expressed specifically in oocytes and is obligatory for the formation of primordial follicles. This factor has been called factor in the germline {alpha} (Fig {alpha}) (22). In mice null for Fig {alpha}, gonadogenesis proceeds normally but primordial follicles are lacking. Because Fig {alpha} is a critical transcription factor for genes encoding zona pellucida (ZP) proteins, it is tempting to speculate that expression of all the zona proteins is needed to provide an extracellular matrix for granulosa cell attachment. This may be critical for initiating or maintaining appropriate ooctye-granulosa cell interactions such as the formation of gap junctions and for establishing specific cell signaling events at the cell surface of granulosa cells and oocytes (23). Fig {alpha} may also regulate the expression of other oocyte-specific genes (Fig. 2Go). Dazla, a germ cell-specific RNA binding protein, is also essential for the maintenance and differentiation of germ cells (24) but is not essential for somatic cell expression of steroidogenic enzymes (25). Although many investigators use an antibody against germ cell nuclear antigen-1 GCNF-1, no one yet knows what this protein does. The orphan nuclear receptor, germ cell nuclear factor (GCNF) is another germ cell specific factor (26). What genes in the oocyte are controlled by GCNF remains unclear although there is some evidence that GCNF has repressor activity (26). Gap junction proteins, connexin 43, and connexin 37 are essential for ovarian cell function. In mice lacking connexin 43, primordial germ cells are absent in embryos and follicles do not form (27). In mice lacking functional connexin 37, junctions between the oocyte and granulosa cells do not form, the oocyte disappears prematurely and luteinized structures are observed (28). New complementary DNA (cDNA) and genomic array analyses will hopefully yield additional oocyte specific genes whose functions will need to be analyzed.

The signal or signals that initiate the growth of primordial follicles to the primary stage remain unknown. However, early follicular growth has been shown to require Kit ligand produced by granulosa cells and c-kit receptor present on oocytes (1) as well as an oocyte-specific member of the TGFß family, growth and differentiation factor 9 (GDF-9) (1) (Figs. 1Go and 2Go). In mice null for GDF-9, theca cells fail to organize appropriately in the stage 3b follicles, oocyte growth is abnormal, and despite the presence of cyclin D2 in granulosa cells of these mice (29), proliferation is impaired (30, 31). The mechanisms by which GDF-9 regulates the organization of the theca are not yet clear and may involve the action of GDF-9 on granulosa/cumulus cells that then produce regulatory molecules (extracellular matrix?) upon which theca cells can attach. In this regard, GDF-9 has been shown to stimulate granulosa cell proliferation and suppress FSH-stimulated granulosa cell differentiation in culture (32). Alternatively, GDF-9 may alter theca cell function directly (33) (Fig. 2Go). The converse may also be true. Once the theca cell layer is formed, it can then impact the function of granulosa cells, establishing another microenvironment within the follicle, the granulosa-theca cell component (Fig. 2Go). Although the TGFß family member MIS is not essential for follicular growth, mice null for MIS exhibit an increase in the number of growing follicles suggesting that the MIS that is expressed in granulosa cells of primary and small antral follicles serves to inhibit growth (34). The specific roles of other TGFß family members TGFß itself, activin and inhibin in ovarian follicular growth are also emerging and will no doubt provide more examples of the intricate regulation of the microenvironments within the follicle (35, 36, 37, 38). The functions of these molecules depend not only on the expression of specific receptors but also on the activation of latent precursor peptides. Thus, this system is complex and regulated at multiple levels.


