Profiling of m6A methylation in porcine intramuscular adipocytes and unravelling PHKG1 represses porcine intramuscular lipid deposition in an m6A-dependent manner.

Intramuscular fat (IMF) content is mainly determined by intramuscular preadipocyte adipogenesis. Epigenetic modifications are known to have a regulatory effect on IMF. As N6-methyladenosine (m6A) is the most abundant epigenetic modification in eukaryotic RNAs. In the present study, we used m6A methylation and RNA sequencing (seq) to identify the m6A-modified RNAs associated with the adipogenic differentiation of intramuscular preadipocytes. Among them, the expression and m6A level of phosphorylase kinase subunit G1 (PHKG1) were found to be significantly changed during adipogenesis. Further studies revealed that knockdown of the methylase METTL3 decreased the m6A methylation of PHKG1 and led to a reduction in PHKG1. Moreover, knockdown of PHKG1 promoted adipogenic differentiation by upregulating the expression of adipogenic genes. In addition, we found that the IMF content in the longissimus thoracis (LT) of Bamei (BM) pigs was greater than that in Large White (LW) pigs, whereas the m6A and PHKG1 expression levels were lower in BM pigs. These findings indicate that the m6A level and expression of PHKG1 were significantly correlated with IMF content and meat quality. In conclusion, this study sheds light on the mechanism by which m6A modification regulates IMF deposition.

Identification of porcine fast/slow myogenic exosomes and their regulatory effects on lipid accumulation in intramuscular adipocytes.

BACKGROUND: Pork quality is affected by the type of muscle fibers, which is closely related to meat color, tenderness and juiciness. Exosomes are tiny vesicles with a diameter of approximately 30-150 nm that are secreted by cells and taken up by recipient cells to mediate communication. Exosome-mediated muscle-fat tissue crosstalk is a newly discovered mechanism that may have an important effect on intramuscular fat deposition and with that on meat quality. Various of adipose tissue-derived exosomes have been discovered and identified, but the identification and function of muscle exosomes, especially porcine fast/slow myotube exosomes, remain unclear. Here, we first isolated and identified exosomes secreted from porcine extensor digitorum longus (EDL) and soleus (SOL), which represent fast and slow muscle, respectively, and further explored their effects on lipid accumulation in longissimus dorsi adipocytes. RESULTS: Porcine SOL-derived exosomes (SOL-EXO) and EDL-derived exosomes (EDL-EXO) were first identified and their average particle sizes were approximately 84 nm with double-membrane disc- shapes as observed via transmission electron microscopy and scanning electron microscopy. Moreover, the intramuscular fat content of the SOL was greater than that of the EDL at 180 days of age, because SOL intramuscular adipocytes had a stronger lipid-accumulating capacity than those of the EDL. Raman spectral analysis revealed that SOL-EXO protein content was much greater than that of EDL-EXO. Proteomic sequencing identified 72 proteins that were significantly differentially expressed between SOL-EXO and EDL-EXO, 31 of which were downregulated and 41 of which were upregulated in SOL-EXO. CONCLUSIONS: Our findings suggest that muscle-fat tissue interactions occur partly via SOL-EXO promoting adipogenic activity of intramuscular adipocytes.

Adipocyte Progenitor Pools Composition and Cellular Niches Affect Adipogenesis Divergence in Porcine Subcutaneous and Intramuscular Fat.

Intramuscular fat (IMF) contributed positively to pork quality, whereas subcutaneous fat (SCF) was often considered to be a detrimental factor impacting growth and carcass traits. Reducing SCF while maintaining optimal IMF levels requires a thorough understanding of the adipogenic differences between these two adipose depots. Our study explored the differences in adipogenesis between porcine IMF and SCF, and the results showed that subcutaneous adipocytes (SCAs) demonstrate a greater potential for adipogenic differentiation, both in vivo and in vitro. Lipidomic and transcriptomic analyses suggested that intramuscular adipocytes (IMAs) are more inclined to biosynthesize unsaturated fatty acids. Furthermore, single-cell RNA sequencing (scRNA-seq) was employed to dissect the intrinsic and microenvironmental discrepancies in adipogenesis between porcine IMF and SCF. Comparative analysis indicated that SCF was enriched with preadipocytes, exhibiting an enhanced adipogenic potential, while IMF was characterized by a higher abundance of stem cells. Furthermore, coculture analyses of porcine intramuscular adipogenic cells and myogenetic cells indicated that the niche of IMAs inhibited its adipogenic differentiation. Cell communication analysis identified 160 ligand-receptor pairs and channels between adipogenic and myogenetic cells in IMF. Collectively, our study elucidated two intrinsic and microenvironmental novel mechanisms underpinning the divergence in adipogenesis between porcine SCF and IMF.

