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Department of Reproductive Medicine, University of California San Diego, School of Medicine, La Jolla, California 92093-0633
Correspondence: Address all correspondence and requests for reprints to: Shunichi Shimasaki, Ph.D., Department of Reproductive Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0633. E-mail: sshimasaki{at}ucsd.edu
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Abstract |
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 Top Abstract I. IntroductionII. The BMP SystemIII. Expression of the...IV. Reproductive Functions of...V. Genetic Mutations of...VI. Perspectives and Future...References |
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I. Introduction |
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TopAbstractI. IntroductionII. The BMP SystemIII. Expression of the...IV. Reproductive Functions of...V. Genetic Mutations of...VI. Perspectives and Future...References |
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), and BMP-3 were described. The deduced amino acid sequences of BMP-2, -3, and -4 closely resemble the members of the TGF-ß family, which at that time was composed of TGF-ß1 (5), TGF-ß2 (6), inhibin/activin subunits (
, ßA, and ßB) (7), decapentaplegic (8), Vgl (product of the Xenopus transcript present in the vegetal pole of eggs) (9), and Müllerian inhibiting substance (MIS) [also called anti-Müllerian hormone (AMH)] (10). Together, these factors represented the founding members of the TGF-ß superfamily, which now consists of more than 35 members (11). Analysis showed that BMP-1 does not share structural homology with the BMPs. Therefore, BMP-1 is not included in the TGF-ß superfamily. Subsequent studies showed that BMP-1 is identical to procollagen C-proteinase, which can cleave procollagen I, II, and III into mature monomers (12). With regard to bone formation, BMP-1 can also cleave the BMP binding protein, chordin, rendering it inactive. Therefore, it is believed that the bone-inducing activities of BMP-1 are manifested through its ability to cleave chordin and thereby eliminate the ability of endogenous chordin to antagonize endogenous BMP activities.
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). Furthermore, because many subsequent BMP family genes were identified by homology-based cDNA cloning, the osteoinductive activity, if any, of many BMPs remains to be established. Consequently, the BMP nomenclature is confusing, and there is a need to develop an appropriate BMP terminology for this family. For the purposes of this review, we have chosen to define the BMP family of ligands to include the BMPs and the GDFs based on the high homology in their amino acid sequences (11). Recent research has suggested that MIS can use BMP-specific type-I receptors and activate BMP intracellular signaling pathways (19, 20, 21, 22). It appears, therefore, that MIS has properties that resemble the BMP/GDF family. However, we have chosen not to focus on MIS in this review because excellent previous reviews have provided a detailed discussion of the MIS field (23, 24, 25, 26). As the tissue expression patterns of the BMP family became known and the recombinant BMPs became available for research, the conclusion emerged that the BMPs regulate growth, differentiation, and apoptosis in a wide variety of tissues in addition to bone (27, 28). After a few key papers that reported the expression of components of the BMP system in mammalian reproductive tissues (16, 29, 30, 31, 32), genetic and experimental studies were quickly performed that demonstrated crucial roles of the BMP system in the regulation of reproductive processes, particularly in the gonads (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42). The continued investigation of the roles of the BMP system in reproductive physiology has provided new insight into our understanding of the physiological regulation of key reproductive processes. Indeed, this information is beginning to provide answers to some of the long-sought questions in the field of reproductive physiology and endocrinology.
In this report, we present a comprehensive review of the literature as it applies to the roles of the BMP system in regulating mammalian reproduction. In a broad sense, we will focus our attention on three main areas: 1) the spatiotemporal expression pattern of BMP ligands, receptors, signaling molecules and regulatory factors in reproductive tissues; 2) the specific biological activities of BMP ligands in regulating reproductive processes; and 3) the genetic studies that have provided particular insight into the role of the BMP system in regulating mammalian fertility.
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II. The BMP System |
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TopAbstractI. IntroductionII. The BMP SystemIII. Expression of the...IV. Reproductive Functions of...V. Genetic Mutations of...VI. Perspectives and Future...References |
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The posttranslational processing of BMPs is important for the secretion of biologically active molecules. All TGF-ß superfamily members are translated as large preproproteins composed of a signal peptide, prodomain, and mature domain. After removal of the signal peptide, the proproteins undergo dimerization. As processing proceeds, specific proteolytic enzymes cleave the dimerized proprotein at the RXXR site, resulting in the generation of the biologically active dimeric mature protein. Some TGF-ß superfamily members are secreted as a complex consisting of the mature dimer noncovalently bound to the prodomain. In the case of TGF-ß, this complex is biologically inactive and can be activated under chaotropic conditions such as low pH (54). There is also evidence that mature BMPs can be secreted as a complex with their prodomains (46, 47, 48, 53, 55); however, the physiological role and biological activities of such BMP complexes are poorly understood.
B. BMP receptors
Crosslinking studies have revealed two major types of membrane-bound receptors for the ligands of the TGF-ß superfamily, type-I and type-II receptors (56, 57). The first of the TGF-ß superfamily receptors to be cloned and characterized was an activin receptor (58). It consists of an extracellular ligand binding domain, a single membrane-spanning domain, and an intracellular domain containing a predicted serine/threonine kinase region. Activin A, activin B, and inhibin A, but not TGF-ß1, bind to this receptor. This receptor was designated as the activin type-II receptor (ActR-II). After the cloning of the ActR-II cDNA, other related type-II receptor cDNAs were cloned, predominantly by homology-based PCR strategies. To date, five mammalian type II receptors have been identified: ActR-II (58), ActR-IIB (59), AMHR-II (MIS/AMH type-II receptor) (60, 61), BMPR-II (BMP type-II receptor) (62, 63, 64, 65), and TßR-II (TGF-ß type-II receptor) (66). In mammals, seven type-I receptors termed activin receptor-like kinase (ALK)-1 to -7 have also been cloned (67, 68, 69, 70, 71, 72).
The ligand-receptor relationships between various BMP ligands and their cognate receptors are not exclusive (Table 2). Originally, ActR-II and ActR-IIB were identified as activin receptors; however, it is now clear that they can also act as receptors for BMP-6 (73), BMP-7 (74), and GDF-5 (75). Conversely, BMPR-II appears to bind exclusively to BMP ligands, including BMP-2 (63), BMP-4 (64, 65), BMP-6 (73), BMP-7 (63, 64), BMP-15 (76), GDF-5 (75), and GDF-9 (77). With respect to type-I receptors, ALK-2 (also called ActR-IA), ALK-3 (BMPR-IA), and ALK-6 (BMPR-IB) have been identified as BMP type-I receptors (73, 74, 75, 78, 79, 80, 81, 82), and overexpression of constitutively active forms of these type-I receptors activates BMP-specific cellular signaling (82, 83, 84). The complexity of the BMPR-ligand interactions is compounded by the fact that there is much cross-reactivity among different BMP ligands and the type-I receptors (Table 2). Furthermore, it has been shown that a particular BMP ligand binds different type-I receptors in different cell types. For example, in MC3T3-E1 and C2C12 cells, BMP-6 strongly binds to ALK-2 and also binds to ALK-3 with lower affinity, whereas in ROB-C26 cells BMP-6 binds most efficiently to ALK-6 and less efficiently to ALK-2 and -3 (73). Nonetheless, some general trends have emerged from a number of studies that have identified preferences of certain BMP ligands for certain BMP type-I receptors (82): BMP-2, BMP-4, and GDF-5 preferentially bind to ALK-3 and/or ALK-6 (75, 78, 82); BMP-6 and -7 most readily bind to ALK-2 and/or ALK-6 (73, 74, 78, 80, 82); BMP-15 most efficiently binds to ALK-6 with very little affinity for ALK-3 (76). Although GDF-9 has been found to bind to BMPR-II (77), the type-I receptor for GDF-9 has yet to be identified.
