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Phosphatase and tensin homolog-induced kinase 1 (PINK1) on the outer membranes of impaired mitochondria promotes mitophagy and regulates mitochondrial morphology. Mammalian oocytes and early embryos are mitochondria rich, but mitochondrial dynamics during preimplantation embryo development is not well-studied. To investigate whether PINK1 is required for mitochondrial dynamics in porcine preimplantation embryos, gene knockdown and inhibitors were used, and mitochondrial dynamics were observed by transmission electron microscopy. PINK1 knockdown significantly impaired blastocyst formation and quality, induced mitochondrial elongation and swelling, and reduced mitochondrial DNA copy number. PINK1 knockdown-induced mitochondrial elongation caused mitochondrial dysfunction, oxidative stress, and ATP deficiency, significantly increasing autophagy and apoptosis. Profission dynamin-related protein 1 overexpression prevented PINK1 knockdown-induced impairment of embryo development, mitochondrial elongation, and dysfunction. Thus, PINK1 promotes mitochondrial fission in porcine preimplantation embryos.-Niu, Y.-J., Nie, Z.-W., Shin, K.-T., Zhou, W., Cui, X.-S. PINK1 regulates mitochondrial morphology via promoting mitochondrial fission in porcine preimplantation embryos.

Thiamethoxam (TMX) is a neonicotinoid insecticide. It has specific high toxicity to insects. Residues of TMX have been detected in various crops. Early embryo quality is vital for fertility. Excessive production of reactive oxygen species (ROS) can override embryonic antioxidant defenses, producing oxidative stress that triggers apoptosis, necrosis, and/or permanent DNA damage responses in the early embryo. Comparative studies have indicated that TMX hepatotoxicity is significant in mammals in acute tests, but little is known about accumulated chronic toxicity in early embryonic development. Porcine embryos were obtained here by the parthenogenetic activation of meiosis II oocytes and cultured in the PZM-5 medium with or without TMX. These embryos were evaluated by various methods. The expansion and hatching of blastocysts treated with TMX decreased by 21.73% and 16.71%, respectively, as compared with controls. In an analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation, the rate of cell proliferation was 44.33% lower as compared with expanded blastocysts of the control group. ROS and γH2AX levels were higher in the TMX group than in the control group. Real-time reverse-transcription polymerase chain reaction showed that Sod1 expression increased and the expression of Mnsod, Gpx1, Igta5, and Cox2 decreased. A CDK1 kinase assay revealed that maturation-promoting factor (MPF) activity diminished by 31.41% in expanding blastocysts. In conclusion, these results suggest that TMX inhibits blastocyst expansion and hatching by ROS-induced DNA damage checkpoint activation, which inhibits the activation of MPF and cell cycle progression in porcine blastocysts., (Copyright © 2018. Published by Elsevier Inc.)

MicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared with in vitro fertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3b and Dnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

Bisphenol A (BPA) is an environmental contaminant widely used in the plastic industry. BPA has been demonstrated to be an endocrine disruptor and has an adverse effect on the embryonic development of mammals. However, the mechanism of action of BPA is limited. In this study, we investigated the role and mechanism of BPA in porcine embryonic development. First, the parthenotes were treated with different concentrations of BPA. We found that blastocyst formation was impaired and the parthenotes were arrested at the 4-cell stage after treatment with 100 μm BPA. Second, ROS increased following the addition of BPA, which further caused mitochondrial damage, and cytochrome c was released from the mitochondria to induce apoptosis. The adaptive response was demonstrated through LC3 immunofluorescence staining and by assessing autophagy-related gene expression. In addition, BPA caused DNA damage through the p53-p21 signaling pathway. Thus, our results indicate that BPA displays an adverse effect on porcine early embryonic development through mitochondrial and DNA damage.