Zn-Mediated Fragmentation of N-Alkoxyphthalimides Enabling the Synthesis of gem-Difluoroalkenes.

Zn-mediated generation of alkoxyl radicals from N-alkoxyphthalimides emerged as an efficient approach for forming diverse and valuable alkyl radicals through ß-scission or a hydrogen atom transfer process. The alkyl radical species can be further trapped by α-trifluoromethyl alkenes to construct a series of gem-difluoroalkenes.

Supplementing Boar Diet with Nicotinamide Mononucleotide Improves Sperm Quality Probably through the Activation of the SIRT3 Signaling Pathway.

Sperm quality is an important indicator to evaluate the reproduction ability of animals. Nicotinamide mononucleotide (NMN) participates in cell energy metabolism and reduces cell oxidative stress. However, the effect and regulatory mechanism of NMN on porcine sperm quality are still unknown. Here, 32 Landrace boars were randomly assigned to four groups (n = 8) and fed with different levels of NMN (0, 8, 16 or 32 mg/kg/d) for 9 weeks, and then serum and semen samples of the boars were collected to investigate the function and molecular mechanism of NMN in sperm quality. The results showed that the dietary NMN supplementation significantly increased sperm volume, density and motility (p < 0.05). Interestingly, NMN apparently improved the antioxidative indexes and increased the levels of testosterone (p < 0.05) in serum. Furthermore, NMN upregulated the protein levels of sirtuin 3 (SIRT3), antioxidation and oxidative phosphorylation (OXPHOS), but downregulated the protein levels of apoptosis in semen. Mechanically, NMN protected sperm from H2O2-induced oxidative stress and apoptosis through SIRT3 deacetylation. Importantly, the SIRT3-specific inhibitor 3-TYP attenuated the antioxidation and antiapoptosis of NMN in sperm. Therefore, NMN exerts antioxidation and antiapoptosis to improve boar sperm quality via the SIRT3 signaling pathway. Our findings suggest that NMN is a novel potential boar antioxidative feed additive to produce high-quality porcine semen.

Kojic acid inhibits pig sperm apoptosis and improves capacitated sperm state during liquid preservation at 17°C.

The parameters of sperm apoptosis and capacitation during liquid storage at 17°C can indicate the quality of pig sperm and the potential development of early embryos. However, the effect of kojic acid (KA) on semen preservation and its mechanism has not been fully understood. In this study, we discovered that adding KA to the diluent improved the antioxidant capacity of sperm mitochondria, maintained the normal structure of sperm mitochondria, and reduced sperm apoptosis. Western blot analysis revealed that KA prevented the release of Cytochrome c from mitochondria to the cytoplasm, reduced the expression of pro-apoptosis proteins cleaved Caspase-3 and cleaved Caspase-9, and increased the expression of the antiapoptosis protein Bcl-XL. Furthermore, KA also enhanced the motility parameters, oxidative phosphorylation level, adenosine triphosphate level, and protein tyrosine phosphorylation of capacitated sperm, while preserving the acrosome integrity and plasma membrane integrity of capacitated sperm. In conclusion, this study offers new insights into the molecular mechanism of how KA inhibits porcine sperm apoptosis and improves capacitated sperm parameters. Additionally, it suggests that KA can serve as an alternative to antibiotics.

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation.

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia†.

