Fertility impairment by maternal stress: Using a novel 3D in vitro system to decipher the impact of cortisol on oviduct epithelium physiology, oviduct-derived extracellular vesicles and early embryonic development

着床前被认为是哺乳动物胚胎发育过程中的一个高度敏感的窗口。母亲暴露在压力下，特别是在胚胎在输卵管中的过渡阶段，对生育和早期胚胎发育产生负面影响，很可能是由输卵管上皮介导的。然而，到目前为止，应激激素(即糖皮质激素)如何局部调节输卵管生理，从而改变早期胚胎环境仍不清楚。细胞外小泡(EVS)最近被认为是胚胎-母体串扰的重要中介。极化细胞，如输卵管上皮，在其管腔和基底外侧细胞表面定向释放不同的EV群体，这是传统2D细胞培养方法无法模仿的。我们最近建立了不同物种(小鼠、猪和牛)输卵管上皮细胞(OEC)的气液界面(ALI)分室培养系统。该系统维持上皮细胞的极化分化，使顶端“输卵管液”的形成和获得，甚至在顶端细胞表面的胚胎发育。在这个项目中，我们将遵循3R原则，利用屠宰场副产品中建立的猪和牛ALI-OEC来研究：1)皮质醇对输卵管上皮生理的直接作用；2)皮质醇与卵巢类固醇激素在整个发情周期中调节输卵管上皮功能的相互作用；3)皮质醇对输卵管来源的EVS的响应；4)皮质醇对输卵管分泌体组成的影响以及(因此)对胚胎发育的影响。我们将应用高通量“组学”和OEC、EV和胚胎水平的综合分析来揭示母体皮质醇过剩影响的复杂生物网络。该项目阐明了皮质醇对早期胚胎微环境的作用机制，从而为哺乳动物应激与不孕之间的联系提供了深刻的见解。

The preimplantation period is considered to be a highly "sensitive window" during embryo development in mammals. Maternal exposure to stress, particularly during the transit phase of the embryo in the oviduct, exerts negative consequences on fertility and early embryonic development, most likely mediated by the oviduct epithelium. However, until now it remains unclear how stress hormones, i.e. glucocorticoids, locally regulate oviduct physiology, thereby altering the early embryonic environment. Extracellular vesicles (EVs) have recently been suggested as important mediators in the embryo-maternal crosstalk. Polarized cells e.g. oviduct epithelia, directionally release distinct EV populations on their luminal and basolateral cell surface, which cannot be mimicked using traditional 2D cell culture methods. We recently established a compartmentalized air-liquid interface (ALI) culture system for oviduct epithelial cells (OEC) from different species (mouse, pig and cattle). This system maintains the polarized differentiation of the epithelial cells, enables formation and acquisition of apical “oviductal fluid” and even embryonic development on the apical cell surface. In compliance with the 3R principle, in this project we will employ porcine and bovine ALI-OEC established from slaughterhouse by-products to study: 1) direct cortisol action on oviduct epithelium physiology; 2) the interplay of cortisol with ovarian steroid hormones regulating oviduct epithelium function throughout the estrous cycle; 3) the modification of oviduct-derived EVs in response to cortisol; 4) the effect of cortisol on the composition of the oviductal secretome and (as a consequence) on developing embryos. We will apply high-throughput “-omics” and integrative analyses at OEC, EV and embryo level to unveil the complex biological networks impacted by maternal cortisol excess. This project elucidates mechanisms of cortisol action on the early embryonic micro-environment, thereby offering profound insights into the connection between stress and infertility in mammals.