Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print December 27, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.057471
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
76/5/848    most recent
biolreprod.106.057471v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dragovic, R. A.
Right arrow Articles by Gilchrist, R. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dragovic, R. A.
Right arrow Articles by Gilchrist, R. B.
Agricola
Right arrow Articles by Dragovic, R. A.
Right arrow Articles by Gilchrist, R. B.
BIOLOGY OF REPRODUCTION 76, 848–857 (2007)
DOI: 10.1095/biolreprod.106.057471
© 2007 by the Society for the Study of Reproduction, Inc.

Oocyte-Secreted Factor Activation of SMAD 2/3 Signaling Enables Initiation of Mouse Cumulus Cell Expansion1

Rebecca A. Dragovic , Lesley J. Ritter , Samantha J. Schulz , Fred Amato , Jeremy G. Thompson , David T. Armstrong , and Robert B. Gilchrist 2

Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, Medical School, University of Adelaide, Adelaide, South Australia 5005, Australia

ABSTRACT

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.

activin, cumulus cells, growth factors, oocyte development, signal transduction

INTRODUCTION

Oocyte paracrine signaling to cumulus cells (CCs) is required for the mucification and expansion of the CCs surrounding the oocyte. Cumulus expansion is a highly coordinated process that occurs a few hours prior to ovulation and involves the production of a complex extracellular matrix (ECM) crucial for ovulation, fertilization and, hence, fertility [1]. Hyaluronan (HA), a nonsulphated glycosaminoglycan, forms the major structural backbone of the matrix and is synthesized by the enzyme hyaluronan synthase 2 (HAS2) [2, 3]. Other important components of the ECM include the HA-binding proteins tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and pentraxin 3 (PTX3). Both TNFAIP6- and PTX3-deficient mice synthesize normal amounts of HAS2, but they are infertile due to their inability to organize HA into a stable matrix [1, 4]. In addition, prostaglandin (PG) signaling is important for assembly of the CC matrix, as knockout mice for the PG rate-limiting enzyme, PG synthase 2 (PTGS2), are infertile, and knockouts for one of the PGE receptors (EP2) have reduced fertility, both due to cumulus expansion defects [5–7].

Expansion of the mouse cumulus-oocyte complex (COC) is critically dependent upon two signaling events: 1) stimulation by gonadotropins or epidermal growth factors (EGFs) and 2) paracrine signals secreted by the oocyte, termed the cumulus expansion-enabling factors (CEEFs), which act on its neighboring CCs, enabling these cells to respond to the aforementioned stimuli [8–11]. While the gonadotropin/EGF signal is now well described, the CEEFs are poorly understood and remain a controversial topic. In vivo, gonadotropin stimulation of cumulus expansion is clearly initiated by the midcycle LH surge. In vitro, when mouse COCs are treated with LH they fail to undergo cumulus expansion, as both CCs and oocytes have low to undetectable levels of LH receptors in the preovulatory follicle and therefore do not respond to direct LH stimulation [12]. Mural granulosa cells, however, express LH receptors, and it has recently been shown that the LH surge induces highly regulated expression of three EGF family members: amphiregulin, epiregulin, and betacellulin [13]. These EGF-like peptides then stimulate cumulus expansion in COCs via the EGF receptor, demonstrating that these molecules are the paracrine mediators transmitting the LH signal to the COCs in the ovarian follicle [13]. Cumulus expansion also can be mimicked in vitro by stimulation with FSH, cAMP analogs, and EGF [8, 14, 15], effects that are blocked by specific mitogen-activated protein kinase (MAPK) inhibitors, suggesting that the gonadotropin cascade is mediated in CCs through MAPK pathways [16, 17].

In addition to gonadotropin/EGF activation of the MAPK cascade, expansion of the mouse COC requires an oocyte-secreted paracrine signal. Microsurgical removal of the oocyte from the COC (generating an oocytectomized [OOX] complex) eliminates FSH-induced CC expansion, and expansion can be restored by coculturing these OOX complexes with denuded oocytes (DOs), demonstrating the secretion of and requirement for oocyte CEEFs [9]. Although the identities of the oocyte-secreted CEEFs remain controversial, it seems likely some combination of transforming growth factor beta (TGFB) superfamily molecules is responsible, as the CEEFs can be mimicked by TGFB1 or growth differentiation factor 9 (GDF9) [18–20]. Recently, oocyte-secreted bone morphogenetic protein 15 (BMP15) also has been implicated in the regulation of cumulus expansion via a mechanism requiring EGF receptor signaling [21, 22]. These studies suggest that oocyte paracrine signaling in CCs via the classical TGFB superfamily signaling cascades is required for cumulus expansion.

TGFB superfamily ligands bind as homodimers or heterodimers to their respective transmembrane-bound serine/threonine kinase type I receptor (activin receptor-like kinase [ALK]) or type II receptor, forming an oligomeric complex. The phosphorylated type I receptor then phosphorylates receptor-regulated SMADs (R-SMADs), which subsequently associate with the co-SMAD (SMAD4). This activated heterodimeric SMAD complex then translocates from the cytoplasm into the nucleus, where it regulates transcription of target genes. TGFB superfamily ligands signal through one of two distinct intracellular cascades—they either activate the SMAD 2/3 or the SMAD 1/5/8 signaling pathway, depending on which type I and type II receptors they use. The TGFBs, activins, GDF9, GDF8 (myostatin), and nodal all activate SMAD 2/3 signal transducers via their respective type II receptors and the recruitment and phosphorylation of ALK 4, 5, or 7 (reviewed in Drummond [23] and Harrison et al. [24]). Conversely, the BMPs activate SMAD 1/5/8 via the BMP type 2 receptor (BMPR2) and ALK 2, 3, or 6.

In the current study we hypothesize that TGFB superfamily signaling, specifically through the SMAD 2/3 pathway, is responsible for mediating the CEEF paracrine signals from the oocyte to CCs, which is required for the initiation of CC expansion. This hypothesis is formulated on the basis that mouse oocyte-secreted factors (OSFs) activate predominately SMAD 2/3 signaling molecules in granulosa cells and CCs [25], and that stimulation of this pathway by TGFB1 [18] or GDF9 [20] mimics the CEEFs and enables CC expansion. In this study we confirm that oocytes express the activins, and so we examined for the first time the role of activins A and B in enabling cumulus expansion, as these growth factors also signal through SMAD 2/3. Our results show that OSF-stimulated expression of Has2, Tnfaip6, Ptx3, and Ptgs2 in CCs is mediated by SMAD 2/3 and that mouse cumulus expansion requires OSF activation of SMAD 2/3 signaling.

MATERIALS AND METHODS

Unless specified, all chemicals and reagents were purchased from Sigma (St. Louis, MO).