Genomic integrity is critical for sexual reproduction, ensuring correct transmission of parental genetic information to the descendant. To preserve genomic integrity, germ cells have evolved multiple DNA repair mechanisms, together termed as DNA damage response. The RNA N6-methyladenosine is the most abundant mRNA modification in eukaryotic cells, which plays important roles in DNA damage response, and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) is a well-acknowledged N6-methyladenosine reader protein regulating the mRNA decay and stress response. Despite this, the correlation between YTHDF2 and DNA damage response in germ cells, if any, remains enigmatic. Here, by employing a Ythdf2-conditional knockout mouse model as well as a Ythdf2-null GC-1 mouse spermatogonial cell line, we explored the role and the underlying mechanism for YTHDF2 in spermatogonial DNA damage response. We identified that, despite no evident testicular morphological abnormalities under the normal circumstance, conditional mutation of Ythdf2 in adult male mice sensitized germ cells, including spermatogonia, to etoposide-induced DNA damage. Consistently, Ythdf2-KO GC-1 cells displayed increased sensitivity and apoptosis in response to DNA damage, accompanied by the decreased SET domain bifurcated 1 (SETDB1, a histone methyltransferase) and H3K9me3 levels. The Setdb1 knockdown in GC-1 cells generated a similar phenotype, but its overexpression in Ythdf2-null GC-1 cells alleviated the sensitivity and apoptosis in response to DNA damage. Taken together, these results demonstrate that the N6-methyladenosine reader YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia, which provides novel insights into the mechanisms underlying spermatogonial genome integrity maintenance and therefore contributes to safe reproduction.

Deubiquitinase UCHL1 regulates estradiol synthesis by stabilizing voltage-dependent anion channel 2.

Lack of estradiol production by granulosa cells blocks follicle development, causes failure of estrous initiation, and results in an inability to ovulate. The ubiquitin-proteasome system plays a critical role in maintaining protein homeostasis and stability of the estrous cycle, but knowledge of deubiquitination enzyme function in estradiol synthesis is limited. Here, we observe that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is more significant in estrous sows and high litter-size sows than in nonestrous sows and low-yielding sows. Overexpression of UCHL1 promotes estradiol synthesis in granulosa cells, and interference with UCHL1 has the opposite effect. UCHL1 binds, deubiquitinates, and stabilizes voltage-dependent anion channel 2 (VDAC2), promoting the synthesis of the estradiol precursor pregnenolone. Cysteine 90 (C90) of UCHL1 is necessary for its deubiquitination activity, and Lys45 and Lys64 in VDAC2 are essential for its ubiquitination and degradation. In vivo, compared with WT and sh-NC-AAV groups, the estrus cycle of female mice is disturbed, estradiol level is decreased, and the number of antral follicles is decreased after the injection of sh-UCHL1-AAV into ovarian tissue. These findings suggest that UCHL1 promotes estradiol synthesis by stabilizing VDAC2 and identify UCHL1 as a candidate gene affecting reproductive performance.

Vitrification induces a focused spindle pole in mouse MI oocytes.

Immature oocyte (germinal vesicle stage, GV) vitrification can avoid a cycle of ovarian stimulation, which is friendly to patients with hormone-sensitive tumors. However, the in vitro maturation of vitrification-thawed GV oocyte usually results in aneuploidy, and the underlying mechanism remains unclear. Stable spindle poles are important for accurate chromosome segregation. Acentriolar microtubule-organizing centers (aMTOCs) undergo fragmentation and reaggregation to form spindle poles. Microtubule nucleation is facilitated via the perichromosome Ran after GVBD, which plays an important role in aMTOCs fragmentation. This study showed that vitrification may reduce microtubule density by decreasing perichromosomal Ran levels, which reduced the localization of pKIF11, thereby decreased the fragmentation of aMTOCs and formed a more focused spindle pole, ultimately resulted in aneuploidy. This study revealed the mechanism of abnormal spindle pole formation in vitrified oocytes and offered a theoretical support to further improve the quality of vitrified oocytes.

Tricaprylin, a medium-chain triglyceride, aggravates high-fat diet-induced fat deposition but improves intestinal health.

