Expression of genes involved in testosterone and 17β-estradiol production in ex vivo cultured prepuberal mouse testes

Abstract

A highly efficient testicular culture system involving a gas-liquid interface has emerged. This method has demonstrated the ability, although a low rate, to differentiate male germ cells from neonatal spermatogonia to haploid cells. However, the model requires thorough examination to facilitate forthcoming experimental approaches in physiological and toxicological reproductive studies. Sequential male germ cell proliferation and differentiation requires the production and the action of steroid hormones. Consequently, our aims were to record production levels of testosterone (Tes) and 17β-estradiol (E2) and to examine the expression of genes encoding proteins that are involved in such hormone production in ex vivo cultured testes. The mRNA expression patterns of 17β-Hydroxysteroid dehydrogenase 1 (17β-Hsd1), 3β-Hydroxysteroid dehydrogenase (3β-Hsd1), Sex hormone binding globulin (Shbg) and Cytochrome P450-Aromatase (Cyp19a1) were followed in 6.5 days old C57BL/6J mouse testis explants cultured during 44 days and were compared to those recorded during in vivo development. Notoriously, 17β-Hsd1 and 3β-Hsd1 had a similar expression pattern ex vivo vs. in vivo, but the levels of Tes decreased from 5 to 20 days in culture and thereafter remained low. Concomitantly, the mRNA expression of Shbg was up-regulated with time in the explants, linked to the low Tes production. Finally, as in vivo, Cyp19a1 expression tended to increase from days 10 to 30, while the E2 levels recorded from 15 to 20 days were the highest. The latter was associated with the appearance of haploid spermatids. Interestingly, when retinoic acid was added to the media, the levels of Tes were increased and mRNA expression levels of 17β-Hsd1 and 3β-Hsd1 were up-regulated, while those of Shbg were down-regulated. Our results demonstrated active production of steroid hormones in the explants and suggest that modulation of these hormones could enhance the ex vivo spermatogenesis process. Supported by SGCyT UNS-PGI-UNS [24/B272 to GMO], FONCyT, [PICT2020- 02056 to GMO] and SGCyT UNS-PGI 24/B341 to GMO and JML.