Collection of COCs

This study was approved by Local Animal Ethics Committees and was conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Ovaries were collected 46 h after injecting 21- to 26-day-old 129/SV mice with 5 IU equine chorionic gonadotropin (eCG; Folligon; Intervet, Castle Hill, Australia). Ovaries were cleaned free of adherent adipose and connective tissues and placed in HEPES-buffered tissue cultured medium-199 (H-TCM-199; ICN Biomedicals Inc., Irvine, CA) supplemented with 0.1% (wt/vol) BSA (H-TCM-199/BSA). COCs were isolated by puncturing antral follicles with 27-gauge needles and were collected in H-TCM-199/BSA. Only COCs with a uniform covering of compacted CCs were used in this study.

Treatment of COCs and OOX Complexes

Stimulation of cumulus expansion. Cumulus expansion was stimulated either with FSH (50 mIU/ml recombinant human FSH; Puregon; Organon, Oss, The Netherlands) as the traditional hormone used to stimulate expansion in vitro [26], or with EGF (10 ng/ml recombinant human EGF; R&D Systems, Minneapolis, MN) to simulate mediators of the LH cascade [13].

Oocyte-secreted factors enabling cumulus expansion. To examine the participation of oocyte paracrine factors in cumulus expansion, native OSFs were eliminated by microsurgically removing the oocyte from COCs by oocytectomy using a micromanipulation apparatus on an inverted microscope as previously described by Buccione et al. [9]. To enable expansion, OOX complexes were cocultured with DOs or treated with various growth factors from the TGFB superfamily. DOs were generated by stripping COCs of their surrounding CCs by rapidly agitating COCs using a vortex mixer for approximately 4 min in 2 ml H-TCM-199/BSA. A total of 40 DOs were cultured per 50-µl drop, resulting in a DO concentration of 0.8/µl. Alternatively, OOXs were treated with the SMAD 2/3-activiating positive controls: recombinant human activin A, activin B (12.5–400 ng/ml; R&D Systems), or recombinant mouse GDF9 (145 ng/ml). The GDF9 was produced in house using a transfected 293 human embryonic kidney cell line and is partially purified by hydrophobic interaction chromatography, as previously described [27, 28]. This preparation of GDF9 has been used in a number of studies [20, 25, 27, 28], and the dose used for the current study was based on a dose-response experiment using this experimental model [20].

Oocyte-secreted factor antagonists. Attempts were made to antagonize recombinant and native oocyte-secreted activin A and B bioactivities using follistatin-288, which was generously donated by S. Shimasaki (University of California San Diego, San Diego, CA). In order to examine the TGFB superfamily signaling pathway used to enable cumulus expansion, oocyte paracrine factors were antagonized by treatment with the small molecule inhibitor, SB-431542 (generously donated by GlaxoSmithKline, Stevenage, UK). SB-431542 acts as competitive ATP binding site kinase inhibitor, potently antagonizing the activities of ALK5, ALK4, and ALK7 [29, 30]. SB-431542 has no effect on ALKs 1, 2, 3, and 6 and has very low affinity for any other cellular kinases, and so it is a highly specific ALK 4/5/7 kinase inhibitor [30]. Consequently, SB-431542 potently antagonizes the ALK 4/5 ligands—TGFB1, the activins, and GDF9—without affecting BMP signaling [25, 30]. We have recently demonstrated that SB-431542 completely antagonizes OSF activation of SMAD3 in granulosa cells, thereby ablating the proliferative effects of native OSFs and GDF9 on these cells [25]. Furthermore, treatment of intact COCs with SB-431542 does not adversely affect oocyte meiotic or cleavage potential [31]. A 10-mM stock solution of SB-431542 was prepared in dimethyl sulfoxide (DMSO) and diluted in culture medium prior to addition to culture drops such that the maximum concentration of DMSO was 0.04% (vol/vol).

Culture of COCs and OOX complexes and assessment of expansion. Complexes were cultured in 50-µl drops of Waymouth MB 752/1 medium (WAY; Sigma) supplemented with penicillin G (100 U/ml; JRH Biosciences, Lenexa, KS), streptomycin sulphate (100 mg/ml; JRH Biosciences), and 5% (vol/vol) fetal calf serum (FCS; Trace Biosciences, Castle Hill, Australia), with or without treatment reagents (see above), and were overlaid with mineral oil in Falcon Petri dishes (Becton Dickinson, Franklin Lakes, NJ). A total of 10 COCs or OOX complexes were cultured per 50-µl drop. Complexes were incubated for 20 h at 37°C, 96% humidity in 5% CO2 in air, followed by blinded assessment of morphological cumulus expansion to eliminate bias. Cumulus expansion was assessed according to a well-established subjective scoring system (0 to +4); in brief, score 0 indicates no expansion, and score +4 indicates maximum expansion [10]. A cumulus expansion index (0.0 to 4.0) was calculated as previously described [32].

CC apoptotic DNA assessment by TUNEL. To determine the effects of SB-431542 on CC viability, COCs were treated with SB-431542, and then CC apoptosis was quantified using TUNEL (Roche Diagnostic, Penzberg, Germany) and confocal microscopy, as recently described [33]. In brief, COCs were cultured in 50-µl microdrops of WAY supplemented with FCS and FSH, either alone or treated with SB-431542 (4 µM), for 6 h. Following culture, intact COCs were washed in PBS, fixed in paraformaldehyde, and then processed for TUNEL as previously described [33]. Positive controls were incubated in DNAse 1 and negative controls in the absence of terminal deoxynucleotidyl transferase. Following TUNEL, the proportion of apoptotic CCs was quantified individually in each COC using confocal microscopy and IPLab software (Scanalytics, Fairfax, VA), as described in detail [33]. In brief, three optical Z sections were acquired for each COC in both a green channel (apoptotic) and a red channel (total DNA), the proportion of apoptotic cells was determined in each section, and then an average was generated from the three sections for each COC. This was repeated for a minimum of 30 COCs in each of the two treatment groups.

RT-PCR of Oocytes

RNA isolation. COCs were collected from mouse ovaries and examined for mRNA expression of the two activin subunits, Inhba and Inhbb, and Gdf8. Mouse ovary and skeletal muscle were collected as positive tissue samples. COCs were collected in H-TCM-199/BSA and denuded of surrounding CCs by mouth pipetting. DOs then were washed three times in H-TCM-199/BSA and transferred to Eppendorf tubes (40 DOs per tube) on ice. The RNA from mouse ovary, skeletal muscle, and DOs was extracted using a micro RNA isolation kit (Qiagen, Doncaster, Australia) as described previously [20].

