Oocytes determine cumulus cell lineage in mouse ovarian follicles

Ovarian Graafian follicles are comprised of somatic cells that exhibit both endocrine and developmental functions. The endocrine functions, including steroidogenesis, are carried out primarily by the mural granulosa cells (Fig. 1). The cumulus cells, which are closely associated with the oocyte, promote oocyte growth and developmental competence through complex bi-directional interactions with the oocyte. In fact, oocyte stimulation of cumulus cell function is crucial for its own subsequent development (Eppig, 2001; Hussein et al., 2006; Su et al., 2004). To fulfil both endocrine and developmental functions, follicle growth must involve the coordinated development of both cumulus and mural granulosa cells, as a deficiency in either hormone production or development of oocyte competence would impair fertility.

Although granulosa cells express many mRNA transcripts and proteins in common, the mural and cumulus cells each express a subset of different transcripts, with markedly different steady state levels (Fig. 1). Preantral granulosa cells are the common precursors of both mural and cumulus cells. Large preantral follicles (secondary follicles) develop into antral follicles (tertiary follicles) after forming a fluid-filled antrum under stimulation of follicle stimulating hormone (FSH). Antrum formation physically separates the preantral granulosa cells into a mural cell compartment along the follicle wall and a cumulus cell compartment surrounding the oocyte (Fig. 1). The mural cells produce increasing amounts of estrogen, which eventually initiates the surge of luteinizing hormone (LH) from the pituitary resulting in oocyte maturation, expansion of the cumulus oophorus and ovulation. The cumulus cells promote oocyte growth and developmental competence (Eppig, 2001; Hussein et al., 2006; Su et al., 2004). A defining feature of cumulus cells is their ability to undergo expansion following the LH surge. Cumulus expansion involves the production of a mucified matrix by the cumulus cells that is necessary for ovulation and therefore required for fertility (Chen et al., 1993).

The mechanisms responsible for differentiation of preantral granulosa cells into mural and cumulus cells are not fully defined, but involve factors secreted by oocytes and FSH produced by the pituitary. FSH is essential, as follicles fail to develop beyond the early antral stage in mice lacking FSH (Kumar et al., 1997) or FSH receptors (Dierich et al., 1998). FSH stimulates expression of Lhcgr (luteinizing hormone receptor) mRNA in mural cells (Fig. 1). Lhcgr mRNA is a key marker of mural cell differentiation (Lei et al., 2001). Both cumulus and mural cells express FSH receptors (Richards and Midgley, 1976); however, oocyte suppression of Lhcgr in cumulus cells restricts Lhcgr mRNA to mural cells (Eppig et al., 1997). In addition to suppressing Lhcgr mRNA, paracrine factors secreted by oocytes have profound effects on follicle growth and development both before and after the LH surge. For example, absence of oocyte-derived growth and differentiation factor 9 (GDF9) results in failure to progress beyond the early preantral stages of follicle development (Dong et al., 1996; Elvin et al., 1999). Lack of bone morphogenetic protein 15 (BMP15), another oocyte-secreted protein, leads to defective cumulus cell differentiation and cumulus expansion (Su et al., 2004; Yan et al., 2001; Yoshino et al., 2006). Oocyte-derived factors are also required to enable cumulus expansion after the preovulatory LH-surge (Buccione et al., 1990; Diaz et al., 2006). The major impact that oocytes have in directing follicle development was demonstrated by recombining mid-growth stage oocytes from large preantral follicles with follicular somatic cells from newborn ovaries. The resulting re-aggregated ovary not only forms follicles when transplanted to recipient mice, but the chronologically more advanced oocytes actually accelerate the development of the newborn somatic cells (Eppig et al., 2002). These observations provided strong evidence that oocyte-derived factors are important mediators of follicle growth, cumulus cell differentiation and cumulus expansion.

