Melatonin regulates the antioxidant capacity of sheep granulosa cells through a novel uORF-Nrf2aa mediated Nrf2/KEAP1 pathway.

Ovarian dysfunction stands as a prevalent contributor to female infertility, with its etiology intertwined with genetic, autoimmune, and environmental factors. Within the ovarian follicles, granulosa cells (GCs) represent the predominant cell population. Alterations in GCs, notably oxidative stress (OS) and the consequential surge in reactive oxygen species (ROS), play pivotal roles in the orchestration of ovarian function. Nrf2aa, a newly identified upstream open reading frame (uORF), is situated within the 5' untranslated region (5'UTR) of sheep Nrf2 mRNA and is regulated by melatonin, a crucial intrafollicular antioxidant. In this study, we have noted that Nrf2aa has the capacity to encode a peptide and exerts a negative regulatory effect on the translation efficiency (TE) of the Nrf2 CDs region. Further in vitro experiments, we observed that interfering with Nrf2aa can enhance the cellular functionality of GCs under 3-np-induced oxidative stress, while overexpressing Nrf2aa has the opposite effect. Furthermore, overexpression of Nrf2aa counteracts the rescuing effect of melatonin on the cellular functions of GCs under oxidative stress conditions, including estrogen secretion, proliferation, apoptosis, and many more. Finally, we confirmed that Nrf2aa, by regulating the expression of key proteins in the Nrf2/KEAP1 signaling pathway, further modulates the antioxidant levels in GCs.

FTO demethylates regulates cell-cycle progression by controlling CCND1 expression in luteinizing goat granulosa cells.

In mammals, N6-methyladenosine (m6A) stands out as one of the most abundant internal mRNA modifications and plays a crucial role in follicular development. Nonetheless, the precise mechanism by which the demethylase FTO regulates the progression of the goat luteinizing granulosa cells (LGCs) cycle remains to be elucidated. In our study, we primarily assessed the protein and mRNA expression levels of genes using Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), cell proliferation via EdU, cell viability with CCK-8, and apoptosis and cell cycle progression through flow cytometry. Here, the results demonstrated that knockdown of FTO significantly enhanced apoptosis, impeded cell proliferation, and increased autophagy levels in goat LGCs. Furthermore, the silencing of FTO substantially reduced cyclin D1 (CCND1) expression through the recognition and degradation of YTHDF2, consequently prolonging the cell cycle progression. This study sheds light on the mechanism by which FTO demethylation governs cell cycle progression by controlling the expression of CCND1 in goat LGCs, underscoring the dynamic role of m6A modification in the regulation of cell cycle progression.

N6-methyladenosine methyltransferase METTL3 modulates the cell cycle of granulosa cells via CCND1 and AURKB in Haimen goats.

N6-methyladenosine (m6A) plays a crucial role in many bioprocesses across species, but its function in granulosa cells during oocyte maturation is not well understood in animals, especially domestic animals. We observed an increase in m6A methyltransferase-like 3 (METTL3) in granulosa cells during oocyte maturation in Haimen goats. Our results showed that knockdown of METTL3 disrupted the cell cycle in goat granulosa cells, leading to aggravated cell apoptosis and inhibition of cell proliferation and hormone secretion. Mechanistically, METTL3 may regulate the cell cycle in goat granulosa cells by mediating Aurora kinase B (AURKB) mRNA degradation in an m6A-YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) manner and participating in AURKB transcription via the Cyclin D1 (CCND1)-Retinoblastoma protein (RB)-E2F transcription factor 1 (E2F1) pathway. Overall, our study highlights the essential role of METTL3 in granulosa cells during oocyte maturation in Haimen goats. These findings provide a theoretical basis and technical means for understanding how RNA methylation participates in oocyte maturation through granulosa cells.

chi-miR-130b-3p regulates the ZEA-induced oxidative stress damage through the KEAP1/NRF2 signaling pathway by targeting SESN2 in goat GCs.

