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.
Supplementary file 1. List of group specific proteins as well as common proteins in all groups as analyzed qualitatively. Detection threshold is at least two unique peptides.
Supplementary file 2. Functional annotation clusters (CC, BP and MF GO terms) of the common proteins among all three stages around ovulation time window by DAVID.
Supplementary file 3. Unique proteins in one or two groups identified by the qualitative analysis of proteomic data. A unique protein is defined as it was detected (by at least 2 unique peptides) in ≥ 3 samples of one group, but undetectable (< 2 unique peptides) in at least one other group.
Supplementary file 4. List of all proteins detected in porcine oviductal EVs around the ovulation window by the mass spectrometry. Note: Estrus 4 sample was removed from the quantitative proteome analysis as protein measurement was found to be flawed.
Supplementary file 5. Full list of significant differentially expressed proteins (DEPs) as detected by the quantitative analysis of mass spectrometry data. The pairs of ATP2B2 and ATP2B2.1, COL15A1 and COL15A1.1, as well as TLN1 and TLN1.1, are peptides double-matched to two different species.
Supplementary file 6. Matrix representation of the Pearson correlation coefficients among estrus, late estrus and post ovulation groups based on the label-free protein abundances (LFQ) of differentially expressed proteins (DEPs). Pearson correlation values were indicated by red color scale.
Supplementary file 7. Western blot analysis of OVGP1 in all EV samples derived from oviductal fluid during the estrus (E), late estrus (LE), and post ovulation stage (PO), respectively.