Blocking T-type calcium channels disrupts spermatogenesis in vivo and adversely affects spermatocytes in vitro by impairing mitochondrial function and autophagic flux

Abstract

T-type calcium channels are pivotal in spermatogenesis. To evaluate the molecular mechanisms by which T-type calcium channels regulate spermatogenesis, we constructed animal and cellular models using T-type calcium channel inhibitor flunarizine (FNZ). Intraperitoneal administration of FNZ (30 mg/kg) significantly impaired sperm motility, inhibited testicular germ cell proliferation, and disrupted sperm mitochondrial function in male mice. FNZ, at concentrations of 7.5 μM, 15 μM, and 30 μM, significantly inhibited mouse spermatocyte (GC-2) cells proliferation. The detrimental effects of FNZ were mediated through the disruption of calcium homeostasis, mitochondrial dysfunction, and the induction of apoptosis. Moreover, FNZ exposure resulted in the accumulation of autophagosomes and an upregulation of P62 protein, which is implicated in autophagic degradation. Notably, the autophagy activator Rapamycin (Rapa) was found to mitigate FNZ-induced cellular damage in GC-2 cells by enhancing autophagy process. Conversely, chloroquine (CQ), an autophagy inhibitor that disrupts lysosomal degradation, corroborated the role of FNZ in autophagy modulation. Our results indicate that FNZ induces mitochondrial damage, impairs sperm motility and spermatocyte proliferation, and is accompanied by obstacles to autophagic flux.