145 OVIDUCTAL EXPRESSION OF GENES INVOLVED IN GROWTH FACTOR, CYTOKINE, HORMONE, AND WNT SIGNALING DURING THE EARLY ESTROUS CYCLE OF THE COW

Abstract

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the pre-implantation embryo, called embryokines, is largely incomplete. To identify possible candidates for this function, an experiment was conducted to assess expression patterns during the first 7 days after ovulation for 92 genes that could be involved in control of development. Included were genes for 27 growth factors, 11 cytokines, 22 interleukins, 3 hormones, 19 WNT ligands, and 9 WNT regulatory molecules. Cows were slaughtered at Days 0, 3, 5, and 7 relative to predicted ovulation. Reproductive tracts were obtained and transversal sections from the isthmus of oviducts ipsi- and contralateral to the corpus luteum were harvested for gene expression analysis. Abundance of specific mRNA molecules was determined using the NanoString nCounter analysis system (NanoString Technologies, Seattle, WA, USA). Data were normalized against 6 housekeeping genes (ACTB, ERK1, GAPDH, RPL19, SLC30A6, SUZ12) and internal positive controls. Genes were considered expressed if the number of reads was greater than 2 standard deviations above the mean of negative controls. Data were analysed by ANOVA using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA) with day, side, and day × side as fixed effects, and cow as random effect. Side did not have any significant effect so data were analysed without side and day × side in the model. In contrast to what was observed earlier for endometrium (P. Tribulo and P. J. Hansen, unpublished data), we found no difference in gene expression between oviducts ipsi- and contralateral to the corpus luteum. Overall, there was wide variation in the magnitude of gene expression. Among the 20 most expressed genes, average reads varied from 164 to over 10 726. All genes were detected at Days 0, 3, and 7 but only 67 of the 92 genes were expressed at Day 5. The 10 highest-expressed genes were CTGF, CXCL3, CXCL10, CXCL12, GRO1, IGF2, IK, SFRP1, WNT5A, and WNT6. Of these, CTGF, CXCL12, IGF2, IK, HDGF, WNT5A, and CXCL3 were within the 10 highest expressed at all days (P. Tribulo and P. J. Hansen, unpublished data). There were only 6 genes whose expression was significantly affected by day. Expression was highest at oestrus (VEGFA), Day 5 (GRO1, SFRP1) or Day 7 (BMP4, IK, WNT16). This experiment identifies some potential maternal regulators of embryonic development. Expression of most of these putative embryokine genes did not vary with stage of the oestrous cycle, suggesting that expression is either not under endocrine control or varies between cell types within the oviduct. Further studies are needed to determine the effect of these maternally secreted molecules on embryonic development.

Study was supported by the National Institutes of Health (HD080855).