Lipid-rich porcine oocytes are extremely sensitive to cryopreservation compared to other low-lipid oocytes. Vitrification has outperformed slowing freezing in oocyte cryopreservation and is expected to improve further by minimizing cellular osmotic and/or oxidative stresses. In this study, we compared the effects of loading porcine cumulus-oocyte complexes with glycine (an organic osmolyte) or glycine plus melatonin (an endogenous antioxidant) during vitrification, thawing and subsequent maturation to mitigate osmotic injuries or osmotic and oxidative damages on the developmental potential of porcine oocytes. Our data demonstrated that glycine treatment significantly increased the vitrification efficiency of porcine oocytes to levels comparable to those observed with glycine plus melatonin treatment. It was manifested as the thawed oocyte viability, oocyte nuclear maturation, contents of reactive oxygen species, translocation of cortical granules and apoptotic occurrence in mature oocytes, levels of ATP and transcripts of glycolytic genes in cumulus cells (markers of oocyte quality), oocyte fertilization and blastocyst development. However, the latter was more likely than the former to increase ATP contents and normal mitochondrial distribution in mature oocytes. Taken together, our results suggest that mitigating osmotic and oxidative stresses induced by vitrification and thawing can further enhance the developmental competency of vitrified porcine oocytes at the germinal vesicle stage.

Background: ADD1 (adducin-1) and TPX2 (targeting protein for Xklp2) are centrosomal proteins and regulate mitotic spindle assembly. Mammalian oocytes that segregate homologous chromosomes in Meiosis I and sister chromatids in Meiosis II with a spindle lacking centrosomes are more prone to chromosome segregation errors than in mitosis. However, the regulatory mechanisms of oocyte spindle assembly and the functions of ADD1 and TPX2 in this process remain elusive.Result: We found that the expression levels and localization of ADD1, S726 phosphorylated ADD1 (p-ADD1), and TPX2 proteins exhibited spindle assembly-dependent dynamic changes during mouse oocyte meiosis. Taxol treatment, which stabilizes the microtubule polymer and protects it from disassembly, made the signals of ADD1, p-ADD1, and TPX2 present in the microtubule organizing centers of small asters and spindles. Knockdown of approximately 60% of ADD1 protein levels destabilized interpolar microtubules in the meiotic spindle, resulting in aberrant chromosome alignment, reduced first polar body extrusion, and increased aneuploidy in metaphase II oocytes, but did not affect K-fiber homeostasis and the expression and localization of TPX2. Strikingly, TPX2 deficiency caused increased protein content of ADD1, but decreased expression and detachment of p-ADD1 from the spindle, thereby arresting mouse oocytes at the metaphase I stage with collapsed spindles.Conclusion: Phosphorylation of ADD1 at S726 by TPX2 mediates acentriolar spindle assembly and precise chromosome segregation in mouse oocytes.

