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

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.


Introduction
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 Ca 2+ concentration levels are essential for both hyperactivated sperm motility and the acrosome reaction [2]. PMCA4, a member of a family of Ca 2+ efflux pumps, maintains low resting cytosolic [Ca 2+ ] ([Ca 2+ ] c ) and a Ca 2+ gradient across the sperm membrane and plays the major role in the maintenance of Ca 2+ homeostasis in murine sperm [3]. The importance of PMCA4's role in sperm is underscored by studies showing that global Pmca4 knockout leads to a critical loss of progressive and hyperactivated motility, and subsequently infertility in mice [4,5].
We have previously shown that the PMCA4b splice variant interacts with CASK (Ca 2+ /CaM-dependent Serine Kinase) in regulating murine sperm Ca 2+ [6]. Recently 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]. Since caput sperm have 2-6 times higher [Ca 2+ ] c than caudal sperm and are incapable of progressive motility [8], the uptake of PMCA4a during epididymal transit may play a direct and vital role in sperm acquisition of progressive motility and in their viability. Similarly, 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 [Ca 2+ ] for hyperactivated motility in the AIJ [9], and for the acrosome reaction [2].
During transit in both the male and female tracts and during ejaculation, the sperm plasma membrane undergoes extensive modifications in which it acquires a variety of proteins [10]. Our laboratory has shown that SPAM1, a glycosyl phosphatidylinositol (GPI)-linked sperm membrane protein, is expressed in the oviduct and uterus where it is secreted and acquired by sperm [11,12]. We hypothesize that PMCA4a is also expressed and secreted in the female reproductive tract where it can be acquired by sperm during capacitation. Of the two splice variants of PMCA4, 4a shows a much higher basal activity and is more effective than 4b in returning Ca 2+ to resting levels [13]. Thus it would readily meet the demands of maintaining homeostasis in conditions of high [Ca 2+ ] c during capacitation. Our results show that PMCA4a is expressed and secreted in the LF in the oviduct, uterus and the vagina, with the secretion highest in the LF of the oviduct where sperm are stored during estrus. Importantly, we detected that it resides in membranous vesicles in the OLF as well as in uterosomes from the ULF. Mature caudal sperm were shown to acquire PMCA4a in vitro after incubation in the LF or from their isolated exosomes. Our findings indicate that oviductal PMCA4a may play an important and crucial role in the regulation of proper Ca 2+ handling during sperm capacitation.

Animals and Reagents
Sexually mature 4-12 week old female and 3-6 month old male mice (C57BL/6 and ICR strains; Harlan, Indianapolis, IN) were used throughout the investigation. In addition to these wild-type (WT) mice, Pmca4 null mice [4] were used to provide epididymal tissue for the Western blotting analysis. These mice were a generous gift from Dr. Gary Shull in whose laboratory they were generated. Breeding and genotyping of these mice were described previously [4]. Studies were approved by the Institutional Animal Care and Use Committee at the University of Delaware and were in agreement with the Guide for the Care and Use of Laboratory Animals published by the National Research Council of the National Academies, 8th ed., Washington, D.C. (publication 85-23, revised 2011).

Antibodies
Rabbit polyclonal antibodies against peptides specific for bovine PMCA4a have been generated and previously validated [14,15]. Sequence analysis suggested a high probability of cross-reactivity with mouse PMCA4a, and studies in male mice revealed the specificity of the antibodies for this species [7]. Thus these antibodies were used in our Western blots, immunofluorescence, immunoelectron microscopy, and flow cytometric studies, all of which further demonstrated their competence to specifically detect mouse PMCA4a. Anti-CD9 antibody (SC-18869) and anti-human HSC70 (heat shock cognate protein 70; SC7298) mouse monoclonal antibody were obtained from Santa Cruz Biotechnology, and β-actin antibody (#4970) from Cell Signaling (Boston, MA).