As a dominant mycotoxin, zearalenone (ZEA) has attracted extensive attention due to its estrogen-like effect and oxidative stress damage in cells. In order to find a way to relieve cell oxidative stress damage caused by ZEA, we treated goat granulosa cells (GCs) with ZEA and did a whole transcriptome sequencing. The results showed that the expression level of Sesterin2 (SESN2) was promoted extremely significantly in the ZEA group (p < .01). In addition, our research demonstrated that SESN2 could regulate oxidative stress level in GCs through Recombinant Kelch Like ECH Associated Protein 1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. The overexpression of SESN2 could reduce the oxidative damage, whereas knockdown of SESN2 would aggravate the oxidative damage caused by ZEA. What's more, microRNA (miRNA) chi-miR-130b-3p can bind to SESN2 3'-untranslated region (3'UTR) to regulate the expression of SESN2. The mimics/inhibition of chi-miR-130b-3p would have an effect on oxidative damage triggered by ZEA in GCs as well. In summary, these results elucidate a new pathway by which chi-miR-130b-3p affects the KEAP1/NRF2 pathway in GCs by modulating SESN2 expression in response to ZEA-induced oxidative stress damage.

Melatonin protects Leydig cells from HT-2 toxin-induced ferroptosis and apoptosis via glucose-6-phosphate dehydrogenase/glutathione -dependent pathway.

HT-2 toxin is a mycotoxin commonly found in food and water that can have adverse effects on male reproductive systems, including testosterone secretion. Ferroptosis and apoptosis are two types of programmed cell death that have been implicated in the regulation of cellular functions. Melatonin, a powerful antioxidant with various physiological functions, has been shown to regulate testosterone secretion. However, the mechanisms underlying the protective effects of melatonin against HT-2 toxin-induced damage in testosterone secretion are not fully understood. In this study, we investigated the effects of HT-2 toxin on sheep Leydig cells and the potential protective role of melatonin. We found that HT-2 toxin inhibited cell proliferation and testosterone secretion of Leydig cells in a dose-dependent manner and induced ferroptosis and apoptosis through intracellular reactive oxygen species accumulation, leading to lipid peroxidation. Exposure of Leydig cells to melatonin in vitro reversed the defective phenotypes caused by HT-2 toxin via a glucose-6-phosphate dehydrogenase/glutathione-dependent mechanism. Interference of glucose-6-phosphate dehydrogenase disrupted the beneficial effect of melatonin on ferroptosis and apoptosis in HT-2 toxin-treated Leydig cells. Furthermore, similar results were observed in vivo in the testes of male mice injected with HT-2 toxin with or without melatonin treatment for 30 days. Our findings suggest that melatonin inhibits ferroptosis and apoptosis by elevating the expression of glucose-6-phosphate dehydrogenase to eliminate reactive oxygen species accumulation in HT-2 toxin-treated Leydig cells. These results provide fundamental evidence for eliminating the adverse effects of HT-2 toxin on male reproduction.

Dietary potential probiotics and enzymes complex modulates the performance and rumen microbiota in weaned goats.

AIMS: This trial was performed to investigate the effects of combined feeding of Candida utilis CICC 31170, Bacillus coagulans R11, and Lactobacillus acidophilus NCFM and a multi-enzyme complex on the growth performance, immune parameters, feed digestibility, and rumen microbiota of weaned goats. METHODS AND RESULTS: Thirty weaned goats were randomly divided into CON, PRB, and COB groups and fed different diets. End weight and ADG increased significantly in the PRB and COB groups (P < 0.05), and ADFI increased significantly in COB (P < 0.05). On day 80, there was a significant increase in IL-10 content in PRB and COB compared to the CON (P < 0.05). Highly significant increases in rumen papilla width, epithelial cell thickness, stratum spinosum+basale thickness, and stratum corneum thickness were found in PRB and COB (P < 0.05). COB group significantly increased the gene expression of HMGCL and MCT1 in rumen epithelium (P < 0.001). The COB group had the tendency to increase the feed digestibility of dry matter and crude fat compared with the CON group (P < 0.10). The abundance of Prevotellaceae_unclassified was significantly higher in PRB (P < 0.05), and the abundance of Fibrobacteres was significantly higher in COB in comparison to those in CON (P < 0.05). CONCLUSIONS: The results indicate that the dietary potential probiotics and enzymes complex could modulate the growth performance, immunity, feed digestibility, and rumen microbiota in weaned goats.

Effects of Zearalenone on Apoptosis and Copper Accumulation of Goat Granulosa Cells In Vitro.

