Expression Of Growth Differentiation Factor-9 mRNA Research Articles

Melatonin Restores the Developmental Competence of Heat Stressed Porcine Oocytes, and Alters the Expression of Genes Related to Oocyte Maturation.

Simple SummaryMelatonin improves the quality and in vitro maturation (IVM) of oocytes under heat stress. Melatonin treatment counteracts the adverse effects induced by heat stress (HS), such as the poor survival rate and maturation rate, distribution of α-tubulin and F-actin, expression of NRF2 and GDF9 mRNA. However, HS and melatonin have similar effects on increasing expression of HSP70 and NRF2 mRNA. Furthermore, HS inhibits expression of GDF9 mRNA.Melatonin enhances the quality and in vitro maturation (IVM) of oocytes under heat stress (HS), but the mechanism of melatonin in reducing HS injury on oocytes is not fully understood. In this study, porcine cumulus-oocyte complexes (COCs) were randomly divided into three groups. The COCs of the control group were cultured at 38.5 °C for 42 h, and the COCs of the HS group were cultured at 41.5 °C for 4 h, and then transferred into 38.5 °C for 38 h. The COCs of the HS + melatonin group were cultured with 10−9 M melatonin under the same conditions as the HS group. The survival rate, maturation rate, distribution of α-tubulin and F-actin of the oocytes were assessed. In addition, the expression profiles for genes related to the oocyte maturation, including heat shock protein 70 (HSP70), nuclear factor erythroid 2-related factor 2 (NRF2), cyclin-dependent kinase 1 (CDK1), growth differentiation factor 9 (GDF9) were analyzed by real-time quantitative PCR. The results showed that HS decreased the survival rate and maturation rate, distribution of α-tubulin and F-actin, but melatonin treatment could partly counteract these adverse effects. In addition, HS increased expression of HSP70 and NRF2 mRNA, and melatonin treatment had a similar effect on HSP70 expression, but had a contrary effect on NRF2 expression. Furthermore, HS inhibited expression of CDK1 and GDF9 mRNA, but melatonin treatment could weaken the effect on GDF9 expression induced by HS. In summary, melatonin treatment could attenuate the unfavorable effects induced by HS to enhance developmental competence of porcine oocytes during IVM.

GDF-9 and BMP-15 mRNA Levels in Canine Cumulus Cells Related to Cumulus Expansion and the Maturation Process.

Simple SummaryThe knowledge of physiological events associated with canine reproduction involving oocyte developmental potential is essential to increase the success of reproductive biotechnologies in this species. In mammals, the oocytes are closely surrounded by a group of cells known as the cumulus cells. Although it is not well-known how these cells interact with the oocyte to promote maturation, they may provide important answers concerning oocyte development. The competence to undergo expansion is a unique characteristic of cumulus cells which is critical for normal oocyte maturation, however, the complete expansion of these cells takes longer in canines, which has been associated with the lengthy maturation process of the oocyte. Growth Differentiation Factor 9 (GDF-9) and Bone Morphogenetic Protein 15 (BMP-15) are described as relevant players in the oocyte–cumulus cells’ regulatory mechanisms. Cumulus cells express many important genes from a very early stage, therefore, we proposed to study the gene expression of GDF-9 and BMP-15 in canine cumulus cells in relation to cumulus expansion and the maturation process. We demonstrate, for the first time, that these genes are differentially expressed in canine cumulus cells throughout the estrous cycle and that this expression is related to cumulus expansion and maturity status, suggesting specific regulation.The competence to undergo expansion is a characteristic of cumulus cells (CCs). The aim was to investigate the expression of GDF-9 and BMP-15 mRNA in canine cumulus cells in relation to cumulus expansion and meiotic development over the estrous cycle. CCs were recovered from nonmatured and in vitro-matured (IVM) dog cumulus oocyte complexes (COCs), which were obtained from antral follicles at different phases of the estrous cycle. Quantitative real-time polymerase chain reaction (q-PCR) was used to evaluate the relative abundance of GDF-9 and BMP-15 transcripts from the CCs with or without signs of expansion. The results were evaluated by ANOVA and logistic regression. The maturity of the oocyte and the expansion process affected the mRNA levels in CCs. There were differences (p < 0.05) in GDF-9 and BMP-15 gene expression in CCs isolated from nonmatured COCs when comparing the reproductive phases. Lower mRNA levels (p < 0.05) were observed in anestrus and proestrus in comparison to those in estrus and diestrus. In contrast, when comparing GDF-9 mRNA levels in IVM COCs, no differences were found among the phases of the estrous cycle in expanded and nonexpanded CCs (p < 0.05). However, the highest (p < 0.05) BMP-15 gene expression in CCs that did not undergo expansion was exhibited in anestrus and the lowest (p < 0.05) expression was observed in estrus in expanded CCs. Although the stage of the estrous cycle did not affect the second metaphase (MII )rates, the expanded CCs obtained at estrus coexisted with higher percentages of MII (p < 0.05). In conclusion, the differential expression patterns of GDF-9 and BMP-15 mRNA transcripts might be related to cumulus expansion and maturation processes, suggesting specific regulation and temporal changes in their expression.

