Exposure to chlorpyrifos interferes with intercellular communication in cumulus-oocyte complexes during porcine oocyte maturation.

Chlorpyrifos (CPF), a widely used organophosphorus pesticide (OP) to control pests has been verified reproductive toxicity on mammalian oocytes. However, limited information exists on its correlation with the dysfunction of the intercellular communication in cumulus-oocyte complexes (COCs). Herein, our study utilized porcine COCs as models to directly address the latent impact of CPF on the communication between cumulus cells (CCs) and oocytes during in vitro maturation. The results demonstrated that CPF exposure decreased the rate of the first polar body (PB1) extrusion and blocked meiosis progression. Notably, the cumulus expansion of CPF-exposed COCs was suppressed significantly, accompanied by the down-regulated mRNA levels of cumulus expansion-related genes. Furthermore, the early apoptotic level was raised and the expression of BAX/BCL2 and cleaved caspase 3 was up-regulated in the CCs of CPF-exposed COCs (p < 0.05). Moreover, CPF exposure impaired mRNA levels of antioxidant enzyme-related genes, induced higher levels of reactive oxygen species (ROS) and reduced the levels of mitochondrial membrane potential (MMP) in CCs (p < 0.05). Additionally, the integrated optical density (IOD) rate (cumulus/oocyte) of calcein and the expression of connexin 43 (CX43) was increased in CPF treatment groups (p < 0.05). As well, CPF exposure reduced the expression levels of FSCN1, DAAM1 and MYO10, which resulted in a significant decrease in the number and fluorescence intensity of transzonal projections (TZPs). In conclusion, CPF inhibited the expansion of cumulus and caused oxidative stress and apoptosis as well as disturbed the function of gap junctions (GJs) and TZPs, which eventually resulted in the failure of oocyte maturation.

PAK1-Dependent Regulation of Microtubule Organization and Spindle Migration Is Essential for the Metaphase I-Metaphase II Transition in Porcine Oocytes.

P21-activated kinase 1 (PAK1) is a critical downstream target that mediates the effect of small Rho GTPase on the regulation of cytoskeletal kinetics, cell proliferation, and cell migration. PAK1 has been identified as a crucial regulator of spindle assembly during the first meiotic division; however, its roles during the metaphase I (MI) to metaphase II (MII) transition in oocytes remain unclear. In the present study, the potential function of PAK1 in regulating microtubule organization and spindle positioning during the MI-MII transition was addressed in porcine oocytes. The results showed that activated PAK1 was co-localized with α-tubulin, and its expression was increased from the MI to MII stage (p < 0.001). However, inhibiting PAK1 activity with an inhibitor targeting PAK1 activation-3 (IPA-3) at the MI stage decreased the first polar body (PB1) extrusion rate (p < 0.05), with most oocytes arrested at the anaphase-telophase (ATI) stage. IPA-3-treated oocytes displayed a decrease in actin distribution in the plasma membrane (p < 0.001) and an increase in the rate of defects in MII spindle reassembly with abnormal spindle positioning (p < 0.001). Nevertheless, these adverse effects of IPA-3 on oocytes were reversed when the disulfide bond between PAK1 and IPA-3 was reduced by dithiothreitol (DTT). Co-immunoprecipitation revealed that PAK1 could recruit activated Aurora A and transform acidic coiled-coil 3 (TACC3) to regulate spindle assembly and interact with LIM kinase 1 (LIMK1) to facilitate actin filament-mediated spindle migration. Together, PAK1 is essential for microtubule organization and spindle migration during the MI-MII transition in porcine oocytes, which is associated with the activity of p-Aurora A, p-TACC3 and p-LIMK1.

PAK1 Is Involved in the Spindle Assembly during the First Meiotic Division in Porcine Oocytes.

