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Throughout estrous cycle the oviduct epithelium undergoes dramatic morphological and functional changes. To elucidate cyclic cellular events and associated regulation mechanisms of estradiol (E2) and progesterone (P4), we mimicked estrous cycle stages in vitro using a culture system of primary porcine oviduct epithelium cells (POEC). Cells were polarized in an air/liquid interface, and then treated with E2 and P4 for physiological time periods: In experiment 1, high concentration of P4 with low concentration of E2 for 10 days resembled diestrus; in experiment 2, following the previous diestrus, sequential high E2 with low P4 for 2.5 days represented estrus. Histomorphometry and electron microscopy showed cyclic changes in cellular height, cell population and cilia density under influence of hormone stimulation. Trans-epithelial electrical resistance was high in simulated diestrus but reduced in estrus. Thus, E2 and P4 affect cellular polarity, transformation of ciliated and secretory cells, as well as electrical conductivity of oviduct epithelium. Simulation of diestrus led to significant decrease in expression of hormone receptors (PGR and ESR1) and other epithelial markers (MUC16, OVGP1 and HSP90B1), while sequential simulated estrus caused an increase in these markers. The hormonal regulation of some marker genes was clearly time dependent. Furthermore, POEC showed increased sperm binding capacity in simulated estrus. In this study we also present a novel approach based on the AndroVision software which can be routinely utilized as a parameter for ciliary activity and for the first time show fluid movement patterns along the epithelium lining in vitro.