Y-box binding protein 1 influences zygotic genome activation by regulating N6-methyladenosine in porcine embryos.

Y-box binding protein 1 (YBX1) is a member of the family of DNA- and RNA-binding proteins that play crucial roles in multiple aspects, including RNA stabilization, translational repression, and transcriptional regulation; however, its roles in embryo development remain less known. In this study, to investigate the function of YBX1 and its mechanism of action in porcine embryo development, YBX1 was knocked down by microinjecting YBX1 siRNA at the one-cell stage. YBX1 is located in the cytoplasm during embryonic development. The mRNA level of YBX1 was increased from the four-cell stage to the blastocyst stage but was significantly decreased in YBX1 knockdown embryos compared with the control. Moreover, the percentage of blastocysts was decreased following YBX1 knockdown compared with the control. Defecting YBX1 expression increased maternal gene mRNA expression and decreased zygotic genome activation (ZGA) gene mRNA expression and histone modification owing to decreased levels of N6-methyladenosine (m6A) writer N6-adenosine-methyltransferase 70 kDa subunit (METTL3) and reader insulin-like growth factor 2 mRNA-binding protein (IGF2BP1). In addition, IGF2BP1 knockdown showed that YBX1 regulated the ZGA process through m6A modification. In conclusion, YBX1 is essential for early embryo development because it regulates the ZGA process.

High Temperature-Induced m6A Epigenetic Changes Affect Early Porcine Embryonic Developmental Competence in Pigs.

N6-methyladenosine (m6A), the most prevalent modification in eukaryotic messenger RNA (mRNA), plays a key role in various developmental processes in mammals. Three proteins that affect RNA m6A modification have been identified: methyltransferases, demethylases, and m6A-binding proteins, known as "writer," "eraser," and "reader" proteins, respectively. However, changes in the m6A modification when early porcine embryos are exposed to stress remain unclear. In this study, we exposed porcine oocytes to a high temperature (HT, 41°C) for 10 h, after which the mature oocytes were parthenogenetically activated and cultured for 7 days to the blastocyst stage. HT significantly decreased the rates of the first polar body extrusion and blastocyst formation. Further detection of m6A modification found that HT can lead to increased expression levels of "reader," YTHDF2, and "writer," METTL3, and decreased expression levels of "eraser," FTO, resulting in an increased level of m6A modification in the embryos. Additionally, heat shock protein 70 (HSP70) is upregulated under HT conditions. Our study demonstrated that HT exposure alters m6A modification levels, which further affects early porcine embryonic development.

ZSCAN4 Regulates Zygotic Genome Activation and Telomere Elongation in Porcine Parthenogenetic Embryos.

Zinc finger and SCAN domain-containing 4 (ZSCAN4), a DNA-binding protein, maintains telomere length and plays a key role in critical aspects of mouse embryonic stem cells, including maintaining genomic stability and defying cellular senescence. However, the effect of ZSCAN4 in porcine parthenogenetic embryos remains unclear. To investigate the function of ZSCAN4 and the underlying mechanism in porcine embryo development, ZSCAN4 was knocked down via dsRNA injection in the one-cell stage. ZSCAN4 was highly expressed in the four- and five- to eight-cell stages in porcine embryos. The percentage of four-cell stage embryos, five- to eight-cell stage embryos, and blastocysts was lower in the ZSCAN4 knockdown group than in the control group. Notably, depletion of ZSCAN4 induced the protein expression of DNMT1 and 5-Methylcytosine (5mC, a methylated form of the DNA base cytosine) in the four-cell stage. The H3K27ac level and ZGA genes expression decreased following ZSCAN4 knockdown. Furthermore, ZSCAN4 knockdown led to DNA damage and shortened telomere compared with the control. Additionally, DNMT1-dsRNA was injected to reduce DNA hypermethylation in ZSCAN4 knockdown embryos. DNMT1 knockdown rescued telomere shortening and developmental defects caused by ZSCAN4 knockdown. In conclusion, ZSCAN4 is involved in the regulation of transcriptional activity and is essential for maintaining telomere length by regulating DNMT1 expression in porcine ZGA.

Mps1 controls spindle assembly, SAC, and DNA repair in the first cleavage of mouse early embryos.