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The pattern of receptor oligomerization for activin and TGF-ß is well known. These ligands first bind to the type-II receptors, which in turn, leads to the recruitment of the type-I receptors. This pattern of receptor oligomerization is supported by the finding that neither activin nor TGF-ß has an affinity for the type-I receptors alone (85), but they do have an affinity for the type-II receptors. Although the pattern of BMP receptor oligomerization is not well understood, there is evidence to suggest that it may be different from that of activin and TGF-ß. This conclusion is based on the fact that BMP ligands have little, if any, affinity for BMPR-II, but they do have an affinity for type-I receptors. However, if BMPR-II is overexpressed together with the appropriate type-I receptor, the affinity of BMP ligands for BMPR-II is dramatically increased (63, 65, 73, 78, 79, 81). These data suggest that BMPR-II and the appropriate type-I receptor may act together to form a high-affinity complex for the BMP ligands (63, 76, 86). Alternatively, it has been proposed that BMP ligands may first bind to type-I receptors, followed by the recruitment of BMPR-II (63, 76, 86). If true, then the process of BMP ligand-receptor binding would be opposite of that for the activins and TGF-ßs.
C. BMP signal transduction
Upon binding of the BMP ligand (Fig. 1), the type-II receptor transphosphorylates the type-I receptor at an intracellular juxtamembrane site termed the GS domain, which is rich in glycine and serine residues (68, 87). The phosphorylated type-I receptor, in turn, transphosphorylates a set of intracellular substrate signaling proteins called Smads (84, 88, 89, 90). The specific Smad proteins whose activities depend on phosphorylation by type-I receptors are called receptor-regulated Smads (R-Smads). These R-Smads include Smad1, 2, 3, 5, and 8. Once activated, the R-Smad molecules interact with another Smad molecule, termed Smad4, which is a common partner for all R-Smads and thus called common Smad (Co-Smad) (91, 92, 93). This Smad complex then translocates to the nucleus to interact with specific transcription factors to regulate the expression of target genes.
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). This evidence, together with the fact that both the BMPs and the activins can bind to ActR-II and ActR-IIB, supports the hypothesis that the specificity of Smad signaling is determined by the type-I receptors, rather than the type-II receptors (106). Further support comes from the finding that overexpression of constitutively active forms of BMP-specific type-I receptors results in the activation of Smad1/5/8, which in turn, evokes BMP-specific biological responses (78, 80, 82, 83, 84, 103, 104). ALK-2 was originally identified as having an affinity for activin (70); however, later studies found that ALK-2 binds BMP ligands and transduces Smad signaling of BMPs but not activin (80, 107) (Table 2). Although there is evidence that individual BMP ligands may differentially activate Smad1, 5, and/or 8 (73, 82), little is currently known about the precise roles of a specific Smad protein in the mechanisms of different BMP ligand signaling (94). Uniquely, there are recent reports demonstrating that GDF-9 phosphorylates Smad2 but not Smad1/5/8 in rat and human granulosa cells (108, 109).
Although most research on BMP signaling has focused on the Smad pathway, there is increasing evidence that other signaling pathways may also be involved in mediating BMP action (86, 110, 111, 112, 113). One line of evidence is that there can be cross-talk in the BMP signal transduction pathway between the Smads and the MAPK family of signaling molecules, i.e., ERK1/2, p38, and stress-activated protein kinase/Jun N-terminal kinase (114, 115). Activated MAPK molecules have been shown to either positively transduce BMP signals (76, 113, 116, 117, 118) or act as inhibitors of Smad signaling (113, 119) (Fig. 1). The identification of the specific roles of other signaling pathways in the regulation of BMP biological activity is likely to progress rapidly.
D. Regulation of BMP action
The biological actions of BMPs can be regulated by 1) the extracellular regulation of ligand access to the receptors by BMP binding proteins as well as other BMP family ligands, and 2) the intracellular modulation of BMP signal transduction in the target cells (Fig. 1).
1. Extracellular regulation.
There are a number of TGF-ß superfamily binding proteins that can influence ligand access to receptors. The prototype of these binding proteins is follistatin, which was first isolated from ovarian follicular fluid as an inhibitor of pituitary FSH secretion, similar to inhibin (120, 121). Subsequent cloning studies, however, revealed that follistatin has no sequence homology with the inhibins (122, 123, 124). Several years later, follistatin was found to be an activin binding protein (125), and this property of follistatin accounts for much of its biological activity in the pituitary (126). Thereafter, the role of follistatin was extended to include the regulation of the activities of BMP-4, -7, -4/7 heterodimers, and BMP-15 (74, 127, 128). Therefore, it is not clear whether a given in vivo effect of follistatin is caused by the inhibition of the activins and/or the BMPs. It is interesting that male and female transgenic mice overexpressing follistatin exhibit numerous defects in reproduction: males exhibit Leydig cell hyperplasia, defective spermatogenesis, and seminiferous tubule degeneration; females exhibit small ovaries due to a block in folliculogenesis (129). These phenotypes are different from those seen in ActR-II knockout mice (130) and mice deficient for the activin subunits (131, 132). One possible interpretation of these results is that the phenotype of the follistatin-overexpressing mice may be caused by the inactivation of additional factors, quite possibly the BMPs. Another binding protein, follistatin-related protein, can also bind both BMPs and activins and inhibit their biological activity (133, 134, 135).
Interestingly, a recent study showed that follistatin can act to augment certain BMP-7 biological actions while inhibiting other BMP-7 biological activities (136). Low concentrations of BMP-7 induce muscle growth during chick embryogenesis through the up-regulation of Pax-3 expression. By contrast, high concentrations of BMP-7 induce apoptosis and muscle loss. It is interesting that the addition of follistatin inhibits the induction of apoptosis and muscle loss by BMP-7, yet augments BMP-7-stimulation of Pax-3 expression and muscle growth. Based on these data, it was proposed that the role of follistatin on BMP-7 action is to escort BMP-7 to the surface of myogenic cells and present it to the receptors at a concentration optimal for the stimulation of embryonic muscle growth (136).
