scholarly journals 251.Regulation of mouse cumulus expansion by oocyte-secreted growth differentiation factor-9 (GDF-9)

2004 ◽  
Vol 16 (9) ◽  
pp. 251
Author(s):  
R. A. Dragovic ◽  
L. J. Ritter ◽  
F. Amato ◽  
S. J. Scott ◽  
M. Cranfield ◽  
...  
Keyword(s):  
Cumulus Cell ◽  
Cumulus Expansion ◽  
Maximum Expansion ◽  
Paracrine Signalling ◽  
Differentiation Factor ◽  
Protein Receptor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Hyaluronan Synthase 2 ◽  
Candidate Molecule

Oocyte paracrine signalling is necessary for mouse cumulus cell expansion, an important preovulatory process. The oocyte-secreted factor growth differentiation factor-9 (GDF-9) signals through the bone morphogenetic protein receptor-II (BMPR-II) and is currently the primary candidate molecule for the cumulus expansion enabling factor (CEEF). The present study was conducted to determine whether in the mouse GDF-9 is the CEEF. Cumulus oocyte complexes (COC) were collected from eCG-primed mice and the oocyte was microsurgically removed to generate an oocytectomised complex (OOX). An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P�<�0.05) induced by either recombinant mouse GDF-9 (score; mean +/– SEM: 2.7 +/– 0.1), recombinant TGF-μ1 (score: 2.6 +/– 0.2) or co-culture with oocytes (score: 2.3 +/– 0.2). A GDF-9 neutralising antibody mAb-53, raised against hGDF-9, was effective in neutralising the response of OOX complexes to GDF-9 (score: 0.1 +/– 0.1), but had no significant effect on the expansion of OOX complexes co-cultured with oocytes (score: 2.3 +/– 0.2). Likewise, a TGF-μ antagonist neutralised (P�<�0.05) TGF-μ-induced, but not oocyte-induced, expansion of OOX complexes. A soluble portion of the BMPR-II ectodomain, a known GDF-9 antagonist, failed to neutralise oocyte-induced cumulus expansion (P�>�0.05) at the highest dose implying that BMPR-II is not a critical receptor involved in regulating cumulus expansion. Using real-time RT-PCR, hyaluronan synthase-2 (HAS2) mRNA expression by OOXs was upregulated 6- to 7-fold by oocytes and GDF-9. The GDF-9 neutralising antibody mAb-53, partially neutralised GDF-9-induced OOX HAS2 expression, but not oocyte-induced HAS2 expression. This study provides evidence that like TGF-μ1, GDF-9 can enable FSH-induced cumulus expansion, however more importantly demonstrates that neither GDF-9 nor TGF-μ1 alone account for the crucial oocyte-secreted factor regulating cumulus expansion in the mouse.

scholarly journals Role of Oocyte-Secreted Growth Differentiation Factor 9 in the Regulation of Mouse Cumulus Expansion

Endocrinology ◽  
2005 ◽  
Vol 146 (6) ◽  
pp. 2798-2806 ◽  
Author(s):  
Rebecca A. Dragovic ◽  
Lesley J. Ritter ◽  
Samantha J. Schulz ◽  
Fred Amato ◽  
David T. Armstrong ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Neutralizing Antibody ◽  
Cumulus Expansion ◽  
Bone Morphogenetic Protein Receptor ◽  
Secreted Factors ◽  
Differentiation Factor ◽  
Protein Receptor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Hyaluronan Synthase 2

