Macroautophagy involved in testosterone synthesis in Leydig cells of male dairy goat (Capra hircus)

Testosterone is an important steroid hormone that is indispensable for male sexual development and the reproductive system. Leydig cells (LCs), where autophagy is extremely active, reside in the testicular interstitium and are the major sites of testosterone production. However, the ultrastructural characteristics and the functional role of autophagy in LCs of livestock remain unknown. In this study, the LCs of the dairy goats were investigated to identify the steroidogenic activity and autophagy levels at different ages of development by light microscopy, immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM). Morphological results showed that the steroidogenic activity (3β-HSD staining) and ultrastructural characteristics of the LCs were changed with increasing age. TEM results demonstrated that the organelles involved in testosterone synthesis, e.g., smooth endoplasmic reticulum, mitochondria, and lipid droplets, were abundantly distributed within the cytoplasm of LCs in pubertal and adult testes. Moreover, autophagy activity was enhanced in the testes at pubertal and adult stages compared with that at the juvenile stage. Several different autophagic vacuoles, including pre-autophagosomes, autophagosomes, and autolysosomes, were observed within the cytoplasm of LCs from pubertal and adult testes. However, immunofluorescent staining and TEM results showed that no typical lipophagic or mitophagic vacuoles were observed in the cytoplasm of LCs. Furthermore, primary LCs from dairy goats were used to study the effect of autophagy on testosterone production. After treatment with 3-methyladenine (3-MA, an autophagy inhibitor), the primary LCs decreased testosterone production. In contrast, treatment with rapamycin (an autophagy activator), enhanced steroidogenesis in LCs. Collectively, these in vivo and in vitro results suggested that autophagy activity is related to steroidogenesis in LCs of dairy goats, which may ultimately influence the spermatogenesis and fertility of these animals.