Background
The adult mammalian ovary is a complex tissue composed of four fundamental cell types: the oocytes, granulosa, interstitial, and corpora luteal cells. Within this basic quartet are many other cell types such as the ovary surface epithelium and blood vessels in addition to the classic subtypes of granulosa (membrana, periantral, cumulus, and corona radiata), theca (interna and externa), and luteal cells (granulosa and theca lutein). The coordinated control of proliferation, differentiation, and apoptosis in these cell types is the underlying basis for primate and non-primate menstrual and estrous cycles, respectively. The process by which each cell type obtains its state of differentiation is the subject of intense study. The role of hormones in this process is clear; however, new results indicate that autocrine/paracrine or growth factor mechanisms also have important roles.
The bone morphogenetic protein (BMP) family including ligands, receptors, and binding proteins, has emerged as a central player in ovary physiology and female fertility [
1]. The BMPs represent a large subclass of the transforming growth factor-β (TGF-β) surperfamily of ligands whose biological responses are mediated by Ser/Thr kinase receptors via the Smad-signaling molecules [
2]. The mRNAs encoding several BMP family members have been identified in the murine ovary, including the ligands, BMP-2 [
3], BMP-3 [
3], BMP-3b [
3], BMP-4 [
4], BMP-6 [
5], BMP-7 [
4], BMP-15 [
6‐
8], the BMP receptors, BMPR-IA, BMPR-IB and BMPR-II [
4] and the BMP binding protein, follistatin (FS) [
9]. In the murine, BMP-4 and -7 mRNA have been identified in the theca interstitial cells [
4], BMP-6 and -15 in oocytes [
5‐
8], BMPR-IA, BMPR-IB and BMPR-II in oocytes and granulosa cells (GCs) [
4], and FS in granulosa and lutein cells [
9,
10].
The direct involvement of the BMP system in regulating ovary function has been established.
In vitro experiments with rat GCs have demonstrated that BMP-4 and -7 inhibit and stimulate FSH-induced progesterone and estradiol production, respectively [
4]. In related studies BMP-6 and BMP-15 were found to block FSH action through inhibiting FSH-dependent adenylate cyclase activity and FSH receptor expression, respectively [
11,
12]. The importance of these findings is underscored by the fact that FSH action is obligatory for normal folliculogenesis and female fertility. In addition, BMP-7 and -15, but not BMP-6 have stimulatory effects on GC DNA synthesis, indicating they may play a mitogenic role in follicle growth [
8,
12,
13]. The importance of this finding is underscored by the fact that the control of GC proliferation is crucial for follicle dominance and atresia. Equally important, other studies have shown that loss-of-function and naturally occurring mutations in BMP ligands and receptors are associated with dysregulation of folliculogenesis and ovulation [
14‐
19]. Of particular interest is the finding that ewes having a mutation in the
bmp15 gene in both alleles are infertile whereas the heterozygotes are superfertile [
16]. Thus, the
bmp15 gene can control ovulation quota through a dosage-sensitive mechanism. Evidence that a mutation at the Ser/Thr kinase domain of BMPR-IB leads to high fecundity in ewes [
17‐
19] provides further support that BMP signaling pathways play a key role in specifying ovary function.
Although it is becoming clear that intrinsic BMPs are important players in the ovary, the cellular localization of BMPs in the ovary is poorly understood, and a proper understanding of how the cell-specific expression of the BMP system changes during the cycle is still lacking. As part of our ongoing investigations of the roles of BMPs in ovary physiology, we have analyzed the spatiotemporal expression patterns of the BMP system in the rat ovary cell types during the normal estrous cycle.
Discussion
To better understand the physiological role of the BMP family in ovarian function, the spatiotemporal expression patterns of a number of relevant BMP ligands, receptors, and a binding protein were determined by in situ hybridization in rat ovaries over the estrous cycle. The general principle to emerge from our data is that the developmental programs of folliculogenesis (recruitment, selection, and atresia), ovulation and luteogenesis (luteinization and luteolysis) are accompanied by precise cell-specific changes in the expression of the genes encoding the BMP family. Therefore, we hypothesize the regulated expression of BMP gene activity forms a part of the controlling network that ensures the proper timing of the developmental events that generates a normal estrous cycle.
The finding that the genes encoding the three types of BMP receptors are expressed in primordial follicles indicates that these fundamental reproductive units are targets for the BMPs. In this regard, we have shown recently that BMP-7 can promote primordial follicle growth [
13]. Thus, it is reasonable to propose that one function of the BMP receptors in the primordial follicle might be to mediate the activation of recruitment by endogenous BMP-7. As there is no evidence that primordial follicles express BMP ligands, the mechanism by which this activation occurs would be paracrine, most likely involving BMP-7 from adjacent tissues such as the theca, secondary interstitial, or sex cords.
