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G-protein-coupled receptor kinase 2 (GRK2) interacts with Gβγ 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 (βi) 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%; βi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; βi: 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 βi decreased Ca 2+ 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., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Rotenone is a commonly used insecticidal chemical in agriculture and it is an inhibitor of mitochondrial complex Ⅰ. Previous studies have found that rotenone induces the production of reactive oxygen species (ROS) by inhibiting electron transport in the mitochondria of somatic and germ cells. However, there is little precise information on the effects of rotenone exposure in porcine oocytes during in vitro maturation, and the mechanisms underlying these effects have not been determined. The Cumulus-oocyte complexes were supplemented with different concentrations of rotenone to elucidate the effects of rotenone exposure on the meiotic maturation of porcine oocytes during in vitro maturation for about 48 hours. First, we found that the maturation rate and expansion of cumulus cells were significantly reduced in the 3 and 5 μM rotenone-treated groups. Subsequently, the concentration of rotenone was determined to be 3 μM. Also, immunofluorescence, western blotting, and image quantification analyses were performed to test the rotenone exposure on the meiotic maturation, total and mitochondrial ROS, mitochondrial function and biogenesis, mitophagy and apoptosis in porcine oocytes. Further experiments showed that rotenone treatment induced mitochondrial dysfunction and failure of mitochondrial biogenesis by repressing the level of SIRT1 during in vitro maturation of porcine oocytes. In addition, rotenone treatment reduced the ratio of active mitochondria to total mitochondria, increased ROS production, and decreased ATP production. The levels of LC3 and active-caspase 3 were significantly increased by rotenone treatment, indicating that mitochondrial dysfunction induced by rotenone increased mitophagy but eventually led to apoptosis. Collectively, these results suggest that rotenone interferes with porcine oocyte maturation by inhibiting mitochondrial function., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Heo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Increased levels of oxidative stress are major factors that drive the process of post-ovulatory oocyte aging. Epigallocatechin-3-gallate (EGCG), which accounts for up to 50% of the catechins, possesses versatile biological functions, including preventing or treating diabetes, cancer, and heart diseases. The aim of this study was to explore whether EGCG can delay porcine oocyte aging by preventing oxidative stress. Metaphase II (MII) oocytes were cultured for 48 h with different concentrations of EGCG (0-100 μM) in vitro as a post-ovulatory aging model. An optimal concentration of 5 μM EGCG maintained oocyte morphology and developmental competence during aging. The oocytes were randomly divided into five groups: fresh, 24 h control, 24 h EGCG, 48 h control, and 48 h EGCG. The results suggest that EGCG significantly prevents aging-induced oxidative stress, glutathione (GSH) reduction, apoptosis, and autophagy. Moreover, mitochondria DNA copy number was decreased, and the number of active mitochondria and adenosine triphosphate (ATP) levels significantly increased by supplementation with EGCG. Thus, EGCG has a preventive role against aging in porcine post-ovulatory oocytes due to its ability to inhibit oxidative stress and promote mitochondrial biogenesis.

Ubiquinol-10, the reduced form of coenzyme Q10, protects mammalian cells from oxidative damage and enhances mitochondrial activity. However, the protective effect of ubiquinol-10 on mammalian oocytes is not well understood. In this study, we investigated the effect of ubiquinol-10 on porcine oocytes during postovulatory aging. Metaphase II oocytes were selected as fresh oocytes and further cultured for 48 h with different concentrations of ubiquinol-10 (0-400 μM) in vitro as a postovulatory aging model. After choosing the optimal concentration of ubiquinol-10 (100 μM) that maintained oocyte morphology and developmental competence during the progression of aging, the oocytes were randomly divided into five groups: fresh, control-24 h, ubiquinol-24 h, control-48 h, and ubiquinol-48 h. The results revealed that ubiquinol-10 significantly prevented aging-induced oxidative stress, GSH reduction, cytoskeleton impairment, apoptosis, and autophagy. Mitochondrial biogenesis (SIRT1 and PGC-1α) and mitophagy (PINK1 and PARKIN)-related proteins were decreased during aging. Addition of ubiquinol-10 prevented the aging-induced reduction of these proteins. Consequently, although mitochondrial content was decreased, the number of active mitochondria and ATP level were significantly increased upon treatment with ubiquinol-10. Thus, ubiquinol-10 has beneficial effects on porcine postovulatory aging oocytes owing to its antioxidant properties and ability to promote mitochondrial renewal.

Tubastatin A (Tub-A), a highly selective histone deacetylase 6 (HDAC6) inhibitor, has been widely used as a cytotoxic anticancer agent, or for the treatment of patients with asthma. However, the potential toxicity of Tub-A on oocyte maturation and asymmetric division is still unclear. Therefore, the present study was designed to examine the effect and potential regulatory role of Tub-A on the meiotic maturation of oocytes. We observed that Tub-A treatment induced an increased level of the acetylation of α-tubulin, and a failure of spindle migration and actin cap formation. Based on the spindle structure, most Tub-A treated oocytes were arrested in an MI-like or a GVBD-like stage and exhibited decondensed chromosomes in a dose dependent manner. Moreover, Tub-A treatment decreased the protein expression of mTOR, a factor responsible for spindle formation, and the expression of mDia1, an inhibitor of actin assembly, in an HDAC6 expression-dependent manner. Importantly, following Tub-A supplementation, most oocytes failed to extrude the first polar body, which indicates that these defects are closely linked to abnormal oocyte maturation. Taken together, our data demonstrates that HDAC6 is one of the essential factors for oocyte maturation and asymmetric division via the HDAC6/mTOR or mDia1 pathway in mice.

Background: Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown., Results: In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell-like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each "blastomere" of the 2-cell-like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each "blastomere" and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure., Conclusion: Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI.