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Abstract: Leptin, a multifunctional hormone, is present in mammalian oocytes and follicular fluids and cumulus cells. While leptin modulates oocyte maturation in vitro which seems to result in enhancement of embryo development, it is unclear whether leptin treatment of oocytes affects cytoplasmic maturation and fertilization processes. In order to gain a better understanding of the role of leptin during oocyte maturation, we examined microtubule and microfilament assembly following oocyte maturation and blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) in leptin-treated oocytes. Addition of 10 or 100ng/ml leptin during oocyte maturation did not increase the proportion of metaphase II oocytes, but enhanced development to blastocyst stage by day 7 (P <0.01) after parthenogenetic activation (PA), accompanied by increased cell number. However there was no effect on the number of apoptotic cells in blastocysts. Following maturation in the presence of leptin, there were more oocytes with normal spindle formation. MAPK activity decreased more rapidly, and pronuclear formation was accelerated after parthenogenetic activation or ICSI of leptin-treated oocytes. These results suggested that exogeneous leptin enhanced spindle assembly and accelerated pronuclear formation following fertilization, possibly via the MAPK pathway. [Copyright &y& Elsevier]

Abstract: To gain insights into the roles the paternal genome and chromosome number play in pre-implantation development, we cultured fertilized embryos and diploid and haploid parthenotes (DPs and HPs, respectively), and compared their development and gene expression patterns. The DPs and fertilized embryos did not differ in developmental ability but HPs development was slower and characterized by impaired compaction and blastocoel formation. Microarray analysis revealed that fertilized blastocysts expressed several genes at higher levels than DP blastocysts; these included the Y-chromosome-specific gene eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked (Eif2s3y) and the imprinting gene U2 small nuclear ribonucleoprotein auxiliary factor 1, related sequence 1 (U2af1-rs1). We also found that when DPs and HPs were both harvested at 44 and 58h of culture, they differed in the expression of 38 and 665 genes, respectively. However, when DPs and HPs were harvested at the midpoints of 4-cell stage (44 and 49h, respectively), no differences in expression was observed. Similarly, when the DPs and HPs were harvested when they became blastocysts (102 and 138h, respectively), only 15 genes showed disparate expression. These results suggest that while transcripts needed for early development are delayed in HPs, it does progress sufficiently for the generation of the various developmental stages despite the lack of genetic components. [Copyright &y& Elsevier]

Abstract: Dicer1, an RNAse III enzyme, is a key factor for the production of microRNAs involved in post-transcriptional gene silencing. To elucidate the roles of Dicer1 and the microRNA pathway in early embryo development, we initially evaluated its gene expression in mouse oocytes and embryos in vitro. The transcript levels in GV stage oocytes steadily decreased up to the 2-cell embryo stage, and expression remained stable during morulae and blastocyst formation. DICER1 protein synthesis was additionally observed in mouse oocytes and early embryos. Silencing of mRNA expression by RNA interference (siRNA) did not inhibit development up to the blastocyst stage. Real-time RT-PCR experiments confirmed the decreased expression of selected transcription factors, including POU domain, class 5, transcription factor 1 (Pou5f1), SRY-box containing gene 2 (Sox2), and Nanog homeobox (Nanog). Moreover, POU5F1 protein expression was suppressed by Dicer1 siRNA. The results suggest that Dicer1 gene expression is associated with the levels of transcription factors, Pou5f1, Sox2, and Nanog which possibly regulate differentiation processes at the blastocyst stage. [Copyright &y& Elsevier]

Abstract: The aim of this study was to determine the effects of IGF-I on the viability and development of porcine presumptive diploid parthenotes developing in vitro. In vitro derived 2-cell parthenotes were cultured to blastocysts with or without exogenous IGF-I in the presence or absence of BSA. The addition of 10 or 100ng/ml IGF-I into NCSU 23 medium did not enhance the development of 2-cell parthenotes to the blastocyst stage. However, IGF-I increased cell numbers (P < 0.001) in the presence of BSA. Similarly, IGF-I alone did not affect apoptosis in the blastocysts but when combined with BSA, it reduced apoptosis (P < 0.05). Real time reverse-transcription polymerase chain reaction revealed that IGF-I significantly decreased the mRNA expression (pro-apoptotic gene Fas or Bak and anti-apoptotic gene Bcl-xL) ratios of both Fas/Bcl-xL (P < 0.01) and Bak/Bcl-xL (P < 0.01) in the presence of 0.4% BSA. These data suggest that IGF-I synergies with BSA to act as a mitogen and apoptotic survival factor in porcine preimplantation parthenotes. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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]

