Plasma Membrane Ca2+‐ATPase 4 Splice Variants are Acquired by Murine Sperm During Epididymal Transit via Epididymosomes

Abstract

Plasma Membrane Ca2+‐ATPase isoform 4 (PMCA4) is the primary Ca2+ efflux pump in murine sperm where it regulates motility. In Pmca4 null sperm hyperactivated motility is lost, resulting in infertility. We have shown that PMCA4b – CASK interaction in sperm is key in regulating Ca2+ homeostasis. However, recent work showed that in bulls the PMCA4a splice variant (not in testis) is epididymally‐expressed, along with 4b, and may be transferred to sperm. Here we show, via conventional and in situ RT‐PCR, that both splice variants of Pmca4 mRNA are expressed in the murine testis and along the epididymis. Immunohistochemistry localized PMCA4a in the epididymal epithelium and Western analysis showed, for the first time, that PMCA4a is secreted in the epididymal luminal fluid (ELF). Flow cytometry indicated the presence of PMCA4a on mature caudal sperm where it was increased ~5‐fold compared to caput sperm (detected by Western) and~2‐fold and ~2.75‐fold after incubation in ELF and epididymosomes, respectively, revealing in vitro uptake. Using pan‐PMCA4 antibodies, CO‐IP assays revealed that both variants associate with CASK, indicating possible function as a complex. Our study suggests a mechanism for combining the different functional attributes of both variants, leading to greater efficiency of the pump in maintenance of Ca2+ homeostasis, which is crucial for sperm motility and male fertility.