P21-activated kinase 1 (PAK1), as a member of the PAK family, has been implicated in various functions during somatic mitosis; however, less is known about its role during oocyte meiosis. Herein, we highlight the indispensable role of PAK1 in regulating spindle assembly and cell cycle progression during the first meiotic division of porcine oocytes. First, we found that the activated PAK1 expressed dynamically, and its subcellular localization was tightly associated with the spindle dynamics during meiosis in porcine oocytes. Specific inhibition of PAK1 activity by inhibitor targeting PAK1 activation-3 (IPA-3) led to impaired extrusion of the first polar body (PB1); with most of the IPA-3-treated oocytes arrested at germinal vesicle breakdown (GVBD) and subjected to failure of bipolar spindle formation. However, the adverse effects caused by IPA-3 on oocytes could be restored by reducing disulfide bonds between PAK1 and IPA-3 with dithiothreitol (DTT) treatment. Furthermore, the co-immunoprecipitation assay revealed that PAK1 interacted directly with Aurora A and transforming acidic coiled coil 3 (TACC3), providing an additional explanation for the similar localization of Aurora A and activated PAK1. Additionally, inhibiting the activity of PAK1 decreased the expression of p-Aurora A and p-TACC3; however, the reduced activity of Aurora A and TACC3 could be restored by DTT. In conclusion, PAK1 plays a crucial role in the proper assembly of the spindle during the first meiotic division of porcine oocytes, and the regulation of PAK1 is associated with its effects on p-Aurora A and p-TACC3 expression.

Effects of Ran-GTP/importin ß inhibition on the meiotic division of porcine oocytes.

The Ran-GTP/importin ß pathway has been implicated in a diverse array of mitotic functions in somatic mitosis; however, the possible meiotic roles of Ran-GTP/importin ß in mammalian oocyte meiosis are still not fully understood. In the present study, importazole (IPZ), a small molecule inhibitor of the interaction between Ran and importin ß was used to explore the potential meiotic roles of Ran-GTP/importin ß in porcine oocytes undergoing meiosis. After IPZ treatment, the extrusion rate of the first polar body (PB1) was significantly decreased, and a higher proportion of the oocytes were arrested at the germinal vesicle breakdown (GVBD) stage. Moreover, IPZ treatment led to severe defects in metaphase I (MI) spindle assembly and chromosome alignment during the germinal vesicle (GV)-to-MI stage, as well as failure of metaphase II (MII) spindle reassembly and homologous chromosome segregation during the MI-to-MII stage. Notably, IPZ treatment decreased TPX2 expression and abnormal subcellular localization. Furthermore, the expression levels of aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3) were significantly reduced after IPZ treatment. Collectively, these data indicate that the interaction of Ran-GTP and importin ß is essential for proper spindle assembly and successful chromosome segregation during two consecutive meiotic divisions in porcine oocytes, and regulation of this complex might be related to its effect on the TPX2 signaling cascades.

TPX2 deficiency leads to spindle abnormity and meiotic impairment in porcine oocytes.

The targeting protein for Xklp2 (TPX2) is a spindle assembly factor, that can stimulate microtubule formation and promote spindle completion during mitosis. However, the role of TPX2 in mammalian oocyte meiotic maturation is still not fully understood. This study was conducted to address the dynamic distribution and potential roles of TPX2 in microtubule nucleation during meiotic maturation in porcine oocytes by microinjecting specific siRNAs. Western blotting results revealed that the expression of TPX2 displayed a lower level from 0 to 22 h of culture, while its expression exhibited a higher level after 28 h of culture. Immunofluorescence staining demonstrated that TPX2 was distributed along the microtubules and enriched in the poles after meiotic spindle formation at the 28 and 44 h of culture. From immunoprecipitation, TPX2 can interact with the microtubule-associated proteins aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3). Meanwhile, the dynamic changes in the expression and localization of AURKA and TACC3 were highly consistent with TPX2 during meiotic maturation. After knocking down TPX2 by siRNA injection, the proportion of oocytes with aberrant spindles and scattered cytoplasmic actin filaments was significantly increased. In addition, TPX2 depletion markedly downregulated the expression of AURKA and TACC3. Thus, these results suggested that TPX2 is essential for meiotic spindle formation in the porcine oocyte, and that this regulation is related to its effect on AURKA and TACC3 expression.

Grape Seed Proanthocyanidin Ameliorates FB1-Induced Meiotic Defects in Porcine Oocytes.

