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.

Transient meiotic arrest maintained by DON (6-diazo-5-oxo-l-norleucine) enhances nuclear/cytoplasmic maturation of porcine oocytes.

The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.

2-Hydroxy-3-methoxybenzoic acid attenuates mast cell-mediated allergic reaction in mice via modulation of the FcεRI signaling pathway.

Mast cells are important effector cells in immunoglobulin (Ig) E-mediated allergic reactions such as asthma, atopic dermatitis and rhinitis. Vanillic acid, a natural product, has shown anti-oxidant and anti-inflammatory activities. In the present study, we investigated the anti-allergic inflammatory effects of ortho-vanillic acid (2-hydroxy-3-methoxybenzoic acid, o-VA) that was a derivative of vanillic acid isolated from Amomum xanthioides. In mouse anaphylaxis models, oral administration of o-VA (2, 10, 50 mg/kg) dose-dependently attenuated ovalbumin-induced active systemic anaphylaxis and IgE-mediated cutaneous allergic reactions such as hypothermia, histamine release, IgE production and vasodilation; administration of o-VA also suppressed the mast cell degranulator compound 48/80-induced anaphylaxis. In cultured mast cell line RBL-2H3 and isolated rat peritoneal mast cells in vitro, pretreatment with o-VA (1-100 µmol/L) dose-dependently inhibited DNP-HSA-induced degranulation of mast cells by decreasing the intracellular free calcium level, and suppressed the expression of pro-inflammatory cytokines TNF-α and IL-4. Pretreatment of RBL-2H3 cells with o-VA suppressed DNP-HSA-induced phosphorylation of Lyn, Syk, Akt, and the nuclear translocation of nuclear factor-κB. In conclusion, o-VA suppresses the mast cell-mediated allergic inflammatory response by blocking the signaling pathways downstream of high affinity IgE receptor (FcεRI) on the surface of mast cells.

Iloprost supports early development of in vitro-produced porcine embryos through activation of the phosphatidylinositol 3-kinase/AKT signalling pathway.

Despite evidence of the presence of prostaglandin (PG) I2 in mammalian oviducts, its role in early development of in vitro-produced (IVP) embryos is largely unknown. Thus, in the present study we examined the effects of iloprost, a PGI2 analogue, on the in vitro developmental competence of early porcine embryos and the underlying mechanism(s). To examine the effects of iloprost on the development rate of IVF embryos, iloprost was added to the in vitro culture (IVC) medium and cultured for 6 days. Supplementation of the IVC medium with iloprost significantly improved developmental parameters, such as blastocyst formation rate, the trophectoderm:inner cell mass ratio and cell survival in IVF and parthenogenetically activated (PA) embryos. In addition, post-blastulation development into the expanded blastocyst stage was improved in iloprost-treated groups compared with controls. Interestingly, the phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway was significantly activated by iloprost supplementation in a concentration-dependent manner (10-1000nM), and the beneficial effects of iloprost on the early development of porcine IVF and PA embryos was completely ablated by treatment with 2.5µM wortmannin, a PI3K/AKT signalling inhibitor. Importantly, expression of the PI3K/AKT signalling pathway was significantly reduced in somatic cell nuclear transfer (SCNT) compared with IVF embryos, and iloprost supported the early development of SCNT embryos, as was the case for IVF and PA embryos, suggesting a consistent effect of iloprost on the IVC of IVP porcine embryos. Together, these results indicate that iloprost can be a useful IVC supplement for production of IVP early porcine embryos with high developmental competence.

Superovulatory responses in cynomolgus monkeys (Macaca fascicularis) depend on the interaction between donor status and superovulation method used.

