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Molecular Endocrinology, doi:10.1210/me.2003-0399
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Molecular Endocrinology 18 (6): 1509-1519
Copyright © 2004 by The Endocrine Society

Interrelationship of Growth Differentiation Factor 9 and Inhibin in Early Folliculogenesis and Ovarian Tumorigenesis in Mice

Xuemei Wu, Lei Chen, Christopher A. Brown, Changning Yan and Martin M. Matzuk

Departments of Pathology (X.W., L.C., C.A.B., C.Y., M.M.M.), Molecular and Cellular Biology (M.M.M.), and Molecular and Human Genetics (M.M.M.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Martin M. Matzuk, M.D., Ph.D, The Stuart A. Wallace Chair, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To investigate the interrelationship of inhibin {alpha} and growth differentiation factor 9 (GDF9) during early folliculogenesis, we generated mice lacking both inhibin {alpha} and GDF9. Our findings on these Inha Gdf9 double-mutant mice are as follows: 1) females develop ovarian tumors and a cachexia-like wasting syndrome, resembling mice lacking inhibin {alpha} alone. This indicates that the granulosa cells are competent to proliferate despite the lack of GDF9; 2) follicular development progresses to multiple-layer follicle stages before tumorigenesis. This demonstrates that the up-regulation of inhibin {alpha} in the Gdf9 knockout ovary directly prevents the proliferation of the granulosa cells at the primary follicle stage, an effect that is released in the absence of inhibin {alpha}; 3) a morphological theca forms around the preantral follicles with no detectable selective theca markers [i.e. 17{alpha}-hydroxylase (Cyp17), LH receptor (Lhr), and Kit]. These results indicate that the theca recruitment can occur independently of GDF9, but the differentiation of thecal cells is blocked; and 4) inhibin/activin subunits ßA, ßB, and Kit ligand (Kitl) mRNA are highly up-regulated, suggesting that the increased activins and KITL play functional roles in early folliculogenesis. Thus, GDF9 appears to function indirectly to regulate early granulosa cell proliferation and theca recruitment in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FEMALE MAMMALS HAVE a fixed number of primordial follicles in their ovaries as neonates, and these primordial follicles are depleted gradually during their reproductive lifespan (1). Initiation of follicular growth is morphologically characterized by an increase in oocyte size and a squamous-to-cuboidal transition of associated granulosa cells. Once follicular growth is initiated, follicles develop through the primary and secondary stages before they reach the antral follicle stage. During this early folliculogenesis, the oocyte grows dramatically, and the granulosa cells proliferate to form multiple layers and acquire functional characteristics such as expression of steroidogenic enzymes and LH receptor (LHR) (2). One of the distinctive features of secondary follicles is the formation of a thecal cell layer. Follicles beyond the primary stage are surrounded by a thin layer of mesenchymal-derived thecal cells that increase in number during follicular development. Thecal cells produce a number of factors that act locally to regulate the proliferation and differentiation of adjacent granulosa cells. In response to LH, thecal cells secrete androgens to serve as substrates for the granulosa cells to secrete estrogens (3). Throughout normal follicular development, oocyte growth and maturation are coordinated with granulosa and thecal cell proliferation and differentiation. Pituitary-derived gonadotropins [i.e. FSH and LH] partially control the process; however, paracrine/autocrine factors derived from the oocyte and its surrounding somatic cells (i.e. granulosa and thecal cells) predominantly modulate early folliculogenesis (2, 4). Many of these paracrine/autocrine factors are TGFß superfamily members (5).

Inhibins are {alpha}:ß heterodimeric members of the TGFß superfamily. Inhibins were initially discovered as gonadal peptides that inhibit the synthesis and release of pituitary FSH (6, 7). Inhibin {alpha} has been detected in multiple tissues in mammals, including the ovary, testis, pituitary, and adrenal gland (8). The major gonadal sites of inhibin {alpha} synthesis are the granulosa cells in females and the Sertoli cells in males. We have previously generated an inhibin {alpha} knockout mouse model that develops sex-cord stromal gonadal tumors in both males and females, and adrenal cortical tumors in gonadectomized mice with nearly 100% penetrance, resulting in death secondary to a cachexia-like wasting syndrome (9, 10, 11). These studies on inhibin {alpha}-deficient mice demonstrated that inhibin {alpha} plays a negative regulatory role in granulosa cell proliferation in mice. However, little is known about the mechanisms by which inhibin functions, although receptors that bind inhibin have been identified recently (12, 13, 14, 15, 16).

