Human Reproduction Update Advance Access originally published online on February 10, 2005
Human Reproduction Update 2005 11(2):144-161; doi:10.1093/humupd/dmh061
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The role of proteins of the transforming growth factor-ß superfamily in the intraovarian regulation of follicular development
AgResearch, Wallaceville Animal Research Centre, P.O.Box 40063, Ward Street, Upper Hutt 6007, New Zealand
1 To whom correspondence should be addressed. Email: ken.mcnatty{at}agresearch.co.nz
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Abstract |
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Ovarian follicular development occurs in a hierarchical manner with each follicle having a unique biochemical composition at any moment in time. It has long been understood that a precise coordination between the growth and maturation of the oocyte and adjacent follicular cells (i.e. somatic cells) is essential in order to produce an oocyte that is fully competent to undergo fertilization and embryo development. In addition to the critical endocrine signalling pathways between the hypothalamus, pituitary and ovary, it is now evident that the oocyte itself is important in influencing the microenvironment of the developing follicle by regulating, via paracrine and autocrine mechanisms, its own maturation as well as somatic cell proliferation, differentiation and ovulation rate. Several of the key oocyte-derived regulating factors are members of the transforming growth factor-ß (TGF-ß) superfamily and to date the best understood are growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and BMP6. Significant species differences appear to exist in the relative importance of these growth factors and much remains to be elucidated about their roles in the human ovary. More information on the roles of these factors during ovarian follicular development is likely to advance new therapeutic applications for management of fertility as well as our understanding of how better to assess oocyte quality.
Key words: bone morphogenetic protein / growth differentiation factor / follicle / ovary / transforming growth factor-ß
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Introduction |
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Traditionally, studies examining the control of ovarian follicular development have focused mainly on the endocrine regulation of the hypothalamic–pituitary–ovarian system. Indeed, the current methods available to regulate ovarian function in order to suppress or enhance fertility ultimately influence this axis. However, it has long been thought that intraovarian factors are also important in regulating follicular development in a paracrine or autocrine manner. In recent years considerable attention has been focused on members of the transforming growth factor-ß (TGF-ß) superfamily and their potential roles as local regulators of ovarian function and fertility. In part, this interest has arisen from several reports showing that growth differentiation factor 9 (GDF9) and/or bone morphogenetic protein 15 (BMP15, also known as GDF9B) are essential for normal follicular development in rodents, sheep and humans (Elvin et al., 2000
; McNatty et al., 2003; Di Pasquale et al., 2004; Shimasaki et al., 2004). Of special interest is that the aforementioned growth factors are produced by oocytes which in turn not only influence normal follicular development but also play a key role in regulating the number of follicles ovulating at each cycle (i.e. ovulation rate), at least in some species. Recognition of the central role of the oocyte in controlling follicular development and ovulation rate is leading to a paradigm shift in the way scientists and clinicians view the regulation of ovarian function and these discoveries are forming the basis for the development of new therapeutics for regulating fertility in mammals. This review focuses on the putative intraovarian roles of TGF-ß 1, 2 and 3, BMP6, GDF9 and BMP15. The purpose of focusing on these particular TGF-ß family members is that the TGF-ß 1, 2 or 3 proteins have been the most extensively studied and that they together with BMP6, GDF9 and BMP15 have been identified in ovarian follicular cell types including oocytes in some species. While we have chosen a limited number of TGF-ß family members, a central theme emerging from several laboratories is that many members of this superfamily are involved in the regulation of ovarian follicular growth. In this context, there are several other recent reviews that may be of interest to the reader (Durlinger et al., 2002; Matzuk et al., 2002; Welt et al., 2002; Knight and Glister, 2003; Shimasaki et al., 2004). |
Overview of the TGF-ß superfamily |
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The TGF-ß superfamily is comprised of >35 proteins that share common structural motifs (Chang et al., 2002
). These proteins include members of the TGF-ß, activin/inhibin, GDF and BMP subfamilies as well as proteins such as anti-Müllerian hormone (AMH). Many members of the TGF-ß superfamily members are pivotal in controlling cellular growth and differentiation during fetal as well as adult life (Heldin et al., 1997; ten Dijke et al., 2000). Structural characteristics
The TGF-ß superfamily members are each synthesized as a preproprotein comprised of a signal peptide, a large proregion, and a smaller biologically active mature region (Massague, 1990; Chang et al., 2002). The proregion is separated from the mature region by a basic amino acid cleavage site which is recognized by members of the proprotein convertase family such as furin. However, the specific proteases involved in processing members of the TGF-ß superfamily in the various ovarian cell types have not been identified. Typically, these proteases recognize a four amino acid cleavage site, with arginine as the first and often last amino acid and minimum recognition site of RXXR (Thomas, 2002; Taylor et al., 2003; Rockwell and Thorner, 2004). The mature region typically contains either seven or nine cysteines of which six form a cystine knot that is characteristic of this family (Vitt et al., 2001; Chang et al., 2002) (Figure 1). The examples shown in Figure 1 represent homodimers of ovine BMP15 and human TGF-ß1. Despite the fact that the mature regions of these two molecules only 20% identity at the amino acid level, their tertiary structures share are similar. Another characteristic of these proteins is that the formation of homo- or heterodimers is thought to be important for biological activity of most, if not all, family members. These dimers are covalently bound through a conserved cysteine residue (Chang et al., 2002). Intriguingly, a few members of the family, including GDF9 and BMP15, are missing this conserved cysteine and thus are unable to form this covalent bond (McPherron and Lee, 1993; Dube et al., 1998; Laitinen et al., 1998) (Figure 1). However, the evidence from in vitro studies suggests that both GDF9 and BMP15 can form homodimers as well as heterodimers through non-covalent interactions (Liao et al., 2003, 2004). In some instances, the heterodimer forms of the proteins appear to be more biologically active (Israel et al., 1996). The role of the proregion of the TGF-ß superfamily members is not well understood. It is thought to be important for the correct folding and dimerization of the molecule as dimerization occurs before proteolytic processing to release the biologically active mature region (Shimasaki et al., 2004). For some members of the family, the proregion also appears to be important for regulating the activity of the mature region through non-covalent association with the mature dimer to either inhibit or enhance biological activity. For example, the proregions of both TGF-ß (Massague, 1990) and myostatin (GDF8) (Jiang et al., 2004) are thought to inhibit the biological activity of the mature regions of these proteins whereas the proregion of AMH appears to enhance the biological activity of the mature region (Wilson et al., 1993).
