The subtle balance of insulin and thyroxine on survival and development of primordial follicles culturedin vitroenclosed in ovarian tissue

Abstract

23Thyroid hormones have presented a positive hormonal interaction on follicular 24 development of secondary follicles and oocytes from antral follicles; however, the effect 25 of thyroid hormones on primordial follicles is unclear. Here we investigated the in vitro 26 effects of combined insulin and thyroxine on caprine primordial follicle survival and 27 development. Ovarian tissues were cultured for 1 or 7 days using 10 ng/ml (low) or 10 28 µg/ml (high) insulin in the absence or presence of thyroxine at 0.5, 1 or 2 µg/ml. 29Thereafter, follicular survival and development, gene expression related to apoptosis 30 (Bcl2/Bax), insulin and thyroid receptors, and estradiol and reactive oxygen species 31 production were evaluated. In low-insulin conditions, supplementation with 2 µg/ml 32 thyroxine maintained follicular survival similar to non-cultured control, while 0.5 µg/ml 33 thyroxine enhanced the survival (P<0.05) in comparison to thyroxine-free treatment. Only 34 treatments containing low-insulin and thyroxine at 0.5 or 2 µg/ml increased (P<0.05) 35 reactive oxygen species production from day 1 to day 7. Contrarily to high-insulin 36 containing medium, the presence of thyroxine in low-insulin medium yielded higher 37 stromal cell density (P<0.05). There were higher (P<0.05) estradiol production and 38 Bcl2/Bax ratio in low-insulin versus high-insulin treatments on day 1 and 7, respectively. 39High levels of both insulin and thyroxine showed better follicular development (P<0.05), 40 yielding great follicle and oocyte diameter. Finally, the high-insulin level led to insulin 41 and thyroid receptors expression reduction as compared to non-cultured control. In 42 conclusion, the combination of low concentrations of insulin and thyroxine better 43 maintained follicle survival, while high levels ensured better follicular development. 44 45 3 46 65 20). Nevertheless, even though few in vitro studies reported the effects of TH on the 66 development of late preantral follicle categories to the best of our knowledge of its in 67 vitro role on primordial follicle survival and development remains unknown. 68 Among the various hormones that act on folliculogenesis, insulin has an important 69 role in follicle development due to its action on cell survival and proliferation (21). Our 4 70team has shown that insulin effects could be influenced by follicular category (22, 23), as 71 well as its interactions with other substances (24)(25)(26). For instance, the use of insulin at 72 10 ng/ml during in vitro culture of either primordial (22), secondary or tertiary follicles 73 (23) yielded greater oocyte growth than using 10 µg/ml insulin. On the other hand, 74 secondary follicles cultured in vitro with insulin at 10 µg/ml in association to FSH (24) 75 and GH (25), or FSH + VEGF (26) enhance meiosis resumption in goat oocytes. 76Although the hormonal network is a complex process, the balance of insulin and 77 thyroxine concentration on in vitro primordial follicles development is unclear. 78Therefore, the novelty of the...