Growth differentiation factor 9 (GDF9) is also a member of the TGFß superfamily and highly expressed in the mammalian oocyte beginning at the primary follicle stage (17, 18). Gdf9 knockout females are infertile due to a block in folliculogenesis at the type 3b primary follicle stage (19). No ovarian or adrenal tumors form in these Gdf9 null mice. Other defects in GDF9-deficient ovaries include an absence of thecal cell layers around the follicles, defects in the meiotic competence of the oocytes, and formation of steroidogenic clusters that structurally resemble corpora lutea with expression of both luteal (e.g. LHR and cytochrome P450 side chain cleavage) and nonluteal (e.g. inhibin {alpha} and cytochrome P450 aromatase) markers in the ovary (20, 21). The primary follicles of Gdf9 null ovaries showed high expression of inhibin {alpha} and kit ligand (Kitl) mRNA (20). We therefore hypothesized that GDF9 may act through down-regulation of inhibin {alpha}. To test our hypothesis and define the interrelationship of GDF9 and inhibin {alpha} in early follicular development, we generated a double-mutant mouse model lacking both GDF9 and inhibin {alpha}. Our findings demonstrate in vivo interactions of inhibin and GDF9 at this early stage of ovarian folliculogenesis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inha–/–Gdf9–/– Mice Develop a Cachexia-Like Wasting Syndrome and Ovarian Tumors
One indicator for ovary tumorigenesis in Inha–/– females is the development of a cachexia-like wasting syndrome that leads to severe weight loss and early death of these animals (9, 10). To monitor tumor development in Inha Gdf9 double-mutant females, we weighed Inha+/–Gdf9+/–, Inha–/–Gdf9+/–, Inha+/–Gdf9–/–, and Inha–/–Gdf9–/– mice weekly at 4–17 wk. Initially, the body weight among these mice is indistinguishable. After 6–7 wk, Inha–/–Gdf9+/– and Inha–/–Gdf9–/– mice begin to lose weight, whereas the body weight of Inha+/–Gdf9+/– and Inha+/–Gdf9–/– mice still increases gradually (Fig. 1Go). There is no significant difference in the rate and extent of weight loss between Inha–/–Gdf9+/– and Inha–/–Gdf9–/– mice. The number of surviving Inha Gdf9 double-mutant females was also determined weekly between the ages of 4 and 20 wk. Similar to Inha–/– mice, most Inha–/–Gdf9+/– and Inha–/–Gdf9–/– females die between 9 and 18 wk of age, whereas 100% of Inha+/–Gdf9+/– and Inha+/–Gdf9–/– mice are still alive at 20 wk of age (Fig. 2Go). These results indicate that both Inha–/–Gdf9+/– and Inha–/–Gdf9–/– mice develop a cachexia-like wasting syndrome, which is similar to mice deficient in inhibin {alpha} only (10).



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  		Fig. 1. Body Weight Curves of Inha Gdf9 Double-Mutant Females

Mice were weighed weekly on the same day between 4 and 17 wk of age. Inha+/–Gdf9+/–, n = 11; Inha+/–Gdf9–/–, n = 10; Inha–/–Gdf9+/–, n = 9; Inha–/–Gdf9–/–, n = 13. *, P < 0.001 vs. Inha+/–Gdf9+/– group; #, P < 0.001 vs. Inha+/–Gdf9–/– group.

 


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  		Fig. 2. Survival Rates of Inha Gdf9 Double-Mutant Females

Mice were counted weekly between 4 and 20 wk of age. Number of mice in each group is as shown in Fig. 1Go. *, P < 0.01 vs. Inha+/–Gdf9+/– group; #, P < 0.01 vs. Inha+/–Gdf9–/– group.

 
Unlike Gdf9 null females, whose ovaries are much smaller than wild-type ovaries (19), gross ovarian enlargement in Inha Gdf9 double-null females is obvious in most cases beginning at 5 wk of age (Fig. 3AGo). Similar to knockout mice lacking inhibin {alpha} alone, ovarian tumors are microscopically evident as early as 4 wk of age in Inha–/–Gdf9–/– females when nodular proliferation of granulosa cells occurs. Histological analysis shows that these ovarian tumors are mixed or incompletely differentiated gonadal sex-cord stromal tumors (Fig. 3BGo). The morphology and histology of these ovarian tumors are indistinguishable between Inha–/–Gdf9+/–, Inha–/–Gdf9–/–, and Inha–/– mice (Ref.9 and data not shown).



