Immunoblotting analysis of HMGCR and SREBP2 in Atg7Flox/Flox, SF1-Atg7−/−, Atg5Flox/Flox, and SF1-Atg5−/− Leydig cells. The decrease in LDs and cholesterol was somewhat similar to that observed in autophagy-deficient adipocytes, accompanied by increased copy numbers of mitochondria (Zhang et al., 2009). BODIPY (green) and Filipin (red) staining were performed in both Atg7Flox/Flox (top) and SF1-Atg7−/− Leydig cells (bottom; C). 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. For NBD-cholesterol staining, cells were stained lively with 1 μg/ml NBD cholesterol along with 100 nM LysoTracker (for detection of lysosomes) for 1 h at 37 °C and 5% CO2. Regarding the evaluation of the effect of hCG stimulation on the steroidogenesis activity of siRNA-treated cells, hCG was added at 24 h post-transfection period to the new complete culture media at a concentration of 10 IU/ml. To study the role of autophagy in testosterone production, human testicular tissue samples were used. And very recently, another group of investigators demonstrated in the porcine ovary that FSH promotes progesterone production by enhancing autophagy through the upregulation of Beclin1 via the PI3K/JNK/c-Jun pathway to accelerate LD degradation in porcine GCs . In the absence of Sirt1, LC3 fails to deacetylate and translocate to the cytoplasm, resulting in disruption of autophagic flux, impaired cholesterol uptake and deficient testosterone biosynthesis . Another study in 2021 demonstrated that autophagic flux and autophagy-mediated testosterone synthesis are defective in SF1-Sirt1−/− mice. In 2019, an elegant study described autophagy-dependent steroid hormone (ecdysone) production in the Drosophila model by demonstrating that autophagy mediates lipid trafficking required for steroid synthesis . A Representative confocal images of the luteinized granulosa cells of the patients with normal and defective luteal function treated with hCG (10 IU/ml) w/wo chloroquine (CQ, 60 μM). Defective co-localization of BODYPY with lysosome became more obvious in the cells with defective LF when they were treated with an hCG+chloroquine combination (Fig. 8A, B, Supplementary Fig. S6 and Supplementary Movies S5 and S6) in comparison to the control cells with normal LF. Notably, the ER-resident chaperone Sig-1R, concentrated at MAMs, governs calcium dynamics and responses to oxidative stress (Gottschalk et al., 2022), suggesting its potential as a therapeutic target for conditions linked to ROS-induced damage in Leydig cells. Mitochondrial contact sites with the ER are vital for regulating ROS in gonadal cells. Notably, there may be additional connections between testosterone and autophagy. Concurrently, there is an increase in total cholesterol (TC) and LD levels in serum-free media. Stress reduces lipid droplets but increases testosterone release, effects counteracted by inhibiting autophagy.
