T(3) preserves ovarian granulosa cells from chemotherapy-induced apoptosis

Infertility is a dramatic and frequent side effect in women who are undergoing chemotherapy. Actual strategies are mainly focused on oocyte cryopreservation, but this is not always a suitable option. Considering the key role that granulosa cells play in follicle life, we studied whether thyroid hormone 3,5,3'-triiodothyronine (T(3)) protects rat ovarian granulosa cells from chemotherapy-induced apoptosis. To this aim, a cell line was established from fresh isolated rat granulosa cells and named rGROV. Cells were exposed to paclitaxel (PTX) and T(3), and apoptosis, cell viability, and cell cycle distribution were analyzed under different conditions. First, the integrity of the steroidogenic pathway was demonstrated, and the presence of thyroid receptors, transporters, and deiodinases was confirmed by quantitative PCR. Cells were then exposed to PTX alone or contemporary to T(3). MTT and TUNEL assays revealed that while there was a relevant percentage of dying cells when exposed to PTX (40-60%), the percentage was sensibly reduced (20-30%) in favor of living cells if T(3) was present. Cell cycle analysis showed that cells exposed to PTX alone were first collected in G2 and then died by apoptosis; on the other hand, the T(3) granted the cells to cycle regularly and survive PTX insult. In addition, western blot and FCM analyses confirmed that caspases activation, casp 3 and Bax, were downregulated by T(3) and that Bcl2 and cyclins A and B together with cdk1 were upregulated by T(3). In conclusion, we demonstrated that thyroid hormone T(3) can counteract the lethal effect of taxol on granulosa cells.