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  		Fig. 3. Morphological and Histological Analysis of Inha Gdf9 Double-Mutant Ovaries

Ovaries from Inha–/–Gdf9–/– and wild-type mice are compared morphologically at 14 wk of age; bilateral ovarian tumors form in Inha–/–Gdf9–/– females (A). Histological analysis of an ovarian tumor from a 15-wk-old mouse shows that the ovarian tumor is a mixed or incompletely differentiated gonadal stromal tumor (B). Histology of ovaries shows that there is no remarkable difference among Inha+/–Gdf9+/– (C), Inha–/–Gdf9+/– (D), and Inha–/–Gdf9–/– (F) ovaries with follicles from primordial to late preantral stages at 3 wk of age; folliculogenesis is arrested at the primary follicle stage in Inha+/–Gdf9–/– ovaries (E). Whereas only primordial and one-layer primary follicles with no surrounding thecal cells are seen in the Inha+/–Gdf9–/– ovary (G, high power), multiple-layer follicles with a morphologically evident theca (arrow, T) form in the Inha–/–Gdf9–/– ovary (H, high power).

 
Ovarian Folliculogenesis in Inha–/–Gdf9–/– Mice Proceeds beyond the One-Layer Primary Follicle Stage and Morphological Thecal Layers Form
Inhibin {alpha} is highly up-regulated in the arrested one-layer primary follicles in Gdf9 null ovaries (20). To test the functions of inhibin {alpha} in these early-stage follicles, ovaries from Inha Gdf9 double-mutant mice were histologically analyzed at 3 wk of age, a time point just before gross ovarian tumor formation in Inha–/–Gdf9+/– and Inha–/–Gdf9–/– mice (Fig. 3Go, D and F). Compared with Inha+/–Gdf9+/– (Fig. 3CGo) and wild-type ovaries, Inha+/–Gdf9–/– ovaries (Fig. 3EGo) are much smaller in size, resembling Gdf9 null ovaries. Similar to ovaries lacking GDF9 alone, follicles in Inha+/–Gdf9–/– ovaries fail to develop beyond the one-layer primary follicle stage, and no thecal cells are visible around these primary follicles (Fig. 3Go, E and G). However, at 3 wk of age, the majority of the ovaries from Inha–/–Gdf9–/– mice appear histologically normal (Fig. 3Go, F and H). Numerous primordial, primary, two-layer secondary, and multiple-layer secondary follicles [type 2 to type 5b (22)] are evident in the ovary, and morphological thecal layers form around follicles beyond the type 3b follicle stage (Fig. 3HGo). Thus, in the absence of inhibin {alpha}, follicular development progresses beyond the one-layer primary follicle stage, and the recruitment of thecal cells surrounding these follicles occurs in Inha–/–Gdf9–/– ovaries, despite the lack of GDF9.

Characterization of Thecal Cell Marker Genes
To determine whether these thecal cells are functional, two thecal layer marker genes were initially examined by in situ hybridization. 17{alpha}-Hydroxylase (CYP17) is a thecal cell-specific enzyme involved in the synthesis of androgens. Similar to wild-type mice, Cyp17 mRNA is exclusively expressed in thecal cells surrounding follicles beyond the type 3b stage in Inha+/–Gdf9+/– ovaries (Fig. 4Go, A and B). In Inha+/–Gdf9–/– ovaries, weak signals are seen in some scattered interstitial cells that are not associated with follicles, and these cells may represent a thecal cell precursor population (Fig. 4Go, E and F). However, no signal is visible in follicles at any stages in Inha–/–Gdf9+/– (Fig. 4Go, C and D) and Inha–/–Gdf9–/– ovaries (Fig. 4Go, G and H). Consistent with previous studies in wild-type mice, Lhr mRNA is expressed in the thecal cells surrounding preantral follicles and some interstitial cells in Inha+/–Gdf9+/– ovaries (Fig. 4Go, I and J), but no signal is detected in Inha–/–Gdf9+/–, Inha+/–Gdf9–/–, and Inha–/–Gdf9–/– ovaries (Fig. 4Go, K–P). Therefore, these thecal cells do not appear to be steroidogenic due to the lack of expression of a key steroidogenic enzyme, CYP17, and LHR.



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  		Fig. 4. In Situ Hybridization Analysis of 17{alpha}-Hydroxylase (Cyp17) and Lhrin Inha Gdf9 Double-Mutant Ovaries

Cyp17 mRNA is expressed exclusively in thecal cells in Inha+/–Gdf9+/– ovaries; a complete ring forms around follicles at the type 4 follicular stage and beyond (A and B). Faint signals are seen in some scattered interstitial cells in Inha+/–Gdf9–/– ovaries, which may represent some theca precursor cells (E and F). No Cyp17 signal is detectable in either Inha–/–Gdf9+/– (C and D) or Inha–/–Gdf9–/– (G and H) ovaries. Lhr is expressed in thecal cells and some interstitial cells in Inha+/–Gdf9+/– ovaries (I and J), but not in Inha–/–Gdf9+/– (K and L), Inha+/–Gdf9–/– (M and N), and double-null ovaries (O and P).

