Interrupting Two-Pore Channels 2 (TPC2) and Sperm Function Enhancement

Abstract

The proper functioning of sperm is essential for successful fertilization. Various physiological regulators control sperm motility, energy balance, capacitation, and the acrosomal reaction. Intracellular calcium ion (Ca2+) concentration and pH levels play a crucial role in regulating sperm physiology. Two pore channels 2 (TPC2), located on lysosomes, are involved in ion fluxes, and play a vital role in reproductive and embryonic development. This thesis aims to investigate the role of TPC2 as a mediator of sperm function. TPC2 knockout (KO) mice and wild type (WT) mice were used to study the role of TPC2 in sperm physiology. TPC2 KO sperm exhibited significantly increased motility and hyperactivation-related parameters associated with capacitation compared to WT sperm. Additionally, TPC2 KO sperm showed higher ATP content than WT controls. GPD2 protein, involved in tyrosine phosphorylation during sperm capacitation, was found to have significantly higher mRNA transcript levels in TPC2 KO sperm using RT-qPCR analysis. We utilized our model to understand specific molecular changes in TPC2 KO mouse sperm by measuring PPARα, which controls GPD2 activity. TPC2 KO sperm showed a shift towards oxidative phosphorylation, increased mitochondrial activity, and biogenesis. The antioxidant effect of TPC2 was more pronounced than in WT sperm, as indicated by the expression of antioxidant enzymes such as superoxide dismutase 1 and 2 (SOD1, SOD2). PGC1α, involved in mitochondrial biogenesis, was significantly increased in TPC2 KO sperm, suggesting a potential mechanism underlying the enhancement of sperm function in the absence of TPC2 activity. TPC2 KO sperm undergo capacitation at a faster rate than WT sperm, as evidenced by increased phosphorylation of protein kinase A substrates (PKAs) in TPC2 KO sperm compared to WT. Similar effects were observed in tyrosine phosphorylation and the acrosome reaction. As sperm capacitation is crucial for fertilization, non-capacitated TPC2 KO sperm demonstrated a higher fertility rate than WT sperm in in vitro fertilization (IVF) studies, supporting the notion that interfering with TPC2 activity enhances sperm physiological function. Moreover, TPC2 deficiency led to a more alkaline intracellular pH (pHi), which activated the CatSper channel and allowed increased influx of Ca2+. Additionally, the alkaline cytosol enhanced the activity of soluble adenylyl cyclase (sAC), leading to sperm capacitation through the activation of the cAMP/PKA signalling pathway, which may contribute to this characteristic. Together this work demonstrates that disturbing the activity of TPC2 improves sperm function and enhances fertilization capability.