Oocyte factors and FSH signal from opposite compartments. The oocyte signals emanate from a central follicular location, whereas FSH signals arrive from outside the follicle. This leads to a gradient of expression of mural and cumulus transcripts. Levels of the mural marker transcript Lhcgr are highest near the basal lamina and lowest in the cumulus cells (Meduri et al., 1992) (Fig. 1). Conversely, expression of Amh mRNA, which is stimulated by oocytes (Salmon et al., 2004) is highest in the cumulus cells (Baarends et al., 1995) (Fig. 1). Oocyte-secreted paracrine factors are also required to enable increases in Has2, Ptgs2, Ptx3 and Tnfaip6 transcripts during cumulus expansion (Diaz et al., 2006). Each of these transcripts is absolutely required for cumulus expansion as the phenotype of null mutations in the Ptgs2, Ptx3 or Tnfaip6 genes or inhibition of HAS2 activity severely compromises cumulus expansion (Chen et al., 1993; Fulop et al., 2003; Ochsner et al., 2003a; Ochsner et al., 2003b; Varani et al., 2002).

The signaling pathways activated by oocytes and required for cumulus cell function remain largely unclear. However, pathways activated by TGF μ-related proteins, such as GDF9 and BMP15, are probably crucial for mediating the effect of oocytes on granulosa cells. BMP15 and GDF9 activate SMAD1/5/8 and SMAD2/3 signaling pathways, respectively, in granulosa cells (Moore et al., 2003; Roh et al., 2003). Signaling through either SMAD1/5/8 or SMAD2/3 pathways requires that these receptor-regulated SMADs bind the common SMAD, SMAD4. Conditional deletion of the Smad4 gene in granulosa cells of preantral follicles leads to severe defects in subsequent follicle development and differentiation owing to a lack of SMAD4-dependent signaling (Pangas et al., 2006). Moreover, mice deficient in Smad3 show increased rates of cell death and abnormal cell differentiation (Tomic et al., 2004). Thus, SMAD signaling is crucial for proper follicle development. More recently, the BMP receptor type II (BMPR2) extracellular domain and an inhibitor of SMAD2/3 activation, SB431542, were used to block oocyte-stimulated proliferation of mural and cumulus cells (Gilchrist et al., 2006). However, the exact TGF μ-related signaling pathways activated in cumulus cells by oocytes that lead to cumulus cell differentiation and expansion remain undefined. The work reported here tests the hypothesis that oocyte-stimulated SMAD2/3 signaling mediates the ability of oocytes to stimulate the cumulus cell phenotype during antral follicle development.

A strong pSMAD2 signal was detected by immunofluorescence staining of tissue sections in preantral granulosa cells of small and large preantral follicles (Fig. 2A,B). In antral follicles, pSMAD2 localized more strongly to cumulus cells than to mural cells (Fig. 2C). Surprisingly, treatment with hCG in vivo resulted in decreased pSMAD2 levels by 8 hours (Fig. 2D). No pSMAD2 signal was detected in ovary sections incubated with secondary antibody alone or when the pSMAD2 antibody was preincubated with a SMAD2 phosphopeptide (pSer465/467, Abcam) (Fig. 2E and data not shown). To verify the results observed in tissue sections, we compared pSMAD2 levels in freshly isolated cumulus-oocyte complexes (COCs) and mural cells by western blot and found that levels of total SMAD2 were similar in mural and COCs, but levels of phosphorylated SMAD2 were higher in COCs compared with mural cells (Fig. 2F). The levels of pSMAD2 observed in cumulus cells were stimulated by the oocyte. Levels of pSMAD2 detected by western blot were high in untreated COCs (Fig. 3). Removal of the oocyte greatly reduced the pSMAD2 signal to an almost undetectable level, which was restored upon co-culture with oocytes. SB431542 suppressed pSMAD2 in COCs and OOX cells co-cultured with oocytes (Fig. 3). Thus, the oocyte potently stimulates pSMAD2 in cumulus cells.

The effect of SB431542 and SIS3 on activation of various signaling molecules in cultured COCs was determined to assess the specificity of the inhibitors. SB431542 blocked SMAD2 and SMAD3 activation in COCs, but had no effect on total SMAD2 levels or levels of pSMAD1/5/8 (Fig. 4A). By contrast, SIS3 blocked pSMAD3 activation without affecting total SMAD2, pSMAD2 or pSMAD1/5/8 levels (Fig. 4A). The efficacy of SB431542 was determined by a dose-response experiment where COCs were treated with concentrations of SB431542 ranging from 0.1 to 100 μM (Fig. 4B). The minimum dose that effectively blocked pSMAD2 levels in COCs was 3 μM (Fig. 4B). Although SB431542 and SIS3 are highly specific for inhibiting the kinase activity of ALK-4, ALK-5 and ALK-7, the possibility exists that these molecules could have effects on other untested cellular kinases or even other non-kinase proteins.

