Supplementation of granulosa cells conditioned medium with pyruvate and testosterone could improve early follicular development in cultured 1-day-old mouse ovaries.

In vitro activation of primordial follicles could serve as a safe method to preserve fertility in patients with cancer subjected to ovarian tissue cryopreservation during oncotherapy, however the culture medium for this purpose requires to be optimized. Granulosa cell conditioned medium (GCCM) has been recognized to enhance primordial follicle activation and the present study was conducted to understand whether addition of pyruvate, a combination of insulin, transferrin and selenium (ITS) or testosterone to GCCM could improve its efficiency in this regard. To this end, 1-day-old mouse ovaries were cultured in four different media including CON (control; containing GGCM only), PYR (containing GCCM plus pyruvate), ITS (containing GCCM plus ITS) or TES (containing GCCM plus testosterone) for 11 days. Furthermore, follicular dynamics and gene expression of factors involved in follicular development were assessed using histological examination and RT-PCR, respectively, on days 5 and 11 of culture. Pyruvate decreased follicular activation, but it enhanced the progression of follicles to the primary stage. Moreover, it upregulated Bmp15 and Cx37 (P < 0.05). In the ITS group, activation of follicles was not affected and total number of follicles was reduced by day 11 of culture. Additionally, ITS downregulated Pi3k, Gdf9, Bmp15 and Cx37 (P < 0.05). Although testosterone did not affect primordial follicle activation, it enhanced the development of follicles up to the preantral stage (P < 0.05). Furthermore, testosterone inhibited the expression of Pten but stimulated the expression of Gdf9 and Cx37 (P < 0.05). In conclusion, the present study revealed that inclusion of pyruvate and testosterone into GCCM could enhance the early development of follicles in cultured 1-day-old mouse ovaries.

Incorporation of arginine, glutamine or leucine in culture medium accelerates in vitro activation of primordial follicles in 1-day-old mouse ovary.

In vitro activation of primordial follicles provides cancer patients subjected to oncotherapy with a safe therapeutic strategy for fertility preservation, however a successful protocol for activation of primordial follicles in prepubertal patients has not yet been defined comprehensively. There is evidence that amino acids such as leucine, arginine and glutamine could stimulate the mammalian target of rapamycin (mTOR) pathway, which plays a pivotal role in primordial follicle activation. Nevertheless, there has been no report that elucidates the effect of these amino acids on in vitro development of ovarian follicles. Therefore, the present study was conducted to evaluate the effects of these amino acids and their combination on the formation and activation of primordial follicles in 1-day-old murine ovaries during an 11-day culture period. The experimental groups consisted of base medium (BM), base medium + arginine (ARG), base medium + glutamine (GLU), base medium + leucine (LEU) and base medium + a combination of arginine, glutamine and leucine (AGL). The proportions of different stages of ovarian follicles and gene expression of regulatory factors were assessed using histology and quantitative real-time PCR on days 5 and 11 of culture. The proportion of transitional and primary follicles was greater in all amino acid-treated groups compared with the BM group (P < 0.05). Moreover, leucine resulted in elevated expression of Gdf9 and Bmp15, and glutamine augmented the expression of Pi3k on day 11 of culture. In conclusion, the present study showed that inclusion of leucine, glutamine, arginine or their combination in the culture medium for murine ovarian tissue could accelerate the activation of primordial follicles and alter the expression of the corresponding factors.