Background: The ovarian follicle is an enclosed microenvironment that ensures oocyte growth and maturation, making it essential for female fertility. Normal folliculogenesis development and oocyte maturation strictly rely on orchestrated intercellular interactions among the mural granulosa cells (MGCs), cumulus cells (CCs), and the oocyte within antral follicles. Despite the importance, how these cell populations communicate remains largely unknown. Due to this knowledge gap, current oocyte in vitro maturation (IVM) systems fail to fully replicate the physiological follicular microenvironment due to the lack of MGC regulation, leading to poor oocyte quality and lower success rates of assisted reproductive technologies. Results: RNA-seq was performed on MGCs, CCs, and oocytes isolated from single bovine antral follicles at different developmental stages (3–5 mm, 6–8 mm and 9–12 mm). Using a ligand-receptor interaction scoring strategy, we constructed a dynamic intrafollicular interaction map. We revealed that MGCs are the most active cell type, acting as either secretors or receivers, based on the number of ligands and receptors it expresses, as well as the number of interaction pairs it establishes with other cell types. Intriguingly, intrafollicular crosstalk exhibits spatiotemporal commonality, characterized by shared ligand-receptor usage and potential functional redundancy across cell types and stages. Notably, the intrafollicular communication map enables the prediction of MGC-secreted factors that can improve the oocyte quality after IVM. Based on the predicted candidate factors, we have confirmed that supplementation with pleiotrophin (PTN) significantly improved in vitro matured oocyte quality and subsequent embryonic development. In addition, we also revealed significant heterogeneity at single-follicle resolution, unhealthy follicles showed aberrant activation or loss of partial core interaction signaling, supporting the hypothesis that these interactions play a critical role in determining follicular fate. Conclusions: By reconstructing a map of intrafollicular interactions, our study provides novel insights and important clues for understanding cellular and molecular mechanisms that fine-tune antral folliculogenesis. We also offer a roadmap for predicting candidate factors that can be used for optimizing mammalian in vitro embryo production systems.

Embryo genomic selection (EGS) is a contemporary breeding strategy that combines genomic selection (GS) methodology with embryo biotechnology. By conducting genotyping and genomic prediction at the pre-implantation stage, embryos with superior breeding value can be identified for transfer, markedly increasing breeding efficiency while reducing the uncertainty and temporal expenditure associated with conventional GS. This study establishes a reliable embryo biopsy-based GS pipeline for sheep, incorporating optimized whole-genome amplification and microcell genotyping techniques. We developed a high-efficiency in vitro sheep embryo production platform compatible with embryo biopsy. Systematic comparison of Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping Based Amplification Cycles (MALBAC) whole-genome amplification systems yielded high-quality genotypes from biopsy samples of embryos containing as few as 10 cells. Imputation using 10× whole-genome sequencing data significantly increased both genotype call rates and accuracy. High concordance was observed between embryo and lamb genotypes, and genomic estimated breeding values (GEBVs) for key growth traits exhibited strong correlations (R²: 0.91–0.98). This system enables accurate preimplantation genomic evaluation and provides an efficient strategy to accelerate genetic improvement in sheep breeding programs.

Ovulation and granulosa cell luteinization are induced by ovulatory signals, including luteinizing hormone (LH) and human chorionic gonadotropin (hCG). Histone modifications enable rapid, signal-responsive transcriptional reprogramming. However, the effects of LH/hCG-induced histone modification changes on the mural granulosa cells (MGCs) function remain to be fully elucidated. By mining public datasets we integrated transcriptomic and histone-modification profiles of MGCs across the ovulatory interval and tracked LH/hCG-driven gene expression at three time points (0, 4, and 12 h after-hCG). During oocyte maturation, the 4 h LH-surge constitutes a critical window for meiotic resumption, during which many genes display rapid transcriptional changes followed by a return to baseline levels. Early-response genes are enriched for cell locomotion, inflammatory responses, the activation of signaling pathways, and histone modifications. Furthermore, LH/hCG-induced transcriptome remodeling is highly correlated with dynamic gains or losses of H3K4me3 and H3K27ac. Notably, we discovered for the first time that H3K27ac marks super-enhancers (SEs) that regulate LH/hCG-induced transcriptional activation in MGCs. Finally, through complementary in vitro and in vivo pharmacological inhibition, we demonstrate that LH/hCG governs oocyte maturation and ovulation by reshaping the MGC transcriptome via H3K4me3- and H3K27ac-dependent chromatin remodeling. In summary, our study advances the understanding of how gonadotropins regulate MGC function and oocyte maturation through histone-modification-mediated transcriptional control.

Although laser‐assisted biopsy has shown advantages in other species, optimised protocols for bovine embryos remain scarce. This study developed a systematically optimised laser biopsy method for bovine embryos, which demonstrated high post‐biopsy survival rates. Key parameters including laser energy, needle diameter, and operating medium were refined to establish a reliable and efficient standard protocol. This technical advance provides critical support for large‐scale embryo genomic selection in cattle, facilitating enhanced genetic breeding outcomes.

In vitro maturation (IVM) of oocytes remains suboptimal due to oxidative stress and disrupted cumulus–oocyte communication. Oocyte-derived factors (ODFs) are key mediators of this crosstalk and crucial for oocyte competence. Here, we provide systematic evidence that ephrin-A5 (EFNA5) is an oocyte-derived membrane ligand capable of regulating oocyte quality during IVM. Cross-species transcriptomic analysis revealed that EFNA5 is stably enriched in mammalian oocytes but markedly reduced in in vitro-matured oocytes compared with in vivo counterparts. Using the ovine IVM model, supplementation with recombinant EFNA5 significantly improved blastocyst formation, increased total cell numbers, and reduced apoptosis. Mechanistically, EFNA5 promoted cumulus–oocyte complex expansion, reduced reactive oxygen species accumulation, activated NRF2-dependent antioxidant signaling, and suppressed NF-κB-driven inflammation. RNA-seq and functional validation further confirmed that EFNA5 enhanced redox homeostasis and decreased DNA damage, collectively improving oocyte developmental potential. These findings establish EFNA5 as a novel and conserved ODF that alleviates oxidative and inflammatory stress to enhance oocyte quality and embryo development, providing mechanistic insight and a potential strategy for improving assisted reproductive technologies.

The implementation of embryo genomic selection (EGS) is constrained by the lack of a standardized workflow that balances high genotyping accuracy with the preservation of embryo viability, particularly given embryonic heterogeneity. To address this, we systematically optimized the critical steps of trophectoderm biopsy and whole‐genome amplification, establishing a diameter‐stratified strategy that links embryo diameter to a biopsy area window designed to protect embryo recovery while ensuring sufficient DNA input for reliable genotyping. Our results demonstrated high concordance (> 98%) between embryo and calf genomic estimated breeding values (GEBVs), supporting the reliability of predictions. Crucially, we defined a diameter‐based biopsy framework: for embryos < 150 μm, biopsy areas < 1000 μm ² supported viability, whereas larger embryos (≥ 150 μm) tolerated up to 1500 μm ² , with a minimum threshold of 840 μm ² guaranteeing > 90% genotype call rates. By preventing oversampling in small embryos and undersampling across embryo sizes, this diameter‐guided control improves both embryo survival and GEBV prediction accuracy. This study provides a validated technical pathway for EGS that addresses the major practical bottleneck in livestock breeding. By ensuring reliable selection without compromising developmental potential, our work facilitates the rapid introgression of superior genetics and marks a significant step toward making EGS scalable, accelerating genetic progress in cattle and other species.