Fumonisin B1 (FB1), as the most prevalent and toxic fumonisin, poses a health threat to humans and animals. The cytotoxicity of FB1 is closely related to oxidative stress and apoptosis. The purpose of this study is to explore whether Grape seed proanthocyanidin (GSP), a natural antioxidant, could alleviate the meiotic maturation defects of oocytes caused by FB1 exposure. Porcine cumulus oocyte complexes (COCs) were treated with 30 µM FB1 alone or cotreated with 100, 200 and 300 µM GSP during in vitro maturation for 44 h. The results show that 200 µM GSP cotreatment observably ameliorated the toxic effects of FB1 exposure, showing to be promoting first polar body extrusion and improving the subsequent cleavage rate and blastocyst development rate. Moreover, 200 µM GSP cotreatment restored cell cycle progression, reduced the proportion of aberrant spindles, improved actin distribution and protected mitochondrial function in FB1-exposed oocytes. Furthermore, reactive oxygen species (ROS) generation was significantly decreased and the mRNA levels of CAT, SOD2 and GSH-PX were obviously increased in the 200 µM GSP cotreatment group. Notably, the incidence of early apoptosis and autophagy level were also significantly decreased after GSP cotreatment and the mRNA expression levels of BAX, CASPASE3, LC3 and ATG5 were markedly decreased, whereas BCL2 and mTOR were observably increased in the oocytes after GSP cotreatment. Together, these results indicate that GSP could exert significant preventive effects on FB1-induced oocyte defects by ameliorating oxidative stress through repairing mitochondrial dysfunction.

Exposure to chlorpyrifos leads to spindle disorganization and mitochondrial dysfunction of porcine oocytes during in vitro maturation.

Chlorpyrifos (CPF), as one of the most extensively applied organophosphorus pesticides (OPs) in agricultural and domestic settings, causes a potential threat to human and animal health. Various reproductive toxicities of CPF have been reported, however, little information is available on whether CPF exposure could exert toxic effects on mammalian oocytes. Herein, the effects of CPF on the meiotic maturation and developmental capability of porcine oocytes were investigated, and the possible toxic mechanisms of CPF were also explored. Porcine cumulus-oocyte complexes (COCs) were treated with 0, 5, 10, or 20 µM CPF for 44 h during in vitro maturation (IVM), and the results showed that the first polar body (PB1) extrusion rate was significantly decreased, and the subsequent developmental competence of the resulting metaphase II (MII) oocytes was also impaired when the concentration of CPF reached 10 µM. In addition, a higher percentage of CPF-exposed oocytes were arrested at the anaphase-telophase I (ATI) stage, accompanied by misaligned chromosomes and aberrant spindles. Furthermore, higher levels of ROS and upregulated antioxidant-related genes (CAT, SOD1, SOD2, GPX) were detected in CPF-treated oocytes. Additionally, CPF treatment led to the depolarization of mitochondrial membrane potential (MMP) and the release of cytochrome c (Cyt c). Simultaneously, the apoptotic rate of the oocytes was significantly increased, and the expression levels of Bax and CASPASE3 were significantly upregulated after CFP exposure. Together, exposure to 10 µM CPF can disrupt the meiotic cycle progression, lead to aberrant spindles and mitochondrial dysfunction, which eventually induce oxidative stress and apoptosis in porcine oocytes.

Involvement of PINK1/Parkin-mediated mitophagy in mitochondrial functional disruption under oxidative stress in vitrified porcine oocytes.

Vitrification is an effective technique for fertility preservation, but is known to lead to mitochondrial dysfunction in porcine oocytes. Mitophagy is induced to rebalance mitochondrial function, a process in which reactive oxygen species (ROS) plays a role. In this study, vitrified-warmed porcine oocytes were incubated for 4 h with the oxidant AAPH or antioxidant α-tocopherol to alter ROS levels. A series of tests suggested that vitrification damaged mitochondrial structure and caused dysfunction, including blurred mitochondrial cristae, decreased mitochondrial membrane potential, decreased mtDNA copy number and increased ROS generation. This dysfunction resulted in mitophagy and the loss of embryonic developmental potential. Incubation with AAPH or α-tocopherol altered mitochondrial function and mitophagy flux status in vitrified oocytes. The PINK1/Parkin pathway was involved in oxidative stress regulation in vitrified oocytes. Under AAPH-induced oxidative stress, increased fluorescence intensity of Parkin, increased expression of PINK1, Parkin, and LC3B-II, and decreased expression of MFN2 and p62 were observed, whereas the opposite effects were induced under α-tocopherol treatment. The inhibition of ROS by α-tocopherol benefitted mitochondrial homeostasis and alleviated PINK1/Parkin-mediated mitophagy, resulting in the recovery of embryonic developmental potential in vitrified porcine oocytes. Therefore, this study provides a new mechanism for the application of antioxidants to aid the cryopreservation of porcine oocytes.