Transcripts encoding the LH receptor, Lhcgr, the steroidogenic enzyme P450 side chain cleavage, Cyp11a1, and the immune molecule Cd34 were higher in mural than in cumulus cells as measured by real-time PCR (Fig. 1). We used these transcripts as markers of the pre-LH-surge mural cell phenotype. Oocyte-derived factors suppress expression of Lhcgr mRNA in cumulus cells (Eppig et al., 1998; Eppig et al., 1997). We hypothesized that oocytes might suppress Lhcgr, Cyp11a1 and Cd34 through activation of the TGF μ signaling pathway. To test this idea, the effects of oocyte extirpation (OOX), FSH (100 ng/ml), SB431542 (an inhibitor of SMAD2 and SMAD3 activation, 10 μM) and SIS3 (a SMAD3-specific inhibitor, 20 μM) on expression of mural transcripts in COC and mural cells were measured. FSH was required to maintain expression of mural cell markers in vitro (Fig. 5A,B). Fully-grown, germinal vesicle (GV) stage oocytes (FGO) potently suppressed the steady-state levels of the three mural transcripts that had been maintained by FSH (Fig. 5A,B). The suppression of mural transcripts by oocytes in mural cells was blocked by treatment with SB431542, but not SIS3 (SMAD3 inhibitor) suggesting that SMAD3 signaling was not involved (Fig. 5B). In COCs, we hypothesized that oocytes would also suppress mural transcripts through TGF μ signaling pathways. Removal of the oocyte (OOX) allowed FSH to stimulate increased expression of mural transcripts in cumulus cells (Fig. 6A). Co-culture with oocytes prevented FSH-induced increases of mural transcripts, but treatment with SB431542, but not SIS3, blocked the ability of oocytes to suppress mural transcript induction by FSH (Fig. 6B,C), indicating that oocyte suppression of mural transcripts in cumulus cells is likely via pSMAD2.

Oocyte-derived factors promote expression of Slc38a3 (Eppig et al., 2005) and Amh mRNA (Salmon et al., 2004) in cumulus cells. These transcripts along with Ar (androgen receptor) mRNA are more highly expressed in COCs than mural cells (Fig. 1). We hypothesized that oocytes may promote expression of the cumulus transcripts Slc38a3, Amh and Ar though the TGF μ signaling pathway. To test this idea, the effects of oocyte extirpation (OOX), FSH (100 ng/ml), SB431542 (an inhibitor of SMAD2 and SMAD3 activation, 10 μM) and SIS3 (a SMAD3-specific inhibitor, 20 μM) on expression of cumulus transcripts was tested. OOX resulted in a decrease in cumulus markers (Slc38a3, Amh and Ar mRNA), which were restored by oocyte co-culture (Fig. 7A) indicating that oocyte factors stimulate these transcripts. SB431542 but not SIS3 (Fig. 7B,C) caused a decrease in cumulus marker transcript levels. Surprisingly, FSH completely suppressed Slc38a3 and decreased Ar transcript levels by ∼50%, even in the presence of oocytes (Fig. 7B). Mural cells express lower levels of Slc38a3, Amh and Ar mRNAs (Fig. 1). Since oocytes can stimulate pSMAD2 levels in mural cells (Gilchrist et al., 2006) and pSMAD2 appears important for expression of cumulus transcripts (Fig. 7B), we hypothesized that oocytes would stimulate cumulus transcripts in mural cells. Levels of Slc38a3 and Ar were not induced by oocytes in mural cells. However, oocytes did stimulate Amh transcript levels in mural cells, which was blocked by SB431542, but not SIS3 (Fig. 8A,B).

Cumulus expansion requires one or more oocyte-secreted factors (Vanderhyden et al., 1990). SB431542 and SIS3 were used to test the effect of blocking both SMAD2 and SMAD3 or SMAD3 only on cumulus expansion. As expected, expansion was stimulated by EGF treatment in vitro (Fig. 9A). Treatment with SB431542 completely blocked cumulus expansion (Fig. 9A). By contrast, expansion was not severely affected by SIS3 treatment, but some cells did attach to the culture dish suggesting that expansion was not completely normal (white arrows Fig. 9A). The minimum effective dose of SB431542 that blocks expansion was 1-3 μM (Fig. 9B) and is similar to the minimum dose that blocks pSMAD2 levels in COC (Fig. 4B).