Study Question: What is the function of Spindlin 1 (Spin1) in metaphase II stage oocytes in pigs?, Summary Answer: Depletion of Spin1 induces spontaneous oocyte activation and overexpression of Spin1 causes multinuclear formation through induction of DNA damage in porcine oocytes., What Is Known Already: Little is known about the function of Spin1 in oocytes and embryos. In mouse oocytes, Spin1 is specifically expressed during gametogenesis and is essential for meiotic resumption. In somatic cells, Spin1 promotes cancer cell proliferation and activates WNT/T-cell factor signaling., Study Design Size, Duration: After knockdown (KD) or overexpression of Spin1 in porcine MII-stage oocytes, MII maintenance was checked following additional culture for 24 h. Investigated parthenotes were cultured up to the four cell (72 h) or blastocyst (7 days) stages., Participants/materials, Setting, Methods: Spin1 was knocked down in porcine oocytes and embryos via microinjection of pig Spin1-targeting siRNA. For Spin1 overexpression, porcine Spin1-eGFP cRNA was generated. Additionally, for rescue experiments, cRNA encoding siRNA-resistant mouse Spin1 was added to the pig Spin1-targeting siRNA. For the overexpression and rescue experiments, microinjection and culture were performed using the same methods as the KD experiments., Main Results and the Role of Chance: KD of Spin1 in MII-stage porcine oocytes reduced metaphase-promoting factor and mitogen-activated protein kinase activities, resulting in spontaneous pronuclear formation without calcium activation. However, the DNA damage response was triggered by Spin1 overexpression, generating the checkpoint protein γH2A.X. Furthermore, Spin1 overexpression blocked metaphase-anaphase transition and led to multinucleation in oocytes and embryos., Large Scale Data: None., Limitations, Reasons for Caution: This study is based on in vitro investigations with abnormal expression levels of Spin1. This may or may not accurately reflect the situation in vivo., Wider Implications of the Findings: Spin1 is essential to maintain MII arrest, but a high level of Spin1 induces DNA damage in oocytes and embryos. Thus, a system to accurately regulate Spin1 expression operates in porcine MII-stage oocytes and embryos., Study Funding and Competing Interest(s): This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2015R1D1A1A01057629). The authors declare no competing financial interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Peroxisome proliferator-activated receptor δ (Pparδ) is a nuclear receptor that plays critical roles in lipid metabolism, glucose metabolism, and cell growth and differentiation. Several recent studies have shown that Pparδ promotes blastocyst hatching in vitro. However, the mechanism by which it promotes preimplantation embryonic development in vitro remains unclear. In this study, oocytes and parthenotes were treated with a specific agonist of PPARδ, GW501516. The activation of PPARδ had no effect on oocyte maturation for 1 μM and 10 μM GW501516 compared with the control group. Additionally, the PPARδ agonist did not affect blastocyst formation (77.79 ± 3.59% [10 μM], 79.00 ± 5.53% [50 μM], and 79.64 ± 6.00% [100 μM] vs. 81.69 ± 2.61% [control]). However, the blastocyst hatching rate was significantly greater for parthenotes treated with 10 and 50 μM agonist, and did not differ between those treated with 100 μM agonist and the control group (61.80 ± 3.03% [10 μM], 65.10 ± 5.25% [50 μM], and 38.85 ± 7.45% [100 μM] vs. 41.77 ± 10.88% [0 μM]). Activation of PPARδ also increased blastocyst quality and cell number, as well as ATP production. There were no clear differences in mitochondrial membrane potential, mitochondrion copy number, or glucose consumption between the treatment and control groups. However, PPARδ activation enhanced lipid accumulation via Fabp3 and Fabp5. Fatty acid oxidation also increased in response to treatment with the agonist via the rate-limiting gene Cpt2. Reactive oxygen species were modified and REDOX maintenance-related gene expression increased significantly in GW501516-exposed blastocysts. In addition, the activation of PPARδ resulted in changes in miRNA content. After treatment with the PPARδ agonist, miR-99 increased and miR-32 decreased. These data showed that PPARδ has a positive impact on blastocyst hatching via the regulation of lipid metabolism., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fatty acid synthase (FASN) is an enzyme responsible for the de novo synthesis of long-chain fatty acids. During oncogenesis, FASN plays a role in growth and survival rather than acting within the energy storage pathways. Here, the function of FASN during early embryonic development was studied using its specific inhibitor, C75. We found that the presence of the inhibitor reduced blastocyst hatching. FASN inhibition decreased Cpt1 expression, leading to a reduction in mitochondria numbers and ATP content. This inhibition of FASN resulted in the down-regulation of the AKT pathway, thereby triggering apoptosis through the activation of the p53 pathway. Activation of the apoptotic pathway also leads to increased accumulation of reactive oxygen species and autophagy. In addition, the FASN inhibitor impaired cell proliferation, a parameter of blastocyst quality for outgrowth. The level of OCT4, an important factor in embryonic development, decreased after treatment with the FASN inhibitor. These results show that FASN exerts an effect on early embryonic development by regulating both fatty acid oxidation and the AKT pathway in pigs., Competing Interests: The authors have declared that no competing interests exist.

