| Transforming growth factor-β (TGFβ) superfamily have been shown as critical regulators of germ cell development, which acted as the extracellular ligand of signaling transduction that regulates the proliferation, differentiation, apoptosis and other cell behaviors. Growth differentiation factor 9 (GDF9) is a member of the TGFβ superfamily, which plays a critical role in ovarian follicular development and ovulation rate in human, goat and mice. In addition, GDF9 and its receptor have been detected in mice, cat and alpaca testis, but the role of GDF9 in testis has not been well elucidated. In vivo studies in mice found GDF9 can affect the expression and localization of the tight-junction-associated protein in tight junctions (TJs) between Sertoli cells. However, studies of GDF9 and bovine reproduction are relatively rare. In this study, we analyzed the associations between GDF9 gene polymorphism and the reproductive traits such as superovulation performance, embryo transfer pregnancy rates and semen quality; secondly, the study was designed to investigate the effects of GDF9 on in vitro development and survival of bovine preantral follicles; Thirdly, we identified the presence and distribution of GDF9 and its receptor in bovine testis, and revealed the effects of GDF9 on Sertoli cell proliferation, differentiation, and apoptosis, as well as on the tight junctions between Sertoli cells. The major results were described as follows:1. Effect of bovine GDF9 gene polymorphisms on the superovulation performance, pregnancy rates after embryo transfer and semen quality.Polymorphisms of GDF9 gene for bovine superovulation performance in 171 Chinese Holstein cows treated for superovulation were detected by PCR-RFLP, PCR-SSCP and sequencing. Furthermore, GDF9 gene was analyzed in 132 Luxi catties, and evaluated its associations with the pregnancy rates after embryo transfer and the concentrations of progesterone (P4) and estrogen (E2). Finally, the GDF9 was chosen as a candidate gene to investigate their effects on 129 Holstein bulls’ semen quality. The major results were as follows:(1) Two mutations of A485T and A625T were found in intron I of bovine GDF9 gene. Three genotypes of each mutation were detected in Chinese Holstein cows and Holstein bulls. These two mutations significantly associated with the number of transferable embryos, and the A625T polymorphism had significant effects on the total number of ova. Furthermore, these two mutations were associated significantly with Holstein bull’s semen quality. These results suggested that GDF9 was major gene controlling reproductive traits of Holsteins.(2) One mutation of A625T was identified in Luxi cattles, and three genotypes were detected. This mutation had no significant effect on the pregnancy rates and concentrations of E2 and P4. These results suggested that GDF9 has no effect on the establishment and maintenance of pregnancy.2. Effects of GDF9 on the survival, activation, and growth of cattle preantral folliclesThis study aims to investigate the effects of growth and differentiation factor-9 (GDF9) in combination with follicle stimulating hormone (FSH) on the activation, survival and growth of cattle preantral follicles by histological analysis and TUNEL staining. The major results were as follows:(1) GDF9 in combination with FSH inhibited apoptosis of follicles and stromal cells after the long-term culture of ovarian cortex:FSH+GDF9 mediums had a lesser proportions of apoptotic follicles than FSH medium, and significantly inhibited stromal cells from becoming apoptotic at 22 days after culture;(2) promoted the primordial to primary and secondary follicles transition:FSH+ GDF9 medium had a lesser proportion of the primordial follicles than FSH medium after 3 days of culture, as a result of a greater proportion of the primary follicles after 3 days of culture and secondary follicles after 14 days of culture compared to FSH medium;(3) promoted the growth of primary and secondary follicles:Tissues cultured in FSH +GDF9 medium resulted in increased follicles diameter for the primary and secondary follicles relative to those in the FSH medium after 7 days of culture.These findings indicate that GDF9 in combination with FSH can improve the survival, activation, and growth of cattle primordial follicles after the long-term culture of ovarian cortex.3. Expression and localization of GDF9 and its receptor in bovine testisThe present study aims to investigate the expression and localization of GDF9 and its receptor (ALK5 and BMPRII) in prepubertal bovine testis and sertoli cell using RT-PCR, Western blot and immunohistochemical staining The major results were as follows:(1) GDF9 mRNA and protein were expressed and located in spermatogonia and Sertoli cells;(2) GDF9 receptors mRNA, ALK5 and BMPRII, have been detected in bovine testis and sertoli cell.These findings indicate that GDF9 can regulate spermatogenesis via directly or indirectly affects sertoli cell function in the testis.4. Effect of GDF9 on the development and functions of Sertoli cellThe study detected the effects of GDF9 on apoptosis, proliferation and the tight junctions between Sertoli cells using real-time-PCR, flow cytometry, transepithelial electrical resistance (TEER). The major results were as follows:(1) GDF9 inhibited apoptosis of Sertoli cells, a significant reduction in the percentage of apoptosis cell was found in all various concentrations of GDF9 compared to control group;(2) GDF9 promoted proliferation of Sertoli cells and significantly increased the proportion of G2 phase cell,100 ng/mL GDF9 medium had a significantly higher proportion of G2 phase cell than control group after cultured for 24h and 48h;(3) Addition of GDF9 significantly decreased the junctional protein claudin-11 mRNA expression, thereby disrupting tight junction integrity.These findings indicate that the ability of GDF9 to regulate testicular development and spermatogenesis is partly via their effects on apoptosis, proliferation and the tight junctions between Sertoli cells. |
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