Sirt1 regulates testosterone biosynthesis in Leydig cells via modulating autophagy

Next, the luteinized GCs were treated with a fixed dose of hCG combined with incremental concentrations of lysosomal proteolysis inhibitor chloroquine. Transcript levels of the steroidogenic enzymes, FSH/LH receptors, hormone levels and the signal intensities in immunoblotting and confocal imaging were continuous variables and, therefore, expressed as the mean ± SD. Estradiol (E2), Progesterone (P4) and buy testosterone enanthate online (T) levels in culture media were determined by using electrochemiluminescence immunoassay “ECLIA” (Elecsys and Cobas immunoassay analyzers, Roche Diagnostics, USA). Total cholesterol isolation and free cholesterol, and cholesteryl esters quantification were performed using a colorimetric method (the Cholesterol/ Cholesteryl Ester Assay Kit, ab65359, Abcam, UK) according to the manufacturer’s instructions. The live-cell imaging process was performed by a confocal microscope (Leica, DMI8) equipped with an incubation chamber (37 °C and 5% CO2).
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.
HCG treatment enhances autophagy to promote NHERF2 clearance and testosterone order synthesis in Leydig cells. These results suggest that the disruption of autophagy in Leydig cells results in the abnormal accumulation of the SR-BI–negative regulator NHERF2, which in turn down-regulates SR-BI, leading to defective cholesterol uptake and a deficiency in testosterone synthesis. (A) The protein levels of SR-BI were extremely reduced in autophagy-deficient Leydig cells. Compared with the control groups, the protein levels of SR-BI in autophagy-deficient Leydig cells were dramatically reduced, whereas the LDLR protein levels were not affected (Fig. 5 A). To further investigate the mechanism of autophagy in cholesterol uptake, we determined the protein levels of SR-BI and LDLR in autophagy-deficient Leydig cells using immunoblotting. After treatment with these inhibitors, the rates and amounts of DiI-HDL absorbed by Leydig cells were significantly reduced compared with the control group (Fig. 4, B and C), indicating that autophagy might be involved in cholesterol uptake. High-density lipoprotein (HDL) cholesterol is a major source of cholesterol for testosterone production in Leydig cells (Landschulz et al., 1996).
Recent research also suggests that FSH, regardless of the traditional steroidogenic pathway, enhances autophagy by upregulating Beclin1 through the PI3K/JNK/c-Jun pathway, promoting LDs breakdown in pig GCs (Liu et al., hedgedoc.eclair.ec-lyon.fr 2021). This mouse model showed a nearly 75% reduction in Becn1 levels, with p62 accumulation observed in GCs. These observations imply that autophagy is a vital player in the proper sexual development. Mutually, BFF HD-sEVs enhance macroautophagy and mitophagy in bGCs, inhibit apoptosis in bGCs, and elevate 17β-estradiol release via the PI3K/Akt/mTOR signaling pathways as shown in Figure 4D (Wang et al., 2023).
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.
Herein, we presented a comprehensive overview on the effects of the regulation of autophagy on male reproduction, including its relation to spermatogenesis, the endocrinology of testis, and the key molecule of autophagy mechanism—the regulation of mTORC1 (Figure 4). Autophagy can maintain the survival of testicular cells or accelerate the apoptosis of some cells, representing a double-edged sword . As a central modulator in stem cell homeostasis, mTORC1 signaling governs stem cells quiescence 127,128. Retinoic acid (RA) is required for the self-renewal of spermatogonial stem cells (SSCs) and for subsequent entry into meiosis . In other words, high mTORC1 activity promotes biomolecular synthesis and simultaneously inhibits autophagy 124,125.