Sex-biased transcriptome in in vitro produced bovine early embryos.

BACKGROUND: Morphologic sex differences between males and females typically emerge after the primordial germ cell migration and gonad formation, although sex is determined at fertilization based on chromosome composition. A key debated sexual difference is the embryonic developmental rate, with in vitro produced male embryos often developing faster. However, the molecular mechanisms driving early embryonic sex differences remain unclear. RESULTS: To investigate the transcriptional sex difference during early development, in vitro produced bovine blastocysts were collected and sexed by PCR. A significant male-biased development is consistently observed in expanded blastocysts. Ultra-low input RNA-seq analysis identified 837 DEGs, 1555 significantly sex-biased differential alternative splicing (AS), and 1151 differentially expressed isoforms (DEIs). Among all of the DEGs, there were 231 upregulated and 606 downregulated in males. Functional enrichment analysis revealed male-biased DEGs were associated with metabolic regulation, whereas female-biased DEGs were related to female gonad development, sex differentiation, inflammatory pathways, and TGF-beta signaling. Comparing X chromosome and autosome expression ratio, we found that female-biased DEGs contributed to the higher X-linked gene dosage, a phenomenon not observed in male embryos. Moreover, we identified the sex-biased transcription factors and RNA-bind proteins, including pluripotent factors such as SOX21 and PRDM14, and splicing factors FMR1 and HNRNPH2. Additionally, we revealed that the significantly sex-biased differential AS were predominantly skipped exons, and they could be mapped to 906 genes, with 59 overlapping with DEGs enriched in metabolic and autophagy pathways. By incorporating novel isoforms from long reads sequencing, the sex-biased DEIs were associated with 1017 genes. Functional analysis showed that female-biased DEIs were involved in the negative regulation of transcriptional activity, while male-biased DEIs were related to energy metabolism. Furthermore, we identified sex-biased differential exon usage in DENND1B, DIS3L2, DOCK11, IL1RAPL2, and ZRSR2Y, indicating their sex-specific regulation in early embryo development. CONCLUSION: This study provided a comprehensive analysis of transcriptome differences between male and female bovine blastocysts, integrating sex-biased gene expression, alternative splicing, and isoform dynamics. Our findings indicate that enriched metabolism processes in male embryos may contribute to the faster developmental pace, providing insights into sex-specific regulatory mechanisms during early embryogenesis.