Maturation-associated changes in Sertoli cells following in vitro culture of frozen-thawed prepubertal mouse testicular tissue

To preserve the fertility of prepubertal boys with cancer, testicular tissue can be cryopreserved in the hope of subsequently inducing their maturation after transplantation or in vitro culture for natural conception or medically assisted reproduction. Testicular tissue maturation procedures are currently at an experimental stage and mainly developed in animal models. Among these, in vitro sperm yield production in cultures of mouse prepubertal testicular explants is relatively low. Despite the essential role of Sertoli cells in the progression of spermatogenesis, the maturation and functionality of these somatic cells under organotypic culture have never been thoroughly studied. This work aims to investigate Sertoli cell maturation and functionality after in vitro maturation of frozen-thawed mouse prepubertal testicular. Frozen-thawed testicular fragments from 6-day-old prepubertal mouse tissues were cultured for 16 days (D16) and 30 days (D30). Immunohistochemical, RT-qPCR analyses as well as measurements of intratesticular AMH and inhibin B levels by ELISA were performed on cultured explants and on postpartum (d pp ) in vivo controls. The number of Sertoli cells was higher at D30 than at 36 d pp . In addition, the expression of immature and mature Sertoli cell markers were similar between in vitro cultured explants and in vivo control tissues. Moreover, inhibin B, a marker of Sertoli cell functionality, was detected in cultured explants at D16 and D30. However, supplementation of culture media with 5 ng/mL of FSH, a pituitary hormone that regulates Sertoli cell functions, had little impact on the progression of the first wave of spermatogenesis from cultured prepubertal explants.