A 5-Methylcytosine Site of Growth Differentiation Factor 9 (GDF9) Gene Affects Its Tissue-Specific Expression in Sheep.

Simple SummaryGrowth differentiation factor 9 (GDF9) is an important gene for ovine fertility. GDF9 is highly expressed in the ovary as opposed to other tissues, but the reason for this is unknown. Our study found this can be caused by the methylation level of the promoter CpG island mC-4 site. This finding contributes to the understanding of the regulatory mechanism of GDF9 gene in reproduction.Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (−1453 to −1854) of GDF9 promoter in ovary and heart was determined. In this region (−1987 to −1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.

Expressions of growth differentiation factor-9 on granulosa cells of infertile women with endometriosis undergoing in vitro fertilization

Endometriosis may cause ovarian physiology disturbances and one of them, by affecting folliculogenesis that leads to a decreased oocyte quality. The oocyte plays an important role in regulating and promoting follicle growth by oocyte growth factors production. Several growth factors have been identified in human oocytes, including growth differentiation factor-9 (GDF-9). However, studies on GDF-9 expression in the granulosa cells of infertile women with endometriosis are sparse. To investigate the expression of GDF-9 mRNA in the granulosa cells of endometriosis patients undergoing in vitro fertilization (IVF) and identify the correlation between GDF-9 expression and oocyte quality. This cross-sectional study was conducted at the Yasmin IVF Clinic and Dr. Sander B Clinic, Jakarta from July 2014 to July 2017. In total, 50 granulosa cell samples were collected from 25 women with endometriosis and 25 controls who were healthy. The granulosa cell samples were collected at the time of oocyte retrieval. GDF-9 mRNA expression was investigated by real-time PCR. The numbers of retrieved oocytes, mature oocytes, and the oocyte morphology score were lower in patients with endometriosis than in controls; the difference was statistically significant. GDF-9 mRNA expression levels were quantitatively lower in the endometriosis groups than in the control group (5.05 [0.00002–3523.0] ng/mL vs. 81.93 [1.47–32450] ng/μL; p = 0.01). However, no correlation was obtained between GDF-9 expression levels and oocyte quality (oocyte morphology score and fertilization rate). The GDF9 mRNA level was lower in the endometriosis group than in the control group. However, no direct relationship was noted between individual GDF-9 level and oocyte quality. Hence, large-scale studies are needed to confirm whether GDF-9 expression is correlated with the oocyte quality and to ascertain whether GDF-9 has the potential for use as a new molecular marker to predict oocyte developmental competency.

The effect of moderate to severe endometriosis on expression of growth differentiation factor-9 mRNA in human granulosa cells under controlled ovarian hyperstimulation.

To investigate the effect of moderate to severe endometriosis on mRNA expression of growth differentiation factor-9 (GDF-9) in the granulosa cells of mature follicles. Follicular fluid (FF) was obtained from 13 patients with moderate to severe endometriosis and 11 without endometriosis, as a control group, and GDF-9 protein levels in both were assayed by western blotting. mRNA expression by GDF-9 and LH receptor (LHR) in granulosa cells obtained from all patients in the study were investigated by StepOne Real-Time PCR. Although GDF-9 in FF from patients with endometriosis was no different from that of controls, GDF-9 mRNA expression in granulosa cells of patients with endometriosis was statistically significantly lower than for the control group. The number of oocytes and high-quality embryos was positively correlated with GDF-9 mRNA expression in controls but not in patients with endometriosis Moreover, a negative correlation was identified between GDF-9 mRNA expression and serum estrogen and progesterone levels in the control group, whereas no correlation was observed for the endometriosis group. Moderate to severe endometriosis can significantly reduce GDF-9 mRNA expression in the granulosa cells of patients with the disease compared with those without, thus causing poor oocyte maturation and lower embryo quality.