   FSH: An external driver that impacts the microenvironments of growing follicles
Top
 Introduction
 Organizers of the ovarian...
 FSH: An external driver...
LH: an external switch...
 When local organizational...
 Summary
 References
 
Although the organization of the primordial follicle and the initiation of follicle growth can occur in the absence of pituitary gonadotropins, small growing follicles are responsive to FSH and the final growth of preovulatory follicles is critically dependent on FSH. The obligatory requirement for FSH appears to coincide with a time when granulosa cell proliferation and follicle growth lead to a spatial separation of the cumulus cells from the mural granulosa cells. This spatial separation may necessitate the regulation mural granulosa cells by factors/hormones other than those emanating from the oocyte-cumulus complex (Figs. 1Go and 3Go). Because the growing follicle is avascular, gradients of factors appear to establish zones and microenvironments within the follicle. Knockout experiments have shown that follicular growth is arrested in mice lacking FSHß (39) or the FSH receptor present on granulosa cells (40, 41). Follicular growth is also dependent on insulin-like growth factor I (IGF-1) and its receptor (42, 43), the synthesis of estradiol by aromatase CYP19 (44), the presence of both estrogen receptor (ER) subtypes {alpha} and ERß (45, 46, 47), and the LH receptor (48, 49) (Fig. 2). As expected from the plethora of studies of these hormones in many mammalian models, each of these knockouts exhibits an abnormal ovarian phenotype. These signaling molecules appear to comprise a self-sustaining interdependence in granulosa cells of growing follicles (Fig. 3Go). For example, although IGF-1 alone cannot induce expression of sterodigenic enzymes (2), it enhances the effects of FSH presumably by increasing energy utilization and cell survival in granulosa cells. IGF may also regulate the levels of ERß (Richards, J. S., unpublished data). Specific genes induced by estradiol have not yet been identified but estradiol also enhances the effects of FSH (50). Estradiol is not required for the expression of IGF-1 that persists in hypophysectomized rats and mice (51). In this light, FSH is a key driver of granulosa cell differentiation and the induction of specific genes with IGF-1 and estradiol exerting supportive roles (Fig. 3Go).



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  		Figure 3. A schematic the interactions of the local organizational factors and impact factors that operative in preovulatory, ovulatory, and ovulating follicles. The transition of small antral follicles to preovulatory follicles is dependent on both the local organizational factors such as IGF-1 and the external impact factors FSH and LH. LH regulates theca cell differentiation. FSH dictates specific changes in granulosa cell gene expression, enhanced proliferation, and the formation of the antrum that clearly separates the oocyte-cumulus component of the follicle from the mural layer of endocrine cells. FSH and IGF pathways interact to control the expression/activation of specific kinase cascades. Two kinases in these cascades are Sgk and PKB, which activate forkhead transcription factors, FKRH, and other cell survival pathways, such as the expression and movement of the glucose transporter Glut-4 to the plasma membrane (see text for further discussion). The LH surge terminates the follicular program of gene expression in granulosa cells and turns on genes controlling cumulus expansion (COX-2, HAS, TSG-6), ovulation (PR, ADAMTS-1), and luteinization (C/EBPß and Egr-1). LH acts directly on granulosa cells and theca cells but likely acts indirectly on the cumulus cells. For example, LH may act on granulosa cells (heavy red arrow) to stimulate the synthesis of a product(s) that is released and impacts cumulus cell function leading to the induction of COX-2 and TSG-6. One oocyte-derived factor that has been implicated in cumulus cell expression of COX-2 is GDF-9. Because GDF-9 is present in ooctyes at all stages of follicular growth, LH may stimulate the production of a specific protease to activate latent GDF-9. COX-2 in turn appears critical for the expression of TSG-6 (Ochsner, S., and J. S. Richards, unpublished observations). Oocytes of ovulating follicles also express mater, a maternal gene product necessary for embryogenesis (117 ). In the mural granulosa cells, LH acts to induce other genes involved in the ovulation process (PR, ADAMTS-1, and Cathepsin L). Genes involved in the establishment of luteinization include C/EBPß, Egr-1 and p27kip1. Once terminal differentiation is complete, luteal cells express steroidogenic enzymes (P450scc and StAR),transcription factors (JunD, Fra2, ER{alpha}) as well as Sgk, a kinase in the PI3-K/PDK1 pathway in a constitutive manner.