RT-PCR analysis. RNA was reverse transcribed using random primers (Boehringer Mannheim, Mannheim, Germany) and a Superscript II RT kit (Life Technologies Inc., Grand Island, NY) according to the manufacturer's instructions. A negative RT control substituting water for reverse transcriptase was included in each experiment. Primer pairs for Inhba, Inhbb, and Gdf8 were designed using Primer Express software (PE Applied Biosystems, Foster City, CA), and synthesized by Geneworks (Adelaide, Australia). Actβ primers were generously donated by S. Robertson (University of Adelaide, Adelaide, Australia). Primer pair sequences are listed in Table 1. Each PCR reaction sample consisted of 2.5 µl Qiagen 10x buffer, 1 mmol/l MgCl2, 0.4 mmol/l each of dATP, dCTP, dGTP, and dTTP, 0.5 units HotStarTaq DNA polymerase, 10 pmol of each primer, and 3 µl diluted cDNA sample (1:5), and was made up to a final volume of 25 µl with ultra-pure water (Biotech, Bentley, Australia). Water was substituted for cDNA in the no-template control sample included in each PCR run. PCR samples were treated at 94°C for 5 min, followed by 40 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 1 min, and a final extension step of 72°C for 7 min. PCR products were analyzed by electrophorsis by running a 2% (wt/vol) agarose gel containing 15 mg ethidium bromide (Boehringer) in Tris borate EDTA buffer (TBE) and visualized with a Kodak 120 digital camera over an ultraviolet light box. PCR product size was determined by comparison with HpaII-digested pUC19. The identities of the Inhba, Inhbb, and Gdf8 PCR products were confirmed by sequencing.


View this table:
[in this window]
[in a new window]
[Download PPT slide]
  		TABLE 1. Primer sets used for RT-PCR and real time RT-PCR.

Real-Time RT-PCR for CC Matrix Transcripts

Experimental design. OOX complexes were cultured in 50-µl microdrops of WAY supplemented with 5% (vol/vol) FCS + FSH (50 mIU/ml) for 6 h in one of the following treatments: 1) no treatment (control), 2) GDF9 (250 ng/ml), 3) GDF9 + SB-431542 (4 µM), 4) activin A (200 ng/ml), 5) activin A + SB-431542 (4 µM), 6) oocytes (0.8/µl), or 7) oocytes + SB-431542 (4 µM). A total of 10 OOX complexes were cultured per treatment group, each treatment group was cultured in quadruplicate, and the experiment was replicated on five separate occasions.

Real-time RT-PCR analysis. After the 6-h incubation, OOX complexes were collected for RNA isolation as previously described [20]. Primer pairs for each transcript were designed using Primer Express software and were synthesized by Geneworks. The sequences for Rpl19, Has2, Ptgs2, Tnfaip6, and Ptx3 primer pairs are listed in Table 1. PCR conditions were the same as previously described [20], with the exception that 80 ng RNA was reverse transcribed and gene expression was calculated using the standard curve method. Standard curves were generated by serial dilution of COC cDNA. Critical threshold (CT) values for each given sample were within the range of the standard curve for each gene of interest. Gene expression was calculated for each sample, then normalized to the housekeeping gene, Rpl19. Finally, PCR products were run on a 2% (wt/vol) agarose gel for confirmation of single, correctly sized products, and the identity of each PCR product was confirmed by sequencing.

Immunoblot Analysis of Phosphorylated Smad2 Protein

Experimental design. COCs were collected in H-TCM-199/BSA supplemented with 4 µM SB-431542, washed thoroughly in inhibitor-free medium, and then cultured in WAY supplemented with 5% (vol/vol) FCS + FSH (50 mIU/ml) alone or treated with an increasing dose of SB-431542 (1–4 µM). Ninety complexes per treatment were cultured in 250 µl for 1 h in Nunc four-well dishes (Nalge Nunc International, Roskilde, Denmark). At the end of the culture period, COCs were collected, washed once in cold PBS, and then stored at –80°C as cell pellets. This experiment was replicated on three separate occasions.

Smad2 Western blot analysis. Thawed complexes were mixed with loading buffer containing 100 mM dithiothreitol (DTT) and subjected to SDS-PAGE (10% polyacrylamide gel). Proteins were subsequently electrotransferred to nitrocellulose membranes (Hybond-ECL; Amersham Life Science) in 25 mM Tris, 19·2 mM glycine containing 20% methanol. Blots were blocked in 20 mM Tris (pH 7.6) containing 137 mM NaCl, 0.1% Tween-20, and 2% blocking agent (provided in the ECL Advance Kit) for 1 h at room temperature, then incubated overnight with an anti-phospho-Smad2 rabbit polyclonal antibody (1:10 000; Chemicon International, Temecula, CA; generously donated by Ann Drummond, Prince Henry's Institute, Melbourne, Australia) at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit antibody (1:200 000; Chemicon International) and detected using the sensitive Enhanced Chemiluminescence (ECL) Advance system (Amersham Biosciences).

Data Analyses

Each experiment was performed three to five times (see figure legends). Treatment effects on cumulus expansion were examined using a Kruskal-Wallis one-way ANOVA on ranks, and differences between means were detected using Dunn method posthoc comparisons or t-tests. Real-time RT-PCR data were log transformed, and treatment effects were examined using a one-way ANOVA followed by Tukey comparisons. A P value of <0.05 was considered statistically significant.

RESULTS

Oocyte Expression of Activin A and Activin B mRNA

We used conventional RT-PCR to determine which of the TGFB superfamily members that signal through SMAD 2/3 are expressed in oocytes. Nodal is not expressed in the oocyte [34], and it is well known that oocytes express GDF9 and TGFB1/B2, but there are conflicting reports as to whether mouse oocytes express the Inhba and Inhbb subunits constituting the activins [35, 36]. Inhba and Inhbb mRNA transcripts were detected in mouse oocytes (Fig. 1). We also examined oocyte mRNA expression of Gdf8 (myostatin), as it also activates the common SMAD 2/3 signaling pathway [37]. Gdf8 mRNA transcripts were not detected in oocytes (Fig. 1).


Figure 01
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1. Expression of inhibin subunits and myostatin mRNA in oocytes. COCs were collected and CCs and oocytes were manually separated by careful mouth pipetting. RNA was extracted from 50 oocytes, reverse transcribed, and amplified by PCR. +ve, positive tissue sample (mouse ovary for Actβ, Inhba, and Inhbb; mouse skeletal muscle for Gdf8); -ve RT, no reverse transcriptase; NTC, no template control (water substituted for cDNA template). Ladder, HpaII-digested pUC19.

Activin A and Activin B Enable Cumulus Expansion

Although the role of activins in the regulation of cumulus expansion is unknown, the activins are related to and use a common intracellular signaling pathway to TGFB and GDF9, both of which can enable cumulus expansion [18, 20]. To examine the effect of activins A and B on cumulus expansion, OOX complexes were treated with FSH and cultured in the presence of an increasing dose of activin A (Fig. 2A) or activin B (Fig. 2B). FSH-treated OOX complexes underwent expansion (P < 0.05), induced by activin A, in a dose-dependent manner. In contrast, FSH-treated OOX complexes cultured without activin failed to expand. Activins A and B mimicked the actions of native OSFs (oocytes in coculture) and GDF9 in enabling CC expansion to a level comparable to intact COCs.