Another interesting study in the field of BMP binding proteins is the recent discovery that connective-tissue growth factor (CTGF) has an affinity for both BMP-4 and TGF-ß (137). An intriguing aspect of this finding is that CTGF inhibits BMP-4 biological activity while enhancing the biological activity of TGF-ß. The underlying mechanism of this differential regulation by CTGF is not yet understood. However, because CTGF has a higher affinity for BMP-4 (Kd = 5 nM) than for TGF-ß (Kd = 30 nM) it was suggested that CTGF may block BMP-4 but facilitate TGF-ß binding to their cognate receptors. Although the impact of this novel finding on reproductive function has not yet been elucidated, there is accumulating evidence that suggests a role for CTGF in ovarian folliculogenesis (138, 139, 140, 141).
There are other binding proteins with specific affinities for BMPs. The prototype of the BMP-specific binding proteins is noggin, which was first identified as a factor regulating normal dorsal development in Xenopus embryos (142). Later studies demonstrated that noggin is an inhibitory binding protein for BMP-2, -4, -7, and -14 and GDF-5 (143, 144, 145, 146). The examination of the crystal structure of the complex of the noggin dimer bound to BMP-7 revealed that noggin blocks the epitopes in which BMP-7 binds to both the type-I and type-II receptors, demonstrating a mechanism for the inhibitory effects of noggin (145). Another binding protein, chordin, exhibits similar BMP-antagonistic properties as noggin in that both of these factors bind BMP-2 and -4 with high affinity, bind BMP-7 with lower affinity, and do not bind activin or TGF-ß (147, 148). A protein that is structurally similar to chordin, thus called chordin-like, binds and antagonizes BMP-4, -5, and -6, with the highest affinity for BMP-6. Although chordin-like does not bind activin, it does have a weak affinity for TGF-ß1 and -2 (149). Gremlin, DAN and cerberus form a family of secreted BMP-binding proteins that share a region of structural homology called a can domain, which closely resembles the cystine knot motif of the TGF-ß superfamily (150, 151). At present, nothing is known about the functional significance of these BMP antagonists in the mammalian reproductive system.
Inhibin, a member of the TGF-ß superfamily, has been known to block activin actions in a number of cell types (152, 153). The mechanism underlying this block is still controversial (154); however, currently available information suggests that inhibin binds to the membrane-bound binding protein, ß-glycan, and the complex then associates with ActR-II or ActR-IIB. In so doing, inhibin sequesters the type II activin receptors, thus preventing activin action (155). A recent study by Wiater and Vale (156) has extended the role of inhibin to include antagonism of BMP-2 and -7 activity. In this study inhibin A was shown to inhibit BMP actions by competitively sequestering ActR-II and ActR-IIB as well as BMPR-II (Fig. 1). The inhibition of BMP action by inhibin A is highly dependent on ß-glycan. It will be interesting to investigate the significance of inhibin A inhibition of BMP action with respect to reproductive physiology.
2. Intracellular regulation.
BMP actions can also be governed by the intracellular regulation of BMP signaling in the target cells (Fig. 1). Smad6 and 7, unlike other members of the Smad family, are inhibitors of TGF-ß superfamily signaling. Hence, they are called inhibitory Smads or I-Smads (157, 158, 159). This inhibition occurs through the ability of Smad6 and 7 to compete with the R-Smads for binding to the activated type-I receptors (160). Smad6 has also been shown to inhibit BMP signaling by forming a complex with Smad1, and thereby competing with Smad4 binding (94). Although both Smad6 and 7 can inhibit the activin/TGF-ß and BMP signaling pathways, it is generally believed that Smad6 preferentially inhibits BMP signaling (113). Administration of TGF-ß superfamily ligands can induce expression of the I-Smads, suggesting that these Smads act as part of an autocrine negative feedback loop (161).
BMP and activin membrane bound inhibitor (BAMBI) has structural features that resemble type-I receptors, except that it lacks the intracellular serine/threonine kinase domain. There is evidence that BAMBI can compete with the type-I receptors for ligand binding and inhibit the signaling of the TGF-ßs, activins, and BMPs (162, 163), suggesting that BAMBI might function as a dominant negative receptor. FK506 binding protein (FKBP)-12 binds TGF-ß superfamily type-I receptors in the GS domain and prevents transphosphorylation by the type-II receptors (164). Smad ubiquitination regulatory factor-1 (SMURF1) is an intracellular signaling inhibitor that targets Smad1 and 5 for ubiquitination and proteosomal degradation (165). This factor appears to specifically target BMP signaling as evidenced by its failure to affect Smad2 and 4. SMURF1 is unique because it reduces the intracellular level of Smads, independent of BMP ligand stimulation (165). The roles of these inhibitory factors in reproductive function have not yet been established. However, as the importance of the BMPs in reproductive physiology becomes better understood, it is likely that the intracellular control of BMP action in target cells will prove to be of importance for reproductive processes.
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III. Expression of the BMP System in Reproductive Tissues |
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TopAbstractI. IntroductionII. The BMP SystemIII. Expression of the...IV. Reproductive Functions of...V. Genetic Mutations of...VI. Perspectives and Future...References |
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A. Primordial germ cell (PGC) formation
The establishment of the germ line is a fundamental aspect of reproduction in all animals. In mice, germ cell determination is induced in epiblast cells by the extraembryonic ectoderm (166, 167), and is not acquired through the inheritance of preformed germ plasma, as it is in nonmammals (168). There is compelling evidence that BMP-4 and -8b play a central role in determining PGC formation in the mouse embryo. The genes encoding BMP-4 and -8b have overlapping expression in the extraembryonic ectoderm before gastrulation, i.e., before PGCs are seen (166, 169). Most BMP-4 knockout mice die in early gastrulation; however, some survive long enough to show that no PGCs develop (170). Thus, PGC formation in mice requires BMP-4 expression. There is also evidence from knockout mice that BMP-8b is required for PGC formation (169, 171). Interestingly, BMP-4 cannot rescue the defects in PGC formation in BMP-4-null mice, whereas the PGC defects can be rescued by BMP-8b (169, 171).
B. The pituitary
There is increasing evidence that locally produced BMPs play a critical role in the differentiation of the pituitary gonadotrope (172, 173). During normal mouse pituitary development, BMP-4 is expressed in the ventral diencephalon, whereas BMP-2 and the BMP binding protein, chordin, are expressed in the ventral condensing mesenchyme. Although the targeted deletion of the Bmp4 gene in mice is embryonic lethal, the analysis of these embryos indicates a failure of the invagination of Rathke’s pouch, the progenitor tissue for the anterior pituitary. According to this evidence, it has been concluded that BMP-4 plays an important role in pituitary development. In another study, the targeted expression of the BMP antagonist, noggin, to Rathke’s pouch caused the arrest of pituitary development resulting in the absence of almost all endocrine cell types, including the gonadotropes that produce FSH and LH (174). These data suggest that the patterning and histogenesis of Rathke’s pouch depend on the tissue-specific expression and regulated action of BMP-2, BMP-4, and chordin.