Monopolar spindle-1 (Mps1) is a critical interphase regulator that also involves into the spindle assembly checkpoint for the cell cycle control in both mitosis and meiosis. However, the functions of Mps1 during mouse early embryo development is still unclear. In this study, we reported the important roles of Mps1 in the first cleavage of mouse embryos. Our data indicated that the loss of Mps1 activity caused precocious cleavage of zygotes to 2-cell embryos; however, prolonged culture disturbed the early embryo development to the blastocyst. We found that the spindle organization was disrupted after Mps1 inhibition, and the chromosomes were misaligned in the first cleavage. Moreover, the kinetochore-microtubule attachment was lost and Aurora B failed to accumulate to the kinetochores, indicating that the spindle assembly checkpoint (SAC) was activated. Furthermore, the inhibition of Mps1 activity resulted in an increase of DNA damage, which further induced oxidative stress, showing with positive γ-H2A.X signal and increased reactive oxygen species level. Ultimately, irreparable DNA damage and oxidative stress-activated apoptosis and autophagy, which was confirmed by the positive Annexin-V signal and increased autophagosomes. Taken together, our data indicated that Mps1 played important roles in the control of SAC and DNA repair during mouse early embryo development.

RAB7 GTPase regulates actin dynamics for DRP1-mediated mitochondria function and spindle migration in mouse oocyte meiosis.

RAB7 is a small GTPase that belongs to the Rab family, and as a vesicle trafficking factor it is shown to regulate the transport to late endocytic compartments, autophagosome maturation and organelle function. In present study, we showed the critical roles of RAB7 GTPase on actin dynamics and mitochondria function in oocyte meiosis. RAB7 mainly accumulated at cortex and spindle periphery during oocyte maturation. RAB7 depletion caused the failure of polar body extrusion and asymmetric division, and Rab7 exogenous mRNA supplement could rescue the defects caused by RAB7 RNAi. Based on mass spectrometry analysis, we found that RAB7 associated with several actin nucleation factors and mitochondria-related proteins in oocytes. The depletion of RAB7 caused the decrease of actin dynamics, which further affected meiotic spindle migration to the oocyte cortex. In addition, we found that RAB7 could maintain mitochondrial membrane potential and the mitochondrial distribution in mouse oocytes, and this might be due to its effects on the phosphorylation of DRP1 at Ser616 domain. Taken together, our data indicated that RAB7 transported actin nucleation factor for actin polarization, which further affected the phosphorylation of DRP1 for mitochondria dynamics and the meiotic spindle migration in mouse oocytes.

Citrinin exposure disrupts organelle distribution and functions in mouse oocytes.

Citrinin (CTN) is a secondary fungal metabolite produced by several species of Aspergillins and Penicillins, and it is widely found in vegetable-derived foods such as cereals and fermented rice-based food supplements. Previous studies indicated that CTN had immunotoxicity, hepatotoxicity, nephrotoxicity, and reproductive toxicity, which caused severe effects on human and animal health. However, the potential toxicity of CTN on the organelles of mouse oocytes is still unclear. In this study, we showed that the exposure to 30 µM CTN significantly reduced the developmental capacity of mouse oocytes. Our results revealed that mitochondria exhibited abnormal distribution and mitochondrial membrane potential decreased under CTN exposure. And the endoplasmic reticulum (ER) failed to accumulate to the spindle periphery, which is accompanied by the occurrence of ER stress, showing with increased GRP78 expression. We also found that similar with ER, the Golgi apparatus showed homogenous localization pattern after CTN exposure, and the vesicle transport was disturbed, showing with aberrant expression and localization of Rab11a. Moreover, our results indicated that CTN exposure increased the expression of LAMP2, indicating the induction of lysosomal damage. In summary, our study showed that CTN exposure to mouse oocytes was toxic to the distribution and functions of organelles, which further led to a decrease of oocyte quality.

Exposure to PBDE47 affects mouse oocyte quality via mitochondria dysfunction-induced oxidative stress and apoptosis.

