220 STEROID-REGULATED mRNA EXPRESSION IN OVIDUCT EPITHELIAL CELLS IN THE MARE

Abstract

It is well known that oviducal physiology undergoes cyclical changes under the influence of steroid hormones associated with the establishment of an optimal microenvironment for fertilization and early embryonic development. Genes associated with the preovulatory phase are likely to be involved in modulation of gamete function and sperm storage, whereas those in the post-ovulatory phase are involved in embryonic development. So far, in the horse, no genes that change activity during the oestrous cycle, which may be important in fertilization, gamete and embryo passage, and early embryonic development, have been identified. Therefore, to identify steroid-regulated genes, we compared the mRNA expression of PAI-1, uPA, TGFA, TIMP-1, MMP-1, CSF, PTGER2, and PTGER4 in oviducal explants from mares in the pre- and the post-ovulatory early luteal stages. The cycle stage of healthy warmblood mares was determined in the slaughterhouse by assessment of ovarian morphology and uterine oedema. Oviducal explants were isolated by scraping the epithelium of oviducts ipsilateral to a preovulatory follicle or a recent corpus luteum and frozen in lysis buffer at –80°C until RNA extraction. The genes were quantified by RT-qPCR and normalized against the geometric mean of 6 validated reference genes (UBB, SDHA, 18S, TUB, ACTB, HPRT) according to the MIQE-guidelines (Bustin et al., 2009). Data were analysed using the independent samples t-test or the Mann–Whitney U-test. The mRNA expression of PAI-1, MMP-1, TGFA (0.01 < P < 0.05), and uPA (P = 0.007) was downregulated in the post-ovulatory phase, whereas no significant change in TIMP-1, CSF, PTGER2, and PTGER4 expression was observed. The results show that mRNA of components of the extracellular matrix turnover, such as PAI-1, uPA, MMP-1, and TIMP-1 as well as growth factors, such as TFGA and CSF and prostaglandin E2 receptors (PTGER2 and PTGER4), are produced. Moreover, down-regulation of PAI-1, MMP-1, TGFA, and uPA in the early luteal stage indicates that mRNA expression of these genes is steroid regulated. In other species, all these components are likely to protect gametes and the early embryo against proteolytic degradation and to stimulate embryonic development. Further transcriptome and proteome analyses are necessary to unravel changes in the oviducal epithelium during the oestrous cycle and early pregnancy in the mare.