These results suggest that an insufficient supply of cholesterol in autophagy-deficient Leydig cells might be the primary reason for the decline in the serum testosterone concentration in autophagy-deficient mice. Immunoblotting data showed that the protein levels of either ATG7 or ATG5 were dramatically reduced in knockout Leydig cells compared with their control groups (Fig. 2, A and B). To further explore the correlation between testosterone production and autophagy in Leydig cells, we characterized the in vivo autophagy activity in mouse Leydig cells at various developmental stages. In response to hormone stimulation, autophagic flux is induced in Leydig cells to promote testosterone synthesis by facilitating the degradation of NHERF2 and up-regulation of SR-BI.
CHX chase assay of NHERF2 in Atg7Flox/Flox and SF1-Atg7−/− (E) as well as Atg5Flox/Flox and SF1-Atg5−/− (G) Leydig cells. CHX chase assay of NHERF2 in HEK293T cells in the absence or presence of 3-MA. (C) NHERF2 was stabilized in autophagic flux–disrupted cells.
(A) The overexpression of NHERF1, NHERF2, and MAP17 down-regulated the protein levels of SR-BI. Usually, selective autophagy substrates have one or more LC3-interacting region (LIR) motifs that are essential for the protein’s interaction with LC3 and further degradation (Rogov et al., 2014). Furthermore, coimmunoprecipitation experiments in HEK293T cells showed that NHERF2 could physically interact with LC3 and the autophagic adapter proteins NBR1 and SQSTM1 but not Toll-interacting protein (TOLLIP; Fig. 6 J; Lu et al., 2014). To determine whether these negative regulators of SR-BI might be regulated by autophagy, 3-MA was added to the media of HEK293T cells transfected with the three genes. Next, we determined how autophagy down-regulates SR-BI in Leydig cells. (F) SR-BI immunofluorescence analysis (red) in Leydig cells of the low-serum testosterone (T) level azoospermia or oligospermia patients and control groups.
It is a fundamental and vital degradation/recycling pathway that removes undesirable components, such as cytoplasmic organelles, misfolded proteins, viruses, and intracellular bacteria, to provide energy and essential materials for organisms. Moreover, to assess the effects of m6A modification on RNA stability, cells were treated with actinomycin D at 5 μg/mL for indicated times. After 36 h of transfection, cells were treated with 2 μg/mL puromycin (Medchemexpress, HY-B1743A) to obtain stable cell lines.
Treatment of the cells with another pharmacological autophagy inhibitor, vinblastine, which inhibits the fusion of the autophagosome with lysosome, yielded similar results. In another set of experiments, the luteinized GCs were treated only with chloroquine at the same concentrations only to see the effect of inhibition of autophagy on basal P4 production. B Representative confocal images of the cells stained for LC3 (green) and lysotracker (red) 24 h after treatment with hCG w/wo CQ at indicated concentrations. D Representative blots of the luteinized granulosa cells before and 24 h after treatment with luteinizing hormone (LH) at the indicated concentration. B Representative confocal images of the cells after staining for the steroidogenic enzymes StAR and 3β-HSD 24 h after treatment with hCG (10 IU/ml). Given that LC3B-II itself is an autophagy substrate that degraded following the fusion of the autophagosome with lysosome, these observations suggest that hCG may induce autophagic flux and, therefore, there might be a link between autophagy and steroidogenesis in these cells. Similar results were obtained after treatment of the cells with recombinant LH (Fig. 1D, E).
Reduction of m6A levels alleviates m6A-mediated Ppm1a translation and upregulates CAMKK2 expression by retarding m6A-mediated decay of the transcript, contributing to activation of PRKAA2 and subsequently autophagy initiation Next, we found that HsCG treatment markedly promoted nuclear translocation of NR5A1, CEBPB, and TFEB in both TM3 cells and primary LCs (Figure 9Fand S17). Therefore, we anticipated that HsCG treatment could upregulate ALKBH5 expression by increasing its transcriptional levels. We noted that HsCG treatment upregulated the mRNA and protein levels of ALKBH5 (Figures 4D and Figures 9A). In this study, we observed reduced METTL14 levels in LCs upon HsCG treatment (Figure 4Dand S9), while we detected no significant changes in mRNA levels (Figure 9A). (C and D) TM3 cells with or without transfection of shRNA targeting Alkbh5 (sh-Alkbh5) were pre-treated with MG-132 (2 μM) for 1.5 h followed by exposure to HsCG for 6 h.

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