Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells.

Malignant melanoma represents a form of skin cancer characterized by a bleak prognosis and heightened resistance to traditional therapies. Quercetin has demonstrated notable anti-carcinogenic, anti-inflammatory, anti-oxidant, and pharmacological effects across various cancer types. However, the intricate relationship between quercetin's anti-cancer properties and ganglioside expression in melanoma remains incompletely understood. In this study, quercetin manifests specific anti-proliferative, anti-migratory, and cell-cycle arrest effects, inducing mitochondrial dysfunction and apoptosis in two melanoma cancer cell lines. This positions quercetin as a promising candidate for treating malignant melanoma. Moreover, our investigation indicates that quercetin significantly reduces the expression levels of ganglioside GD3 and its synthetic enzyme. Notably, this reduction is achieved through the inhibition of the FAK/paxillin/Akt signaling pathway, which plays a crucial role in cancer development. Taken together, our findings suggest that quercetin may be a potent anti-cancer drug candidate for the treatment of malignant melanoma.

Optimizing Embryo Collection for Application of CRISPR/Cas9 System and Generation of Fukutin Knockout Rat Using This Method.

Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized-egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the fukutin gene without any off-target effect detected. The production of fukutin knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats.

Deoxynivalenol leads to endoplasmic reticulum stress-mediated apoptosis via the IRE1/JNK/CHOP pathways in porcine embryos.

The cytotoxic mycotoxin deoxynivalenol (DON) reportedly has adverse effects on oocyte maturation and embryonic development in pigs. Recently, the interplay between cell apoptosis and endoplasmic reticulum (ER) stress has garnered increasing attention in embryogenesis. However, the involvement of the inositol-requiring enzyme 1 (IRE1)/c-jun N-terminal kinase (JNK)/C/EBP homologous protein (CHOP) pathways of unfolded protein response (UPR) signaling in DON-induced apoptosis in porcine embryos remains unknown. In this study, we revealed that exposure to DON (0.25 µM) substantially decreased cell viability until the blastocyst stage in porcine embryos, concomitant with initiation of cell apoptosis through the IRE1/JNK/CHOP pathways in response to ER stress. Quantitative PCR confirmed that UPR signaling-related transcription factors were upregulated in DON-treated porcine blastocysts. Western blot analysis showed that IRE1/JNK/CHOP signaling was activated in DON-exposed porcine embryos, indicating that ER stress-associated apoptosis was instigated. The ER stress inhibitor tauroursodeoxycholic acid protected against DON-induced ER stress in porcine embryos, indicating that the toxic effects of DON on early developmental competence of porcine embryos can be prevented. In conclusion, DON exposure impairs the developmental ability of porcine embryos by inducing ER stress-mediated apoptosis via IRE1/JNK/CHOP signaling.

Ochratoxin A triggers endoplasmic reticulum stress through PERK/NRF2 signaling and DNA damage during early embryonic developmental competence in pigs.

Ochratoxin A (OTA), a mycotoxin found in foods, has a deleterious effect on female reproduction owing to its endocrine-disrupting activity mediated through endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production. However, the mechanisms of OTA-induced ER stress in pig embryos during in vitro culture (IVC) are not yet fully understood. In the present study, porcine embryos were cultured for two days in an IVC medium supplemented with 0.5, 1.0, and 5.0 µM OTA, which led to an OTA-induced reduction in the developmental rate of blastocysts. The mRNA-seq transcriptome analysis revealed that the reduced blastocyst development ability of OTA-exposed porcine embryos was caused by ER stress, ultimately resulting in the accumulation of ROS and the occurrence of apoptosis. The expression levels of some UPR/PERK signaling-related genes (DDIT3, EIF2AK3, EIF2S1, NFE2L2, ATF4, EIF2A, and KEAP1) were found to differ in OTA-exposed pig embryos. OTA induces DNA damage by triggering an increase in RAD51/γ-H2AX levels and suppressing p-NRF2 activity. This effect is mediated through intracellular ROS and superoxide accumulation in the nuclei of porcine embryos. The cytotoxicity of OTA increased the activation of the PERK signal pathways (p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP) in porcine embryos, with abnormal distribution of the ER observed around the nucleus. Collectively, our findings indicate that ER stress is a major cause of decline in the development of porcine embryos exposed to OTA. Therefore, OTA exposure induces ER stress and DNA damage via oxidative stress by disrupting PERK/NRF2 signaling activity in the developmental competence of porcine embryos during IVC.

