Bovine ampullary and isthmic epithelial spheroids: proteomic profile and physiological features for in vitro studies of gamete-oviduct interactions.

After mating or insemination, spermatozoa reach the first part of the oviduct, known as the isthmus, where a subpopulation binds to the epithelial cilia, forming a reservoir. Then, spermatozoa migrate toward the ampulla, where additional interactions with cilia occur and fertilization takes place. Exploring sperm-oviduct interactions requires physiologically relevant in vitro models. Our goal was to characterize isthmic and ampullary epithelial spheroids in terms of cellular, proteomic and sperm binding properties. Spheroids measuring 100-150 µm in diameter, formed after a 3-day culture of pre-ovulatory oviduct mucosa fragments, were selected and analyzed for (i) epithelial, fibroblast and cilia markers by immunostaining, (ii) proteomic content by dia-PASEF nanoLC-MS/MS, and (iii) ability to interact with bull spermatozoa during a 6-h co-culture, assessed by confocal and scanning electron microscopy. Cells within both isthmic and ampullary spheroids showed positive immunostaining for cytokeratin and no staining for vimentin. Ampullary spheroids consistently exhibited a higher proportion of ciliated cells compared to isthmic spheroids (24.5 % vs 20 %; p < 0.05; variation coefficients among replicates of 15 and 11 %, respectively). A total of 6914 proteins were quantified, of which 1010 (14 %) were differentially abundant between isthmic and ampullary spheroids. Following co-culture, 95-97 % of bound spermatozoa were attached by their head to cilia of both spheroid groups. The density of bound spermatozoa was on average 33-45 % higher on ampullary compared to isthmic spheroids (p < 0.001). This study provides a well-differentiated oviduct epithelium model for in vitro investigations of gamete and embryo interactions with the oviduct.