Expression and Secretion of Plasma Membrane Ca2+-ATPase 4a (PMCA4a) during Murine Estrus: Association with Oviductal Exosomes and Uptake in Sperm

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.

Oviductosome-Sperm Membrane Interaction in Cargo Delivery

“…Collection of Oviductal Luminal Fluids from Females after Hormonal Induction of Estrus-Females were induced into estrus as previously described (2,6). Oviductal luminal fluids (OLF) were obtained from 10 superovulated females per experiment.…”

“…Oviductal luminal fluids (OLF) were obtained from 10 superovulated females per experiment. The methodology of collecting the fluid is similar to that described previously (2,6).…”

“…Recently, we discovered that the murine oviductal fluid contains EVs, which we termed "oviductosomes" (OVS), and that during the proestrus/estrus stage they carry and deliver to sperm an essential fertility-modulating protein, plasma membrane Ca 2ϩ -ATPase 4 (PMCA4) (6). PMCA4 with its two isoforms (4a and 4b), is a 10-pass transmembrane protein that has functional domains on the cytoplasmic side of the membrane and interacts with membrane-associated proteins (7).…”

“…This underscores its functional and maturational roles in sperm. Thus, in the female tract the acquisition of additional PMCA4 from the OVS might be important for maintaining sperm viability during capacitation (the final sperm maturational stage), as well as in hyperactivation, and acrosome reaction (AR) (6), which all require elevated levels of Ca 2ϩ (13)(14)(15)(16).…”

Gelatin Binding Proteins in Reproductive Physiology

“…Most proteins associated with motility are usually located within the mid piece of spermatozoa and BSP proteins have been detected on the mid piece of spermatozoa in several species, which hints at a biological function in the motility of spermatozoa [reviewed in [5]. The motility of spermatozoa is known to be regulated by intracellular calcium signaling molecules, indicating a regulatory function of Ca ?2 -ATPase in spermatozoa motility through this signaling pathway [58]. Further, it was reported that PDC-109 interacts with Ca ?2 -ATPase and enhances the motility of spermatozoa in the bovine model by increasing the pumping efficiency of Ca ?2 -ATPase in an irreversible and cooperative way across the plasma membrane [reviewed in [28].…”