Next, we asked whether the accumulation of NHERF2 was the major cause of the cholesterol uptake defect in autophagy-deficient Leydig cells. Next, we characterized NHERF2 degradation in autophagy-deficient Leydig cells. We cooverexpressed these genes with constant amounts of SR-BI in HEK293T cells and detected that increased expression of these proteins was accompanied by decreased expressions of SR-BI protein (Fig. 6 A). The mRNA levels of SR-BI showed no changes in autophagy-deficient Leydig cells compared with the control groups. The disruption of autophagy in Leydig cells inhibits cholesterol uptake by down-regulating SR-BI. Collectively, these results suggest that autophagy plays a role in cholesterol uptake in Leydig cells. Mechanistic insights into autophagy-mediated steroidogenesis in testes. Mechanistic insights into autophagy-mediated steroidogenesis… Mechanistic depiction of autophagy. Major research discoveries in autophagy. The ABP levels in the supernatants were assessed by an ELISA kit for rats (CSB-E12118r, Cusabio) according to the manufacturer's instructions. Total RNA isolation, cDNA synthesis and qPCR were performed as previously described44,45. Chloroquine (CQ), oil red O, testosterone and rapamycin were obtained from Sigma-Aldrich, MO, USA (C6628, O0625, T6147, 37094). Primary LCs were pre-treated with 3-methyladenine (3-MA, 5.0 mM) for 1 h followed by exposure to HsCG for 6 h. YTHDF1 promotes translation of the target m6A-modified mRNA, while YTHDF2 decrease mRNA stability and mediate subcellular localization and alternative splicing of mRNA 23,34–36. One class of m6A readers is the YTHDF (YTH N6-methyladenosine RNA binding protein), which includes YTHDF1 and YTHDF2. M6A exerts many of its functions through "reader" proteins that selectively recognize and bind to the m6A-modified transcripts 28–33. M6A modification is a dynamic and reversible process, which is mediated through "writer" and "eraser" proteins 21,23. With aging, the ability to synthesize testosterone in LCs progressively declines, resulting in a reduction of serum testosterone and concomitant alterations in physical function . This is just the tip of the iceberg, and there are still many gaps between autophagy and male reproduction that are worthy of exploration. Despite the cumulative gains revealed, autophagy is blossoming in many aspects of male reproduction. On the other hand, testosterone inhibits autophagy in a negative feedback loop. In conclusion, the review presents the double-edged characteristics of autophagy in the most important processes involved in male reproduction. Additionally, FSH causes the breakdown of LDs in porcine GCs (Liu et al., 2021), leading to the synthesis of progesterone via the Beclin1 protein. The exact mechanism through which Becn1 downregulation increases autophagosome quantities in luteal cells remains unclear. To generate mice with ovarian-specific conditional knockout (cKO), Beclin1-knockout mice were employed, where Beclin1 was selectively deleted in granulosa and luteal cells (Gawriluk et al., 2014). HD-sEVs elevate the protein and mRNA levels of VDAC1, CTSD, and HSP60, subsequently promoting mitophagy in bGCs (as shown in Figure 4D). These findings signify that in bGCs, LDL stimulates StAR expression, progesterone production, and lysosome development while lysosomes facilitate this process via secreting FC molecules from the breakdown of LDL (Zhang et al., 2015). The m 6 A mRNA methylation regulates AMPK activity in Leydig cells (LCs).… Thus, m6A modification resulted in reduced AMPK activity and subsequent autophagy inhibition. In the process of spermatogenesis, autophagy is crucial for the formation of specific structures that guarantee successful spermatogenesis, as well as for the degradation of certain constituents. S3 shows that the steroidogenic cell–specific disruption of autophagy did not affect the concentrations of TGs and TC in testes and serum. S2 shows that the lipids in the adrenal cortex and corticosterone in the serum decreased in the steroidogenic cell–specific autophagy disruption mice. S1 shows that the disruption of autophagy did not affect the fetal–adult Leydig cell transition, proliferation, and Gn secretion. 24 h after transfection, the expression of these proteins was confirmed by immunoblotting. HEK293T cells were maintained at 37°C and 5% CO2 in DMEM supplemented with penicillin, streptomycin, and 10% FBS. The protein lysates (25 µg) were separated by SDS-PAGE and electrotransferred onto a nitrocellulose membrane.