Preparation of Murine Female Reproductive Tissues and Isolation of Caudal Sperm
Vagina, uterus and oviduct were dissected immediately after sacrifice and frozen at -80°C for subsequent expression analysis, or they were flushed with PBS to remove the LF and immediately frozen at -80°C. Sperm were harvested from the caudal epididymides of 4-6 sexually mature males per experiment, by mincing the caudae in PBS with Protease inhibitor as described earlier [16] and kept at 37°C for 10 min to allow sperm to swim out of the tissue. After gravity settling of the tissues the supernatant was collected and subjected to centrifugation at 500 x g for 10 min to pellet the sperm which were then washed with PBS.

Total RNA Isolation and Reverse Transcriptase (RT)
PCR-Reproductive tissues (vagina, uterus, oviduct) were collected from two virgin females after superovulation, and total RNAs were extracted using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's directions and RNA samples were further treated with DNAase (Turbo DNA-Free, Ambion Inc., Austin, TX). Total RNA (1μg) was used to synthesize cDNA using the iScript TM cDNA synthesis kit (BioRad, Hercules, CA). Samples without the addition of reverse transcriptase served as controls. To amplify cDNA samples, PCR reactions (25 µl final volume) were performed with 2 µl cDNA for 30 cycles (ABI Veriti Thermocycler), using Pmca4a and 4b primers as follows: forward primer sequence was 5'-GGA CGA GAT TGA CCT TGC CG -3' and the reverse 5'-CAC CAT CCA ACA GGA GCA CAC T-3'. PCR amplification was performed for 35 cycles at denaturation for 30 sec at 96°C, annealing for 30 sec at 59°C, extension for 1 min at 72°C, and a final extension of 5 min at 72°C. Gapdh was used as an internal control, Using commercial mouse Gapdh primers (Qiagen, Valencia, CA), PCR amplification was performed for 34 cycles at denaturation for 10 sec at 95°C, annealing for 30 sec at 55°C, extension for 90 sec at 72°C, and a final extension of 10 min at 72°C. The total RNA extracted from testis was used as positive control. RT-PCR products (10 µl) were run on 2% Nusieve agarose gel in TAE (40 mM Trisacetate, 2 mM EDTA, pH 8.5) containing ethidium bromide (10 µg/ml), along with a 100 bp ladder (Invitrogen).

Protein Localization by Immunofluorescence (IF)
Mouse vaginal, uterine, and oviductal tissues were collected from superovulated females and immediately embedded in optimum cutting temperature media (OCT) (Tissue Tek, Torrance, California) and frozen at -80°C, or were frozen immediately after flushing to remove he luminal fluids. Cryostat PMCA4a in the Female Reproductive Tract PLOS ONE | www.plosone.org sections (20μm) were made and kept in -80°C until processing. Slides were fixed in 1:1 acetone:methanol pre-chilled for 20 min at -20°C and then allowed to air-dry for 10 min before being placed in protein blocker [1% bovine serum albumin (BSA) in PBS] for 1-2 h at RT. They were then incubated overnight at 4°C with the anti-PMCA4a primary antibody diluted at 1:50 in blocking solution, or with PBS or rabbit IgG for the negative controls, followed by washing with PBS (2X, 20 min). Sections were then incubated for 1h at RT with Alexafluor 568conjugated goat anti-rabbit IgG (Molecular Probes, Eugene Oregon, 1:200) containing 1:2000 Draq-5 as a nuclear stain (Biostatus Limited, Leicestershire, United Kingdom), followed by (2X, 20 min) washing with PBS. Finally, the samples were mounted with mounting media and cover-slipped. Slides were visualized using a Zeiss LSM 780 confocal microscope (Carl Zeiss, Inc, Gottingen, Germany) using a plan-Apochromatic 20x objective.

Analysis of PMCA4a Expression during Physiological Estrous Cycle
Eight to 12 week-old virgin females were used in this investigation. They were categorized by the stages of estrous cycle based on the proportion of different cell types observed in the vaginal secretion [17]. Uterine tissues were collected from females at diestrus, proestrus, estrus, and metestrus. Tissues were then processed for IF as described above.