Study Question: What are the mechanisms by which the preimplantation restraint stress (PIRS) impairs embryo development and pregnancy outcome?, Summary Answer: PIRS impairs embryo development by triggering apoptosis in mouse oviducts and embryos,and this involves activation of the Fas system., What Is Known Already: Although it is known that the early stages of pregnancy are more vulnerable than later stages to prenatalstress, studies on the effect of preimplantation stress on embryo developmentare limited. Furthermore, the mechanisms by which psychological stress impairs embryo development are largely unknown. These issues are worth exploring using the mouse PIRS models because restraint of mice is an efficient experimental procedure developed for studies of psychogenic stress., Study Design, Size and Duration: Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in FasL in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female and male mice were used 8-10 weeks and 10-12 weeks after birth, respectively. Female mice showing vaginal plugs were paired by weight and randomly assigned to restraint treatments or as controls. For restraint treatment, an individual mouse was put in a micro-cage with food and water available. Control mice remained in their cages with food and water during the time treated females were stressed., Participants/materials, Setting, Methods: Female mice were exposed to PIRS for 48 h starting from 16:00 on the day of vaginal plug detection. At the end of PIRS, levels of glucorticoids (GC), corticotropin-releasing hormone (CRH)and redox potential were measured in serum, while levels of GC, GC receptor (GR), CRH, CRH receptor (CRHR), Fas and Fas ligand (FasL) protein, mRNAs for brain derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1), oxidative stress (OS) and apoptosis were examined in oviducts. Preimplantation development and levels of GR, Fas, redox potential and apoptosis were observed in embryos recovered at different times after the initiation of PIRS. The gld mice were used to confirm a role for the Fas system in triggering apoptosis of embryos and oviducts., Main Results and the Role of Chance: Compared to those in control mice, while the number of blastocysts/mouse (5.0 ± 0.7 versus 11.1 ± 0.5), cell number/blastocyst (49.1 ± 1.3 versus 61.5 ± 0.9), percentages of term pregnancy (37.5% versus 90.9%) and litter size (3.7 ± 0.1versus 9.6 ± 0.6) decreased, blood CRH (560 ± 23 versus 455 ± 37 pg/ml), cortisol (27.3 ± 3.4 versus 5 ± 0.5 ng/ml) and OS index (OSI: 2.8 versus 1.7) increased significantly (all P < 0.05) following PIRS. In the oviduct, while levels of CRH (1175 ± 85 versus 881 ± 33 pg/100 mg), cortisol (28.9 ± 1.7 versus14 ± 4 ng/g), CRHR (2.3 ± 0.3 versus 1.0 ± 0.0), FasL (1.31 ± 0.06 versus 1.08 ± 0.05 ng/g), Fas (1.42 ± 0.13 versus 1.0 ± 0.0) and apoptotic cells (19.1 ± 0.5% versus 8.4 ± 0.4%) increased, levels of GR proteins (0.67 ± 0.14 versus 1.0 ± 0.0) and Igf-1 (0.6 ± 0.09 versus 1.0 ± 0.0) and Bdnf (0.73 ± 0.03 versus 1.0 ± 0.0) mRNAs decreased significantly (all P < 0.05 versus control) after PIRS. Mouse embryos expressed GR and Fas at all stages of preimplantation development and embryo OS (GSH/GSSG ratio: 0.88 ± 0.03 versus 1.19 ± 0.13) and annexin-positive cells (blastocysts: 31.4 ± 3.8% versus 10.96 ± 3.4%) increased significantly (P < 0.05) following PIRS. Furthermore, the detrimental effects of PIRS on embryo development and oviductal apoptosis were much reduced in gld mice. Thus, PIRS triggered apoptosis in oviductal cells with activation of the Fas/FasL system. The apoptotic oviductal cells promoted embryo apoptosis with reduced production of IGF-1 and BDNF and increased production of FasL., Limitations, Reasons for Caution: Although important, the conclusions were drawn from limited results obtained using a single model in one species and thus they need further verification using other models and/or in other species. Furthermore, as differences in stressed samples were modest and sometimes not significant between gld and wild-type mice whereas differences between control and stressed samples were always present within gld mice, it is deduced that signaling pathways other than the Fas/FasL system might be involved as well in the PIRS-triggered apoptosis of oviducts and embryos., Wider Implications of the Findings: The data are important for studies on the mechanisms by which psychological stress affects female reproduction, as FasL expression has been observed in human oviduct epithelium., Large Scale Data: Not applicable., Study Funding and Competing Interests: This study was supported by grants from the National Basic Research Program of China (Nos. 2014CB138503 and 2012CB944403), the China National Natural Science Foundation (Nos. 31272444 and 30972096) and the Animal breeding improvement program of Shandong Province. All authors declare that their participation in the study did not involve factual or potential conflicts of interests., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: journals.permissions@oup.com.)

