G-Protein-Coupled Receptor Kinase 2 Inhibition Induces Meiotic Arrest by Disturbing Ca2+ Release in Porcine Oocytes.

G-protein-coupled receptor kinase 2 (GRK2) interacts with Gßγ and Gαq, subunits of G-protein alpha, to regulate cell signalling. The second messenger inositol trisphosphate, produced by activated Gαq, promotes calcium release from the endoplasmic reticulum (ER) and regulates maturation-promoting factor (MPF) activity. This study aimed to investigate the role of GRK2 in MPF activity during the meiotic maturation of porcine oocytes. A specific inhibitor of GRK2 (ßi) was used in this study. The present study showed that GRK2 inhibition increased the percentage of oocyte arrest at the metaphase I (MI) stage (control: 13.84 ± 0.95%; ßi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; ßi: 65.40 ± 1.14%). The level of phospho-GRK2 decreased in the treated group, suggesting that GRK2 activity was reduced upon GRK2 inhibition. Furthermore, the addition of ßi decreased Ca2+ release from the ER. The protein levels of cyclin B and cyclin-dependent kinase 1 were higher in the treatment group than those in the control group, indicating that GRK2 inhibition prevented a decrease in MPF activity. Collectively, GRK2 inhibition induced meiotic arrest at the MI stage in porcine oocytes by preventing a decrease in MPF activity, suggesting that GRK2 is essential for oocyte meiotic maturation in pigs.

Transcriptome analysis of the effects of high temperature on zygotic genome activation in porcine embryos.

Damage to the development of porcine gametes and embryos caused by high temperatures (HT) is one of the main reasons for the decline in the economic benefits of the livestock industry. Zygotic genome activation (ZGA) marks the beginning of gene expression programs in mammalian pre-implantation embryos. In pigs, ZGA occurs at the 4-cell (4 C) stage, indicating that correct gene expression at this stage plays an important regulatory role in embryonic development. However, the effect of the HT environment on early porcine embryonic development and the RNA expression profile of ZGA remain unclear. In this study, we compared the RNA transcription patterns of porcine 4 C embryos under normal and HT conditions using RNA-seq and identified 326 differentially expressed genes (DEGs). These changes were mainly related to DNA polymerase activity, DNA replication, and nucleotidyltransferase activity. In addition, entries for reverse transcription and endonuclease activity were enriched, indicating that ZGA interfered under HT conditions. Further comparison of the experimental results with the porcine ZGA gene revealed 39 ZGA genes among the DEGs. KEGG and GSEA analysis showed that the oxidative phosphorylation pathway was significantly enriched and signaling pathways related to energy metabolism were significantly downregulated. We also found that NDUFA6 and CDKN1A were located at the center of the protein-protein interaction network diagram of the DEGs. In summary, HT conditions affect mitochondrial function and oxidative phosphorylation levels, and lead to changes in the expression pattern of ZGA in early porcine embryos, with its hub genes NDUFA6 and CDKN1A.

MAT2A is essential for zygotic genome activation by maintaining of histone methylation in porcine embryos.

Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme in the methionine cycle that generates S-adenosylmethionine (SAM) by reacting with methionine and ATP. SAM acts as a methyl donors for histone and DNA methylation, which plays key roles in zygotic genome activation (ZGA). However, the effects of MAT2A on porcine ZGA remain unclear. To investigate the function of MAT2A and its underlying mechanism in porcine ZGA, MAT2A was knocked down by double-stranded RNA injection at the 1-cell stage. MAT2A is highly expressed at every stage of porcine embryo development. The percentages of four-cell-stage embryos and blastocysts were lower in the MAT2A-knockdown (KD) group than in the control group. Notably, depletion of MAT2A decreased the levels of H3K4me2, H3K9me2/3, and H3K27me3 at the four-cell stage, whereas MAT2A KD reduced the transcriptional activity of ZGA genes. MAT2A KD decreased embryonic ectoderm development (EED) and enhancer of zeste homolog 2 (EZH2) expression. Exogenous SAM supplementation rescued histone methylation levels and developmental arrest induced by MAT2A KD. Additionally, MAT2A KD significantly increased DNA damage and apoptosis. In conclusion, MAT2A is involved in regulating transcriptional activity and is essential for regulating histone methylation during porcine ZGA.