Luteolin supplementation during porcine oocyte maturation improves the developmental competence of parthenogenetic activation and cloned embryos.

Luteolin (Lut), a polyphenolic compound that belongs to the flavone subclass of flavonoids, possesses anti-inflammatory, cytoprotective, and antioxidant activities. However, little is known regarding its role in mammalian oocyte maturation. This study examined the effect of Lut supplementation during in vitro maturation (IVM) on oocyte maturation and subsequent developmental competence after somatic cell nuclear transfer (SCNT) in pigs. Lut supplementation significantly increased the proportions of complete cumulus cell expansion and metaphase II (MII) oocytes, compared with control oocytes. After parthenogenetic activation or SCNT, the developmental competence of Lut-supplemented MII oocytes was significantly enhanced, as indicated by higher rates of cleavage, blastocyst formation, expanded or hatching blastocysts, and cell survival, as well as increased cell numbers. Lut-supplemented MII oocytes exhibited significantly lower levels of reactive oxygen species and higher levels of glutathione than control MII oocytes. Lut supplementation also activated lipid metabolism, assessed according to the levels of lipid droplets, fatty acids, and ATP. The active mitochondria content and mitochondrial membrane potential were significantly increased, whereas cytochrome c and cleaved caspase-3 levels were significantly decreased, by Lut supplementation. These results suggest that Lut supplementation during IVM improves porcine oocyte maturation through the reduction of oxidative stress and mitochondria-mediated apoptosis.

Cadmium exposure impairs oocyte meiotic maturation by inducing endoplasmic reticulum stress in vitro maturation of porcine oocytes.

Cadmium (Cd) is toxic metal that can induce various diseases, such as cardiovascular, nervous, and reproductive systems. This study investigated the effect of Cd exposure on porcine oocyte maturation and the underlying mechanism. Porcine cumulus-oocyte complexes were exposed various Cd concentration and tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum (ER) stress during in vitro maturation (IVM). After IVM, we evaluated meiotic maturation, ER stress, and oocyte quality by Cd exposure. Cd exposure inhibited cumulus cell expansion and meiotic maturation, increased oocyte degeneration, and induced ER stress. The levels of spliced XBP1 and ER stress-associated transcripts, markers of ER stress, were elevated in Cd-treated cumulus-oocyte complexes and denuded oocytes during IVM. Moreover, Cd-induced ER stress impaired oocyte quality by disrupting mitochondrial function and elevating intracellular reactive oxygen species levels while decreasing ER function. Interestingly, TUDCA supplementation significantly decreased the expression of ER stress-related genes and increased the quantity of ER compared with the Cd treatment. Additionally, TUDCA was also able to rescue excessive levels of ROS and restore normal mitochondrial function. Moreover, the addition of TUDCA under Cd exposure greatly ameliorated Cd-mediated detrimental effects on meiotic maturation and oocyte quality, including cumulus cell expansion and MII rate. These findings suggest that Cd exposure during IVM impairs the meiotic maturation of oocytes by inducing of ER stress.

Rapamycin encourages the maintenance of mitochondrial dynamic balance and mitophagy activity for improving developmental competence of blastocysts in porcine embryos in vitro.

Rapamycin induces autophagosome formation and activity during oocyte maturation, improved fertilization ability of matured oocytes, and early embryonic developmental competence. However, potential changes in mitochondrial fission and mitophagy via regulation of autophagy in early porcine embryonic development have not been previously studied. Here, we investigated embryonic developmental ability and quality of porcine embryos 2 days after in vitro fertilization and following treatment with 1 and 10 nM rapamycin. As a results, 1 nM rapamycin exposure significantly improved (p < 0.05) blastocyst developmental competence compared to that in nontreated embryos (nontreated: 26.2 ± 5.7% vs. 1 nM rapamycin: 35.3 ± 5.1%). We observed autophagic (LC3B) and mitochondrial fission protein expression (dynamin-related protein-1 [DRP1] and pDRP1-Ser616) at the cleavage stage of 1 and 10 nM rapamycin-treated porcine embryos, using Western blot and immunofluorescence analyses. Interestingly, 1 nM rapamycin treatment significantly improved autophagy formation, mitochondrial activation, and mitochondrial fission protein levels (p < 0.05; p-DRP1 [Ser616]) at the cleavage stage of porcine embryos. Additionally, mitophagy was significantly increased in blastocysts treated with 1 nM rapamycin. In conclusion, our results suggest that rapamycin promotes blastocyst development ability in porcine embryos through mitochondrial fission, activation, and mitophagy in in vitro culture.