Liver receptor homolog 1 (Lrh1, also known as Nr5a2) belongs to the orphan nuclear receptor superfamily and has diverse functions in development, metabolism, and cell differentiation and death. Lrh1 regulates the expression of Oct4, which is a key factor of early embryonic differentiation. However, the role of Lrh1 in early development of mammalian embryo is unknown. In the present study, the localization, Lrh1 mRNA expression, and LRH1 protein levels in porcine early parthenotes were examined by immunofluorescence and real-time reverse-transcription polymerase chain reaction. To determine the role of Lrh1 in porcine early embryo development, the parthenotes were treated with the specific LRH1 antagonist 505601. The immunofluorescence signal for LRH1 was only observed in the nucleus of blastocysts. The blastocyst developmental rate in the presence of 50 and 100 μM 505601 was significantly lower than that in the control group. The blastocyst hatching rate was also reduced in the presence of 50 and 100 μM 505601 than that under control conditions. The latter effect was possibly due to the decreased expression of hatching-related genes such as Fn1, Itgα5, and Cox2 upon the inhibition of Lrh1. Incubation with the LRH1 antagonist also increased the number of apoptotic cells among the blastocysts. Moreover, LRH1 inhibition enhanced the expression of the pro-apoptotic genes Bax and Casp3, and reduced the expression of the anti-apoptotic gene Bcl2. Lrh1 inhibition also led to significant decrease in the expression levels of Oct4 mRNA and octamer-binding transcription factor 4 (OCT4) protein in the blastocysts. In conclusion, Lrh1 affects blastocyst formation and hatching in porcine embryonic development through the regulation of OCT4 expression and cell apoptosis.

Iron is an essential trace element that plays important roles in the cellular function of all organs and systems. However, the function of Fe(II) in mammalian embryo development is unknown. In this study, we investigated the role of Fe(II) during preimplantation embryo development. Depletion of Fe(II) using thiosemicarbazone-24 (TSC24), a specific Fe(II) chelator, rescued quenching of the Fe(II)-sensitive fluorophore phen green-SK. After in vitro fertilization, TSC24 significantly reduced the cleavage rate as well as blastocyst formation. The hatch rate of blastocysts was also reduced with 1 pM TSC24 treatment (20.25±1.86 versus 42.28±12.96%, p<0.05). Blastocysts were cultured in leukemia inhibitory factor-free mouse embryonic stem cell culture medium with or without TSC24, and those with depleted Fe(II) displayed delayed attachment and lost the ability to induce embryoid body formation. To further explore the mechanism of Fe(II) in embryo development, we assessed the expression of 5-hydroxymethylcytosine (5hmC) and OCT4 in the pronuclear and blastocyst stages, respectively. We observed that Fe(II) reduced 5hmC and OCT4 expression, which could be explained by low ten-eleven translocation (TET) enzyme activity induced by TSC24 treatment. These findings demonstrate that Fe(II) is required for mammalian embryo development and that it facilitates the process via regulation of TET activity.