To better define the molecular effects of SB431542 and SIS3 on cumulus expansion, an analysis of cumulus-expansion-related transcripts at several time points during cumulus expansion was undertaken. EGF stimulated expression of Has2, Ptgs2, Ptx3 and Tnfaip6 mRNA in COCs with the highest level measured occurring 6 hours after EGF treatment (Fig. 9C). The effect of the inhibitors on EGF-induced expression of expansion-related transcripts was dependent on the specific transcript and on the time after EGF treatment. Levels of Ptgs2 mRNA were not affected by SIS3, whereas treatment with SB431542 suppressed levels of Ptgs2 mRNA at 8 and 10 hours (Fig. 9C). Both SIS3 and SB431542 reduced levels of Ptx3 mRNA by 75-95% (Fig. 9C). Levels of Tnfaip6 mRNA were not affected by either SB431542 or SIS3 at 6 or 8 hours after EGF, but by 10 hours, both inhibitors suppressed Tnfaip6 mRNA levels (Fig. 9C). Levels of Has2 mRNA were not affected by SIS3, but were completely suppressed by SB431542 at all times examined (Fig. 9C). Thus, regulation of expansion transcripts is likely controlled through a complicated network of pathways which includes pSMAD2 and pSMAD3 signaling.

Cumulus expansion requires both activation of MAPK3/1 and MAPK14 and oocyte-stimulated SMAD2/3 signaling. SB431542 can inhibit MAPK14 activation in a cell-free assay system (Inman et al., 2002). Therefore, we analyzed the effect of SB431542 on phosphorylation of MAPK3/1 and MAPK14 by EGF and the effect of EGF on pSMAD2 levels in order to begin to identify at what level the MAPK and SMAD signaling pathways interact. EGF stimulated MAPK3/1 and MAPK14 phosphorylation in COCs (Fig. 10A). SB431542 treatment completely blocked pSMAD2 levels in COCs, but lack of SMAD2 phosphorylation did not prevent MAPK3/1 or MAK14 activation 4 hours after EGF treatment (Fig. 10A). Similar to in vivo hCG-treated follicles (Fig. 2E), levels of pSMAD2 began to decrease significantly (P>0.05) by 8 hours with a further decrease by 12 hours after EGF treatment in vitro (Fig. 10B). The levels of total SMAD2 did not change during EGF stimulation indicating that the decrease in SMAD2 activation observed at 8 and 12 hours was due to decreased pSMAD2 activation and not to loss of SMAD2 protein. Thus, activation of MAPK is independent of SMAD2/3 activation during cumulus expansion. However, activation of MAPK pathways led to decreased pSMAD2 signaling during cumulus expansion.

Evidence is presented here that oocyte-stimulated SMAD signaling is crucial for defining the differentiation and function of cumulus cells. Moreover, oocyte-secreted factors oppose the action of FSH, which would, in the absence of the oocyte, promote the expression in cumulus cells of characteristics more typical of mural granulosa cells. Soluble oocyte-derived factors strongly promoted phosphorylation of SMAD2 in cumulus cells. Activated SMAD2 promoted expression of marker cumulus transcripts and suppressed expression of mural transcripts in cumulus cells, and together with pSMAD3, enabled cumulus expansion. The SMAD3-specific inhibitor, SIS3, had no effect on expression of mural or cumulus marker transcripts, but did block increases in Ptx3 and Tnfaip6 mRNA, two transcripts induced by EGF during cumulus expansion, suggesting that SMAD2 and SMAD3 have partially divergent effects on cumulus cell function. By contrast, FSH stimulated expression of mural marker transcripts and suppressed the expression of some cumulus marker transcripts. In the presence of oocytes, FSH was unable to induce mural transcripts, but did suppress cumulus transcripts in vitro. Thus, we present a model (Fig. 11) where oocyte-stimulated pSMADs and FSH establish opposing gradients of influence that define the cumulus and mural granulosa cell phenotypes, respectively. These mechanisms are important for promoting optimal endocrine and developmental functions in the ovarian follicle.