Figure 02
View larger version (28K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2. Effect of activins A and B on cumulus expansion and of follistatin on oocyte-induced expansion. A and B) COCs and OOX complexes were cultured in media supplemented with 50 mIU/ml rhFSH and 5% FCS, and OOX complexes were treated with oocytes (0.8/µl), GDF9 (145 ng/ml), and an increasing dose (12.5–400 ng/ml) of activin A (A) or activin B (B). Asterisk indicates significant difference to OOX alone (P < 0.05). C and D) OOX complexes cocultured with oocytes (0.8/µl) and treated with either 200 ng/ml activin A (C) or 200 ng/ml activin B (D) were all treated with an increasing dose of follistatin-288 (400–1000 ng/ml). Negative controls included OOX complexes alone or OOX complexes treated with activin A or activin B together with SB-431542 (4 µM). Means within a line graph with different lowercase letters are significantly different (P < 0.05). Asterisks indicate the two means at that dose of follistatin are significantly different (P < 0.05). After 20 h of culture, the degree of cumulus expansion was assessed using the subjective scoring system, 0 (no expansion) to +4 (maximal expansion), and the cumulus expansion index calculated. Results show the mean ± SEM of three individual experiments, each with a total of 10 complexes (OOX or COC) per treatment group.

Neutralization of Activins A and B Does Not Prevent Oocyte-Induced Cumulus Expansion

Having discovered that exogenous activins A and B enable cumulus expansion, we next assessed whether oocyte-secreted activin A and/or B contribute essential components of the mouse CEEF. A functional neutralization experiment was designed using the native activin-binding protein, follistatin, which is a well-characterized activin antagonist (reviewed in Harrison et al. [24]). Activin A- and B-induced cumulus expansions were significantly (P < 0.05) ablated when treated with follistatin-288 or the ALK 4/5/7 inhibitor, SB-431542 (Fig. 2, C and D). In contrast, follistatin-288 had no significant (P > 0.05) effect on cumulus expansion induced by oocytes (Fig. 2, C and D). Failure of follistatin-288 to neutralize oocyte-induced cumulus expansion was not due to insufficient antagonist. In a separate experiment, increasing the dose of follistatin-288 to 2 µg/ml also did not prevent oocyte-induced cumulus expansion (mean ± SEM: 3.1 ± 0.1).

Requirement of SMAD 2/3 Signaling for CC Expansion

It is now known that FSH and EGF stimulation of cumulus expansion is mediated by MAPK signaling in CCs [16, 17, 38], and mouse cumulus expansion also requires signaling by the oocyte-secreted CEEFs, but the signaling pathway(s) for the latter is unknown. To characterize TGFB superfamily signaling pathways involved in the regulation of cumulus expansion, the ALK 4/5/7 inhibitor, SB-431542, was used to determine the involvement of SMAD 2/3. First, the capacity of SB-431542 to block SMAD2 phosphorylation in intact COCs was examined by Western blot. FSH-stimulated COCs exhibited activated SMAD2 phosphorylation, which was dose dependently decreased by treatment with SB-431542 (Fig. 3A), although it was not completely eliminated at the highest dose. To exclude the possibility of toxic effects of SB-431542, we next evaluated CC viability by quantifying the apoptotic incidence following a 6-h exposure to SB-431542. Treatment of COCs with SB-431542 did not alter the incidence of CC apoptosis (Fig. 3B).


Figure 03
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3. Effect of the ALK 4/5/7 inhibitor SB-431542 on COCs. A) Effect of SB-431542 on SMAD2 phosphorylation in COCs. Groups of 90 FSH-stimulated COCs were cultured for 1 h alone or treated with an increasing dose of SB-431542 (1–4 µM). Proteins were subjected to 10% SDS-PAGE and Western blotting using a phospho-SMAD2 antibody. Each lane contains an equal number (90) of COCs. B) Effect of SB-431542 on CC apoptosis. FSH-stimulated COCs (15–20 per treatment per experiment) were cultured alone or treated with SB-431542 (4 µM) for 6 h followed by assessment of apoptosis by TUNEL. CC apoptosis was unaffected by treatment with SB-431542. Columns represent the average (±SEM) percentage of apoptotic CCs within individual COCs.

Oocytes and GDF9 stimulated cumulus expansion of OOX complexes, which in turn were both neutralized by SB-431542 in a dose-dependent manner (Fig. 4). Oocyte-induced cumulus expansion was completely abolished at 2 µM SB-431542. The SB-431542 carrier, DMSO, at a vol/vol dose equivalent to 4 µM SB-431542, had no significant (P > 0.05) effect on GDF9- or ooctye-induced cumulus expansion (Figs. 4 and 5A). SB-431542 also completely abolished cumulus expansion of intact COCs, whether stimulated to expand by FSH (Fig. 5A) or EGF (Fig. 5C). To determine whether the prevention of cumulus expansion by SB-431542 is reversible, an experiment was conducted in which FSH-stimulated COCs were exposed to SB-431542 for just 6 h, washed free of the inhibitor, and then returned to control culture medium for a further 14 h (Fig. 5B). The 6-h time point was chosen, as the results in Figure 6 demonstrate that ECM genes are substantially upregulated from 0–6 h of culture and that this is prevented by SB-431542. After removal from the inhibitor and 14 h of culture, these COCs had undergone cumulus expansion, although the overall degree of expansion was somewhat reduced compared with that of the controls (Fig. 5B).


Figure 04
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4. Effect of the ALK 4/5/7 inhibitor SB-431542 on GDF9- and OSF-induced cumulus expansion. FSH-treated OOX complexes were cultured with either oocytes (0.8/µl) or GDF9 (145 ng/ml) and treated with an increasing dose of SB-431542 (0.5–4 µM). The SB-431542 carrier, DMSO, at a vol/vol dose equivalent to 4 µM SB-431542, did not affect cumulus expansion. The degree of cumulus expansion was determined after 20 h of culture using a scale of 0 (no expansion) to +4 (maximal expansion). Means within a line graph with different lowercase letters (a–d, x–z) are significantly different (P < 0.05). Asterisks indicate the two means at that dose of antagonist are significantly different (P < 0.05).


Figure 05
View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5. Effect of the ALK 4/5/7 inhibitor SB-431542 on FSH- and EGF-stimulated expansion of intact COCs. FSH-stimulated (A) or EGF-stimulated (C) COCs were cultured alone or treated with an increasing dose of SB-431542 (0.5–4 µM). The degree of cumulus expansion was determined after 20 h of culture using a scale of 0 (no expansion) to +4 (maximal expansion). Means within a line graph with different superscript letters are significantly different (P < 0.05). COCs without EGF stimulation were cultured as a negative control (C). The SB-431542 carrier, DMSO, at a vol/vol dose equivalent to 4 µM SB-431542, did not affect cumulus expansion (A). B) FSH-stimulated COCs were either cultured alone for 20 h, treated with SB-431542 for 20 h, or exposed to SB-431542 for just 6 h, then washed and returned to control medium for 14 h. Cumulus expansion was assessed after 20 h of culture. Bars with different superscript letters are significantly different (P < 0.05).


