128 THE ROLE OF ENDOTHELINS IN REGULATING BOVINE GRANULOSA CELLS STEROIDOGENESIS

Endothelins are a group of vasoactive 21 amino acid peptides reported to play roles in steroidogenesis, folliculogenesis, and ovulation (Bridges et al. 2012 Life Sci. 91, 501–506). Nevertheless, the role of endothelins in regulating steroidogenesis in the bovine species requires further investigation. Thus, the objective of this study was to investigate the effects of endothelin 1 (ET-1) and endothelin 2 (ET-2) on bovine granulosa cell (GC) steroidogenesis. Bovine ovaries were obtained from a local abattoir. Follicular fluid was aspirated from small (1–5 mm) follicles and GC were isolated and exposed to various treatments (ET-1, ET-2, or ET-1 plus ET-2 with FSH and with or without insulin-like growth factor-1). In replicated experiments, culture medium was removed and analysed for steroid production via radioimmunoassay. Granulosa cells were either harvested with trypsin and counted using a Coulter Counter or collected with Trizol for RNA extraction and quantification via real-time PCR (18S rRNA was used as a housekeeping gene). Steroid production was expressed as nanograms (in the case of progesterone) and picograms (in the case of oestradiol) per 105 cells per 24 h. Relative quantity of target gene mRNA was expressed as 2–ΔΔCt using the relative comparative threshold cycle (Ct) method. Data were analysed via ANOVA and the general linear models (GLM) procedure of SAS for Windows (SAS Institute Inc., Cary, NC). If a significant main effect was identified, differences among means were determined by Fisher’s protected least significant differences test. The values were reported as least squares means ± standard error of the mean. In the presence of insulin-like growth factor-1, ET-1 significantly inhibited oestradiol production at 300 ng mL–1 (100.30 ± 11.05; P < 0.05), but not at 30 ng mL–1 (114.47 ± 11.05; P > 0.05) in comparison to the control (141.21 ± 11.05), whereas no differences were observed for progesterone production at 300 ng mL–1 (60.11 ± 7.11; P > 0.05) or at 30 ng mL–1 (64.02 ± 7.11; P > 0.05) in comparison to control (76.75 ± 7.11). ET-2 also significantly inhibited oestradiol production at 300 ng mL–1 (91.08 ± 11.87; P < 0.01), but not at 30 ng mL–1 (112.77 ± 11.87; P > 0.05) in comparison to the control in the presence of insulin-like growth factor-1. No significant effect of ET-1 and ET-2 was observed on steroidogenesis of granulosa cells cultured without insulin-like growth factor-1. Consistent with steroids production data, real-time PCR results indicated that, in the presence of IGF-1, ET-1 (5.66 ± 1.05) and ET-2 (5.65 ± 1.05) inhibited (P < 0.05) aromatase gene expression compared to controls (11.33 + 1.05), and ET-1 plus ET-2 (2.42 ± 1.05) reduced (P < 0.05) expression below that observed with either alone. No effect of ET-1 (4.38 ± 0.95; P > 0.05), ET-2 (5.94 ± 0.95; P > 0.05), or ET-1 plus ET-2 (4.57 ± 0.95; P > 0.05) was observed for side-chain cleavage enzyme (CYP11A1) in comparison to controls (4.4 ± 1.07). Altogether, these results indicate that endothelins are involved in the regulation of steroidogenesis of bovine GC.