Mural and cumulus cells both originate from preantral granulosa cells. The divergent functions (endocrine vs developmental) of these two lineages develop within the context of a follicle and thus the specification of the cumulus vs mural cell fate must depend in part on the local microenvironment. In the antral follicle, the oocyte is emerging as a central regulator of cumulus cell function. We hypothesized that SMAD2/3 signaling pathways might be involved in specifying the cumulus cell phenotype because of two recent reports showing: (1) an inhibitor of SMAD2/3 activation, SB431542, blocks the ability of the oocyte to stimulate cumulus cell proliferation (Gilchrist et al., 2006) and (2) deletion of the Smad4 gene in granulosa cells results in defective cumulus cell differentiation (Pangas et al., 2006). In SMAD4-deficient granulosa cells, activated forms of SMAD2/3 and SMAD1/5/8 would be unable to alter target gene transcription because of the absence of the co-SMAD, SMAD4, effectively ablating signaling through these pathways. As a first step in analyzing SMAD2/3 signaling in the follicle, activated SMAD2 was localized in tissue sections by immunofluorescence and immunoblot analyses. Strong pSMAD2 immunostaining was evident in granulosa cells of preantral follicles. However, in large antral follicles, 48 hours after eGC treatment, pSMAD2 levels were much higher in cumulus cells compared with mural cells, similar results were obtained by immunoblotting. The pattern of pSMAD2 staining suggested that the oocyte is responsible for activating this pathway in cumulus cells. Oocytectomy completely ablated pSMAD2 levels in cumulus cells, which were restored upon oocyte co-culture showing that oocytes stimulate pSMAD2 signaling in cumulus cells. These observations are consistent with a role of SMAD2/3 signaling in cumulus cell function.

The TGF μ signaling pathways are emerging as a key regulator of ovarian function. In this report, two inhibitors of TGF μ-activin-GDF9 signaling, SB431542 and SIS3 were used. The activin-like kinase (ALK) inhibitor, SB431542, specifically inhibits the activity of ALK4, ALK5 and ALK7 without effect on other cellular kinases in living cells (Inman et al., 2002; Laping et al., 2002) (Figs 4, 10). ALK4 and ALK5 are the type 1 receptors involved in GDF9 and activin signaling. When activated by a ligand, ALK4 and ALK5 pair with a specific type II receptor, resulting in phosphorylation of SMAD2 and SMAD3 signaling molecules (Moore et al., 2003; Roh et al., 2003). SIS3 specifically blocks SMAD3 activity by inhibiting ALK5-mediated phosphorylation of SMAD3 and association with SMAD4 (Jinnin et al., 2005). In COCs, SB431542 is a specific inhibitor of SMAD2 and SMAD3 activation, whereas SIS3 specifically blocks SMAD3 activation. Neither inhibitor had any effect on total SMAD2 levels or on phosphorylation of the related SMAD1/5/8 pathway.

Increasing evidence supports a direct role of the oocyte in promoting cumulus cell function before the LH surge. Oocytes are known to promote glycolysis (Sugiura et al., 2005), proliferation (Gilchrist et al., 2006; Vanderhyden et al., 1992), amino acid transport (Eppig et al., 2005) and cell survival (Hussein et al., 2005), and to suppress inappropriate expression of mural transcripts such as Lhcgr in cumulus cells (Eppig et al., 1997). Results presented in this study confirm and extend these observations to include oocyte regulation of other cumulus and mural transcripts before the LH surge. For example, we show that oocytes promote expression of three cumulus transcripts, Slc38a3, Amh and Ar. Each of these transcripts has important functions. Expression of SLC38A3 in cumulus cells probably mediates, at least in part, the transport of specific amino acids from the cumulus cells to the oocyte in support of oocyte growth (Eppig et al., 2005). Expression of AR in the follicle is required for full fertility because Ar null mice are severely subfertile and show defects in cumulus cell morphology and differentiation (Hu et al., 2006). In addition, androgens augment the ability of oocytes to stimulate cellular proliferation (Hickey et al., 2005). AMH in early antral follicles antagonizes FSH-induced follicle growth (Durlinger et al., 2001; Durlinger et al., 1999). Moreover, we show that treatment with SB431542, but not SIS3, resulted in reduced cumulus marker transcript levels. These observations suggest that SMAD2 and SMAD3 have divergent functions in the regulation of the steady-state levels of these transcripts. Surprisingly, FSH suppressed Slc38a3 and Amh mRNA levels in cultured COCs and is likely to be one reason why Slc38a3 and Amh are not expressed in mural cells.