Collection of Luminal Fluids in Naturally Cycling Females and from Males
Caudal luminal fluids, used as controls, were collected from the epididymal caudae pooled from 2-3 WT ICR mice, using a method that was previously described [7,18]. Reproductive luminal fluids were collected from 4 females in proestrus and estrus (combined), and from 4 in metestrus and diestrus (combined), as previously described [12,18]. Briefly, vaginas, uterine horns and oviducts were flushed with PBS plus protease inhibitors by inserting a 26-gauge needle attached to a 1 ml syringe into the lumen and fluids were collected. Centrifugation at 3,500 x g for 10 min was used to exclude tissue fragment and cells and to clarify the female fluids.

Collection of Vaginal, Uterine and Oviductal Luminal Fluids from Females after hormonal induction of Estrus
VLF, ULF and OLF were obtained from 6-8 superovulated female mice per experiment. The methodology of collecting the fluid is similar to described above [18].

Fractionation of ULF and OLF
Clarified uterine and oviductal luminal fluids, were collected from 6-8 superovulated female mice as described above. To fractionate the female LF (FLF) we used a methodology similar to previously described [12]. Briefly, the clarified FLFs were subjected to ultracentrifugation at 120,000 x g for 2 h, at 4°C using a Beckman Optima 2-70 k ultracentrifuge and a Ti60 rotor. The resulting pellets were re-suspended in homogenization buffer and protease inhibitor, for Western blot analysis, or in PBS to a final concentration of 2 mg/ml proteins to be used for co-incubation with sperm or transmission electron microscopy (TEM). The supernatants were collected and the proteins precipitated with 10% TCA and recovered in sample buffer, before being subjected to SDS-PAGE and Western blotting.

SDS-PAGE and Western Blot Analysis
Preparation of protein extracts from vagina, uterus, and oviduct was performed as described previously [11], using caudal sperm as a positive control. Total protein concentration in the lysates was determined using the bicinchoninic acid protein assay Kit (Pierce), according to the manufacturer's protocol. Samples for electrophoresis were diluted in 2x Laemmli sample buffer with DTT (final concentration 100 mM) and urea (125 mg/ml) and incubated for 10 min at 37°C. Twenty to 60 µg of proteins from tissues and fluids, respectively, were loaded per lane on 10% polyacrylamide gels and transferred onto a nitrocellulose membrane (Amersham Biosciences). Western blotting was performed with the WesternBreeze Chemiluminescent Immunodetection Kit (Invitrogen) according to the manufacturer's instructions. Blots were blocked for 1h at RT and incubated in anti-PMCA4a primary antibody (1: 500) or in anti-CD9 (1:1000) primary antibody overnight at 4°C. Non-specific binding of antibody was removed using 5X washes of TBST (20 mM Tris, pH 8.0, containing 150 mM NaCl and 0.5% Tween 20) before incubation in the alkaline phosphatase (AP)-conjugated antirabbit IgG (Invitrogen, diluted 1:2000) or AP-conjugated anti-rat IgG (Sigma-Aldrich, diluted 1:32,000) for 1 h at 4°C. The membrane was again washed (6X, 15 min) using TBST before chemiluminescence was detected by using the ECL kit (Bio-Rad, Hercules, CA). The membrane was re-probed with HSC70 or β-actin antibody which was used as internal loading control and for normalization.

Negative Staining for TEM
Nickel TEM grids (Electron Microscopy Sciences), 400 mesh with a formvar/carbon film, were floated on a drop of the fractions of purified OLF pellet suspension. The grids were then washed with several drops of water and then stained with 1% uranyl acetate, a phospholipid stain, before being subjected to microscopic analysis. Membrane vesicles were imaged using the TEM (Zeiss LIBRA 120).

