FTO regulates testosterone secretion in Leydig cells: insights into the role of m6A modifications and the therapeutic potential of hCG

Testosterone plays a pivotal role in male reproductive health and is synthesized primarily by Leydig cells (LCs) in the testes. Alterations in testosterone levels can lead to sexual dysfunction, reduced fertility, and various systemic health issues. FTO, an m6A demethylase, has been implicated in the regulation of RNA modification and has significant roles in various biological processes. However, its influence on testosterone secretion in LCs remains unclear. This study aims to investigate the role of FTO in regulating testosterone secretion by LCs and to explore the potential impact of hCG treatment in rescuing the effects of FTO inhibition. In this study, we assessed the mRNA and protein expression levels of FTO in LCs from 39 male patients diagnosed with obstructive azoospermia. Additionally, FTO knockdown was performed in TM3 cells, followed by analysis of cell proliferation, apoptosis, and testosterone secretion. The effect of hCG on rescuing FTO inhibition-induced changes was also evaluated. We identified a positive correlation between FTO expression levels and testosterone concentrations in LCs from 39 male patients with obstructive azoospermia. FTO knockdown in TM3 cells significantly reduced testosterone secretion, cell proliferation, and increased apoptosis. Specifically, 48 h post-transfection, the apoptosis rate in shRNA-FTO-transfected TM3 cells was 6.26%, significantly higher than in mock-transfected cells (3.03%, P = 0.013). FTO inhibition also markedly suppressed cell proliferation by 26.2% (P < 0.0001) at 24 h, 34.3% (P = 0.0006) at 48 h, and 21.5% (P = 0.002) at 72 h, as measured by CCK-8 assay. However, the addition of 10 IU hCG significantly rescued the proliferation and reduced the apoptosis rate in the FTO knockdown group. Testosterone secretion in the FTO inhibition group was also significantly lower than in controls at all time points (6, 24, 48, and 72 h), but hCG treatment restored testosterone levels by 26.4% (P = 0.003) at 6 h, 29.4% (P = 0.0026) at 24 h, 18.8% (P = 0.028) at 48 h, and 36.6% (P = 0.0005) at 72 h. Our study provides new evidence that FTO plays a critical role in regulating testosterone secretion in LCs. Additionally, we demonstrate that hCG treatment can restore testosterone production impaired by FTO inhibition. These findings offer valuable insights into the molecular mechanisms underlying testosterone secretion and may inform therapeutic strategies for male infertility and hypogonadism.