Porcine Early Embryonic Development Research Articles

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.

Melatonin alleviates defects induced by zearalenone during porcine embryo development

Zearalenone (ZEA), which is produced by several fusarium mycotoxins, is found in animal feed and food products, and can exert estrogen-like activity. Melatonin (MT) is emerging as a supplement that can fight the toxic effects of mycotoxins. With a variety of physiological functions that play crucial roles in the development of animal germ cells and embryos, melatonin regulates circadian rhythms and has an anti-inflammatory and anti-oxidative role. This study investigated the protective effects of melatonin against ZEA in porcine early embryonic development. Our results showed that ZEA adversely affected this development, while melatonin supplementation ameliorated the toxic effects. ZEA exposure increased oxidative stress and impaired mitochondrial function, which may affect blastocyst formation. Moreover, we found that ZEA exposure promotes apoptosis, DNA damage, and autophagy in porcine blastocysts. The toxic effects of ZEA on early embryos may be the result of oxidative stress-mediated early apoptosis, while melatonin treatment significantly improved these phenotypes in ZEA-exposed porcine early embryos. Taken together, our results indicate that melatonin has a protective effect on defects caused by ZEA during early porcine embryonic development.

Carnosic acid improves porcine early embryonic development by inhibiting the accumulation of reactive oxygen species.

Carnosic acid (CA), a natural catechol rosin diterpene, is used as an additive in animal feeds and human foods. However, the effects of CA on mammalian reproductive processes, especially early embryonic development, are unclear. In this study, we added CA to parthenogenetically activated porcine embryos in an in vitro culture medium to explore the influence of CA on apoptosis, proliferation, blastocyst formation, reactive oxygen species (ROS) levels, glutathione (GSH) levels, mitochondrial membrane potential, and embryonic development-related gene expression. The results showed that supplementation with 10 μM CA during in vitro culture significantly improved the cleavage rates, blastocyst formation rates, hatching rates, and total numbers of cells of parthenogenetically activated porcine embryos compared with no supplementation. More importantly, supplementation with CA also improved GSH levels and mitochondrial membrane potential, reduced natural ROS levels in blastomeres, upregulated Nanog, Sox2, Gata4, Cox2, Itga5, and Rictor expression, and downregulated Birc5 and Caspase3 expression. These results suggest that CA can improve early porcine embryonic development by regulating oxidative stress. This study elucidates the effects of CA on early embryonic development and their potential mechanisms, and provides new applications for improving the quality of in vitro-developed embryos.

The toxic effect of aflatoxin B1 on early porcine embryonic development

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.

Selected steps of swine ICSI method to overcome seasonal effect and achieve acceptable early embryonic development, a preliminary study.

Porcine intra cytoplasmic sperm injection's (ICSI) efficacy by selected protocol steps was investigated. Three trials per year's period (hot, medium, cold) were carried out. Only large size follicles (6-8mm) were aspirated, brilliant cresyl blue (BCB) test was performed and only the BCB+ oocytes were in vitro maturated (40h) and involved to ICSI process. The presumptive embryos were in vitro cultured (15h). Raw boar semen and SpermCatch® as slowing medium were used. No differences were observed between periods regarding early embryonic development and maturation competence. ICSI achieves acceptable porcine early embryonic development rates under the investigated conditions.

The possible molecular mechanisms of bisphenol A action on porcine early embryonic 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.

CDK2 Is Required for the DNA Damage Response During Porcine Early Embryonic Development.

Cyclin-dependent kinase (CDK) 2 inhibition plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine embryos. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or meiosis II arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage, as demonstrated by the formation of distinct gammaH2AX foci in nuclei of Day-3 and Day-5 embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activation of the ataxia telangiectasia mutated (ATM)-P53-P21 pathway. However, the mRNA expression of genes involved in nonhomologous end joining or homologous recombination pathways for double-strand break repair were reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in Day-7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression.

Cloning of Porcine Pituitary Tumor Transforming Gene 1 and Its Expression in Porcine Oocytes and Embryos.

The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis.

Fe(III) Is Essential for Porcine Embryonic Development via Mitochondrial Function Maintenance.