In vitro embryo culture is essential for human assisted reproduction and livestock breeding, yet its efficiency remains limited owing to developmental arrest triggered by suboptimal media composition and environmental stressors. Preimplantation embryos are highly sensitive to a minor increase in osmolarity under organic osmolyte deficiency, which disrupts cell volume homeostasis to cause developmental block. However, the osmosensing mechanisms and the causal link between volume dysregulation and developmental arrest remain undefined. Elucidating these mechanisms will identify targeted osmoregulatory interventions to enhance in vitro culture efficiency. This study established a porcine two-cell embryo developmental arrest model under physiological-range hyperosmotic stress (330 mOsm) and organic osmolyte deficiency, which disrupts cell volume homeostasis. Through single-embryo RNA-seq, Real-time quantitative polymerase chain reaction (RT-qPCR), H3K4me3/H3K27ac/H3K9me3/m6A/BrdU immunofluorescence, mitochondrial assays (MitoTracker Red and reactive oxygen species (ROS) staining), and metabolic analysis (pyruvate dehydrogenase (PDH) activity by Western blotting, fatty acid oxidation by FAOBlue staining), we identified hyperosmosis-induced developmental impairments. Rescue experiments via organic osmolyte supplementation, PDH modulation, and epigenetic interventions further defined the molecular basis of embryonic arrest. Here, we reveal that physiological-range hyperosmolarity in the absence of organic osmolytes disrupts cell volume homeostasis in porcine two-cell embryos, triggering developmental arrest at the S phase of the four-cell stage. This arrest coincides with aberrant maternal-to-zygotic transition, characterized by impaired maternal transcript degradation, compromised zygotic genome activation (ZGA), and coordinated dysregulation of nuclear and mitochondrial DNA transcription. Mechanistically, arrested embryos exhibit disrupted metabolic–epigenetic crosstalk, including PDH inactivation via S293 p-PDH accumulation that blocks pyruvate-to-acetyl-coenzyme A (CoA) conversion, fatty acid β-oxidation inhibition, alongside elevated mitochondrial membrane potential (MMP), increased ROS accumulation, and reduced H3K4me3 and H3K27ac modifications. Critically, while pharmacological modulation of H3K4me3/H3K27ac fails to rescue developmental defects, restoring volume homeostasis with organic osmolytes (e.g., glycine/betaine) or reactivating PDH via dichloroacetate (DCA) treatment completely reverses hyperosmotic stress-induced developmental arrest. These findings identify that mitochondria in porcine preimplantation embryos act as osmotic stress sensors. Under conditions of extracellular organic osmolyte deficiency and elevated osmolarity, they drive metabolic reprogramming and nuclear epigenetic dysregulation, ultimately disrupting mitochondrial–nuclear communication, compromising ZGA, and inducing developmental arrest. These findings provide mechanistic insights for optimizing in vitro culture systems in reproductive technologies.

Previous studies have demonstrated that lipopolysaccharide (LPS), as a central toxic factor of gram‐negative bacteria, can induce oxidative stress and cellular inflammation to result in the impairment of female fertility in different organisms. Particularly, it has harmful effects on the oocyte quality and subsequent embryonic development. However, the approach concerning how to prevent oocytes from LPS‐induced deterioration still remains largely unexplored. We assessed the effective influences of velvet antler water extract (VAWE) by immunostaining and fluorescence intensity quantification on the meiotic maturation, mitochondrial function and sperm binding ability of oocytes under oxidative stress. Here, we report that VAWE treatment restores the quality of porcine oocytes exposed to LPS. Specifically, LPS exposure contributed to the failed oocyte maturation, reduced sperm binding ability and fertilization capability by disturbing the dynamics and arrangement of meiotic apparatuses and organelles, including spindle assembly, chromosome alignment, actin polymerization, mitochondrial dynamics and cortical granule distribution, the indicators of oocyte nuclear and cytoplasmic maturation. Notably, VAWE treatment recovered these meiotic defects by removing the LPS‐induced excessive ROS and thus inhibiting the apoptosis. Collectively, our study illustrates that VAWE treatment is a feasible strategy to improve the oocyte quality deteriorated by the LPS‐induced oxidative stress.

Introduction: Early preimplantation embryos of mammals exhibit pronounced sensitivity to hyperosmotic conditions, which results in an embryonic developmental block. The reduction of medium osmolarity or the supplementation with organic osmolytes can eliminate this arrest. Therefore, cell volume homeostasis is essential for embryonic development in vitro. Oocytes become capable of independent volume regulation after detaching from the follicle microenvironment. Whether the efficiency and quality of oocyte maturation can be improved by optimizing cell volume regulation by adjusting the osmolality of the culture medium in the presence of the organic osmolyte of glycine remains to be determined.Methods: The IVM of porcine oocytes was divided into two stages, i.e. the first 22 h as the first stage, and the last 22 h as the second stage. In the presence of 1 mM glycine, we adjusted the osmolality of the culture medium from low to high (290 mOsM for the first 22 h and 320 mOsM thereafter) by adding raffinose, which cannot be used by animal cells, in a culture stage-dependent manner.Results: Stage-dependent adjustment of simplified medium PZM-3 osmolarity (290 mOsM for the first 22 h and 320 mOsM thereafter) in the presence of 1 mM glycine significantly improved the quality of porcine oocyte maturation in vitro, manifested by the oocyte maturation rate, functional mitochondrial distribution and activity, the transcript levels of glycolysis genes in granulosa cells, and subsequent embryonic developmental ability and ROS levels.Conclusion: Our study demonstrates that optimizing cell volume regulation can further enhance the developmental potential of oocytes cultured in vitro.