Medium-chain triglycerides (MCTs) are absorbed and metabolized more rapidly than long-chain triglycerides (LCTs) and therefore are considered to have obesity-prevention potential in foods. The effect of adding tricaprylin, an MCT, to food on fat deposition and intestinal health is uncharted. In this study, mice were randomly divided into four groups and fed a normal diet (ND), ND with tricaprylin, a high-fat diet (HFD), or HFD with tricaprylin. Supplementation of 2% tricaprylin in HFD significantly increased the body weight, fat mass, liver weight, adipocyte size in adipose tissue and liver, and upregulated genes related to fat deposition. Metabolomic analysis of serum and adipose tissue revealed that tricaprylin significantly increased the contents of metabolites related to lipid metabolism, triglyceride storage, and fat deposition related signaling pathways. In vitro experiments and molecular docking analysis suggest that octanoic acid, a primary decomposition product of tricaprylin, may promote adipogenic differentiation of preadipocytes by acting as a PPARγ ligand to activate the expression of lipogenesis-related genes. Although supplementation with 2% tricaprylin in HFD cannot reduce fat deposition, it has a beneficial effect on intestinal health. Tricaprylin improved intestinal morphology, digestive enzyme activity, short-chain fatty acid concentration, and intestinal barrier function-related protein expression, while reducing inflammatory factor levels and the abundance of harmful intestinal microorganisms.

miR-10a-5p inhibits steroid hormone synthesis in porcine granulosa cells by targeting CREB1 and inhibiting cholesterol metabolism.

During growth, proliferation, differentiation, atresia, ovulation, and luteinization, the morphology and function of granulosa cells (GCs) change. Estrogen and progesterone are steroid hormones secreted by GCs that regulate the ovulation cycle of sows and help maintain pregnancy. miR-10a-5p is highly expressed in GCs and can inhibit GC proliferation. However, the role of miR-10a-5p in the steroid hormone synthesis of porcine GCs is unclear. In this study, miR-10a-5p agomir or antagomir was transfected into GCs. Overexpression of miR-10a-5p in GCs inhibited steroid hormone secretion and significantly downregulated steroid hormone synthesis via 3ß-hydroxy steroid dehydrogenase and cytochrome P450 family 19 subfamily A member 1. Interference with miR-10a-5p had the opposite effect. Bodipy and Oil Red O staining showed that overexpression of miR-10a-5p significantly reduced the formation of lipid droplets. Overexpression significantly inhibited the content of total cholesterol esters in GCs. The mRNA and protein levels of 3-hydroxy-3-methylglutaryl-CoA reductase and scavenger receptor class B member 1 decreased significantly, and the opposite effects were seen by interference with miR-10a-5p. Bioinformatic analysis of potential targets identified cAMP-responsive element binding protein 1 as a potential target and dual-luciferase reporter system analysis confirmed that miR-10a-5p directly targets the 3' untranslated region. These findings suggest that miR-10a-5p inhibits the expression of 3ß-hydroxy steroid dehydrogenase and cytochrome P450 family 19 subfamily A member 1 to inhibit the synthesis of steroid hormones in GCs. In addition, miR-10a-5p inhibits the cholesterol metabolism pathway of GCs to modulate steroid hormone synthesis.

Structural variation and introgression from wild populations in East Asian cattle genomes confer adaptation to local environment.

BACKGROUND: Structural variations (SVs) in individual genomes are major determinants of complex traits, including adaptability to environmental variables. The Mongolian and Hainan cattle breeds in East Asia are of taurine and indicine origins that have evolved to adapt to cold and hot environments, respectively. However, few studies have investigated SVs in East Asian cattle genomes and their roles in environmental adaptation, and little is known about adaptively introgressed SVs in East Asian cattle. RESULTS: In this study, we examine the roles of SVs in the climate adaptation of these two cattle lineages by generating highly contiguous chromosome-scale genome assemblies. Comparison of the two assemblies along with 18 Mongolian and Hainan cattle genomes obtained by long-read sequencing data provides a catalog of 123,898 nonredundant SVs. Several SVs detected from long reads are in exons of genes associated with epidermal differentiation, skin barrier, and bovine tuberculosis resistance. Functional investigations show that a 108-bp exonic insertion in SPN may affect the uptake of Mycobacterium tuberculosis by macrophages, which might contribute to the low susceptibility of Hainan cattle to bovine tuberculosis. Genotyping of 373 whole genomes from 39 breeds identifies 2610 SVs that are differentiated along a "north-south" gradient in China and overlap with 862 related genes that are enriched in pathways related to environmental adaptation. We identify 1457 Chinese indicine-stratified SVs that possibly originate from banteng and are frequent in Chinese indicine cattle. CONCLUSIONS: Our findings highlight the unique contribution of SVs in East Asian cattle to environmental adaptation and disease resistance.