 
Despite the intensive amount of research on the structure and function of the ER, the specific role(s) of estradiol and its receptors in the ovary remain unexpectedly elusive. The discovery of ERß and it elevated expression in granulosa cells was a major surprise (52). Its presence in granulosa cells of growing follicles complicated the interpretation and approach for doing ER knockout experiments. The ER knockouts have clearly shown that neither the absence of ER{alpha} alone nor the absence of ERß alone completely prevents follicular growth or the response of ovarian cells to gonadotropins (46, 47). This is probably best explained because granulosa cells express both ER{alpha} and ERß, clearly demonstrable at the RNA and protein levels (53, 54, 55). However, the ER{alpha}/ß double knockout does show impaired follicular growth and surprisingly a trend toward a reversal of the female phenotype (45A ). The ovaries of adult ER{alpha}/ß mice begin to exhibit Sertoli-like seminiferous tubules that lack germ cells and express genes associated with testis formation, SOX-9 and MIS. Similar structures and the sex-reversed expression of these genes have been reported in the Wnt4 (56) and aromatase CYP19 knockout mice (44). Conversely, disruption of SOX-9 leads to sex reversal of XY males to a female phenotype, indicating that expression of SOX-9 is suppressed in female gonadal development (57). Sex reversal of XY male gonads to a female phenotype also occurs in mice null for fibroblast growth factor 9 (Fgf9), indicating that Fgf9 may control expression of SOX-9 and MIS (57A ). What is the role of estradiol and its receptors in the ovary? Does estradiol and its receptors block expression of male-related genes such as Fgf-9, SOX-9 and MIS? Evidence in support of this is the decrease in expression of MIS in large antral, estrogen-producing follicles. Equally intriguing is the observation that estrogens are weak activators of consensus ERE-reporter constructs transfected into granulosa cells compared with the robust activation of these same constructs in granulosa cells by cAMP inducing agents such as FSH and forskolin. Does this suggest that granulosa cells have specific coactivators or corepressors of ER{alpha}/ß that might also be regulated by gonadotropins? What is the role of the nuclear receptor corepressor RIP140 that leads to female infertility and anovulation (58). Is it a corepressor of ER or other nuclear receptors? The mystery around estrogens in the ovary remains.

Although the actions of FSH have been exhaustively studied and thought to be well defined, recent studies indicate that there are some novel twists to FSH signal transduction as well. Specifically, FSH stimulates the phosphorylation of two closely related kinases that are downstream targets of the IGF-1/PI3-K/PDK1 pathway (59, 60). These kinases are Sgk (serum and glucocorticoid-induced kinase) and protein kinase B (PKB) (Fig. 3Go). FSH-induced phosphorylation of these kinases occurs by mechanisms that are independent of cAMP-dependent protein kinase A (PKA) but require PI3-K phophoinositide induced kinase. Importantly, the effects of FSH are mimicked by forskolin, 8-bromo-cAMP as well as IGF-1. Potential mediators of this new avenue of cAMP action in granulosa cells are the cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) (61, 62) that are expressed in granulosa cells and likely activate Ras-like small GTPases that impact PI3-K (60). Important targets of PKB and Sgk include members of the forkhead family (FOX) of transcription factors (63). At least one of these (FKHR) is expressed in mouse and rat granulosa cells (Richards, J. S., unpublished data) and is a distant relative of human FOXL2 mutations of which are associated with abnormal ovarian development (64, 65). The activation of this pathway by FSH may explain why IGF can enhance the actions of FSH on granulosa cells when FSH is present at low concentrations (synergy between PKA and PI3-K pathways) but not when FSH is at higher concentrations. At higher concentrations, FSH can activate not only the PKA pathway but also the PI3-K pathway. In this way, FSH has direct entry into the cell survival pathway usually associated with IGF/PDK1/PKB (60). Therefore, FSH has the potential to maintain cell survival pathways and at the same time, via PKA, impact differentiation events obligatory for induction of specific genes (Fig. 3Go).

