Arrangement of PMCA4 in bovine sperm membrane fractions

Abstract

The plasma membrane Ca(2+) -ATPase (PMCA) is the main restorer of Ca(2+) balance in sperm. Particularly, PMCA isoform 4 has an essential function in sperm fertility by its participation in gaining sperm hypermotility. PMCA activity is influenced by its lipid environment. Sperm membranes exhibit lipid raft microdomains or detergent-resistant membrane domains, enriched in sphingolipids and cholesterol, forming functional specialized areas. Lipid and protein composition of lipid rafts alters during the capacitation process, which is characterized by a cholesterol efflux. In this study, the localization of PMCA4 in lipid membrane fractions of the sperm plasma membrane was investigated. We identified PMCA4 in both the detergent-resistant membrane (DRM) and in the detergent-soluble (DS) fraction of caput and cauda sperm, respectively. Capacitation did not influence PMCA4 localization. In immunocytochemical studies PMCA4 was co-localized with the lipid raft/DRM marker caveolin in the mid piece of caput and cauda sperm. Functional studies with seminal vesicle major protein PDC-109 showed that the Ca(2+) -ATPase activity in DS fractions of cauda sperm and capacitated cauda sperm was stronger enhanced than in the DRMs. In both fractions the effect was statistically significant. In contrast, in lipid overlay experiments PDC-109 interacted stronger with the lipids extracted from DRMs than with lipids extracted from DS. Our results indicate a possible functional compartmentalization of PMCA in bull sperm membranes and point to a presumptive, yet unknown interaction partner of Ca(2+) -ATPase and PDC-109, mediating the PDC-109 action from DRMs to the DS fraction of sperm plasma membrane.

Switch of PMCA4 Splice Variants in Bovine Epididymis Results in Altered Isoform Expression during Functional Sperm Maturation

“…It has also been shown in mice that PMCA4 is the major isoform in testis (90%) and is located in the principal piece of sperm tail (7). In bovine sperm we could show that PMCA4 localizes to the mid piece of caput and cauda sperm (42). Using in situ hybridization and immunohistochemistry to study the gene expression and distribution of PMCA4 protein in bovine germ cell stages, we found clear evidence that PMCA4 mRNA expression is restricted to spermatogonia and early primary spermatocytes.…”

Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

“…Cholesterol inclusion works to increase the thickness and order of plasma membranes [51] and it has been suggested that some membrane domains are formed only in the presence of certain concentrations of cholesterol [52]. Lipid rafts are enriched in cholesterol, sphingomyelin, lipids with saturated long chain acyl chains, [1] and in sperm, the plasma membrane which lays over the acrosome displays rafts enriched in gangliosides and cholesterol [53, 54]. Cholesterol content of membranes both above and below native membrane content (16 moL% of total lipids) decreases Na + K + -ATPase activity in PC liposomes using pig kidney ATPase [47].…”

“…Lipid rafts contain lipids, glycosylphosphatidylinositol- (GPI-) anchored proteins and various signal transduction proteins [1, 2] that engage the microdomain in cell signaling and cell adhesion events [3]. Capacitation is a process whereby spermatozoa gain the ability to fertilize through a series of membrane changes including a reorganization of membrane lipids and proteins [4, 5].…”

CASK interacts with PMCA4b and JAM‐A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility

“…The homogenate was centrifuged for 10 min at 14,000 × g at 4°C, and the supernatant subjected to ultracentrifugation at 100,000 × g for 60 min at 4°C to yield microsomes. The pellet was re-suspended for 15 min at 4°C in 100 μl of homogenization buffer containing 1.5% n-octyl-β-d-glucopyranoside as described (Post et al, 2010), and protein concentration was determined using the BCA kit mentioned above. Ca 2+ -ATPase activity was determined as described (Kosk-Kosicka, 1999) for the enzymatic hydrolysis rate of ATP via quantitation of the resulting inorganic phosphate (P i ), as a function of time.…”