The current study was performed to investigate the effect of oocyte donor status, including age and body weight, on metaphase II (MII) oocyte recovery using two superovulation methods in cynomolgus monkeys. The use of Method A [recombinant gonadotrophin (75 IU/kg, 3 ×, 3-day intervals) and human chorionic gonadotropin (hCG)] led to great increases in ovary size and the mean number of MII oocytes retrieved in age- and body-weight-dependent manner; in contrast, both the parameters were similar in Method B [recombinant gonadotrophin (60 IU, twice daily, 6 days), recombinant gonadotropin and recombinant human luteinizing hormone (rhLH) (60 IU, twice daily, 3 days), and hCG]. Importantly, Method A showed maximal MII oocyte recovery rate in > 60-month-old or 4.5-5.0-kg female monkeys, whereas Method B was equally effective regardless of the donor age and body weight. These results indicate that superovulatory responses depend on the interaction between oocyte donor status and the superovulation method used in cynomolgus monkeys.

Native plants (Phellodendron amurense and Humulus japonicus) extracts act as antioxidants to support developmental competence of bovine blastocysts.

OBJECTIVE: Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. METHODS: After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense (0.01 µg/mL) and H. japonicus (0.01 µg/mL). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. RESULTS: We observed that the blastocysts rate was significantly increased (p<0.05) in P. amurense (28.9%±2.9%), H. japonicus (30.9%±1.5%), and a mixture of P. amurense and H. japonicus (34.8%± 2.1%) treated groups compared with the control group (25.4%±1.6%). We next confirmed that the intracellular levels of reactive oxygen species (ROS) were significantly decreased (p<0.01) in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05) in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos. CONCLUSION: These results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

Developmental competence of bovine early embryos depends on the coupled response between oxidative and endoplasmic reticulum stress.

The stress produced by the coupling of reactive oxygen species (ROS) and endoplasmic reticulum (ER) has been explored extensively, but little is known regarding their roles in the early development of mammalian embryos. Here, we demonstrated that the early development of in vitro-produced (IVP) bovine embryos was governed by the cooperative action between ROS and ER stress. Compared with the tension produced by 5% O2, 20% O2 significantly decreased the blastocyst formation rate and cell survival, which was accompanied by increases in ROS and in levels of sXBP-1 transcript, which is an ER stress indicator. In addition, treatment with glutathione (GSH), a ROS scavenger, decreased ROS levels, which resulted in increased blastocyst formation and cell survival rates. Importantly, levels of sXBP-1 and ER stress-associated transcripts were reduced by GSH treatment in developing bovine embryos. Consistent with this observation, tauroursodeoxycholate (TUDCA), an ER stress inhibitor, improved blastocyst developmental rate, trophectoderm proportion, and cell survival. Moreover, ROS and sXBP-1 transcript levels were markedly decreased by supplementation with TUDCA, suggesting a possible mechanism governing the mutual regulation between ROS and ER stress. Interestingly, knockdown of XBP-1 transcripts resulted in both elevation of ROS and decrease of antioxidant transcripts, which ultimately reduced in vitro developmental competence of bovine embryos. Based on these results, in vitro developmental competence of IVP bovine embryos was highly dependent on the coupled response between oxidative and ER stresses. These results increase our understanding of the mechanism(s) governing early embryonic development and may improve strategies for the generation of IVP embryos with high developmental competence.

Valproic acid enhances early development of bovine somatic cell nuclear transfer embryos by alleviating endoplasmic reticulum stress.

Despite the positive roles of histone deacetylase inhibitors in somatic cell nuclear transfer (SCNT), few studies have evaluated valproic acid (VPA) and its associated developmental events. Thus, the present study was conducted to elucidate the effect of VPA on the early development of bovine SCNT embryos and the underlying mechanisms of action. The histone acetylation level of SCNT embryos was successfully restored by VPA, with optimal results obtained by treatment with 3mM VPA for 24h. Importantly, the increases in blastocyst formation rate and inner cell mass and trophectoderm cell numbers were not different between the VPA and trichostatin A treatment groups, whereas cell survival was notably improved by VPA, indicating the improvement of developmental competence of SCNT embryos by VPA. Interestingly, VPA markedly reduced the transcript levels of endoplasmic reticulum (ER) stress markers, including sXBP-1 and CHOP. In contrast, the levels of GRP78/BiP, an ER stress-alleviating transcript, were significantly increased by VPA. Furthermore, VPA greatly reduced cell apoptosis in SCNT blastocysts, which was further evidenced by the increased levels of the anti-apoptotic transcript Bcl-xL and decreased level of the pro-apoptotic transcript Bax. Collectively, these results suggest that VPA enhances the developmental competence of bovine SCNT embryos by alleviating ER stress and its associated developmental damage.

