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UBC Theses and Dissertations

Investigation of the presence of GnRH and GnRH-R system in bovine oocytes, sperm and early embryos and their functional role in reproduction Perera, Uswatteliyanaralalage Felicia Ruwanie


The objectives of this study were to investigate: 1) the effect of GnRH agonist on oocyte maturation, sperm function and fertilization, 2) the presence of GnRH-R in bovine oocytes, sperm and early embryos. To examine the effect of GnRH agonist on sperm function, sperm were incubated in modified Tyrode’s medium with 0, 0.2, 0.4, 0.8 and 1.2 µgmL⁻¹ of buserelin and with1 µgmL⁻¹ P₄ for 3 h. Acrosome status in each group was assessed at 0 h and 3 h. For zona-binding assay, in vitro matured oocytes were co-incubated with sperm in different concentrations of buserelin and P₄ for 4 h and the zona-bound sperm in each treatment group was determined. Acrosome reacted percentage were higher in sperm treated with 0.4, 0.8 µgmL⁻¹ buserelin than the control group (p<0.001). The number of zona-bound sperm were higher in 0.8 µgmL⁻¹ buserelin compared to negative control (p<0.01). Effect of buserelin was blocked by antide. To investigate the effect of GnRHa on maturation of bovine oocytes 0.8 µgmL⁻¹ buserelin was added to the in vitro maturation media and maturation rate was obtained after 24 h against control groups with FSH and without FSH. To assess the effect on fertilization rate one group of oocytes undergoing in vitro fertilization was treated with 0.8 µgmL⁻¹ buserelin, while in vitro fertilization without buserelin served as control. Maturation rate was not different among the groups and were 55.14 ± 0.39, 50.69 ± 0.42 and 43.5 ± 0.46 % for oocytes treated with FSH, buserelin and negative control, respectively. No difference was observed on fertilization rates, with fertilization rates being 55.25 ± 0.89 and 51.25 ± 0. 51% for buserelin treated and the control group, respectively. Presence of GnRH-R protein and mRNA on bovine sperm, oocytes, cumulus cells and early embryos were studied using immunostaining technique and RT-PCR. No receptors for GnRH were detected in bovine sperm, oocytes or early embryos studied and expression of GnRH-R mRNA was also not evident. GnRH-R mRNA was expressed by cumulus cells, immature and mature COC. GnRH receptor mRNA expression by mature COC was higher than immature COC (2.01±0.12 and 0.89±0.2).

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