It has become evident that BMP ligands and receptors are expressed in the adult pituitary gland. In terms of ligands, BMP-6, -7, and -15 mRNAs are expressed in mice (175, 176); GDF-9 mRNA in humans (177); BMP-15 and GDF-9 mRNAs in brushtail possums (178); and BMP-15 mRNA in sheep (38). In terms of receptors, BMPR-II, ActR-II, ALK-2, and ALK-3 mRNAs are expressed in mice (176); ALK-6 mRNA in sheep (40); and ActR-II and -IIB mRNAs in rats (179). Of potential physiological importance are the findings that BMP-6, -7, and -15 can act directly on pituitary gonadotropes to regulate FSH synthesis and secretion (175, 176).
C. The hypothalamus
Northern blotting experiments have identified GDF-9 mRNA in the hypothalamus of the mouse and rat (177). Presumably, the GDF-9 is expressed in neurons; however, the cellular site of GDF-9 expression in the hypothalamus remains to be determined. There is also evidence for the expression of ActR-II and -IIB mRNAs in the rat hypothalamus; these receptors are found in areas involved in neuroendocrine regulation including the suprachiasmatic, supraoptic, paraventricular, and arcuate nuclei (179). In terms of reproduction, this evidence begs the question: to what extent might a hypothalamic BMP system contribute to the regulation of the GnRH neuron?
D. The female reproductive organs
1. The ovary.
Research performed in many laboratories in the last few years has provided important information about the expression of the BMP system in the mammalian ovary. The most comprehensive study on the expression of BMPs in the ovary has been reported recently using adult cycling rats (180) (Table 3). This section reviews the spatiotemporal expression patterns of the BMP system in the mammalian ovary with special emphasis on their implications for folliculogenesis and luteogenesis. It should be stressed that the pattern of BMP expression in the rat ovary may not necessarily apply to other species. Furthermore, given the importance of the mouse as a genetic model system, a thorough understanding of the tissue-specific expression of the BMP system in the mouse could help integrate the information on the functional roles of BMPs collected from experiments in other animals with the information that can be collected from genetic studies using the mouse model.
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i. Primordial/primary transition.
The initial event in folliculogenesis is the activation of a dormant primordial follicle to begin growing, a process termed the primordial/primary transition (181). The first indication that a primordial follicle has been activated is that the granulosa cells begin to transform from a squamous to a cuboidal shape. As this occurs, the granulosa cells begin to proliferate, and follicular growth is initiated. When more than 90% of the granulosa cells are cuboidal, there is an increase in RNA synthesis in the immature oocyte.
The mechanisms of the primordial/primary transition are poorly understood, but there is evidence that BMPs are involved. In rat primordial follicles, in situ hybridization and immunohistochemistry studies have demonstrated the expression of ALK-2, -3, -6, BMPR-II, ActR-II, ActR-IIB, ß-glycan, Smad5, and Smad8 in oocytes and low variable expression of ALK-3 and -6 in the surrounding squamous granulosa cells (179, 180, 182) (Table 3). In sheep primordial follicles, the expression of ALK-6 has also been reported in oocytes, and BMPR-II is detectable in both oocytes and granulosa cells (40, 183). It should be emphasized that there appear to be species differences in the expression of BMP system components in primordial follicles. For example, unlike the rat and sheep, no ALK-6 is detected in any cell types of mouse primordial follicles (184), and no ActR-II is detectable in human primordial follicles (185).
Collectively, these results suggest that primordial follicles are a target tissue for BMPs. What are the functional ligands for these BMP receptors? Lee et al. (186) have shown that BMP-7 can activate primordial follicles in the rat ovary. Because rat primordial follicles do not express BMP-7 (180), the BMP-7 ligand must originate from another site. The finding that BMP-7 is expressed in the theca, secondary interstitial cells, and sex cords (180) suggests that BMP-7 produced by these tissues may promote the primordial/primary transition in the rat. Evidence supporting a role for GDF-9 in this process has also been presented by Vitt et al. (187). Because primordial follicles are activated to grow in GDF-9-deficient animals (33), it can be concluded that GDF-9 is not essential for this basic developmental event in mice. It is perhaps noteworthy that, in the rat, primordial follicles do not appear to express BMP-2, -3, -3b, -4, and -6 (180). GDF-9 and BMP-15 are predominantly expressed in the oocytes of growing follicles (i.e., primary follicles throughout folliculogenesis); however, it appears that in some species mRNA transcripts of these genes can be detected in primordial follicles. In particular, there is accumulating evidence that in cattle, sheep, and humans, expression of GDF-9 precedes that of BMP-15 and can be detected in oocytes of primordial follicles, whereas BMP-15 expression can first be detected in oocytes of primary follicles (38, 188, 189, 190, 191). This pattern of expression appears to be different from the mouse and the rat, in which neither GDF-9 nor BMP-15 expression can be detected in oocytes of primordial follicles and the onset of expression occurs during the primary stage of folliculogenesis (30, 31, 35, 36, 37, 189, 192, 193, 194). In the brushtail possum, both GDF-9 and BMP-15 transcripts are detectable in oocytes of primordial follicles (178). The significance of the species differences in the expression of these genes remains to be elucidated.
ii. The primary/secondary transition.
A major event in mammalian folliculogenesis is the development of a second layer of granulosa cells. Among vertebrates, this step, termed the primary-to-secondary transition, is unique to mammals (195). Although the mechanisms involved in this basic event remain unknown, several lines of evidence indicate a functional role for BMPs. Studies in GDF-9-deficient mice (33) have demonstrated that folliculogenesis is blocked at the primary/secondary follicle transition stage in the absence of GDF-9. Therefore, the concept that oocyte-derived GDF-9 is essential for eliciting the primary/secondary transition in mice is clear. In sheep (38), but not mice (196), BMP-15 plays an essential role in evoking the primary/secondary transition. Considering this striking species difference in the physiological role of BMP-15 in the primary/secondary transition, much caution is needed in extrapolating the findings of BMP research from one species to another. An important question is how the BMPs regulate the primary/secondary transition. In the rat, the expression of BMP-6, BMP-15, and GDF-9 in the oocyte, BMP-2, -6, ALK-3, -6, and BMPR-II in the granulosa cells, and BMP-4 and -7 in the theca cells increases significantly during the primary/secondary transition (Table 3). GDF-9 and BMP-15 are expressed in human oocytes during the primary/secondary transition stage (189, 191). Questions concerning the role of BMPs in this process are of interest in light of the paper by Teixeira et al. (191) showing that the expression of GDF-9 mRNA is abnormally low in oocytes at the primary/secondary transition in women with polycystic ovary syndrome.
iii. Theca development.