Mito-TEMPO protects preimplantation porcine embryos against mitochondrial fission-driven apoptosis through DRP1/PINK1-mediated mitophagy.

AIMS: Mdivi-1 (Md-1) is a well-known inhibitor of mitochondrial fission and mitophagy. The mitochondrial superoxide scavenger Mito-TEMPO (MT) exerts positive effects on the developmental competence of pig embryos. This study aimed to explore the adverse effects of Md-1 on developmental capacity in porcine embryos and the protective effects of MT against Md-1-induced injury. MAIN METHODS: We exposed porcine embryos to Md-1 (10 and 50 µM) for 2 days after in vitro fertilization (IVF). MT (0.1 µM) treatment was applied for 4 days after exposing embryos to Md-1. We assessed blastocyst development, DNA damage, mitochondrial superoxide production, and mitochondrial distribution using TUNEL assay, Mito-SOX, and Mito-tracker, respectively. Subsequently, the expression of PINK1, DRP1, and p-DRP1Ser616 was evaluated via immunofluorescence staining and Western blot analysis. KEY FINDINGS: Md-1 compromised the developmental competence of blastocysts. Apoptosis and mitochondrial superoxide production were significantly upregulated in 50 µM Md-1-treated embryos, accompanied by a downregulation of p-DRP1Ser616, PINK1, and LC3B levels and lower mitophagy activity at the blastocyst stage. We confirmed the protective effects of MT against the detrimental effect of Md-1 on blastocyst developmental competence, mitochondrial fission, and DRP1/PINK1-mediated mitophagy activation. Eventually, MT recovered DRP1/PINK1-mediated mitophagy and mitochondrial fission by inhibiting superoxide production in Md-1-treated embryos. SIGNIFICANCE: MT protects against detrimental effects of Md-1 on porcine embryos by suppressing superoxide production. These findings expand available scientific knowledge on improving outcomes of IVF.

Stabilization of F-Actin Cytoskeleton by Paclitaxel Improves the Blastocyst Developmental Competence through P38 MAPK Activity in Porcine Embryos.

Changes in F-actin distribution and cortical F-actin morphology are important for blastocyst developmental competence during embryogenesis. However, the effect of paclitaxel as a microtubule stabilizer on embryonic development in pigs remains unclear. We investigated the role of F-actin cytoskeleton stabilization via P38 MAPK activation using paclitaxel to improve the developmental potential of blastocysts in pigs. In this study, F-actin enrichment and adducin expression based on blastomere fragment rate and cytokinesis defects were investigated in cleaved embryos after in vitro fertilization (IVF). Adducin and adhesive junction F-actin fluorescence intensity were significantly reduced with increasing blastomere fragment rate in porcine embryos. In addition, porcine embryos were cultured with 10 and 100 nM paclitaxel for two days after IVF. Adhesive junction F-actin stabilization and p-P38 MAPK activity in embryos exposed to 10 nM paclitaxel increased significantly with blastocyst development competence. However, increased F-actin aggregation, cytokinesis defects, and over-expression of p-P38 MAPK protein by 100 nM paclitaxel exposure disrupted blastocyst development in porcine embryos. In addition, exposure to 100 nM paclitaxel increased the misaligned α-tubulin of spindle assembly and adhesive junction F-actin aggregation at the blastocyst stage, which might be caused by p-P38 protein over-expression-derived apoptosis in porcine embryos.

RPL17 Promotes Colorectal Cancer Proliferation and Stemness through ERK and NEK2/ß-catenin Signaling Pathways.

