The subtle balance of insulin and thyroxine on survival and development of primordial follicles cultured in vitro enclosed in ovarian tissue

Abstract

Thyroid hormones have presented a positive hormonal interaction on follicular development of secondary follicles and oocytes from antral follicles; however, the effect of thyroid hormones on primordial follicles is unclear. Here we investigated the in vitro effects of combined insulin and thyroxine on caprine primordial follicle survival and development. Ovarian tissues were cultured for 1 or 7 days using 10 ng/ml (low) or 10 {micro}g/ml (high) insulin in the absence or presence of thyroxine at 0.5, 1 or 2 {micro}g/ml. Thereafter, follicular survival and development, gene expression related to apoptosis (Bcl2/Bax), insulin and thyroid receptors, and estradiol and reactive oxygen species production were evaluated. In low-insulin conditions, supplementation with 2 {micro}g/ml thyroxine maintained follicular survival similar to non-cultured control, while 0.5 {micro}g/ml thyroxine enhanced the survival (P<0.05) in comparison to thyroxine-free treatment. Only treatments containing low-insulin and thyroxine at 0.5 or 2 {micro}g/ml increased (P<0.05) reactive oxygen species production from day 1 to day 7. Contrarily to high-insulin containing medium, the presence of thyroxine in low-insulin medium yielded higher stromal cell density (P<0.05). There were higher (P<0.05) estradiol production and Bcl2/Bax ratio in low-insulin versus high-insulin treatments on day 1 and 7, respectively. High levels of both insulin and thyroxine showed better follicular development (P<0.05), yielding great follicle and oocyte diameter. Finally, the high-insulin level led to insulin and thyroid receptors expression reduction as compared to non-cultured control. In conclusion, the combination of low concentrations of insulin and thyroxine better maintained follicle survival, while high levels ensured better follicular development.