Dynamic changes of the ratio of GDF9 and BMP15 mrna expression in normal and PCOS oocytes during maturation

Oocyte secreted factors, namely growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play crucial roles in the regulation of follicular development and oocyte maturation, and the ratio of GDF9 and BMP15 expression differs across a large rang of species. The aim of this study was to investigate the ratio of GDF9 and BMP15 mRNA expression in human oocytes during maturation, and to compare the differences between normal and PCOS oocytes.

Effect of soothing liver therapy on oocyte quality and growth differentiation factor-9 in patients undergoing in vitro fertilization and embryo transfer

ObjectiveTo investigate the effect of Soothing liver therapy on infertile women undergoing in vitro fertilization and embryo transfer (IVF-ET) and to explore its mechanism. MethodsFifty-eight women with tubal infertility were randomized into two groups: 30 in an experimental group treated with Xiaoyao powder (Shugan) plus gonadotropin-releasing hormone analog (GnRHa)/follicle-stimulating hormone (FSH)/human chorionic gonadotropin (hCG) and 28 in the control group who were treated with GnRHa/FSH/hCG only. The total gonadotropin (Gn) doses required, endometrial thickness, oocyte numbers, high quality embryo production rate and pregnancy rate of the two groups were compared. The concentration of growth differentiation factor-9 (GDF-9) in follicular fluid was detected by western blotting and the expression of GDF-9 mRNA in granulosa cells was measured using reverse transcription-polymerase chain reaction amplification. ResultsIn the experimental group, the Gn dose was significantly lower than that in the control group; the endometrial thickness, high quality embryo production and pregnancy rates were significantly higher and the expression of GDF-9 mRNA was also significantly higher than in the control group (all P<0.05). ConclusionShugan treatment can improve the pregnancy rate of women with tubal infertility; its mechanism is possibly related to the increased expression of GDF-9 in granulosa cells.

The ratio of growth differentiation factor 9: Bone morphogenetic protein 15 mRNA expression is tightly co-regulated and differs between species over a wide range of ovulation rates

Recent evidence suggests that the species-specific ovulation-rate phenotypes may be influenced by differences in the expression levels of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) mRNA and protein. The aim of this study was to compare GDF9 and BMP15 mRNA levels in individual denuded oocytes (DO) from a range of single (i.e. cow, red deer), single-to-triple (i.e. sheep) and high (i.e. pig, mouse, rat) ovulation-rate species. Compared to all other species studied, GDF9 mRNA levels were lower in DO of cows and deer, whilst BMP15 levels were highest in DO of pigs. There was no detectable expression of either GDF9 or BMP15 mRNA in CC from any species. The ratio of GDF9:BMP15 mRNA expression was highly correlated ( R 2 > 0.80) within each species but differed markedly between species ( P < 0.01). Thus, we conclude that the ratio of GDF9:BMP15 mRNA is species-specific across a wide range of ovulation-rate phenotypes.

Molecular characterization of growth differentiation factor 9 and its spatio-temporal expression pattern in gibel carp (Carassius auratus gibelio)

Growth differentiation factor 9 (GDF9) is a member of the transforming growth factor β (TGF-β) superfamily with a key role in regulating follicle development. In this study, the GDF9 full-length genomic DNA and cDNA were isolated and characterized from the gibel carp ovary using rapid-amplification of cDNA ends (RACE) and LD-PCR. The full-length genomic DNA and cDNA sequences of GDF9 are 3979 and 2044bp which code 428 amino acid residues with a specific RKKR protease cleavage site of TGF-β superfamily. Sequence analysis showed that gibel carp was similar to zebrafish and other fish species. Spatio-temporal expression analysis using real-time quantitative PCR revealed that GDF9 mRNA was largely expressed in ovary and testis. GDF9 is mainly present at stage I follicles indicating its important role in early follicles development. The same result was obtained in immunohistochemistry localization of GDF9 protein. Within the follicle, the follicle layer cells were barely expressed whereas GDF9 mRNA was mostly expressed in the oocytes. Supplemented with human chorionic gonadotropin (hCG) in isolated follicles, the expression of GDF9 mRNA was increased firstly and then decreased. The results of this study indicated that GDF9 gene played a role in fish during development of follicles, especially in the early stage follicles.