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Receptors
Members of the TGF-ß superfamily signal through receptor complexes composed of two types of membrane-bound serine-threonine kinases. Currently, seven type I receptors [activin receptor-like kinase (ALK) 1–7] and five type II receptors (ActRII, ActRIIB, BMPRII, TGFßRII and AMHRII) have been identified (de Caestecker, 2004; ten Dijke and Hill, 2004). Multiple associations appear to occur between receptor subtypes and thus a large number of potentially unique receptor complexes with signalling specificity are possible (ten Dijke et al., 2003; Shimasaki et al., 2004). Typically, binding of the TGF-ß/activin family members occurs to the type II receptor which then facilitates binding of the type I receptor to the complex, whereas affinity of the BMP family members is typically higher for type I receptors (Shi and Massague, 2003; ten Dijke et al., 2003; de Caestecker, 2004). An example of a BMP homodimer bound to the extracellular domains of a type II and type I receptor complex is shown in Figure 2 (McNatty et al., 2004). Binding of ligand activates the kinase of the type II receptor which then phosophorylates the type I receptor leading to activation of its kinase (Shi and Massague, 2003; ten Dijke et al., 2003; de Caestecker, 2004). As a consequence, the active type I receptor phosphorylates and thereby activates certain intracellular signalling molecules known as Smads (Miyazawa et al., 2002; ten Dijke and Hill, 2004) (Figure 2). A third receptor type, namely the TGF-ßRIII or betaglycan, has also been identified; in some cases this facilitates binding of TGF-ß2 to the type II receptor, thus enhancing the ability of the TGF-ß2 to signal (Shi and Massague, 2003). Similarly, betaglycan facilitates inhibin's ability to associate with the activin type II receptor and thereby antagonize the actions of activin (Gray et al., 2002) and potentially a number of BMPs (Wiater and Vale, 2003).
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Cellular signalling
As indicated above, binding of the TGF-ß superfamily members to its receptor complex results in phosphorylation of the Smad signalling molecules (Miyazawa et al., 2002; Derynck and Zhang, 2003; Shi and Massague, 2003; ten Dijke and Hill, 2004). There are eight Smad proteins in total, Smad 1–8. Typically, Smad 2 and 3 are activated by members of the TGF-ß/activin subfamilies and Smad 1, 5 and 8 are activated by members of the BMP subfamilies. Once phosphorylated, these Smads combine with a common Smad, namely Smad 4, and then translocate to the nucleus where they regulate transcription through interactions with transcriptional regulation elements (Figure 2). Smad 6 and 7 are inhibitory Smads that prevent the activation of the Smad signalling pathway. There is growing evidence that activation of the TGF-ß receptor complex can also regulate other signalling pathways such as the mitogen-activated protein kinase (MAPK) pathways (Derynck and Zhang, 2003).
Binding proteins
Another mechanism involved in regulating TGF-ß superfamily action is through interactions with binding proteins. Binding of a ligand to a binding protein may cause inhibition of ligand bioactivity (Figure 2). Follistatin is one of the best characterized representatives of these binding proteins (Welt et al., 2002; Lin et al., 2003). This protein binds activin with very high affinity and has also been shown to bind other TGF-ß superfamily members, including BMP 2, 4, 7 and 15 but with lower affinity (
10% or less of that shown for activin) (Otsuka et al., 2001a; Glister et al., 2004). Other proteins, including some related to follistatin, have also been shown to have the ability to bind TGF-ß superfamily members: these include Noggin, Chordin, Chordin-like protein, Follistatin-related protein (FSRP), the differential screening-selected gene aberrative in neuroblastoma (DAN) Cerberus protein family, Sclerostin and bone morphogenetic protein-binding endothelial cell precursor-derived regulator (BMPER) (Balemans and Van Hul, 2002; Moser et al., 2003). The TGF-ß subfamily is known to be secreted as a latent protein consisting of the TGF-ß mature region dimer non-covalently associated with a dimer of the proregion which is covalently associated with a latent TGF-ß binding protein (LTBP) (Oklu and Hesketh, 2000; Kohli et al., 2003). Removal of these latent binding proteins is essential for activation of TGF-ß as they inhibit the biological activity of the TGF-ß. However, both the LTBP and the proregion are also important for the correct folding of the TGF-ß. In addition to the binding proteins, a membrane-bound protein (Bambi) with structural similarities to the type I receptors, but lacking the serine-threonine kinase activity, has also been identified. This protein can act as an inhibitor of signalling through association with the ligand-bound type II receptor, thus preventing activation of the Smad pathway (Balemans and Van Hul, 2002) (Figure 2). Finally, naturally occurring soluble forms of the extracellular region of some type I receptors have been identified that are thought to inhibit signalling by binding to their ligand (Choi, 1999).
