89 Transcriptomic difference of in vitro-produced male and female early embryos in bovine

M. Shi; G. Li; H. Araujo; A. Lee; J. Zhang; S. H. Cheong; J. E. Duan

doi:10.1071/RDv37n1Ab89

RESEARCH ARTICLE

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89 Transcriptomic difference of in vitro-produced male and female early embryos in bovine

M. Shi ^A , G. Li ^A , H. Araujo ^A , A. Lee ^A , J. Zhang ^A , S. H. Cheong ^B and J. E. Duan ^A

+ Author Affiliations

- Author Affiliations

^A Department of Animal Science, College of Agriculture and Life Science, Cornell University, Ithaca, NY, USA

^B Department of Clinical Science, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

Reproduction, Fertility and Development 37, RDv37n1Ab89 https://doi.org/10.1071/RDv37n1Ab89

Morphologic sex differences between male and female embryos typically begin after the primordial germ cell migration and sex gonad formation, although the sex of the embryo is determined at fertilization based on the sex chromosome composition. One of the most debated sexual differences is the speed of development, with male embryos produced in vitro developing faster. However, this observation remains controversial due to inconsistent findings. Moreover, the underlying mechanisms driving sex-specific differences in early embryo development are not yet fully understood. To investigate the transcriptional sex difference in early development, bovine blastocysts (BLs) produced in vitro with commercial media (IVF Bioscience) were collected and sexed using sex-specific primers by PCR (Xiao et al. 2021 J. Dairy Sci. 104, 3722–3735). Ultra-low input RNA-seq libraries were constructed using the half-cut expanded BLs (pool of two half-cut embryos) via Smart-Seq® HT Plus Kit (Takara Bio). Significant (chi-square test) male-biased development was observed in Day 7 expanded BLs (103/148, P < 1.87e-06) and in Day 8 non-expanded (80/99, P < 8.75e-10), expanded (85/104, P < 9.68e-11), and hatched (29/34, P < 3.86e-05) BLs. Transcriptomic analysis was conducted on five male and six female expanded BL RNA-seq samples. On average, 36 ± 3.2 million raw reads with pair-ended 150 bp were generated by NovaSeq for each sample. We identified 837 differentially expressed genes (DEGs; fold change > 1.5 and adjusted P-value < 0.1) between sexes: 231 were upregulated and 606 were downregulated in males compared with females. Gene Ontology (GO) analysis revealed that DEGs upregulated in male blastocysts were enriched in metabolic processes, while those upregulated in female blastocysts were enriched in female gonad development and female sex differentiation. KEGG pathway analysis indicated that DEGs upregulated in male blastocysts were associated with metabolic pathways, while those upregulated in female blastocysts were linked to pathways involving inflammatory mediators, regulation of TRP channels, TGF-β signaling, mRNA surveillance, and tight junction. Additionally, using rMATs (Wang et al. 2024 Nat. Protoc. 19, 1083–1104), 28 527 alternative splicing events were identified, with 1167 differentially alternative spliced (DAS; FDR ≤ 0.01 and delta percent spliced in index value ≥ 0.05) between males and females. The most common splicing category was skipped exon. These events were annotated to 873 genes, with 59 overlapping with DEGs. The overlapping genes were significantly enriched in metabolic and autophagy pathways. The top DEGs and DAS genes were further validated by qPCR, and PCR and gel electrophoresis, respectively. In summary, these findings suggest that the faster development of male embryos was driven by upregulated metabolism, partly mediated by alternative splicing of metabolism-related genes.