Dynamic profile of EVs in porcine oviductal fluid during the periovulatory period

Abstract

In mammals, around the time of ovulation, the hormonal profile dynamically changes in synchrony with reproductive events occurring in the oviduct, that is, sperm arrival, fertilization, and early embryo development. Extracellular vesicles (EVs) have been recently recognized as key components of the embryonic milieu; however, composition and function of oviductal EVs during this crucial period remains to be further explored. Therefore, we initially characterized EVs from porcine oviductal fluid specifically around the critical ovulation window: that is, estrus (E), late estrus (LE, day of expected ovulation), post ovulation (PO), and additionally diestrus (D). Total EV numbers gradually rose from D to E, LE and PO (P < 0.05), which corresponded to the total EV protein amount (P < 0.05). Strikingly, the mean size of EVs in PO was significantly smaller than in E and LE groups, which also had a lesser proportion of small EVs (P < 0.05). The EV protein cargoes during the periovulatory period were further analyzed by mass spectrometry. Qualitative analysis detected 1118 common proteins, which are most enriched in the cellular component of EVs/exosomes. Hierarchical clustering indicated similar protein profile within the biological replicates, but large discrepancy among stages. Further quantitative analysis discovered 34 and 4 differentially expressed proteins in the comparison between E and PO and in the comparison between E and LE, respectively. The dynamic EV protein profile together with the quick adaption in EV size and quantity suggests that porcine oviductal EV secretion are under the hormonal influence during the estrus cycle.

Extracellular Vesicles and the Oviduct Function

“…This would suggest that OLFM3 and TLN-1 expression is stimulated by E2 and/or suppressed by P4. Contrary to other studies, MYH9 and CLTC (clathrin heavy chain) were most abundant during the pre-ovulatory phase rather than the post-ovulatory phase [50]. These data may suggest differences in oEV protein content among species.…”

“…In pigs, oEV concentration, size, and protein content change across the estrous cycle [50]. This is different than cows as there are no observable differences in concentration or size distribution across the estrous cycle [22].…”

“…However, OLFM3 is absent in oEVs isolated from post-ovulatory stage. Talin-1 (TLN1), a cytoskeletal protein, levels are the lowest during the post-ovulatory stage [50]. This would suggest that OLFM3 and TLN-1 expression is stimulated by E2 and/or suppressed by P4.…”

Extracellular Vesicles, the Road toward the Improvement of ART Outcomes

“…Differences were also found at the protein level, and the major differences were found between OVS recovered at the post-ovulatory and pre-ovulatory stages [71]. Similarly, differences were found along the estrus cycle when the protein cargo of porcine OVS was analyzed [62]. Due to the variations found during the estrous cycle, the authors hypothesized that OVS cargo changes are regulated by hormonal changes during the estrous cycle [62,71].…”

“…Interestingly, EVs isolated from ovarian follicular fluid used as an additive during bovine oocyte maturation and embryo development in in vitro conditions enhanced blastocyst rate and decreased global DNA methylation and hydroxymethylation levels [59]. Nevertheless, caution needs to be taken when using follicular fluid EVs as additive to enhance oocyte competence, as it was shown that the EVs cargo vary along the estrus cycle [60][61][62], the size of the follicle [63], and with the age of the female [56].…”