Iron is an important trace element involved in several biological processes. The role of iron in porcine early embryonic development remains unknown. In the present study, we depleted iron (III, Fe3+) with deferoxamine (DFM), a specific Fe3+ chelator, in cultured porcine parthenotes and monitored embryonic development, apoptosis, mitochondrial membrane potential, and ATP production. Results showed biphasic function of Fe3+ in porcine embryo development. 0.5 μM DFM obviously increased blastocyst formation (57.49 ± 2.18% vs. control, 43.99 ± 1.72%, P < 0.05) via reduced (P < 0.05) production of reactive oxygen species (ROS), further increased mitochondrial membrane potential and ATP production in blastocysts (P < 0.05). 0.5 μM DFM decreased mRNA expression of Caspase 3 (Casp3) and increased Bcl-xL. However, results showed a significant reduction in blastocyst formation in the presence of 5.0 μM DFM compared with the control group (DFM, 21.62 ± 3.92% vs. control, 43.99 ± 1.73%, P < 0.05). Fe3+ depletion reduced the total (DFM, 21.10 ± 8.78 vs. control, 44.09 ± 13.65, P < 0.05) and increased apoptotic cell number (DFM, 11.10 ± 5.24 vs. control, 2.64 ± 1.43, P < 0.05) in the blastocyst. An obvious reduction in mitochondrial membrane potential and ATP level after 5.0 μM DFM treatment was observed. Co-localization between mitochondria and cytochrome c was reduced after high concentration of DFM treatment. In conclusion, Fe3+ is essential for porcine embryonic development via mitochondrial function maintenance, but redundant Fe3+ impairs the function of mitochondria.

Junction-mediating and regulatory protein (JMY) is essential for early porcine embryonic development.

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.

Dynamic reprogramming of 5-hydroxymethylcytosine during early porcine embryogenesis

DNA active demethylation is an important epigenetic phenomenon observed in porcine zygotes, yet its molecular origins are unknown. Our results show that 5-methylcytosine (5mC) converts into 5-hydroxymethylcytosine (5hmC) during the first cell cycle in porcine in vivo fertilization (IVV), IVF, and SCNT embryos, but not in parthenogenetically activated embryos. Expression of Ten-Eleven Translocation 1 (TET1) correlates with this conversion. Expression of 5mC gradually decreases until the morula stage; it is only expressed in the inner cell mass, but not trophectoderm regions of IVV and IVF blastocysts. Expression of 5mC in SCNT embryos is ectopically distinct from that observed in IVV and IVF embryos. In addition, 5hmC expression was similar to that of 5mC in IVV cleavage-stage embryos. Expression of 5hmC remained constant in IVF and SCNT embryos, and was evenly distributed among the inner cell mass and trophectoderm regions derived from IVV, IVF, and SCNT blastocysts. Ten-Eleven Translocation 3 was highly expressed in two-cell embryos, whereas TET1 and TET2 were highly expressed in blastocysts. These data suggest that TET1-catalyzed 5hmC may be involved in active DNA demethylation in porcine early embryos. In addition, 5mC, but not 5hmC, participates in the initial cell lineage specification in porcine IVV and IVF blastocysts. Last, SCNT embryos show aberrant 5mC and 5hmC expression during early porcine embryonic development.

Stage-Specific Gene Expression Profile Differences During Early Porcine Embryonic Development.

Stage-Specific Gene Expression Profile Differences During Early Porcine Embryonic Development.

Regulation of H3K27me3 and H3K4me3 during early porcine embryonic development

The epigenetic marks H3K27me3 and H3K4me3 are important repressive and permissive histone modifications, respectively, which are involved in gene regulation such as Hox gene expression during embryonic development. In this study, we investigated the global levels of these two histone modifications. We also investigated the expression of H3K27me3's methyltransferase (EZH2), EZH2 co-factors (EED and SUZ12) and demethylases (JMJD3 and UTX), as well as H3K4me3's methylases (ASH1L and MLL1) and demethylase (RBP2) in porcine pre-implantation embryos. In addition, the expression of Hox genes, HOXA2, HOXA3, HOXA7, HOXA10, HOXB4, HOXB7, HOXC8, HOXD8, and HOXD10 was investigated. We found that global levels of H3K27me3 decreased from the 1- to the 4-cell stage, corresponding to the time of major embryonic genome activation. Subsequently, the levels increased in hatched blastocysts, particularly in the trophectoderm. The expression levels of EZH2, EED, SUZ12, JMJD3, and UTX correlated well with these findings. The global levels of H3K4me3 decreased from the 1-cell to the morula stage and increased in hatched blastocysts, especially in trophectoderm. A peak in expression of ASH1L was seen at the 4-cell stage, but overall, expression of ASH1L, MLL1, and RBP2 correlated poorly with H3K4me3. HOXA3, A7, and B4 were expressed in 4-cell embryos, and HOXA7, A10, B4, and D8 were expressed in hatched blastocysts, and did not correlate well to global methylation of H3K27me3 or H3K4me3. Thus, H3K4me3 may play a role in early porcine embryonic genome activation, whereas, H3K27me3 may be involved in initial cell lineage segregation in the blastocyst.