Induction of autophagy during in vitro maturation improves the nuclear and cytoplasmic maturation of porcine oocytes.

While a critical role of autophagy in mammalian early embryogenesis has been demonstrated, few studies have been conducted regarding the role of autophagy in in vitro maturation (IVM) of immature oocytes. In the present study we investigated the effect of rapamycin, a chemical autophagy inducer, on the nuclear and cytoplasmic maturation of porcine oocytes. Rapamycin treatment led to increased expression of LC3-II, an autophagy marker. Compared with the control group, as well as the 5 and 10nM rapamycin treatment groups, the rate of MII oocyte production was higher in the 1nM rapamycin treatment group, indicating improvement in nuclear maturation. In the analyses of cytoplasmic maturation, we found that the level of p34(cdc2), a cytoplasmic maturation marker, and the monospermic fertilisation rate were higher in the 1nM rapamycin treatment group than in the other groups. Moreover, the beneficial effect of 1nM rapamycin on cytoplasmic maturation of MII oocytes was further evidenced by increases in blastocyst formation rate, total cell number and cell survival. In the blastocyst embryos, anti-apoptotic Bcl-xL transcript levels were elevated in the 1nM rapamycin-treated group, whereas pro-apoptotic Bax transcript levels were decreased. Collectively, these results suggest that induction of autophagy during IVM contributes to enhancement of the nuclear and cytoplasmic maturation of porcine oocytes.

Application of ultrasonographic human estimated foetal weight formulas to cynomolgus monkeys (Macaca fascicularis) at 129-132 days of gestation: A comparative study of estimated and actual birthweight.

BACKGROUND: Cynomolgus monkeys (Macaca fascicularis) are essential in biomedical research, including reproductive studies. However, the application of human estimated foetal weight (EFW) formulas using ultrasonography (USG) in these non-human primates is not well established. OBJECTIVES: This study aims to evaluate the applicability of human EFW formulas for estimating foetal weight in cynomolgus monkeys at approximately 130 days of gestation. METHODS: Our study involved nine pregnant cynomolgus monkeys. We measured foetal parameters, including biparietal diameter, head circumference, abdominal circumference and femur length using USG. The EFW was calculated using 11 human EFW formulas. The actual birthweight (ABW) was recorded following Cesarean section, the day after the EFW calculation. For comparing EFW and ABW, we employed statistical methods such as mean absolute percentage error (APE) and Bland-Altman analysis. RESULTS: The ABW ranged between 200.36 and 291.33 g. Among the 11 formulas, the Combs formula showed the lowest APE (4.3%) and highest correlation with ABW (p < 0.001). Notably, EFW and ABW differences for the Combs formula were ≤5% in 66.7% and ≤10% in 100% of cases. The Bland-Altman analysis supported these results, showing that all cases fell within the limits of agreement. CONCLUSIONS: The Combs formula is applicable for estimating the weight of cynomolgus monkey fetuses with USG at approximately 130 days of gestation. Our observations suggest that the Combs formula can be applied in the prenatal care and biomedical research of this species.

Subcutaneous leiomyosarcoma in a cynomolgus macaque (Macaca fascicularis).

Leiomyosarcoma, a malignant tumour originating from smooth muscle cells, has rarely been documented in non-human primates. In this case study, a 7-year-old female cynomolgus macaque (Macaca fascicularis) presented with a rapidly growing mass overlying the left elbow joint. Radiographs indicated the presence of a soft tissue neoplasm without any associated bone involvement. The mass was surgically resected. Histological and immunohistochemical analyses revealed spindle-shaped cells with eosinophilic cytoplasm that resembled smooth muscle cells, exhibiting positive immunoreactions for vimentin, desmin and smooth muscle actin and a negative reaction for pan-cytokeratin. This is the first reported case of subcutaneous leiomyosarcoma in a cynomolgus macaque and provides important insights into the incidence and characteristics of this condition in this species.

