92 Extracellular vesicles from oviductal spheroids and uterine horn epithelial cells show different uptake times by equine spermatozoa and act upon capacitation

In vivo, uterine and tubal epithelial cells and their secretions provide a microenvironment supporting and regulating the interaction with spermatozoa during their transit in the female tract. Unfortunately, in equine species, the nature of this interaction is not fully understood for IVF purposes, despite the trial of different in vitro models such as monolayers, explants, and apical plasma membranes. Since extracellular vesicles (EVs) are important mediators of intercellular communication, the aim of the present study was to investigate the interaction of EVs, secreted by oviducal spheroids and apical uterine horn epithelial cells, on in vitro equine spermatozoa capacitation. Three genital tracts were collected at a slaughterhouse from mares in late oestrus. Uterine explants were digested with collagenase and trypsin, and the cells obtained were cultured on an air–liquid interface (ALI) system to allow their polarisation. Oviducts were squeezed out to obtain spheroids. To produce EVs, epithelial cells and spheroids were cultured for 3 days in serum-free medium. Supernatants were centrifuged at 100 000 × g for 1 h at 4°C, and the resulting pellets were tested for EV concentration and size by Nanosight. Samples of ejaculated spermatozoa from three stallions were pooled and centrifuged at 100 × g for 1 min to remove particulate matter and dead spermatozoa. The supernatant was centrifuged at 600 × g for 5 min to obtain the pellet, which was resuspended in Whitten’s non-capacitating medium (McPartlin et al. 2009 Biol. Reprod. 81, 199) to a final concentration of 10 × 10⁶ spermatozoa mL⁻¹. To trace interaction between spermatozoa and EVs by fluorescence microscopy, uterine EVs were labelled with PKH26 dye and oviducal EVs with PKH67. Aliquots of 500 µL of spermatozoa were incubated for 6 h in the presence of 400 × 10⁶ EVs mL⁻¹ of each type. Incorporation was monitored every hour by confocal microscopy. To evaluate capacitation-related events, sperm were incubated in capacitating-type medium (CM) (McPartlin et al. 2009 Biol. Reprod. 81, 199) for 6 h and for another 6 h in CM supplemented with different types of EVs alone or in combination. A control was performed in the absence of EVs. Sperm motility was assayed by computer-assisted cell sorting, and rates of acrosomal reaction (AR) and apoptosis by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA)/propidium iodide staining by fluorescence microscopy. Our results showed that the size of EVs from uterus was 181.9 ± 70.6 nm, and from oviduct was 158.4 ± 59.6 nm. The concentration of EVs from uterus was 1.07 × 10¹¹ ± 7.9 × 10⁶ particles mL⁻¹, while for oviduct was 3.8 × 10¹⁰ ± 2.59 × 10⁶. Based on size, these EVs can be categorised as shedding vesicles. After 4 h of incubation, uterine EVs were detected in the head of spermatozoa, whereas oviducal EVs were detected after 1 h in the middle piece. The rate of AR for sperm incubated with uterine and oviducal EVs was, respectively, 50.25% and 57.14%. When EVs were added in combination, the AR rate was 71.42%. In the control, the rate of AR was 15%. The motility was characterised by an increase in ALH with a concomitant decrease of LIN and/or STR (P < 0.05). This change in motility is consistent with hyperactivaction (McPartlin et al. 2009 Biol. Reprod. 81, 199). We concluded that EVs from the female genital tract stimulate equine sperm hyperactivation in vitro and induce AR.