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Regular Article |
a Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, granulosa cells, growth factors, kinases, signal transducers
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GDF-9 expression has been found in oocytes from rodent [9, 11], bovine/ovine [18], and human [19] follicles at early stages of follicular development. Mice deficient in GDF-9 show an arrest of follicle development beyond the primary stage [12]. Treatment with GDF-9 enhances primary and preantral follicular growth in vitro and in vivo [11, 20] and promotes granulosa cell proliferation and modifies granulosa cell differentiation in vitro [21]. In addition, treatment with GDF-9 enables cumulus expansion [22] and increases thecal cell androgen production [23].
Other TGFß family proteins closely related to GDF-9 also modulate follicle development. GDF-9B/BMP-15, which is expressed exclusively by the oocyte [17, 19], partially mimics GDF-9 effects on granulosa cell proliferation and progesterone production [24]. The crucial role of GDF-9B in follicle development was revealed by the Inverdale sheep that carry a point mutation of the GDF-9B gene [25]. In addition, treatment with either BMP-7 or BMP-4, known to be secreted by ovarian theca cells, reduces FSH-induced progesterone production by granulosa cells [13]. Thus, several TGFß superfamily members originating from different follicular compartments have overlapping effects on granulosa cell proliferation and differentiation, indicating that these ligands may use one or more common receptors in their signaling pathways.
Members of the TGFß superfamily signal through single transmembrane serine/threonine kinase receptors [26–29]. Based on their sequence divergence, these receptors can be classified into two groups, designated type I and type II receptors. Formation of a heterodimeric complex between type I and type II receptors is required for signal transduction [30–32]. Type I receptors are designated activin-like receptor kinases (ALKs). Studies on TGFß proteins and activins have shown that these ligands bind to their respective type II receptors before associating with type I receptors [27, 33, 34], leading to type I receptor phosphorylation followed by phosphorylation of receptor-regulated signal transducers and transcription factors (Smads). Mammalian Smads are related to Drosophila-derived MADs (mothers against dpp) and the nematode SMA proteins. Formation of heteromeric complexes between receptor-regulated Smads and co-Smads such as Smad-4 results in their translocation to the nucleus to regulate target gene transcription [35].
BMPs, which are the closest paralogs (related proteins of the same species) to GDF-9, interact with specific BMP type II and type I receptors. BMP-2 and BMP-4 interact with ALK-3 and ALK-6 and with BMP receptor type II (BMPRII) [13, 31, 36–40]. In addition to interaction with these BMP receptors, BMP-7 can also signal through activin type I and type II receptors [32, 38]. In contrast to TGFß ligands however, BMPs can bind to the type I receptor in the absence of the type II receptor [28, 36, 41]. The BMPRII can bind the ligand weakly, but this binding is enhanced by the presence of type I receptors [31, 38, 40].
To identify potential GDF-9 receptors, we screened the entire human genome for the presence of novel serine/threonine kinase receptors and analyzed sequence alignments between GDF-9 and other TGFß superfamily ligands [42]. After identifying potential candidates, we tested the soluble ectodomains (N-terminal extracellular region) of several receptors for their potential to bind GDF-9 and therefore interfere with GDF-9 modulation of granulosa cell function in vitro. In combination with the use of antisense BMPRII oligomers, we found that BMPRII is essential for GDF-9 signaling in granulosa cells and that the BMPRII ectodomain directly interacts with GDF-9 and completely blocks GDF-9 actions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature female rats (Sprague-Dawley) were obtained from Simonsen Laboratories (Gilroy, CA). Animals (23 days old) were anesthetized and killed using CO2 72 h after insertion of diethylstilbestrol (DES) implants [43]. All animals were housed under controlled humidity, temperature, and light conditions and fed ad libitum with standard rat chow. Animal care was consistent with institutional and National Institutes of Health (NIH) guidelines.