In addition to this novel cAMP signaling pathway, newly identified variants of the FSH receptor are expressed in the ovary and can activate Ca2+/protein kinase C signaling activities (66, 67). These variants have the external ligand binding domain of the FSH GPCR, but this is then spliced to a single pass growth factor-type transmembrane receptor. Of note, this altered form of FSH receptor is increased in ovaries of mice treated with PMSG. What is the functional role of this variant FSH receptor in granulosa cell differentiation? What about the LH receptor? Does it also have variant forms? Does LH also stimulate phosphorylation of PKB and Sgk? Probably yes. Since LH receptor knockouts do not form corpora lutea, can FSH substitute for LH? What other defects are there in these FSH and LH receptor knockout mice? Are theca cells normal? Is the interstitial cell compartment normal?

Follicular growth involves increased proliferation of granulosa cells and the regulation/activation of cell cycle kinase cascades. One activator of the cell cycle cyclin D2 is selectively expressed and shown to be critical for granulosa cell proliferation (29, 68). Both estradiol and FSH appear to impact the expression of cyclin D2 but the cellular signaling pathways that alter transcription factor activity on the cyclin D2 promoter in granulosa cells have not been examined. In addition to estradiol and FSH, members of the TGFß family, namely MIS, inhibin and activin, also appear to impact granulosa cell proliferation. Mice null for inhibin {alpha} develop tumors at the time of puberty; tumor formation depends on the presence of an intact pituitary, FSH/LH (69). Likewise, mice overexpressing the {alpha}-subunit of the glycoprotein hormones develop ovarian tumors (70), indicating that the gonadotropins, activins/inhibin and steroids all impact cell cycle regulation in granulosa cells. Whether or not the effect of the gonadotropins is mediated in part by steroids (estradiol and its receptors?) is not known. The rare occurrence of granulosa cell tumors indicates that this system is normally under very stringent controls—apoptosis and luteinization, both of which remove granulosa cells from the cell cycle and both of which are critical to the controlled cessation of ovarian follicular growth.

FSH and LH receptors are not the only GPCR present on granulosa cells. The pituitary adenylyl cyclase activating peptide (PACAP) and its receptors are present and regulated in a manner suggestive of a key role in follicular development or ovulation (71, 72, 73). There are new GPCR that have been identified and that are expressed in ovarian tissues (74, 75). The roles of these novel GPCR need to be analyzed, and the ligands that activate them need to be identified. There are also many other kinases that are involved in granulosa cell function, one of which is calmodulin kinase IV (76). Mice null for CaMKIV exhibit impaired ovarian follicular development and luteinization (76).


   LH: an external switch that terminates ovarian follicular growth
Top
 Introduction
 Organizers of the ovarian...
 FSH: An external driver...
 LH: an external switch...
When local organizational...
 Summary
 References
 
The main function of the ovary is to release a fertilizable oocyte. During follicular growth, the oocyte acquires functions to make it competent for fertilization (77). What these functions are is not entirely clear, but they appear to require FSH and insulin (or endogenous IGF-1?). During the growth of preovulatory follicles, aromatase and the LH receptor are induced in granulosa cells. Increased levels of serum estradiol then trigger the LH surge. The LH surge rapidly acts on granulosa cells of the preovulatory follicles to terminate the follicular program of gene expression while at the same time stimulating the expression of genes required for ovulation and luteinization (50, 78) (Figs. 1Go and 3Go). Genes that are induced rapidly but transiently have been linked to the ovulation process (79). These include two transcription factors the progesterone receptor (PR) (80) and CAAT enhancer binding protein ß C/EBPß (81) and the enzyme COX-2 (82, 83). Female mice null for each of these genes are infertile and fail to ovulate (78, 79). Because follicular growth in these mice is normal, the mutation of these genes provides a closer link to steps specifically involved in the ovulation process compared with genes regulating follicular growth (Fig. 3Go).

