Abstract
PMCA4, a membrane protein, is the major Ca2+ efflux pump in murine sperm where its deletion leads to a severe loss of hyperactivated motility and to male infertility. We have previously shown that the PMCA4b splice variant interacts with CASK (Ca2+/CaM-dependent serine kinase) in regulating sperm Ca2+. More recently we detected that PMCA4a isoform, in addition to its presence in testis, is secreted in the epididymal luminal fluid and transferred to sperm. Here we show that Pmca4 mRNA is expressed in both the 4a and 4b variants in the vagina, uterus, and oviduct. Immunofluorescence reveals that PMCA4a is similarly expressed and is elevated during estrus, appearing in the glandular and luminal epithelia. Western analysis detected PMCA4a in all tissues and in the luminal fluids (LF) of the vagina (VLF), uterus (ULF), and the oviduct (OLF) collected during estrus. It was ~9- and 4-fold higher in OLF than in VLF and ULF, and only marginally present in LF collected at metestrus/diestrus. Fractionation of the LF collected at estrus, via ultracentrifugation, revealed that 100% of the PMCA4a resides in the vesicular fraction of the ULF and OLF. Transmission electron microscopy (TEM) revealed that OLF vesicles have an exosomal orientation (with the cytoplasmic-side inward), a size range of 25-100 nm, with the characteristic CD9 biomarker. Thus, we dubbed these vesicles “oviductosomes”, to which PMCA4a was immunolocalized. Incubation of caudal sperm in the combined LF or exosomes resulted in up to a ~3-fold increase of sperm PMCA4a, as detected by flow cytometry, indicating in vitro uptake. Our results are consistent with the increased requirement of Ca2+ efflux in the oviduct. They show for the first time the presence of oviductal exosomes and highlight their role, along with uterosomes and vaginal exosomes, in post-testicular sperm acquisition of PMCA4a which is essential for hyperactivated motility and fertility.
Highlights
Capacitation which occurs in the female reproductive tract is the final maturational process that mammalian sperm undergo before they are competent to effect fertilization
We showed that PMCA4 splice variants 4a and 4b are secreted in the mouse epididymal luminal fluid and that 4a is transferred to the sperm membrane during epididymal maturation, with caudal sperm having a 5-fold increase over that in caput sperm [7]
Our results show that Plasma Membrane Ca2+-ATPase 4a (PMCA4a) is expressed and secreted in the luminal fluids (LF) in the oviduct, uterus and the vagina, with the secretion highest in the LF of the oviduct where sperm are stored during estrus
Summary
Capacitation which occurs in the female reproductive tract is the final maturational process that mammalian sperm undergo before they are competent to effect fertilization. At the fertilization site in the ampullary-isthmic junction (AIJ) [1], high intracellular Ca2+ concentration levels are essential for both hyperactivated sperm motility and the acrosome reaction [2]. We showed that PMCA4 splice variants 4a and 4b are secreted in the mouse epididymal luminal fluid and that 4a is transferred to the sperm membrane during epididymal maturation, with caudal sperm having a 5-fold increase over that in caput sperm [7]. It is likely that sperm may need to acquire additional PMCA4a in order to: a) preserve sperm fertility in the storage reservoir of the oviduct by avoiding premature capacitation, b) maintain viability after the demand for high intracellular [Ca2+] for hyperactivated motility in the AIJ [9], and for the acrosome reaction [2]
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