After the primary/secondary transition, the basal lamina of the follicle becomes surrounded by mesenchymal cells that will eventually develop into an inner theca interna and an outer theca externa (197). The terminal differentiation of the theca externa is characterized by the expression of contractile proteins (i.e., actin and myosin) typical of smooth muscle cells. The terminal differentiation of the theca interna is characterized by the development of theca interstitial cells that produce androgens. Understanding theca development is important because theca-derived androgens are obligatory precursors for follicle estrogen production and have been implicated in atresia (197).
Genetic studies in mice have provided evidence that oocyte-derived GDF-9 may be necessary for attracting prospective theca cells to the basal lamina of developing preantral follicles (33, 198). Further evidence supporting a role of GDF-9 in theca development comes from the finding that recombinant GDF-9 stimulates the expression of a theca interstitial cell marker, CYP17, in the rat (187). Thus, rat theca interstitial cells appear to be targets for GDF-9. Analysis of theca development in adult cycling rats has revealed that the theca interstitial cells express BMP-3b, -4, -7, ALK-6, and follistatin (Table 3). One intriguing observation is that the theca interna of healthy follicles appears to be composed of two distinct populations of theca interstitial cells: one group, which expresses BMP-4, is present as an outer layer of cells juxtaposed to the theca externa; and another, which expresses BMP-7, is present at the proximal side of the theca interna near the basal lamina of the follicle (180). These differences are found throughout folliculogenesis, beginning during the secondary stage. This is intriguing because it implies, for the first time, that there is functional heterogeneity among the theca interstitial cells during follicular growth and development.
iv. Selection and follicle dominance.
A hallmark of estrous and menstrual cycles is the selection of a dominant follicle that will ovulate a fertilizable egg and then develop into a corpus luteum that secretes progesterone. A fundamental concept in ovary physiology is that selection is critically dependent on the secondary rise in plasma FSH. Despite considerable effort, the mechanism by which FSH causes selection remains poorly understood.
There is increasing evidence that the development of dominant follicles in mammals is accompanied by the tissue-specific expression of the BMP system, replete with ligands, receptors, and binding proteins (Fig. 2 and Table 3). In a broad sense, the studies of a variety of species (31, 32, 179, 182, 183, 184, 191, 194, 199, 200), most notably the rat (201), indicate the following: the oocytes of Graafian follicles express BMP-6, -15, GDF-9, ALK-2, -3, -6, and ActR-II; the granulosa cells express BMP-2, ALK-3, -6, BMPR-II, and follistatin; and the theca cells express BMP-3b, -4, -7, ALK-3, and -6. In the rat, one particularly interesting observation is the dramatic loss of BMP-6 mRNA in the granulosa cells at or about the time the dominant follicle is selected during the normal estrous cycle (180). This observation suggests that a rapid loss of BMP-6 expression could be an incipient marker for the newly selected dominant follicle in the rat. Because BMP-6 can prevent FSH action (39), one could speculate that the rapid loss of BMP-6 expression in the granulosa cells may be required for FSH to exert its obligatory functions during the selection process. Because FSH induces the loss of BMP-6 mRNA in rat granulosa cells (G. F. Erickson and S. Shimasaki, unpublished data), one might predict that the secondary rise of FSH may down-regulate BMP-6 expression and this change may have a central role in selection. Finally, with regard to FSH action, follistatin has been shown to neutralize the bioactivity of BMP-15, which inhibits FSH receptor expression (128). It is possible, therefore, that the strong expression of follistatin by the dominant follicle (Table 3) may play a role in inhibiting BMP-15 action such that sufficient FSH receptors are expressed in the granulosa cells to permit FSH action during follicle development.
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). Indeed, the highest levels of BMP-2 expression in the rat ovary are found in the granulosa cells of atretic follicles. These findings, together with the fact that atresia is accompanied by a dramatic decrease in follistatin expression (Table 3), have led to the proposal that atresia might be governed by the high activity of BMP-2 and -6.
b. Corpus luteum formation.
After ovulating the oocyte-cumulus complex, the dominant follicle transforms into a corpus luteum by a process termed luteinization. As luteinization proceeds, the corpus luteum secretes large quantities of progesterone. If embryo implantation does not occur, the corpus luteum stops synthesizing progesterone and degenerates, a process termed luteolysis. Despite enormous effort, the mechanisms underlying luteogenesis remain unclear. The idea that intrinsic BMPs might be involved is gaining support.
i. Luteinization.
The classic view of luteinization is deeply rooted in the principle of luteinization inhibitors (208, 209). In this principle, oocyte-derived regulatory molecules, luteinization inhibitors, act within the follicle to inhibit premature luteinization and limit progesterone biosynthesis (208, 210, 211, 212). In terms of the oocyte, three BMPs with luteinization inhibitor activity in the granulosa cells have been identified: BMP-6 (39), BMP-15 (37, 213), and GDF-9 (214). Therefore, it is reasonable to conclude that the release of the oocyte at ovulation plays a prominent role in activating luteinization by virtue of the loss of these luteinization inhibitors. Further insight into the role of the BMP system in the rat corpus luteum has been reported recently (180). One key finding is that the level of expression of BMP ligands and receptors is quite low in the corpus luteum during the process of luteinization. Most notably, BMP-2 expression is rapidly down-regulated after ovulation, and there is no detectable BMP-2 in the corpus luteum during luteinization (Table 3). Importantly, luteinization is accompanied by a high level of follistatin expression. Taken as a whole, these data fit the hypothesis that a loss of BMP responses may have an important role in evoking and maintaining luteinization and progesterone production by the rat corpus luteum. These basic findings rest at the foundation of our present hypothesis that BMPs may be the long-sought luteinization inhibitors (32).
ii. Luteolysis.
The spatiotemporal pattern of expression of the BMP system in the rat corpus luteum undergoes important changes during luteolysis (180). In a broad sense, these changes are consistent with an increase in the expression of the BMP system, suggesting the interesting hypothesis that the increased expression and action of BMPs may be a part of the physiological mechanism of luteolysis. There are three main lines of evidence to support this hypothesis. The first is that the expression of the Bmp2 gene is reactivated at the time of luteolysis, being strongly expressed in clusters of endothelial cells throughout the corpus luteum (180). The abundant evidence linking BMP-2 to the induction of apoptosis in other cell types (111, 215, 216, 217, 218) raises the question: does BMP-2 play a role in luteolysis by promoting apoptosis and in inhibiting progesterone production? The second is that follistatin, which is strongly expressed in healthy corpora lutea, becomes undetectable in the corpus luteum during early luteolysis (180) (Table 3). The loss of follistatin could be physiologically relevant because it may serve to allow BMP-2 to function during early luteolysis. It should be noted that follistatin mRNA remains undetectable in the corpus luteum until proestrus 2000 h when few lutein cells appear strongly positive (Table 3). The functional significance of this follistatin expression during the late luteolytic phase is unknown. A third line of evidence is the reactivation of ALK-6 expression during luteolysis. When luteolysis is initiated, the ALK-6 expression increases in the corpus luteum, being most abundant in the theca externa and vascular endothelial cells. Interestingly, a functional link between the expression of ALK-6 and luteolysis was provided by loss-of-function studies showing that the corpus luteum of the cycle continues to secrete progesterone in the absence of ALK-6 (184). The mechanisms by which the increase in BMP system activity may cause luteolysis have not yet been elucidated. The general principle to emerge from this study is that the expression of the BMP system in the rat corpus luteum appears to decrease in association with luteinization and increase during luteolysis.
c. Secondary interstitial cells.