In Vivo-Matured Oocyte Resists Post-Ovulatory Aging through the Hub Genes DDX18 and DNAJC7 in Pigs.

Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the "electron transport chain" and "cell redox homeostasis" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in "RNA binding", "mRNA metabolic process", etc., as well as in GO terms, and "spliceosome" and "nucleotide excision repair" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.

The effects of repeated freezing and thawing on bovine sperm morphometry and function.

Refreezing the remaining genetic resources after in vitro fertilization (IVF) can conserve genetic materials. However, the precise damage inflicted by repeated freezing and thawing on bovine sperm and its underlying mechanism remain largely unexplored. Thus, this study investigates the impact of repeated freeze-thaw cycles on sperm. Our findings indicate that such cycles significantly reduce sperm viability and motility. Furthermore, the integrity of the sperm plasma membrane and acrosome is compromised during this process, exacerbating the advanced apoptosis triggered by oxidative stress. Additionally, transmission electron microscopy exposed severe damage to the plasma membranes of both the sperm head and tail. Notably, the "9 + 2" structure of the tail was disrupted, along with a significant decrease in the level of the axonemal protein DNAH10, leading to reduced sperm motility. IVF outcomes revealed that repeated freeze-thaw cycles considerably impair sperm fertilization capability, ultimately reducing the blastocyst rate. In summary, our research demonstrates that repeated freeze-thaw cycles lead to a decline in sperm viability and motility, attributed to oxidative stress-induced apoptosis and DNAH10-related dynamic deficiency. As a result, the utility of semen is compromised after repeated freezing.

Nobiletin enhances mitochondrial function by regulating SIRT1/PGC-1α signaling in porcine oocytes during in vitro maturation.

Nobiletin is a natural flavonoid found in citrus fruits with beneficial effects, including anti-inflammatory, anti-cancer and anti-oxidation effects. The aim of this study was to investigate whether nobiletin improves mitochondrial function in porcine oocytes and examine the underlying mechanism. Oocytes enclosed by cumulus cells were cultured in TCM-199 for 44 h with 0.1% dimethyl sulfoxide (control), or supplemented with 5, 10, 25, and 50 µM of nobiletin (Nob5, Nob10, Nob25, and Nob50, respectively). Oocyte maturation rate was significantly enhanced in Nob10 (70.26 ± 0.45%) compared to the other groups (control: 60.12 ± 0.47%; Nob5: 59.44 ± 1.63%; Nob25: 63.15 ± 1.38%; Nob50: 46.57 ± 1.19%). The addition of nobiletin reduced the levels of reactive oxygen species and increased glutathione levels. Moreover, Nob10 promoted mitochondrial biogenesis by upregulating the protein levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α). This resulted in an increase in the number of active mitochondria, mitochondrial DNA copy number, mitochondrial membrane potential, and ATP production, thereby enhancing mitochondrial function. The protein level of p53 decreased, followed by the phosphorylation of B-cell lymphoma 2, suggesting a reduction in mitochondria-mediated apoptosis in the Nob10 group. Additionally, the release of cytochrome c from the mitochondria was significantly diminished along with a decrease in the protein expression of caspase 3. Thus, nobiletin has a great potential to promote the in vitro maturation of porcine oocytes by suppressing oxidative stress and promoting mitochondrial function through the upregulation of the SIRT1/PGC-1α signaling pathway.

ß-hydroxybutyrate impairs bovine oocyte maturation via pyruvate dehydrogenase (PDH) associated energy metabolism abnormality.