Comparative analysis of superovulated versus uterine-embryo synchronized recipients for embryo transfer in cynomolgus monkeys (Macaca fascicularis).

Introduction: Assisted reproductive technologies (ARTs), such as intracytoplasmic sperm injection and embryo transfer, are essential for generating genetically edited monkeys. Despite their importance, ARTs face challenges in recipient selection in terms of time and the number of animals required. The potential of superovulated monkeys, commonly used as oocyte donors, to serve as surrogate mothers, remains underexplored. The study aimed to compare the efficacy of superovulated and uterine-embryo synchronized recipients of embryo transfer in cynomolgus monkeys (Macaca fascicularis). Methods: This study involved 23 cynomolgus monkeys divided into two groups-12 superovulated recipients and 11 synchronized recipients. The evaluation criteria included measuring endometrial thickness on the day of embryo transfer and calculating pregnancy and implantation rates to compare outcomes between groups. Results: The study found no statistically significant differences in endometrial thickness (superovulated: 4.48 ± 1.36 mm, synchronized: 5.15 ± 1.58 mm), pregnancy rates (superovulated: 30.8%, synchronized: 41.7%), and implantation rates (superovulated: 14.3%, synchronized: 21.9%) between the groups (p > 0.05). Conclusion: The observations indicate that superovulated recipients are as effective as synchronized recipients for embryo transfer in cynomolgus monkeys. This suggests that superovulated recipients can serve as viable options, offering an efficient and practical approach to facilitate the generation of gene-edited models in this species.

Disruption of early embryonic development in mice by polymethylmethacrylate nanoplastics in an oxidative stress mechanism.

Polymethylmethacrylate (PMMA) has been used in many products, such as acrylic glass, and is estimated to reach 5.7 million tons of production per year by 2028. Thus, nano-sized PMMA particles in the environment are highly likely due to the weathering process. However, information on the hazards of nanoplastics, including PMMA in mammals, especially reproductive toxicity and action mechanism, is scarce. Herein, we investigated the effect of PMMA nanoplastics on the female reproductive system of mice embryos during pre-implantation. The treated plastic particles in embryos (10, 100, and 1000 µg/mL) were endocytosed into the cytoplasm within 30 min, and the blastocyst development and indices of embryo quality were significantly decreased from at 100 µg/mL. Likewise, the transfer of nanoplastic-treated embryos at 100 µg/mL decreased the morula implantation rate on the oviduct of pseudopregnant mice by 70%, calculated by the pregnant individual, and 31.8% by the number of implanted embryos. The PMMA nanoplastics at 100 µg/mL significantly increased the cellular levels of reactive oxygen species in embryos, which was not related to the intrinsic oxidative potential of nanoplastics. This study highlights that the nanoplastics that enter systemic circulation can affect the early stage of embryos. Thus, suitable action mechanisms can be designed to address nanoplastic occurrence.

Efficient production of transgenic mice by intracytoplasmic injection of streptolysin-O-treated spermatozoa.