Figure 06
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6. Effect of the ALK 4/5/7 inhibitor SB-431542 on CC Has2, Ptx3, Tnfaip6, and Ptgs2 mRNA expression. OOX complexes (40 per treatment per experiment) were cultured for 6 h in media supplemented with 50 mIU/ml rhFSH and 5% FCS. Real-time RT-PCR analysis was performed using primer sets for Has2, Ptx3, Tnfaip6, Ptgs2, and Rpl19, using RNA from CCs (OOX) cultured alone (control), treated with GDF9 (145 ng/ml) or activin A (200 ng/ml), or cocultured with DOs (0.8/µl), either in the absence or presence of 4 µM SB-431542. The levels of Has2, Ptx3, Tnfaip6, and Ptgs2 mRNA were normalized to Rpl19. Columns are means ± SEM of five independent experiments and are expressed as a fold change from mRNA levels in OOX complexes cultured alone (control; set to a value of 1). Asterisks denote a significant effect of SB-431542 for that treatment (P < 0.05).

To confirm that the cumulus expansion-inhibiting effects of SB-431542, as assessed above by morphological criteria, are actually caused by inhibition of expression of matrix genes, we examined CC Has2, Tnfaip6, Ptgs2, and Ptx3 mRNA expression using real-time RT-PCR. FSH-treated OOX complexes were cultured alone, treated with GDF9 or activin A, or cocultured with oocytes, in the presence or absence of 4 µM SB-431542 for 6 h. CC Has2, Tnfaip6, Ptgs2, and Ptx3 all were upregulated notably by GDF9 and oocytes, and to a lesser extent by activin A, compared with the control (Fig. 6). The ALK 4/5/7 inhibitor, SB-431542, significantly (P < 0.05) antagonized activin A- and oocyte-induced OOX mRNA expression of all four genes. SB-431542 also significantly (P < 0.05) antagonized GDF9-induced OOX Has2 and Ptgs2 mRNA expression. These results verify that the dramatic morphological changes in cumulus expansion observed after treatment with SB-431542 correlate with functional changes in the expression of key matrix genes regulating expansion.

DISCUSSION

The release of a fertilizable oocyte from the ovary is dependent upon expansion of the CCs. Formation of the ECM surrounding the oocyte requires both gonadotropin stimuli and unidentified OSFs, the CEEFs, which most likely are a combination of growth factors belonging to the TGFB superfamily. In vitro, stimulation of COC expansion is most commonly achieved by treatment with FSH or EGF. Recently, Diaz et al. [16] demonstrated that both FSH and EGF activate MAPK3/1 (formerly ERK1/2) and MAPK14 (formerly p38) in CCs, a process required for cumulus expansion. However, it is well known that an oocyte paracrine signal also is required to enable gonadotropin/EGF-stimulated expansion. We have previously demonstrated that both TGFB1 and GDF9 can mimic the oocyte CEEF and enable FSH-induced cumulus expansion, but neither one alone nor the two together accounts for the mouse CEEF [20]. More recently, we have shown that OSFs primarily activate the SMAD 2/3 signaling cascade to stimulate granulosa cell proliferation [25]. This led us to hypothesize that TGFB superfamily signaling via the SMAD 2/3 pathway is responsible for mediating the CEEF paracrine signal from the oocyte to CCs, enabling expansion.

Results from this study provide evidence that the CEEFs signal through the SMAD 2/3 pathway and that this is essential for the induction of cumulus expansion. We used an ALK 4/5/7 inhibitor, SB-431542, to completely neutralize GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. In addition, expression of CC genes involved in the formation of the ECM, including Has2, Tnfaip6, Ptx3, and Ptgs2, was substantially attenuated by treatment with SB-431542. Our results also demonstrate that the activin subunits are expressed in the oocyte and that activins A and B can mimic the actions of the oocyte CEEFs and enable FSH-induced cumulus expansion.

Intracellular SMAD proteins mediate the signal transduction of TGFB superfamily ligands, which control cell proliferation, differentiation, and apoptosis [39, 40]. Activation of specific SMAD proteins is dependent on which type I and type II receptors the ligands use. SMAD 2/3 is activated by ligands that signal through ALK 4, 5, or 7, whereas SMAD 1/5/8 is activated by molecules (mainly BMPs) that use ALK 2, 3, or 6 [39, 40]. Using SMAD reporter constructs and Western blotting, we have very recently demonstrated that OSFs activate granulosa cell SMAD 2/3 [25]; however, prior to the current study, it was unknown whether the SMAD signaling pathways play a role in the regulation of cumulus expansion. In this study we have used the small molecule inhibitor, SB-431542, in the cumulus expansion assay. SB-431542 acts as a competitive ATP binding site kinase inhibitor that is highly specific for ALKs 4, 5, and 7, the kinases that activate SMAD 2/3 [30]. Accordingly, SB-431542 prevents oocyte-induced activation of granulosa cell SMAD 2/3 phosphorylation and oocyte-stimulated granulosa cell proliferation [25], and it substantially reduces SMAD2 phosphorylation in intact COCs, as shown here. Our current results demonstrate that FSH- and EGF-stimulated COC expansions were eliminated by the kinase inhibitor. In addition, SB-431542 effectively inhibited FSH-stimulated recombinant GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. These data demonstrate that oocyte paracrine signaling through the SMAD 2/3 pathway in CCs is required for mouse cumulus expansion.

The CC morphological changes that were observed after treatment with the ALK 4/5/7 inhibitor were supported by changes in CC Has2, Tnfaip6, Ptx3, and Ptgs2 gene expression, all of which are necessary for cumulus expansion. Our results demonstrate that GDF9, activin A, and oocytes can enable FSH-stimulated CC Has2, Tnfaip6, Ptx3, and Ptgs2 mRNA expression. Oocyte-induced expression of all four genes was substantially reduced by treatment with SB-431542, and while not completely eliminated, expression levels were reduced to those of the controls (OOXs, which do not expand). Hence, these gene expression data support the morphological observations and demonstrate that oocyte activation of SMAD 2/3 is required for FSH-stimulated CC Has2, Tnfaip6, Ptx3, and Ptgs2 mRNA expression.

The kinase inhibitor SB-431542 was originally characterized as a specific inhibitor of ALK5 [29]. Because the kinase domains of ALK4 and ALK7 are similar to that of ALK5, the inhibitor was later tested and described as a specific inhibitor of ALKs 4, 5, and 7 [30]. The effect of SB-431542 also was tested on the activities of ALKs 1, 2, 3, and 6, along with various other protein kinases. The results clearly demonstrated that 10 µM SB-431542 had no significant effect on the kinase activities of ALK 1 or 2; neither did it affect components of the ERK, JNK, or MAP kinase signaling pathways [30]. ALKs 3 and 6 and the p38{alpha} MAPK signaling pathways were weakly affected, but only when using SB-431542 at a concentration of 10 µM [30]. Accordingly, we also have recently demonstrated that SB-431542 does not affect BMP activation of SMAD1 in granulosa cells [25]. Hence, SB-431542 is specific for ALK 4/5/7 when used at <10 µM, and it is important to note that in the current study the highest concentration used was 4 µM, suggesting that the antagonistic effects of SB-431542 on CC expansion are indeed due to antagonism specifically of ALK 4/5/7 and not that of other protein kinases. In a previous study we showed that doses as low as 0.5 µM SB-431542 were sufficient to block oocyte-induced activation of a SMAD3-responsive reporter construct [25]; however, in the current study there was still some SMAD2 phosphorylation in intact COCs exposed to 2 or 4 µM (doses that prevented cumulus expansion). The significance of this low-level SMAD2 activity is unclear, although it may be at a level too low to allow cumulus expansion to proceed.