2, 2', 4, 4'-Tetrabrominated diphenyl ether (PBDE47) is the most abundant homologues in polybrominated diphenyl ethers (PBDEs) and is used widely in daily life as a brominated flame retardant. It has been shown that PBDE47 has neurotoxicity, thyroid toxicity, and also is reported to have reproductive toxicity, which can impair the follicular function of female rats and activate ovarian endoplasmic reticulum stress, leading to ovarian damage. However, the toxicity of PBDE47 to the quality of mammalian oocyte has not been reported. In this study, we reported that PBDE47 exposure affected the mouse ovarian and uterine. We also showed that oocyte number reduced and meiotic maturation competence decreased. Moreover, PBDE47 exposure disrupted actin filaments distribution, and also changed the level of histone methylation. Based on the genome-wide sequencing we showed that PBDE47 altered multiple gene expression in mouse oocytes. PBDE47 exposure caused mitochondria dysfunction, showing with aberrant distribution and mitochondrial membrane potential. In addition, our results suggested that PBDE47 induced early apoptosis, which was caused by oxidative stress, showing with increased ROS level and positive Annexin-V signal. In conclusion, our results indicated that PBDE47 exposure affected the maturation of mouse oocyte via its effects on mitochondria function, ROS level and its related apoptosis.

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.

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

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.

Three-Dimensional Mirror-Assisted and Concave Pyramid-Shaped Solar-Thermal Steam Generator for Highly Efficient and Stable Water Evaporation and Brine Desalination.

Although significant advances have been achieved in developing solar-driven water evaporators for seawater desalination, there is still room for simultaneously enhancing water evaporation efficiency, salt resistance, and utilization of solar energy. Herein, for the first time, we demonstrate a highly efficient three-dimensional (3D) mirror-assisted and concave pyramid-shaped solar-thermal water evaporation system for high-yield and long-term desalination of seawater and brine water, which consists of a 3D concave pyramid-shaped solar-thermal architecture on the basis of polypyrrole-coated nonwoven fabrics (PCNFs), a 3D mirror array, a self-floating polystyrene foam layer, and a tail-like PCNF for upward transport of water. The 3D concave pyramid-shaped solar-thermal architecture enables multiple solar light reflections to absorb more solar energy, while the 3D mirror-assisted solar light enhancement design can activate the solar-thermal energy conversion of the back side of the concave pyramid-shaped PCNF architecture to improve the solar-thermal energy conversion efficiency. Crucially, selective accumulation of the precipitated salts on the back side of the concave pyramid-shaped architecture is realized, ensuring a favorable salt-resistant feature. The 3D mirror-assisted and concave pyramid-shaped solar-driven water evaporation system achieves a record high water evaporation rate of 4.75 kg m-2 h-1 under 1-sun irradiation only and exhibits long-term desalination stability even when evaporating high-salinity brine waters, demonstrating its great applicability and reliability for high-yield solar-driven desalination of seawater and high-salinity brine water.

ATF6 aggravates apoptosis in early porcine embryonic development by regulating organelle homeostasis under high-temperature conditions.

Activating transcription factor 6 (ATF6), one of the three sensor proteins in the endoplasmic reticulum (ER), is an important regulator of ER stress-induced apoptosis. ATF6 resides in the ER and, upon activation, is translocated to the Golgi apparatus, where it is cleaved by site-1 protease (S1P) to generate an amino-terminal cytoplasmic fragment. Although recent studies have made progress in elucidating the regulatory mechanisms of ATF6, its function during early porcine embryonic development under high-temperature (HT) stress remains unclear. In this study, zygotes were divided into four groups: control, HT, HT+ATF6 knockdown, and HT+PF (S1P inhibitor). Results showed that HT exposure induced ER stress, which increased ATF6 protein expression and led to a decrease in the blastocyst rate. Next, ATF6 expression was knocked down in HT embryos under microinjection of ATF6 double-stranded RNA (dsRNA). Results revealed that ATF6 knockdown (ATF6-KD) attenuated the increased expression of CHOP, an ER stress marker, and Ca 2+ release induced by HT. In addition, ATF6-KD alleviated homeostasis dysregulation among organelles caused by HT-induced ER stress, and further reduced Golgi apparatus and mitochondrial dysfunction in HT embryos. AIFM2 is an important downstream effector of ATF6. Results showed that ATF6-KD reduced the occurrence of AIFM2-mediated embryonic apoptosis at HT. Taken together, our findings suggest that ATF6 is a crucial mediator of apoptosis during early porcine embryonic development, resulting from HT-induced ER stress and disruption of organelle homeostasis.

E2F4 regulates cell cycle to mediate embryonic development in pigs.