Quantitative analysis of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) gene expression in calf and adult bovine ovaries

BackgroundIt has been reported that calf oocytes are less developmentally competent than oocytes obtained from adult cows. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) play critical roles in folliculogenesis, follicular development and ovulation in mammalian ovaries. In the present study, we attempted to compare the expression patterns of BMP15 and GDF9 in the cells of calf and cow ovaries to determine a relationship between the level of these genes and the low developmental competence of calf oocytes.MethodsBovine tissues were collected from 9-11 months-old calves and from 4-6 years-old cows. We characterized the gene expression of BMP15 and GDF9 in calf and adult bovine oocytes and cumulus cells using quantitative real-time reverse transcriptase polymerase chain reaction (QPCR) and in situ hybridization. Immunohistochemical analysis was also performed.ResultsThe expression of BMP15 and GDF9 in cumulus cells of adult ovaries was significantly higher than that in calf ovaries, as revealed by QPCR. GDF9 expression in the oocytes of calf ovaries was significantly higher than in those of the adult ovaries. In contrast, BMP15 expression in the oocytes of calf and adult ovaries was not significantly different. The localization of gene expression and protein were ascertained by histochemistry.ConclusionsOur result showed for the first time BMP15 and GDF9 expression in bovine cumulus cells. BMP15 and GDF9 mRNA expression in oocytes and cumulus cells was different in calves and cows.

Expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in the oocytes from unstimulated polycystic ovaries

OBJECTIVE: This study was designed to investigate the expression of GDF9 and BMP15 mRNA in the oocytes from unstimulated ovaries of women with PCOS, and explore whether they are associated with aberrant folliculogenesis in PCOS.

Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-100 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (10 ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 116.5 +/- 3.5 microm) compared to the control (0 ng/ml; 110.5 +/- 2.8 microm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (10 ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway.

SPATIAL AND TEMPORAL REGULATION OF BMP-2, -6, -15 AND GDF-9 mRNA EXPRESSION IN DEVELOPING PORCINE OVARIAN FOLLICLES

Members of the transforming growth factor beta (TGF-beta) superfamily, namely the bone morphogenetic proteins (BMPs) and growth differentiation factor 9 (GDF-9), have been shown to play important roles during follicular development and ovulation. However, despite the wealth of information available in rodent and ruminant species, very little is known about the role of these genes in the porcine ovary. Therefore, the aims of the present study were to investigate the expression of BMP-2, -6, -15 and GDF-9 mRNA in the oocyte, granulosa cells and theca cells of porcine follicles and to determine if changes occur in the levels of expression of these genes during follicular growth. Oocyte, granulosa cells and theca cells were recovered from antral follicles (2-10 mm in diameter) of first parity sows at day 1, 2 and 4 after weaning or at day 14, 16 and 20 of their subsequent estrous cycle. These time points correspond to specific phases of follicular growth; the recruitment of a growing cohort of follicles, the mid-selection phase and the final selection of the pre-ovulatory follicle population. Plasma estrogen and progesterone analysis was used to confirm the expected physiological status of each animal. Follicular fluid estradiol concentration in day 4 and day 20 animals was also used to determine whether the follicles had been exposed to the LH surge. Total RNA was extracted, reverse transcribed and the levels of expression for these genes were determined using real-time PCR. The delta Ct values were calculated using cyclophilin as the internal control and the data were analysed using the general linear model procedure of SAS (SAS Institute Inc., Cary, NC). It is particularly interesting to notice that both BMP-2 and BMP-15 were expressed in all three cell types but were expressed at considerably higher levels in the oocyte (p < 0.001). BMP-2 mRNA levels increased in granulosa cells (P = 0.018) and theca cells (P < 0.001) from the recruitment phase to the mid-selection phase, remained constant until the final selection phase and decreased after the LH surge. BMP-15 mRNA levels also increased in both cell types from the recruitment phase to the mid-selection phase, however the levels decreased in the granulosa cells of the final pre-ovulatory follicle population while remaining constant during this phase in the theca cells (P < 0.05). As opposed to BMP-2, the level of BMP-15 mRNA did not change in response to the LH surge. Finally, BMP-15 mRNA levels showed a strong tendency to decrease in the oocytes from recruitment to the final selection phase (P = 0.054). These results confirm that the oocyte is the main source of BMP-2 and BMP-15 but contrary to other studies, their expression was also found in granulosa cells and theca cells. These differences suggest that follicular growth and development may be regulated differently in swine. (poster)