Reagents and Hormones
McCoy's 5a medium (modified) was obtained from Cellgro (Mediatech, Herndon, VA), and L-15 Leibovitz medium was obtained from Life Technologies (Gaithersburg, MD). Recombinant human FSH was acquired from Organon N.V. (Oss, The Netherlands), and recombinant BMP-7 was a gift from Dr. D. Lin (Creative Biomolecules, Boston, MA). The ectodomains of BMP receptors were shown to be suitable for studies of receptor-ligand interactions by demonstrating binding of these ectodomains to BMPs [41, 44] and the ligand specificity of a soluble form of mouse ALK-3 [45]. Recombinant ALK-2 (activin receptor type I), ALK-3 (BMP receptor type IA), ALK-6 (BMP receptor type IB), BMPRII, and activin receptor type IIA (ActIIA) ectodomains fused to the human IgG-Fc region were obtained from R&D Systems (Minneapolis, MN).
BMPRII morpholino antisense oligomers and control oligomers were synthesized by Gene Tools LLC (Corvallis, OR). Morpholino oligomers have a 6-member morpholine ring linked to each nucleotide base, thus rendering the oligomers more stable for transfection studies. Androstenedione and BSA were obtained from Sigma Chemical Co. (St. Louis, MO), and L-glutamine, penicillin, and streptomycin were purchased from BioWhittaker (Wakersville, MD). Recombinant GDF-9 and N-terminal-tagged GDF-9 was generated in transfected mammalian cells and characterized as previously described [9]. Conditioned medium containing GDF-9 was concentrated 100-fold using Biomax-10 (Millipore Corp., Bedford, MA), thus removing proteins with a lower molecular weight. Purified N-terminal-tagged GDF-9 was used as a standard for the quantitation of wild-type GDF-9 in the conditioned medium of 293T cells by immunoblots using specific GDF-9 antibodies.
Preparation and Culture of Granulosa Cells
Granulosa cells were obtained from small antral follicles of DES-treated rats. Ovaries were punctured in L-15 Leibovitz medium. Ovarian debris, oocytes, and small follicles were removed, and the remaining medium containing granulosa cells was collected following low-speed centrifugation at 500 x g for 10 min. Granulosa cells were dispersed by repeated washing and suspension into the culture medium (McCoy's 5a) supplemented with 10-7 M androstenedione, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Thymidine Incorporation into Granulosa Cells
Granulosa cells (2 x 105 viable cells/500 µl) were cultured in 5-ml polypropylene Falcon tubes (Becton Dickinson, Franklin Lakes, NJ) with conditioned medium containing GDF-9 or BMP-7 with or without different concentrations of the ectodomains of different receptors fused to human IgG-Fc. The highest dose tested was 3 µg/ml for all receptor ectodomains, which corresponds to approximately a 10-fold molar excess over the GDF-9 concentration.
The doses used for GDF-9 (200 ng/ml) and BMP-7 (500 ng/ml) correspond to the minimum doses necessary to obtain maximum bioactivity as tested in prior dose-response studies. The volume of conditioned medium containing GDF-9 added to the cells in culture was <5 µl in 1 ml culture medium (0.5 %). Cultures were maintained at 37°C under 5% CO2 in air. Conditioned medium from nontransfected 293T cells served as a negative control. After 24 h of culture with 1 µCi/tube of [methyl-3H]thymidine (Amersham Life Science, Arlington Heights, IL), cells were washed once and resuspended in ice-cold PBS by centrifugation at 2000 x g for 30 min. The radioactivity in the cells was determined after resuspension by scintillation counting in a ß-photomultiplier.
Blockage of BMPRII Receptor Biosynthesis
Granulosa cells (4 x 105 viable cells/500 µl) were cultured in 5-ml polypropylene Falcon tubes (Becton Dickinson) with 200 ng/ml GDF-9 with or without different concentrations of the BMPRII morpholino antisense oligomers using the manufacturer's protocol found to be suitable for ALK-2 antisense studies in urogenital ridge explants [46]. The sequence of the BMPRII oligomer (5'-TGCAGCGAGGAAGTCATCCCTGGGC-3') was derived from a rat EST (GenBank accession BF554751), which is 91% and 95% identical to human and mouse BMPRII mRNAs, respectively. Furthermore, granulosa cells were cultured with GDF-9 with or without a control oligomer (5'-cctcttacctcagttacaatttata-3'). Granulosa cells were preincubated for 30 min followed by transfection with the morpholino oligomers using 0.4 µM EPEI delivery solution supplied by the manufacturer (Gene Tools LLC). After 3 h of incubation, the supernatant was removed and washed once, and fresh culture medium containing [methyl-3H]thymidine and GDF-9 was added. Thymidine incorporation was assessed 48 h later as described above.