A concern that may arise when using inhibitors is cell toxicity of the kinase inhibitor itself or the carrier employed, in this case DMSO. To exclude the possibility of CC toxicity, we examined the effect of SB-431542 on CC apoptosis by TUNEL and found it did not increase CC death. Furthermore, we also conducted a washout experiment in which COCs were exposed to SB-431542 for 6 h, followed by 14 h without the inhibitor. Our real-time RT-PCR results demonstrate that this 6-h exposure to SB-431542 is sufficient to completely prevent expression of the key matrix genes required for oocyte-induced expansion. The antagonistic effects of SB-431542 on CC expansion were reversible, as these COCs had reinitiated cumulus expansion after 14 h of inhibitor-free culture. Together, these results demonstrate that the kinase inhibitor has no overt adverse effect on cell viability, and this is consistent with our recent observation that COCs treated with SB-431542 during oocyte maturation have normal meiotic and cleavage potential [31].

Growth factors identified to date that can mimic the oocyte and enable cumulus expansion are all TGFB superfamily members. With the exception of BMP15 [22], which activates SMAD 1/5/8, all other family members that can enable CC expansion are those that activate SMAD 2/3 [18–20], consistent with the main finding of this study that the CEEFs act through SMAD 2/3 to initiate cumulus expansion. In this study we examined additional members of the TGFB superfamily that activate SMAD 2/3 as candidate molecules that might contribute to the CEEFs. The TGFB superfamily members that activate SMAD 2/3 are TGFB1, TGFB2, TGFB3, activin A, activin B, GDF9, nodal, and GDF8 (reviewed in Drummond [23] and Harrison et al. [24]). Of these growth factors, GDF9 [20] and the TGFBs [18, 19] have been studied in terms of their roles in cumulus expansion, nodal is not expressed in the oocyte [34] and, using RT-PCR, the current results demonstrate that mouse oocytes express both Inhba and Inhbb subunits but do not express Gdf8. Previous reports have been contradictory as to oocyte Inhba and Inhbb expression [35, 36]. Activins are secreted as homodimers or heterodimers of the inhibin β subunits, and activins use the type I receptor, ALK4, to activate the SMAD 2/3 signaling pathway [40].

We next went on to investigate the effect of activin A and activin B on cumulus expansion. This study demonstrates for the first time that both activin A and activin B enable FSH-induced cumulus expansion of mouse OOX complexes. Further studies are required to demonstrate whether or not oocytes actually secrete dimeric, biologically active activin(s); however, it is perhaps noteworthy that the activin-binding protein, follistatin, effectively inhibited activin A- and activin B-induced cumulus expansion but did not antagonize oocyte-induced cumulus expansion. Hence, activin A and activin B appear to act in the same manner as TGFB1 and GDF9 [18, 20]: all activate SMAD 2/3 and can mimic the paracrine actions of the oocyte and enable cumulus expansion, but none of these, in isolation at least, account for the CEEF activity of oocytes. In support of this, activin βB knockout mice are fertile [41], implying that these mice have normal cumulus expansion.

Given that: 1) OSF activation of SMAD 2/3 is required for cumulus expansion; 2) TGFBs, GDF9, and activins all signal through SMAD 2/3; 3) all of these growth factors enable FSH-stimulated cumulus expansion; and yet 4) none of them account for the CEEF in isolation—then it seems apparent that the CEEFs consist of some combination of these factors and possibly others, cooperatively enabling CC expansion. This hypothesis might seem inconsistent with the notion that oocyte-secreted BMP15 plays an important role in CC expansion [21, 22, 42], as BMP15 signals through the SMAD 1/5/8 pathway and probably not through SMAD 2/3 [43]. BMP15-null mice are subfertile, displaying decreased ovulation and fertilization rates as well as a decrease in the expression of CC Has2, and this is compounded on a GDF9 heterozygous background [42]. These characteristics imply some sort of GDF9/BMP15 interaction in the regulation of cumulus expansion. This could be manifested in many ways and may include the involvement of a putative GDF9/BMP15 heterodimer, which modeling has predicted could activate SMAD 2/3 and SMAD 1/5/8 [44]. Furthermore, Yi et al. [45] found that ALK6-deficient female mice are infertile, primarily due to defects in cumulus expansion.

Yoshino et al. [22] recently demonstrated that recombinant human BMP15 stimulates expansion of intact mouse COCs in vitro without the need for exogenous FSH or EGF. In this scenario, it appears the obligatory activation of MAPK required for CC expansion may come from an EGF signal generated within the COC, as BMP15-stimulated COC expression of the EGF-like peptides, epiregulin, amphiregulin and betacellulin, and BMP15-stimulated expansion was blocked by an EGF receptor antagonist [22]. Another significant observation from Yoshino et al. [22] is that mouse oocytes do not appear to express processed (bioactive) BMP15 prior to the preovulatory gonadotropin surge, nor do they for at least the first 5 h after hCG treatment, by which time CC expansion is well underway, as evidenced in this study by expression of the key ECM transcripts within 6 h. Oocyte BMP15 expression is upregulated by the gonadotropins [21], and functional, mature BMP15 protein only appears in the oocyte 9 h after hCG treatment [22], suggesting that mouse oocytes first secrete BMP15 toward the end of oocyte maturation. If this proves to be the case, then it seems unlikely that BMP15 signaling through SMAD 1/5/8 could contribute to the oocyte signal(s) enabling the initiation of expansion. This is consistent with our current results, including the observation that SB-431542 prevented EGF-stimulated COC expansion, and that follistatin, which is an activin- and BMP15-binding protein [46], did not block oocyte-induced cumulus expansion. Hence, while OSF activation of SMAD 2/3 appears obligatory for the initiation of expansion, BMP15 signaling may play a key role in organization of the ECM later in maturation and during ovulation.

In conclusion, the results of this study provide evidence to support the hypothesis that the initiation of cumulus expansion requires oocyte-secreted factor activation of the SMAD 2/3 pathway in CCs (see the model in Fig. 7). Oocyte activation of SMAD 2/3 does not stimulate CC expansion in and of itself, but instead enables FSH or EGF-like peptides to do so. FSH/EGF stimulate cumulus expansion through MAPK, and it has been postulated that an oocyte-secreted paracrine factor is required to enable FSH activation of CC MAPK [16, 17, 38]. The results of the current study support and extend this hypothesis, whereby the oocyte-secreted CEEFs activate SMAD 2/3 signaling in the CCs, and this then initiates an obligatory signal to the MAPK pathway that enables FSH/EGF to activate MAPK and eventually stimulate cumulus expansion (Fig. 7). Further studies are required to determine the interaction between SMAD 2/3 and SMAD 1/5/8 signaling after the initiation of cumulus expansion and during ovulation.


