JoAnne S. Richards

MAPK3/1 (ERK1/2) in Ovarian Granulosa Cells Are Essential for Female Fertility

Regulating Oocyte Maturation Understanding exactly how ovarian follicles mature to generate fertile eggs is key to many aspects of fertility treatment. When the pituitary surge of luteinizing hormone (LH) binds to its receptor on granulosa cells of preovulatory follicles, a cascade of signaling events triggers granulosa cells to become luteal cells and the oocyte to resume meiosis. Fan et al. (p. 938; see the Perspective by Duggavathi and Murphy), using the mouse as a model system, targeted disruption of the kinases ERK1 and ERK2 selectively in granulosa cells. The kinases were essential in vivo mediators of LH induction of ovulation and luteinization. Targeted disruption of the kinases derails the molecular events that mediate induction of female reproductive development. A surge of luteinizing hormone (LH) from the pituitary gland triggers ovulation, oocyte maturation, and luteinization for successful reproduction in mammals. Because the signaling molecules RAS and ERK1/2 (extracellular signal–regulated kinases 1 and 2) are activated by an LH surge in granulosa cells of preovulatory follicles, we disrupted Erk1/2 in mouse granulosa cells and provide in vivo evidence that these kinases are necessary for LH-induced oocyte resumption of meiosis, ovulation, and luteinization. In addition, biochemical analyses and selected disruption of the Cebpb gene in granulosa cells demonstrate that C/EBPβ (CCAAT/Enhancer-binding protein–β) is a critical downstream mediator of ERK1/2 activation. Thus, ERK1/2 and C/EBPβ constitute an in vivo LH-regulated signaling pathway that controls ovulation- and luteinization-related events.

The ovary: basic biology and clinical implications

The classical view of ovarian follicle development is that it is regulated by the hypothalamic-pituitary-ovarian axis, in which gonadotropin-releasing hormone (GnRH) controls the release of the gonadotropic hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and that ovarian steroids exert both negative and positive regulatory effects on GnRH secretion. More recent studies in mice and humans indicate that many other intra-ovarian signaling cascades affect follicular development and gonadotropin action in a stage- and context-specific manner. As we discuss here, mutant mouse models and clinical evidence indicate that some of the most powerful intra-ovarian regulators of follicular development include the TGF-beta/SMAD, WNT/FZD/beta-catenin, and RAS/ERK1/2 signaling pathways and the FOXO/FOXL2 transcription factors.

Perspective: The Ovarian Follicle—A Perspective in 20011

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 oocytecumulus 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.

Ovulation : New factors that prepare the oocyte for fertilization

Ovulation is a complex LH-induced process that allows the release of a fertilizable oocyte. Critical to ovulation is the proper formation of an extracellular hyaluronan (HA) rich matrix by the cumulus oocyte complex (COC), a process called expansion. During expansion genes associated matrix formation such as hyaluronan synthase 2 (HAS-2) are induced rapidly in COCs. To stabilize the long hyaluronan polymers, various HA binding proteins are covalently (or non-covalently) linked with hyaluronan. Some of the hyaluronan binding factors that have been identified in the COC matrix are the serum derived factor inter-alpha trypsin inhibitor (IalphaI) and tumor necrosis factor stimulated gene-6 (TSG-6). The latter is dependent on the induction in cumulus cells of cyclooxygenase-2 (COX-2) the limiting enzyme in the synthesis of prostaglandins (primarily PGE) that bind the PG receptor subtype EP2, leading to increased cAMP. TSG-6 and the heavy chains of I(alpha)I interact with each other and HA in a manner that is critical for the formation and/or stabilization of the expanded matrix. Another hyaluronan binding component of the expanded COC is the proteoglycan versican. Versican is induced by the LH surge and is a preferred substrate of the protease, a disintegrin and metalloproteinase with thrombospondin like repeats (ADAMTS-1), which co-localizes with versican and is coordinately induced in granulosa cells and COCs of ovulating follicles by LH and the progesterone receptor (PR). Mice null for COX-2 and EP2 fail to ovulate and exhibit impaired COC expression of TSG-6. Progesterone receptor knockout (PRKO) mice are also anovulatory and present impaired expression of ADAMTS-1. Thus, HA binding proteins and associated factors appear to be essential components of the matrix that is obligatory for release of the COCs through the ovulation pore.