The anovulatory phenotype of mice null for PR is associated with impaired expression of two proteases. These proteases are ADAMTS-1 (a disintegrin and matrix metalloproteinase with thrombospondin-like repeats) (84, 85) and Cathepsin L (85). Whether or not these proteases directly activate MMP13 (collagenase) to break down extracellular collagen in the stroma of the ovary or exert other specific functions is not yet known. Based on the different structural domains of ADAMTS-1, it appears to be a multifunctional protein. METH-1, the human homolog of ADAMTS-1 has been shown to have potent antiangiogenic activity (86). This functional activity is likely conferred by the thrombospondin-like repeats of the molecule because thrombospondin is a potent antiangiogenic factor. Based on similarities to the aggrecanase ADAMTS-4, ADAMTS-1 may degrade proteoglycans. Its disintegrin domain may allow it to bind integrins, leading to the activation of cell signaling. Which of these functions if any are critical for ovulation needs to be determined. The specific role of cathepsin L in ovulation is also unclear at this time. In these PR null mice, the expression of COX-2 is normal, indicating that the effects of PR are not mediated by COX-2. The identification of other PR regulated genes will provide further insight into this mutant mouse model.

In mice null for then prostaglandin synthesizing enzyme COX-2 (87, 88) or the prostaglandin E2 receptor, EP2 (89, 90, 91) follicular growth is normal and LH induces the expression of PR in granulosa cells of ovulatory follicles. However, expansion of the cumulus cells around the oocyte is abnormal indicating that the formation of this special matrix is obligatory for successful extrusion of the cumulus-cell ooctye complex. In the COX-2 KO mice, ovulation can be restored by treating mice with PGE2 (92). A new twist to this process is the evidence that the oocyte-derived growth factor, GDF9, appears to be involved in the expression of COX-2 and cumulus expansion that are initiated by the LH surge (30) (Fig. 3Go). The role of GDF9 in this process raises an interesting dilemma. Because GDF9 is expressed in the oocyte throughout follicular development one wonders why this action of GDF9 is dependent on the LH surge. Because GDF9, like other TGFß family members, is released as an inactive prohormone, it is possible that the LH surge leads to the synthesis/activation of a mural granulosa-derived protease that in turn activates GDF9 localized to the cumulus complex. Alternatively, LH may indirectly or directly (?) induce proteases or specific receptors for GDF9 that would be critical for its function on cumulus cells. Answers to these important questions will surely be forthcoming once more is known about follicular proteases and GDF9 receptors (Fig. 3Go). The specific defects relating to ovulation failure in mice null for C/EBPß remain to be resolved. In fact, C/EBPß null mice also appear to have abnormal corpora lutea (93).

Other genes recently shown by differential display RT-PCR to be induced by LH include carbonyl reductase (94), 3{alpha}-hydroxysteroid dehydrogenase 3{alpha}HSD (95), regulator of G protein signaling RGS-2 (96), tumor necrosis factor induced gene-6 TSG-6 (97), and early growth regulator-1 (Egr-1) (98). Their diverse functions and spatial expression in the ovary illustrate the global effect of the LH surge on ovarian cells during the ovulation and luteinization processes. Both carbonyl reductase, which is induced exclusively in the theca/stroma regions of the ovary, and 3{alpha}HSD, which is induced in theca, granulosa, and luteal cells, can metabolize (detoxify?) physiologically active steroids and prostanoids. The importance of these activities to the ovulation or luteinization processes is not yet known. RGS-2 (96) and TSG-6 (99) are selectively expressed in cumulus cells and granulosa cells on the inner antral surface. Because RGS-2 null mice are fertile, a critical role for this regulator of GPCR signaling in the ovary seems unlikely (100). TSG-6 is a hyaluron binding protein that appears to be important for cumulus expansion because it binds intera-inhibitor (IaI) to form a stable matrix complex that characterizes cumulus expansion (99) (Figs. 1Go and 3Go). TSG-6 mutant mice have not yet been reported. Mice null for Egr-1 are infertile and may have ovarian as well as pituitary defects (101).

Cyclin D2 null mice are also anovulatory for reasons that are not entirely clear (102). Because granulosa cell proliferation is impaired in these mutant mice, ovarian follicles are small and contain fewer granulosa cells. However, these follicles can respond to exogenous hormone treatments and express the follicular and luteal patterns of gene expression in a normal temporal pattern (102). Thus, the underlying molecular or cellular defect that prevents ovulation in these mice remains to be determined. One possibility is that a reduced number of cumulus cells may preclude normal cumulus-oocyte expansion and thereby prevent extrusion of the ooctye.

