Stabley, Deborah L.

Plasma Membrane Ca 2+ ATPase isoform 4b (PMCA4b) is the primary Ca 2+ 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 interacts with CASK in regulating Ca 2+ homeostasis and motility. Recently, it has been shown that PMCA4a and 4b are expressed in all bovine epididymal (EPL) regions, with increasing amounts of 4a distally and only 4b in testis. These findings suggest that PMCA4a and b are acquired by sperm during EPL maturation. Here we show, via RT‐PCR, that murine Pmca4b mRNA is expressed uniformly in testis and epididymis (EP) and 4a differentially in the EP. Western confirmed the RT‐PCR results showing that PMCA4a is most abundant in testis and cauda EP. Immunoprecipitation using pan‐PMCA4 antibody showed 4b and 4a in caput and corpus EP and 4a in cauda, seminal vesicle (SV), and vas deferens (VD). Western revealed an ~4 fold increase of 4a in caudal, compared to, caput sperm and confirmed its presence in the VD and SV. Importantly, PMCA4a was seen in sperm‐free EPL luminal fluid, suggesting that it is secreted, and acquired by sperm via epididymosomes. Since PMCA4a and 4b have different kinetic properties for Ca 2+ transport, our study suggests for the first time that murine sperm possess PMCA4a which has a higher Ca 2+ turnover, better facilitating capacitation events in the female tract. Funding: NIH‐RO3 HD061637.

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.