Expression of Growth Differentiation Factor 9 in the Oocytes Is Essential for the Development of Primordial Follicles in the Hamster Ovary

Postnatal growth differentiation factor 9 (GDF-9) expression in the hamster oocytes precedes the formation of primordial follicles. We examined the functional significance of GDF-9 in primordial folliculogenesis in the hamster ovary using RNA interference knockdown of GDF-9 mRNA and protein expression. Fifteen-day-old fetal ovaries were cultured for 9 d with or without 1 ng FSH, 1 microl Metafectane, 100 nM control nontargeting small interfering RNA (siRNA), GDF-9 siRNA, or GDF-9 siRNA + FSH, and the development of primordial follicles examined. The efficiency of siRNA transfecting ovarian cells in the organ culture was tested by culturing ovaries with siGlo, a nontargeting control siRNA labeled with Cy3. More than 90% of cells in the ovary were siGlo positive, and neither the Metafectane nor the siRNA-induced cellular apoptosis. Control siRNA did not affect the basal levels of GDF-9 mRNA, but GDF-9 siRNA slightly but significantly reduced the level. FSH markedly up-regulated the levels of GDF-9 mRNA and protein, and the effect was completely suppressed by GDF-9 siRNA. However, GDF-9 siRNA did not affect the levels of bone morphogenetic protein receptor IA or beta-actin mRNA. GDF-9 siRNA alone also reduced GDF-9 protein expression. Concurrent with GDF-9 expression, FSH significantly augmented primordial follicle formation, but the effect was abolished by GDF-9 siRNA. These results suggest that endogenous GDF-9 plays an important role in somatic cell differentiation and the formation of primordial follicles. Furthermore, FSH, by virtue of regulating GDF-9 expression, modulates oocyte regulation of primordial follicles formation.

Aberrant expression of growth differentiation factor-9 in oocytes of women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a major cause of female infertility. Despite substantial effort, the etiology and pathogenesis of PCOS and polycystic ovaries (PCO) in women remain unknown. Recent studies in laboratory animals have documented a link between dysfunction of two oocyte growth factors, growth differentiation factor-9 (GDF-9) and bone morphogenetic factor-15 (BMP-15), and aberrant folliculogenesis. Because aberrant follicle development is a hallmark of PCOS, we wondered whether the expression patterns of these growth factors might be disrupted in PCOS and PCO oocytes. To address this issue, we compared the pattern and level of expression of GDF-9 and BMP-15 mRNA in ovaries from normal cycling (n = 12), PCOS (n = 5), and PCO (n = 7) patients. In situ hybridization studies showed that the expression of GDF-9 and BMP-15 is restricted to the oocytes in all ovaries examined. Interestingly, a decreased level of GDF-9 signal was observed in developing PCOS and PCO oocytes, compared with normal. This difference was evident throughout folliculogenesis, beginning at recruitment initiation and continuing through the small Graafian follicle stage. By contrast, there were no qualitative or quantitative changes in the expression of BMP-15 mRNA in PCOS oocytes during folliculogenesis. There were also no significant differences between normal and PCOS and PCOs in the levels of the mRNA encoding the housekeeping gene, cyclophilin. Together, these results indicate that the expression of GDF-9 mRNA is delayed and reduced in PCOS and PCO oocytes during their growth and differentiation phase. Because oocyte-derived GDF-9 is crucial for normal folliculogenesis and female fertility, we suggest that a dysregulation of oocyte GDF-9 expression may contribute to aberrant folliculogenesis in PCOS and PCO women.

Molecular cloning of a cDNA encoding a bovine growth differentiation factor-9 (GDF-9) and expression of GDF-9 in bovine ovarian oocytes and in vitro-produced embryos.