 
Examination of Two Upstream Transcription Factors: Steroidogenic Factor 1 (Nr5a1) and Liver Receptor Homolog 1 (Nr5a2)
Two orphan members of the nuclear hormone receptor family, steroidogenic factor 1 (NR5A1) and liver receptor homolog (LRH1; NR5A2), are involved in the regulation of many steroidogenic enzyme-encoding genes in the ovary. Similar to previous reports using wild-type rats and mice (23, 24, 25), Nr5a1 mRNA is expressed throughout the ovary and appears more abundant in thecal/interstitial cells (Fig. 5Go, A and B), whereas Nr5a2 mRNA is selectively detected in granulosa cells in Inha+/–Gdf9+/– ovaries (Fig. 5Go, I and J). Expression level of Nr5a1 shows no apparent change in Inha–/–Gdf9+/– (Fig. 5Go, C and D), Inha+/–Gdf9–/– (Fig. 5Go, E and F), and Inha–/–Gdf9–/– (Fig. 5Go, G and H) ovaries, which is consistent with our RT-PCR results (data not shown). Similarly, the expression pattern and level of Nr5a2 in Inha–/–Gdf9+/– (Fig. 5Go, K and L) and Inha–/–Gdf9–/– (Fig. 5Go, O and P) ovaries mimic those in Inha+/–Gdf9+/– mice. However, in addition to the single-layer granulosa cells, Nr5a2 mRNA also appears highly expressed in oocytes in Inha+/–Gdf9–/– ovaries (Fig. 5Go, M and N). Therefore, the absence of inhibin {alpha} or GDF9 does not affect the expression of Nr5a1 and Nr5a2 dramatically.



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  		Fig. 5. In Situ Hybridization Analysis of Steroidogenic Factor 1 (SF1; NR5A1) and LRH1 (NR5A2) in Inha Gdf9 Double-Mutant Ovaries

All ovaries are from 3-wk-old mice. Nr5a1 mRNA is expressed throughout the ovary and most abundant in the thecal/interstitial cells in Inha+/–Gdf9+/– ovaries (A and B). In general, the expression level of Nr5a1 does not change dramatically in Inha–/–Gdf9+/– (C and D), Inha+/–Gdf9–/– (E and F), and Inha–/–Gdf9–/– (G and H) ovaries. T (A and B) refers to thecal layers indicated by arrows. Whereas Nr5a2 is specifically expressed in granulosa cells in Inha+/–Gdf9+/– ovaries (I and J), its expression pattern and levels remain essentially unchanged in Inha–/–Gdf9+/– (K and L) and Inha–/–Gdf9–/– ovaries (O and P). Interestingly, in addition to the one-layer granulosa cells, Nr5a2 also appears highly expressed in oocytes in Inha+/–Gdf9–/– ovaries (M and N). Tu (K and L) denotes an early-formed granulosa cell tumor. Magnification: A–H, x125; I–P, x31.25.

 
Analysis of Kit and Kit Ligand (Kitl) Expression
Because KITL, via signaling through its receptor KIT on oocytes and thecal cells, plays important roles for germ cell proliferation and follicular development (26), we performed in situ hybridization to examine Kitl and Kit mRNA in these Inha Gdf9 double-mutant ovaries. As previously demonstrated in wild-type ovaries, only faint expression of Kitl is detected in granulosa cells of follicles at all stages in Inha+/–Gdf9+/– ovaries (Fig. 6Go, A and B); the expression of Kitl increases remarkably in the single-layer granulosa cells in primary follicles in Inha+/–Gdf9–/– ovaries and reaches the highest level in type 3b follicles containing larger oocytes (Fig. 6Go, E and F). No signal above background is seen in Inha–/–Gdf9+/– ovaries (Fig. 6Go, C and D). Surprisingly, in Inha–/–Gdf9–/– ovaries, Kitl mRNA level is similarly up-regulated in the granulosa cells compared with Inha+/–Gdf9–/– ovaries, and increases dramatically compared with Inha–/–Gdf9+/– ovaries (Fig. 6Go, G and H). Kit is expressed in oocytes from primordial to antral follicles and thecal cells in preantral and antral follicles in Inha+/–Gdf9+/– ovaries (Fig. 6Go, I and J). The expression level of Kit in the oocyte is decreased in Inha–/–Gdf9+/– ovaries, especially in follicles beyond the primary stage, and not detectable in thecal cells (Fig. 6Go, K and L). Resembling its expression pattern in Gdf9 null ovaries, Kit is slightly increased in the oocytes in primordial and primary follicles in Inha+/–Gdf9–/– ovaries (Fig. 6Go, M and N). Interestingly, even though Kit mRNA is highly expressed in the oocytes in Inha–/–Gdf9–/– ovaries, no signal is detected in thecal cells in these mice (Fig. 6Go, O and P).