Assessment of Progesterone Production
Granulosa cells (1 x 105 viable cells/ml) were cultured in 48-well plates (Corning, Corning, NY) in the presence of FSH (1 ng/ml) with or without GDF-9 (200 ng/ml) in combination with different concentrations (0.03–3 µg/ml) of BMPRII ectodomains fused to IgG-Fc. Conditioned medium from nontransfected 293T cells served as a negative control. After 48 h of culture, the supernatant was collected and stored at -80°C until measurement for progesterone content using RIA [21]. The within-assay coefficient of variation, between-assay coefficient of variation, and sensitivity of the progesterone assay were <2%, 5%, and 4 pg, respectively.
Direct Binding of GDF-9 to Receptor Ectodomains Fused to IgG-Fc
Ectodomains of different receptors fused to the Fc region of human IgG were used to precipitate GDF-9. After overnight incubation of receptor ectodomains (0.2 µM) with equimolar concentrations of GDF-9 (12.8 ng/ml), the samples were mixed with agarose beads coated with Protein G (Amersham Pharmacia, Piscataway, NJ). The reaction mixture was gently rocked at 4°C for 3 h. After centrifugation, the beads were washed three times with buffer containing 50 mM Tris, pH 7.4, 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, and 0.1% SDS. Protein bound to the beads was removed by heating the beads in SDS-PAGE Laemmli buffer containing ß-mercaptoethanol (3 min, 95°C) before analysis for GDF-9 content using immunoblotting [11]. Rabbit anti-GDF-9 was used as the primary antibody [11], and horseradish peroxidase-conjugated donkey IgG was used as the secondary antibody. Signals were detected using the ECL system (Amersham Pharmacia).
Expression Analysis of Different Type II and Type I Receptors in the Neonatal Ovary
Analysis of the expression of BMPRII, ALK-2, ALK-3, ALK-6, and ActIIA in neonatal rat ovaries was performed using 12 ovaries, which were collected at 1 and 5 days postpartum and frozen rapidly on dry ice. Heart tissue also was collected from these animals, and ovaries were collected from adult rats. After thawing, the tissues were homogenized and DNA was synthesized from 3 µg total RNA using oligo(dT) primers. Reverse transcriptase (Clontech, Palo Alto, CA) was used in buffer containing 3 mM MgCl2 for 1 h at 42°C followed by 5 min at 94°C to stop cDNA synthesis. Following reverse transcription, the polymerase chain reaction (PCR) was carried out using HotStarTaq DNA polymerase (Qiagen, Valencia, CA). Primers for rat ALK-2, ALK-3, and ActIIA and mouse ALK-6 were derived from mRNA sequences available in GenBank (accessions NM_024486, D38082, L10639, and NM_007560, respectively). Primer sequences were as follows: ALK-2, 5'-ATGGTCGATGGAGCAATGATCCTTTCT-3' and 5'-AATCTTTACATCCTGGGATTCAACCAT-3'; ALK-3, 5'-ATGACTCAGCTATACACTTACATCAGA-3' and 5'-AATCTTTACATCCTGGGATTCAACCAT-3'; ALK-6, 5'-ATGCTCTTACGAAGCTCTGGAAAATTA-3' and 5'-GAGTTTAATGTCCTGGGACTCTGACAT-3'; ActIIA, 5'-ATGGGAGCTGCTGCAAAGTTGGCGTTC-3' and 5'-TAGACTAGATTCTTTGGGAGGAAAGTC-3'. Rat EST sequences (accessions BF554751 and BF400292) with 90% homology to human BMPRII were used for primer design (primers: 5'-ATGACTTCCTCGCTGCAGCGGCCCTTT-3' and 5'-TTCACCTGGGAAGAGGTCTGTGCACCT-3'). The extracellular, transmembrane, and one third of the intracellular domain of BMPRII were amplified. The PCR consisted of initial incubation for 15 min at 95°C followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. After the PCR, the cDNA samples were sequenced with the primers mentioned above with their identity verified.