Immunogold Labeling of PMCA4a or CD9 in Exosomes
The methodology of immunogold labeling is similar to previously described [19]. Briefly, following ultracentrifugation, vesicle suspensions were mixed 1:1 with 4% paraformaldehyde in 0.15 M Sorensen's phosphate buffer pH 7.4 then were applied to 400-mesh nickel grids. The grids were then washed on 3 drops of PBS then incubated with 0.05 M glycine in PBS for 15 min. After blocking with goat block (Aurion, Wageningen, Netherlands) for 30 min and washing, the grids were incubated with primary anti-PMCA4a antibody diluted 1:10 in 0.1 % BSAc TM containing 0.02 % Triton X-100 (to permeabilize the vesicle membranes) for 2 h or CD9 antibody diluted 1:10 in 0.1 % BSA-c TM . Grids were then washed 6X, 5 min each, with 0.1 % BSA-c TM (Aurion) and then were exposed to goat anti-rabbit or PMCA4a in the Female Reproductive Tract PLOS ONE | www.plosone.org goat anti-rat secondary antibodies conjugated to 6 nm gold particles (Aurion) diluted 1:20 in 0.1 % BSA-c TM (Aurion) for 2 h. The grids were then washed 6X, 5 min each, with 0.1% BSA-c and then with PBS 3x, 5 min each. Grids were post-fixed with 2% glutaraldehyde in PBS for 5 min and then washed with PBS for 5 min, followed with filtered double de-ionized water 5X, 2 min each. The membrane vesicles on the grids underwent negative staining with 1% uranyl acetate. After drying, the grids were examined with a Zeiss LIBRA 120 electron microscope operated at 120 kV. Control samples were processed identically, except that rabbit IgG or rat IgG was substituted for the primary antibody.

Analysis of In Vitro Sperm Uptake of PMCA4a from Female Luminal Fluids (FLF)
In Vitro sperm uptake assay of PMCA4a was similar to that previously described from our lab [7,12,16,18]. Briefly, caudal sperm were incubated in exosomes or unfractionated FLF (combined VFL, OFL and ULF), recovered as described above in PBS supplemented with 200 μM zinc acetate and protease inhibitor, pH 7.0. This PBS-zinc acetate solution was used for incubating equal aliquots of control sperm samples. After coincubation of sperm and FLF for periods of 2-3 h at 37°C, control and test samples were washed (3X, collected with centrifugation at 500 x g for 15 min) using PBS with 200 μM zinc acetate. They were then fixed with 1.5% paraformaldehyde for 1 hr at RT, washed (3X, 15 min) with PBS and then permeabilized with 0.1% Triton X-100 for 10 min at RT. After washing cells with blocking buffer (2% BSA in PBS) they were incubated in blocking buffer for 30 min at RT. Permeabilized cells were then incubated in anti-PMCA4a primary antibody (1:200 dilution in blocking solution, applied to the cells at 4°C, for 1h), followed by washing (3X, 15 min) with PBS. Alexafluor 488-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene Oregon) diluted 1:200 in blocking solution was used as a secondary antibody in which cells were incubated at RT for 30 min in the dark, followed by washing (3X, 20 min) with PBS. Stained sperm were analyzed by flow cytomety on a FACSCalibur or a FACSAria TM II (BD Sciences, San Jose, CA), equipped with an argon laser at 488 nm excitation. Analysis was performed using standard protocols and quantification criteria to assess PMCA4a uptake by sperm.

Statistical Analysis
Two-way ANOVA and Student's t-tests were performed on the means ± ±SEM for 3 replicates, to compare the statistical significance between unpaired data or for multiple groups, respectively. P values were calculated and *P<0.05 was considered significant.

Analysis of Pmca4 Transcripts in Murine Female Reproductive Tissues
To obtain information on the expression of transcripts from the C-terminal splice variants of Pmca4 in the murine female reproductive tract, we performed RT-PCR using specific primers. Testis tissues were used as a positive control. As shown in Figure 1 A, mRNA for Pmca4a and 4b variants was present throughout the female tract in tissues recovered following superovulation. The expected PCR products of 469 and 278 bp were observed in all three regions: the oviduct, uterus, and vagina. The mRNA distribution varied in the tissues. Of the two variants Pmca4b mRNA is more abundant in all three regions ( Figure 1B). Sequence analysis of the 469 and 278 bp PCR products matched the cDNA sequence of PMCA4a and the alternatively spliced PMCA4b, respectively. A negative control for the PCR showed no product for the reaction in the absence of reverse transcriptase.

