Hormonal Control of the Cell Cycle in Ovarian Cells: Proliferation Versus Differentiation

^a Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030

	INTRODUCTION

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The growth of ovarian follicles, ovulation, and the formation of the corpus luteum are complex processes that involve dramatic changes in granulosa cell function. The changes are sequential and are dictated by specific, tightly regulated responses to gonadotropins, steroids, and growth factors [1–3]. One of the most dramatic changes in granulosa cell function is the rapid switch from the highly proliferative stage characterizing granulosa cells of preovulatory follicles to the nonproliferative, terminally differentiated phase of luteal cells. Some of the cell cycle regulatory mechanisms mediating this switch, as well as their control by hormones, are the topics of this minireview.

In primordial follicles, the oocyte is surrounded by a single layer of nondividing granulosa cells arrested in Go phase of the cell cycle. Primordial follicles leave this quiescent state and initiate a phase of slow growth in which the granulosa cells have entered the cell cycle but proliferation is exceedingly slow [1]. However, as these slowly dividing granulosa cells acquire enhanced responsiveness to FSH and LH and begin producing estradiol [4, 5], exposure to these hormones triggers a rapid burst of proliferation that results in the formation of large preovulatory follicles [6]. This rapid phase of growth is characterized by a marked increase in the labeling of granulosa cells by tritiated thymidine [1, 6], as well as by 5-bromodeoxyuridine (BrdU) [7]. Granulosa cells of these preovulatory follicles not only are highly proliferative but are also differentiating and acquire LH receptors [8]. The LH surge then triggers dramatic changes in both follicular structure and function. LH terminates follicular growth by causing granulosa cells of preovulatory follicles to exit the cell cycle [1, 6] and rapidly initiates a program of terminal differentiation (luteinization) in which the cells cease to divide [9, 10]. As shown herein, the exit of preovulatory granulosa cells from the cell cycle occurs within about 4 h after the LH surge (Fig. 1), and this related to dramatic changes in specific molecules regulating cell cycle progression (see Figs. 4 and 5). In addition, follicular rupture (ovulation) occurs and granulosa cells luteinize to form mature corpora lutea. Interestingly, granulosa cells are completely reprogrammed to luteinize by 7 h after exposure to the LH surge [4, 10].

View larger version (97K): [in this window] [in a new window]   FIG. 1. Changes in granulosa cell proliferation in the ovary detected by BrdU labeling. Preovulatory follicles in mice 48 h after treatment with 4 IU eCG have many proliferating granulosa cells, seen as cells with black nuclei. In response to the LH surge (5 IU hCG), these cells exit the cell cycle, as seen by the absence of labeled granulosa cells in large preovulatory follicles. Methods as described previously [7]. x200 (reproduced at 88%).

View larger version (74K): [in this window] [in a new window]   FIG. 4. Expression of cyclin D2 and p27Kip1 in the ovary. In situ hybridization of rat ovarian sections shows that cyclin D2 mRNA is expressed at high levels in the granulosa cells of preovulatory follicles and subsequently down-regulated by hCG treatment. Expression of p27 mRNA is initially low following an hCG surge, but then is elevated to high levels by 24 h. Western analysis performed on protein from granulosa cells and luteal cells isolated from ovaries of these same animals shows similar results. Data are modified from results presented in [34] with permission from The Endocrine Society. x100 (reproduced at 92%).

View larger version (42K): [in this window] [in a new window]   FIG. 5. Regulation of cyclin E in granulosa cells. Western analysis of cyclin E expression in granulosa/luteal cells isolated from ovaries of hormone-treated H rats.