Data Analysis
All experimental data are presented as the mean ± SEM of duplicate measurements of triplicate cultures. Significance of differences was determined by the Student paired t-test or ANOVA for multiple group comparisons. Significance was accepted at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used the known type II and type I serine/threonine kinase receptors to search the human genome and excluded the presence of additional unknown type II or type I receptors homologous to the known TGFß/activin/BMP receptors. Thus, GDF-9 most likely signals through known serine/threonine kinase receptors. Previously, we screened predicted open reading frames to retrieve human proteins containing a cystine knot signature [42]. Representative members of the TGFß, activin, and BMP subgroups [47, 48] were used for sequence comparison (Fig. 1A). Because of the characteristic cystine knot motif, proteins of the TGFß superfamily show two finger-like projections (fingers 1 and 2) and a region between these fingers commonly referred to as a "heel." GDF-9 has the typical WXXW signature in the tip of finger 1 (Fig. 1A, underlined), which is absent in the TGFß subgroup but present in BMPs and activins. In addition, the alignment to the TGFß subgroups requires introduction of several gaps to accommodate the longer sequence of the BMPs (Fig. 1A, shading). The alignment between GDF-9 and the activin subgroup requires the insertion of a larger gap region in the heel region (Fig. 1A, solid). In contrast, GDF-9 aligns without gaps to all BMPs. Sequence comparison using a distance matrix [49] revealed that GDF-9 is most closely related to GDF-9B/BMP-15 and that this subgroup is closer to the BMPs than to the activin/TGFß-like ligands. Most of the BMPs interact with BMPRII except for BMP-7, which also interacts with ActIIA [28, 32, 38] (Fig. 1B). The type I receptors for BMPs include ALK-2, ALK-3, and ALK-6 [28, 31]. Based on these data, we predicted that GDF-9 might interact with BMPRII, ActIIA, and one or more of these BMP type I receptors.
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Coincubation with Ectodomains of Type II and Type I BMP Receptors Block GDF-9 Action
GDF-9-induced granulosa cell proliferation was inhibited by the BMPRII ectodomain in a dose-dependent fashion; 1 µg/ml of the ectodomain completely blocked the action of 200 ng/ml GDF-9 (Fig. 2A). In contrast, ActIIA did not block GDF-9 granulosa cell proliferation completely at any of the doses tested. Of the type I receptors tested, only ALK-3 and ALK-6 soluble ectodomains reduced GDF-9 effects in a dose-dependent manner but only up to approximately 30%, whereas the ALK-2 ectodomain had no effect (Fig. 2B).
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The BMPRII ectodomain inhibited GDF-9 effects on granulosa cell progesterone production in a dose-dependent manner (Fig. 3). At 1 µg of BMPRII ectodomain, the FSH-induced progesterone production was restored to levels seen without addition of GDF-9. Restoration of FSH-induced progesterone production by the addition of the ectodomains suggested a lack of any cytotoxic effects of the ectodomain preparations.
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Because the receptors tested are known to interact with BMP-7, we investigated whether they interfere with BMP-7-induced granulosa cell proliferation (Fig. 4). In contrast to GDF-9, the effects of BMP-7 on granulosa cells could not be blocked completely by the type II receptor ectodomains at the highest dose tested (3 µg), which is more than 5-fold the BMP-7 concentration used. In contrast, the type I receptor ectodomains had a significant effect on BMP-7 action.
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Direct Interactions Between GDF-9 and the BMPRII Ectodomain
The observed antagonistic effects of different soluble receptor ectodomains are presumably due to the ability of these proteins to bind GDF-9. To confirm the direct interactions between GDF-9 and the ectodomains of different BMP receptors, we took advantage of the ability of the IgG-Fc region of the fusion protein to interact with protein G. As shown in Figure 5 (lane 2), GDF-9 could be precipitated by the BMPRII ectodomain Fc fusion protein incubated with protein G-coated beads, thus showing that this receptor ectodomain directly interacted with GDF-9. In contrast, ActIIA-Fc protein was only minimally effective in binding GDF-9 (Fig. 5, lane 6), whereas none of the type I receptors interacted with GDF-9 (Fig. 5, lanes 3–5).
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Blockage of BMPRII Biosynthesis with Antisense Oligomers Suppressed the GDF-9 Stimulation of Granulosa Cell Proliferation
To determine whether BMPRII is essential for GDF-9 signaling in follicular cells, we used specific morpholino antisense oligomers to block the biosynthesis of this receptor in rat granulosa cells. Addition of BMPRII morpholino oligomers reduced the GDF-9 stimulation of thymidine incorporation by cultured granulosa cells in a dose-dependent manner (Fig. 6). At 1.4 µM, BMPRII completely blocked the stimulatory effects of GDF-9 on granulosa cell proliferation. In contrast, addition of the control oligomers at different doses did not lead to a suppression of granulosa cell proliferation.
