The effects of equine growth hormone on the in vitro maturation of equine oocytes

The objective of this dissertation was to evaluate the effect of equine growth hormone (eGH) on the in vitro maturation (IVM) of equine oocytes.;We evaluated the effect of eGH on the IVM of equine oocytes and the levels of steroidogenesis by cumulus-oocyte complexes (COCs) matured for 30 h at 38.5°C in air with 5% CO2. Oocytes were stained for nuclear and cytoplasmic evaluation via confocal microscopy. Following incubation with 400 ng/ml of eGH, 41.47% of oocytes were classified as mature vs. 30.56% of oocytes in the control group (p=0.08). On average, media in which COCs had been cultured with eGH had significantly higher testosterone (T4) and progesterone (P4) levels compared to the control group (p=0.04). This suggests that eGH has an effect on the nuclear and cytoplasmic maturation and increases P4 and T4 production during oocyte maturation.;The second objective was to investigate the localization of equine eGH-receptor (eGH-R) in the follicular wall, the oocyte and the COCs. Real-time reverse transcription-PCR and immunohistochemistry methods were used to localize eGH-R. Immunohistochemical showed positive immunostaining for eGH-R in the cumulus cells, the oocyte and the granulosa cells of the follicle. Quantification by TaqMan PCR showed that relative gene transcription for eGH-R was higher in cumulus cells and COCs at the start of IVM than in COCs cultured for 30h. The presence of eGH-R in equine ovarian follicular structures might mediate a positive effect of eGH on oocyte maturation.;The final objective was to investigate the interactions between eGH, insulin-like growth factor-I, estradiol (E2), gonadotropins, and fetal calf serum (FCS) on the IVM and cytoskeleton organization of equine oocytes. Oocytes were in vitro matured and stained with markers for microtubules, microfilaments and DNA. Following incubation with eGH, E 2, gonadotropins and FCS, 37.93% of oocytes were classified as mature vs. 17.65% of oocytes in the control group (p=0.05). Microtubules were detected at the metaphase spindle having a symmetrical barrel shaped structure with the chromosomes aligned along its midline. We conclude that the uses of E 2, gonadotropins and FCS in the presence of eGH increase the number of oocytes reaching M-II stage of development.