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Effects of members of the TGF-ß superfamily on follicular development |
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TGF-ß
Localization of TGF-ß, their receptors, regulators and signalling pathways
In mammals, the TGF-ß subfamily is comprised of three proteins, namely TGF-ß1, TGF-ß2 and TGF-ß3. The mRNA/proteins have been observed in granulosa and thecal cells as well as oocytes of some species (Chegini and Flanders, 1992; Teerds and Dorrington, 1992; Schmid et al., 1994; Juneja et al., 1996; Nilsson et al., 2003; Bristol and Woodruff, 2004) (Figure 3). However, the expression patterns of TGF-ß seem to vary between species. While some of these differences among species are likely to be real, others may be due to the use of different methodologies with different sensitivities and specificities. In humans (Chegini and Flanders, 1992), TGF-ß1 was observed in oocytes of primary follicles and in thecal and granulosa cells of antral follicles. Expression intensity appeared to increase in the thecal and granulosa cell layers as the follicle matured. In contrast, TGF-ß2 was restricted to thecal cells of large preantral/antral follicles with increased expression in larger follicles. In addition, granulosa cells of larger antral follicles also expressed TGF-ß2 protein. The pattern of expression of TGF-ß3 has not been determined. In sheep, expression of mRNA encoding TGF-ß1 and 2 was observed in thecal cells of preantral and antral follicles (Juengel et al., 2004a). The TGF-ß3 mRNA was not detected in ovine follicles, indicating that TGF-ß3 is unlikely to regulate follicular cells in a paracrine/autocrine role in this species (Juengel et al., 2004a). In rats, expression of TGF-ß1 protein was first detected in oocytes and some granulosa cells of preantral follicles (Teerds and Dorrington, 1992). Expression was first observed in the theca at the time of antrum formation and signal appeared to increase as the follicle matured with consistent expression in the granulosa layer. TGF-ß2 protein was first detected in oocytes of preantral follicles. Granulosa cells of antral follicles, particularly the pre-ovulatory follicle, express TGF-ß2 protein but thecal cells show little or no expression. TGF-ß3 mRNA was observed in oocytes as well as granulosa and thecal cells of both preantral and antral follicles in mice (Schmid et al., 1994). In cows, TGF-ß1, 2 and 3 protein was localized to oocytes of all sizes of follicles. TGF-ß1 protein was also localized to the granulosa cells of primordial through to small antral follicles but was not present in granulosa cells of larger follicles or in thecal cells of any follicular size. In contrast, TGF-ß2 and 3 were present in granulosa cells of all sizes of follicles and in thecal cells from the early antral stage and thereafter (thecal cells of preantral follicles were not examined) (Nilsson et al., 2003).
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In summary, in some species (e.g. rodents and humans) TGF-ß bioactivity is likely to be derived from both the thecal and granulosa cells with expression first observed during preantral follicular growth and intensifying as the follicle matures. However, in some other species (e.g. sheep, cows and pigs) follicular TGF-ß bioactivity originates mainly from the thecal cells. For example, in sheep follicles, expression of TGF-ß mRNA was restricted to the theca of preantral and antral follicles (Juengel et al., 2004a
). In pigs, the theca interna appears be the primary source of TGF-ß, as granulosa cells express TGF-ß1 mRNA but not protein (May et al., 1996). Cultured granulosa cells isolated from cattle and pigs exhibit little if any TGF-ß bioactivity (Mulheron et al., 1992; May et al., 1996). Expression of TGF-ß2 mRNA has been identified in bovine oocytes, but no TGF-ß bioactivity was observed (Gilchrist et al., 2003) notwithstanding the presence of immunoreactive protein (Nilsson et al., 2003).
The TGF-ßRII and ALK5 (also known as TGF-ßRI) have been identified as the receptors for the TGF-ß with the presence of betaglycan necessary for binding of TGF-ß2 in many cell types (Table I). Expression of ALK5 mRNA/protein was observed in many cells of the ovary including the oocyte, granulosa cells and thecal cells of many species including humans (Juneja et al., 1996; Roy and Kole, 1998; Qu et al., 2000; Juengel et al., 2004a) (Figure 3). ALK5 was noted in oocytes of all follicular sizes in humans, sheep and mice (Juneja et al., 1996; Roy and Kole, 1998; Qu et al., 2000; Juengel et al., 2004a). In granulosa cells, ALK5 was observed in preantral follicles of all species with expression first observed at the primordial stage in mice (Juneja et al., 1996), primordial to primary stage in humans (Roy and Kole, 1998; Qu et al., 2000) and in small preantral follicles in sheep (Juengel et al., 2004a). In some studies, TGF-ßRII mRNA was restricted primarily to thecal cells of both preantral and antral follicles (Schmid et al., 1994; Juengel et al., 2004a) (Figure 3). In other studies using immunocytochemistry, strong staining for TGF-ßRII was observed in granulosa cells of preantral and antral follicles with variable staining in theca and little or no staining in oocytes (Roy and Kole, 1995; Juneja et al., 1996; Roy and Kole, 1998; Wehrenberg et al., 1998; Bristol and Woodruff, 2004). The reasons for these discrepancies are uncertain but may be due to species differences or to the techniques employed (i.e. in situ hybridization versus immunohistochemistry). Betaglycan, which facilitates binding of TGF-ß2 to the type II TGFß receptor, is expressed in granulosa and thecal cells as well as oocytes of preantral and antral follicles in rats (Drummond et al., 2002; MacConell et al., 2002). A similar expression pattern has been observed in sheep ovaries (J.L. Juengel et al., unpublished data). In human ovaries, betaglycan expression was strongest in the theca of antral and pre-ovulatory follicles with weak immunostaining observed in late stage granulosa cells (Liu et al., 2003). Thus in general, thecal cells of most species would be able to respond to TGF-ß as both type I and II receptors are present (Figure 3). However, responsiveness of the granulosa cells may be dependent on the species as some species appeared to have little if any TGFßRII (Figure 3). A direct effect on oocytes is unlikely due to the absence of TGF-ßRII in oocytes of most species (Figure 3).
