Lei An

In brief: Embryo biopsy and cryopreservation compromise pregnancy rates, limiting genetic testing in livestock and humans. This study shows that Y‑27632 improves post‑thaw embryo quality and increases pregnancy rates in biopsied sheep embryos. Preimplantation genetic testing relies on embryo biopsy, yet biopsied embryos exhibit reduced cryotolerance and compromised post-thaw viability. In this study, we optimized biopsy parameters and explored cryoprotective strategies in sheep embryos. Trophectoderm biopsy at a position two-thirds distant from the inner cell mass yielded optimal cell numbers (21.96 ± 1.21 cells) while maintaining high postbiopsy re-expansion rates (92.56%). An 8-hr postbiopsy recovery period prior to vitrification was identified as optimal, achieving complete re-expansion with minimal apoptosis while avoiding progression to hatched stages unsuitable for transfer. Supplementation with 10 μM Y-27632 at different vitrification stages was assessed for effects on re-expansion, cell viability, apoptosis, and pregnancy rates after transfer. Transcriptomic profiling revealed that Y-27632 effectively suppressed lipid and sterol biosynthesis, shifting metabolism toward energy conservation pathways that enhance cryoresistance. Treated embryos also showed significantly reduced lipid droplet accumulation, confirming metabolic remodeling. Y-27632 administration, particularly before and after vitrification, markedly improved cryotolerance, developmental capacity, and pregnancy outcomes of biopsied embryos, increasing pregnancy rates from 24.3% to 37.2%. These findings establish an optimized biopsy and recovery protocol and identify molecular changes associated with the protective effects of Y-27632 by which Y-27632 enhances embryo cryotolerance, presenting a practical strategy to improve cryopreservation efficacy in livestock and human-assisted reproduction.

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.

Bovine embryonic stem cells (bESCs) are a cornerstone for next-generation applications such as cell-cultured meat, large-animal gene editing, and embryo-stem cell breeding systems. Although primed bESCs and bovine expanded potential stem cells have recently been established, their routine maintenance commonly relies on mouse embryonic fibroblast or bovine fetal fibroblast feeder layers, which are complex, labor-intensive, poorly standardized, and incompatible with scalable manufacturing. Here, we report a simplified feeder-free culture platform for bESCs in which a commercially available basal medium is combined with Growth Factor Reduced Matrigel to generate a three-dimensional, niche-mimetic substrate. Using this approach, we successfully derived a novel feeder-free bESCs (FF-bESCs) that can be robustly expanded for more than 60 passages without ROCK inhibitors, while maintaining a normal karyotype, high proliferation rates, and stable expression of core pluripotency markers. Functional assays confirmed that FF-bESCs possess bona fide pluripotency, as evidenced by their ability to form embryoid bodies in vitro and generate teratomas containing derivatives of all three germ layers in vivo. Transcriptomic profiling further revealed that FF-bESCs align with a formative pluripotent state. Notably, these cells can be directed to differentiate into primordial germ cell-like cells. Our feeder-free culture system provides a scalable, reproducible foundation for advancing bESC-based technologies in bovine precision genome editing and germ cell differentiation for embryo-stem cell breeding systems.

Global warming poses a public health risk, and heat-induced reproductive defects are a growing concern for both humans and animals. Disrupted estrous cycle and reduced estrogen synthesis are notable changes in females exposed to heat stress (HS); however, the underlying mechanism remains largely unknown, thus effective preventive or interventional strategies are still lacking. Here, by focusing on Cyp19a1, the gene encoding the rate-limiting enzyme for estrogen synthesis in ovarian granulosa cells, we identify HS as a trigger for Ca2+ release from the endoplasmic reticulum (ER). The increased intracellular Ca2+ flux functions as a high-temperature sensor and activates STAT3 phosphorylation. The activated Ca2+-pSTAT3 cascade disrupts the expression of H3K27me3-modifying enzymes, thus elevating H3K27me3 levels and finally repressing Cyp19a1. Moreover, we demonstrate that retinoic acid (RA), the primary active metabolite of vitamin A, rescues CYP19A1 expression by antagonizing HS on multiple layers of the cascade. In vivo, RA administration rescues estrogen synthesis and corrects the estrous cycle in female mice under HS. Together, our study establishes a mechanistic link between HS and impaired estrogen synthesis and identifies the antagonistic function of RA. Finally, we propose a nutritional strategy to prevent or alleviate high temperature-induced estrus disorders in females.

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.

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.

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.

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 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.

C-type natriuretic peptide (CNP), encoded by the NPPC (Natriuretic Peptide Precursor C), has been recognized as the principal endogenous factor sustaining oocyte meiotic arrest in mammalian follicles. Yet its influence on porcine ovarian granulosa cell fate and the regulatory mechanism of NPPC expression within these cells remain poorly understood. Here, utilizing an in vitro culture model of primary porcine ovarian granulosa cells and immature oocytes, we examined the impact of CNP on granulosa cell apoptosis and oocyte meiotic resumption, and elucidated the molecular circuitry governing NPPC expression. We found that follicular atresia in pigs was accompanied by a marked decline in the CNP receptor NPR2 (natriuretic peptide receptor 2). Correspondingly, exogenous CNP suppressed apoptosis in cultured porcine granulosa cells. Estradiol can significantly promote the expression level of NPPC in porcine ovarian granulosa cells and, by enhancing NPR2 levels, it can synergize with CNP to inhibit oocyte meiotic resumption in vitro. Conversely, EGF signaling can significantly downregulate NPPC mRNA expression in porcine granulosa cells, an effect likely mediated by ERK-activated tristetraprolin (TTP). Collectively, these findings broaden our understanding of CNP in follicular development and delineate the endocrine network that controls NPPC transcription in the porcine ovary.

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.