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  		Fig. 6. In Situ Hybridization Analysis of Kit-Ligand (KITL) and Kit in Inha Gdf9 Double-Mutant Ovaries

All ovaries are from 3-wk-old mice. Low-level expression of Kitl is detected in granulosa cells throughout folliculogenesis in Inha+/–Gdf9+/– ovaries (A and B). Whereas no signal is detectable in Inha–/–Gdf9+/– ovaries (C and D), Kitl is highly up-regulated in granulosa cells in the single-layer primary follicles in Inha+/–Gdf9–/– ovaries (E and F) and follicles from the primordial to secondary stages in Inha–/–Gdf9–/– ovaries (G and H). White arrows in G and H denote an early-formed granulosa cell tumor that shows no expression of Kitl, indicating that these tumor cells are undifferentiated. Kit is expressed in both oocytes and thecal/interstitial cells in Inha+/–Gdf9+/– ovaries (I and J). Whereas Kit is down-regulated in the oocytes in follicles beyond the primary stage in Inha–/–Gdf9+/– ovaries, no expression is detectable in the theca (K and L). In Inha+/–Gdf9–/– (M and N) and Inha–/–Gdf9–/– (O and P) ovaries, Kit mRNA is slightly up-regulated in the oocytes in all stage follicles, and no thecal cell expression is visible.

 
Expression of Activin Subunits
As natural antagonists of inhibins, activins are homodimers of inhibin/activin ß subunits (ßA and ßB), which are encoded by the Inhba and Inhbb genes (27). Expression of inhibin/activin ßA and ßB subunits was also tested in Inha Gdf9 double-mutant ovaries. Both ßA and ßB are expressed in granulosa cells in late preantral and antral follicles in Inha+/–Gdf9+/– ovaries, and neither of these genes is expressed at appreciable levels in type 4 follicles (two-layer secondary follicles) and primordial or primary follicles (Fig. 7Go, A, B, I, and J). As expected, expression of ßA and ßB is increased dramatically in Inha–/–Gdf9+/– ovaries (Fig. 7Go, C, D, K, and L). In Inha+/–Gdf9–/– ovaries, ßA is expressed in follicles with degenerating oocytes and follicle nests (Fig. 7Go, E and F), whereas ßB is barely detectable (Fig. 7Go, M and N). The expression levels and patterns of ßA and ßB in Inha+/–Gdf9–/– ovaries resemble those seen in Gdf9 null ovaries (20). Despite the absence of GDF9, both ßA and ßB are highly up-regulated in Inha Gdf9 double-null ovaries (Fig. 7Go, G, H, O, and P), which is similar to the findings in Inha–/–Gdf9+/– ovaries.



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  		Fig. 7. In Situ Hybridization Analysis of Inhibin/Activin ßA and ßB Subunits in Inha Gdf9 Double-Mutant Ovaries

All ovaries are from 3-wk-old mice. Both ßA and ßB are expressed in granulosa cells in multiple-layer secondary follicles and beyond in Inha+/–Gdf9+/– ovaries (A, B, I, and J). In the absence of inhibin {alpha}, both ßA and ßB are increased dramatically in Inha–/–Gdf9+/– (C, D, K, and L) and Inha–/–Gdf9–/– (G, H, O, and P) ovaries. Whereas ßA is detectable only in the follicles with degenerated oocytes (white arrows), ßB is not detectable at all in Inha+/–Gdf9–/– ovaries (E, F, M, and N).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have previously shown that GDF9 is required for the development of follicles beyond the type 3b follicle stage (19). Our current studies presented in this manuscript indicate that the block at the primary follicle stage in Gdf9 null ovaries can be overcome by deletion of the Inha gene. Early folliculogenesis is primarily regulated by intraovarian paracrine factors. So far, only a few factors have been identified that play important roles during early follicular recruitment and growth, such as KITL, anti-Müllerian hormone, connexin 43, sheep bone morphogenetic protein 15 (BMP15), and GDF9 (19, 28, 29, 30, 31). Among them, only GDF9 appears to be critical for the progression from primary to secondary follicles in the mouse. It was reported that in vivo and in vitro treatment with GDF9 could induce preantral follicular growth in immature rats (32, 33). However, in our Inha Gdf9 double-null ovary studies, follicular growth proceeds normally until the type 5b (late preantral) follicle stage before the formation of granulosa cell tumors despite the absence of GDF9. Therefore, GDF9 is not essential as a direct promoter for early follicular growth in vivo. In contrast to the data of Roh et al. (34) showing that GDF9 increases inhibin {alpha} production in cultured neonatal rat ovarian explants and rat granulosa cells in vitro, our results suggest that suppression of inhibin {alpha} mediates, at least in part, some functions of GDF9 in vivo.