Localization and Detection of PMCA4a in the Female Reproductive Tract and in its Secretions
To determine the expression and localization pattern of the PMCA4a protein in the female reproductive tract, sections of vaginal, uterine, and oviductal tissues from superovulated virgins were subjected to indirect IF analysis using the polyclonal PMCA4a antibody. The results in Figure 2A show that PMCA4a is mostly present in the luminal and glandular epithelial cells of the endometrium of the uterus. In the vagina a strong signal of PMCA4a was detected in the mucosa and within the epithelial layers at the apical membrane near the PMCA4a in the Female Reproductive Tract PLOS ONE | www.plosone.org lumen (yellow arrowhead) and a weaker staining at the basement membrane (white arrowhead). In the oviduct the signal for PMCA4a is strongest in the luminal epithelia of the convoluted epithelial lining on the apical membrane. The presence of PMCA4a on the apical membrane of the luminal epithelial cells suggests that PMCA4a is secreted in the lumen.
The uterine expression pattern of PMCA4a in naturally cycling females was determined after vaginal smears were examined to identify the stages of the estrus cycle. Uterine tissues collected from virgins at the four stages of the estrus cycle were analyzed. Interestingly, only in the pro-estrus and estrus phases was expression of PMCA4a detectable in both the luminal and glandular epithelium, while it was absent or marginally present in the metestrus and diestrus phases in these regions (Figure 3, insets). In contrast, the myometrium showed strong expression of PMCA4a in all four phases of the estrus cycle (Figure 4).
To confirm the IF data, we performed Western blotting analysis on tissues recovered after superovulation. In all three regions of the female reproductive tract the ~128 kDa PMCA4a band was detected ( Figure 2B), corroborating the IF findings. Western analysis also revealed that while females in proestrus/ estrus secreted PMCA4a in the luminal fluids combined (FLF) those in metestrus/diestus had only marginal levels of the protein ( Figure 5A). As expected, females brought into estrus by superovulation also displayed PMCA4a in the luminal fluids from the vagina (VLF), uterus (ULF), and the oviduct (OLF), as shown in Figure 5B. Densitometric analysis showed that the level of expression of PMCA4a in the LFs varied, being lowest in the VLF and highest in the OLF which is present at the site of fertilization. The 9-fold higher level of PMCA4a in OLF over VLF ( Figure 5C) represents a significant increase (P = 0.03).

Characterization of membrane vesicles from Oviductal Luminal Fluids as Exosomes
Using antibodies which recognize the extracellular domains of CD9 tetraspanin, one of the most abundant proteins found on the exosomal membrane [20,21], we detected the presence of the 24 kDa CD9 by Western analysis of the OLF pellets as well as of the uterosomes ( Figure 6C). The band was however absent in the supernatant. The presence of CD9 in the OLF membrane vesicles was confirmed by TEM analysis and immunogold labeling on the membrane where the gold particles were seen on the exterior ( Figure 6D), an orientation that typifies exosomes [19]. Therefore, consistent with the requirements for defining membrane vesicles as exosomes, we show that vesicles in the OLF: 1) are within a size range of 25-100 nm in diameter, as detected by negative staining and TEM ( Figure 6B), 2) enriched with CD9 tetraspanin, a biomarker of exosomes [20,21] (Figure 6C), and 3) have membrane proteins in the exosomal orientation. Thus, we defined these vesicles, purified from oviductal fluid, as exosomes and termed them"oviductosomes"

