Extracellular vesicles from oviductal spheroids and uterine horn epithelial cells show different uptake times by equine spermatozoa and act upon capacitation
Abstract
Data di Pubblicazione:
2022
Citazione:
Extracellular vesicles from oviductal spheroids and uterine horn epithelial cells show different uptake times by equine spermatozoa and act upon capacitation / A. Lange Consiglio, S. Canesi, F. Funghi, G. Bosi, F. Cremonesi. - In: REPRODUCTION FERTILITY AND DEVELOPMENT. - ISSN 1031-3613. - 34:2(2022), pp. 92.283-92.283. ((Intervento presentato al 48. convegno Annual Conference of the International Embryo Technology Society (IETS) tenutosi a Savannah (Georgia) nel 2022.
Abstract:
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 noncapacitating
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.
Tipologia IRIS:
01 - Articolo su periodico
Elenco autori:
A. Lange Consiglio, S. Canesi, F. Funghi, G. Bosi, F. Cremonesi
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