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor-beta superfamily, is known to be expressed specifically in ovaries of various mammalian species and to be important for normal follicular development in mice. In the present study, a cDNA encoding for bovine GDF-9 was isolated and characterized, and expression of GDF-9 mRNA in ovarian oocytes and in-vitro-derived embryos was examined. Isolation of bovine GDF-9 was achieved using the polymerase chain reaction (PCR) with primers based on an ovine GDF-9 cDNA sequence. A 1385 bp cDNA encodes a deduced 453-amino acid sequence which contains the proregion (318 residues) and the mature protein (135 residues) portion. The deduced amino acid sequence of bovine GDF-9 is 98 and 93% identical to ovine GDF-9 and human GDF-9 in the mature portion of the molecule, respectively. Results from reverse transcription (RT)-PCR analysis detected bovine GDF-9 mRNA in preantral follicles (150-200 microm in diameter), early antral follicles (400-800 microm in diameter), and immature oocytes, whereas no detectable PCR signal was observed in cumulus/granulosa cells. In addition, bovine GDF-9 mRNA continued to be expressed in developing embryos up to the 8-cell stage, but was undetectable at the blastocyst stage. These findings give rise to new possibilities regarding an additional physiological role of GDF-9 in early embryonic development as well as in the development of follicles.

Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis.

Growth differentiation factor 9 (GDF-9) is a transforming growth factor-beta family member that is required for normal folliculogenesis in female mice, but its role as a regulator of human fertility is still unclear. We determined here by in situ hybridization and immunohistochemical analyses the localization of the GDF-9 messenger ribonucleic acid (mRNA) and protein during human folliculogenesis. The GDF-9 transcripts were not detected in primordial follicles, but they are abundantly expressed in primary follicles in frozen sections of ovarian cortical tissue material obtained at laparoscopic surgery. We raised antipeptide antibodies against GDF-9 and showed by immunohistochemical studies on paraffin sections of whole human ovaries that the GDF-9 protein is most abundantly expressed in primary follicles. We recently demonstrated that a novel GDF-9-related factor, GDF-9B, is coexpressed with GDF-9 during murine folliculogenesis. We now isolated human GDF-9B complementary DNA and genomic clones and report the unusually restricted expression pattern of human GDF-9B. The human GDF-9B transcript can be detected only in the gonads by RT-PCR analysis, and in situ hybridization studies indicate that it is not expressed in small primary follicles but, rather, in the oocytes of late primary follicles. Functional studies using the Xenopus laeuis embryo model indicate that unlike the transforming growth factor-beta family members activin and bone morphogenetic protein-4, neither GDF-9 nor GDF-9B affects mesoderm induction, suggesting that they may use signaling pathways distinct from those well defined for activin and bone morphogenetic protein-4. We conclude that 1) both GDF-9 mRNA and protein are abundantly expressed in oocytes of primary follicles in human ovary, suggesting that the GDF-9 transcript is translated at this early stage of folliculogenesis; 2) human GDF-9B is specifically expressed in gonads at low levels; and 3) the expression of GDF-9 mRNA begins slightly earlier than that of GDF-9B in the human oocytes during follicular development. Our results are consistent with the suggestion that GDF-9 and GDF-9B may regulate human folliculogenesis in a manner specific to the ovary.

Expression of growth differentiation factor-9 messenger ribonucleic acid in ovarian and nonovarian rodent and human tissues.

Growth differentiation factor-9 (GDF-9) is a member of the transforming growth factor-beta family that is reported to be expressed exclusively in the ovary, specifically in the oocyte. Female mice deficient in GDF-9 are infertile, suggesting that GDF-9 receptor agonists and antagonists may specifically modulate fertility. We now report that GDF-9 messenger RNA (mRNA) is expressed in nonovarian tissues in mice, rats, and humans. GDF-9 mRNA was detected in mouse and rat ovary, testis, and hypothalamus by Northern blot and RT-PCR analyses. The localization of GDF-9 mRNA specifically in oocytes of the mouse ovary was confirmed by in situ hybridization histochemistry. In mouse testis, although localization in Sertoli cell cytoplasm could not be ruled out, mRNA expression was observed in large pachytene spermatocytes and round spermatids. The expression of GDF-9 mRNA in human tissues was assessed by Northern blot and RT-PCR analyses. GDF-9 mRNA was observed in ovary and testis and, surprisingly, in diverse nongonadal tissues, including pituitary, uterus, and bone marrow. Therefore, GDF-9 mRNA expression in rodents is not exclusive to the ovary, but includes the testis and hypothalamus. Furthermore, human GDF-9 mRNA is expressed not only in the gonads, but also in several extragonadal tissues. The function and relevance of nongonadal GDF-9 mRNA are not known, but may affect strategies for contraception and fertility that are based on GDF-9 activity.