During atresia, the oocyte and granulosa cells undergo apoptosis, whereas the theca remains in the stroma as islands of secondary interstitial cells that continue to exhibit the potential for androgen synthesis (197). The physiological role of the secondary interstitial cells is not understood in any species. The secondary interstitial cells of the adult rat ovary express BMP-2 and -7, the receptors ALK-3 and -6, and the BMP binding protein, follistatin (Table 3). A particularly interesting feature of this system is the temporal pattern of follistatin mRNA expression in the secondary interstitial cells during the preovulatory period. Follistatin mRNA is absent in all the secondary interstitial cells during most of the cycle except at proestrus 2000 h when it is very strongly expressed (see footnote in Table 3) (180). Because secondary interstitial cells are target cells for LH (197), it is not unreasonable to propose that the preovulatory surge of LH during proestrus may be involved in the burst of follistatin gene expression in these cells at proestrus 2000 h. Based on the antiluteinization properties of the BMP system (32), it is possible that this burst of follistatin expression by the secondary interstitial cells may be involved in neutralizing ovarian BMP activities, which in turn, leads to the preovulatory surge of progesterone production, resulting in inducing ovulation and mating behavior in the rat. If this hypothesis proves correct, then the follistatin expression in the secondary interstitial cells in the rodent would be raised to a new level of prominence.
d. The ovarian surface epithelium.
The mammalian ovary is covered by a layer of epithelial cells affixed to a basement membrane. A characteristic feature of the ovarian surface epithelium is the potential to undergo cyclic apoptosis and regeneration, respectively, during the ovulatory and luteal phases of the cycle. In normal cycling rats, the genes encoding BMP-3b, -4, and -6 are expressed in the surface epithelium (180). The cells appear to express BMP-3b and -6 constitutively over the cycle, a finding consistent with a maintenance-type role for these BMPs. Notably, the expression of BMP-4 is cell- and cycle-specific. Namely, Bmp4 gene expression occurs in a subset of epithelial cells covering the freshly ovulated follicle and the newly formed corpus luteum on estrus and diestrus (180). Therefore, the spatiotemporal pattern of BMP-4 expression is tightly correlated with the replenishment of the surface epithelium that is exfoliated from the ovulating follicle. This raises the interesting hypothesis that inducible and cell-specific BMP-4 expression may have a role in the regeneration of the ovarian surface epithelium during postovulatory repair. Evidence from epidemiological studies indicates that the risk of epithelial ovarian cancer is decreased by factors that suppress ovulation, such as pregnancy, breast-feeding, and the oral contraceptive pill (219, 220). Considering the relationship between ovulation and the induction of Bmp4 gene expression, it will be interesting to explore a possible link between the risk of ovarian cancer and the ovulation-inducible and epithelial cell-specific Bmp4 gene expression.
e. The sex cords.
The sex cords are vestiges of the cranial portion of the mesonephric tubules and ducts, and in the adult ovary they appear as a cluster of blind tubules in the hilar region. Although the physiological importance of ovarian sex cords is unknown, they have been linked to the genesis of ovarian cancer through distinct TGF-ß signaling pathways (221, 222). Furthermore, a role for follistatin in the genesis of this cancer has been established (129). In addition, the ovarian sex cords in adult rat ovaries express an intrinsic BMP system replete with ligands (BMP-3b, -4, and -7) and receptors (ALK-3, -6, and BMPR-II) (180) (Table 3). Because BMPs are potent growth factors and follistatin can modulate their bioactivity, it is possible that this intrinsic BMP system could be involved in the pathogenesis of sex cord tumors. The fact that sex cords are sometimes found juxtaposed to developing follicles, blood vessels, and secondary interstitial cells (180) suggests that the sex cord-derived BMPs may have paracrine actions on these tissues.
2. The uterus.
The uterus is prepared for embryo implantation by estrogen and progesterone. Implantation involves a complex pattern of cellular changes resulting in placenta formation. These changes occur in the uterine epithelium, stroma, and embryonic trophoblasts. The general course of events involves cell proliferation, cytodifferentiation, and apoptosis. There is increasing evidence for a role of the BMP family in this important process.
a. The nonpregnant uterus.
The mRNAs encoding a number of BMP family members have been identified in the nonpregnant uterus of both rodents and humans. In normal cycling mice, BMP-6 (223), BMP-7 (224), GDF-10 (225), and ALK-6 (184) are expressed in the uterus. In situ hybridization studies revealed that BMP-6 and -7 are coexpressed in the endometrial epithelial and subjacent stromal cells (223, 224), and ALK-6 mRNA is restricted to the epithelial and glandular cells (184). In mice, BMP-7 expression in the uterus is abolished in response to estrogen treatment (224). Because endometrial cells proliferate in response to estrogen, the reduced BMP-7 expression might be involved in the mechanism for estrogen-induced endometrial cell proliferation. In humans, but not rodents, GDF-9 mRNA has been identified by Northern blotting in the nonpregnant uterus (177). Evidence that the BMP system might have a functional role in the uterus comes from studies of ALK-6-deficient mice (184). In the absence of ALK-6, the uterine linings are thin, and endometrial glands are absent. This suggests that ALK-6 has an important role in the development of mouse endometrial glands.
b. Decidualization.
In the mouse, the deciduum develops from uterine stromal cells and eventually completely surrounds the implanted embryo. During the decidual reaction, the adjacent endometrial stromal cells undergo hyperplasia and hypertrophy as they convert from mesenchymal to fully differentiated decidual cells. In addition, some cells undergo apoptosis, thereby creating space for the growing embryo. There is evidence from in situ hybridization studies that members of the BMP family are expressed during decidualization. The first is that BMP-7 mRNA is lost from the uterine epithelium shortly after implantation; however, it continues to be expressed in the decidualizing stromal cells, being distributed in a gradient with the highest expression in the stromal cells nearest the uterine epithelium (226). Secondly, when embryo attachment to the epithelium occurs, BMP-2 expression is induced in the subjacent stromal cells (227, 228). After implantation, BMP-2 continues to be expressed in the dividing decidual cells farthest from the embryo. A similar pattern of expression has been reported for BMP-8a (29). The loss of BMP-8a does not alter female fertility (229); thus, BMP-8a is not required for decidualization in mice. Thirdly, several other BMP family members have been identified in and around the mouse deciduum. BMP-4 mRNA and Smad1 are predominantly expressed in the vascular endothelial cells, suggesting a role of these components of the BMP system in the angiogenesis that accompanies decidualization (226). BMP-5 mRNA shows low levels of expression in the stroma close to the myometrium and in the myometrial connective tissue (228). There is also evidence that several BMP antagonists colocalize with these BMP ligands in mouse decidual tissue, including follistatin (230), noggin (228), and DAN/Dante (228).
c. The placenta.
