Single-blastomere transcriptomics identifies NR5A2-associated metabolic genes during porcine zygotic genome activation.
NR5A2 is a key regulator of zygotic genome activation (ZGA), which primarily takes place at the 4-8-cell stage in porcine embryos; however, its associated metabolic programs remain largely uncharacterized. In this study, single-blastomere RNA sequencing was performed using Smart-seq2 in 8-cell-stage porcine embryos derived from in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Based on the bimodal expression pattern of NR5A2, individual blastomeres were categorized into NR5A2-active and NR5A2-inactive groups. Differential expression analysis identified 46 differentially expressed genes (DEGs) in IVF embryos, which were mainly enriched in steroid hormone and lipid metabolism. In SCNT embryos, the top-ranked DEGs were functionally associated with mitochondrial respiration and ribosome biogenesis. A total of 14 NR5A2-associated DEGs were consistently identified in both embryo types and clustered into three core metabolic modules: glycolysis and energy metabolism (represented by HK2), lipid and steroid metabolism (including STAR, LIPG, PLCD1 and DLC1), and inositol metabolism (represented by MIOX). Single-blastomere qRT-PCR in parthenogenetic embryos further validated the bimodal expression of NR5A2, HK2, STAR and MIOX. Collectively, this study reveals an NR5A2-associated metabolic transcriptional signature in blastomeres of both IVF and SCNT embryos. These findings identify candidate metabolic genes that may inform future optimization of in vitro culture media to enhance the developmental competence of porcine embryos and warrant subsequent functional validation.