This pattern of granulosa cell proliferation and differentiation that characterizes the natural growth of follicles can be mimicked in hypophysectomized rats by a specific hormonal regimen [4, 5, 11]. Injections of estradiol (1.5 mg for 3 days) followed by FSH (1.0 µg for 2 days) stimulate granulosa cell proliferation and follicle growth to the preovulatory stage. A subsequent ovulatory dose of hCG (10 IU) triggers ovulation and luteinization. In the hypophysectomized rat [4, 11], as well as in mutant mice lacking either gonadotropins (hypogonadal [12, 13]) or FSH [14], follicular development is arrested at the preantral stage (Fig. 2). Mice lacking estrogen receptor (ER) also exhibit impaired follicular growth and infertility [15], but the follicles develop to the antral stage presumably due to the presence of estrogen receptor ß (ERß) in these cells [16]. These models illustrate that although the early, slow stages of granulosa cell proliferation and preantral follicle growth occur in the absence of gonadotropins and estradiol, these hormones (and their receptors) are required for normal growth—especially the final rapid stages of development that form preovulatory follicles and permit these cells to luteinize.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Schematic of ovarian follicular development and models that disrupt this process. Granulosa cell proliferation leads to follicular growth and the formation of large preovulatory follicles. LH triggers ovulation and the formation of corpora lutea. Hypophysectomized rats, as well as mice null for GnRH (hypogonadal), FSHß subunit, and cyclin D2, exhibit impaired follicular growth with follicles arrested at the preantral stage of growth. Mice null for ER, but not ERß, have antral follicles that do not ovulate or luteinize. Mice lacking p27Kip1 exhibit abnormal corpus luteum formation.

Cell cycle progression and proliferation are controlled by a balance of positive and negative regulators converging on cell cycle kinase cascades (reviewed in [17–23]; see schematics, Figs. 3 and 7). Interestingly, specific roles for cell cycle regulatory molecules in the control of granulosa cell proliferation and differentiation during follicular development have been elucidated by the altered ovarian phenotypes described in mice null for cyclin D2 [24] and p27^Kip1 [25–27]. Cyclin D2 [28, 29] acts as a positive regulator of cell cycle progression by its ability to bind cyclin-dependent kinases (cdks) 4 or 6 and thereby activate a cascade of events that permits progression through G1 phase of the cell cycle ([30]; Fig. 3). Cyclin E also acts as a positive regulator of cell cycle progression [31, 32]. By binding and activating cdk 2, it regulates the G1 to S phase transition. In contrast, p27^Kip1 blocks cell cycle progression by inactivating these same cdk cascades, and cells remain in G1 phase [33]. In mice null for cyclin D2, granulosa cell proliferation is impaired, the ovarian follicles remain small, and ovulation fails to occur [24]. In mice null for p27^Kip1, follicular growth is not compromised but granulosa cells do not luteinize properly in response to LH [25–27]. Therefore, in order to better understand the control of cellular proliferation in the ovary, we analyzed the expression of these cell cycle regulatory molecules and their regulation by hormones during follicular development, specifically when granulosa cell proliferation is rapid and during luteinization when cell division has terminated. The results presented highlight the ability of the LH surge to acutely regulate cyclin D2 and p27^Kip1 in an inverse manner. Methods for the analysis of cyclin E protein done in the studies presented herein were the same as previously described [34].

View larger version (61K): [in this window] [in a new window]   FIG. 3. Cyclin D2/cyclin E and p27Kip1 exert opposing actions on cdk activity and cell cycle progression. Increased synthesis and binding of cyclin D2 activate cdk4/6 activity. Subsequently, cyclin E binds and activates cdk2, enabling progression through G1 phase of the cell cycle. Conversely, binding of p27KIP1 blocks cdk activity, and the cell cycle is arrested in G1.

View larger version (44K): [in this window] [in a new window]   FIG. 7. Schematic of known cell cycle events and factors regulated in ovarian cells by gonadotropins and estradiol. Solid arrows depict events shown herein to be regulated by FSH, LH, and estrogen during follicular growth and luteinization. Dashed arrows show events likely to be controlled by FSH/cAMP and estrogen on the basis of studies described in the text. Other arrows indicate the sequence of regulatory steps in the cell cycle as summarized from numerous reviews cited in the text. T-shaped lines indicate inhibitory actions.

	REGULATION OF CYCLIN D2, CYCLIN E, AND P27Kip1

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To characterize the hormonal regulation of cyclin D2, cyclin E, and p27 in the ovary, we used the hypophysectomized rat, in which the effects of estradiol, FSH, and LH on granulosa cell proliferation [4–6] and differentiation [4, 5, 9, 11] have been well characterized. In situ hybridization of ovarian sections from hormone-treated hypophysectomized (H) rats showed that cyclin D2 and p27 exhibit distinctly regulated patterns of expression in granulosa cells (Fig. 4). The results were verified by Western analysis of protein obtained from granulosa cells or luteal cells of these same animals. Specifically, cyclin D2 mRNA and protein are expressed at high levels in granulosa cells of preovulatory follicles of H rats treated with estradiol and FSH (HEF). When HEF rats are injected with hCG, cyclin D2 is down-regulated within 4 h and remains low throughout luteinization. As shown by in situ localization, the down-regulation of cyclin D2 mRNA occurs specifically in granulosa cells of preovulatory follicles that are destined to ovulate and undergo terminal differentiation or luteinization. Cyclin D2 mRNA continues to be expressed in smaller growing follicles that lack LH receptors [3, 4, 8].

