Lipid droplets-mitochondria interaction involved in testosterone synthesis in Leydig cells of dairy goats (Capra hircus)

Leydig cells (LCs) are the primary testosterone-producing cells. Their steroidogenic capacity is heavily influenced by the postnatal development of the LC lineage. The final developmental stage of LCs involves transition from immature LCs (ILCs) to adult LCs (ALCs) during puberty. However, the morphological and functional changes that occur during this transition in most mammals remain unclear. In this study, we conducted a comparative analysis of morphological and functional changes in developing LCs in dairy goats. The results revealed that during the transition from ILC to ALC, the ovoid LC transformed into an irregular round shape with a well-developed smooth endoplasmic reticulum (sER), numerous mitochondria, and small lipid droplets (LDs) distributed in the cytoplasm. Subsequently, the isolated primary ILCs were matured using chorionic gonadotropin (CG). CG stimulation increases the expression of steroidogenic genes and decreases the levels of testosterone-metabolizing enzymes. In vitro studies have shown that small LDs are the preferred source of cholesterol substrates in steroidogenesis, and that their interaction with mitochondria facilitates free cholesterol transport. Further analyses using confocal microscopy, ultrastructural analysis, and co-immunoprecipitation consistently demonstrated that vimentin filaments contributed to the interaction between LDs and mitochondria. Specifically, the knockdown of vimentin using siRNA substantially reduced the physical contact between LDs and mitochondria, resulting in inadequate cholesterol transport and ultimately impaired steroidogenesis. Overall, our findings indicate that significant morphological and functional changes occurred during the transition from ILCs to ALCs in goats. The cytoskeleton, primarily composed of vimentin filaments, is an important component of the interactions between LDs and mitochondria and contributes to cholesterol transport during testosterone synthesis.