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TGF-ßs are known to activate both Smad 2 and 3 (Table I) and, in the rat ovary, Smad 2 mRNA is expressed at a much greater abundance than Smad 3 mRNA (Drummond et al., 2002
). Nevertheless, there is evidence of impaired fertility with altered patterns of follicular growth in mice rendered deficient in Smad 3 activity through targeted disruption of exon 8 (Tomic et al., 2002). Using immunohistochemical techniques, Smad 2 and 3 proteins have been localized to the oocytes, granulosa and thecal cells of both preantral and antral follicles in rats (Drummond et al., 2002; Xu et al., 2002) and mice (Gueripel et al., 2004). In mice, oocytes and granulosa cells had moderate to weak staining for Smad 3 with thecal cells sometimes also containing a weak signal (Gueripel et al., 2004). In human ovarian tissue, oocytes and granulosa cells of primordial, primary, preantral and antral follicles (no oocytes from antral follicles available for analysis) expressed Smad 2 protein. In addition, Smad 2 protein was also consistently observed in the theca of antral follicles >1 mm in diameter (Pangas et al., 2002). Granulosa–luteal cells from pre-ovulatory follicles in human ovaries have also been shown to express both Smad 2 and 3 mRNA (Jaatinen et al., 2002). Thus, the receptor-mediated second messenger system for TGF-ß, namely the Smad 2/3 pathway, appears to be present in granulosa and thecal cells as well as in oocytes of follicles at all stages of follicular growth.
In vitro effects of TGF-ß on function of ovarian cells
When studying the actions of TGF-ß on cellular function, it is common to use a single representative of the TGF-ß subfamily, usually TGF-ß1. When the actions of TGF-ß1 and TGF-ß2 have been compared in ovarian cell cultures, their effects have been very similar. In contrast, studies using TGF-ß3 have not been as extensive, so its effects are less well understood. The actions of TGF-ß on granulosa cell function are known to vary between species. However, it is important to recognize that granulosa cells, when placed in culture, often undergo a luteinization process so that some of the effects observed with granulosa cells in vitro might be more related to a potential role for TGF-ß in luteal function rather than in follicular function. In rodents, TGF-ßs are potent stimulators of granulosa cell proliferation (Dorrington et al., 1988; Roy, 1993; Saragueta et al., 2002). However, in other species, such as cattle (TGF-ß2), sheep (TGF-ß1 and 2) and pigs (TGF-ß1 and TGF-ß2), these growth factors have only mild stimulatory or even inhibitory effects on granulosa cell proliferation/survival (Skinner et al., 1987; May et al., 1988; Gangrade and May, 1990; Gilchrist et al., 2003; Juengel et al., 2004a). Likewise, under in vitro conditions, TGF-ßs stimulate progesterone synthesis from rodent granulosa cells (Dodson and Schomberg, 1987; Hutchinson et al., 1987; Knecht et al., 1987) whereas inhibitory effects are observed in granulosa cells collected from cattle, sheep (TGF-ß1 and 2) and pigs (Mondschein et al., 1988; Kubota et al., 1994; Fabre et al., 2003; Gilchrist et al., 2003; Juengel et al., 2004a). TGF-ß augmented FSH-stimulated estradiol production from rodent granulosa cells (Adashi et al., 1989; Zachow et al., 1999). However, no effect was observed on aromatase mRNA expression in human granulosa–lutein cells (McAllister et al., 1994). Moreover, species differences were also observed in the regulation of FSH-mediated expression of gonadotrophin receptors where TGF-ß1 and 2 stimulated expression in granulosa cells from rats but inhibited expression in pigs. In human granulosa–luteal cells, TGF-ß1 and 2 have also been shown to stimulate the expression of the inhibin/activin ßB subunit mRNA without affecting the inhibin 
or inhibin/activin ßA subunit mRNA (Eramaa and Ritvos, 1996).
In contrast to the effects of TGF-ß on granulosa cells, those on theca/interstitial cells with respect to steroidogenesis appear similar between species. TGF-ß suppressed LH or forskolin-stimulated androgen production in rat (Magoffin et al., 1989; Hernandez et al., 1990), porcine (Caubo et al., 1989; Engelhardt et al., 1992), bovine (Demeter-Arlotto et al., 1993) and human (Attia et al., 2000) theca/interstitial cells. Expression of 17-hydroxylase, a key protein controlling androgen production which catalyses the conversion of progestagens to androgens, and steroidogenic acute regulatory protein (StAR), which facilitates the transport of cholesterol into the mitochondria for steroid synthesis, are both down-regulated by TGF-ß in thecal cells, indicating some common underlying mechanisms in the regulation of androgen production between species (Demeter-Arlotto et al., 1993; Attia et al., 2000). However, differing effects have been observed with respect to the actions of TGF-ß in the regulation of [3H]thymidine incorporation and cell numbers in thecal cells. For example, TGF-ß increased [3H]thymidine incorporation and cell numbers in the rat (Pehlivan et al., 2001) whereas [3H]thymidine incorporation decreased and no effect was observed on cell numbers in bovine thecal cells (Roberts and Skinner, 1991). In addition, TGF-ß, when given with TGF-, was shown either to enhance or to suppress apoptosis of theca/interstitial cells of rats (Foghi et al., 1997, 1998; Pehlivan et al., 2001). These discrepancies may be species differences and/or variation of methodologies in some of the studies. Overall, it seems likely that TGF-ß suppresses steroidogenesis in thecal cells from most species (Table II). However, the physiological effects on granulosa cells are less clear (Table II). In rodents, TGF-ß appears to be synthesized by the granulosa cells and strongly stimulates both cell proliferation and steroidogenesis. In contrast, in granulosa cells of cows, sheep and pigs, whose granulosa cells seem less active in the synthesis of TGF-ß, the effects are only mildly stimulatory or even inhibitory in respect to both cell proliferation and steroidogenesis. Whether these observed differences are truly differences between species or result from differences in methodologies, such as the use of different culture conditions, remains to be fully resolved.