Figure 07
View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 7. Model depicting the two signaling pathways controlling the initiation of cumulus expansion. Initiation of mouse cumulus expansion requires two signaling events: oocyte-secreted paracrine factors act on CCs, thereby enabling CCs to respond to stimulation by FSH (in vitro) or the gonadotropin-induced EGF cascade (in vivo). FSH and EGF both activate the MAPK signaling cascade. The major finding of this study is that the oocyte-secreted CEEFs signal through the SMAD 2/3 pathway to enable FSH- or EGF-stimulated expansion. The SMAD 2/3 and MAPK signaling pathways probably cross talk to upregulate the expression of various important CC ECM genes, leading to the formation of a stable and expanded cumulus matrix.

ACKNOWLEDGMENTS

SB-431542 was generously donated by GlaxoSmithKline, and we are grateful to David Froiland for undertaking the TUNEL assessment, to Ann Drummond (Prince Henry's Institute, Melbourne, Australia) for donating the SMAD2 antibody, and to Shunichi Shimasaki (University of California San Diego) for the follistatin.

FOOTNOTES

1Supported by a National Health and Medical Research Council (NHMRC) of Australia Program Grant and the Research Centre for Reproductive Health. R.A.D. was supported by the Faculty of Health Sciences, Adelaide University, and The Queen Elizabeth Hospital Research Foundation. Back

Correspondence: 2FAX: 61 08 8303 8177; e-mail: robert.gilchrist{at}adelaide.edu.au

Received: 19 September 2006.

First decision: 20 October 2006.

Accepted: 26 December 2006.

