(D and E) Relative mRNA level of SR-BI in autophagy-deficient Leydig cells. (B and C) SR-BI was decreased in the autophagy-deficient Leydig cells. (E and G) The absorption curve of DiI-HDL in autophagy-deficient Leydig cells and control groups. D Representative confocal images of the luteinized granulosa cells stained for Beclin1 (green) and Oil Red O (red). Consistent with the qRT-PCR and immunoblot results, the signal intensity of Beclin1 was significantly weaker in the cells with defective LF in confocal image analysis (Fig. 7D). By contrast, there was a significant reduction in the expression of LC3B-I and LC3B-II along with a concomitant increase in StAR and Beclin1 expression (Fig. 7B) and a robust increase in P4 output after hCG treatment in the luteinized GCs of the patients with normal LF (Fig. 7C). First, we simply compared the transcripts of certain autophagy genes and found that mRNA expressions of AMBRA1, ATG16-L1, ATG4, ATG5, BECLIN1, GABARAP, GABARAP-L1/L2 were significantly downregulated in the luteinized GCs of the patients with defective luteal function. D Testosterone (T) production of the samples 24 h after treatment with hCG (10 IU/ml) w/wo CQ (60 μM). Classic macroautophagy is initiated from an isolated membrane (phagophore), which is followed by the formation of a double-membrane autophagosome that then delivers the engulfed proteins and fuses with lysosomes for degradation. Autophagy is a lysosomal degradation pathway that eliminates defective organelles and proteins from the cell. In humans, an alternative ABP transcript accumulates in germ cells in a highly regulated manner throughout the spermatogenic cycle3 and its expression is positively correlated with sperm motility4. ABP can also directly up-regulate the expression of aromatase in germ cells and oestrogen receptor beta2. ABP can promote germ cell differentiation and it has been reported that ABP up-regulates the expression of transition protein 1 (TP1), which is involved in nuclear chromatin condensation in rodent spermatids1. These data demonstrate that testosterone up-regulates ABP expression at least partially by suppressing the autophagic degradation. Our data demonstrated that HsCG treatment increased the degradation rate of METTL14 (Figure 9B,C). To investigate the molecular mechanism underlying HsCG-induced decreased expression of METTL14, we assessed the effects of HsCG on METTL14 protein stability. Following HsCG treatment, m6A enrichment at both site 4 and site 5 became significantly reduced; however, ALKBH5 knockdown effectively suppressed these changes (Figure 8G). (L) TM3 cells were transfected with sh-Mettl14 followed by transfection with indicated vectors, and cell lysate was subjected to western blotting Our data revealed that METTL14 knockdown enhanced phosphorylation of PRKAA2, upregulated expression of LC3B-II, and decreased expression of SQSTM1 (Figure 6A); however, compound C effectively attenuated these changes (Figure 6A). Given that we identified a negative correlation between m6A methylation and autophagy, we next evaluated the possible relationship between m6A methylation and activation of AMPK in LCs. (B) m6A levels in LCs were assessed by immunofluorescence assay. We further demonstrated that HsCG decreased m6A levels and increased HSD3B levels in LCs using immunofluorescence analyses (Figure 4F). (A) m6A levels in LCs were assessed by immunofluorescence staining. Thus, autophagy participates in testosterone production by providing SR-BI, the receptor of high-density lipoproteins (HDLs), to promote the selective uptake of lipoproteins . Previous research demonstrated that 3-MA rescues apoptosis by partially aggravating the reduction in the autophagy flux in cadmium-treated mouse spermatogonia and rescued apoptosis by inhibiting autophagy in spermatocyte cells . On the whole, testosterone may act through a negative feedback loop regarding autophagy to sustain cellular homeostasis, while autophagy participating in testosterone production and ABP metabolism regulates the process of spermatogenesis indirectly . A previous study demonstrated that inhibiting autophagy in primary rat LCs with chloroquine (CQ) or siAtg7 reduced testosterone production and decreased the level of FC ; here, the type of autophagy was lipophagy. To extend our observations from cell culture, we performed intratesticular injection of siRNA for ATG7 or MTOR and we found that in vivo results were consistent with those in primary cells (Fig. 1H, 1I, 1J). Lipid droplets were found in nearly all cells, but only a few cells were alkaline phosphatase positive (Fig. 1A), indicating a very high purity of the Sertoli cells used in this study. Primary Sertoli cells were isolated from 18–22-day-old rats and the cells isolated at this age had almost negligible somatic (i.e., Leydig cells) cell contamination17. However, it is still not clear how the process of ABP metabolism works in Sertoli cells or how the process is regulated. The scavenger receptor class B type I (SR-BI) aids the selective intake of cholesteryl esters having its source in lipoproteins. Therefore, its manipulation in the current scenario was recently accompanied by Chen et al. (2020) when they investigated m6A methylation in regulation of Leydig cell differentiation and their subsequent physiology. Membrane carriers, e.g., the steroidogenic acute regulatory (StAR) protein, are important in relocating FCs to mitochondria (Galano et al., 2022). While our knowledge of the molecular entities in mammalian autophagy continues to expand and solidify, much remains to be elucidated. More comprehensive mechanistic insights into autophagy are available (Levine and Klionsky, 2004; Klionsky, 2005; Mizushima et al., 2008; Vargas et al., 2023). In case of prolonged starvation-induced autophagy, lysosomes undergo recycling through a process called autophagic lysosome reformation, regulated by an mTOR-dependent pathway.