The mural cell phenotype is produced to a great extent by FSH stimulation of transcripts involved in steroidogenesis (Cyp11a1), ovulation (Lhcgr) and immune function (Cd34). However, oocytes potently suppressed mural transcripts in mural cells even in the presence of FSH. Because oocyte factors and FSH form opposing concentration gradients in the follicle, a gradient of Lhcgr transcript levels is observed in antral follicles, where granulosa cells farther away from the oocyte express higher levels of mural transcripts (Eppig et al., 2002; Lei et al., 2001). Cumulus cells are closely associated with the oocyte and therefore it is not surprising that expression of mural transcripts is low in cumulus cells because of the suppressive action of oocyte-derived factors. This helps to explain why FSH did not promote expression of the mural marker transcripts in cultured COCs even though cumulus cells robustly express FSH receptors (Richards and Midgley, 1976). However, FSH is able to stimulate mural transcript expression in cumulus cells in the absence of oocytes (OOX) or in COCs treated with SB431542, but not SIS3 indicating that SMAD2 activity is required for suppression of mural transcripts by oocyte factors. That FSH suppressed cumulus marker transcripts and stimulates mural transcripts in vitro, suggests that oocytes must continuously overcome these two potential effects of FSH on cumulus cells before the LH surge in vivo. It is remarkable that oocyte-stimulated SMAD2 signaling promotes elevated levels of cumulus marker transcripts and suppresses mural transcripts in cumulus cells, while only suppressing mural transcripts in mural cells. These observations might reflect recruitment of different co-activators or co-repressors to the promoter regions of these genes. However, further studies are needed to define how a common signal can generate both positive and negative responses in the same cell type.

In addition to regulating the levels of cumulus transcripts before the LH surge, oocyte-secreted factors also modify cumulus cell function after the LH surge by enabling cumulus expansion. The process of cumulus expansion requires the presence of cumulus expansion enabling factors (CEEFs) secreted by the oocyte. The nature of the CEEFs has remained elusive, but could include GDF9 (Elvin et al., 1999; Gui and Joyce, 2005) and/or BMP15 (Su et al., 2004; Yoshino et al., 2006) since both recombinant proteins stimulate expansion. Here, we demonstrated that SB431542 completely blocks EGF-induced expansion, whereas the effect of SIS3 is more subtle, expansion still occurs in SIS3 treated COCs, but many cells from attachments to the culture dish, indicating an abnormal expansion. Cumulus expansion is dependent on increases in transcript levels of Has2, Ptx3, Ptgs2 and Tnfaip6 (Chen et al., 1993; Fulop et al., 2003; Ochsner et al., 2003a; Ochsner et al., 2003b; Varani et al., 2002). The potential requirement of pSMAD2 and pSMAD3 in driving the increased levels of these transcripts in cumulus cells was unknown. Here we demonstrate a complex pattern of transcript regulation during cumulus expansion. One transcript, Tnfaip6, was largely unaffected by either inhibitor during EGF-induced expansion. At the other extreme, EGF-induced increase in Ptx3 mRNA was suppressed by both inhibitors at all time points examined. Ptgs2 mRNA levels were not suppressed at 6 hours, but by 8-10 hours, SB431542, but not SIS3, suppressed levels of this transcript. Finally, Has2 was suppressed by SB431542, but not by SIS3. These results indicate that the oocyte-enabled increase in Ptx3 requires activation of both SMAD2 and SMAD3, whereas SMAD2 activation is sufficient for enabling increases in Ptgs2 and Has2 mRNA. However, the CEEF activity responsible for enabling increases in Tnfaip6 mRNA signal through pathways that are not acutely dependent on either pSMAD2 or pSMAD3. This suggests the possible existence of one or more CEEF factors secreted by the oocyte.