REFERENCES

  1. Fulop C, Szanto S, Mukhopadhyay D, Bardos T, Kamath RV, Rugg MS, Day AJ, Salustri A, Hascall VC, Glant TT, Mikecz K. Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice. Development 2003; 130:2253–2261[Abstract/Free Full Text]
  2. Eppig JJ. FSH stimulates hyaluronic acid synthesis by oocyte-cumulus cell complexes from mouse preovulatory follicles. Nature 1979; 281:483–484[CrossRef][Medline]
  3. Fulop C, Salustri A, Hascall VC. Coding sequence of a hyaluronan synthase homologue expressed during expansion of the mouse cumulus-oocyte complex. Arch Biochem Biophys 1997; 337:261–266[CrossRef][Medline]
  4. Salustri A, Garlanda C, Hirsch E, De Acetis M, Maccagno A, Bottazzi B, Doni A, Bastone A, Mantovani G, Peccoz PB, Salvatori G, Mahoney DJ, et al. PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization. Development 2004; 131:1577–1586[Abstract/Free Full Text]
  5. Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC, Langenbach R. Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1beta. Endocrinology 1999; 140:2685–2695[Abstract/Free Full Text]
  6. Matsumoto H, Ma W, Smalley W, Trzaskos J, Breyer RM, Dey SK. Diversification of cyclooxygenase-2-derived prostaglandins in ovulation and implantation. Biol Reprod 2001; 64:1557–1565[Abstract/Free Full Text]
  7. Hizaki H, Segi E, Sugimoto Y, Hirose M, Saji T, Ushikubi F, Matsuoka T, Noda Y, Tanaka T, Yoshida N, Narumiya S, Ichikawa A. Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP(2). Proc Natl Acad Sci U S A 1999; 96:10501–10506[Abstract/Free Full Text]
  8. Salustri A, Yanagishita M, Hascall VC. Mouse oocytes regulate hyaluronic acid synthesis and mucification by FSH-stimulated cumulus cells. Dev Biol 1990; 138:26–32[CrossRef][Medline]
  9. Buccione R, Vanderhyden BC, Caron PJ, Eppig JJ. FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte. Dev Biol 1990; 138:16–25[CrossRef][Medline]
  10. Vanderhyden BC, Caron PJ, Buccione R, Eppig JJ. Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev Biol 1990; 140:307–317[CrossRef][Medline]
  11. Eppig JJ, Wigglesworth K, Chesnel F. Secretion of cumulus expansion enabling factor by mouse oocytes: relationship to oocyte growth and competence to resume meiosis. Dev Biol 1993; 158:400–409[CrossRef][Medline]
  12. Eppig JJ, Wigglesworth K, Pendola F, Hirao Y. Murine oocytes suppress expression of luteinizing hormone receptor messenger ribonucleic acid by granulosa cells. Biol Reprod 1997; 56:976–984[Abstract]
  13. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 2004; 303:682–684[Abstract/Free Full Text]
  14. Downs SM and Hunzicker-Dunn M. Differential regulation of oocyte maturation and cumulus expansion in the mouse oocyte-cumulus cell complex by site-selective analogs of cyclic adenosine monophosphate. Dev Biol 1995; 172:72–85[CrossRef][Medline]
  15. Downs SM. Specificity of epidermal growth factor action on maturation of the murine oocyte and cumulus oophorus in vitro. Biol Reprod 1989; 41:371–379[Abstract]
  16. Diaz FJ, O'Brien MJ, Wigglesworth K, Eppig JJ. The preantral granulosa cell to cumulus cell transition in the mouse ovary: development of competence to undergo expansion. Dev Biol 2006; 299:91–104[CrossRef][Medline]
  17. Su YQ, Wigglesworth K, Pendola FL, O'Brien MJ, Eppig JJ. Mitogen-activated protein kinase activity in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse. Endocrinology 2002; 143:2221–2232[Abstract/Free Full Text]
  18. Salustri A, Ulisse S, Yanagishita M, Hascall VC. Hyaluronic acid synthesis by mural granulosa cells and cumulus cells in vitro is selectively stimulated by a factor produced by oocytes and by transforming growth factor-beta. J Biol Chem 1990; 265:19517–19523[Abstract/Free Full Text]
  19. Vanderhyden BC, Macdonald EA, Nagyova E, Dhawan A. Evaluation of members of the TGFbeta superfamily as candidates for the oocyte factors that control mouse cumulus expansion and steroidogenesis. Reprod Suppl 2003; 61:55–70[Medline]
  20. Dragovic RA, Ritter LJ, Schulz SJ, Amato F, Armstrong DT, Gilchrist RB. Role of oocyte-secreted growth differentiation factor 9 in the regulation of mouse cumulus expansion. Endocrinology 2005; 146:2798–2806[Abstract/Free Full Text]
  21. Gueripel X, Brun V, Gougeon A. Oocyte bone morphogenetic protein 15, but not growth differentiation factor 9, is increased during gonadotropin-induced follicular development in the immature mouse and is associated with cumulus oophorus expansion. Biol Reprod 2006; 75:836–843[Abstract/Free Full Text]
  22. Yoshino O, McMahon HE, Sharma S, Shimasaki S. A unique preovulatory expression pattern plays a key role in the physiological functions of BMP-15 in the mouse. Proc Natl Acad Sci U S A 2006; 103:10678–10683[Abstract/Free Full Text]
  23. Drummond AE. TGFbeta signalling in the development of ovarian function. Cell Tissue Res 2005; 322:107–115[CrossRef][Medline]
  24. Harrison CA, Gray PC, Vale WW, Robertson DM. Antagonists of activin signaling: mechanisms and potential biological applications. Trends Endocrinol Metab 2005; 16:73–78[CrossRef][Medline]
  25. Gilchrist RB, Ritter LJ, Myllymaa S, Kaivo-Oja N, Dragovic RA, Hickey TE, Ritvos O, Mottershead DG. Molecular basis of oocyte-paracrine signaling that promotes granulosa cell proliferation. J Cell Sci 2006; 119:3811–3821[Abstract/Free Full Text]
  26. Eppig JJ. Gonadotropin stimulation of the expansion of cumulus oophori isolated from mice: general conditions for expansion in vitro. J Exp Zool 1979; 208:111–120[CrossRef][Medline]
  27. Gilchrist RB, Ritter LJ, Cranfield M, Jeffery LA, Amato F, Scott SJ, Myllymaa S, Kaivo-Oja N, Lankinen H, Mottershead DG, Groome NP, Ritvos O. Immunoneutralization of growth differentiation factor 9 reveals it partially accounts for mouse oocyte mitogenic activity. Biol Reprod 2004; 71:732–739[Abstract/Free Full Text]
  28. Hickey TE, Marrocco DL, Amato F, Ritter LJ, Norman RJ, Gilchrist RB, Armstrong DT. Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells. Biol Reprod 2005; 73:825–832[Abstract/Free Full Text]
  29. Laping NJ, Grygielko E, Mathur A, Butter S, Bomberger J, Tweed C, Martin W, Fornwald J, Lehr R, Harling J, Gaster L, Callahan JF, et al. Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542. Mol Pharmacol 2002; 62:58–64[Abstract/Free Full Text]
  30. Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 2002; 62:65–74[Abstract/Free Full Text]
  31. Hussein TS, Thompson JG, Gilchrist RB. Oocyte-secreted factors enhance oocyte developmental competence. Dev Biol 2006; 296:514–521[CrossRef][Medline]
  32. Fagbohun CF and Downs SM. Maturation of the mouse oocyte-cumulus cell complex: stimulation by lectins. Biol Reprod 1990; 42:413–423[Abstract]
  33. Hussein TS, Froiland DA, Amato F, Thompson JG, Gilchrist RB. Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins. J Cell Sci 2005; 118:5257–5268[Abstract/Free Full Text]
  34. Wang H, Jiang JY, Zhu C, Peng C, Tsang BK. Role and regulation of nodal/Alk7 signaling pathway in the control of ovarian follicular atresia. Mol Endocrinol 2006; 20:2469–2482[Abstract/Free Full Text]
  35. Albano RM, Groome N, Smith JC. Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation. Development 1993; 117:711–723[Abstract]
  36. Sidis Y, Fujiwara T, Leykin L, Isaacson K, Toth T, Schneyer AL. Characterization of inhibin/activin subunit, activin receptor, and follistatin messenger ribonucleic acid in human and mouse oocytes: evidence for activin's paracrine signaling from granulosa cells to oocytes. Biol Reprod 1998; 59:807–812[Abstract/Free Full Text]
  37. Rebbapragada A, Benchabane H, Wrana JL, Celeste AJ, Attisano L. Myostatin signals through a transforming growth factor beta-like signaling pathway to block adipogenesis. Mol Cell Biol 2003; 23:7230–7242[Abstract/Free Full Text]
  38. Su YQ, Denegre JM, Wigglesworth K, Pendola FL, O'Brien MJ, Eppig JJ. Oocyte-dependent activation of mitogen-activated protein kinase (ERK1/2) in cumulus cells is required for the maturation of the mouse oocyte-cumulus cell complex. Dev Biol 2003; 263:126–138[CrossRef][Medline]
  39. Kaivo-Oja N, Jeffery LA, Ritvos O, Mottershead DG. Smad signalling in the ovary. Reprod Biol Endocrinol 2006; 4:21.[CrossRef][Medline]
  40. Shimasaki S, Moore RK, Otsuka F, Erickson GF. The bone morphogenetic protein system in Mammalian reproduction. Endocr Rev 2004; 25:72–101[Abstract/Free Full Text]
  41. Schrewe H, Gendron-Maguire M, Harbison ML, Gridley T. Mice homozygous for a null mutation of activin beta B are viable and fertile. Mech Dev 1994; 47:43–51[CrossRef][Medline]
  42. Su YQ, Wu X, O'Brien MJ, Pendola FL, Denegre JN, Matzuk MM, Eppig JJ. Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Dev Biol 2004; 276:64–73[CrossRef][Medline]
  43. Moore RK, Otsuka F, Shimasaki S. Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells. J Biol Chem 2003; 278:304–310[Abstract/Free Full Text]
  44. McNatty KP, Moore LG, Hudson NL, Quirke LD, Lawrence SB, Reader K, Hanrahan JP, Smith P, Groome NP, Laitinen M, Ritvos O, Juengel JL. The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology. Reproduction 2004; 128:379–386[Abstract/Free Full Text]
  45. Yi SE, LaPolt PS, Yoon BS, Chen JY, Lu JK, Lyons KM. The type I BMP receptor BmprIB is essential for female reproductive function. Proc Natl Acad Sci U S A 2001; 98:7994–7999[Abstract/Free Full Text]
  46. Otsuka F, Moore RK, Iemura S, Ueno N, Shimasaki S. Follistatin inhibits the function of the oocyte-derived factor BMP-15. Biochem Biophys Res Commun 2001; 289:961–966[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
Y. S. Lee, K. E. Latham, and C. A. VandeVoort
Effects of in vitro maturation on gene expression in rhesus monkey oocytes
Physiol Genomics, October 7, 2008; 35(2): 145 - 158.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
K. T. Jones
Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age
Hum. Reprod. Update, March 1, 2008; 14(2): 143 - 158.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
R. B. Gilchrist, M. Lane, and J. G. Thompson
Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality
Hum. Reprod. Update, March 1, 2008; 14(2): 159 - 177.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Cecconi, A. Mauro, G. Capacchietti, P. Berardinelli, N. Bernabo, A. R. Di Vincenzo, M. Mattioli, and B. Barboni
Meiotic Maturation of Incompetent Prepubertal Sheep Oocytes Is Induced by Paracrine Factor(s) Released by Gonadotropin-Stimulated Oocyte-Cumulus Cell Complexes and Involves Mitogen-Activated Protein Kinase Activation
Endocrinology, January 1, 2008; 149(1): 100 - 107.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
C. X. Yeo, R. B. Gilchrist, J. G. Thompson, and M. Lane
Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice
Hum. Reprod., January 1, 2008; 23(1): 67 - 73.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
Y.-Q. Su, K. Sugiura, K. Wigglesworth, M. J. O'Brien, J. P. Affourtit, S. A. Pangas, M. M. Matzuk, and J. J. Eppig
Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells
Development, January 1, 2008; 135(1): 111 - 121.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
76/5/848    most recent
biolreprod.106.057471v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend