The oviduct is a pivotal and central reproductive organ that provides the finely tuned micromilieu for gametes’ maturation and early embryonic development. In vitro models of primary oviductal epithelium cells, derived from human clinical samples or food industry byproducts, not only comply with the 4Rs (replacement, reduction, refinement, and rehabilitation) framework for addressing ethical concerns on animal experimentation but also build a platform for functional experimental science. Culture conditions, such as media, have been optimised to allow for the investigation of oviduct functions. Since 5% CO2 and atmospheric O2 are the standard conditions for the vast majority of cell cultures, the actual O2 availability for the cells in the cultures and its comparability with the physiological conditions informing a specific cell environment have often been neglected variables in cell culture procedures. In the oviduct, O2 levels range from 4 to 10%, varying among mammalian species. Additionally, maternal stress and/or elevated glucocorticoid levels are associated with impaired fertility in both humans and animals. Gametes and embryos are particularly vulnerable to the environment in which they develop, especially in stressful conditions. Although previous studies have shown that reproductive performances were impaired due to the elevation of cortisol, the direct actions of physiological cortisol on oviduct epithelial cells have been elucidated less well. This study therefore aimed to explore the potential effects of O2 and cortisol on the oviduct epithelium. First, the effect of O2 tension on the oviduct epithelium was investigated using an established air-liquid interface (ALI) culture model based on primary porcine oviduct epithelial cells (POEC). The O2 levels in the incubator were set to 5% or 18% O2 during culture, representing physiological and supraphysiological conditions, respectively. Both conditions were applied with two different media regimes and cells were cultured for approximately 4 weeks in the respective conditions. Regardless of the O2 tensions or media conditions, the cultured cells developed a differentiated epithelial monolayer containing both ciliated and secretory cells. The supraphysiological level of O2 (18%) enhanced the differentiation and ciliogenesis in POEC. Notably, O2 tensions dramatically affected the production and content of the apical fluid, altered cellular composition, and influenced the expression of key oviductal function genes in POEC. Secondly, to explore the long-term effect of physiological stress-induced cortisol levels on the oviduct epithelium, an in vitro ALI model based on primary bovine oviduct epithelial cells (BOEC) was used, with basolateral administration of cortisol for 3 weeks after differentiation. Chronic exposure to cortisol significantly disrupted the architectural integrity of the oviduct epithelium. In addition to activating glucocorticoid signalling genes, cortisol also considerably reduced gene expression associated with oviduct functionality, apoptosis, and inflammation in BOEC. Moreover, the expression of the gene HSD11B2, coding for a cortisol-metabolising enzyme which converts bioactive cortisol to inactive cortisone, was downregulated dramatically, suggesting that the oviduct was less capable of metabolising cortisol under constant treatment. To summarise, differential O2 exposure using the ALI system mimics physiological conditions, providing valuable insights into how oviduct epithelial cells adapt to their environment and maintain homeostasis. The use of both one- and/or two-step media regimes resulted in high-quality outcomes in the oviduct epithelial cells (OEC) study, underscoring the critical role of O2 levels in cell culture. Maintaining physiologically representative conditions is essential not only for the health and longevity of cell culture but also for improving reproducibility. The BOEC study results indicated that chronic exposure to cortisol impairs oviduct epithelium functions at multiple levels, including adverse effects on cellular integrity and gene regulation. This indicates that stress-induced perturbation of the oviduct potentially impairs fertility. Comparing the effect of chronic physiological cortisol on the oviduct epithelium in pigs and cattle revealed species-specific responses, particularly in the regulation of HSD11B2, indicating differing capabilities for metabolising cortisol. These findings highlight the importance of finely tuned oviduct regulation as a prerequisite for creating the microenvironment essential for gamete maturation, fertilisation and embryo development.