Insulin regulates gap junction intercellular communication in porcine granulosa cells through modulation of connexin43 protein expression.

Gap junction intercellular communication (GJIC) among granulosa cells plays an important role in folliculogenesis, and it is temporal-spatially regulated during follicular development. Connexin (Cx) proteins predominantly form the basal structure of gap junctions in granulosa cells. In our study, immunohistochemical analysis revealed that Cx43 is the most widely expressed connexin in porcine follicles, especially among the large antral follicles. With application of insulin on porcine granulosa cells, we found that insulin significantly facilitated the protein level of Cx43, not mRNA level. This process is dependent on the phosphorylated activities of AKT and Erk since selective AKT and Erk inhibitors, LY294002 and U0126, respectively, hampered the potential of insulin to up-regulate Cx43 protein expression. As a consequence, the insulin-enhanced Cx43-couple GJIC activity in porcine granulosa cells was corresponding attenuated by the administration of LY294002 and U0126. Our findings provide a new insight into the molecular mechanisms by which insulin mediates cell-cell communication in porcine granulosa cells and sheds light on nutrition-reproduction interactions.

Effects of Alginic Acid on the Porcine Granulosa Cells and Maturation of Porcine Oocytes.

SCOPE: Alginic acid (AA) from brown algae is a marine organic compound. There is extensive use of AA in the food industry and healthcare, suggesting a high probability of AA exposure. The present study investigates the effects of AA on porcine ovarian granulosa cells (GCs) and oocytes to explore its mechanism in female reproduction because of its adverse effects on reproduction. METHODS AND RESULTS: The study adds 20 µM AA to the porcine primary ovarian GCs medium and porcine oocyte in vitro maturation (IVM) medium. Estrogen and progesterone levels are downregulated in GCs. Reactive oxygen species are excessive, and the antioxidant capacity declines. Then mitochondria-mediated apoptosis pathway is involved in GCs apoptosis. In addition, scores of autophagosomes are found in the experimental cells. Furthermore, AA significantly inhibits the proliferation of GCs around cumulus-oocyte complexes (COCs) accompanied by abnormal spindle assembly, chromosome arrangement disorder, and aberrant cortical granules distribution in oocytes, leading to a decreased oocyte maturation rate. CONCLUSION: These findings suggest that 20 µM AA is toxic to sow reproduction by interfering with estrogen production, oxidative stress, mitochondria-mediated apoptosis, autophagy in GCs of sows, and oocyte maturation.

Melatonin promotes gut anti-oxidative status in perinatal rat by remodeling the gut microbiome.

Gut health is important for nutrition absorption, reproduction, and lactation in perinatal and early weaned mammals. Although melatonin functions in maintaining circadian rhythms and preventing obesity, neurodegenerative diseases, and viral infections, its impact on the gut microbiome and its function in mediating gut health through gut microbiota remain largely unexplored. In the present study, the microbiome of rats was monitoring after fecal microbiota transplantation (FMT) and foster care (FC). The results showed that FMT and FC increased intestinal villus height/crypt depth in perinatal rats. Mechanistically, the melatonin-mediated remodeling of gut microbiota inhibited oxidative stress, which led to attenuation of autophagy and inflammation. In addition, FMT and FC encouraged the growth of more beneficial intestinal bacteria, such as Allobaculum, Bifidobacterium, and Faecalibaculum, which produce more short-chain fatty acids to strengthen intestinal anti-oxidation. These findings suggest that melatonin-treated gut microbiota increase the production of SCFAs, which improve gut health by reducing oxidative stress, autophagy and inflammation. The transfer of melatonin-treated gut microbiota may be a new and effective method by which to ameliorate gut health in perinatal and weaned mammals.

