Response of the bovine oviduct epithelium to sperm binding and proximity: a region-specific proteomic approach using spheroids

Following uterine migration, spermatozoa reach the isthmus, where a subpopulation binds to luminal oviduct epithelial cells (OECs) to establish a functional reservoir. From there, spermatozoa continue toward the ampulla, maintaining interactions with OECs prior to fertilization. This study investigated the proteomic response of OECs to sperm binding and proximity in both regions. Isthmic and ampullary epithelial spheroids were incubated either alone (controls), in direct co-culture with frozen-thawed bull spermatozoa (contact groups), or separated by a 0.4-µm insert (non-contact groups) for 6 h at 38.5 °C. The membrane and acrosome integrity of free spermatozoa were assessed by flow cytometry. After 6 h, bound spermatozoa were removed and spheroids were analyzed by nanoLC-MS/MS. Co-culture with ampullary spheroids in the non-contact groups tended to increase the proportion of membrane- and acrosome-intact spermatozoa compared with controls (p = 0.07). Proteomic data showed that direct sperm contact elicited a greater number of differentially abundant proteins (DAPs) in spheroids than the non-contact groups, regardless of OEC origin (up to 195 vs. 135 DAPs compared with controls, respectively). When comparing contact and non-contact groups, 168 and 218 DAPs were identified as specifically associated with sperm binding in isthmic and ampullary OECs, respectively. Up to 37% of overabundant DAPs were predicted to be secreted. The DAPs had predicted functions in fertilization and embryo development in ampullary OECs, as well as ciliary beating, response to external stimulus and the immune response in both regions. Altogether, these findings demonstrate a region-dependent sperm–oviduct dialogue shaped by the type of sperm interaction. • Co-culture with ampullary epithelial spheroids had a positive effect on sperm membrane and acrosome integrity over time • Sperm binding and proximity differentially altered the proteome of oviduct epithelial spheroids • Sperm induced specific proteomic changes according to the isthmic or ampullary origin of spheroids • Proteins altered by sperm binding had functions in ciliary beating, immune response and fertilization