RNA-Seq study of in vivo-produced single male and female bovine blastocysts

ABSTRACT The objective of this study was to identify differentially expressed genes (DEG) between females and males in early bovine embryos. Superovulated cattle (n = 4) in the age span of 16–18 months were artificially inseminated with semen from the same bull that has been proven to be fertile. Blastocysts were collected by routine non-surgical uterine flushing on day 7 after insemination. This study determines the sex of embryos using a micro-injection pipette to collect a few blastomeres through the Zona pellucida. The remaining blastomeres were used for single-cell RNA sequencing (scRNA-seq) analysis on the Illumina platform, followed by differential expression analysis, Gene Ontology (GO) function analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The criteria for identifying DEG will be set at |log2(Fold change)| ≥ 1 and P<0.05. A total of 8004 DEG were detected in female (Denoted as BLX, n = 3) and male (Denoted as BLY, n = 3) blastocysts. Transcripts highly expressed in the female embryos were related to catalytic activity, nucleotide binding, and catabolic process, while transcripts highly expressed in the male embryos were linked to oxidative phosphorylation, mitochondrion, and the ribosome. Nine genes that may be involved in blastocyst growth and development were screened, suggesting that their differential expression may be responsible for the differences in the development of male and female embryos. This study provides important information on potential genes and pathways associated with differences in early female and male embryonic development.