Mitophagy is involved in the mitochondrial dysfunction of vitrified porcine oocytes.

Mitochondrial dysfunction is considered a crucial factor aggravating oocyte viability after vitrification-warming. To clarify the role of mitophagy in mitochondrial extinction of vitrified porcine oocytes, mitochondrial function, ultrastructural characteristics, mitochondria-lysosomes colocalization, and mitophagic proteins were detected with or without chloroquine (CQ) treatment. The results showed that vitrification caused mitochondrial dysfunction, including increasing reactive oxygen species production, decreasing mitochondrial membrane potential, and mitochondrial DNA copy number. Damaged mitochondrial cristae and mitophagosomes were observed in vitrified oocytes. A highly fused fluorescence distribution of mitochondria and lysosomes was also observed. In the detection of mitophagic flux, mitophagy was demonstrated as increasing fluorescence aggregation of microtubule-associated protein light chain 3B (LC3B), enhanced colocalization between LC3B, and voltage-dependent anion channels 1 (VDAC1), and upregulated LC3B-II/I protein expression ratio. CQ inhibited the degradation of mitophagosomes in vitrified oocytes, manifested as decreased mitochondria-lysosomes colocalization, increased fluorescence fraction of VDAC1 overlapping LC3B, increased LC3B-II/I protein expression ratio, and p62 accumulation. The inhibition of mitophagosomes degradation by CQ aggravated mitochondrial dysfunction, including increased oxidative damage, reduced mitochondrial function, and further led to loss of oocyte viability and developmental potentiality. In conclusion, mitophagy is involved in the regulation of mitochondrial function during porcine oocyte vitrification.

Fumonisin B1 exposure adversely affects porcine oocyte maturation in vitro by inducing mitochondrial dysfunction and oxidative stress.

Fumonisin B1 (FB1), as the most toxic fumonisin, is a common Fusarium mycotoxin contaminant of feed stuff and food, posing a potential health hazard to animals and humans. FB1 has been reported to cause hepatotoxicity, neurotoxicity, nephrotoxicity, immunotoxicity and embryotoxicity; however, little information is available on whether FB1 has toxic effects on mammalian oocytes. Herein, we adopted porcine oocytes as models to explore the effects and potential mechanisms of FB1 on mammalian oocytes during in vitro maturation. Porcine cumulus oocyte complexes (COCs) were exposed to 0, 20, 30 and 40 µM FB1 for 44 h during in vitro maturation, and the results reported that first polar body (PB1) extrusion was significantly inhibited when the FB1 concentration reached 30 (P < 0.01) or 40 µM (P < 0.001). Further cell cycle analysis revealed that meiotic progression was disrupted, with a larger proportion of the 30 µM FB1-treated oocytes being arrested at the germinal vesicle breakdown (GVBD) stage (P < 0.01). After being treated with 30 µM FB1 for 28 h, the percentage of oocytes with aberrant spindle assembly was observably increased (P < 0.01), and the distribution of actin filaments on the plasma membrane was significantly reduced (P < 0.05). Furthermore, an observably higher rate of abnormal mitochondrial distribution (P < 0.05) and significantly decreased mitochondrial membrane potential (MMP) (P < 0.05) were observed in FB1-exposed oocytes. In addition, ROS generation in FB1-treated oocytes was rapidly increased (P < 0.05), while the transcriptional levels of antioxidant-related genes (CAT, SOD2 and GSH-Px) were sharply decreased compared with those in the control group. Additionally, the incidence of early apoptosis in FB1-treated oocytes was also significantly increased (P < 0.05), suggesting that FB1 exposure induced oxidative stress and further triggered apoptosis in porcine oocytes. Thus, these results suggested that FB1 adversely affected oocyte maturation by disturbing cell cycle progression, destroying cytoskeletal dynamics and damaging mitochondrial function, which eventually induced oxidative stress and apoptosis in porcine oocytes.

Microcystin-LR exposure results in aberrant spindles and induces apoptosis in porcine oocytes.

