Zinc improves the developmental ability of bovine in vitro fertilization embryos through its antioxidative action.

Zinc, an essential trace mineral, exerts a pivotal influence in various biological processes. Through zinc concentration analysis, we found that the zinc concentration in the bovine embryo in vitro culture (IVC) medium was significantly lower than that in bovine follicular fluid. Therefore, this study explored the impact of zinc sulfate on IVC bovine embryo development and investigated the underlying mechanism. The results revealed a significant decline in zygote cleavage and blastocyst development rates when zinc deficiency was induced using zinc chelator N, N, N', N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) in culture medium during embryo in vitro culture. The influence of zinc-deficiency was time-dependent. Conversely, supplementing 0.8 µg/mL zinc sulfate to culture medium (CM) increased the cleavage and blastocyst formation rate significantly. Moreover, this supplementation reduced reactive oxygen species (ROS) levels, elevated the glutathione (GSH) levels in blastocysts, upregulated the mRNA expression of antioxidase-related genes, and activated the Nrf2-Keap1-ARE signaling pathways. Furthermore, 0.8 µg/mL zinc sulfate enhanced mitochondrial membrane potential, maintained DNA stability, and enhanced the quality of bovine (in vitro fertilization) IVF blastocysts. In conclusion, the addition of 0.8 µg/mL zinc sulfate to CM could enhance the antioxidant capacity, activates the Nrf2-Keap1-ARE signaling pathways, augment mitochondrial membrane potential, and stabilizes DNA, ultimately improving blastocyst quality and in vitro bovine embryo development.

Bioinformatics identification of potential biomarkers and therapeutic targets for ischemic stroke and vascular dementia.

Ischemic stroke and vascular dementia, as common cerebrovascular diseases, with the former causing irreversible neurological damage and the latter causing cognitive and memory impairment, are closely related and have long received widespread attention. Currently, the potential causative genes of these two diseases have yet to be investigated, and effective early diagnostic tools for the diseases have not yet emerged. In this study, we screened new potential biomarkers and analyzed new therapeutic targets for both diseases from the perspective of immune infiltration. Two gene expression profiles on ischemic stroke and vascular dementia were obtained from the NCBI GEO database, and key genes were identified by LASSO regression and SVM-RFE algorithms, and key genes were analyzed by GO and KEGG enrichment. The CIBERSORT algorithm was applied to the gene expression profile species of the two diseases to quantify the 24 subpopulations of immune cells. Moreover, logistic regression modeling analysis was applied to illustrate the stability of the key genes in the diagnosis. Finally, the key genes were validated using RT-PCR assay. A total of 105 intersecting DEGs genes were obtained in the 2 sets of GEO datasets, and bioinformatics functional analysis of the intersecting DEGs genes showed that GO was mainly involved in the purine ribonucleoside triphosphate metabolic process，respiratory chain complex，DNA-binding transcription factor binding and active transmembrane transporter activity. KEGG is mainly involved in the Oxidative phosphorylation, cAMP signaling pathway. The LASSO regression algorithm and SVM-RFE algorithm finally obtained three genes, GAS2L1, ARHGEF40 and PFKFB3, and the logistic regression prediction model determined that the three genes, GAS2L1 (AUC: 0.882), ARHGEF40 (AUC: 0.867) and PFKFB3 (AUC: 0.869), had good diagnostic performance. Meanwhile, the two disease core genes and immune infiltration were closely related, GAS2L1 and PFKFB3 had the highest positive correlation with macrophage M1 (p < 0.001) and the highest negative correlation with mast cell activation (p = 0.0017); ARHGEF40 had the highest positive correlation with macrophage M1 and B cells naive (p < 0.001), the highest negative correlation with B cell memory highest correlation (p = 0.0047). RT-PCR results showed that the relative mRNA expression levels of GAS2L1, ARHGEF40, and PFKFB3 were significantly elevated in the populations of both disease groups (p < 0.05). Immune infiltration-based models can be used to predict the diagnosis of patients with ischemic stroke and vascular dementia and provide a new perspective on the early diagnosis and treatment of both diseases.

METTL3-mediated m6A methylation regulates granulosa cells autophagy during follicular atresia in pig ovaries.