Aims: Ribosomal protein L17 (RPL17), a 60S subunit component, is up-regulated in colorectal cancer (CRC). However, its oncogenic role in CRC progression remains unexplored. Thus, we aimed to investigate the effect of RPL17 targeting on CRC in vitro and in vivo and whether RPL17 gained an extra-ribosomal function during CRC development. Methods: RPL17-specific siRNAs complexed with cationic lipids were transfected to CRC cells to silence target gene expression and then real-time RT-PCR and western blotting were applied to observe the change of expression or activity of genes or proteins of interest. Cell proliferation assay, clonogenic assay and cell cycle analysis were used to determine the in vitro effects of RPL17siRNAs on CRC cell growth, and a subcutaneous xenograft assay was applied to test the effect of RPL17siRNAs on in vivo tumor growth. RNA sequencing and western blotting were used to investigate the underlying mechanisms. Sphere-forming assay, invasion assay and migration assay were used to evaluate the effects of RPL17siRNAs on CRC stemness. Results: siRNA-mediated inhibition of RPL17 expression suppressed CRC cell growth and long-term colony formation by inducing apoptotic cell death. Similarly, targeting RPL17 effectively suppressed tumor formation in a mouse xenograft model. RNA sequencing of RPL17-silenced CRC cells revealed the same directional regulation of 159 (93 down- and 66 up-regulated) genes. Notably, NIMA-related kinase 2 (NEK2), which functionally cooperates with extracellular-regulated protein kinase (ERK) and plays a pivotal role in mitotic progression and stemness maintenance, was down-regulated. RPL17 silencing reduced NEK2, ß-catenin, and p-ERK protein levels. These molecular alterations reflected the reduction in sphere-forming capacity, expression of stem cell marker genes, migration, and invasion. Reversely, RPL17 overexpression increased the ability of long-term colony formation, migration, and invasion. Conclusion: Our findings indicate that RPL17 promotes CRC proliferation and stemness via the ERK and NEK2/ß-catenin signaling axis, and targeting RPL17 could be the next molecular strategy for both primary CRC treatment and prevention of secondary tumor formation.

SESN2/NRF2 signaling activates as a direct downstream regulator of the PERK pathway against endoplasmic reticulum stress to improve the in vitro maturation of porcine oocytes.

Nuclear erythroid 2-related factor 2 (NRF2) is a critical regulator of oxidative stress in mammalian oocytes. Our previous study described the protective effects of Sestrin-2 (SESN2) as a stress regulator against endoplasmic reticulum (ER) stress in porcine oocytes during in vitro maturation (IVM). However, their roles in unfolded protein response-related signaling pathways in porcine oocyte maturation capacity remain unknown. The purpose of this study was to evaluate the role of SESN2/NRF2 signaling in H2O2-induced oxidative stress and ER stress via protein kinase-like ER kinase (PERK) downstream factor during porcine oocyte maturation. Here, we found that the p-NRF2(Ser40) activation in the nucleus of porcine oocytes was accompanied by PERK signaling downregulation using western blot and immunofluorescence staining at 44 h after IVM. The total and nuclear NRF2 protein expression was also induced in porcine oocytes following H2O2 and tunicamycin (Tm) exposure. Notably, the upregulation of PERK signaling significantly increased the SESN2 and NRF2 signaling in H2O2-and Tm-exposed porcine cumulus oocyte complexes. Interestingly, inducing the knockdown of the SESN2 gene expression by siRNA interrupted the NRF2 signaling activation of porcine oocyte maturation, whereas NRF2 expression blockade by ochratoxin A, an NRF2 inhibitor, did not affect the expression level of the SESN2 protein. Moreover, a defect in SESN2 completely blocked the activity of nuclear NRF2 on spindle assembly in porcine oocytes. These findings suggest that the PERK/SESN2/NRF2 signaling pathway may play an important role against ER stress during meiotic maturation and oocyte maturation capacity.

Combined Chaetocin/Trichostatin A Treatment Improves the Epigenetic Modification and Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos.