Zearalenone (ZEA), also known as F-2 toxin, is a mycotoxin. Despite numerous reports of ZEA impairing livestock production performance and fertility, little information is available, including information about the mechanism underlying damage to cell metal ion transport. Copper, which is essential for cell survival as a metal ion, can consist of a variety of enzymes that facilitate abundant metabolic processes. However, the accumulation of copper in cells can have toxic effects. Here, we intended to determine whether ZEA could impair goat granulosa cells (GCs) and alter the cellular copper concentration. GCs were divided into a negative control (NC) group (cells cultured with 0.1% dimethyl sulfoxide (DMSO) for 8 h) and a ZEA group (cells cultured with 200 µmol/L ZEA diluted in DMSO for 8 h). The results showed that ZEA could inhibit GC proliferation and impair cell viability. GCs showed significant increases in the apoptosis rate and oxidative stress levels, while their ability to synthesize estrogen decreased. In addition, RNA-seq results showed dramatic changes in the expression of copper transport-related genes. The expression levels of ATPase copper transporting alpha (ATP7A) and ATPase copper transporting beta (ATP7B) were significantly downregulated (p < 0.01), while the expression of solute carrier family 31 member 1 (SLC31A1) was not modified in the ZEA group compared with the NC group. In accordance with these trends, the copper concentration increased significantly in the ZEA group (p < 0.01). In summary, our results show that ZEA can negatively affect GCs and cause copper accumulation. This finding may provide a prospective line of research on the relationship between ZEA and the transport of copper ions in GCs.

YBX1 mediates alternative splicing and maternal mRNA decay during pre-implantation development.

BACKGROUND: In mammals, maternal gene products decay and zygotic genome activation (ZGA) during maternal to zygotic transition (MZT) is critical for the early embryogenesis. Y-box binding protein YBX1 plays vital roles in RNA stabilization and transcriptional regulation, but its roles remain to be elucidated during pre-implantation development. METHODS: In the present study, we re-analyzed transcriptional level of YBX1 in mice, human, bovine, and goat embryos using public RNA-seq datasets. We further performed siRNA microinjection to knock down the expression of YBX1, and RNA sequencing of the 8-cell stage embryos in the control and YBX1 knockdown group. To reveal the regulation mechanisms of YBX1, we conducted differentially expression analysis, alternative splicing (AS) analysis, enrichment analysis, and 5-EU staining using DESeq2, rMATs, clusterProfiler, and immunofluorescence technique, respectively. RESULTS: The expression of YBX1 was increased during MZT in goat, bovine, human, and mice, but significantly decreased in YBX1 knockdown embryos compared with the controls, suggesting successfully knockdown of YBX1. The percentage of blastocyst was decreased, while embryos blocked at the 2- and 4-cell stage were increased in YBX1 knockdown embryos compared to the controls. Using RNA-seq, we identified 1623 up-regulated and 3531 down-regulated genes in the 8-cell stage YBX1 knockdown embryos. Of note, the down-regulated genes were enriched in regulation of RNA/mRNA stability and spliceosome, suggesting that YBX1 might medicate RNA stability and AS. To this end, we identified 3284 differential AS events and 1322 differentially expressed maternal mRNAs at the 8-cell stage YBX1 knockdown embryos. Meanwhile, the splicing factors and mRNA decay-related genes showed aberrant expression, and the transcriptional activity during ZGA in goat and mice was compromised when YBX1 was knocked down. CONCLUSION: YBX1 serves an important role in maternal mRNA decay, alternative splicing, and the transcriptional activity required for early embryogenesis, which will broaden the current understanding of YBX1 functions during the stochastic reprogramming events.

The function of the m6A methyltransferase METTL3 in goat early embryo development under hypoxic and normoxic conditions.

It has been reported that N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) plays an important role in zygote genome activation during embryonic development, but the effects of METTL3 under oxidative stress in the early development of goat embryos remain largely unknown. In this study, zygotes were monitored at 72 and 168 h after fertilization, and they developed to the 8-cell stage and blastocyst stage under hypoxic conditions and normoxic conditions. Single-cell transcriptome sequencing was performed at the 8-cell stage and the blastocyst stage in the goat embryos, the differentially expressed METTL3 was screened from the sequencing results. We found that microinjection of small interfering RNA (siRNA) against METTL3 caused developmental arrest, both 8-cell rates (37.45 ± 2.21% vs. 47.09 ± 1.38%; P < 0.01) and blastocyst rates of Si-METTL3 (12.17% ± 2.84 vs. 20.83 ± 3.61%; P < 0.01) in Si-METTL3 group were significantly decreased compared with that of control under hypoxic conditions, significant changes were found in the m6A-related genes and the expression levels of critical transcription factors, such as, NANOG, GATA3, CDX2 and SOX17, were decreased. This study revealed the key role of METTL3 in the regulation of embryonic development under oxidative stress, and laid the foundation for further study of the crucial mechanism of oxidative stress during the early embryonic development of goats.