Microcystin-LR (MC-LR), as a well-known hepatotoxin, was recently found to accumulate in gonads and induce a variety of reproductive damages in zebrafish, mice and other model organisms, however, little information is available on whether MC-LR has toxic effects on the mammalian oocytes, especially in livestock species. In this study, the effects of MC-LR on meiotic maturation of porcine oocytes were investigated, and the potential mechanism of MC-LR toxicity was explored. Germinal vesicle (GV)-stage oocytes were exposed to 0, 20, 40 and 60 µM MC-LR, respectively, during the in vitro maturation for 44 h, and the results showed that the first polar body (PbI) extrusion rate of the oocytes decreased significantly when the MC-LR concentration reached 40 (P < 0.01) or 60 µM (P < 0.001). After treated with 60 µM MC-LR for 44 h, a significant higher percentage of the oocytes arrested at anaphase-telophase I (ATI) stage (P < 0.01). Laser scanning confocal results further confirmed that a significantly larger proportion of the 60 µM MC-LR-treated oocytes exhibited aberrant spindles and misaligned chromosomes, suggesting a failure of spindle assembly and homologous chromosome segregation during the ATI stage. Furthermore, the ROS levels in the 60 µM MC-LR-exposed oocytes were significantly higher than the control group (P < 0.01), while the expression of antioxidant related genes (SOD1, CAT and GPX) were much lower compared with control group, indicating that oxidative stress was induced and the antioxidant capacity of oocytes was depleted by 60 µM MC-LR treatment. Additionally, markedly decreased mitochondrial membrane potential (MMP) (P < 0.01) and significantly higher incidence of early apoptosis (P < 0.01) were observed in the 60 µM MC-LR-treated oocytes, suggesting that MC-LR exposure induced apoptosis in porcine oocytes. Moreover, the protein expression of PP2A was remarkably inhibited, whereas the expression of p53, BAX, Caspase3 and Cleaved-caspase3 were prominently increased in the 60 µM MC-LR-exposed oocytes. Together, these results suggested that 60 µM of MC-LR exposure can induce oxidative stress, and lead to aberrant spindles, impaired MMP, and trigger apoptosis, which eventually result in failure of porcine oocyte maturation.

Aurora A inhibition disrupts chromosome condensation and spindle assembly during the first embryonic division in pigs.

As common overexpression of Aurora A in various tumours, much attention has focused on its function in inducing cancer, and its value in cancer therapeutics, considerably less is known regarding its role in the first cleavage division of mammalian embryos. Here, we highlight an indispensable role of Aurora A during the first mitotic division progression of pig embryos just after meiosis. The expression and spatiotemporal localization of Aurora A were initially assessed in pig embryos during the first mitotic division by Western blot analysis and indirect immunofluorescent staining. Then, the potential role of Aurora A was further evaluated using a highly selective Aurora A inhibitor, MLN8054, during this mitotic progression in pig embryos. Aurora A was found to express and exhibit a specific dynamic intracellular localization pattern during the first mitotic division in pig embryos. Aurora A was diffused in the cytoplasm at the prophase stage, and then exhibited a dynamic intracellular localization which was tightly associated with the chromosome and spindle dynamics throughout subsequent mitotic phases. Inhibition of Aurora A by MLN8054 treatment led to the failure of the first cleavage, with the majority of embryos being arrested in prophase of the mitotic division. Further subcellular structure examination showed that Aurora A inhibition not only led to the failure of spindle microtubule assembly, but also resulted in severe defects in chromosome condensation, accompanied by an obvious decrease in p-TACC3(S558) expression during the prophase of the first mitosis. Together, these results illustrated that Aurora A is crucial for both spindle assembly and chromosome condensation during the first mitotic division in pig embryos, and that the regulation of Aurora A may be associated with its effects on p-TACC3(S558) expression.

Plk1 is essential for proper chromosome segregation during meiosis I/meiosis II transition in pig oocytes.