Developmental defects in somatic cell nuclear transfer (SCNT) embryos are principally attributable to incomplete epigenetic reprogramming. Small-molecule inhibitors such as histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) have been used to improve reprogramming efficiency of SCNT embryos. However, their possible synergistic effect on epigenetic reprogramming has not been studied. In this study, we explored whether combined treatment with an HMTi (chaetocin) and an HDACi (trichostatin A; TSA) synergistically enhanced epigenetic reprogramming and the developmental competence of porcine SCNT embryos. Chaetocin, TSA, and the combination significantly increased the cleavage and blastocyst formation rate, hatching/hatched blastocyst rate, and cell numbers and survival rate compared to control embryos. In particular, the combined treatment improved the rate of development to blastocysts more so than chaetocin or TSA alone. TSA and combined chaetocin/TSA significantly reduced the H3K9me3 levels and increased the H3K9ac levels in SCNT embryos, although chaetocin alone significantly reduced only the H3K9me3 levels. Moreover, these inhibitors also decreased global DNA methylation in SCNT embryos. In addition, the expression of zygotic genome activation- and imprinting-related genes was increased by chaetocin or TSA, and more so by the combination, to levels similar to those of in vitro-fertilized embryos. These results suggest that combined chaetocin/TSA have synergistic effects on improving the developmental competences by regulating epigenetic reprogramming and correcting developmental potential-related gene expression in porcine SCNT embryos. Therefore, these strategies may contribute to the generation of transgenic pigs for biomedical research.

Peroxiredoxin 1 Controls Ovulation and Ovulated Cumulus-Oocyte Complex Activity through TLR4-Derived ERK1/2 Signaling in Mice.

Peroxiredoxins (PRDXs) are expressed in the ovary and during ovulation. PRDX1 activity related to the immuno-like response during ovulation is unknown. We investigated the roles of Prdx1 on TLR4 and ERK1/2 signaling from the ovulated cumulus-oocyte complex (COC) using Prdx1-knockout (K/O) and wild-type (WT) mice. Ovulated COCs were collected 12 and 16 h after pregnant mare serum gonadotropin/hCG injection. PRDX1 protein expression and COC secretion factors (Il-6, Tnfaip6, and Ptgs2) increased 16 h after ovulated COCs of the WT mice were obtained. We treated the ovulated COCs in mice with LPS (0.5 µg/mL) or hyaluronidase (Hya) (10 units/mL) to induce TLR4 activity. Intracellular reactive oxygen species (ROS), cumulus cell apoptosis, PRDX1, TLR4/P38/ERK1/2 protein expression, and COC secretion factors' mRNA levels increased in LPS- and Hya-treated COCs. The ERK inhibitor (U0126) and Prdx1 siRNA affected TLR4/ERK1/2 expression. The number and cumulus expansion of ovulated COCs by ROS were impaired in Prdx1 K/O mice but not in WT ones. Prdx1 gene deletion induced TLR4/P38/ERK1/2 expression and cumulus expansion genes. These results show the controlling roles of PRDX1 for TLR4/P38/ERK1/2 signaling activity in ovulated mice and the interlink of COCs with ovulation.

Melatonin Protects Against Mdivi-1-Induced Abnormal Spindle Assembly and Mitochondrial Superoxide Production During Porcine Oocyte Maturation.

Mitochondrial division inhibitor 1 (Mdivi-1) reportedly provides a close connection between oocyte maturation and mitochondrial function in pigs. N-acetyl-5-methoxy-tryptamine (melatonin) is known to be a representative antioxidant with the ability to rehabilitate meiotic maturation of porcine oocytes. However, the ability of melatonin to recover Mdivi-1-mediated disruption of spindle formation during meiotic maturation of porcine oocytes during in vitro maturation (IVM) has not been studied. Here, we first investigated changes in mitochondrial length, such as fragmentation and elongation form, in mature porcine oocytes during IVM. Mature oocytes require appropriate mitochondrial fission for porcine oocyte maturation. We identified a dose-dependent reduction in meiotic maturation in porcine oocytes following Mdivi-1 treatment (50, 75, and 100 µM). We also confirmed changes in mitochondrial fission protein levels [dynamin-related protein 1 phosphorylation at serine 616 (pDRP1-Ser616) and dynamin-related protein 1 (DRP1)], mitochondrial membrane potential, and ATP production in 75 µM Mdivi-1-treated oocytes. As expected, Mdivi-1 significantly reduced mitochondrial function and DRP1 protein levels and increased spindle abnormalities in porcine oocytes. In addition, we confirmed that melatonin restores abnormal spindle assembly and reduces meiotic maturation rates by Mdivi-1 during porcine oocyte maturation. Interestingly, the expression levels of genes that reduce DNA damage and improve tubulin formation were enhanced during porcine meiotic maturation. Taken together, these results suggest that melatonin has direct beneficial effects on meiotic maturation through tubulin formation factors during porcine oocyte maturation.