Background: Ketosis is one of the most frequent and costly metabolic disorders in high-producing dairy cows, and negatively associated with the health and reproductive performance of bovine. Ketosis is mainly caused by the accumulation of ketone body ß-hydroxybutyric acid and its diagnosis is based on ß-hydroxybutyrate (ßHB) concentration in blood. Methods: In this study, we investigated the effects of ßHB on bovine oocyte maturation in the concentration of subclinical (1.2 mM) ßHB and clinical (3.6 mM). Results: The results showed ßHB disrupted bovine oocyte maturation and development capacity. Further analysis showed that ßHB induced oxidative stress and mitochondrial dysfunction, as indicated by the increased level of reactive oxygen species (ROS), disrupted mitochondrial structure and distribution, and depolarized membrane potential. Furthermore, oxidative stress triggered early apoptosis, as shown by the enhanced levels of Caspase-3 and Annexin-V. Moreover, 3.6 mM ßHB induced the disruption of the pyruvate dehydrogenase (PDH) activity, showing with the decrease of the global acetylation modification and the increase of the abnormal spindle rate. Conclusion: Our study showed that ßHB in subclinical/clinical concentration had toxic effects on mitochondrial function and PDH activity, which might affect energy metabolism and epigenetic modification of bovine oocytes and embryos.

Tetrabromobisphenol Exposure Impairs Bovine Oocyte Maturation by Inducing Mitochondrial Dysfunction.

Tetrabromobisphenol (TBBPA) is the most widely used brominated flame retardant in the world and displays toxicity to humans and animals. However, few studies have focused on its impact on oocyte maturation. Here, TBBPA was added to the culture medium of bovine cumulus-oocyte complexes (COCs) to examine its effect on oocytes. We found that TBBPA exposure displayed an adverse influence on oocyte maturation and subsequent embryonic development. The results of this study showed that TBBPA exposure induced oocyte meiotic failure by disturbing the polar-body extrusion of oocytes and the expansion of cumulus cells. We further found that TBBPA exposure led to defective spindle assembly and chromosome alignment. Meanwhile, TBBPA induced oxidative stress and early apoptosis by mediating the expression of superoxide dismutase 2 (SOD2). TBBPA exposure also caused mitochondrial dysfunction, displaying a decrease in mitochondrial membrane potential, mitochondrial content, mtDNA copy number, and ATP levels, which are regulated by the expression of pyruvate dehydrogenase kinase 3 (PDK3). In addition, the developmental competence of oocytes and the quality of blastocysts were also reduced after TBBPA treatment. These results demonstrated that TBBPA exposure impaired oocyte maturation and developmental competence by disrupting both nuclear and cytoplasmic maturation of the oocyte, which might have been caused by oxidative stress induced by mitochondrial dysfunction.

Presence of ginsenoside re during in vitro maturation protects porcine oocytes from heat stress.

Heat stress (HS) affects the development of porcine gametes and embryos negatively, induces the decrease of reproductive ability significantly, threatens global pig production. Ginsenoside Re (GRe), is a main bioactive component of ginseng, shows very specific anti-apoptotic, antioxidant and anti-inflammatory activities. To investigate the alleviating effect of GRe on the in vitro maturation of porcine oocyte under the HS, the polar body extrusion rate, intracellular levels of reactive oxygen species (ROS) and glutathione (GSH), ATP content, mitochondrial membrane potential (MMP) were assessed. For the current study, porcine cumulus-oocyte complexes (COCs) randomly divided into four groups: the control, GRe, HS and HS + GRe group. The results showed that HS inhibited the cumulus cell expansion and polar body extrusion rate, the levels of GSH and MMP, the ATP content, the gene expression of Nrf2 of porcine oocytes and the parthenogenetic activation (PA) embryo development competence, but GRe treatment could partly neutralize these adverse effects. Furthermore, HS increased the ROS formation and percentage of apoptosis, the gene expression of HSP90, CASP3 and CytoC of porcine oocytes, but GRe could weaken the effect on Cyto C and BAX expression induced by HS. Taken together, these results showed that the presence of GRe during in vitro maturation protects porcine oocytes from HS. These findings lay a foundation for GRe may be used as a potential protective drug to protect porcine oocytes against HS damage.