BACKGROUND: Polo-like kinase 1 (Plk1), as a characteristic regulator in meiosis, organizes multiple biological events of cell division. Although Plk1 has been implicated in various functions in somatic cell mitotic processes, considerably less is known regarding its function during the transition from metaphase I (MI) to metaphase II (MII) stage in oocyte meiotic progression. METHODS: In this study, the possible role of Plk1 during the MI-to-MII stage transition in pig oocytes was addressed. Initially, the spatiotemporal expression and subcellular localization pattern of Plk1 were revealed in pig oocytes from MI to MII stage using indirect immunofluorescence and confocal microscopy imaging techniques combined with western blot analyses. Moreover, a highly selective Plk1 inhibitor, GSK461364, was used to determine the potential role of Plk1 during this MI-to-MII transition progression. RESULTS: Upon expression, Plk1 exhibited a specific dynamic intracellular localization, and co-localization of Plk1 with α-tubulin was revealed in the meiotic spindle of pig oocyte during the transition from MI to MII stage. GSK461364 treatment significantly blocked the first polar body (pbI) emission in a dose-dependent manner and resulted in a failure of meiotic maturation, with a larger percentage of the GSK461364-treated oocytes arresting in the anaphase-telophase I (ATI) stage. Further subcellular structure examination results showed that inhibition of Plk1 with GSK461364 had no visible effect on spindle assembly but caused a significantly higher proportion of the treated oocytes to have obvious defects in homologous chromosome segregation at ATI stage. CONCLUSIONS: Thus, these results indicate that Plk1 plays an essential role during the meiosis I/meiosis II transition in porcine oocytes, and the regulation is associated with Plk1's effects on homologous chromosome segregation in the ATI stage.

Phosphorylation of histone H3 on Ser-10 by Aurora B is essential for chromosome condensation in porcine embryos during the first mitotic division.

Phosphorylation of histone H3 on Ser-10 (H3S10ph) is involved in regulating mitotic chromosome condensation and decondensation, which plays an important regulatory role during mitotic cell cycle progression in mammalian cells. However, whether H3S10ph plays a similar role in early porcine embryos during the first mitotic division remains uncertain. In this study, the subcellular localization and possible roles of H3S10ph were evaluated in the first mitotic cell cycle progression of porcine embryos using western blot, indirect immunofluorescence and barasertib (H3S10ph upstream regulator Aurora-B inhibitor) treatments. H3S10ph exhibited a dynamic localization pattern and was localized to chromosomes from prometaphase to anaphase stages. Treatment of porcine embryos with barasertib inhibited mitotic division at the prophase stage and was associated with a defect in chromosome condensation accompanied by the reduction of H3S10ph. These results indicated that H3S10ph is involved in the first mitotic division in porcine embryos through its regulatory function in chromosome condensation, which further affects porcine embryo cell cycle progression during mitotic division.

Plk1 inhibition leads to a failure of mitotic division during the first mitotic division in pig embryos.

PURPOSE: This study was conducted to examine the dynamic distribution of polo-like 1 kinase (Plk1) and the possible role it plays in first mitotic division during early porcine embryo development. METHODS: Indirect immunofluorescence and confocal microscopy imaging techniques combined with western blot analyses were used to study the dynamic expression and subcellular localization of Plk1 protein in pig parthenogenetic embryos. Finally, a selective Plk1 inhibitor, GSK461364, was used to evaluate the potential role of Plk1 during this special stage. RESULTS: The results showed that Plk1 upon expression exhibited specific dynamic intracellular localization, which closely correlated with the α-tubulin distribution during the first mitotic division. GSK461364 treatment resulted in cleavage failure, with majority of the GSK461364-treated embryos being arrested in prometaphase. Further results of the subcellular structure examination showed that GSK461364 treatment led to a significantly higher proportion of the treated embryos having abnormal spindles and misarranged chromosomes at the prometaphase stage. CONCLUSIONS: Thus, these results indicated that Plk1 is essential for porcine embryos to complete the first mitotic division. Furthermore, Plk1 regulation was associated with effects on spindle assembly and chromosome arrangement.

Aurora B inhibitor barasertib prevents meiotic maturation and subsequent embryo development in pig oocytes.