Follicular atresia is a normal physiological event in mammals, yet its mechanism remains to be studied. Granulosa cell (GC) autophagy is closely associated with follicular atresia. The N6-methyladenosine (m6A) modification is the most common post-transcriptional modification in eukaryotes, but its role in follicular atresia is still unknown. In this study, the possible relationship amongst follicular atresia, GC autophagy and m6A modification was studied. Our results showed that the level of autophagy in GCs increased with the degree of follicle atresia, whereas the overall m6A level decreased. Rapamycin treatment induced atresia in vitro cultured follicles, whereas 3-Methyladenine inhibited follicular atresia. Progressed atretic follicle (PAF) GCs had significantly lower METTL3 levels and significantly higher FTO levels than healthy follicle (HF) GCs. Differential gene expression analysis of GCs in PAF and HF by RNA sequencing was showed that the autophagy-related genes ULK1, ULK2, ATG2A, and ATG2B were significantly elevated in the PAF. In cultured GCs, overexpression of METTL3 significantly decreased the mRNA level of ULK1, as well as the autophagy level, whereas knockdown of METTL3 by RNAi significantly increased the mRNA level of ULK1, as well as the autophagy level. Our results indicate that m6A modification can regulate autophagy in GCs and play a role in the process of porcine follicular atresia.

Blastocyst Stage Affects the Isolation and Culture of Buffalo Naive/Primed Embryonic Stem-Like Cells.

Since embryonic stem cells (ESCs) were first identified, significant progress has been achieved. However, the establishment of buffalo ESCs (bESCs) is still unclear. This study was undertaken to explore the effect of the blastocyst stage on the isolation of bESCs. Firstly, our results indicated that the pluripotent genes were mainly expressed at the early stages of blastocyst, and the attachment and colony formation rates of bESCs derived from expanded blastocyst and hatched blastocyst were significantly higher than early blastocyst and blastocyst. In the meantime, bESCs showed positive alkaline phosphatase activity and expressed genes like OCT4, NANOG, SOX2, c-MYC, CDH1, KLF4, and TBX3. Immunofluorescence also confirmed the expression of OCT4, SOX2. Embryoid bodies expressing three marker genes were generated from the differentiation experiment, and fibroblast, epithelial, and neuron-like cells were induced. Moreover, naive-related genes KLF4, TBX3, primed-related genes FGF5, ACTA2 were expressed in the cells, but not REX-1. Immunofluorescence and western blot confirmed the FGF5 expression. Furthermore, bESCs could maintain pluripotency with the signal of LIF and bFGF. In summary, our results indicated that expanded blastocyst and hatched blastocyst are more suitable for bESCs isolation.

RNAi-mediated knockdown of Xist improves development of the female buffalo (Bubalus bubalis) nuclear transfer embryos.

Xist plays a critical role in the X-chromosome inactivation (XCI), an important epigenetic reprogramming of somatic cell nuclear transfer (SCNT) embryos. Modulation of Xist expression enhanced the developmental ability of mouse cloned embryos. However, the roles of Xist in buffalo SCNT embryos remain unknown. In this study, we investigated the methylation and expression status of Xist in different genders of buffalo donor cells and various stages (two-cell, eight-cell, morula and blastocyst) of in vitro fertilization (IVF) and SCNT embryos. The methylation of Xist in SCNT-♀ and SCNT-♂ embryos was aberrant hypomethylation compared with the buffalo foetal fibroblast (♀-BFF and ♂-BFF), IVF-♀ and IVF-♂ embryos. At the eight-cell stage, Xist expression was significantly higher in SCNT-♀ embryos compared with those in SCNT-♂, IVF-♀ and IVF-♂ embryos (P < 0.05). Meanwhile, no significant difference was found between IVF-♀ and IVF-♂ embryos (P > 0.05). Accordingly, we suppressed Xist expression by RNAi-Xist in SCNT-♀ embryos. Results showed that injection of Xist-shRNA significantly improved the morula and blastocyst rates (P < 0.05). These results indicated that correcting the abnormal expression of the Xist gene contributed to the development of SCNT-♀ embryos.

Histone Demethylase KDM4D Could Improve the Developmental Competence of Buffalo (Bubalus Bubalis) Somatic Cell Nuclear Transfer (SCNT) Embryos.