Ovarian Cell Differentiation: A Cascade of Multiple Hormones, Cellular Signals, and Regulated Genes

During the development of preovulatory follicles, tonic levels of FSH (and steroid) induce expression of aromatase, the LH receptor, and RII beta in a coordinate manner. Despite the similar temporal increase in steady-state levels of mRNA encoding these proteins, the cis-acting DNA elements and trans-acting factors regulating each gene are distinct (Richards, 1993). Whereas the aromatase gene has a TATA motif and a single transcriptional initiation site (Fitzpatrick and Richards, 1993), both the LH receptor (Wang et al., 1992; Tsai-Morris et al., 1993) and RII beta (Kurten et al., 1992; Luo et al., 1992) genes have promoters that are GC rich, lack TATA motifs, and initiate transcription at multiple sites. The aromatase promoter appears to be regulated, in part, by SF-1, a CRE-like region, and possibly another or overlapping region binding an Ad3BP-like factor. The RII beta promoter has a region that binds several nuclear proteins, whose identity is not yet known. Likewise, the LH receptor promoter elements have yet to be clearly defined (Figures 2, 4, and 25; Kurten et al., 1992). FSH can also induce the expression of at least three immediate-early genes that encode novel kinases or kinase-like proteins (Figure 25). One of these is called serum-inducible kinase (snk) (Simmons et al., 1992), another is serum and glucocorticoid regulated kinase (sgk) (Webster et al., 1993), and a third is called pole kinase (Clay et al., 1993). Steady-state levels of snk and sgk mRNA are induced rapidly (within a few hours) by FSH in granulosa cells prior to the appearance of transcripts for aromatase, LH receptor, and RII beta (T. Alliston and J. S. Richards, in preparation). The functional role of these kinases in the initial response of granulosa cells to tonic (not surge) levels of FSH remains to be elucidated. The cellular signaling pathways mediating the effects of the LH surge appear equally or more complex (Fig. 25). Based on data presented herein, as well as on analyses of the cloned and expressed LH receptor (Guderman et al., 1992), it is clear that low concentrations of LH stimulate adenylyl cyclase, cAMP production, and activation of protein kinase A. Higher (surge) concentrations of LH also increase IP3 and activation of protein kinase C. GnRH has been used in several studies to examine the ability of the protein kinase C pathway to mimic effects of high LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian Follicular Development: From Physiology to Molecular Biology

Publisher Summary Theca cell differentiation and the synthesis of aromatizable androgens are obligatory for follicular estradiol production and the differentiation of granulosa cells. Previous studies indicated that theca cell differentiation was dependent on subtle increases in serum luteinizing hormone (LH) concentrations. To determine whether the effects of LH on theca cell 17 α-hydroxylase in vivo were mediated by cAMP, a theca cell culture system using theca explants from small antral (SA), preovulatory (PO), and luteinizing follicles was developed. SA theca produces only low amounts of androstenedione when cultured in medium alone. However, either when LH was added at the beginning of culture or when forskolin was added on day 4 of culture androstenedione, production was increased to 5–10 ng ml/theca and was maintained for 20 days of culture. PO theca initially produced more androstenedione than SA theca but failed to maintain steroid synthesis unless either LH or forskolin was added to the cultures.

MOLECULAR MECHANISMS OF OVULATION AND LUTEINIZATION

Ovulation is a complex process initiated by the mid-cycle surge of luteinizing hormone (LH). Once initiated, a cascade of events occurs that culminates in the release of a fertilizable oocyte. The complex series of events involves specific ovarian cell types, diverse signaling pathways and temporally controlled expression of specific genes. This review will focus on several genes shown to control the ovulation process.

