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. 2018 Mar 1;24(3):143-157.
doi: 10.1093/molehr/gay003.

Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility

Pradeepthi Bathala  1 , Zeinab Fereshteh  1 , Kun Li  1   2 , Amal A Al-Dossary  1   3 , Deni S Galileo  1 , Patricia A Martin-DeLeon  1
Affiliations

Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility

Pradeepthi Bathala et al. Mol Hum Reprod. .

Abstract

Study questions: Are extracellular vesicles (EVs) in the murine oviduct (oviductosomes, OVS) conserved in humans and do they play a role in the fertility of Pmca4-/- females?

Summary answer: OVS and their fertility-modulating proteins are conserved in humans, arise via the apocrine pathway, and mediate a compensatory upregulation of PMCA1 (plasma membrane Ca2+-ATPase 1) in Pmca4-/- female mice during proestrus/estrus, to account for their fertility.

What is known already: Recently murine OVS were identified and shown during proestrus/estrus to express elevated levels of PMCA4 which they can deliver to sperm. PMCA4 is the major Ca2+ efflux pump in murine sperm and Pmca4 deletion leads to loss of sperm motility and male infertility as there is no compensatory upregulation of the remaining Ca2+ pump, PMCA1. Of the four family members of PMCAs (PMCA1-4), PMCA1 and PMCA4 are ubiquitous, and to date there have been no reports of one isoform being upregulated to compensate for another in any organ/tissue. Since Pmca4-/- females are fertile, despite the abundant expression of PMCA4 in wild-type (WT) OVS, we propose that OVS serve a role of packaging and delivering to sperm elevated levels of PMCA1 in Pmca4-/- during proestrus/estrus to compensate for PMCA4's absence.

Study design, size, duration: Fallopian tubes from pre-menopausal women undergoing hysterectomy were used to study EVs in the luminal fluid. Oviducts from sexually mature WT mice were sectioned after perfusion fixation to detect EVs in situ. Oviducts were recovered from WT and Pmca4-/- after hormonally induced estrus and sectioned for PMCA1 immunofluorescence (IF) (detected with confocal microscopy) and hematoxylin and eosin staining. Reproductive tissues, luminal fluids and EVs were recovered after induced estrus and after natural cycling for western blot analysis of PMCA1 and qRT-PCR of Pmca1 to compare expression levels in WT and Pmca4-/-. OVS, uterosomes, and epididymal luminal fluid were included in the comparisons. WT and Pmca4-/- OVS were analyzed for the presence of known PMCA4 partners in sperm and their ability to interact with PMCA1, via co-immunoprecipitation. In vitro uptake of PMCA1 from OVS was analyzed in capacitated and uncapacitated sperm via quantitative western blot analysis, IF localization and flow cytometry. Caudal sperm were also assayed for uptake of tyrosine-phosphorylated proteins which were shown to be present in OVS. Finally, PMCA1 and PMCA4 in OVS and that delivered to sperm were assayed for enzymatic activity.

Participants/materials, setting, methods: Human fallopian tubes were flushed to recover luminal fluid which was processed for OVS via ultracentrifugation. Human OVS were negatively stained for transmission electron microscopy (TEM) and subjected to immunogold labeling, to detect PMCA4. Western analysis was used to detect HSC70 (an EV biomarker), PMCA1 and endothelial nitric oxide synthase (eNOS) which is a fertility-modulating protein delivered to human sperm by prostasomes. Oviducts of sexually mature female mice were sectioned after perfusion fixation for TEM tomography to obtain 3D information and to distinguish cross-sections of EVs from those of microvilli and cilia. Murine tissues, luminal fluids and EVs were assayed for PMCA1 (IF and western blot) or qRT-PCR. PMCA1 levels from western blots were quantified, using band densities and compared in WT and Pmca4-/- after induced estrus and in proestrus/estrus and metestrus/diestrus in cycling females. In vitro uptake of PMCA1 and tyrosine-phosphorylated proteins was quantified with flow cytometry and/or quantitative western blot. Ca2+-ATPase activity in OVS and sperm before and after PMCA1 and PMCA4 uptake was assayed, via the enzymatic hydrolysis rate of ATP.