As in the deciduum, there is evidence for an intrinsic BMP system in the mouse and human placenta. In mouse embryos, the expression of BMP-4 in the epiblast-derived tissues has been implicated in the development of the vascular connection between the placenta and embryo (231). This could be of primary importance in maintaining the viability of the embryo. In the mouse placenta, BMP-8b is expressed in trophoblasts in the labyrinthine region (29), BMP-4 is expressed in the spongiotrophoblasts, and BMP-7 is highly expressed in the giant trophoblasts (224). Thus, the hypothesis has emerged that the expression of BMP-4, -7, and -8a in the mouse placenta may be involved in trophoblast proliferation differentiation. In humans, BMP-7 can act on cultured trophoblasts to inhibit the secretion of human chorionic gonadotropin (hCG) (232). This could be clinically relevant because an appropriate level of hCG production is critical for maintaining early pregnancy in women. Little is known about the expression of the BMP family in the human placenta. In this regard, GDF-15, also called placental bone morphogenetic protein (PLAB) or prostate-derived factor (PDF) (Table 1), is highly expressed in the terminal villae of the human placenta (233, 234). Thus, GDF-15 may be involved in regulating functions in the human placenta. It should be noted that placental calcification commonly increases with gestational age during pregnancy and is often observed in placentas associated with preeclampsia and/or fetal growth restriction (235).
3. The mammary gland.
Studies conducted in mice indicate that BMP-2 and -4 are expressed within the developing mammary gland (236). In the early stages of mammary gland development, BMP-2 and -4 are expressed in the epithelium and underlying mesenchymal cells, respectively (236). BMP-2 and -4 often show this tissue-specific pattern of epithelial-mesenchymal expression during morphogenesis and primary induction. Consequently, it has been proposed that the localized expression of these BMPs play a role in the induction and invagination of the mammary epithelium. In the postnatal mammary gland, both BMP-2 and -4 mRNAs are expressed within the cells of the stromal fat pad. Therefore, the transition of the mammary gland from fetal to postnatal development involves the transition of Bmp2 gene expression from the epithelium to a zone of mesenchymal cells in which the mammary fat cells ultimately differentiate. There is evidence for the expression and function of BMPs in breast cancer. For example, BMP-2 is expressed in primary breast tumor tissue, and BMP-2 can act on the breast cancer cells to regulate growth (237). The question of the involvement of the BMP system in mammary tumorigenesis is a focus of current research.
E. The male reproductive organs
1. The testis.
In situ hybridization studies have established that BMP receptors and ligands are expressed in testicular germ cells during spermatogenesis. In murine seminiferous tubules, ALK-3 (238) and ActR-II (179) are expressed in the pachytene spermatocytes and round spermatids (179). There is no detectable ALK-6 in the murine testis (238). A variety of BMP ligands have also been identified in male germ cells. Before puberty, both Bmp8a and 8b genes are expressed at low levels in mouse spermatogonia and primary spermatocytes (29). After puberty, BMP-8a and -8b expression is shut off in spermatogonia, but high levels of expression continue in stage 6–8 round spermatids. The observation that there is an inverse relationship between the level of BMP-8a and -8b expression and germ cell apoptosis suggests that these BMPs may function to inhibit germ cell degeneration. Indeed, gene knockout studies have demonstrated that BMP-8b is required for germ cell survival and fertility in adult male mice (34). In addition to BMP-8a and -8b, GDF-9 has been shown to be strongly expressed in the mouse testis, being present in pachytene spermatocytes and early round spermatids (177). Because GDF-9-deficient male mice are fertile (33), one can conclude that testicular GDF-9 is not essential for normal spermatogenesis and male fertility in this species. It is notable that BMP-15 mRNA is not detectable in mouse, human, and sheep testes by Northern blot hybridization (30, 189); however, BMP-15 transcripts have been detected in human (189) and fetal ovine (38) testes by RT-PCR followed by Southern blot hybridization.
2. The epididymis.
Mammalian sperm acquire the capacity for forward motility, which is essential for male fertility, during passage through the epididymis. This is accomplished through functional interactions between spermatozoa and the epididymal epithelial cells. In situ hybridization studies have demonstrated an intrinsic BMP system in the mouse epididymis. BMP ligands (BMP-7 and -8a), BMP receptors (ALK-3 and -6, BMPR-II), and Smad1 have been identified in the epithelial cells of the mouse epididymis (29, 34, 229, 238, 239, 240). The BMP ligands are coexpressed in the initial segment or caput (229), whereas the receptors and Smad1 are ubiquitously expressed throughout the epithelium of the epididymis (229). There is evidence that the cell-specific expression of BMP-7 is developmentally regulated. During early postnatal development, BMP-7 transcripts are expressed uniformly in the epithelial cells throughout the epididymis (240). As the mice age, BMP-7 expression becomes gradually restricted to the initial segment with an ascending gradient of expression in the epididymal epithelial cells in the transition from the cauda epididymal tubules to the vas deferens.
3. The prostate.
The prostate is composed of alveoli and ducts embedded in fibromuscular tissue. Its secretion together with that of the seminal vesicle serves as a diluent and vehicle for transport of sperm to the female. The prostate is of great medical interest because 1) prostatic hyperplasia is a common disease in the aging male; and 2) prostate cancer is the most common malignant tumor in the elderly male in the United States (241).
In mice, BMP-4 and -7 are expressed in the prostate (242). BMP-7 mRNA is significantly decreased after orchidectomy and increased by testosterone and dihydrotestosterone treatment, indicating that BMP-7 expression in the mouse prostate is androgen dependent; this is in contrast to BMP-4 that is constitutively expressed (242). The authors postulate that the expression of BMP-7 in the mouse prostate may be involved in the stimulation of bone formation and osteosclerosis observed in metastatic prostate adenocarcinomas.