Luteinization is the LH-induced process, separate from cumulus expansion, in which mural granulosa cells and theca cells are completely reprogrammed (Figs. 1Go and 3Go). They cease to divide, undergo hypertrophy, and become highly steroidogenic. Expression of the follicular program of gene expression, including the cell cycle activator cyclin D2, is terminated; expression of cell cycle inhibitors p21CIP and p27KIP are increased (29, 68). Genes selectively expressed at high levels in luteal cells are induced. These include P450scc (50, 78, 103), StAR (2), Sgk (104), and the AP1 factors Fra2 and JunD. Although luteinization appears to be impaired in mice null for either p27KIP (105, 106, 107) or the transcription factor Egr-1(101), the molecular and cellular basis for the effects of these factors remains unclear. With the exception of Egr-1, which is only transiently induced in response to the LH surge, the constitutive expression of the other genes indicates a major switch in the molecular and signaling mechanisms the underlie the process of luteinization. Understanding the events that reprogram the granulosa cells to luteinize and that maintain their steroidogenic functions is critical to understanding successful implantation and pregnancy in all mammals.


   When local organizational factors and impact factors fail
Top
 Introduction
 Organizers of the ovarian...
 FSH: An external driver...
 LH: an external switch...
 When local organizational...
Summary
 References
 
Less than 1% of the oocytes present in the ovaries of mammals at birth ever ovulate. The remaining oocytes and follicles become atretic and undergo programmed cell death because local regulatory factors or impact factors such as FSH and LH are insufficient to sustain growth. Follicles must be in the right place at the right time and receive the right signal to maintain growth and ultimately ovulate. Thus, tremendous evolutionary pressures operate to restrict the number of ovulating follicles in mammals. In general, the apoptotic mechanisms that control oocyte and follicular cell death are similar to those that regulate other cells and are being intensely studied by several groups (108, 109, 110, 111). If programmed cell death could be obviated, the number of ovulating follicles might be enhanced. This would certainly be beneficial for many domestic animal breeding programs as well as in assisted fertilization in women.


   Summary
Top
 Introduction
 Organizers of the ovarian...
 FSH: An external driver...
 LH: an external switch...
 When local organizational...
 Summary
References
 
One of the major issues facing the world today is the exponential growth in world population. New approaches to fertility control are needed and will depend on our ability to selectively regulate ovarian cell functions, such as ovulation. The applications of DD-RT-PCR, cDNA array technology, and subtractive tissue-specific cDNA libraries (112) combined with increased access to genomic information (74) and tissue-specific data bases (113) should lead to many new insights into factors that control ovarian function. The next decades promise new insights into how the ovary is formed and how the microenvironments within the growing follicles interact. Our knowledge of the early stages of gonadogenesis is still minimal despite some of the exciting recent discoveries. Several areas that will continue to develop rapidly are the identification of TGFß family members, their receptors and the specific events within the ovary that they control. We need to know more about theca cell functions and genes that regulate these cells. Little is known about the cofactors, coregulators, and corepressors of transcription factor activity in the ovary. We need to understand molecular and cellular bases of premature ovarian failure (114), polycystic ovarian syndrome (115), and ovarian cancer (116). For the latter, we need markers of early stages of tumor formation. To understand and regulate these conditions of female infertility, we also need to know the molecular, cellular, and endocrine events that regulate the normal processes of follicle growth and ovulation. The focus on LH-induced genes will no doubt provide new targets for contraceptive research and also should identify additional genes that control the transformation of proliferating granulosa cells of the follicle into terminally differentiated, nondividing cells of the corpus luteum. The new age of molecular reproductive endocrinology will be exciting and hopefully new ways to improve women’s health and fertility will be forthcoming.


   Footnotes
 
1 This work was supported in part by NIH Grants HD-16229 and 16272, and Specialized Cooperative Program in Reproductive Research Grant 02495. Back

Received March 2, 2001.


   References
Top
 Introduction
 Organizers of the ovarian...
 FSH: An external driver...
 LH: an external switch...
 When local organizational...
 Summary
 References
 

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