Effects of culture media and inhibitors on biology of porcine early embryonic development in vitro

In vitro-production of porcine embryos is crucial for biomedical and agricultural research, however, current culture systems for porcine embryos are sub-optimal, and the developmental potential of in vitro-produced embryos is not well studied. The objectives of this study were to evaluate whether PZM-3 or NCSU-23 better support porcine embryo development and the effects of α-amanitin and cycloheximide on porcine embryonic development. Briefly, porcine presumptive zygotes were produced after in vitro maturation/fertilization (IVM/IVF) and cultured either in NCSU-23 or PZM-3 culture media. Transcript levels of BAX and BCL2L1 genes from blastocysts were detected by using Real-time PCR. The effects of α-amanitin and cycloheximide were evaluated for the role of inhibiting transcription and translation during early porcine embryogenesis. Results showed that both cleavage and blastocyst rates decreased significantly in NCSU-23 group compared as PZM-3 group. However, BAX and BCL2L1 transcript levels were similar in blastocysts cultured in both PZM-3 and NCSU-23 media. When porcine embryos cultured in PZM-3, cleavage rates were significantly decreased in the present of cycloheximide and both α-amanitin and cycloheximide treatments completely inhibited the blastocyst formation. Results showed that PZM-3 medium better supported porcine early embryonic development than NCSU-23 medium, and the inhibition of embryonic genome activation does not completely stop embryo cleavage but prevents development to the blastocyst stage.

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos

BackgroundIn the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction.ResultsIn order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated.ConclusionGood reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either GAPDH or PGK1, together with ribosomal protein S18 (S18), and UBC is proposed as reference genes, but the use of B2M, BACT, or H2A is discouraged.

Ratio of inner cell mass and trophoblastic cells in blastocysts derived from porcine 4- and 8-cell embryos and isolated blastomeres cultured in vitro in the presence or absence of protein and human leukemia inhibitory factor.

In this study we investigated effects of developmental stage at onset of individual in vitro culture on the progress of development in intact embryos and individual blastomeres derived from 4-cell and 8-cell porcine embryos (referred to as 1/4 and 1/8, respectively), the necessity of serum or BSA supplementation in culture media for embryos and blastomeres (in contrast to development in a defined medium), and the role of two concentrations of human leukemia inhibitory factor (hLIF) on development of blastomeres and embryos. More (p < 0.05) 1/4 blastomeres developed to the blastocyst stage than did 1/8 blastomeres. In the serum-supplemented medium, the percentage of inner cell mass (ICM)/total cells in 8-cell- and 1/8-derived blastocysts was higher (p < 0.05) than that in 4-cell- and 1/4-derived embryos. Development to blastocysts was similar in BSA-supplemented and defined medium as compared to that in serum-enriched medium in intact 4-cell and 8-cell embryos, and 1/4 blastomeres. More (p < 0.05) 1/8 blastomeres developed to the blastocyst stage in serum-supplemented medium than in defined medium. The high hLIF concentration (1000 IU/ml) decreased (p < 0.05) blastocyst development in 1/4 blastomeres in defined medium, but fewer blastocysts (p > 0.05) lacked an ICM (blastocyst-like vesicles) than in defined medium without hLIF. It is concluded that 1) porcine intact embryos and isolated blastomeres can be cultured individually in defined medium up to the blastocyst stage from the 4-cell stage onwards; 2) more 1/4 isolated blastomeres develop to blastocysts with more total cells but with a lower ratio of ICM to total cells than blastocysts derived from 1/8 blastomeres; 3) the effects of hLIF are dependent on proteins present in the culture medium and on the embryonic stage; and 4) in defined medium, high concentrations of hLIF are inhibitory to blastocyst formation, but fewer blastocyst-like vesicles are formed. The defined culture system employed in this study allows examination of the effects of growth factors or cytokines in porcine early embryonic development.