The p27 cdk inhibitor is expressed in the preovulatory granulosa cells of HEF rats, and like cyclin D2, is reduced to low levels after 4 h of hCG treatment (Fig. 4). However, after this time its expression pattern differs dramatically from that of cyclin D2. By 24 h after hCG, p27 mRNA and protein are expressed at very high levels and, as shown by the in situ hybridization, are localized to the luteinizing granulosa cells of the corpus luteum.

On the basis of the observations that both FSH and estradiol induce cyclin D2 mRNA and protein expression in granulosa cells [34], we sought to determine whether these hormones also affect the expression of cyclin E, a downstream mediator critical for cell cycle progression through the G1 checkpoint. Western analysis (Fig. 5) of whole cell extracts showed that levels of cyclin E were low in granulosa cells isolated from ovaries of H rats. Treatment in vivo with FSH increased cyclin E levels within 2 h, after which cyclin E remained elevated at this level. Interestingly, treatment with estradiol caused a greater increase in cyclin E within 2 h than did FSH. Cyclin E levels then continued to increase progressively in the estradiol-treated rats for 48 h. Additional experiments showed that cyclin E expression is also regulated as cells luteinize in response to an ovulatory stimulus of hCG. As in the previous experiment, granulosa cells from untreated H rats contained low levels of cyclin E that were increased in response to estradiol (HE) or estradiol followed by FSH (HEF). When HEF rats were injected with an ovulatory dose of hCG, cyclin E levels remained high at 4 h and 24 h; however, by 48 h after hCG, cyclin E was low in the luteinized granulosa cells.

Collectively, these data suggest that one putative mechanism by which the LH surge terminates granulosa cell proliferation involves the rapid inhibition of cyclin D2 transcription. As shown in Figure 1, granulosa cell exit from the cell cycle occurs within 4 h of the LH surge, coinciding with the drastic down-regulation of cyclin D2, but prior to the down-regulation of cyclin E and the induction of p27. Regulation of cyclin D2 is highly probable as the primary regulatory event controlling granulosa cell proliferation, since 1) cyclin D2, but not cyclin D1 or cyclin D3 (which have redundant functions [30]), is expressed in granulosa cells [34] and 2) the absence of cyclin D2, but not the absence of cyclin D1 [35], markedly impairs granulosa cell proliferation [24]. Additionally, cyclin E, a downstream mediator of cell cycle progression, continues to be expressed in luteinizing granulosa cells long after the rapid disappearance of cyclin D2. Therefore, the down-regulation of cyclin D2 in response to LH would presumably prevent the first step in cell cycle progression, thereby initiating granulosa cell exit from the cell cycle before reaching the cyclin E-regulated checkpoint. The temporal expression pattern for p27 suggests that a second mechanism by which LH terminates granulosa cell proliferation is by increasing the level of this cdk inhibitor. In addition, the increase in p27 may control some aspect of granulosa cell differentiation or maintenance of luteal cell differentiation [25–27]. In summary, these data indicate that the LH surge terminates granulosa cell proliferation and initiates differentiation by inverting the balance of these positive and negative regulators of cell cycle progression (Fig. 6). LH coordinately down-regulates expression of cyclin D2, followed by cyclin E, as it increases the levels of p27. The ovarian phenotypes of the mice lacking cyclin D2 or p27 support such a model. The loss of cyclin D2 results in the absence of FSH- and estradiol-stimulated granulosa cell proliferation [24], which normally leads to large, preovulatory follicles. The loss of p27 results in impaired differentiation as seen by the inability of granulosa cells to luteinize normally and produce sufficient progesterone to support pregnancy [25–27].

View larger version (48K): [in this window] [in a new window]   FIG. 6. The LH surge inverts the balance of positive versus negative cell cycle regulators and triggers granulosa cell exit from the cell cycle concurrent with luteinization.

	MECHANISMS OF CELL CYCLE CONTROL

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Regulation of the cell cycle within any cell type is complex, involves the balance of many regulatory molecules, and can be altered by numerous external signals acting at multiple steps in the cycle. In the ovary, estradiol, FSH, and LH are essential signals for the growth of preovulatory follicles and their subsequent terminal differentiation as corpora lutea. Each hormone acts via specific receptors and intracellular signaling pathways. Additionally, FSH and LH act by controlling distinct levels of cAMP [36] and the activation of the A-kinase pathway [37]. The pivotal roles of cyclin D2 and p27 in ovarian follicular growth and differentiation are indicated by their selective expression and regulation in the ovary and their critical and opposing effects on cdk activity that controls entry and progression through G1 of the cell cycle. However, as briefly summarized below (see reviews [17–23]), other regulatory molecules control progression through additional checkpoints of the cell cycle, and some of these are likely to be critical in the ovary as well (Fig. 7).

Early in G1 phase, the presence of D-type cyclins activates cdk4 or cdk6, while the accumulation of cyclin E later in G1 phase activates cdk2. Progression through S phase is regulated by cyclin A complexes followed by the initiation of M by cyclin B-cdc2 complexes. The binding of cyclin D and cdk4/6 results in the formation of a complex that is then phosphorylated by cdk-activating kinase (CAK). The active cyclin-cdk complex in turn phosphorylates cellular substrates that regulate DNA synthesis. The best-known example is the retinoblastoma protein, Rb, which in a hypophosphorylated state acts as a suppressor of cell division by binding to DP/E2F transcription factors, thereby preventing the transcription of genes necessary for replication and cell division. The repression of transcription by Rb appears to be mediated by its ability to recruit histone deacetylase [38, 39]. However, hyperphosphorylation of Rb by cyclin D-cdk4/6 and cyclin E-cdk2 relieves its suppressive abilities, and the cell cycle is able to progress past the restriction point (R), from G1 to S phase, at which time the cell is irreversibly committed to divide.

The Cip/Kip family of cdk inhibitors, which includes p27, acts to block cell cycle progression by binding and inhibiting the activity of cdks. The Cip/Kips (p21^Cip1, p27^Kip1, p57^Kip2) have relatively broad specificity and are able to bind not only cdk4/6, but also cdk2 and cdc2, enabling them to inhibit the activity of several kinase cascades and thereby block cell cycle progression at multiple points. In contrast, the Ink4 family of cdk inhibitors bind only cdk4 and cdk6, making them specific inhibitors of G1 phase.

Even in this basic scheme it is clear that regulation of the cell cycle is complex and occurs at many levels. Not only are many different types of molecules involved, but many are families of molecules that are expressed in overlapping as well as tissue-specific patterns. In the ovary, cyclin D2 is highly expressed specifically in granulosa cells, as is cdk4 [40], while cyclin D1 and cyclin D3 are barely detectable [34] and mice lacking cyclin D1 exhibit normal ovarian function [35]. In contrast, cyclin D1 and cyclin D3 are expressed in an overlapping pattern, with both expressed at the highest levels in theca cells [34]. Both p27 and a related family member, p21^Cip1, are highly expressed in the corpus luteum with only slightly different patterns of induction [34]. However, they appear to play unique roles in this tissue, since mice lacking p27 exhibit an altered ovarian phenotype [25–27] while mice lacking p21 do not [41]. Rb is also a member of a family of molecules (including p107 and p130), and there are five mammalian isoforms of E2F. Mice lacking p107 [42], p130 [43], or E2F-1 [44, 45] are fertile; thus there may exist overlapping expression and functional redundancy within members of these families in granulosa cells. Precedence for this comes from the observation that phosphatase cdc25, which activates the cyclin B-cdc2 complex, is also expressed as three isoforms that are differentially expressed within the ovary [46].

Another layer of complexity is added to this scenario when one considers the fact that hormonal control of expression and activity of cell cycle regulators not only is achieved by different hormones, but also depends on the dose of hormone. Our studies have shown that cAMP and protein kinase A play a role in accelerating granulosa cell proliferation as well as in terminating follicle growth and initiating terminal differentiation. Specifically, the low levels of cAMP generated in response to FSH induce high levels of cyclin D2 [34] and also increase cyclin E (Fig. 5). In contrast, the LH surge and high levels of cAMP rapidly turn off expression of cyclin D2 followed by cyclin E. Conversely, the LH surge markedly increases levels of p27 as well as p21 [34]. High levels of cAMP have been known to terminate cell division in other cell types [47], and in glial cells this is associated with the induction of p27 expression [48]. Additionally, the cAMP/protein kinase A cascade controls the exit from mitosis by regulating the degradation of cyclin B [49]. Thus, not only can the presence or level of hormone, i.e., FSH versus LH, alter the amount of a regulatory molecule; it can also exert effects at multiple points within the cell cycle machinery, at G1-S and M-G1 (Fig. 7).

