Optimization of oxygen concentration and glycosphingolipid effects on spermatogenesis in mouse testicular culture

Our previous research highlighted the importance of hormones, free fatty acids, lysophospholipids, and antioxidants in supporting in vitro spermatogenesis in mice through the development of a chemically defined medium (CDM). While it was possible to induce round spermatids through the culture of testicular tissue in medium containing these factors, challenges remained with the low efficiency of spermatogenesis and the differentiation into elongating spermatids. This study aimed to further improve the in vitro spermatogenesis system by exploring optimal oxygen concentrations and identifying additional factors necessary for spermiogenesis. In addition to the conventional oxygen concentration of 20%, three hypoxic environments (15%, 10%, and 7%) were tested, and an oxygen concentration of 10% was found to be optimal for the maintenance and differentiation of germ cells in vitro. To address the limited tissue growth observed under low oxygen conditions, we further supplemented the culture medium with glucose and insulin, which led to a significant increase in tissue size. However, this enhancement in growth did not translate into improved spermatogenic differentiation. Following this, we explored factors involved in the induction of elongating spermatids. The addition of glycosphingolipids to the culture medium modestly promoted the formation of elongating spermatids, suggesting a potential role of glycosphingolipids in haploid cell differentiation. This study offers new insights into the environmental conditions and factors that influence spermatogenesis in mice.