Additionally, HD-sEVs decrease the p-mTOR/mTOR ratio and boost autophagosome production and mitochondrial structural alterations in bGCs (Wang et al., 2023). Flow cytometry results indicate that HD-sEVs diminish bGC apoptosis rates by upregulating steroidogenic proteins and mRNAs, including CYP19A and HSD3B in bGCs, HD-sEVs stimulate estradiol secretion. Moreover, in cultured bGCs, previous trials have shown that LDL upregulates StAR mRNA and protein (Figure 4B). These insights contribute to a more comprehensive grasp of the function of autophagy in the luteal changes of mammalian ovary in vivo in Sprague-Dawley rats (Tang et al., 2019). Moreover, numerous studies have confirmed reactive oxygen species-dependent nuclear translocation of TFEB and autophagy activation . TFEB, as a master gene during the autophagic process , has been reported to regulate Alkbh5 promoter activity following hypoxia/reoxygenation treatment in cardiomyocytes . Moreover, it has been reported that ALKBH5 is most highly expressed in the testes but has low expression in the heart and brain . In this study, we found that knock down of ALKBH5 but not FTO could effectively reverse HsCG-induced autophagy. Further studies demonstrated that m6A could inhibit PRKAA2 activity by inducing m6A-dependent translation of Ppm1a and m6A-dependent degradation of the Camkk2 transcript. Following HsCG treatment, we could confirm the dependence of AMPK-ULK1 for the autophagic process in LCs. (I), Cells were treated with CQ (50 uM) in the presence or absence of testosterone (10 nM) and the ABP and LC3 expression levels were assessed with double immunofluorescence after 24 h. We speculated that testosterone regulates ABP clearance via autophagy in Sertoli cells. In addition, both treatments changed the ABP levels that Sertoli cells secreted to the supernatant (Fig. 1E). (B–E), Rat primary Sertoli cells were treated with CQ (50 uM) or rapamycin (10 nM) for 24 h and ABP expression in the cells was assessed with Western blot (B) and immunocytochemistry (D). We report that autophagy degrades ABP in rat Sertoli cells and the autophagic clearance of ABP is regulated by testosterone, which prolongs the ABP biological half-life by inhibiting autophagy. For cellular immunofluorescence assay, the cells plated onto cover glasses were fixed with 4% paraformaldehyde, and incubated antibodies according to protocols above. After being washed with IP buffer for three times, the beads were then treated with proteinase K (Millipore Sigma, 71,049) at 65°C for 0.5 h with occasional shaking. Briefly, cells were lysed with RIP lysis buffer containing protease and RNase inhibitor, after which cell lysate supernatant was incubated with anti-YTHDF1/YTHDF2 antibody-conjugated beads overnight at 4°C. To further confirm this result, an immunofluorescence analysis of SR-BI was performed in autophagy-deficient testes. Two autophagy/lysosome inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ; Wang et al., 2014), were added to inhibit the autophagy–lysosome pathway. Absorption of DiI-HDL (red) was examined using live-cell imaging. To further confirm these results, we crossed SF1-Atg5Flox/− mice with mitochondrial-DsRed transgenic mice (CAG/su9-DsRed2 mice; Hasuwa et al., 2010), and progeny showed that the number of mitochondria was dramatically reduced as characterized with fluorescence microscopy (Fig. S1 J). Thus, the decrease in LDs and cholesterol might reflect a superactive metabolism resulting from increased numbers of mitochondria. With the development of LCs from stem LCs to adult LCs, we observed a gradual decrease of m6A levels (Figure 4A). Furthermore, we also recorded upregulated expression of CAMKK2 and downregulated expression of PPM1A in adult LCs in comparison to stem LCs by western blotting and immunofluorescence assays (Figure 3Fand S5). The cell extracts were subjected to western blotting and quantitative analysis. Consistently, we confirmed the increased expression of p-PRKAA2 Thr172 in mouse LCs during development, peaking in adult LCs, by immunofluorescence assay (Figure 3D). The expression of LC3B-II gradually diminished and perilipin3 gradually increased along with StAR and 3β-HSD expression in immunoblot analysis (Fig. 4B), and P4 production increased (Fig. 4C) in a dose-dependent manner after treatment with hCG at indicated concentrations. We observed a marked increase in the intensity of oil red O staining in the confocal images of the cells treated with chloroquine. Therefore it is a suitable cell line to analyze the effect of autophagy inhibition on E2 production.
