Because the different developmental stages of Leydig cells show distinct steroidogenic capacities, abnormal Leydig cell proliferation or differentiation might influence testosterone levels (Chen et al., 2009). (A) The expression level of LC3 was decreased in Leydig cells of the low-serum testosterone (T) level azoospermia patients. The cholesterol uptake defect could be partially rescued by Nherf2 knockdown in autophagy-deficient Leydig cells. In the testicular interstitium (Purvis et al., 1981), testosterone is primarily produced in Leydig cells, where autophagy has been reported to be extremely active (Tang, 1988; Tang and Zhang, 1990; Yi and Tang, 1991, 1995, 1999; Tang et al., 1992). (F and H) Quantification of the relative NHERF2 levels in E and G. Collectively, these results suggest that NHERF2 is degraded via the autophagy–lysosome pathway. We then mutated this LIR motif to F236A/L239A and found that once this LIR motif was disrupted, the interaction between NHERF2 and LC3 was impaired (Fig. 6, L and M), and its degradation was dramatically delayed (Fig. 6 N and O). After protein sequence analysis, we identified a potential LIR motif in NHERF2 (Fig. 6 K), which is highly conserved in different species. LAMP2 is a lysosome membrane–integrated protein (Fig. S5; Saftig and Klumperman, 2009), suggesting the presence of lysosomes or autolysosomes. Autophagy, a finely tuned and multi-step mechanism, is primarily regulated by autophagy-related (ATG) proteins that have remained evolutionarily conserved from yeast to mammals (Tsukada and Ohsumi, 1993; Yang and Klionsky, 2010). Earlier, we have discussed autophagy in relation to lipolysis (Khawar et al., 2019; Khawar et al., 2021a) in the liver (Nazeer et al., 2023) and reproduction (Gao et al., 2019; Gao et al., 2020; Khawar et al., 2022). Additionally, cells engage other specialized mechanisms for selective targeting, such as lipophagy, zymophagy, mitophagy, and crinophagy; to target specific substrates (Vargas et al., 2023). Herein, we provide the functional role of autophagy in testicular and ovarian steroidogenesis to date, highlighting its modulation in testicular steroidogenesis and its impact on hormone synthesis, follicle development, and fertility therapies. Autophagy regulates sex steroid hormone synthesis through lysosomal degradation of lipid droplets in human ovary and testis. Therefore, a detailed investigation of the role of autophagy-mediated sex steroid production in the pathogenetic mechanisms of the diseases described above is of paramount importance. Although we tried to substantiate our findings using as many different tissue and cell types as possible, there are still other types of cells that produce steroid hormones. Noticeably, mTORC1 is the main gateway to autophagy, connecting cellular nutrient sensing with environmental cues to preserve cellular homoeostasis. Additionally, unfavorable circumstances, such as hypoxia, UV, starvation, ROS, and the accumulation of unfolded proteins, can also provoke autophagy to become a cytoprotective mechanism . LCs can synthesize the estrogen, germ cells, and epididymal spermatozoa-expressed P450 aromatase (CYP19A1) and can actively synthesize estrogens from androgens as well . Interestingly, autophagy is also affected by the concentration of testosterone 54,109. More precisely, the autophagic degradation of ABP is only effective at the protein level. In a detailed study, both in vitro and in vivo experiments demonstrated that autophagy regulates ABP expression. HCG treatment significantly upregulated the expression of StAR and perilipin3 in confocal imaging (Fig. 6B) and immunoblot analysis (Fig. 6C) and resulted in a drastic increase in testosterone (T) production of the samples (Fig. 6D). In support of our hypothesis that lipophagy regulates steroidogenesis, we monitored cholesterol trafficking in the cells and demonstrated that the uptake of NBD cholesterol, a fluorescent analog of cholesterol, is markedly enhanced after hCG treatment. In order to see if perilipin3/ LAMP2A co-localization occur in a dose-dependent fashion, the cells were treated in another set of experiment with chloroquine or vinblastine at different concentrations without hCG co-treatment. C Representative graphic bars indicate progesterone (P4) production of the luteinized granulosa cells treated with hCG at indicated concentrations. By contrast, FSH-induced upregulation in aromatase expression was not accompanied by increased E2 production in the Beclin1 silenced cells (Fig. 3E, F). In order to investigate the role of autophagy in the estrogen synthesis arm of steroidogenesis, we used mitotic non-luteinizing granulosa cells (HGrC1), which express aromatase enzyme and are capable of converting T to E2 when culture medium is supplemented with exogenous T. Following fusion with a lysosome, the autophagosome exposes its cargo to lysosomal hydrolases, helping with degradation. In 1955, Christian de Duve coined the term "autophagy," (Sabatini and Adesnik, 2013) paving the way for later discoveries of 1963 (Klionsky, 2008), G. Non-selective autophagy, also known as mTORC1-dependent autophagy, is triggered by nutrient stress and a low ATP/AMP ratio (Whitmarsh-Everiss and Laraia, 2021). Following initiation, various regulators come into play at the phagophore assembly site, guiding the progression through subsequent stages, including 1) nucleation, 2) expansion and closure of the phagophore, 3) maturation, and 4) degradation. Therefore, we, herein, explore the involvement of autophagy in ovarian and testicular steroidogenesis, along with the scrutiny of its regulatory mechanisms. Interestingly, extensive research on autophagy in normative and pathological endocrine settings has yielded promising knowledge, whereby a unified understanding of steroidogenesis in reproductive organs remains enigmatic. The recognition of autophagy’s substantial implications in various diseases has heightened research interest in exploring its physiological and pathological aspects.