Induction of autophagy protects against extreme hypoxia-induced damage in porcine embryo.

In the mammalian female reproductive tract, physiological oxygen tension is lower than that of the atmosphere. Therefore, to mimic in vivo conditions during in vitro culture (IVC) of mammalian early embryos, 5% oxygen has been extensively used instead of 20%. However, the potential effect of hypoxia on the yield of early embryos with high developmental competence remains unknown or controversial, especially in pigs. In the present study, we examined the effects of low oxygen tension under different oxygen tension levels on early developmental competence of parthenogenetically activated (PA) and in vitro-fertilized (IVF) porcine embryos. Unlike the 5% and 20% oxygen groups, exposure of PA embryos to 1% oxygen tension, especially in early-phase IVC (0-2 days), greatly decreased several developmental competence parameters including blastocyst formation rate, blastocyst size, total cell number, inner cell mass (ICM) to trophectoderm (TE) ratio, and cellular survival rate. In contrast, 1% oxygen tension did not affect developmental parameters during the middle (2-4 days) and late phases (4-6 days) of IVC. Interestingly, induction of autophagy by rapamycin treatment markedly restored the developmental parameters of PA and IVF embryos cultured with 1% oxygen tension during early-phase IVC, to meet the levels of the other groups. Together, these results suggest that the early development of porcine embryos depends on crosstalk between oxygen tension and autophagy. Future studies of this relationship should explore the developmental events governing early embryonic development to produce embryos with high developmental competence in vitro.

Effect of evaporation-induced osmotic changes in culture media in a dry-type incubator on clinical outcomes in in vitro fertilization-embryo transfer cycles.

OBJECTIVE: This study investigated whether adding outer-well medium to inhibit osmotic changes in culture media in a dry-type incubator improved the clinical outcomes of in vitro fertilization-embryo transfer (IVF-ET) cycles. METHODS: In culture dishes, the osmotic changes in media (20 µL)-covered oil with or without outer-well medium (humid or dry culture conditions, respectively) were compared after 3 days of incubation in a dry-type incubator. One-step (Origio) and G1/G2 (Vitrolife) media were used. RESULTS: The osmotic changes in the dry culture condition (308 mOsm) were higher than in the humid culture conditions (285-290 mOsm) after 3 days of incubation. In day 3 IVF-ET cycles, although the pregnancy rate did not significantly differ between the dry (46.2%) and humid culture (52.2%) groups, the rates of abortion and ongoing pregnancy were significantly better in the humid culture group (2.3% and 50.2%, respectively) than in the dry culture group (8.3% and 37.8%, respectively, p<0.05). In day 5 IVF-ET cycles, the abortion rate was significantly lower in the humid culture group (2.2%) than in the dry culture group (25.0%, p<0.01), but no statistically significant difference was observed in the rates of clinical and ongoing pregnancy between the dry (50.0% and 25.0%, respectively) and humid culture groups (59.5% and 57.3%, respectively) because of the small number of cycles. CONCLUSION: Hyperosmotic changes in media occurred in a dry-type incubator by evaporation, although the medium was covered with oil. These osmotic changes were efficiently inhibited by supplementation of outer-well medium, which resulted in improved pregnancy outcomes.

Effect of Triclosan Exposure on Developmental Competence in Parthenogenetic Porcine Embryo during Preimplantation.