PMCA4a is Present in the Exosomes of the Luminal Fluids
When the nitrocellulose membranes used for the detection of CD9 in the exosomes from ULF and OLF ( Figure 6C) were re-probed with anti-PMCA4a antibodies in Western blotting analysis, the ~128 kDa PMCA4a band was detected, but was absent from the supernatant ( Figure 7A). For the uterosomes (10), there was a cross-reactive ~100 kDa band that was not seen in the OLF and whose origin is unknown. Our findings indicate that 100% of PMCA4a in the luminal fluids resides in the particulate fraction, or the exosomes. More importantly, our results indicate that PMCA4a is carried on CD9-positive exosomes.
We confirmed the presence of PMCA4a on CD9-positive exosomes by TEM and immunogold labeling. Control samples incubated in the presence of rabbit IgG showed randomly dispersed 6 nm immunogold particles on the grids, but none localized to vesicles ( Figures 7B, 1-4). In test samples treated with the primary antibody, immunogold particles detecting PMCA4a were observed on both the oviductosomes ( Figures  7B, 5-8) and the uterosomes (Figures 7B, 9-12). It should be noted that gold particles were not randomly distributed and were rarely localized outside of the vesicles on the grids. Importantly, on the vesicles, the particles detecting the localization of PMCA4a ( Figure 7B) required gentle permeabilization of the vesicles in order for the PMCA4a antibodies to gain access to, and recognize an epitope on, the cytoplasmic-side of the molecule. This further confirms that the oviductosomes are exosomes, and not endosomes which have the cytoplasmic side of the molecule in the exterior orientation [19].

In Vitro Uptake of PMCA4a on Caudal Sperm from Unfractionated Luminal Fluids and Exosomes
The presence of PMCA4a in the luminal fluid during estrus, the only phase when sperm traverse the female tract, suggested that the pump may be transferred to the sperm surface during their transit and storage in the oviduct. Fresh caudal epididymal sperm were incubated for 2-3 h in PBS supplemented with Zn 2+ (carrier control), which is known to prevent the acrosome reaction [22], or in FLF suspended in PBS containing Zn 2+ (added in the form of zinc acetate). Sperm were then fixed and stained with anti-PMCA4a primary and Alexafluor-488 conjugated secondary antibodies as described in the Materials and Methods. Flow cytometry, used to quantify fluorescence intensity as a measure of PMCA4a uptake in 50,000 sperm/group, revealed that after 2 h incubation in FLF there was a right peak shift in fluorescence intensity, compared to the PBS control ( Figure 5D). After 3 h co-incubation of sperm and reconstituted exosomes, there was a remarkablẽ 3-fold increase in uptake compared to the PBS control ( Figure  8). In general, the flow cytometric data reveal in vitro acquisition of PMCA4a on caudal sperm and suggest that the plasma membrane in these cells is not all fully saturated with this transmembrane protein when sperm leave the male.