Formation of a theca is a hallmark of follicular development. In response to LH, thecal cells synthesize androgen substrates that are converted to estrogen by aromatase in granulosa cells under the stimulation of FSH (35). Even though morphological thecal layers are evident in Inha Gdf9 double-null ovaries, the lack of selective theca markers (i.e. CYP17, LHR, and KIT) in these cells indicates that they are not functional. The recruitment of thecal layers is more likely a result of follicular development beyond the secondary follicle stage, although mechanisms underlying thecal cell differentiation still remain to be elucidated. Some locally produced factors that mediate somatic cell-somatic cell interactions have been identified in the testis. For example, Sertoli cell (counterpart of ovarian granulosa cells in the testis)-derived desert hedgehog (DHH) signals through its receptor Patched 1, which is localized in Leydig cells (counterpart of ovarian thecal cells in the testis) (36). Mice lacking DHH are sterile due to disrupted spermatogenesis (36). In addition, the differentiation of fetal Leydig cells is defective, and the adult Leydig cells are completely absent (37, 38). Moreover, platelet-derived growth factor A, the main source of which is Sertoli cells from prenatal life until puberty in both the rat and mouse, also plays crucial roles in fetal and adult Leydig cell development (39). Even though DHH is not expressed in the ovary and platelet-derived growth factor A may not function the same way in females, there is evidence showing that factor(s) derived from granulosa cells are responsible for development and differentiation of the theca (40). One potential candidate is KITL, whose receptor, KIT, is expressed in oocytes and theca-interstitial cells. By in situ hybridization, Kitl mRNA is detectable in granulosa cells beginning at the primary follicle stage and increases in three-layered preantral follicles, whereas the expression of Kit begins in primordial follicles and continues throughout all of the subsequent follicle stages. Previous studies have shown that KITL stimulates bovine thecal cell growth and androgen production directly in vitro (41, 42). Kitl is highly up-regulated in Gdf9–/– and Inha–/–Gdf9–/– granulosa cells, but Kit is not detectable in thecal cells in both Inha–/– and Inha–/–Gdf9–/– ovaries. Although it is unlikely that KITL plays key roles during thecal cell recruitment, the lack of its receptor, KIT, might be responsible for the block of thecal cell differentiation in these Inha Gdf9 double-null ovaries.

KIT-KITL interactions are believed to be relevant for initiation of follicular growth from the primordial follicle pool, oocyte growth, theca and antrum formation, and protection of antral follicles from apoptosis (31, 41, 42, 43, 44, 45). Our results further confirm that GDF9 suppresses the expression of KITL that may account for the enlargement of oocytes in Gdf9 null (21) and Inha Gdf9 double-null ovaries. Furthermore, both Kitl and Kit are down-regulated in Inha–/–Gdf9+/– ovaries, an effect that seems to be secondary to inhibin {alpha} deficiency and can be reversed in the absence of GDF9. In vitro studies have demonstrated that FSH alone promotes KITL expression in preantral follicles (46), but the elevated FSH in Inha null mice (9) surprisingly does not seem to increase the KITL expression in vivo. Although some other defects on these granulosa cells may compromise their response to FSH, it is more likely that oocyte-derived GDF9 is directly responsible for the regulation of granulosa cell-derived KITL (46, 47).