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BMP Type II and Type I Receptors Are Expressed in Postnatal Ovaries
GDF-9 mutant mice are characterized by the arrest of folliculogenesis at the primary stage because GDF-9 enhances primordial and primary follicle growth. To analyze the expression of different type II and type I receptors during early folliculogenesis, we performed reverse transcription PCR assays on ovaries from neonatal rats. The expected size of PCR products from trancsripts of ActIIA, BMPRII, ALK-2, ALK-3, and ALK-6 are 1539 base pairs (bp), 1257 bp, 1527 bp, 1538 bp, and 1506 bp, respectively. As shown in Figure 7, all five receptors were expressed in neonatal ovaries at Day 1 postpartum, and their expression increased between Day 1 and Day 5 postpartum (compare lanes 1 and 2, respectively). In addition, all receptors studied were expressed in adult ovaries (lane 3). In the heart (lane 4), ActIIA and ALK-3 were not expressed, but ALK-2 was expressed at low levels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sequence alignment of GDF-9 with other TGFß family members revealed that GDF-9 is most closely related to the BMPs. Although the binding of BMPs to the type II receptor is not an essential requirement for the signaling of several BMPs [28, 38], the BMPRII ectodomain was most efficient in interacting with GDF-9 in the present study. In contrast, none of the type I receptors tested were capable of completely blocking GDF-9 function in vitro or of interacting with GDF-9 in the coprecipitation test. In addition, the blockage of BMPRII biosynthesis using an antisense approach completely blocked GDF-9 action in cultured granulosa cells. Thus, BMPRII receptor expression is essential for GDF-9 signaling. These data suggest that the type II receptor is the primary binding receptor for GDF-9, and heterodimerization with type I receptors might be a secondary event. This model is comparable to the signaling pathways of the TGFß and activin ligands, in which binding to type II receptors is essential for the recruitment of different type I receptors and subsequent Smad activation [27, 30, 50]. This finding corroborates a phylogenetic position of GDF-9 between the BMPs and the activins. Because BMPRII is capable of binding GDF-9 and is required for GDF-9 signaling in granulosa cells, GDF-9 function could be modified by responsive cells through the regulation of BMPRII expression. Thus, even if related ligands (e.g., BMPs) could also interact with the BMPRII expressed in granulosa cells, the cellular responses to the different ligands throughout folliculogenesis in vivo might be different and might depend upon expression levels of these related receptors.
In vitro studies using BMP-4, BMP-7, and BMP-15 have revealed that these ligands mimic some of the GDF-9 effects on FSH-induced steroidogenesis and granulosa cell proliferation [13, 24]. Both BMP-4 and BMP-7 interact with BMPRII [31, 38], and BMP-7 also interacts with ActIIA [51]. Although BMP-7 binds BMPRII expressed on the cell surface [31], our studies indicate that the BMPRII ectodomain is only a weak antagonist of BMP-7 action in granulosa cells. In contrast, the potent antagonistic effect of the BMPRII ectodomain on GDF-9 action indicates that GDF-9 interacts more efficiently with BMPRII than does BMP-7. Several mutations of the BMPRII gene in humans are known to cause primary pulmonary hypertension [52, 53], but a disruption of ovarian physiology has not been reported in these patients, indicating that other receptors might compensate for the loss of BMPRII function or that these mutations lead to a modification but not complete loss of receptor signaling.
Of the type I receptors tested, ALK-3 and ALK-6 inhibited GDF-9 action. Thus, despite the lack of direct interaction between them and GDF-9 in coprecipitation tests, GDF-9 could use a combination of type I receptors as secondary receptors in addition to BMPRII. The role of ALK-6 in follicle development was demonstrated in both mice and sheep mutants. Mice deficient in ALK-6 are infertile because of a lack of cumulus expansion prior to fertilization [54], indicating defects in the final stage of follicle maturation and premature ovulation. A single point mutation of the ALK-6 gene in Booroola sheep is associated with enhanced ovulation rates [55]. In contrast to ALK-6, ALK-3 function in the ovary is not well studied because of the embryonic lethal phenotype of ALK-3-null mice [56]. However, ALK-3 is expressed in granulosa cells of antral follicles in adult ovaries and could mediate the action of thecal cell-derived BMP-4 and BMP-7 [13].