Expression of PMCA4a in the murine female reproductive tract
Of the two major splice variants of PMCA4, 4a and 4b, 4a is faster in extruding Ca 2+ and returning cytosolic Ca 2+ PMCA4a in the Female Reproductive Tract PLOS ONE | www.plosone.org concentration to resting levels [13]. Since sperm in the oviduct, particularly the AIJ, have high levels of intracellular Ca 2+ [23], they need effective efflux mechanisms to prevent Ca 2+ overload in order to maintain their viability. Our mRNA data (Figure 1) revealed that both Pmca4a and 4b isoforms are expressed in reproductive tissues collected from all three regions of the tract in females undergoing estrus. As estrus is the only time when females are receptive to the male and sperm are likely to be   . The ~128 kDa PMCA4a is seen in pro-estrus and estrus and is marginally present at metestrus and diestrus. Caudal epididymal luminal fluid was used as a positive control. The membrane was stripped and re-probed for HSC70 as a loading control. B) Western blots of VLF, ULF, and OLF recovered after superovulation demonstrate the presence of the ~128 kDa PMCA4a. Sperm protein was used as a positive control. The membrane was stripped and re-probed for β-actin as a loading control. C) Quantitation of Western blot data shown in B; the relative expression was determined using VLF as 1. The data represent the mean (±SEM) of a minimum of three independent experiments, and the intensity was quantified by Image J software. ANOVA and t-tests were performed on the mean and P values were calculated. *P = 0.03 indicates a significantly increase amount of PMCA4a in OLF compared to that in VLF. D) A peak shift of fluorescence intensity to the right, indicates increase amounts of PMCA4a in sperm incubated in FLF compared to PBS for 2 h and treated as described in Materials and Methods.   may be necessary to maintain Ca 2+ homeostasis when the demand for efflux is great.
When PMCA4a protein was examined by IF in naturally cycling females, it was found to be expressed in the uterine myometrium in all four stages of the estrus cycle ( Figure 4). This suggests that the pump plays a key role in the maintenance of uterine function by helping to regulate membrane potential and intracellular Ca 2+ . However, during the estrus and pro-estrus phases, unlike metestrus and diestrus, in addition to its presence in the myometrium there was upregulation of PMCA4a in the uterine endometrium, as seen in the luminal and glandular epithelia. This suggests that the transcription of the Pmca4 gene and/or the alternative splicing of its primary RNA transcripts are/is under the control of female sex hormones. Indeed, a Blast search of the promoter region of the Pmca4 gene for the presence of estrogen response elements (EREs) revealed at -313-335 a potential sequence, 5'-GGGCTgacTGACC-3'. Although this sequence contains two mismatches compared to the consensus ERE [25], it fulfills the requirement for ER-ERE binding for estrogen responsive genes [25]. Further studies will be needed to determine the mechanism of estrogen-mediated up-regulation of PMCA4a expression in the female reproductive tract.
To study PMCA4a during estrus, virgins were superovulated and sections of all three regions of the female tract analyzed. IF revealed that in addition to the uterus, the vagina and the oviduct showed high expression of PMCA4a. Importantly, all three ductal regions had the strongest PMCA4a staining on the luminal side of the apical membrane of the epithelium. This finding parallels that seen in the epididymis [7] and is consistent with the secretion of PMCA4a from these tissues into the lumen, similar to the secretion of other PMCAs in other tissues [26][27][28][29].
Western analysis corroborated the IF results and revealed the presence of the ~128 kDa PMCA4a band in all female reproductive tissues as well as in the luminal fluids. Interestingly, in naturally cycling females while fluids collected during metestrus/diestrus had only marginal levels of PMCA4a, those collected during proestrus/estrus had elevated levels of the protein, as seen after induction of superovulation. Here, PMCA4a was most abundant in OLF. These findings are similar to that for SPAM1, a GPI-linked sperm protein which was found to be present on uterosomes, vesicular particles in the uterine fluid [30] during estrus. However, unlike GPI-linked proteins that are found in both the particulate and the soluble fraction of the LF [11,12], we found PMCA4a exclusively in the particulate fraction, the oviductosomes and the uterosomes. This localization is consistent with the PMCA4a's transmembrane structure.
Oviductosomes were identified for the first time, and characterized as exosomes based on negative staining, size (25-100 nm), membrane orientation (cytoplasmic-side inward), and the CD9 biomarker. These oviductosomes are likely to originate as internal vesicles of multivesicular bodies (MVBs) with a unique orientation (cytoplasmic-side inward) [19,31]. It is also possible that they could result from an apocrine pathway resulting from blebbing of the apical membrane of the epithelial lining, as reported for epididymosomes in the male reproductive tract [32]. Whatever their origin, they are likely to play a role in cell-cell communication.