The mitochondrial genome is maternally inherited in animals, despite the fact that paternal mitochondria enter oocytes during fertilization. Autophagy and ubiquitin-mediated degradation are responsible for the elimination of paternal mitochondria in Caenorhabditis elegans; however, the involvement of these two processes in the degradation of paternal mitochondria in mammals is not well understood. We investigated the localization patterns of light chain 3 (LC3) and ubiquitin in mouse and porcine embryos during preimplantation development. We found that LC3 and ubiquitin localized to the spermatozoon midpiece at 3 h post-fertilization, and that both proteins were colocalized with paternal mitochondria and removed upon fertilization during the 4-cell stage in mouse and the zygote stage in porcine embryos. Sporadic paternal mitochondria were present beyond the morula stage in the mouse, and paternal mitochondria were restricted to one blastomere of 4-cell embryos. An autophagy inhibitor, 3-methyladenine (3-MA), did not affect the distribution of paternal mitochondria compared with the positive control, while an autophagy inducer, rapamycin, accelerated the removal of paternal mitochondria compared with the control. After the intracytoplasmic injection of intact spermatozoon into mouse oocytes, LC3 and ubiquitin localized to the spermatozoon midpiece, but remnants of undegraded paternal mitochondria were retained until the blastocyst stage. Our results show that paternal mitochondria colocalize with autophagy receptors and ubiquitin and are removed after in vitro fertilization, but some remnants of sperm mitochondrial sheath may persist up to morula stage after intracytoplasmic spermatozoon injection (ICSI).

Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos.

Researchers have exerted sustained efforts to improve the viability of somatic cell nuclear transfer (SCNT) embryos, testing their experimental designs and probing the resultant embryos. However, the lack of a reliable method to estimate the efficacy of these experimental attempts is a chief hindrance to tackling the low-viability problem in SCNT. Here, we introduce a procedure that assesses the degree of difference in gene expression profiles (GEPs) of blastocysts from each other as a representative control of good quality. We first adapted a multiplex reverse transcription-polymerase chain reaction strategy to obtain GEPs for 15 reprogramming-related genes from single mouse blastocysts. GEPs of individual blastocysts displayed a broad range of variations, the extent of which was calculated using a weighted root mean square deviation (wRMSD). wRMSD-based quantitation of GEP difference (qGEP) found that GEP difference between in vivo-derived blastocysts (in vivo) and SCNT blastocysts was greater than the difference between in vivo blastocysts and in vitro-produced (IVP) blastocysts, demonstrating that the SCNT group was more distantly related to the in vivo group than the IVP group. Our qGEP approach for grading individual blastocysts would be useful for selecting a better protocol to derive embryos of better quality prior to field applications.

Cathepsin B, a lysosomal cysteine protease of the papain family, has recently been implicated in the quality and developmental competence of bovine preimplantation embryos. In this study, to determine whether inhibition of cathepsin B activity can improve porcine oocyte maturation and early embryo developmental competence, we supplemented in vitro maturation or embryo culture media with E-64, a cathepsin B inhibitor. Cathepsin B activity was high in poor quality germinal vesicle stage oocytes, but no differences in mRNA expression or protein localization were observed between good and poor quality oocytes, which were categorized based on morphology. Following treatment with 1 μM E-64, cathepsin B activity sharply decreased in 4-cell and blastocyst stage embryos. E-64 had no effect on cell number but significantly (P < 0.05) increased blastocyst formation and decreased the number of apoptotic cells in blastocysts. It also significantly (P < 0.05) enhanced mitochondrial membrane potential in blastocysts, reducing the release of cytochrome c and resulting in decreased expression of Caspase-3 and Caspase-9. In conclusion, inhibition of cathepsin B activity in porcine parthenotes using 1 μM E-64 resulted in attenuation of apoptosis via a reduction in the release of cytochrome c from mitochondria.

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway.