As a transcription factor, NR5A1 is not only a key determinant of the expression of the cytochrome P450 steroid hydroxylases, but also an important regulator of many other genes that are involved in gonadal specification and development (48) including inhibin {alpha} (49, 50). NR5A2 (LRH1) most closely resembles NR5A1, particularly within the DNA binding domain, suggesting that these two orphan nuclear receptor family members may regulate overlapping target genes (51). NR5A2 is expressed in granulosa cells and corpora lutea in the ovary and shows the ability to activate the promoters of a number of steroidogenic enzymes including CYP11A (cholesterol side-chain cleavage), CYP17, aromatase, and steroidogenic acute regulatory protein in humans (52, 53). In our studies, the absence of either inhibin or GDF9 does not affect Nr5a1 and Nr5a2 mRNA remarkably, indicating the loss of CYP17 expression in the thecal cells may not due to the regulation of NR5A1 and NR5A2, at least not at the mRNA levels. Inhibition of the expression of CYP17 in thecal cells in Inha–/–Gdf9+/– and Inha–/–Gdf9–/– mice is more likely due to the down-regulation of LHR in the absence of inhibin {alpha}. Expression of LHR is one of the major markers of FSH-induced somatic cell differentiation. In granulosa cells, the stimulatory functions of FSH on LHR are mediated by cAMP and modified by many local growth factors (54, 55). However, this is unlikely the case in thecal cells because no Lhr mRNA is detectable by in situ hybridization despite presumed high serum FSH and local activins in Inha–/–Gdf9+/– and Inha–/–Gdf9–/– mice. Although Lhr gene transcription could be regulated by some nuclear orphan receptors and histone deacetylase complexes (56), relevant mechanisms in the ovary remain to be determined. Interestingly, there is no detectable CYP17 expression in the thecal cells in Inha–/–Gdf9+/– ovaries even though Gdf9 expression is fairly normal (data not shown); this contradicts previous findings (32, 57) that showed GDF9 directly stimulated CYP17 expression and steroidogenesis in rat thecal cells in vivo and in vitro. Therefore, GDF9 is neither a direct regulator of theca cell recruitment nor differentiation, although other local growth factors, receptors, or second messengers may affect steroidogenesis in these double-mutant mice.

Activins, homodimers of inhibin/activin ß subunits, antagonize the functions of inhibins on FSH secretion, follicular development, and steroidogenesis (58). Activins have been shown to play a key role early in folliculogenesis to promote granulosa cell growth and differentiation (58, 59). When follicles progress beyond the small antral follicle stage, activins potentiate FSH actions by stimulating FSH receptor formation in granulosa cells (60). There is also evidence showing that activin A enhances FSH-induced aromatase activity (61, 62), LH binding sites, and progesterone production in cultured granulosa cells from estrogen-treated immature rats (63). In the absence of inhibin {alpha}, the high levels of activins may account for the progression of follicular growth beyond the type 3b follicle stage in Gdf9–/– ovaries. However, whether elevated activins are related to the absence of LHR and CYP17 expression in Inha–/–Gdf9+/– and Inha–/–Gdf9–/– thecal cells still remains a question.

Inhibins, activins, and BMPs are structurally related TGFß superfamily members, and most ligands from the TGFß superfamily signal through single-transmembrane serine/threonine kinase receptors that can be classified into type I and type II receptors based on their sequence divergence (5). Whereas activins can bind only to activin type II receptors (ACVR2 and ACVR2B), BMPs can bind to both activin receptors and BMP receptors type II (BMPR2) (64, 65, 66). Inhibins can bind to ACVR2 (67) under the facilitation of betaglycan (12), which acts as a coreceptor to increase the affinity of inhibins for ACVR2s. This is believed to be the mechanism that mediates the antagonistic modulation of inhibins and activins in pituitary and gonadal systems. Recently Wiater and Vale (16) discovered that inhibin blocks cellular responses to BMP family members in different BMP-responsive cell types by competing not only for ACVR2, but also for BMPR2. Betaglycan also enables the binding of inhibin to BMPR2. Being a close paralog to BMPs, GDF9 is postulated to signal in a similar way as BMPs. Recent studies have shown that the BMPR2 mediates the effects of GDF9 on granulosa cells from small antral follicles, and BMPR1A, BMPR1B, and ACVR2 might be partially involved in the signaling of GDF9 as well (68). If that is the case, then inhibin might function as an antagonist of GDF9 during follicular development by competing for binding to BMPR2.

Taken together, studies using Inha Gdf9 double-mutant mice reveal an interrelationship between inhibin {alpha} and GDF9 in vivo, both of which are important parts of an intraovarian paracrine/autocrine network within the follicle. Briefly, oocyte-derived GDF9 functions through suppression of granulosa cell-derived factors such as inhibin {alpha} and KITL. In turn, inhibin {alpha} and KITL regulate the development and differentiation of multiple cell types within the follicle, although additional granulosa cell-derived factor(s) must be involved in the theca recruitment, differentiation, and steroidogenesis. Our hypothesis, summarized in Fig. 8Go, is based on studies of the Inha Gdf9 double-mutant mouse model. Notably, all the gene expression data in this manuscript are at mRNA level. Future studies on meiotic competence of the oocyte and steroidogenetic pathways in the theca, in the presence and absence of inhibin {alpha}, will further elucidate the effects of GDF9 on later stages of folliculogenesis. Inha Gdf9 double-knockout mice provide a unique model for research on mechanisms underlying follicular development, steroidogenesis, and interactions of ovarian factors in vivo.