The factor(s) responsible for pSMAD2/3 activation in cumulus cells are not known. Candidate molecules include oocyte-derived GDF9 or TGFB1 or the granulosa-cell-derived activin (Knight and Glister, 2006). GDF9 is an attractive candidate because mice lacking this protein do not form antral follicles (Dong et al., 1996), rGDF9 activates SMAD2 in granulosa cells in vitro (Roh et al., 2003) and RNAi inhibition of GDF9 production in oocytes abrogates cumulus expansion (Gui and Joyce, 2005), which we now show is dependent on pSMAD2/3. Thus, GDF9 might have roles during both cumulus cell differentiation and cumulus expansion. However, not all the data support a role of GDF9 during expansion, since a GDF9-neutralizing antibody did not block cumulus expansion enabled by oocytes (Dragovic et al., 2005). TGFB1 is another possible candidate since it is expressed by the oocyte and activates the SMAD2/3-signaling pathway. Mice with null mutations in the Tgfb1 gene have reduced fertility (Ingman et al., 2006) suggesting a possible involvement in follicle development. However, a TGFB1-neutralizing antibody does not block cumulus expansion enabled by oocytes (Salustri et al., 1990). The possible involvement of activin in cumulus cell differentiation or cumulus expansion has not been investigated. Regardless of which ligand is responsible for SMAD2/3 activation in cumulus cells, oocyte-stimulated pSMAD2/3 in cumulus cells is part of a differentiation mechanism required to specify the cumulus cell phenotype.

Communication between the cumulus cells and oocyte is crucial for subsequent fertility. Disruption of these signaling pathways between oocytes and cumulus cells leads to poor developmental potential or even infertility. Thus, resolving how cumulus cells and oocytes signal each other is key to understanding the mechanisms responsible for a fundamental process in biology, the production of a mature female gamete. Fig. 11 summarizes our working model and highlights the crucial role of oocyte-stimulated SMAD2/3 signaling in determining aspects of the cumulus cell phenotype, including expression of cumulus transcripts, suppression of mural transcripts before the LH surge and promotion of cumulus expansion after the LH surge. Two opposing radial gradients within the follicle are created by FSH signaling from outside the follicle and oocyte-stimulated SMAD2 signaling from within the follicle. Together, they specify characteristics and functions of the mural and cumulus cell compartments within the follicle to foster an optimal microenvironment for proper endocrine (mural) and developmental (cumulus-oocyte complex) functions.

Female B6SJLF1 mice (Mus musculus) were produced and raised in the research colony of the investigators. Ovaries were collected from mice on day 22, 48 hours after I.P. injection of 5 IU eCG (National Hormone and Peptide Program, NIDDK). For some experiments, ovaries from 12-day-old animals were used for analysis of preantral granulosa cells. Animals were maintained according to the Guide for the Care and Use of Laboratory Animals (Institute for Learning and Animal Research).

COCs and fully grown denuded oocytes at the GV (germinal vesicle) stage were collected from antral follicles. Following COC collection, clumps of mural granulosa cells from approximately one primed ovary were equally divided into four groups. Mural granulosa cells, COCs (25 for mRNA or 50 for protein), OOX complexes (25 or 50) and co-cultures of mural or OOX complexes with fully grown oocytes (FGOs 2 oocytes/μl) were cultured in bicarbonate-buffered MEM-μ (Life Technologies, Grand Island, NY) with Earles salts, supplemented with 75 mg/l penicillin G, 50 mg/l streptomycin sulfate, 10 μM milrinone (to prevent GVB in FGOs), 0.23 mM pyruvate, and 3 mg/ml crystallized lyophilized bovine serum albumen. COC, mural cells or OOX complexes were treated for one or more of the following time points 0, 4, 6, 8, 12 or 15 hours with control medium, FSH (100 ng/ml) or EGF (10 ng/ml). Some cultures were pre-incubated with SB431542 (0.1 to 100 μM, Calbiochem) or SIS3 (20 μM, Calbiochem) to block both SMAD2 and SMAD3 (SB431542) or SMAD3 (SIS3) activation for 1 hour before beginning treatment. Expansion was stimulated in media (MEM-μ) containing EGF 10 ng/ml) and 5% FBS. Samples were immediately frozen in liquid nitrogen and stored at –70°C until analyzed for protein or mRNA levels. All reagents were purchased from Sigma Chemical Company (St Louis, MO) unless otherwise noted. All experiments were repeated at least three times.