In mammals, E2 factor (E2F) acts as a cell cycle regulator. E2F transcription factor 4 (E2F4) is a member of the E2F family of transcription factors and usually represents predominant E2F activity in cells. The E2F4 gene has been extensively studied in animals and is associated with multiple functions, such as cell cycle regulation and apoptosis; however, little is known about its role during embryonic development. In this study, we investigated the function of E2F4 and its mechanism of action in porcine embryo development. For this purpose, we knocked down E2F4 by microinjecting double-stranded RNA of E2F4 at the 1-cell stage. The results showed that E2F4 knockdown in porcine embryos led to a significant decrease in the blastocyst rate and total cell number. Defective E2F4 expression reduced the level of G1/S checkpoints (cyclin E-cyclin-dependent kinase 2) and cell cycle-related gene expression at the 4-cell embryo stage and blastocyst. Moreover, a decrease in E2F4 expression increased phosphorylated H2A.X variant histones and activated ataxia telangiectasia mutated (ATM) and p53-p21 pathway. In addition, E2F4 depletion caused a significant decrease in histone acetylation. Taken together, E2F4 plays a critical role as a transcriptional activator in the development of porcine embryos, an observation that contradicts its well-established role as a transcription repressor.

Alpha-lipoic acid attenuates heat stress-induced apoptosis via upregulating the heat shock response in porcine parthenotes.

Heat stress (HS) is a long-standing hurdle that animals face in the living environment. Alpha-lipoic acid (ALA) is a strong antioxidant synthesized by plants and animals. The present study evaluated the mechanism of ALA action in HS-induced early porcine parthenotes development. Parthenogenetically activated porcine oocytes were divided into three groups: control, high temperature (HT) (42 °C for 10 h), and HT + ALA (with 10 µM ALA). The results show that HT treatment significantly reduced the blastocyst formation rate compared to the control. The addition of ALA partially restored the development and improved the quality of blastocysts. Moreover, supplementation with ALA not only induced lower levels of reactive oxygen species and higher glutathione levels but also markedly reduced the expression of glucose regulatory protein 78. The protein levels of heat shock factor 1 and heat shock protein 40 were higher in the HT + ALA group, which suggests activation of the heat shock response. The addition of ALA reduced the expression of caspase 3 and increased the expression of B-cell lymphoma-extra-large protein. Collectively, this study revealed that ALA supplementation ameliorated HS-induced apoptosis by suppressing oxidative and endoplasmic reticulum stresses via activating the heat shock response, which improved the quality of HS-exposed porcine parthenotes.

Knock-down of YME1L1 induces mitochondrial dysfunction during early porcine embryonic development.

YME1L1, a mitochondrial metalloproteinase, is an Adenosine triphosphate (ATP)-dependent metalloproteinase and locates in the mitochondrial inner membrane. The protease domain of YME1L1 is oriented towards the mitochondrial intermembrane space, which modulates the mitochondrial GTPase optic atrophy type 1 (OPA1) processing. However, during embryonic development, there is no report yet about the role of YME1L1 on mitochondrial biogenesis and function in pigs. In the current study, the mRNA level of YME1L1 was knocked down by double strand RNA microinjection to the 1-cell stage embryos. The expression patterns of YME1L1 and its related proteins were performed by immunofluorescence and western blotting. To access the biological function of YME1L1, we first counted the preimplantation development rate, diameter, and total cell number of blastocyst on day-7. First, the localization of endogenous YME1L1 was found in the punctate structures of the mitochondria, and the expression level of YME1L1 is highly expressed from the 4-cell stage. Following significant knock-down of YME1L1, blastocyst rate and quality were decreased, and mitochondrial fragmentation was induced. YME1L1 knockdown induced excessive ROS production, lower mitochondrial membrane potential, and lower ATP levels. The OPA1 cleavage induced by YME1L1 knockdown was prevented by double knock-down of YME1L1 and OMA1. Moreover, cytochrome c, a pro-apoptotic signal, was released from the mitochondria after the knock-down of YME1L1. Taken together, these results indicate that YME1L1 is essential for regulating mitochondrial fission, function, and apoptosis during porcine embryo preimplantation development.

ATF7-dependent epigenetic changes induced by high temperature during early porcine embryonic development.

