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

Abstract

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.