In vitro porcine embryo production systems have been established and well characterized. However, the efficiency of embryo development during IVC is still very low. In the present study, we have investigated the development of parthenogenetic porcine embryos in the well-known PZM-5 medium for porcine embryos, which was modified by replacing glutamine with the GlutaMAX supplement. We revealed that blastocyst apoptosis was significantly lower in the presence of GlutaMAX, which reduced the release of mitochondrial cytochrome c. Furthermore, the expression of apoptosis genes was significantly lower during GlutaMAX treatment (P < 0.05). The modified medium was also examined for the eventual loss of its efficacy in the presence of GlutaMAX. Three, 6, and 12 months after medium preparation, blastocyst formation in the GlutaMAX-supplemented medium was significantly higher than the number of blastocysts in the medium containing glutamine. After a long period of storage, ammonia concentration was significantly increased in the glutamine medium, whereas it was not statistically different in the GlutaMAX medium. Elevated ammonia concentrations reduced the mitochondrial membrane potential and ATP content of blastocysts in the glutamine medium. These results demonstrate that GlutaMAX can reduce blastocyst apoptosis via inhibition of the cytochrome c pathway and significantly extend the shelf life of the culture medium to at least 1 year. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) is a type of mycotoxin produced by the fungi Aspergillus flavus and Aspergillus parasiticus . AFB1 is considered as the most toxic mycotoxin owing to its toxic effect on health. In the present study, the toxic effect of AFB1 on early porcine embryonic development and its possible mechanism were investigated. Blastocyst formation was impaired with treatment of 1 nM AFB1 compared with control, 0.01, 0.1 group (40.13 ± 2.10%, 28.21 ± 1.62%, 32.34 ± 2.07% vs 19.01 ± 1.06%). Further study showed that the presence of AFB1 induced the generation of reactive oxygen species (ROS). And excessive ROS caused DNA damage which confirmed by the comet assay. Additionally, AFB1 also disrupted the DNA damage repair through the regulation of 53BP1. The AFB1 treatment significantly increased the γH2A foci and decreased the 53BP1 foci. TUNEL assay confirmed the generation of apoptosis, further resulting in the occurrence of autophagy. Moreover, AFB1 significantly increased the expression of pro-apoptosis genes Bax and Casp3 and reduced the expression of anti-apoptotic genes Bcl2 and Bcl-xl . In addition, the AFB1 also significantly increased the expression of autophagy related genes Lc3 and Beclin1. The presence of AFB1 significantly impaired the cell proliferation, a parameter of blastocyst quality for outgrowth. These results showed that the presence of AFB1 impaired porcine early embryonic development through oxidative stress, as well as DNA damage and repair, apoptosis, autophagy. [ABSTRACT FROM AUTHOR]

T-2 toxin is a type A mycotoxin produced by various Fusarium species, while HT-2 toxin is a major metabolite of T-2 toxin. Both T-2 toxin and HT-2 toxin are known to have deleterious effects on animals. Our previous work showed that HT-2 treatment caused the failure of porcine oocyte maturation. In this study, we reported that HT-2 also affected porcine embryo development. In HT-2 toxin treated group, all the percentages of embryos in 2-cell, 4-cell and blastocyst stage were significantly lower compared with those in control groups. We then explored the causes from the epigenetic modification aspect of the oocytes. The analysis of fluorescence intensity showed that 5-methyl cytosine (5 mC) level was increased after exposure to HT-2 toxin in porcine oocytes, indicating that the general DNA methylation level increased in the treated porcine oocytes. In addition, histone modifications were also affected, since our results showed that H3K4me2 and H3K9me2 levels were increased in the oocytes from HT-2-treated group. Therefore, our results indicated that HT-2 toxin decreased porcine embryo developmental competence through altering the epigenetic modifications of oocytes. [ABSTRACT FROM AUTHOR]

Thiamethoxam (TMX) is a neonicotinoid insecticide, the residues of which have been detected on various crops. In addition to its specific acetylcholine toxicity to insects, TMX was also found to be toxic to mammals. Moreover, oocytes are vulnerable to reactive oxygen species (ROS). Excessive ROS production can override antioxidant defenses and produce oxidative stress and DNA damage that trigger apoptosis and necrosis in organisms. In this study, we exposed bovine oocytes to TMX during maturation. Microscopic examination showed that 1.6 mM TMX significantly inhibited maturation at the germinal vesicle (GV) and metaphase I (MI) stages. Immunofluorescence staining and enzyme activity analysis revealed that TMX induced a reduction in CDC25 and CDC2 activity. Furthermore, time-lapse tracking and immunofluorescence staining indicated the maintenance of cyclin B in the cytoplasm, persistence of Bub3 at kinetochores, and absence of actin caps after TMX-exposed oocytes reached the MI stage. In addition, metaphase II (MII) oocytes exposed to TMX showed disordered chromosomes and spindles. These oocytes accumulated excess ROS and showed significantly decreased mitochondrial membrane potential and increased apoptotic signals. Parthenogenetic embryos from these oocytes showed decreased percentages of morulae and blastocysts. These results indicate that TMX delays bovine oocyte progression to MI stage, blocks them at the MI stage, triggers disordered chromosomes and spindles at MII stage, and ultimately results in MII oocytes with poor cleavage ability and inhibited development to morulae and blastocysts. [ABSTRACT FROM AUTHOR]