Many methods for efficient production of transgenic animals for biomedical research have been developed. Despite great improvements in transgenesis rates resulting from the use of intracytoplasmic sperm injection (ICSI), the ICSI-based sperm-mediated gene-transfer (iSMGT) technique is still not optimal in terms of sperm permeabilization efficiency and subsequent development. Here, we demonstrate that streptolysin-O (SLO) can efficiently permeabilize mouse spermatozoa, leading to improved developmental competence and high transgenesis rates in iSMGT embryos and pups. In particular, the most efficient production of iSMGT-transgenic embryos resulted from pretreatment with 5 U/ml SLO for 30 min and co-incubation with 1.0 ng/µl of an EGFP expression vector. By incubating spermatozoa with Cy-3-labelled DNA, we found that fluorescence intensity was prominently detected in the head region of SLO-treated spermatozoa. In addition, blastocyst development rate and blastomere survival were greatly improved by iSMGT using SLO-treated spermatozoa (iSMGT-SLO) as compared to freeze-thawed spermatozoa. Consistent with this, a high proportion of transgenic offspring was obtained by iSMGT-SLO after transfer into foster mothers, reaching 10.6% of the number of oocytes used (42.3% among pups). Together with successful germline transmission of transgenes in all founders analyzed, our data strongly suggest that SLO makes spermatozoa amenable to exogenous DNA uptake, and that the iSMGT-SLO technique is an efficient method for production of transgenic animals for biomedical research.

Particulate matter 10 induces oxidative stress and apoptosis in rhesus macaques skin fibroblast.

Background: Particulate matter (PM) is a major air pollutant that affects human health worldwide. PM can pass through the skin barrier, thus causing skin diseases such as heat rash, allergic reaction, infection, or inflammation. However, only a few studies have been conducted on the cytotoxic effects of PM exposure on large-scale animals. Therefore, herein, we investigated whether and how PM affects rhesus macaque skin fibroblasts. Methods: Rhesus macaque skin fibroblasts were treated with various concentrations of PM10 (1, 5, 10, 50, and 100 µg/mL) and incubated for 24, 48, and 72 h. Then, cell viability assay, TUNEL assay, and qRT-PCR were performed on the treated cells. Further, the reactive oxygen species, glutathione, and cathepsin B levels were determined. The MTT assay revealed that PM10 (>50 µg/mL) proportionately reduced the cell proliferation rate. Results: PM10 treatment increased TUNEL-positive cell numbers, following the pro-apoptosis-associated genes (CASP3 and BAX) and tumor suppressor gene TP53 were significantly upregulated. PM10 treatment induced reactive oxidative stress. Cathepsin B intensity was increased, whereas GSH intensity was decreased. The mRNA expression levels of antioxidant enzyme-related genes (CAT, GPX1 and GPX3) were significantly upregulated. Furthermore, PM10 reduced the mitochondrial membrane potential. The mRNA expression of mitochondrial complex genes, such as NDUFA1, NDUFA2, NDUFAC2, NDUFS4, and ATP5H were also significantly upregulated. In conclusion, these results showed that PM10 triggers apoptosis and mitochondrial damage, thus inducing ROS accumulation. These findings provide potential information on the cytotoxic effects of PM10 treatment and help to understand the mechanism of air pollution-induced skin diseases.

Induction of autophagy promotes preattachment development of bovine embryos by reducing endoplasmic reticulum stress.

The coupling of autophagy and endoplasmic reticulum (ER) stress has been implicated in a variety of biological processes; however, little is known regarding the involvement of the autophagy/ER stress pathway in early embryogenesis or the underlying mechanism(s). Here, we showed that the developmental competence of in vitro-produced (IVP) bovine embryos was highly dependent on the autophagy/ER stress balance. Although relative abundances of autophagy-associated gene transcripts, including LC3, Atg5, and Atg7 transcripts, were high in oocytes and throughout the early stages of preattachment development, extensive autophagosome formation was only detected in fertilized embryos. Using an inducer and inhibitor of autophagy, we showed that transient elevation of autophagic activity during early preattachment development greatly increased the blastocyst development rate, trophectoderm cell numbers, and blastomere survival; these same parameters were reduced by both inhibition and prolonged induction of autophagy. Interestingly, the induction of autophagy reduced ER stress and associated damage, while the developmental defects in autophagy-inhibited embryos were significantly alleviated by ER stress inhibitor treatment, indicating that autophagy is a negative regulator of ER stress in early embryos. Collectively, these results suggest that early embryogenesis of IVP bovine embryos depends on an appropriate balance between autophagy and ER stress. These findings may increase our understanding of important early developmental events by providing compelling evidence concerning the tight association between autophagy and ER stress, and may contribute to the development of strategies for the production of IVP bovine blastocysts with high developmental competence.