Unlike lower vertebrates, the developing mammalian follicle is composed of more than one layer of GC. One of the most important concepts generated in the ovary BMP field is that oocyte derived GDF-9 [
14] plays a role in the mechanisms governing the development of the second layer of GC i.e. the primary /secondary follicle transition. The question of how GDF-9 controls this step has yet to be answered. Our data show that the primary/secondary transition stage is accompanied by some rather dramatic changes in BMP expression including increased expression of: i) BMP-6, BMP-15, and BMPR IB in the oocyte; ii) BMP-2, BMP-6, and BMPR-IA, -IB and -II in the GC; and iii) BMP-4 and BMP-7 in the theca. Because folliculogenesis in the murine proceeds to the preovulatory stage in the absence of BMP-6 [
30], BMP-15 [
15], and BMPR-IB [
31], one can assume that the marked increases in these regulatory molecules are not essential for the development of a second layer of GC. The question of whether the increased expression of BMPR -II and BMP-2, -4, and -7 contribute to this important developmental event remains to be answered. One interesting implication of our BMP-2 data is that the GC have already attained different states of differentiation by the early stages of secondary follicle development Based on previous work [
32,
33], it is not unreasonable to propose that oocyte morphogens may have control over GC fate beginning as early as the primary/secondary follicle transition. It will be interesting to investigate how this phenomenon fits into the mechanisms underlying the primary/secondary follicle transition.
Our results demonstrate for the first time that the theca interna of healthy follicles is composed of two distinct populations of theca interstitial cells (TIC). One group, which expresses BMP-4, is present as an outer layer of cells juxtaposed to the theca externa (TE), and another, which expresses BMP-7, is present at the proximal side of the theca interna near the basal lamina of the follicle. These differences can be found throughout folliculogenesis, beginning during the secondary stage. This, together with the failure to detect BMP-4 and 7 in the theca of atretic follicles, supports the idea that the cellular sites and coordinate expression of these BMP genes may have important regulatory roles in maintaining folliculogenesis. In this regard, a recent study has demonstrated the ability of BMP-4 to inhibit cyclic AMP-stimulated androgen production by the cultured human theca cell line, presumably by activating BMP receptors (BMPR-IA, -IB, and -II) which are expressed in these cells [
34]. Because theca derived androgen is required for follicle estrogen biosynthesis and has been implicated in ovary pathology [
35], it will be interesting to determine how the expression and functions of these TIC-specific BMPs are integrated into the overall processes of physiology and pathophysiology. Besides the TI, a high level of BMP expression (BMP-3b and 4) is found within the smooth muscle cells of the TE during the process of follicle formation. Very little is known about the role of the TE in folliculogenesis: however this new finding raises the possibility that BMP-3b and -4 could have autocrine actions on TE histogenesis and/or paracrine actions on theca/follicle morphogenesis.
The rapid cessation of mitosis and the expression of apoptosis in the GC is the sinequanone of follicle atresia. Our results suggest a possible role for BMPs in follicle atresia. The supporting data are as follow. First, a striking feature of atresia in the rat ovary is the cessation of BMP expression (BMP-3b, -4, and -7) within the theca compartment. This observation is particularly interesting with respect to BMP-7. In previous work, we demonstrated that BMP-7 could stimulate DNA synthesis in rat GC [
13], as it does in human osteoblasts [
36]. Equally relevant, several reports have demonstrated the role for BMP-7 in preventing apoptosis in other cell systems [
37,
38]. Together, these data suggest that theca-derived BMP-7 might be a follicle survival factor through its ability to enhance GC proliferation and suppress apoptosis. Second, atresia is accompanied by a relatively high level of expression of BMP-2 and -6 and the BMPR-IA, -IB, and -II. Significantly, this occurred in the absence of the BMP antagonist, FS. Thus, it is possible that GC BMP receptor signaling pathways are under maximal stimulation by BMP-2 and -6 during atresia. How this phenomenon contributes to atresia is unknown, but our finding [
12] that BMP-6 inhibits FSH signaling is relevant because there is compelling evidence that continuous FSH stimulation is essential for follicle survival.
The expression of the family of BMPs (ligands, receptors and a binding protein) in the fundamental cell types of the dominant follicles (DF) argues for a physiological role of an intrinsic BMP in directing growth and cell fates in the DF. This is supported by two main lines of evidence. First, we found that the genes encoding the BMP receptors (BMPR-IA, -IB, -II) are highly expressed in the DF throughout its course of development. And second, the BMP ligands (BMP-4, -6, -7, and -15) act on the GC to control the level of mitosis and FSH receptor signaling [
1]. In this regard, one particularly interesting observation is the dramatic loss of BMP-6 mRNA in the GC when the DF is selected at E 0200 h. Because BMP-6 can prevent FSH action [
12], the rapid loss of BMP-6 expression may be required for FSH to exert its crucial functions during DF-development. The secondary rise of FSH on estrous morning is obligatory for DF selection, and we have preliminary data showing that FSH induces the loss of BMP-6 mRNA in GC. Thus, the secondary rise of FSH may down regulate BMP-6 expression in the DF. It is notable that the absence of GC BMP-6 is a marker for the DF. Finally, with regard to FSH action, FS has been shown to neutralize the bioactivity of the FSH inhibitor, BMP-15 [
39]. It is possible, therefore, that the strong expression of FS by the DF may serve to modulate BMP-15 action such that sufficient FSH receptors are expressed in the GC to permit the development of a preovulatory follicle.
