63 Maternal microenvironment-induced, sex-specific microRNA expression patterns during the early elongation stage of bovine embryo development
D. Salilew-Wondim A B , C. Blaschka A , E. Tholen B , D. Tesfaye C , U. Besenfelder D , V. Havlicek D and M. Hoelker A BA Department of Farm Animal Sciences, Biotechnology & Reproduction of Farm Animals, University of Göttingen, Göttingen, Germany
B Institute of Animal Sciences, Animal Breeding, University of Bonn, Bonn, Germany
C Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA
D Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria
Reproduction, Fertility and Development 35(2) 158-158 https://doi.org/10.1071/RDv35n2Ab63
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Several pieces of evidence have shown a skewed male to female ratio at birth in cattle. These sex-biased embryonic losses are believed to be more frequent in high-producing dairy cows than heifers. This condition could be associated with different signalling molecules secreted by male and female embryos in response to the maternal microenvironment. Therefore, this study was conducted to understand the effect of physiological conditions of animals on the miRNA expression patterns of the male and female embryo during the early elongation stage. For this, oocytes collected from ovaries of the slaughterhouse were in vitro matured and in vitro fertilised with sexed semen. The male and female zygotes were in vitro developed until 4–8 cell stages and transferred into the oviduct of cows and heifers using endoscopic embryo tubal transfer. The elongated embryos were recovered on Day 13 of the gestation period after targeted slaughter. Male and female embryos derived from cows were classified as CM and CF, respectively and male and female elongated embryos derived from heifers were classified as HM and HF, respectively. Following this, total RNA was isolated from each sample using AllPrep DNA/RNA/miRNA Universal Kit (Qiagen). Samples with >500 ng of total RNA and RIN > 6 were subjected to small RNA sequencing using Illumina HiSeq. The quality of raw sequencing data was evaluated using FastQC (Babraham Bioinformatics). Detection and quantification of miRNAs was done using mapper.pl and quantifier.pl script of the miRdeep 2 software package. Differentially expressed miRNA were identified using the edgeR package. MiRNAs with absolute fold change >1.5, P-value < 0.05, and FDR < 0.1 were considered as differentially expressed. The miRNA target genes were identified using miRNet 2.0 (https://www.mirnet.ca/miRNet/home.xhtml) and functional enrichment of the target gene was performed using gprofiler (https://biit.cs.ut.ee/gprofiler/gost). The results indicated that 376 miRNAs were commonly detected in CM, CF, HM, and HF samples. Of these, the expression level of 8 miRNAs, including bta-miR-6119-5p, bta-miR-148a, bta-miR-10b and bta-miR-378 exhibited read counts > 25,000, suggesting that these miRNAs could be involved in both male and female embryo elongation. Pairwise comparison between HM and HF showed the upregulation of bta-miR-10a, bta-miR-155, and bta-miR-100 and downregulation of bta-miR-6119-5p in the former group. Similarly, the upregulation of 25 miRNAs, including bta-let-7a-5p, bta-miR-181c, bta-miR-1949, bta-let-7 g, bta-miR-2411-3p, and bta-miR-31, and downregulation of 7 miRNAs, including bta-miR-2320-5p and bta-miR-2307, were detected in CM compared with CF shown. These miRNAs were found to be involved in several developmental and immune related pathways including, cytokine-cytokine receptor interaction, TNF signalling pathway, apoptosis, NF-kappa B signalling pathway, and chemokine signalling pathways. Thus, this study indicates that the physiological condition of the animal has a direct effect on the miRNA expression patterns of the male and female elongated embryos.