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The effects of TGF-ß in vivo
Few studies have examined the effects of TGF-ß in vivo. Intrabursal administration of TGF-ß1 just before ovulation in rats undergoing superovulation treatments inhibited, in a dose-dependent manner, the number of oocytes recovered. Furthermore, the fertilization ability of the oocytes recovered from the TGF-ß1-treated rats was also reduced (Juneja et al., 1996
). Histological observation of the ovaries revealed an increase in the number of unruptured follicles with varying degrees of luteinization. In addition, 30–40% of the oocytes recovered from treated rats showed fewer or no cumulus cells. In hyperstimulated human granulosa cells, high levels of expression of TGF-ßRI and TGF-ßRII were negatively correlated with cleavage potential of zygotes (Roy et al., 1998), again supporting a critical role of TGF-ß in regulating fertility. Interestingly, patients undergoing IVF receiving hMG or FSH showed differing levels of TGF-ß1 in their follicular fluid, with levels in the FSH group higher than those observed in the hMG group. While not statistically significant with the relatively few patients included in the FSH group, the percentage of pregnancies was 25% lower in this group (23 versus 29.8%) (Fried et al., 1998). BMP6
Localization of BMP6, its receptors, regulators and signalling pathways
BMP6 mRNA and protein have been localized to oocytes and granulosa cells of antral follicles in several species (Erickson and Shimasaki, 2003; Campbell et al., 2004; Glister et al., 2004) (Figure 3). Expression of BMP6 protein has also been observed in thecal cells of cows and sheep (Campbell et al., 2004; Glister et al., 2004). In sheep, expression of BMP6 mRNA was observed in oocytes of all sizes of follicles but could not be detected in granulosa or thecal cells (McNatty et al., 2005a). In rats (Erickson and Shimasaki, 2003), mRNA encoding BMP6 was first observed in the oocytes and granulosa cells of preantral follicles but was not observed in primordial or primary follicles. Interestingly, BMP6 mRNA could not be localized to granulosa cells of dominant rat follicles, indicating a potential role for down-regulation of this mRNA in the selection/maintenance of the dominant follicle (Erickson and Shimasaki, 2003). Thus, the oocytes appear to be a primary source of follicular BMP6 in many species and expression likely begins very early in follicular development. The potential for expression of BMP6 in granulosa and particularly thecal cells is less clear as detection of BMP6 varied between species and even within the same species between experiments. One potential explanation for the differences observed between experiments could relate to the detection method utilized, namely in situ hybridization versus immunocytochemistry. These techniques can vary in sensitivity and specificity and it is also possible that the protein (i.e. detection by immunocytochemistry) may localize to a cell that has not expressed it.
BMP6 appears able to signal through multiple type I and type II receptors (de Caestecker, 2004; Shimasaki et al., 2004) (Table I) and the choice of receptors appears to be dependent on the cell type (Ebisawa et al., 1999). Currently, ALK2, ALK3 and ALK6 have been identified as potential type I receptors with ALK2 and ALK6 having the strongest affinity for BMP6 (Ebisawa et al., 1999; Shimasaki et al., 2004). BMPRII, ActRII and ActRIIB have been identified as potential type II receptors (de Caestecker, 2004; Shimasaki et al., 2004) (Table I). The preference of BMP6 for receptors in various ovarian cell types has not been determined. In postnatal rat follicles, ALK2 was localized in oocytes and granulosa cells of all sizes of follicles from primordial to the antral development stages (Drummond et al., 2002) (Figure 3). However, expression in the oocyte was not consistently observed in all ages of rats (Drummond et al., 2002). Expression of ALK2 was also observed in oocytes and granulosa and thecal cells of bovine antral follicles (Glister et al., 2004) and in oocytes and cumulus cells in pre-ovulatory follicles of humans (Sidis et al., 1998). However, the ontogeny of ALK2 expression in smaller follicles in these species has not been determined. The expression of ALK3 has been observed in oocytes, granulosa and thecal cells of rats (Erickson and Shimasaki, 2003) and sheep (Souza et al., 2002) and also granulosa–luteal cells from human follicles (Jaatinen et al., 2002). In rats and sheep, expression of ALK3 in oocytes and granulosa cells has been observed from the primordial to antral stages of development (Souza et al., 2002; Erickson and Shimasaki, 2003; McNatty et al., 2005a) (Figure 3). In the theca, the expression of ALK3 in the rat and sheep begins in small preantral follicles (Souza et al., 2002; Erickson and Shimasaki, 2003; McNatty et al., 2005a). For ALK6, expression is also observed in oocytes, granulosa cells and theca of rats (Erickson and Shimasaki, 2003), sheep (Wilson et al., 2001; Souza et al., 2002) and cattle (Glister et al., 2004) (Figure 3). In the rat and sheep, expression of ALK6 was observed in the oocytes from the primordial to antral stages of development (Wilson et al., 2001; Souza et al., 2002; Erickson and Shimasaki, 2003) although it appeared to decrease in large antral follicles (Wilson et al., 2001; Erickson and Shimasaki, 2003). Expression