Main results and the role of chance: TEM revealed that human oviducts contain EVs (exosomal and microvesicular). These EVs contain PMCA4 (immunolabeling), eNOS and PMCA1 (western blot) in their cargo. TEM tomography showed the murine oviduct with EV-containing blebs which typify the apocrine pathway for EV biogenesis. Western blots revealed that during proestrus/estrus PMCA1 was significantly elevated in the oviductal luminal fluid (OLF) (P = 0.02) and in OVS (P = 0.03) of Pmca4-/-, compared to WT. Further, while PMCA1 levels did not fluctuate in OLF during the cycle in WT, they were significantly (P = 0.02) higher in proestrus/estrus than at metestrus/diestrus in Pmca4-/-. The elevated levels of PMCA1 in proestrus/estrus, which mimics PMCA4 in WT, is OLF/OVS-specific, and is not seen in oviductal tissues, uterosomes or epididymal luminal fluid of Pmca4-/-. However, qRT-PCR revealed significantly elevated levels of Pmca1 transcript in Pmca4-/- oviductal tissues, compared to WT. PMCA1 could be transferred from OVS to sperm and the levels were significantly higher for capacitated vs uncapacitated sperm, as assessed by flow cytometry (P = 0.001) after 3 h co-incubation, quantitative western blot (P < 0.05) and the frequency of immuno-labeled sperm (P < 0.001) after 30 min co-incubation. Tyrosine phosphorylated proteins were discovered in murine OVS and could be delivered to sperm after their co-incubation with OVS, as detected by western, immunofluorescence localization, and flow cytometry. PMCA1 and PMCA4 in OVS were shown to be enzymatically active and this activity increased in sperm after OVS interaction.

Large scale data: None.

Limitations reasons for caution: Although oviductal tissues of WT and Pmca4-/- showed no significant difference in PMCA1 levels, Pmca4-/- levels of OVS/OLF during proestrus/estrus were significantly higher than in WT. We have attributed this enrichment or upregulation of PMCA1 in Pmca4-/- partly to selective packaging in OVS to compensate for the lack of PMCA4. However, in the absence of a difference between WT and Pmca4-/- in the PMCA1 levels in oviductal tissues as a whole, we cannot rule out significantly higher PMCA1 expression in the oviductal epithelium that gives rise to the OVS as significantly higher Pmca1 transcripts were detected in Pmca4-/-.

Wider implications of the findings: Since OVS and fertility-modulating cargo components are conserved in humans, it suggests that murine OVS role in regulating the expression of proteins required for capacitation and fertility is also conserved. Secondly, OVS may explain some of the differences in in vivo and in vitro fertilization for mouse mutants, as seen in mice lacking the gene for FER which is the enzyme required for sperm protein tyrosine phosphorylation. Our observation that murine OVS carry and can modulate sperm protein tyrosine phosphorylation by delivering them to sperm provides an explanation for the in vivo fertility of Fer mutants, not seen in vitro. Finally, our findings have implications for infertility treatment and exosome therapeutics.

Study funding and competing interest(s): The work was supported by National Institute of Health (RO3HD073523 and 5P20RR015588) grants to P.A.M.-D. There are no conflicts of interests.