Granulosa cell responses can be regulated differently by ovarian GDF-9 and BMP proteins. For example ovarian FSH-induced estradiol production is suppressed by GDF-9, not affected by BMP-15 [24], and increased by BMP-4 and BMP-7 [13]. The receptors for BMP-15 are not known; however, this ligand is the closest paralog to GDF-9, suggesting an interaction with a similar subgroup of receptors. Functional aspects of GDF-9 and BMP-15 overlap only partially. Unlike treatment with GDF-9 [22], treatment with BMP-15 did not affect the steroidogenic acute regulatory protein or LH receptor mRNA expression in granulosa cells [57]. Future studies are needed to determine whether these discrepancies are the results of disparate utilization of different combinations of type II and type I BMP receptors.
Complete elucidation of the regulation of folliculogenesis by different BMP and GDF ligands with overlapping and sometimes antagonistic effects and divergent expression patterns is difficult. In contrast to these ligands, all of the receptors were expressed in neonatal ovaries. They are present before and throughout primary follicle development and are present in the granulosa cells of adult ovaries [13]. Therefore, paracrine actions of BMP and GDF are likely to be controlled by differential ligand expression and secretion rather than divergent receptor expression. Oocyte-derived GDF-9 and BMP-15 are secreted by the oocyte at the onset of follicle development and throughout all stages of follicle development [11, 16, 19], whereas BMP-4, in addition to its role in primordial germ cell migration [57], is secreted, as is BMP-7, by thecal cells during the later stages of folliculogenesis [13]. Thus, these ligands could act sequentially throughout follicle maturation. Furthermore, the phenotypes of BMP-4- and GDF-9-deficient mice and the Inverdale sheep carrying a BMP-15 mutation [12, 25, 58] suggest that these ligands cannot be completely replaced by other ligands of the same family.
In this study, we demonstrated the interactions between GDF-9 and serine/threonine kinase receptors known to be essential for BMP signaling. In contrast to BMPs, GDF-9 mainly interacted with the BMPRII ectodomain and to a lesser degree with type I BMP receptors. Furthermore, BMPRII biosynthesis was shown to be essential for GDF-9 action on granulosa cells from small preantral follicles. Thus, GDF-9 might signal use of BMPRII as the primary binding receptor followed by heterodimerization with type I receptors. Determination of the exact relationship between BMPRII and different type I receptors or the weakly interacting ActIIA requires future studies using ovarian cells deficient in one or more of these genes. Folliculogenesis is controlled by the partially overlapping and sequential expression and actions of GDF-9 and BMPs. By combinatorial and sequential utilization of two types of receptors, multiple ligands belonging to the BMP family could transduce different cellular responses by signaling through a limited number of receptors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by NIH grant HD31398 and the NIH Specialized Cooperative Centers Program in Reproduction Research. U.A.V. is supported by the Lalor Foundation, and S.M. is supported by the Association pour la Recherche sur le Cancer, Villejuif, France. 
2 Correspondence. FAX: 650 725 7102; aaron.hsueh{at}stanford.edu
Accepted: February 13, 2002.