A role for the BMPs in regulating luteogenesis is strongly suggested our data. Some key findings are as follows. First, the classical view of CL development is that luteinization is inhibited by oocyte-derived luteinization inhibitors [
32,
40,
41], two of which have been identified as BMP-6 and -15 [
8,
12]. Therefore, the loss of oocyte BMP-6 and -15 at ovulation undoubtedly plays an especially prominent role in activating luteinization. Second, the fact that CL BMP-2 expression is suppressed and activated during luteinization and luteolysis, respectively, suggests that BMP-2 might be a novel luteinization inhibitor. Further work is necessary to investigate this possibility. Third, FS is strongly expressed during luteinization, but is undetectable at luteolysis. Thus, it is conceivable that the cycle-specific expression of FS within the CL may control the level of progesterone production through anti-BMP mechanisms. And fourth, a role of BMPR-IB in luteogenesis was suggested by our finding that luteinization and luteolysis, respectively are characterized by the absence and presence of BMPR-IB expression in the CL. A functional link between the expression of BMPR-IB and luteolysis comes from loss-of-function studies showing that the CL of the cycle continues to secrete progesterone in the absence of BMPR-IB [
31]. This work, together with our finding that BMPR-IB is selectively expressed in the TE of the CL at luteolysis, supports the novel hypothesis that the regulation of inducible and TE specific BMPR-IB gene expression may control luteolysis.
The observation that the ovarian sex cords (SC) express an intrinsic BMP system replete with ligands (BMP-3b, -4, -7) and receptors (BMPR-IA, -IB, -II) is novel. The SC are vestiges of the cranial portion of the mesonephric tubules and ducts, and in the adult ovary appear as a cluster of blind tubules in the hilar region. Although little is known about the physiology and biology of ovarian SC, they have been linked to the genesis of ovary cancer through distinct TGF-β signaling pathways [
42,
43]. Significantly, the importance of FS in the genesis of this cancer has been established [
44]. Given the evidence that BMPs are potent growth factors and that FS can modulate their bioactivity, it will be interesting to investigate the possibility that this intrinsic BMP system may be involved to pathogenesis of SC tumors. Because SC were occasionally found juxtaposed to developing follicles, blood vessels and secondary interstitial cells, one cannot exclude the possibility that the SC-derived BMPs may have paracrine functions.
A functional role for the BMPs in the ovary surface epithelium (OSE) is suggested by the strong expression of BMP-3b, -4, and -6. Based on our data, BMP-3b and -6 appear to be constitutively expressed throughout the epithelium over the cycle, a finding consistent with a maintenance-type role for these OSE BMPs. An interesting finding is the cell-specific induction of BMP-4 gene expression in a subset of epithelial cells covering the ovulated follicle and the newly formed CL-I. Although the function of this inducible and cell-specific gene expression is unclear, it is tightly correlated with the replenishment of the surface epithelium that is exfoliated from the surface of ovulating follicle. Thus, the importance of this highly specific expression of BMP-4 may relate to the regeneration of the OSE during postovulatory repair. Although BMP-4 expression appears tightly regulated over the cycle, it remains to be determined how the regulation is controlled. Because the OSE does not appear to express BMP receptors, the OSE-derived BMP-4 probably acts as a paracrine growth factor in the underlying stroma. It is noteworthy that epidemiological studies have shown that the risk of epithelial ovarian cancer is decreased by factors that suppress ovulation, i.e., pregnancy, breast-feeding, and oral contraceptive pill. [
45,
46]. Given the relationship between LH-dependent ovulation and the induction of BMP-4 expression, it will be important to explore a possible link between the risk of ovary cancer and the ovulation-inducible and OSE cell-specific BMP-4 gene expression.
It is clear from this work that the regulation of inducible and cell-specific expression of the BMP family is a general property of the mammalian ovary. Consequently, the potential for independent and coordinated contributions of different BMPs to control growth, differentiation, and apoptosis is widely distributed throughout all the histological units of the ovary. The current challenges are to understand how specific hormone/growth factor interactions regulate BMP expression in the fundamental ovary cell types, and how these interactions are integrated into the morphological and histological events that generate the estrous cycle. The clinical relevance is emphasized by our previous finding that the expression of GDF-9 is dysregulated in oocytes of women with Polycystic Ovary Syndrome [
47].