Barasertib, a highly selective Aurora B inhibitor, has been widely used in a variety of cells to investigate the role of Aurora B kinase, which has been implicated in various functions in the mitotic process. However, effects of barasertib on the meiotic maturation process are not fully understood, particularly in porcine oocyte meiotic maturation. In the present study, the effects of barasertib on the meiotic maturation and developmental competence of pig oocytes were investigated, and the possible roles of Aurora B were also evaluated in porcine oocytes undergoing meiosis. Initially, we examined the expression and subcellular localization of Aurora B using Western blot analysis and immunofluorescent staining. Aurora B was found to express and exhibit specific dynamic intracellular localization during porcine oocyte meiotic maturation. Aurora B was observed around the chromosomes after germinal vesicle breakdown. Then it was transferred to the spindle region after metaphase I stage, and was particularly concentrated at the central spindles at telophase I stage. barasertib treatment resulted in the failure of polar body extrusion in pig oocytes, with a larger percentage of barasertib-treated oocytes remaining at the pro-metaphase I stage. Additional results reported that barasertib treatment had no effect on chromosome condensation but resulted in a significantly higher percentage of the treated oocytes with aberrant spindles and misaligned chromosomes during the first meiotic division. In addition, inhibition of Aurora B with lower concentrations of barasertib during pig oocyte meiotic maturation decreased the subsequent embryo developmental competence. Thus, these results illustrate that barasertib has significant effects on porcine oocyte meiotic maturation and subsequent development through Aurora B inhibition, and this regulation is related to its effects on spindle formation and chromosome alignment during the first meiotic division in porcine oocytes.

Effects of histone acetylation status on the early development of in vitro porcine transgenic cloned embryos.

The purpose of this study was to investigate the effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on transgene expression and development of porcine transgenic cloned embryos, specifically focusing on effects derived from TSA-treated donor cells or TSA-treated reconstructed embryos. The results showed that TSA treatment on reconstructed embryos modified the acetylation status, which significantly improved the development of porcine somatic cell nuclear transfer (SCNT) embryos in vitro, but not donor cells. Furthermore, the treatment of reconstructed embryos with TSA enhanced expression of the pluripotency-related gene POU5F1 and stimulated expression of the anti-apoptotic gene BCL-2. Enhanced green fluorescent protein (EGFP) mRNA expression of every group dropped drastically from donor cells to blastocysts. Interestingly, TSA is likely to prevent a decline in EGFP expression in nuclear reprogramming of porcine SCNT embryos. However DNA hypomethylation induced by modified histone acetylation of donor cells treated with TSA was significantly more effective in increasing EGFP expression in SCNT blastocysts. In conclusion, the acetylation status of both donor cells and reconstructed embryos modified by TSA treatment increased transgene expression and improved nuclear reprogramming and the developmental potential of porcine transgenic SCNT embryos.

A possible strategy to produce pigs resistant to porcine reproductive and respiratory syndrome virus.

The purpose of this study was to enhance the production of transgenic cloned embryos with porcine reproductive and respiratory syndrome virus (PRRSV) shRNA expression cassettes. To construct transgenic vector with expression targeting against PRRSV, PRRSV shRNA expression cassettes were inserted into pEGFP-N1 and the ability of resulting recombinant plasmid pEGFP-G1 inhibiting virus replication was examined in Marc-145 cells. Results showed that PRRSV replication could be significantly inhibited by pEGFP-G1 in Marc-145 cells compared with the control. The pEGFP-G1 plasmid was used to deliver a transgene expressing EGFP and the PRRSV shRNA into porcine fetal fibroblasts (PFF). Fluorescent-positive cells were used as nuclear donors for somatic cell nuclear transfer (SCNT) to produce shRNA-EGFP transgenic cloned embryos. The effects of trichostatin A (TSA) on production of transgenic cloned embryos were investigated. Reconstructed embryos were designed into 4 groups: Donor cells of Group A were treated with 50nM TSA for 24h before SCNT. Reconstructed embryos of Group B were treated with 50nM TSA for 24h after activation. Both donor cells and reconstructed embryos in Group C were treated with TSA and Group D were the control without TSA treatment. The results showed no difference (p>0.05) in cleavage rates among the 4 groups; however, blastocyst developmental rates of Group B and C (30.9% and 42.0%, respectively) were higher than for Group A and D (21.2% and 22.1%, respectively) with Group C highest among groups (p<0.05). Interestingly, EGFP expression intensity of transgenic cloned blastocysts of Group A was the highest. Our results provide promising evidence toward a new approach for production of transgenic cloned pigs with resistance to PRRSV and possibly a wide variety of other porcine diseases.

Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro.

PURPOSE: The purpose of this study was to investigate the relationship of porcine somatic cell nuclear transfer (SCNT) embryo developmental competence with embryonic cell apoptosis and DNA methylation. METHODS: The apoptotic incidence was examined via comet assay, and the mRNA expression of genes implicated in apoptosis (Bcl-2) and DNA methylation (Dnmt1, Dnmt3a) was determined using real-time RT-PCR. RESULTS: Comet assay showed that the SCNT embryos exhibited significantly higher apoptotic rate at 2-cell stage (8.3% versus 2.1%, P<0.05), 16-cell stage (27.3% versus 19.2%, P<0.05) and morula (37.5% versus 26.9, P<0.05) compared with IVF embryos. Compared with IVF embryos, a higher Bcl-2 mRNA expression pattern was observed in SCNT embryos before the 8-cell stage and differed significantly at 2- and 4-cell stages (P<0.05). After the 16-stage, Bcl-2 mRNA expression pattern became significantly lower in SCNT group (P<0.05). The relative expression level of Dnmt1 mRNA showed a higher expression level in oocytes, then sharply decreased and started to increase slightly after the 8-cell (IVF embryos) or 16-cell stage (SCNT embryos). Dnmt1 mRNA expression in IVF embryos appeared to have been lower than that of SCNT group before 16-cell stage embryos, especially at 4- and 8-cell stages (P<0.05). Although a trend for a similar increase of Dnmt3a expression was observed in IVF and SCNT embryos after 8-cell embryos, SCNT group resulted in much higher Dnmt3a mRNA abundance compared with the IVF group, particularly after 16-cell embryos (P<0.05). CONCLUSIONS: The results showed that low efficiency of porcine SCNT technology may be associated with either embryonic apoptosis or incomplete reprogramming of donor nuclear caused by abnormal Dnmts mRNA expression.

Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes.

The purpose of this study was to determine ultrastructural and cytoskeletal changes that result from vitrification of porcine germinal vesicle- (GV-) and meiosis II- (MII-) stage oocytes. To investigate the effects of vitrification on developmental competence, oocytes were divided into three groups: fresh GV-oocytes (control), vitrified GV-oocytes, and vitrified MII-oocytes. In both GV- and MII-oocytes, vitrification resulted in a high proportion with normal morphology (92.4 vs. 94.2%, P > 0.05), while vitrified GV-oocytes yielded a higher survival rate than did vitrified MII-oocytes (56.8 vs. 41.9%, P < 0.05). In vitrified GV-oocytes, 12 of 154 oocytes underwent cleavage after fertilization in vitro, and 6 of these developed to the 8-cell stage, 3 developed to the 16-cell stage, and 3 developed into morulae. No cleavage was obtained from vitrified MII-oocytes. For ultrastructural analysis of oocytes, fresh and vitrified-warmed GV- and MII-oocytes were randomly selected for transmission electron microscopy (TEM). Results showed that vitrification caused various degrees of cryodamage in GV-oocytes. Cumulus cells of some oocytes were separated from the cumulus-oocyte complex (COC), and the zona pellucida adjacent to cumulus cells was fractured. The gap junctions between cumulus cells were ruptured, and many microvilli were disrupted or disappeared. Only homogeneous lipid droplets were observed. After vitrification, cortical granules still lined the oolemma of MII-oocytes. Only morphologically irregular, nonhomogeneous lipid droplets surrounding large vacuoles were found. To examine cytoskeletal structures, fresh and vitrified-warmed MII-oocytes were analyzed by laser-scanning confocal microscopy (LSCM); vitrified-warmed GV-oocytes were cultured for 42-44 hr before LSCM. Of 58 control oocytes, 79.5% displayed normal spindles with chromosomes aligned along the equatorial plate. In vitrified oocytes the percentage with normal spindle organization was decreased significantly in both vitrified GV-oocytes and MII-oocytes (10.1 and 12.9%, respectively, P < 0.05). The proportion of oocytes with normal distribution of F-actin was lower for vitrified GV- and MII-oocytes than for controls (16.9 and 37.2% vs. 72.3%). Results of this experiment suggest that irreversible damage to the cytoskeleton of porcine GV- and MII-oocytes after vitrification could be an important factor affecting developmental competence.