Abstract Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex, which is necessary for ovulation. Oocyte-secreted growth differentiation factor 9 (GDF9) signals through the bone morphogenetic protein receptor II and is currently the primary candidate molecule for the cumulus-expansion enabling factor. This study was conducted to determine whether GDF9 is the mouse cumulus-expansion enabling factor. Cumulus-oocyte complexes were collected from mice, and the oocyte was microsurgically removed to generate an oocytectomized (OOX) complex. OOX complexes treated with FSH alone or recombinant mouse GDF9 alone failed to expand, whereas expansion was induced in the presence of FSH by GDF9, TGFβ1, or coculture with oocytes. A specific GDF9-neutralizing antibody, mAb-GDF9–53, neutralized the expansion of OOX complexes in response to GDF9 but not the expansion of OOX complexes cocultured with oocytes. Using real-time RT-PCR, hyaluronan synthase 2 (HAS2) mRNA expression by OOXs was up-regulated 4- to 6-fold by oocytes and GDF9. Monoclonal neutralizing antibody-GDF9–53 attenuated GDF9-induced OOX HAS2 expression but not oocyte-induced HAS2 expression. A TGFβ antagonist neutralized TGFβ-induced, but not oocyte-induced, expansion of OOX complexes, and when combined with monoclonal neutralizing antibody-GDF9–53 also failed to neutralize oocyte-induced expansion. Furthermore, a soluble portion of the bone morphogenetic protein receptor II extracellular domain, which is a known GDF9 antagonist, completely antagonized GDF9-induced expansion but only partially neutralized oocyte-induced expansion. This study provides further evidence that like TGFβ, GDF9 can enable FSH-induced cumulus expansion, but more importantly, demonstrates that neither GDF9 nor TGFβ alone, nor the two in unison, account for the critical oocyte-secreted factors regulating mouse cumulus expansion.

scholarly journals Differences in the participation of TGFB superfamily signalling pathways mediating porcine and murine cumulus cell expansion

Reproduction ◽  
2011 ◽  
Vol 142 (5) ◽  
pp. 647-657 ◽  
Author(s):  
Robert B Gilchrist ◽  
Lesley J Ritter
Keyword(s):  
Cumulus Cell ◽  
Luciferase Reporter ◽  
Signalling Pathways ◽  
Reporter Assay ◽  
Paracrine Signalling ◽  
Secreted Factors ◽  
Morphogenetic Protein ◽  
Mapk Signalling ◽  
Growth Differentiation Factor 9

It is widely held that mammalian cumulus cell (CC) expansion requires oocyte-paracrine signalling, however in three of the four species studied to date, CC expansion occurs in the absence of the oocyte. This study was conducted to examine the paracrine and SMAD/MAPK intracellular signalling mechanism mediating porcine CC expansion, and to compare these to the mouse. Cumulus–oocyte complexes (COCs) and oocyte-free complexes (OOXs) from pigs and eCG-primed mice were treated in vitro with FSH and a broad range of TGFB superfamily antagonists. Expansion of porcine COCs and OOXs was unaffected by neutralisation of growth differentiation factor 9, TGFB, activin A, activin B and a broad spectrum bone morphogenetic protein antagonist. A SMAD-responsive luciferase reporter assay confirmed that porcine oocytes secreted factors that activate SMAD3 and SMAD1/5/8 in granulosa cells, but murine oocytes activated SMAD3 only. Treatment of COCs with a SMAD2/3 phosphorylation inhibitor (SB431542) partially inhibited porcine CC expansion and expression of TNFAIP6, but ablated murine CC expansion. SB431542 was equally effective at attenuating porcine CC expansion in the presence or absence of the oocyte. By contrast, a SMAD1/5/8 phosphorylation inhibitor (dorsomorphin) had no effect on porcine or murine CC function. Inhibition of ERK1/2 and p38 MAPK signalling pathways prevented porcine COC expansion and expression of most matrix genes examined. The activation of CC SMAD signalling by oocytes, and the requirement of SMAD2/3 signalling for expansion, is notably contrasted in pigs and mice. Nonetheless, porcine CC SMAD2/3 signalling is likely to be needed for optimal matrix formation, possibly by facilitating essential MAPK signals.

231. Mouse oocyte paracrine signalling to cumulus cells by TGF-β superfamily molecules is indispensable for cumulus expansion

2005 ◽  
Vol 17 (9) ◽  
pp. 90
Author(s):  
R. A. Dragovic ◽  
L. J. Ritter ◽  
S. J. Schulz ◽  
D. T. Armstrong ◽  
R. B. Gilchrist
Keyword(s):  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Cumulus Cells ◽  
Transforming Growth Factor Β ◽  
Type I ◽  
Dependent Manner ◽  
Cumulus Expansion ◽  
Maximum Expansion ◽  
Paracrine Signalling ◽  
Growth Differentiation Factor 9

Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex (COC), which is necessary for ovulation. Members of the transforming growth factor-β (TGF-β) superfamily are prime candidates for the mouse cumulus expansion-enabling factor (CEEF), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. This study was conducted to examine TGF-β superfamily processes regulating cumulus expansion. COCs were collected from eCG-primed mice and the oocyte microsurgically removed to generate oocytectomised (OOX) complexes. An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P < 0.05) induced by either GDF9 (score: mean ± SEM, 3.7 ± 0.1), activin A (2.6 ± 0.1), or co-culture with oocytes (3.2 ± 0.2). The type-I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively. We tested the ability of the ALK4/5/7 kinase inhibitor, SB431542, to neutralise cumulus expansion. SB431542 completely neutralised (P < 0.05) the response of OOX complexes to GDF9, activin and oocyte-induced cumulus expansion. SB431542 also neutralised (P < 0.05) COC expansion in a dose dependent manner. Follistatin, an activin antagonist was effective at neutralising the response of OOX complexes to activin (score: 0), but had no significant effect (P > 0.05) on the expansion of OOX complexes co-cultured with oocytes (score: 2.7 ± 0.2). This study provides evidence that activin is not the sole CEEF, but signalling through the ALK4/5/7 pathway is indispensable for mouse cumulus expansion.

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

Endocrinology ◽  
2006 ◽  
Vol 147 (4) ◽  
pp. 1725-1734 ◽  
Author(s):  
Cheng Wang ◽  
Shyamal K. Roy
Keyword(s):  
Protein Expression ◽  
Primordial Follicle ◽  
Postnatal Growth ◽  
Primordial Follicles ◽  
Ovarian Cells ◽  
Differentiation Factor ◽  
Protein Receptor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Interfering Rna

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 μl 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 β-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.

scholarly journals Genetic polymorphisms of fecundity genes in Watish Sudanese desert sheep

2020 ◽  
Vol 13 (4) ◽  
pp. 614-621
Author(s):  
Sara E. Ibrahim Mohamed ◽  
Romaz M. Ahmed ◽  
Khaleel I. Z. Jawasreh ◽  
M. A. M. Salih ◽  
Dalia Mursi Abdelhalim ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Litter Size ◽  
Goodness Of Fit ◽  
Chi Square ◽  
Association Testing ◽  
Genotype Frequencies ◽  
Protein Receptor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Chi Square Test

Background and Aim: The Watish sheep is a strain of desert sheep of smaller size compared to other desert sheep ecotypes, and there is anecdotal evidence that it is endowed with high litter size. The present study was designed for screening for polymorphisms in the known fecundity genes (bone morphogenetic protein receptor type 1B A<G in exon 6, bone morphogenetic protein 15 (BMP15) (FecXB, FecXG, FecXH, and FecXI) in exon2, growth differentiation factor 9 (GDF9) – G1 in exon1 and G8 in exon2 and PRLG<A in intron2) and their association with litter size in Watish. Materials and Methods: The study involved 156 Watish ewes of 2-6 years of age, along with data on litter size in the first, second, and third parity from Sinnar state and contiguous Blue Nile State. Genomic DNA was isolated and genotyped using polymerase chain reaction-restriction fragment length polymorphism. Allele and genotype frequencies were calculated by direct counting. Chi-square test for goodness of fit was performed for agreement with Hardy-Weinberg expectations and association testing. Results: The results demonstrated that all individuals were non-carriers for the target mutations of FecB, BMP15 (FecXB, FecXH, and FecXI), and GDF9-G8. With regard to the GDF9-G1 gene, the genotypic frequencies were 0.07% (G+) and 0.93% (++), in FecXG gene they were 0.993% (++) and 0.006% (B+), in PRL gene 0.516(++), 0.347(B+), and 0.137(BB). The Chi-square test showed a non-significant association between ewe's type of birth and the detected mutations genotypes. Conclusion: These results preliminarily indicated that GDF9-G1, BMP15 (FecXG), and PRL genes might have had some contribution for improving litter size in Watish Sudanese sheep. However, further studies using larger samples are needed to detect the effects of those mutations on Watish sheep litter size.

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