In humans, the prostate expresses BMP-4 mRNA as do the human prostate cancer cell lines, PC-3 and DU-145. Additionally, PC-3 cells also express BMP-2 and BMP-3 in fairly large amounts (243). By comparison, normal and neoplastic rat prostate tissue expresses predominantly BMP-3 mRNA. In humans and rats, the cellular sites of expression and the physiological importance of these BMPs in the prostate gland are unknown. Three possible functions of these BMPs in the prostate have been proposed: 1) normal prostate growth and morphogenesis; 2) neoplastic prostate cell behavior; and 3) the capacity of prostate cancer cells to stimulate new bone formation at metastasis tumor sites in bone. In this regard, Paralkar et al. (234) found that GDF-15, also called PDF or PLAB (Table 1), is strongly expressed in the epithelium of the normal human prostate and induces ectopic cartilage formation and the early stages of endochondral bone formation. Studies in rats have established the concept that prostate PDF expression is regulated by androgens.
Another study has investigated the expression of BMP-6 mRNA and protein in normal and malignant rat and human prostate tissues (244). In the rat, BMP-6 was found at similar levels in both normal and malignant prostate tissues, and the level of BMP-6 expression appeared androgen-independent. In humans, BMP-6 mRNA and protein are expressed in normal and malignant prostate tissues, but interestingly, its expression is higher in prostate cancer as compared with adjacent normal prostate tissue. Taken together, these results have suggested that BMP-6 may contribute to prostate tumorigenesis.
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IV. Reproductive Functions of BMPs |
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TopAbstractI. IntroductionII. The BMP SystemIII. Expression of the...IV. Reproductive Functions of...V. Genetic Mutations of...VI. Perspectives and Future...References |
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A. The ovary
1. BMP-4 and BMP-7.
The presence of a functional BMP system in the mammalian ovary was first reported by Shimasaki et al. (32) in 1999. In this study, BMP-4 and -7 mRNAs were identified predominantly in theca cells, and BMPR-II, ALK-3, and ALK-6 predominantly in granulosa cells of rat ovaries. This finding led to the obvious question: what is the functional role of the intrinsic ovary BMP system? Using primary cultures of rat granulosa cells grown in serum-free medium, recombinant BMP-4 and -7 were found to modulate FSH signaling in a way that promotes estradiol production (aromatization) while inhibiting progesterone biosynthesis (32). It is well established that granulosa cells in growing follicles respond to FSH stimulation in vivo by synthesizing estradiol, but not progesterone until the preovulatory period. On the other hand, when granulosa cells are cultured in vitro they synthesize both estradiol and progesterone in response to FSH stimulation. These results suggested that a selective inhibitor of progesterone synthesis, i.e., a luteinization inhibitor, must act in vivo to modulate FSH bioactivity in a way that reflects normal follicle steroidogenesis during the estrous cycle (208, 209). The challenge has been to identify the nature of this putative luteinization inhibitor. In this regard, theca cell-derived BMP-4 and -7 are the first factors identified with biological activities that are consistent with the long-sought luteinization inhibitor (32). Notably, BMP-4 and -7 do not affect granulosa cell steroidogenesis in the absence of FSH in the rat (32). In a separate study, BMP-4 was also shown to inhibit FSH-stimulated progesterone synthesis by granulosa cells collected from sheep ovaries; however, in contrast to the rat model, BMP-4 also inhibited basal progesterone synthesis in sheep granulosa cells in the absence of FSH (41).
Progress has been made in our understanding of how BMP-7 differentially regulates FSH action. In rat granulosa cells, BMP-7 augments FSH-induced expression of P450 aromatase (P450arom) mRNA, while inhibiting FSH-induced expression of the mRNA encoding the rate-limiting factor in steroidogenesis, steroidogenic acute regulatory protein (StAR) (186). BMP-7 had no effect on the levels of FSH-stimulated P450 side-chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNAs. These results argue that the ability of BMP-7 to differentially regulate FSH-dependent estradiol and progesterone production is mediated by differential expression of P450arom and StAR.
Mice with null mutations in the Bmp4 and Bmp7 genes die either before birth (245) or shortly after birth, respectively (206). Therefore, knockout mouse technology has failed to provide direct information on the role of these BMPs in the ovary in vivo. To address this question, Lee et al. (186) injected recombinant BMP-7 into the rat ovarian bursa and characterized changes in folliculogenesis, ovulation, and steroidogenesis. These experiments revealed that BMP-7 decreases the number of primordial follicles but increases the number of primary, secondary, and antral follicles. Thus, BMP-7 can promote the recruitment of primordial follicles into the growing follicle pool. The finding that BMP-7 stimulates granulosa cell mitosis suggests a possible mechanism by which BMP-7 stimulates this process. Because BMP-7 is not expressed in rat primordial follicles, it is possible that BMP-7 derived from adjacent, larger follicles is responsible for this action of BMP-7 in vivo. In this study, administration of BMP-7 was also shown to inhibit ovulation and progesterone production (186). Given that progesterone is essential for ovulation (246, 247, 248, 249, 250, 251, 252, 253), the inhibition of progesterone by BMP-7 could be causally connected to the mechanisms of ovulation inhibition.
The results of studies using human ovarian theca-like tumor (HOTT) cells have provided additional evidence for a role of BMP-4 in regulating steroidogenesis (254). These cells, which express ALK-3, -6, and BMPR-II, were established from an ovarian tumor that produces excessive androgens. Although HOTT cells do not respond to LH, they do exhibit increases in steroidogenesis in response to stimulation by forskolin and dibutyryl cAMP (255). In this system, BMP-4 inhibits forskolin-stimulated increases of CYP17 levels without changing the levels of StAR, P450scc, and 3ß-HSD. One possible interpretation of this result is that BMP-4 may be involved in switching the androgen-secreting theca cell into a progesterone-secreting theca lutein cell.
2. BMP-2.
Studies with cultured sheep granulosa cells have shown that recombinant BMP-2 can amplify FSH-induced estradiol and inhibin A production (183). To what degree BMP-2 modulates FSH-dependent progesterone production is unknown. In another study, BMP-2 was found to stimulate the expression of inhibin ßB subunit mRNA and the secretion of dimeric inhibin B by cultured human granulosa-lutein cells (256). These stimulatory effects of BMP-2 were inhibited by cotreatment with hCG. Similar results were obtained when these human cells were challenged with BMP-6, but not BMP-3 or BMP-3b.
3. BMP-6.
Unique biological functions of BMP-6 have been identified in the ovary using primary cultures of rat granulosa cells (39) (Fig. 3). Like BMP-4 and -7, BMP-6 is effective in inhibiting FSH-induced progesterone synthesis. However, whereas BMP-4 and -7 augment FSH-induced estradiol production, BMP-6 has no effect on FSH-induced estradiol production. Consistent with its steroidogenic regulation by granulosa cells, BMP-6 inhibits FSH stimulation of StAR and P450scc mRNA expression without affecting the expression of P450arom mRNA. Furthermore, BMP-6 inhibits progesterone synthesis and the corresponding expression of StAR and P450scc induced by forskolin. In contrast, BMP-6 has no effect on these steroidogenic parameters induced by 8-
