(42) PRAMEY-Mediated Epigenetic Regulation of Bovine Preimplantation Development

Chandlar Kern¹; Tae Kim¹; Wan-Sheng Liu¹

1. Department of Animal Science, The Pennsylvania State University, University Park, USA

 

Abstract Text:

Infertility and subfertility are widespread issues in cattle and other species. PRAMEY, a bovid-specific, Y-linked gene family, has been implicated in germ cell formation, fertilization, and early embryonic development. This study aimed to investigate PRAMEY’s role in sperm–egg binding, acrosome integrity, and epigenetic regulation during fertilization and early embryogenesis.

Using IVF with bovine oocytes and caudal epididymal spermatozoa treated with either anti-PRAMEY antibody (ab) or rabbit IgG control, we assessed sperm–egg binding and acrosome integrity at 2-, 4-, and 6-hours post-fertilization (hpf). PRAMEY ab treatment resulted in a two-fold increase in sperm binding per oocyte across all time points, with a significant difference at 6 hpf (P ≤ 0.05), while acrosome integrity was unaffected (P ≥ 0.05). To evaluate PRAMEY’s role in epigenetic reprogramming, DNA and histone methylation dynamics were analyzed. Immunostaining for 5-methylcytosine (5-mC) revealed that zygotes derived from PRAMEY ab-treated sperm exhibited significantly reduced methylation in the paternal pronucleus at 10 hpf and maternal pronucleus at 25 hpf compared to controls (P ≤ 0.01). Both groups showed two global methylation waves: 5-mC levels peaked at the 2-cell stage, declined to a minimum at the 8-cell stage, and increased again at the blastocyst stage. H3K9me3 levels did not differ significantly, but H3K27me3 levels were significantly lower in the PRAMEY ab group at the 8-cell and morula stages (P ≤ 0.05).

We then performed whole-genome bisulfite sequencing (WGBS) on 24 IVF-derived embryonic samples (10 hpf, 8-cell, morula, and blastocyst stages; n = 3 per treatment per stage). Libraries were generated using the ultra-low input scSPLAT pipeline and sequenced on the Illumina NovaSeq X platform (150 bp paired-end reads; ~600 million reads per sample). Reads were aligned to the ARS-UCD1.3 bovine reference genome using Bismark. Initial analysis revealed widespread differences in global and locus-specific methylation between PRAMEY ab and IgG control embryos. A stage-dependent shift in GC content was observed, with single-cell zygotes averaging 31% GC versus 22% in multicellular embryos. Differentially methylated regions (DMRs) were particularly enriched at regulatory elements controlling transcriptional and chromatin remodeling genes. Gene lists derived from these DMRs were subjected to DAVID analysis, revealing functional enrichment in categories related to DNA binding, chromatin organization, and developmental regulation. These WGBS results provide single-base resolution evidence that PRAMEY influences methylation at promoters, enhancers, and imprinted regions. For example, at the morula stage, a total of 45 DMR-related DAVID unique IDs were identified, 22 of which were associated with TATA-box binding protein-associated factor (TAF), Histone H4, and transition nuclear proteins (TNPs), suggesting PRAMEY's involvement in transcriptional activation and nucleosome assembly during embryonic genome activation (EGA).

Together, these data suggest that PRAMEY not only enhances sperm–oocyte binding but also regulates genome reprogramming in bovine embryos. By coordinating DNA and histone methylation dynamics, PRAMEY likely contributes to transcriptional competence and developmental progression. These findings position PRAMEY as a multifaceted regulator of epigenetic programming in the bovine embryo and highlight the importance of sperm-delivered proteins in shaping embryonic development.