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  		Fig. 8. A Model for Early Intraovarian Network of Factors

Oocyte-derived GDF9 suppresses the expression of granulosa cell-derived inhibin and KITL (20 46 ). Inhibin is known to antagonize activin in granulosa cells, and activin is a promoter for granulosa cell growth and differentiation. KITL stimulates oocyte and possibly thecal cell growth, although additional granulosa cell-derived factor(s) must be involved in the recruitment of the theca. The interactions of KITL signaling, LHR expression, thecal cell differentiation, and steroidogenesis in thecal layers remain to be determined. The hypothesis summarized in this model is based on our studies of Inha Gdf9 double-mutant mice. Gene regulation is analyzed at mRNA level.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of Inha Gdf9 Double-Knockout Mice
All the experimental mice were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Inhatm1Zuk (herein called Inha–) and Gdf9tm1Zuk (herein called Gdf9–) mutant mice were generated as described previously (9, 19). Initially, Gdf9–/– (null) males were bred with Inha+/– females to produce Inha+/–Gdf9+/– mice. These mice were later intercrossed to obtain Inha–/–Gdf9–/– mice at a 1:16 frequency. Eventually, Inha+/–Gdf9–/– male mice were bred with double-heterozygous females to increase the frequency of generating double homozygous mutant mice to 1:8. Sex ratio of their offspring is nearly 1:1, and genotype distribution of the pups follows a Mendelian ratio (data not shown). Double-homozygous mutant mice and their control littermates were weighed and counted weekly on the same day.

Histology and in Situ Hybridization Analysis
Ovaries were collected from 3-wk-old or adult C57BL/6;129S6/SvEv mice. Tissue procession and analysis were performed as described elsewhere (69). Specific mouse probes for the 17{alpha}-hydroxylase (Cyp17), LHR (Lhr), kit ligand (Kitl), Kit, and activin/inhibin ßA and ßB were obtained as described previously (20). A 277bp Nr5a1-specific probe containing sequences from Nr5a1 gene 3'-untranslated region (24) and a 452bp Nr5a2-specific probe containing sequences encoding the ligand binding domain (25) were amplified from mouse ovary cDNA pool by PCR (see below). {alpha}-[35S]UTP-labeled antisense and sense probes were generated by the Riboprobe T7/T3 or Riboprobe T7/SP6 combination systems (Promega Corp., Madison, WI). Hybridization was carried out as described previously (20, 70). For each probe, at least three animals from each group were analyzed, and the most representative sections were used for photography.

RNA Isolation and RT-PCR Analysis
Total RNA was extracted from wild-type C57BL/6;129S6Sv/Ev mouse ovaries using STAT-60 (Leedo Medical Laboratories, Houston, TX) as described by the manufacturer and quantitated on a spectrophotometer. Oligo-dT-primed cDNA from 2 µg of total ovarian RNA from wild-type mouse was synthesized using Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. One microliter (1/20 of total) was used in each 25-µl PCR primed with Nr5a1-specific oligonucleotides: 5'-ATCAGAGGCAAGGAAGGTCTAC-3' (sense, nucleotides 1900–1921 of NM_139051) and 5'-TTAGGG-CAGGAATGTTGGCTAC-3' (antisense, nucleotides 2155–2176 of NM_139051), and Nr5a2-specific oligonucleotides: 5'-CGATCACATTTACCGACAAGTG-3' (sense, nucleotides 1379–1400 of M31835) and 5'-TGGCATGCAGCATCTCA-ATGA-3' (antisense, nucleotides of 1809–1830 of M31835), respectively. Products were separated on a 1% agarose gel, visualized by ethidium bromide staining, and cloned into pGEM-T vector (Promega).

Statistics
Student’s t test was used to analyze the mean ± SEM for the body weight and survival rate data.


   ACKNOWLEDGMENTS
 
We thank Drs. T. Rajendra Kumar, Stephanie A. Pangas, and Kathleen H. Burns for their critical review of the manuscript.


   FOOTNOTES
 
This work was supported in part by National Institutes of Health Grants CA60651 and HD33438 (to M.M.M.).

Abbreviations: BMP, Bone morphogenetic protein; CYP17,17{alpha}-hydroxylase; DHH, desert hedgehog; GDF9, growth differentiation factor 9; KITL, KIT ligand; LHR, LH receptor; LRH1, liver receptor homolog 1.

Received for publication October 10, 2003. Accepted for publication March 2, 2004.


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