Transport of PMCA4a from the Female Reproductive Luminal Fluids to Sperm during Capacitation
Exosomes are thought to be the vehicle of transport of proteins from luminal fluids to the sperm surface [33,34]. Accordingly, we detected the transfer of PMCA4a from unfractionated FLF and from exosomes to caudal sperm in vitro with increases of up to ~3-fold. Our finding strongly suggests that PMCA4a is acquired by sperm in vivo during their transit in the female. Figure 8 provides some initial insight into the mechanism of uptake, as we detected that exosomes are not only necessary but are sufficient for the transfer of PMCA4a to the sperm. The presence of PMCA4a's uptake from reconstituted exosomes, in the absence of soluble proteins in the FLF, is supportive of this conclusion.
Studies on the mechanism(s) by which vesicles transport proteins to the sperm surface are limited. Griffiths et al. [12,18] have shown that vesicles dock on the sperm membrane and have postulated that hydrophobic interactions may underlie the transfer of GPI-linked proteins which are attached to the outer leaflet of the lipid bilayer of the membrane. However, this mechanism is unlikely to be involved in the transfer of transmembrane proteins, such as PMCA4a which has its catalytic domain on the cytosolic side of the membrane [35]. In the study by Griffiths et al. [12], the data revealed that the vesicles fuse with the sperm membrane, although the authors did not comment on this. It is likely that such sperm-vesicle fusion is the mechanism by which PMCA4a is transferred to sperm. Recently, Caballero et al. [20] have shown that CD9positive microvesicles mediate the transfer of molecules to bovine sperm during epididymal maturation. It is likely that the CD-9 positive oviductosomes and uterosomes might also play this role in mammalian sperm. Further studies will be required to elucidate the precise mechanism involved. Whatever mechanism is involved, the in vitro uptake of PMCA4a on caudal sperm after co-incubation with female LF indicates that the sperm membrane is not saturated with this protein after epididymal maturation. During progressive maturation in the female, sperm acquire their fertilizing ability while transiting the oviduct [36] where they are stored in the sperm reservoir compartment and remain viable until ovulation occurs [2]. During this period they interact with the oviductal epithelial lining and the oviductal secretions [23,37] and their viability is maintained. To date, very little is known of the component(s) of the secretion that are responsible for sperm viability and maturation. The present study has identified PMCA4a, a key element of the Ca 2+ handling toolkit, as one of these components and suggests that it is highly likely to play a role in the final maturation of sperm in the oviduct.
Of interest, the distribution of PMCA4a in the FLF is most abundant in OLF. This is consistent with an increased volume of OLF during the estrus phase [1] when sperm are likely to be present and undergoing hyperactivated motility. Since the latter is accompanied by activation of Ca 2+ influx via the CatSper channel [24,38], PMCA4a, and likely PMCA4b, could play an essential role in maintaining Ca 2+ homeostasis and sperm viability. Indeed, deletion of the PMCA4a and 4b pumps causes loss of hyperactivated motility and infertility in mice [4,5] and reduced PMCA4 activity in Jam-A null mice has a similar effect [6,39]. On the other hand, lethal calcium concentrations were diminished when bovine sperm were exposed to the apical plasma membrane of oviductal epithelial cells [40]. The authors concluded that anchored proteomic factors on the oviductal epithelial cells are responsible for maintaining sperm motility and viability [40]. The present study now allows us to identify PMCA4a as a potential major proteomic factor involved in diminishing toxic Ca 2+ levels in bovine sperm co-incubated with oviductal epithelial cells. Thus further studies will focus on whether or not PMCA4a that is transferred to sperm has a functional impact.
Although there is evidence that epididymal PMCA4a is acquired in vivo by sperm [7], it is unknown if this is the case for oviductal PMCA4a. It is also unknown how acquisition of epididymal PMCA4a impacts sperm function. Although Pmca4 null females are not sterile their level of fertility, compared to WT, has never been investigated. In this vein, the fertilizing competence of in vitro capacitated sperm in the presence/ absence of oviductal or uterine PMCA4 has yet to be studied. Pmca4 null mice provide the opportunity for investigating the impact of sperm acquisition of PMCA4 from both the male and female reproductive tracts on Ca 2+ -ATPase activity and ultimately fertilizing competence, investigations which are underway in our laboratory.
In conclusion our study reveals for the first time the expression and secretion of PMCA4a in the female reproductive tissues and luminal fluids during estrus. The secretion occurs via exosomes from which PMCA4a, and likely 4b, can be acquired by sperm. The acquisition of this important Ca 2+ handling tool, primarily from the oviductal fluid via oviductosomes which are identified for the first time, is likely to be central to the maintenance of Ca 2+ homeostasis and sperm viability during their storage in the oviduct and during capacitation and the acrosome reaction, both of which are important steps in fertilization.