Inactivated Sendai-virus-mediated fusion improves early development of cloned bovine embryos by avoiding endoplasmic-reticulum-stress-associated apoptosis.

Somatic cell nuclear transfer (SCNT) is a powerful tool, not only for producing cloned animals, but also in revealing various early developmental events. However, relatively little is known regarding the biological events and underlying mechanism(s) directly associated with early development of SCNT embryos. Here, we show that production of high-quality bovine SCNT blastocysts is dependent on the method used for fusion and the associated reduction in endoplasmic reticulum (ER) stress. During fusion between the donor cell and the enucleated oocyte, electrofusion triggers spontaneous oocyte activation, accompanied by an increase in intracellular Ca(2+) and improper nuclear remodelling. These events can be greatly reduced by the use of Sendai virus (SV)-mediated fusion. Moreover, SV-SCNT improves the blastulation rate and blastocyst quality, defined by the number and ratio of inner cell mass and trophectoderm cells in each blastocyst, in comparison with electrofusion-mediated SCNT (E-SCNT). Interestingly, expression of ER-stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos. These increases could be reversed by inhibition of ER stress or by using the SV-mediated fusion method. Collectively, these results indicate that SV-mediated fusion improves the developmental competence and quality of SCNT blastocysts, by reducing ER-stress-associated apoptosis.

WHAMM is essential for spindle formation and spindle actin polymerization in maturing mouse oocytes.

WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is involved in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation-promoting factor, which activates the actin-related protein 2/3 complex (the Arp2/3 complex). However, the role of WHAMM in mammalian oocyte maturation is poorly understood. The presence of WHAMM mRNA and protein during all stages of mouse oocyte maturation has been verified. It is mainly co-localized with the actin cage permeating the spindle during mouse oocyte maturation. Through the knockdown of WHAMM, we confirmed that it regulates spindle formation and affects the localization of the microtubule-organizing center (MTOC) during the early stages of spindle formation. Moreover, depletion of WHAMM impaired the formation of the spindle actin and chromosome alignment, which might be the cause of chromosomal aneuploidy and abnormal, asymmetric division. Treatment with brefeldin A (BFA), an inhibitor of vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus, induced abnormal and dispersed localization of WHAMM. Taken together, these findings show that WHAMM is an essential component of the actin cytoskeleton machinery and plays a crucial role in oocyte maturation, presumably by controlling the formation of spindles with normal length by activating the formation of the spindle actin via the Arp2/3 complex.

Particulate Matter Exposure During Oocyte Maturation: Cell Cycle Arrest, ROS Generation, and Early Apoptosis in Mice.

Particulate matter (PM) is a general atmospheric pollutant released into the air by an anthropogenic and naturally derived mixture of substances. Current studies indicate that fine dust can result in different health defects, including endothelial dysfunction, asthma, lung cancer, cardiovascular diseases, uterine leiomyoma, deterioration in sperm quality, and overall birth impairment. However, the most prominent effects of PM10 (diameter < 10 µM) exposure on the female reproductive system, especially with respect to oocyte maturation, remain unclear. In the present study, maturing mouse oocytes were treated with PM10 and the phenotypes of the resulting toxic effects were investigated. Exposure to PM10 led to impairment of maturation capacity by inducing cell cycle arrest and blocking normal polar body extrusion during in vitro maturation and activation of fertilization of mouse oocytes. Additionally, defects in tubulin formation and DNA alignment were observed in PM10-treated oocytes during metaphase I to anaphase/telophase I transition. Moreover, PM10 induced reactive oxygen species generation, mitochondrial dysfunction, DNA damage, and early apoptosis. Taken together, these results indicate that PM10 exposure leads to a decline in oocyte quality and affects the subsequent embryonic development potential of mammalian oocytes.

Embryo aggregation regulates in vitro stress conditions to promote developmental competence in pigs.

Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in in vitro developmental competence and the relationship of stress conditions in porcine early embryogenesis. We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.