BACKGROUND: Activating transcription factor 7 (ATF7) is a member of the ATF/cAMP response element (CRE) B superfamily. ATF2, ATF7, and CRE-BPa are present in vertebrates. Drosophila and fission yeast have only one homologue: dATF2 and Atf1, respectively. Under normal conditions, ATF7 promotes heterochromatin formation by recruiting histone H3K9 di- and tri-methyltransferases. Once the situation changes, all members are phosphorylated by the stress-activated kinase P38 in response to various stressors. However, the role of ATF7 in early porcine embryonic development remains unclear. RESULTS: In this study, we found that ATF7 gradually accumulated in the nucleus and then localized on the pericentric heterochromatin after the late 4-cell stage, while being co-localized with heterochromatin protein 1 (HP1). Knockdown of ATF7 resulted in decreases in the blastocyst rate and blastocyst cell number. ATF7 depletion resulted in downregulation of HP1 and histone 3 lysine 9 dimethylation (H3K9me2) expression. These effects were alleviated when P38 activity was inhibited. High temperatures increased the expression level of pP38, while reducing the quality of porcine embryos, and led to ATF7 phosphorylation. The expression level of H3K9me2 and HP1 was decreased and regulated by P38 activity. CONCLUSION: Stress-induced ATF7-dependent epigenetic changes play important roles in early porcine embryonic development.

High Temperature Disrupts Organelle Distribution and Functions Affecting Meiotic Maturation in Porcine Oocytes.

Heat stress (HS) has been known to cause reproductive failure in animals, especially in summer. HS severely affects the developmental potential of oocytes and leads to low fertility rates. Previous studies have reported that HS compromises embryo development in bovine oocytes, and reduces ovarian development in mice, thereby impairing reproductive function in animals. However, the effect of high temperature (HT) on the organelles of porcine oocytes is unknown. In this study, we reported that exposure to HT for 24 h (41°C) significantly decreased meiotic maturation in porcine oocytes (p < 0.05). Further experiments on organelles found that HT induced mitochondrial dysfunction, increased abnormal mitochondrial distribution, and decreased mitochondrial membrane potential (MMP). We also found that HT induced abnormal endoplasmic reticulum (ER) distribution and higher expression of glucose regulatory protein 78 (GRP78), suggesting that HT exposure induces ER stress. Our results also indicated that exposure to HT induced abnormal distribution and dysfunction of the Golgi apparatus, which resulted from a decrease in the expression of the vesicle transporter, Ras-related protein Rab-11A (RAB11A). In addition, we found that HT exposure led to lysosomal damage by increasing the expression of lysosome-associated membrane protein 2 (LAMP2) and microtubule-associated protein 1A/1B-light chain 3 (LC3). In summary, our study revealed that HT exposure disrupts organelle dynamics, which further leads to the failure of meiotic maturation in porcine oocytes.

Rotenone causes mitochondrial dysfunction and prevents maturation in porcine oocytes.

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.

Epigallocatechin-3-gallate protects porcine oocytes against post-ovulatory aging through inhibition of oxidative stress.

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.

ROMO1 is required for mitochondrial metabolism during preimplantation embryo development in pigs.

BACKGROUND: Reactive oxygen species (ROS) modulator 1 (ROMO1) is a mitochondrial membrane protein that is essential for the regulation of mitochondrial ROS production and redox sensing. ROMO1 regulates ROS generation within cells and is involved in cellular processes, such as cell proliferation, senescence, and death. Our purpose is to investigates the impact of ROMO1 on the mitochondria during porcine embryogenesis. RESULTS: We found that high expression of ROMO1 was associated with porcine preimplantation embryo development, indicating that ROMO1 may contribute to the progression of embryogenesis. Knockdown of ROMO1 disrupted porcine embryo development and blastocyst quality, thereby inducing ROS production and decreasing mitochondrial membrane potential. Knockdown of ROMO1 induced mitochondrial dysfunction by disrupting the balance of OPA1 isoforms to release cytochrome c, reduce ATP, and induce apoptosis. Meanwhile, ROMO1 overexpression showed similar effects as ROMO1 KD on the embryos. Overexpression of ROMO1 rescued the ROMO1 KD-induced defects in embryo development, mitochondrial fragmentation, and apoptosis. CONCLUSIONS: ROMO1 plays a critical role in embryo development by regulating mitochondrial morphology, function, and apoptosis in pigs.