of ALK6 was also observed in granulosa cells from the primordial/primary stage of development as well as in thecal cells of small preantral and larger follicles (Wilson et al., 2001; Souza et al., 2002; Erickson and Shimasaki, 2003). For human ovaries, the ontogeny of expression and location of ALK6 mRNA in different cell types is not currently known. Expression of BMPRII was observed in oocytes (primordial through antral), granulosa cells (primordial through antral) and thecal cells (small preantral through antral) of sheep (Wilson et al., 2001) and cattle (antral follicles only examined) (Glister et al., 2004). However, in rats, expression has only consistently been observed in granulosa cells from the primary stage of development. No BMPRII mRNA was observed in the theca and localization to oocytes was not consistently evident (Erickson and Shimasaki, 2003). The ActRII (also called ActRIIA) and ActRIIB has been localized to the oocytes, granulosa cells and theca of antral follicles in cattle (Glister et al., 2004). In human ovarian tissue, the ActRII protein has been localized to oocytes of preantral follicles (no oocytes from antral follicles were available for analysis) as well as granulosa cells and thecal cells of antral follicles. Strongest expression in both granulosa and thecal cells was observed in follicles expressing aromatase (Pangas et al., 2002) (Figure 3). Granulosa and thecal cells also expressed ActRIIB protein and expression in the theca was also strongest in those follicles expressing P450 aromatase (Pangas et al., 2002) (Figure 3). In addition, both the oocytes and cumulus cells of pre-ovulatory follicles in humans contained ActRII and ActRIIB mRNA (Sidis et al., 1998). In sheep, we have localized ActRIIB mRNA to oocytes and granulosa cells from the primary to antral stages of development and also in thecal cells of both small preantral and larger follicles (J.L.Juengel et al., unpublished data). Faint expression of ActRII mRNA was also observed in granulosa cells of antral follicles (J.L.Juengel et al., unpublished data). In rat follicles, expression of ActRII and ActRIIB was observed in oocytes of primordial and primary follicles; however, only ActRII was observed in oocytes of preantral and larger follicles (Drummond et al., 2002). In rats, granulosa cells of primary and larger follicles expressed both ActRII and ActRIIB, whereas there was no expression observed in thecal cells.
In summary, receptor complexes capable of responding to BMP6 seem likely to be present in oocytes, granulosa and thecal cells of growing follicles of most species including humans. One exception to this would be thecal cells of rat follicles, as no type II receptor known to be involved in BMP6 signalling has been detected in these cells.
In general, the Smads activated by BMP are from the Smad 1, 5 and 8 group (Table I) (Miyazawa et al., 2002). However, in a mouse myoblast cell line, BMP6 only activated Smad 1 and 5 (Aoki et al., 2001). In rats, Smad 5 and 8 were observed in oocytes of follicles of all sizes. However, neither Smad was observed in granulosa or thecal cells (Drummond et al., 2002). Localization of Smad 1 has not been determined in this species but it would seem likely to be the mediator of the BMP6 signal as granulosa cells are known to be responsive to BMP. In cattle, granulosa cells of antral follicles contained the phosphorylated form of the Smad-1 protein and the intensity of signal increased following treatment with BMP6, further supporting a role for Smad-1 in transducing the BMP6 signal (Glister et al., 2004). In human granulosa/luteal cells, the mRNAs for both Smad 1 and Smad 5 were observed (Jaatinen et al., 2002). Thus, a second messenger system capable of transducing the BMP6 signal appears to be present in oocytes as well as granulosa and thecal cells of several species.
Many different binding proteins have been shown to interact with BMP6 (Table I). Those expressed in the ovary include follistatin (Welt et al., 2002; Lin et al., 2003), protein related to DAN and cerberus (PRDC) (Sudo et al., 2004) and follistatin-related protein (FSRP) (Tortoriello et al., 2001). Follistatin has been shown to be expressed in the granulosa cells of many species, with expression being first observed in small preantral follicles (Shimasaki et al., 1989; Juengel et al., 2000). While expression of follistatin in human preantral follicles has not been examined, granulosa cells of small antral as well as dominant follicles expressed follistatin (Roberts et al., 1993). Expression of PRDC has only been examined in rodents where it was localized to granulosa cells of early antral to pre-ovulatory follicles but absent in oocytes and theca–interstitial cells. While FSRP (also known as follistatin-related gene, FLRG) has been shown to be expressed in the ovary of some species, little is known regarding its expression pattern. Human granulosa and thecal cells were shown to express FSRP mRNA (Liu et al., 2002). In preliminary studies with sheep, the FSRP mRNA was first observed in small preantral follicles in the granulosa cells and theca but expression was not consistently observed in oocytes (J.L.Juengel et al., unpublished). Thus, it seems likely that regulation of BMP6 activity by binding proteins produced by the granulosa cells, and potentially the theca, is likely to occur in growing follicles of most, if not all, species.