Somatic cell nuclear transfer (SCNT) holds vast potential in agriculture. However, its applications are still limited by its low efficiency. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as an epigenetic barrier for this. Histone demethylase KDM4D could regulate the level of H3K9me3. However, its effects on buffalo SCNT embryos are still unclear. Thus, we performed this study to explore the effects and underlying mechanism of KDM4D on buffalo SCNT embryos. The results revealed that compared with the IVF embryos, the expression level of KDM4D in SCNT embryos was significantly lower at 8- and 16-cell stage, while the level of H3K9me3 in SCNT embryos was significantly higher at 2-cell, 8-cell, and blastocyst stage. Microinjection of KDM4D mRNA could promote the developmental ability of buffalo SCNT embryos. Furthermore, the expression level of ZGA-related genes such as ZSCAN5B, SNAI1, eIF-3a, and TRC at the 8-cell stage was significantly increased. Meanwhile, the pluripotency-related genes like POU5F1, SOX2, and NANOG were also significantly promoted at the blastocyst stage. The results were reversed after KDM4D was inhibited. Altogether, these results revealed that KDM4D could correct the H3K9me3 level, increase the expression level of ZGA and pluripotency-related genes, and finally, promote the developmental competence of buffalo SCNT embryos.

Wuzang Wenyang Huayu decoction regulates differentially expressed transcripts in the rats' hippocampus after cerebral hypoperfusion.

The modified Wenyang Huayu decoction has been widely used to treat vascular dementia in China for thousands of years. We have previously proved that a modified version, Wuzang Wenyang Huayu decoction has the potential to be a more effective clinical treatment that can attenuate cerebral ischaemic injury. However, the global transcript profile and signalling conduction pathways regulated by this recipe remains unclear. This study established a two-vessel occlusion rat model by bilateral common carotid artery occlusion. Two groups of rats were intragastrically treated Wuzang Wenyang Huayu 2.5 g/kg vs or Piracetam 0.15 g/kg for 2 weeks. Learning and memory abilities were measured with Morris water maze. Neuronal plasticity was observed by HE staining. Differentially expressed transcripts of rat hippocampus were analysed by transcriptomics with Illumina HiSeq2500 platform. Results showed that Wuzang Wenyang Huayu decoction significantly alleviated learning, memory deficits, coordination dysfunction and prevented hippocampus cellular injury; Results further revealed the increased gene expression in KEGG metabolic pathways (MT-ND2. MT-ND3, MT-ND4, MT-ND4L, MT-ND5 and MT-ATP8) and genes involved in signal transduction, carcinogenesis, immune system, endocrine system, nervous system etc (Results further revealed differential expression of genes involved in various systems, including MT-ND2) Our discovery is likely to provide new insights to molecular mechanisms of Wuzang Wenyang Huayu regarding hippocampal transcripts in a murine vascular dementia model.

The Role of 5-aza-2'-Deoxycytidine on Methylation Status of Xist Gene in Different Genders of Buffalo (Bubalus bubalis) Bone Marrow Mesenchymal Stem Cells.

Previous studies have demonstrated that proper concentration of 5-aza-2'-deoxycytidine (5-aza-CdR) treatment was advantageous to decrease DNA methylation level, but the relationships between 5-aza-CdR treatment and methylation status of imprinted genes are seldom detected. The aim of this study was to investigate the effect of low concentration 5-aza-CdR treatment on the methylation status of imprinted gene Xist in different genders of buffalo bone marrow mesenchymal stem cells (BMSCs). BMSCs were isolated and the cell gender was identified through polymerase chain reaction (PCR). Then different concentrations of 5-aza-CdR (0, 0.02, 0.1 µM) were applied for the treatment. The results showed cellular morphology, growth, Xist gene expression pattern, and adherent ability were not significantly affected with the treatment of 5-aza-CdR for 24 hours. Meanwhile, immunofluorescence analysis indicated that the expression of 5-methylcytosine (5-mC) was also not influenced after the treatment. However, bisulfite sequence PCR (BS-PCR) analysis revealed that the methylation level of Xist differentially methylated region (DMR) decreased significantly when the concentration of 5-aza-CdR increased to 0.1 µM in the ♀BMSCs group (p < 0.05), while there was no significant difference among the ♂BMSCs-treated groups. Our results implied that low concentrations of 5-aza-CdR treatment had little impacts on cellular morphology, growth Xist gene expression pattern, adherent ability, and global DNA methylation level of BMSCs in both genders, but the treatment could significantly decrease the methylation level of Xist DMR in ♀BMSCs. Thus, we conclude 5-aza-CdR treatment can affect the methylation status of Xist DMR, furthermore, the influence is also related to sex differences.

Effect of sex differences in donor foetal fibroblast on the early development and DNA methylation status of buffalo (Bubalus bubalis) nuclear transfer embryos.