Received: November 30, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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M. Orisaka, S. Orisaka, J.-Y. Jiang, J. Craig, Y. Wang, F. Kotsuji, and B. K. Tsang Growth Differentiation Factor 9 Is Antiapoptotic during Follicular Development from Preantral to Early Antral Stage Mol. Endocrinol., October 1, 2006; 20(10): 2456 - 2468. [Abstract] [Full Text] [PDF]
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 R. B. Gilchrist, L. J. Ritter, S. Myllymaa, N. Kaivo-Oja, R. A. Dragovic, T. E. Hickey, O. Ritvos, and D. G. Mottershead Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation J. Cell Sci., September 15, 2006; 119(18): 3811 - 3821. [Abstract] [Full Text] [PDF]
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S. Mazerbourg and A. J.W. Hsueh Genomic analyses facilitate identification of receptors and signalling pathways for growth differentiation factor 9 and related orphan bone morphogenetic protein/growth differentiation factor ligands Hum. Reprod. Update, July 1, 2006; 12(4): 373 - 383. [Abstract] [Full Text] [PDF]
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 S. Assou, T. Anahory, V. Pantesco, T. Le Carrour, F. Pellestor, B. Klein, L. Reyftmann, H. Dechaud, J. De Vos, and S. Hamamah The human cumulus-oocyte complex gene-expression profile Hum. Reprod., July 1, 2006; 21(7): 1705 - 1719. [Abstract] [Full Text] [PDF]
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 L J Spicer, P Y Aad, D Allen, S Mazerbourg, and A J Hsueh Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells. J. Endocrinol., May 1, 2006; 189(2): 329 - 339. [Abstract] [Full Text] [PDF]
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C. Wang and S. K. Roy Expression of Growth Differentiation Factor 9 in the Oocytes Is Essential for the Development of Primordial Follicles in the Hamster Ovary Endocrinology, April 1, 2006; 147(4): 1725 - 1734. [Abstract] [Full Text] [PDF]
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 B. C Jayawardana, T. Shimizu, H. Nishimoto, E. Kaneko, M. Tetsuka, and A. Miyamoto Hormonal regulation of expression of growth differentiation factor-9 receptor type I and II genes in the bovine ovarian follicle. Reproduction, March 1, 2006; 131(3): 545 - 553. [Abstract] [Full Text] [PDF]
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C. J. Guigon and S. Magre Contribution of Germ Cells to the Differentiation and Maturation of the Ovary: Insights from Models of Germ Cell Depletion Biol Reprod, March 1, 2006; 74(3): 450 - 458. [Abstract] [Full Text] [PDF]
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A J W Hsueh, P Bouchard, and I Ben-Shlomo Hormonology: a genomic perspective on hormonal research J. Endocrinol., December 1, 2005; 187(3): 333 - 338. [Abstract] [Full Text] [PDF]
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T. S. Hussein, D. A. Froiland, F. Amato, J. G. Thompson, and R. B. Gilchrist Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins J. Cell Sci., November 15, 2005; 118(22): 5257 - 5268. [Abstract] [Full Text] [PDF]
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S. A. Pangas and M. M. Matzuk The Art and Artifact of GDF9 Activity: Cumulus Expansion and the Cumulus Expansion-Enabling Factor Biol Reprod, October 1, 2005; 73(4): 582 - 585. [Abstract] [Full Text] [PDF]
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 S. Mazerbourg, K. Sangkuhl, C.-W. Luo, S. Sudo, C. Klein, and A. J. W. Hsueh Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses J. Biol. Chem., September 16, 2005; 280(37): 32122 - 32132. [Abstract] [Full Text] [PDF]
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R. A. Dragovic, L. J. Ritter, S. J. Schulz, F. Amato, D. T. Armstrong, and R. B. Gilchrist Role of Oocyte-Secreted Growth Differentiation Factor 9 in the Regulation of Mouse Cumulus Expansion Endocrinology, June 1, 2005; 146(6): 2798 - 2806. [Abstract] [Full Text] [PDF]
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J.L. Juengel and K.P. McNatty The role of proteins of the transforming growth factor-{beta} superfamily in the intraovarian regulation of follicular development Hum. Reprod. Update, March 1, 2005; 11(2): 144 - 161. [Abstract] [Full Text] [PDF]
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 N. Kaivo-Oja, D. G. Mottershead, S. Mazerbourg, S. Myllymaa, S. Duprat, R. B. Gilchrist, N. P. Groome, A. J. Hsueh, and O. Ritvos Adenoviral Gene Transfer Allows Smad-Responsive Gene Promoter Analyses and Delineation of Type I Receptor Usage of Transforming Growth Factor-{beta} Family Ligands in Cultured Human Granulosa Luteal Cells J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 271 - 278. [Abstract] [Full Text] [PDF]