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Figures

Figure 1
Figure 1
Oviductosomes (OVS) are evolutionarily conserved in humans and carry PMCAs. (A) TEM of negatively stained human OVS showing immunogold labeling of PMCA4. (a) The IgG control shows a microvesicle (**), larger OVS (0.1–1 μm) and an exosomes (*) <100 nm. (b) Immunogold labeling (6 nm gold particles) of PMCA4 (arrows) showing gold particles on OVS. These particles are seen only on the background in (a). The inset in (b) is enlarged in (c) to show the particles (bar = 100 nm). (B, C) Western blots show the presence of HSC70, an extracellular vesicle (EV) biochemical marker, after stripping and re-probing of the membrane, as well as PMCA1 and eNOS (in dimeric and monomeric forms) in human OVS (HOVS) and human oviductal tissues (HOVD).
Figure 2
Figure 2
OVS arise from the apocrine pathway. (A) TEM of murine oviductal epithelium. Red arrows show blebs, containing OVS, in the lumen (LUM) among sections of cilia from the ciliated cells (CEC) and microvilli (MV) of the secretory cells (SEC). Blebs are typically found when EVs arise via the apocrine pathway (bar = 0.2 μm). (B) TEM tomographic series reveals OVS arising from the apocrine pathway. Small black arrows show different planes (B (A–F)) of an OVS-containing bleb, released from the epithelium. Large black arrows show different planes of the 3D structure of an EV, not seen for microvilli (green arrow) or cilia (yellow arrow). B (G) An enlarged 3D surface rendering overlaid on the last slide (B (F)) of the tomogram with the bleb (containing blue EVs) seen in red, an isolated exosome (blue) is at the top right, microvilli (green) and a cilium (yellow) are seen (bar = 0.5 μm).
Figure 3
Figure 3
PMCA1 expression levels in murine reproductive tissues during estrus show no difference in WT and Pmca4 KO oviducts. (A) Indirect immunofluorescence shows a strong PMCA1 signal (red) in the oviduct and uterus throughout, including both the apical and basement membranes. A weaker signal is seen in the vagina. Insets are shown in A (c, g and k). Negative controls (NC; rabbit IgG) do not show the staining (d, h and l) and validate the specificity of the PMCA1 antibody. (B) PMCA1 staining shows no difference in intensity of the signal between Pmca4 KO (f) and WT (b) oviduct. Enlarged images are seen in the insets (d and h) and the DNA staining is blue. Bar = 100 μm (200 μm for all insets and the same scale for all micrographs). (C) Hematoxylin and eosin staining of oviductal sections shows no difference in histology between WT and KO. (D) Representative western blot of estrus tissue lysates showed the 139 kDa PMCA1, with testis as a positive control. The membrane was stripped and re-probed with HSC70, as a loading control. (E) Quantification of western blot data in (D) represents the mean (±SEM, n = 3) of band intensities, using Image Lab 6.0 software. Statistical analysis showed no difference between WT and KO tissues.
Figure 4
Figure 4
Oviductal luminal fluid (OLF) of WT and Pmca4 KO shows elevated levels of PMCA1 in estrus during natural cycling in KO only. (A, B) Western blot of WT and KO OLF after hormonally induced estrus s of PMCA1 in Pmca4 KO, compared to WT (P = 0.07, n = 3). Epididymal luminal fluids from Pmca4 KO and WT were included and showed no difference (P = 0.1, n = 3). (C, D) Pmca4 KO OLF shows a significant (*P = 0.02) increase during proestrus/estrus vs WT OLF and a significant decrease (*P = 0.02) during metestrus/diestrus of PMCA1, using One-way ANOVA and Student t-tests.
Figure 5
Figure 5
Compared to WT, estrus Pmca4−/− OVS (unlike uterosomes) and oviductal tissues have significantly elevated PMCA1 and Pmca1 mRNA levels, respectively. Representative Western of PMCA1 in OVS (A) and uterosomes (C) of WT and Pmca4 KO females, with the HSC70 loading controls, and testis used as positive control. (B) Student t-tests (n = 3) show a significant increase (*P = 0.03) of PMCA1 in Pmca4 KO, compared to WT OVS, but not in uterosomes (D). (E) Pmca1 mRNA in oviductal tissues normalized to Gapdh (n = 3) shows a significant increase (P = 0.02) in Pmca4−/−, compared to WT. Similar results were seen for both pairs of primer. Agarose gel electrophoresis of the RT-PCR products are shown in Supplemental Fig. S1.
Figure 6
Figure 6
Murine OVS carry in their cargo PMCA4 partners (nNOS and CASK) which interact with PMCA1. (A) WT and Pmca4−/− OVS show the 155 kDa nNOS band and ~100 kDa CASK band, with testis as positive controls. (B) PMCA1 (139 kDa) co-immunoprecipitates (co-IP) with nNOS in OLF. (C) PMCA1 co-IP with CASK in OLF. For both (B and C), Total protein (TP), obtained from OLF and rabbit IgG were used as a positive and negative control.
Figure 7
Figure 7