Estrogens are known to be potent mitogens and are often associated with cancer. Indeed, most studies that have been designed to determine the mechanism by which estrogens, principally estradiol, regulate proliferation have been performed in breast cancer cell lines, in which the cell cycle machinery and the signals impinging on the cell cycle may be abnormal. In these cell lines, estradiol is able to increase the activity of cdk4 and cdk2 [50, 51] by inducing expression of cyclin D1 [50] as well as by decreasing the levels of cdk inhibitors [51]. Similar mechanisms have also been observed in the uterus, where estradiol induces cyclins D1, D3, E, and A [52]. We have shown that estradiol induces the expression of cyclin D2 and cyclin E in granulosa cells, concurrent with a reduction in levels of p27 [34]. Simultaneous down-regulation of a cdk inhibitor and induction of cyclins may account for the greater mitogenicity of estradiol compared to FSH in these cells [5, 6]. Studies have also shown that cyclin D1 can directly bind the estrogen receptor and enhance transcription of specific genes [53]. It would be interesting to determine whether such a mechanism occurs in granulosa cells, which selectively express the beta subtype of the estrogen receptor (ERß; [16]) and in which estradiol induces cyclin D2 [34].

Lastly, there are many additional hormones that act in the ovary to affect cellular proliferation and differentiation. For instance, activin, which is produced at high levels in preovulatory granulosa cells, has been shown to stimulate granulosa cell DNA synthesis [54]. Therefore, it is possible that activin along with estradiol and FSH regulates cyclin D2. Paradoxically, activin has been shown to down-regulate cyclin D2 in plasmacytic cells [55], and targeted deletion of the activin ßB subunit in mice did not result in an overt ovarian phenotype [56]. Conversely, mice null for the heterodimeric molecule inhibin (/ßA or /ßB) develop ovarian tumors at the time of puberty [57] that are dependent on gonadotropin support [58]. These observations indicate that the unopposed actions of activin in the presence of cAMP or steroid are tumorigenic. Insulin-like growth factor-1 (IGF-1) has also been implicated in granulosa cell proliferation [59]. However, large antral follicles are present in the ovaries of mice null for IGF-1, indicating that IGF-1 may be more important for differentiation than for proliferation [60].

In summary, the relationship of cell proliferation to differentiation is fundamental to all biological processes. Proliferation precedes differentiation; differentiation often precludes further cell division (exceptions are metastasis—tissue repair); nonproliferating and nondifferentiated cells are usually excluded by apoptosis (programmed cell death, a topic not covered herein). The ovarian phenotypes of the cyclin D2 and p27 null mice provide intriguing insights into the relationship between proliferation and differentiation of follicular granulosa cells. In the absence of p27, differentiation characteristic of luteinization appears impaired. However, despite the key role of p27 in checking cell cycle progression and its presence in granulosa cells and luteal cells, the absence of p27 does not lead to rampant uncontrolled proliferation of these cells. Thus, other inhibitors of cell division appear to be more critical during follicular growth. Conversely, in the absence of cyclin D2, the mitotic activity of granulosa cells is markedly impaired, and growing follicles remain small with few (usually only one or two) layers of granulosa cells. Despite the reduced number of cells, these cells respond to LH in a normal pattern of differentiation. For instance, progesterone receptor and prostaglandin synthase-2, two regulators of ovulation [9, 61, 62], are induced by the LH surge in a pattern similar to that of normal ovulating follicles [34]. Yet the "preovulatory" follicles of the cyclin D2 mice do not ovulate. This raises the intriguing question whether or not the number of granulosa cells is critical for stimulating some event associated with ovulation. Are there other situations in which cell number dictates some physiological process? These and many other questions will be answered as we learn more about the control of the cell cycle and cell differentiation in the ovary and other tissues.

	FOOTNOTES

 
¹ Correspondence: JoAnne S. Richards, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. FAX: (713) 790–1275; joanner{at}bcm.tmc.edu

Accepted: March 26, 1998.

Received: January 22, 1998.

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