The involvement of growth hormone in equine oocyte maturation, receptor localization and steroid production by cumulus–oocyte complexes in vitro

Abstract

The objectives of this study were to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, and expression and subcellular localization of eGH-receptors (eGH-R) on equine ovarian follicles. Cumulus–oocyte complexes (COCs) were recovered by aspirating follicles <30mm in diameter from abattoir-derived ovaries. The COCs were morphologically evaluated and randomly allocated to be cultured in either a control maturation medium or supplemented with 400ng/mL eGH, for 30h at 38.5°C in air with 5% CO2. The COCs were stained with 10μg/mL propidium iodide and 10μg/mL fluorescein isothiocyanate-labeled Lens culinaris agglutinin. Chromatin configuration and distribution of cortical granules were assessed via confocal microscopy. Compared to control, COCs incubated with eGH had: more oocytes that reached metaphase II (35/72, 48.6% vs. 60/89, 67.4%, respectively; P=0.02); greater concentrations of testosterone (0.21±0.04 vs. 0.06±0.01ng/mL; P=0.01), progesterone (0.05±0.01 vs. 0.02±0.00ng/mL; P=0.04), and oestradiol (76.80±14.26 vs. 39.58±8.87pg/mL; P=0.05) in the culture medium, but no significant differences in concentration of androstenedione. Based on Real Time RT-PCR analyses, expression of the eGH-R gene was greater in cumulus cells and COCs at the start than at the end of in vitro maturation. Positive immunostaining for eGH-R was present in cumulus cells, the oocytes and granulosa cells. In conclusion, addition of eGH to maturation medium increased rates of cytoplasmic maturation and had an important role in equine oocyte maturation, perhaps mediated by the presence of eGH-R in ovarian follicles.