Insulin promotes preantral follicle growth and antrum formation through temporal expression of genes regulating steroidogenesis and water transport in the cat

Abstract

The aims of the present study were to determine the effects of insulin, in vitro, on: (1) the viability and growth of domestic cat ovarian follicles; (2) mRNA expression of genes regulating steroidogenesis (cytochrome P450 family 17 subfamily, A polypeptide 1 (Cyp17a1), cytochrome P450 family 19 subfamily, A polypeptide 1 (Cyp19a1) and steroidogenic acute regulatory protein (Star)) and water transport (aquaporins (AQPs) Aqp1, Aqp3, Aqp7, Aqp9); and (3) steroid production (17β-oestradiol (E2), progesterone (P4), androstenedione (A4)). Cat secondary follicles were isolated from ovarian cortices and cultured in 0 (Control), 1 or 10 µg mL−1 insulin for 14 days (Day 0 = culture onset). Follicle and oocyte viability (based on neutral red staining), diameter and antrum formation were assessed every 72 h and at the end of incubation (Day 14). Expression of steroidogenic and water transport genes was evaluated on Days 0, 6 and 12, and E2, P4 and A4 concentrations in the culture medium were determined on Day 12. By Day 14, 1 and 10 µg mL−1 insulin had significantly promoted (P < 0.05) both antrum formation in a mean (± s.e.m.) 26.9 ± 9.0% and 78.0 ± 10.0% of follicles respectively, and follicle growth (diameter 151.4 ± 4.5 and 169.9 ± 10.5 µm respectively) compared with Control (antrum formation in 3.3 ± 3.3% of follicles and follicle diameter 129.1 ± 6.6 µm). High insulin (10 µg mL−1) treatment increased follicle viability compared with Control (86.0 ± 9.8% vs 38.1 ± 10.9% respectively; P < 0.05). However, insulin had no beneficial effect (P > 0.05) on oocyte diameter. Cyp17a1 expression on Days 6 and 12 was higher (P < 0.05) in follicles cultured in the low (1 µg mL−1) compared with high (10 µg mL−1) insulin treatment, with no significant difference between low or high insulin vs Control groups. Star expression was higher (P < 0.01) in the low insulin compared with Control group on Day 6, but Star was undetectable in the high insulin group by Day 12. Compared with high insulin, low insulin increased (P < 0.05) Aqp1 expression on Day 6, but there were no significant differences between these two groups on Day 12. In contrast, high insulin decreased (P < 0.05) Aqp9 transcript levels compared with Control. Only P4 production was affected by insulin, with P4 concentrations in the medium being higher (P < 0.05) in the low compared with high insulin and Control groups. In summary, the findings indicate that insulin promotes cat ovarian follicle growth and survival in vitro, including enhanced antrum formation, with the likely mechanism involving temporal expression of Cyp17a1, Star and Aqp9 genes.