OVS deliver elevated levels of PMCA1 to capacitated, compared to uncapacitated, sperm following co-incubation with OLF/OVS. (A) Uncapacitated (uncap) and capacitated (cap) sperm were co-incubated with OLF or PBS (control) for 3 h and after washing processed for indirect IF. Flow cytometry was performed after validating the specificity of the anti-PMCA1 antibody, using the IgG isotype (Supplemental Fig. S2). (a) For uncap sperm in PBS (control), only 12.7% showed fluorescence intensity (FI) at the highest level (marked region), while in OLF there was 27% (a > 2-fold increase) (b). (c) For OLF with cap sperm, 67.7% of the cells had FI in the marked region, >2-fold increase, compared to uncap in OLF. (B) The average frequencies of sperm with the highest FIs in three experiments showed significant differences between co-incubation in PBS and OLF (P < 0.05) for uncap, and for cap and uncap in OLF (P = 0.001). (C) Confocal images of cap and uncap sperm immuno-labeled for PMCA1 following co-incubation in PBS (a, d) and OLF (b, e), IgG isotype control is seen in (c). The PMCA1 signal is most intense in (b, e). The sperm heads are stained with DAPI in the middle panel followed by the merged image. (f) Analysis of ~200 sperm/group show significant (χ2 = 5.33; P = 0.02) differences between the number of immuno-labeled cap and uncap cells in OLF, while highly significantly more positive cells were seen for uncap (χ2 = 112.58; P < 0.001) and cap (χ2 = 789.95; P < 0.001) in OLF vs PBS. (Original mag. = ×630).
Figure 8
Figure 8
Quantitative western reveals that OVS deliver enzymatically active PMCA1 and HSC70 to sperm in significantly higher levels to capacitated, compared to uncapacitated ones. (A) Western blot of PMCA1 in proteins of uncapacitated (uncap) and capacitated (cap) sperm after co-incubation with OVS or oviductal luminal fluid (OLF) supernatant (SUP) for 30 min and 3 h shows the highest band intensities in cap sperm. The IgG isotype control was negative. The membrane was stripped and re-probed with β-actin, as a loading control. (B) One-way ANOVA and Student t-tests on the means of band intensities in (A) show significant differences for cap and uncap sperm in OVS compared to SUP for both time periods (P < 0.001, P < 0.01). (C) Western blot of PMCA1 and HSC70 levels after co-incubation of sperm with PBS, human tubal fluid (HTF), and OVS for 30 min, 90 min and 3 h show significantly elevated levels for OVS compared to PBS and HTF as seen in (D and E). The levels of HSC70 for sperm in HTF was notably significantly lower than those in OVS and PBS. Quantification of the western blot data (B, D, E) represents the mean (±SEM, n = 3) of band intensities, using Image Lab 6.0 software. Significant levels are *<0.05, **<0.01, ***<0.001. (F) Mg2+-dependent Ca2+-ATPase assays of microsomes from OVS and cap sperm reveal that WT and KO OVS carry enzymatically active PMCA1/4 and that sperm–OVS (WT + OVS) co-incubation significantly increased the level of activity in sperm (P = 0.01). Activity in KO OVS was significantly (P = 0.03) higher than that in WT (±SEM, n = 3).
Figure 9
Figure 9
OVS carry tyrosine-phosphorylated proteins (PY) which are markedly increased in sperm following co-incubation with OVS. (A) Sperm were co-incubated with OVS re-constituted in PBS, or HTF capacitating medium, or PBS alone, for 30 min or 3 h. Proteins extracted from washed sperm were analyzed by western blot and membranes re-probed for HSC70. PY markers (first lane), positive control, and the usual 95 kDa sperm band are seen in all samples. The number and intensity of PY bands increased markedly in OVS samples, where there was a prominent 55 kDa band, faintly seen in HTF. The data are representative of four experiments. (B) Samples of OVS re-constituted in PBS were subjected to western blot analysis. Sample #1 was fresh (unused) and #2 and 3# were spent samples recovered after sperm–OVS co-incubation for 15 and 90 min, respectively. The amount of proteins loaded were 6.2 μg (#1), 12.7 μg (# 2), and 12.9 μg (#3). Sample #1 had at least four bands, while samples #2 and 3 had only two bands, documenting the presence of PY in OVS. (C) Flow cytometry showed the levels of PY in aliquots of sperm co-incubated with PBS, HTF and OVS re-constituted in PBS for 2 h. The graph shows a right peak shift in FI with peak channel values of 88 (PBS), 162 (HTF) and 259 (OVS), reflecting an increase in FI of PY in sperm following incubation in capacitating medium and more markedly after their interaction with OVS. The IgG control is seen in Supplemental Fig. S2. (D, E) Indirect IF shows the localization and uptake of PY in the sperm head and flagellum, with significantly higher numbers of immuno-labeled cells seen after co-incubation with OVS vs HTF (χ2 = 19.27; P < 0.0001) and HTF vs PBS (χ2 = 10.67; P < 0.001). For each treatment ~200 sperm were analyzed. The absence of a signal in the IgG isotype control (E) shows the specificity of the primary antibody (original mag. = ×630).
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