55 Identification of microRNAs associated with sex determination in bovine amniotic fluid and maternal blood plasma

In most eutherian mammals, sex determination is the process through which a bipotential gonad (also known as genital ridges) develops into a testis or ovary depending on the sex chromosome content of the embryo, specifically by the presence of the SRY/Sry gene (sex-determining region of the Y chromosome). MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression and are involved in diverse functional roles including development, differentiation, apoptosis, and immunity. We hypothesised that the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) would be affected by the sex of the embryo around the time of sex determination. Amniotic fluid and MP were collected from 6 crossbred beef pregnant heifers (3 carrying a single male and 3 carrying a single female embryo) following slaughter on Day 39 (when the peak of SRY expression occurs in cattle). All heifers had been synchronized and inseminated with semen from the same beef bull. A total of 12 samples (6 AF and 6 MP) were profiled using the miRCURY LNA miRNA Serum/Plasma Focus PCR Panel (Qiagen; 179 assays targeting relevant miRNAs). Data were analysed by GeneGlobe Data Analysis Center (Qiagen). A threshold cycle cut-off of 35 was applied and data were analysed using an unpaired t-test. Gene ontology enrichment analysis was performed using the WebGestaltR package to explore the possible functions of differentially expressed (DE) miRNAs. In this study, DE miRNAs were identified in male vs. female AF (n=5; 3 upregulated and 2 downregulated; P<0.05) and MP (n=57; 54 upregulated and 3 downregulated; P<0.05). Although no enrichment was detected for DE miRNAs in AF (in either sex) or in MP in heifers carrying a female embryo, 37 biological processes were enriched by DE miRNAs in MP of heifers carrying a male embryo (false discovery rate<0.05). Interestingly, the top five most enriched biological processes were male gonad development, development of primary male sexual characteristics, signal transduction in absence of ligand, actomyosin structure organisation, and male sex differentiation, suggesting a potential role of these miRNAs in reproductive traits. Results from this study highlight unique aspects of sex determination in cattle such as the role of miRNAs in gonad development. Moreover, although it is well known that AF provides a protective space around the developing embryo/fetus that allows its movement and growth; here we provide evidence suggesting that its components may play important roles in fetal development. Finally, miRNAs in MP may offer new opportunities to investigate biomarkers for early prediction of embryo/fetal sex in commercial practice. This research was supported by the Science Foundation Ireland (13/IA/1983) and the European Union H2020 Marie Sklodowska-Curie Innovative Training Network project Biology and Technology of Reproductive Health - REP-BIOTECH - 675526.