Abstract 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. Highlights • TMX results in oxidation-reduction reaction out of balance and increased ROS which induces subsequent DNA damage. • The DNA damage accumulates greatly because of rapid cell proliferation of blastocysts at score 3. • The ROS-dependent DNA damage response arrests cell cycle of blastocysts by ATM-CHK1-CDC25C and ATM-p53-p21 pathways. [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]

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. [ABSTRACT FROM AUTHOR]

ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effects of various durations of dibutyryl cyclic AMP (dbcAMP) treatment on the in vitro maturation (IVM) and subsequent development of parthenogenetically activated embryos. Immature porcine oocytes were cultured with or without 1 mM dbcAMP during the first 20, 28, or 36 h of culture, and then incubated for an additional 24 h without dbcAMP. The expression ofWee1B,Myt, andCdc25Band the level of maturation promoting factor (MPF) in metaphase II oocytes were analyzed by real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The distribution of actin microfilaments in oocytes was also assessed. Subsequently, apoptotic cells in blastocysts from each group were visualized by transferase-mediated dUTP nick-end labeling staining. Results showed that oocytes extruded the first polar body between 12 and 18 h after being released from dbcAMP. MPF activity in oocytes at 28 + 24 h and 36 + 24 h after dbcAMP treatment was higher than that in the control group. Significantly more blastocysts were present among embryos in 28 + 24 h (54.28% vs. 39.11%,P< 0.05) and 36 + 24 h (47.24% vs. 32.94%,P< 0.05) groups than among embryos cultured in the absence of dbcAMP. However, the number of total and apoptotic cells was not significantly different between groups. The distribution of actin microfilaments was abnormal in oocytes cultured for 60 h without dbcAMP. In addition, the expression ofWee1B,Myt, andCdc25Bwas higher in the control group at 44 h than in the dbcAMP group, but there were no differences in expression at the other time points. In conclusion, dbcAMP treatment delays oocyte maturation and maintains oocyte quality. [ABSTRACT FROM AUTHOR]

Abstract: Successful in vitro development of embryos is dependent upon maintenance of cellular function in the embryonic microenvironment. However, the molecular aspects involved in the thermoprotection of embryos, against heat and cold stress it is not clear. The aim of this study was to determine the effects of heat and cold shock on the viability and development of porcine diploid parthenotes developing in vitro. Exposure of two-cell stage embryos to 41°C did not affect further cleavage. However, prolonged heat shock, greater than 12h, reduced the percentage of blastocysts that developed from two-cell stage parthenotes, as well as the total number of nuclei in the blastocysts that formed. Furthermore, the degree of apoptosis was increased (P <0.05) in these blastocyst stage parthenotes. In contrast, exposure of two-cell parthenotes to cold (30°C) for 24h did not affect the cleavage rates, development to blastocyst, nor the total cell numbers per blastocyst. Real time PCR revealed that quantitative expression of the Bcl-xL gene was not different, but amounts of HSP 70.2, Bak, and Caspase 3mRNA were significantly increased in the heat shocked embryos, as compared with untreated controls. These results suggest that porcine embryos are more tolerant to cold shock than to heat shock. Heat stress seems to induce apoptosis related gene expression in porcine parthenotes developing in vitro, which results in diminished parthenote viability. [Copyright &y& Elsevier]

Abstract: High mobility group box 1 (HMGB1) is considered a component of chromatin and membranes with a role in a variety of biologically important processes. The aim of this study was to determine the effects of HMGB1 on the viability and development of porcine diploid parthenotes cultured in vitro. In vitro derived 4-cell parthenotes were cultured to blastocysts, with or without recombinant HMGB1, in the presence or absence of BSA. The addition of 1, 10, 100 or 1000ng/mL HMGB1 into NCSU 23 medium containing 0.4% BSA did not enhance the development of 4-cell parthenotes to the blastocyst stage and did not change the total number of nuclei in the blastocysts. However, addition of 10 or 100ng/mL HMGB1 into NCSU 23 medium in the absence of BSA increased (P <0.05) both the development rate of parthenotes to the blastocyst stage and total cell numbers. When cultured in NCSU23 medium supplemented with 10 or 100ng/mL HMGB1 and without BSA, apoptosis in parthenotes at the blastocyst stage was decreased (P <0.05). Based on real time RT-PCR, the addition of HMGB1 to the culture medium in the absence of BSA decreased mRNA expression of pro-apoptotic genes Bak (P <0.005) or Caspase3 (Casp3, P <0.01), but not Bcl-xL (Bcl2l2). In conclusion, we inferred that recombinant HMGB1 in the culture medium in the absence of BSA prevented apoptosis of porcine parthenotes and enhanced porcine embryo viability. [Copyright &y& Elsevier]

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]