Temporal and Spatial Patterns of Ovarian Gene Transcription Following an Ovulatory Dose of Gonadotropin in the Rat

Abstract In recent years, there have been a number of efforts to identify genes that are expressed in mature ovarian follicles in response to an ovulatory dose of LH or its homologue hCG. This review keys on 20 ovulation-specific genes that we have identified by the molecular procedure known as differential display. The objective is to use this sampling of genes to illustrate the diversity in the temporal and spatial patterns of expression of genes in the ovary following the stimulus of this gonadal target tissue by a single glycoprotein hormone. The specific genes that are surveyed include 5-aminolevulinate synthase; early growth response protein-1; γ-glutamylcysteine synthetase; cyclooxygenase-2; epiregulin; pituitary adenylate cyclase-activating polypeptide; tumor necrosis factor-stimulated gene-6; regulator of G-protein signaling protein-2; adrenodoxin; steroidogenic acute regulatory protein; 3α-hydroxysteroid dehydrogenase; CD63, a disintegrin and metalloproteinase with thrombospondin motifs; tissue inhibitor of metalloproteinase-1; carbonyl reductase, a G-protein-coupled receptor; pancreatitis-associated protein-III; glutathione S-transferase; and metallothionein-1. The ovulatory expression of these different genes is predominantly within the granulosa layer of mature follicles. However, there were also instances of expression in the thecal and stromal tissue of the ovary, as well as in vascular endothelial cells and in luteal tissue. The overwhelming impression is that the molecular events of ovulation are far more complex, and therefore more highly ordered, than originally imagined.

Hormonal control of ovarian follicular development: a 1978 perspective.

Publisher Summary This chapter discusses the hormonal control of ovarian follicular development. The growth of a follicle appears to begin with the growth of the primary oocyte, its enclosure within a single layer of granulosa cells, the formation of a basement membrane, and the attachment of surrounding theca cells. In the presence of gonadotropins, however, follicular growth continues until large preantral and antral follicles are developed. Most stages of follicular growth appear to proceed in the presence of basal concentrations of gonadotropins. Thus, during pregnancy antrai follicles are continuously present as new groups of follicles acquire antra and others undergo atresia. The hormonal requirements for the continued growth of follicles to the preovulatory stage appear to involve small but significant increases in gonadotropins, leading to increased synthesis of follicular androgens and estradiol. During the maturation of preovulatory follicles, receptor for LH in theca cells is increased 2- to 3-fold and is accompanied by an increased synthesis of androgens. As some aromatase activity is present in the small antral follicles, the increase in androgen leads automatically to an increase in some estradiol production. Thus, a change in theca cell function and responsiveness to LH appears to precede increased follicular estradiol synthesis. During the estrous cycle and at the end of pregnancy, the final stages of preovulatory follicular growth are dependent on increased estradiol synthesis. This appears to be dependent on increased responsiveness of theca cells to LH and increased production of theca-derived androgens. Thus, the theca cell may hold the clue to what initiates preovulatory follicular growth.

Ovulation: a multi-gene, multi-step process.

The luteinizing hormone (LH) surge initiates a cascade of proteolytic events that control ovulation. One of the genes induced by LH is the progesterone receptor (PR). Because mice with a mutant PR gene (PRKO) fail to ovulate and are infertile, we have used them as a model in which to determine PR target genes that might mediate the ovulatory process. The matrix metalloproteinases (MMPs: MMP2, MMP9, and MMP13) appear to be expressed in ovaries of PRKO mice in a manner similar to that in their wild-type littermates. However, the expression of two other types of proteases, cathepsin L (a member of the papain family) and ADAMTS-1 (A Disintegrin And Metalloproteinase with Thrombospondin-like motifs), are selectively induced in granulosa cells of preovulatory follicles by the LH surge. Maximal levels of these proteases are observed at 12-16 h after an LH surge, the time of ovulation. Furthermore, mRNAs encoding cathepsin L and ADAMTS-1 are reduced in the PRKO mice compared to their wild-type littermates. These novel observations indicate that these two proteases regulate some key step(s) controlling ovulation.