In vitro effects of BMP6 on function of ovarian cells
The effects of BMP6 on granulosa cell function have been examined in rats, cows and humans. In granulosa cells from diethylstilbestrol (DES)-treated rats, BMP6 was not mitogenic and had no effect on basal estradiol or progesterone production. However, BMP6 inhibited FSH-stimulated progesterone production without affecting estradiol production (Otsuka et al., 2001b). Treatment with BMP6 specifically reduced concentrations of StAR and P450scc mRNA without effecting P450arom (enzyme required for conversion of androgens to estrogens) mRNA. The effects of StAR and P450scc mRNA were likely mediated, at least in part, through inhibition of adenylate cyclase activity and subsequent reduction in cAMP levels. In bovine granulosa cells, BMP6 stimulated basal and insulin-like growth factor 1 (IGF-1) stimulated estradiol production with a concomitant decrease in both basal and IGF-1-stimulated progesterone production (Glister et al., 2004). Furthermore, BMP6 was able to increase viable cell numbers, either through a mitogenic effect or through increased cell survival. BMP6 also stimulated production of inhibin-A, activin-A and follistatin (Glister et al., 2004). In human granulosa–luteal cells, BMP6 increased expression of inhibin/activin ßB subunit mRNA without affecting inhibin- subunit mRNA (Jaatinen et al., 2002). Therefore, BMP6 may act to prevent premature luteinization of the follicle while potentially enhancing numbers of granulosa cells (Table II). This proposed function is in agreement with the observed loss of signal for BMP6 in granulosa cells of the dominant follicle in rats (Erickson and Shimasaki, 2003).
In vivo effects of BMP6 on function of ovarian cells
Mice lacking active BMP6 are fertile with no overt phenotype (Solloway et al., 1998). However, a mutation in the ALK6 receptor has been identified in Booroola sheep which is associated with increased ovulation rates (Mulsant et al., 2001; Souza et al., 2001; Wilson et al., 2001) (Table III). The two known mutations in ALK6 in humans, which cause the brachydactyly A2 condition, have no reported effects on reproductive activity (Lehmann et al., 2003). However, in another family with brachydactyly A2, fertility appeared to be increased in those with the condition when compared to normal sibs (Freire-Maia et al., 1980). Whether the underlying cause of this condition in this family is related to mutations in ALK6 has not been examined. The mutation in Booroola sheep appears to increase the basal activity of the receptor-mediated signalling system while inhibiting responsiveness to BMP (Fabre et al., 2003). Intriguingly, follicles from Booroola ewes that were heterozygous or homozygous for the ALK6 mutation mature at a smaller size and have fewer granulosa cells per ovulatory follicle that show an increased responsiveness to FSH as measured by increased cAMP concentrations (McNatty et al., 2001). The increased responsiveness to FSH was not associated with increased numbers or altered binding properties of the FSH receptor. In addition, the cells expressed similar amounts of estradiol but had increased amounts of progesterone (McNatty et al., 2001). Given that ALK6 is known to bind BMP6, these changes in granulosa cell function in Booroola ewes are consistent with a reduced level of downstream signalling after interaction with BMP6 or other BMP ligands.
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GDF9 and BMP15
Localization of GDF9 and BMP15, their receptors, regulators and signalling pathways
Within the ovary, mRNA and protein for both GDF9 and BMP15 are found exclusively in the oocyte in most species (McGrath et al., 1995; Dube et al., 1998; Fitzpatrick et al., 1998; Laitinen et al., 1998; Aaltonen et al., 1999; Bodensteiner et al., 1999; Jaatinen et al., 1999; Galloway et al., 2000; Eckery et al., 2002; Juengel et al., 2002; Wang and Roy, 2004) suggesting that the oocyte is the primary source of both GDF9 and BMP15 (Figure 3). However, in some primates, granulosa cells adjacent to the oocyte as well as the oocyte have also been shown to express GDF9 mRNA and protein (Sidis et al., 1998; Duffy, 2003). The expression pattern of these two growth factors differs among species with GDF9 expression first observed in primordial follicles of sheep, cattle, possum and hamster (Bodensteiner et al., 1999; Eckery et al., 2002; Wang and Roy, 2004) and in primary follicles in rats, mice and humans (Dube et al., 1998; Laitinen et al., 1998; Aaltonen et al., 1999; Jaatinen et al., 1999). In sheep, GDF9 mRNA was present in germ cells prior to follicular formation (Mandon-Pepin et al., 2003) and GDF9 mRNA and protein was localized to oocytes that were not fully encapsulated with granulosa cells (i.e. prior to follicular formation) (Juengel et al., 2004b). BMP15 was first observed in oocytes in primary follicles of sheep, humans, rats, mice (Dube et al., 1998; Laitinen et al., 1998; Aaltonen et al., 1999; Jaatinen et al., 1999; Galloway et al., 2000) and in primordial follicles in the brushtail possum (Eckery et al., 2002). However, BMP15 mRNA expression was not observed in oocytes prior to follicular formation, suggesting that this factor does not have a role in this process. Thus, GDF9 and BMP15 have been localized to the oocytes of growing follicles in all mammalian species examined to date, indicating that these growth factors likely have a central role in the regulation of follicular development in mammals. In addition, GDF9, but not BMP15, may play a role in follicular formation in some species.
It is also worth noting that GDF9 and BMP15 mRNA have been found in the pituitary, testis and other tissues of some species, indicating that these factors may not have actions exclusive to the ovary (Fitzpatrick et al., 1998; Aaltonen et al., 1999; Galloway et al., 2000; Eckery et al., 2002; Otsuka and Shimasaki, 2002b)
Recently, GDF9 was shown to signal through the ALK5 receptor (Mazerbourg et al., 2004) and BMPRII was identified as a type II receptor (Vitt et al., 2002) (Table I). BMP15 has been shown to signal through ALK6 (Moore et al., 2003) and BMPRII (Table I) (Moore et al., 2003). However, it is possible that additional type I and type II receptors will be identified for these ligands. The expression patterns for these receptors have been detailed in the sections on TGF-ß and BMP6. GDF9 has been shown to activate the Smad 2 and 3 pathway (Kaivo-Oja et al., 2003; Roh et al., 2003) whereas BMP15 activates the Smad 1, 5 and 8 pathway (Table I) (Moore et al., 2003). Expression of these Smad pathways during follicular development is detailed in the sections on TGF-ß and BMP6. Thus, it would seem likely that granulosa cells from small growing to mature follicles would be able to respond to GDF9 and BMP15 across a wide range of mammalian species. Whether oocytes and thecal cells would be capable of responding to GDF9 and BMP15 remains unclear. BMPRII is expressed in oocytes and theca of numerous species (e.g. cows and sheep) and complementary components thought to be necessary for GDF9 and BMP15 signalling that have been examined were present. However, the lack of consistent expression of BMPRII in oocytes and thecal cells in rodents indicates that they may not be responsive. Recent data indicate that follistatin, which is expressed by granulosa cells in small growing follicles of most mammalian species, can also bind BMP15 and thereby block its bioactivity (Otsuka et al., 2001a) providing an additional pathway for regulating the actions of BMP15.
In vitro effects of GDF9 and BMP15 on function of ovarian cells
Follicular formation. GDF9 may play a role in follicular formation in some species. Wang and Roy have recently observed that the culture of perinatal ovaries from hamsters with GDF9-conditioned media for 9 days increased the proportion of oocytes in follicles as opposed to isolated oocytes (Wang and Roy, 2004). This effect appeared to be mediated, at least in part, by stem cell factor (SCF).
Granulosa and thecal cells. GDF9 and BMP15 have been shown to regulate a variety of granulosa and thecal cell functions in vitro (Table II). Conflicting results between species and even between experiments involving the same or closely related species have been noted. The reasons for these differences are unclear but are most likely related to culture conditions, maturational stage of cells being studied or inherent species differences of the cells as well as of the growth factors themselves (McNatty et al., 2005b,c). Overall, GDF9 and BMP15 both appeared to be mitogenic for granulosa and thecal cells (Hayashi et al., 1999; Otsuka et al., 2000; Vitt et al., 2000a,b; Hreinsson et al., 2002; Nilsson and Skinner, 2002; Di Pasquale et al., 2004; McNatty et al., 2005b,c), although this affect was not always observed (Nilsson and Skinner, 2002; Yamamoto et al., 2002; McNatty et al., 2005b,c). BMP15 and GDF9 stimulated expression of SCF, which partly accounts for the ability of BMP15 to regulate mitosis (Otsuka and Shimasaki, 2002a), whereas the effects for GDF9 were not always consistent (Joyce et al., 2000; Nilsson and Skinner, 2002). GDF9 has been shown to stimulate inhibin production (Hayashi et al., 1999; Kaivo-Oja et al., 2003; Roh et al., 2003). The effects of BMP15 on inhibin production have been reported to show a suppression of FSH-stimulated mRNA encoding inhibin-, inhibin/activin ßA and ßB subunits in rat granulosa cells with human BMP15 (Otsuka et al., 2000), while other studies report no effect of recombinant ovine BMP15 on inhibin production (i.e. free inhibin- subunit and inhibin protein) in FSH-stimulated rat, ovine or bovine granulosa cells (McNatty et al., 2005b,c). Recently, GDF9 was shown to stimulate expression of Gremlin, a BMP antagonist in rat granulosa cells (Pangas et al., 2004). However, this binding protein was not able to antagonize the effects of GDF9 in vitro (Pangas et al., 2004).
In general, GDF9 and BMP15 both seem to inhibit gonadotrophin-stimulated progesterone production from granulosa cells (Otsuka et al., 2000; Vitt et al., 2000a; Yamamoto et al., 2002) although this effect has not been observed consistently (Elvin et al., 1999a; McNatty et al., 2005b,c). These effects may be mediated, at least in part, through regulation of gonadotrophin action as GDF9 suppresses binding of hCG (Vitt et al., 2000a) and expression of LH-R mRNA (Elvin et al., 1999a) whereas BMP15 suppresses FSH-R mRNA expression and thereby LH-R mRNA expression (Otsuka et al., 2001c). Like BMP6, BMP15 is able to suppress FSH-stimulated progesterone production without affecting estradiol secretion (Otsuka et al., 2000). However, GDF9 suppressed expression of P450arom mRNA in human granulosa cells, suggesting that GDF9 likely would affect estradiol secretion (Yamamoto et al., 2002). Both GDF9 and BMP15 have been shown to decrease FSH-stimulated expression of mRNA encoding P450scc and StAR (Otsuka et al., 2000; Yamamoto et al., 2002). Inconsistancies also exist for the effects of GDF9 on thecal cell function where stimulation (Solovyeva et al., 2000) and inhibition (Yamamoto et al., 2002) of steroidogenesis have been observed.
The combined administration of GDF9 and BMP15 often resulted in effects that were altered or potentiated from those observed when these growth factors were added separately (McNatty et al., 2005b,c). The potentiating effect was evident in rat granulosa cells, where ovine (o)GDF9 and oBMP15 alone were unable to stimulate [3H]thymidine uptake but a 4–9-fold increase in uptake was observed when the growth factors were added together. Similarly, oGDF9 and oBMP15 together strongly inhibited progesterone production and stimulated inhibin- production even though these factors on their own had no effects (Figure 4). Similar, albeit less potent, co-operating effects were observed for some, but not all, parameters in ovine and bovine granulosa cells (McNatty et al., 2005c). As both of these proteins are secreted from oocytes during follicular growth, the recent evidence suggests that they interact and should be regarded as a functional unit. Overall, it seems that GDF9 and BMP15 act to stimulate cell proliferation and to regulate the level of gonadotrophin-induced differentiation (Table II).
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