Cytokines and exosomal miRNAs in skeletal muscle-adipose crosstalk.

Skeletal muscle and adipose tissues (ATs) are secretory organs that release secretory factors including cytokines and exosomes. These factors mediate muscle-adipose crosstalk to regulate systemic metabolism via paracrine and endocrine pathways. Myokines and adipokines are cytokines secreted by skeletal muscle and ATs, respectively. Exosomes loaded with nucleic acids, proteins, lipid droplets, and organelles can fuse with the cytoplasm of target cells to perform regulatory functions. A major regulatory component of exosomes is miRNA. In addition, numerous novel myokines and adipokines have been identified through technological innovations. These discoveries have identified new biomarkers and sparked new insights into the molecular regulation of skeletal muscle growth and adipose deposition. The knowledge may contribute to potential diagnostic and therapeutic targets in metabolic disease.

CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

BACKGROUND: Clock circadian regulator (CLOCK) is a core factor of the mammalian biological clock system in regulating female fertility and ovarian physiology. However, CLOCK's specific function and molecular mechanism in porcine granulosa cells (GCs) remain unclear. In this study, we focused on CLOCK's effects on GC proliferation. RESULTS: CLOCK significantly inhibited cell proliferation in porcine GCs. CLOCK decreased the expression of cell cycle-related genes, including CCNB1, CCNE1, and CDK4 at the mRNA and protein levels. CDKN1A levels were upregulated by CLOCK. ASB9 is a newly-identified target of CLOCK that inhibits GC proliferation; CLOCK binds to the E-box element in the ASB9 promoter. CONCLUSIONS: These findings suggest that CLOCK inhibits the proliferation of porcine ovarian GCs by increasing ASB9 level.

[The role and regulatory mechanism of tissue and organ crosstalk on skeletal muscle development: a review].

Skeletal muscle is one of the most important organs in animal, and the regulatory mechanism of skeletal muscle development is of great importance for the diagnosis of muscle-related diseases and the improvement of meat quality of livestock. The regulation of skeletal muscle development is a complex process, which is regulated by a large number of muscle secretory factors and signaling pathways. In addition, in order to maintain steady-state and maximum use of energy metabolism in the body, the body coordinates multiple tissues and organs to form the complex and sophisticated metabolic regulation network, which plays an important role for the regulation of skeletal muscle development. With the development of omics technologies, the underlying mechanism of tissue and organ communication has been deeply studied. This paper reviews the effects of crosstalk among adipose tissue, nerve tissue and intestinal tissue on skeletal muscle development, with the aim to provide a theoretical basis for targeted regulation of skeletal muscle development.

Ru360 protects against vitrification-induced oocyte meiotic defects by restoring mitochondrial function.

Oocyte vitrification has been widely application in female fertility preservation. Recent studies found that vitrification of immature (germinal vesicle stage, GV) oocytes increased the risk of aneuploidy during meiotic maturation; however, the underlying mechanisms and the strategies to prevent this defect remain unexplored. In this study, we found that vitrification of GV oocytes decreased the first polarbody extrusion rate (90.51 ± 1.04% vs. 63.89 ± 1.39%, p < 0.05) and increased the aneuploid rate (2.50% vs. 20.00%, p < 0.05), accompanied with a series of defects during meiotic maturation, including aberrant spindle morphology, chromosome misalignment, incorrect Kinetochore-Microtubule attachments (KT-MTs) and weakened spindle assembly checkpoint protein complex (SAC) function. We also found that vitrification disrupted mitochondrial function by increasing mitochondrial Ca2+ levels. Importantly, inhibition of mitochondrial Ca2+ entry by 1 µM Ru360 significantly restored mitochondrial function and rescued the meiotic defects, indicating that the increase of mitochondrial Ca2+, at least, was a cause of meiotic defects in vitrified oocytes. These results shed light on the molecular mechanisms of oocyte vitrification-induced adverse effects of meiotic maturation and provided a potential strategy to improve oocyte cryopreservation protocols further.