Low efficiency of somatic cell nuclear transfer (SCNT) embryos is largely attributable to imperfect reprogramming of the donor nucleus. The differences in epigenetic reprogramming between female and male buffalo cloned embryos remain unclear. We explored the effects of donor cell sex differences on the development of SCNT embryos. We and then compared the expression of DNA methylation (5-methylcytosine-5mC and 5-hydroxymethylcytosine-5hmC) and the expression level of relevant genes, and histone methylation (H3K9me2 and H3K9me3) level in SCNT-♀ and SCNT-♂ preimplantation embryos with in vitro fertilization (IVF) counterparts. In the study, we showed that developmental potential of SCNT-♀ embryos was greater than that of SCNT-♂ embryos (p < 0.05). 5mC was mainly expressed in SCNT-♀ embryos, whereas 5hmC was majorly expressed in SCNT-♂ embryos (p < 0.05). The levels of DNA methylation (5mC and 5hmC), Dnmt3b, TET1 and TET3 in the SCNT-♂ embryos were higher than those of SCNT-♀ embryos (p < 0.05). In addition, there were no significant differences in the expression of H3K9me2 at eight-stage of the IVF, SCNT-♀ and SCNT-♂embryos (p < 0.05). However, H3K9me3 was upregulated in SCNT-♂ embryos at the eight-cell stage (p < 0.05). Thus, KDM4B ectopic expression decreased the level of H3K9me3 and significantly improved the developmental rate of two-cell, eight-cell and blastocysts of SCNT-♂ embryos (p < 0.05). Overall, the lower levels of DNA methylation (5mC and 5hmC) and H3K9me3 may introduce the greater developmental potential in buffalo SCNT-♀ embryos than that of SCNT-♂ embryos.

Effects of Scriptaid on the Histone Acetylation, DNA Methylation and Development of Buffalo Somatic Cell Nuclear Transfer Embryos.

The present study was undertaken to examine the effect of Scriptaid treatment on histone acetylation, DNA methylation, expression of genes related to histone acetylation, and development of buffalo somatic cell nuclear transfer (SCNT) embryos. Treatment of buffalo SCNT embryos with 500 nM Scriptaid for 24 h resulted in a significant increase in the blastocyst formation rate (28.2% vs. 13.6%, p<0.05). Meanwhile, treatment of buffalo SCNT embryos with Scriptaid also resulted in higher acetylation levels of H3K18 and lower methylation levels of global DNA at the blastocyst stage, which was similar to fertilized counterparts. The expression levels of CBP, p300, HAT1, Dnmt1, and Dnmt3a in SCNT embryos treated with Scriptaid were significantly lower than the control group at the eight-cell stage (p<0.05), but the expression of HAT1 and Dnmt1a was higher than the control group at the blastocyst stage (p<0.05). When 96 blastocysts developed from Scriptaid-treated SCNT embryos were transferred into 48 recipients, 11 recipients (22.9%) became pregnant, whereas only one recipient (11.1%) became pregnant following transfer of 18 blastocysts developed from untreated SCNT embryos into nine recipients. These results indicate that treatment of buffalo SCNT embryos with Scriptaid can improve their developmental competence, and this action is mediated by resulting in a similar histone acetylation level and global DNA methylation level compared to in vitro-fertilized embryos through regulating the expression pattern of genes related to histone acetylation and DNA methylation.

Effects of scriptaid on the histone acetylation of buffalo oocytes and their ability to support the development of somatic cell nuclear transfer embryos.

The present study was undertaken to investigate the effect of scriptaid treatment on histone H3 on lysine 18 (H3K18) acetylation and relative expression levels of genes related to histone acetylation (HAT1, CBP, and p300) in buffalo oocytes during IVM. Meanwhile, the embryonic developmental ability of buffalo oocytes after SCNT was also examined. The H3K18 acetylation in oocytes increased from the germinal vesicle (GV) stage to the GV breakdown (GVBD) stage and arrived at a high acetylation level at the GVBD stage. Then, the H3K18 deacetylated completely at the metaphase I (MI) and acetylated again at the MII stage. However, addition of 500-nM scriptaid to the maturation medium resulted in a significant increase in the H3K18 acetylation at the MI stage. The expression profiles of genes related to histone acetylation (HAT1, CBP, and p300) in the meiosis stages of oocytes matured in the medium supplemented with 500-nM scriptaid were significantly higher than those of the oocytes matured in the medium without scriptaid (P < 0.05) with the exception of p300 at the GVBD stage. More SCNT embryos reconstructed with oocytes matured in the medium supplemented with 500-nM scriptaid developed to blastocysts (23.1%) in comparison with oocytes matured in the medium without scriptaid (13.8%, P < 0.05). These results indicate that scriptaid can increase the histone acetylation of buffalo oocytes during meiotic maturation and improve their ability to support the development of SCNT embryos.