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J. E. I. Gittens, K. J. Barr, B. C. Vanderhyden, and G. M. Kidder Interplay between paracrine signaling and gap junctional communication in ovarian follicles J. Cell Sci., January 1, 2005; 118(1): 113 - 122. [Abstract] [Full Text] [PDF]
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K P McNatty, L G Moore, N L Hudson, L D Quirke, S B Lawrence, K Reader, J P Hanrahan, P Smith, N P Groome, M Laitinen, et al. The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology Reproduction, October 1, 2004; 128(4): 379 - 386. [Abstract] [Full Text] [PDF]
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 T. Hamatani, G. Falco, M. G. Carter, H. Akutsu, C. A. Stagg, A. A. Sharov, D. B. Dudekula, V. VanBuren, and M. S.H. Ko Age-associated alteration of gene expression patterns in mouse oocytes Hum. Mol. Genet., October 1, 2004; 13(19): 2263 - 2278. [Abstract] [Full Text] [PDF]
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R.B. Gilchrist, L.J. Ritter, M. Cranfield, L.A. Jeffery, F. Amato, S.J. Scott, S. Myllymaa, N. Kaivo-Oja, H. Lankinen, D.G. Mottershead, et al. Immunoneutralization of Growth Differentiation Factor 9 Reveals It Partially Accounts for Mouse Oocyte Mitogenic Activity Biol Reprod, September 1, 2004; 71(3): 732 - 739. [Abstract] [Full Text] [PDF]
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S. A. Pangas, C. J. Jorgez, and M. M. Matzuk Growth Differentiation Factor 9 Regulates Expression of the Bone Morphogenetic Protein Antagonist Gremlin J. Biol. Chem., July 30, 2004; 279(31): 32281 - 32286. [Abstract] [Full Text] [PDF]
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X. Wu, L. Chen, C. A. Brown, C. Yan, and M. M. Matzuk Interrelationship of Growth Differentiation Factor 9 and Inhibin in Early Folliculogenesis and Ovarian Tumorigenesis in Mice Mol. Endocrinol., June 1, 2004; 18(6): 1509 - 1519. [Abstract] [Full Text] [PDF]
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J. P. Hanrahan, S. M. Gregan, P. Mulsant, M. Mullen, G. H. Davis, R. Powell, and S. M. Galloway Mutations in the Genes for Oocyte-Derived Growth Factors GDF9 and BMP15 Are Associated with Both Increased Ovulation Rate and Sterility in Cambridge and Belclare Sheep (Ovis aries) Biol Reprod, April 1, 2004; 70(4): 900 - 909. [Abstract] [Full Text] [PDF]
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S. Mazerbourg, C. Klein, J. Roh, N. Kaivo-Oja, D. G. Mottershead, O. Korchynskyi, O. Ritvos, and A. J. W. Hsueh Growth Differentiation Factor-9 Signaling Is Mediated by the Type I Receptor, Activin Receptor-Like Kinase 5 Mol. Endocrinol., March 1, 2004; 18(3): 653 - 665. [Abstract] [Full Text] [PDF]
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 S. Shimasaki, R. K. Moore, F. Otsuka, and G. F. Erickson The Bone Morphogenetic Protein System In Mammalian Reproduction Endocr. Rev., February 1, 2004; 25(1): 72 - 101. [Abstract] [Full Text] [PDF]
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O. M. Onagbesan, V. Bruggeman, P. Van As, K. Tona, J. Williams, and E. Decuypere BMPs and BMPRs in chicken ovary and effects of BMP-4 and -7 on granulosa cell proliferation and progesterone production in vitro Am J Physiol Endocrinol Metab, November 1, 2003; 285(5): E973 - E983. [Abstract] [Full Text] [PDF]
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D. M. Duffy Growth Differentiation Factor-9 Is Expressed by the Primate Follicle Throughout the Periovulatory Interval Biol Reprod, August 1, 2003; 69(2): 725 - 732. [Abstract] [Full Text] [PDF]
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N. Kaivo-Oja, J. Bondestam, M. Kamarainen, J. Koskimies, U. Vitt, M. Cranfield, K. Vuojolainen, J. P. Kallio, V. M. Olkkonen, M. Hayashi, et al. Growth Differentiation Factor-9 Induces Smad2 Activation and Inhibin B Production in Cultured Human Granulosa-Luteal Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 755 - 762. [Abstract] [Full Text] [PDF]
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J.-S. Roh, J. Bondestam, S. Mazerbourg, N. Kaivo-Oja, N. Groome, O. Ritvos, and A. J. W. Hsueh Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells Endocrinology, January 1, 2003; 144(1): 172 - 178. [Abstract] [Full Text] [PDF]
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