Triclosan (TCS) is included in various healthcare products because of its antimicrobial activity; therefore, many humans are exposed to TCS daily. While detrimental effects of TCS exposure have been reported in various species and cell types, the effects of TCS exposure on early embryonic development are largely unknown. The aim of this study was to determine if TCS exerts toxic effects during early embryonic development using porcine parthenogenetic embryos in vitro. Porcine parthenogenetic embryos were cultured in in vitro culture medium with 50 or 100 µM TCS for 6 days. Developmental parameters including cleavage and blastocyst formation rates, developmental kinetics, and the number of blastomeres were assessed. To determine the toxic effects of TCS, apoptosis, oxidative stress, and mitochondrial dysfunction were assessed. TCS exposure resulted in a significant decrease in 2-cell rate and blastocyst formation rate, as well as number of blastomeres, but not in the cleavage rate. TCS also increased the number of apoptotic blastomeres and the production of reactive oxygen species. Finally, TCS treatment resulted in a diffuse distribution of mitochondria and decreased the mitochondrial membrane potential. Our results showed that TCS exposure impaired porcine early embryonic development by inducing DNA damage, oxidative stress, and mitochondrial dysfunction.

Chaetocin Improves Pig Cloning Efficiency by Enhancing Epigenetic Reprogramming and Autophagic Activity.

Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation (H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases (suv39h1 and suv39h2) and DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) were higher in SCNT embryos. Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.

Exposure of Triclosan in Porcine Oocyte Leads to Superoxide Production and Mitochondrial-Mediated Apoptosis During In Vitro Maturation.

While triclosan (TCS) exerts detrimental effects on female reproduction, the effect of TCS-derived toxins on porcine oocytes during in vitro maturation (IVM) is unclear. This study investigated the effects of TCS on mitochondrion-derived reactive oxygen species (ROS) production and apoptosis pathways during porcine oocyte maturation. Porcine oocytes were treated with TCS (1, 10, and 100 µM) and triphenylphosphonium chloride (Mito-TEMPO; 0.1 µM), and matured cumulus oocyte complexes (COCs) were stained with orcein, dichlorofluorescein diacetate (DCF-DA), and Mito-SOX. Proteins and mRNA levels of factors related to cumulus expansion and mitochondrion-mediated apoptosis and antioxidant enzymes were analyzed by western blotting and reverse-transcription polymerase chain reaction (RT-PCR), respectively. Meiotic maturation and cumulus cell expansion significantly decreased for COCs after TCS treatment along with an increase in mitochondrial superoxide levels at 44 h of IVM. Further, mitochondrion-related antioxidant enzymes and apoptosis markers were significantly elevated in porcine COCs following TCS-mediated oxidative damage. The protective effect of Mito-TEMPO as a specific superoxide scavenger from TCS toxin improved the maturation capacity of porcine COCs. Mito-TEMPO downregulated the mitochondrial apoptosis of TCS-exposed porcine COCs by reducing superoxide level. In conclusion, our data demonstrate that TCS mediates toxicity during porcine oocyte maturation through superoxide production and mitochondrion-mediated apoptosis.

Butylparaben Is Toxic to Porcine Oocyte Maturation and Subsequent Embryonic Development Following In Vitro Fertilization.

Parabens are widely used in personal care products due to their antimicrobial effects. Although the toxicity of parabens has been reported, little information is available on the toxicity of butylparaben (BP) on oocyte maturation. Therefore, we investigated the effects of various concentrations of BP (0 µM, 100 µM, 200 µM, 300 µM, 400 µM, and 500 µM) on the in vitro maturation of porcine oocytes. BP supplementation at a concentration greater than 300 µM significantly reduced the proportion of complete cumulus cell expansion and metaphase II oocytes compared to the control. The 300 µM BP significantly decreased fertilization, cleavage, and blastocyst formation rates with lower total cell numbers and a higher rate of apoptosis in blastocysts compared to the control. The BP-treated oocytes showed significantly higher reactive oxygen species (ROS) levels, and lower glutathione (GSH) levels than the control. BP significantly increased the aberrant mitochondrial distribution and decreased mitochondrial function compared to the control. BP-treated oocytes exhibited significantly higher percentage of γ-H2AX, annexin V-positive oocytes and expression of LC3 than the control. In conclusion, we demonstrated that BP impaired oocyte maturation and subsequent embryonic development, by inducing ROS generation and reducing GSH levels. Furthermore, BP disrupted mitochondrial function and triggered DNA damage, early apoptosis, and autophagy in oocytes.