Total RNA was isolated from frozen samples and reverse transcribed into cDNA as described previously (Diaz et al., 2006). Quantification of mural and cumulus and expansion-related transcripts was conducted using gene-specific primers as described previously using Rpl19 as an internal control (Diaz et al., 2006). Only one product of the appropriate size was identified for each set of primers and all amplification products were sequenced to confirm specificity. All experiments were repeated three to four times and values shown are the mean ± s.e.m.

Samples were prepared from 50 COCs or OOX complexes cultured alone or with FGOs (2 oocytes/μl). Some samples were pretreated with SB431542 (10 μM) or SIS3 (20 μM) for 1 hour. Groups were treated with medium only (control), FSH (100 ng/ml) or EGF (10 ng/ml) for 1, 4 or 15 hour(s). Samples were simultaneously denatured by boiling in 1 μ loading buffer for 5 minutes followed by quenching on ice for 5 minutes. Proteins were separated on a 10% SDS PAGE gel and transferred to PVDF membrane. Membranes were blocked in 1 μ blocking buffer (Odyssey Blocking buffer, Licor Bioscience, Lincoln, NE) for 1 hour with shaking at room temperature followed by incubation with specific anti-pMAPK3/1 antibody (1:2000, Sigma), anti-pMAPK14 antibody (1:1000, Cell Signaling Technology, Danvers, MA), anti-pSMAD2 antibody (1:1000, Invitrogen), anti-total SMAD2 antibody (1:2000, Cell Signaling Technology, Danvers, MA), anti-pSMAD1/5/8 antibody (1:1000, Cell Signaling Technology, Danvers, MA), anti-pSMAD3 antibody (1:1000, Invitrogen) or μ-actin (ACTB) antibody (1:6000, Sigma) diluted in blocking buffer with 0.1% Tween-20 for 2-12 hours at room temperature. Following incubation, blots were washed three times for 10 minutes each with wash buffer (PBS, 0.1% Tween-20). Fluorescently labeled secondary antibodies (IRDye™ 800 anti-mouse or anti-rabbit, Rockland Immunochemicals, Gilbertsville, PA) were diluted at 1:5000 and incubated with the blots for 1 hour at room temperature. Blots were washed as above with an additional final wash in PBS without Tween-20. Detection was accomplished with an infrared scanner (Licor Bioscience, Lincoln, NE). Representative blots of three to four independent experiments are shown in Figs 2, 3, 4 and 10.

Ovaries from 12-day-old mice, 22-day-old mice primed with eCG (44 hours) and 22-day-old mice primed with eCG plus hCG (5 IU, 8 hours) were fixed overnight in 4% paraformaldehyde and embedded in paraffin wax. Ovarian sections were dewaxed in xylene (2 μ 5 minutes) followed by incubation for 5 minutes in each of the following: xylene:ethanol (1:1), 100% ethanol (2 μ), 95% ethanol, 85% ethanol, 75% ethanol and ddH₂O. Slides were then incubated in 1 μ antigen retrieval solution (DakoCytomation Retrieval solution) at 95°C for 25 minutes followed by washing with ddH₂O (2 μ) for 10 minutes and PBS (3 μ) for 5 minutes. Slides were then incubated in blocking buffer (PBS, pH 7.4, 3% BSA, 10% goat serum and 0.05% Triton X-100) for 1 hour at room temperature followed by incubation with anti-pSMAD2 (1:400, Invitrogen) diluted in blocking buffer for 12 hours at 4 C. Slides were then washed three times for 15 minutes with wash buffer (PBS 0.1% Triton-X 100), followed by incubation with anti-rabbit IgG Alexa Fluor 594 conjugate (1:1500, Molecular Probes) for 1 hour at room temperature, counterstained with DAPI and mounted with anti-fade solution (Slow-fade, Molecular Probes). Slides were imaged using in an Olympus BX60 upright fluorescent microscope connected to a 3CCD camera and computer.

Results from real-time PCR and immunoblot experiments were analyzed by two-way ANOVA followed by Tukeys HSD post-hoc test or Dunnetts HSD (Fig. 10B) if a positive F-test was detected. Results in Fig. 1 were analyzed by Student's t-test. The JMP 6.0 statistical analysis software was used for all analysis (SAS, Cary, NC). P<0.05 was considered statistically significant.

We thank Susan Ackerman, Robert Burgess and Mary Ann Handel for their helpful suggestions in preparing this manuscript. This work was supported by grant HD23839 from the NICHD (J.J.E.).