Expression pattern of alkB homolog 5 in goat testis and its role in spermatogonial stem cells.

RNA N6-methyladenosine (m6A) is essential for many bioprocesses in many species, but its role in goat testis development remains elusive, especially alkB homolog 5 (ALKBH5), one of the m6A demethylases. To this end, nine healthy Haimen goats of different ages were chosen randomly to provide testes. The results showed that the expression level of ALKBH5 was increased significantly (P < 0.05) in the 9-month group compared with the 0-day and 3-month groups, and ALKBH5 was located in goat spermatocytes with the highest expression level compared with Leydig cells and Sertoli cells. Thus, pcDNA3.1-ALKBH5 was constructed to explore the influences of the ALKBH5 increase in goat spermatogonial stem cells (SSC) in vitro. The results showed that the expression level of ALKBH5 in SSC transfected with pcDNA3.1-ALKBH5 (OE_ALKBH5) was significantly increased (P < 0.001) compared with that in SSC transfected with pcDNA3.1-EGFP (EGFP). With ALKBH5 overexpression in SSC, flow cytometry analysis showed that cells at G1 phase were significantly reduced (P < 0.01), while cells at S phase significantly increased (P < 0.01), and cell apoptosis was inhibited. Accordingly, the mRNA degradation of CCND1, CCNE1, and BCL2 was suppressed with ALKBH5 overexpression in SSC after treatment with actinomycin D. Furthermore, the mRNA levels of pluripotency maintenance- and cell differentiation-associated genes were changed between the two groups. Overall, the results indicated the crucial role of ALKBH5 during Haimen goat testis development. The results of this study provide a theoretical basis and technical means for RNA methylation participating in goat testis development.

Melatonin alleviated oxidative stress induced by energy restriction on sheep Leydig cells through Sirt1/Sod2 pathway.

Energy balance is essential for normal reproduction of ram. However, the effect of energy restriction (ER) on reactive oxygen species (ROS) of sheep Leydig cells (LCs) and the rescuee methods are still unclear. To investigate the in vitro effect of melatonin on cellular ROS in fER-treated sheep LCs and explore the underlying mechanism, Hu sheep LCs were restricted energy using no serum culture medium and resaved with 10 ng/ml melatonin, respectively. The results showed that ER significantly increased MDA level, while decreased CAT, GHS-px expression and ΔΨm (p < 0.05). Meanwhile, ER decreased testosterone concentration and cell proliferation rate (p < 0.05). And the expression of testosterone synthesis-related enzymes was also down-regulated by ER (p < 0.05). Furthermore, we revealed that melatonin reversed the defective phenotypes in ER-treated LCs via Sirt1/Sod2 pathway. The interference of Sirt1 abolished the melatonin-mediated improvement of cellular ROS and testosterone secretion. Taken together, our study firstly indicated that melatonin could alleviate the excessive ROS accumulation and promote testosterone biosynthesis in ER-treated sheep LCs via the activation of Sirt1/Sod2 pathway.

Characterization of transcriptional activity during ZGA in mammalian SCNT embryo†.

Developmental arrest of somatic cell nuclear transfer (SCNT) embryos first occurs at zygotic/embryonic genome activation (ZGA/EGA), which is critical for preimplantation development. However, study on transcriptome of SCNT embryos during ZGA/EGA is limited. In the present study, we performed RNA sequencing (RNA-seq) of the eight-cell SCNT embryos in goat and provide cross-species analysis of transcriptional activity of SCNT embryos during ZGA/EGA in mice, human, bovine, and goat. RNA-seq data revealed 3966 differentially expressed genes (DEGs) failed to be reprogrammed or activated during EGA of SCNT embryos in goat. Series test of cluster analysis showed four clusters of DEGs and similar changes of the clusters in the four species. Specifically, genes in cluster 3 were somehow upregulated compared with the donor cells and the in vitro fertilization embryo. Moreover, the histone methylation key players and N6-methyladenosine modifiers (SUV39H1, SETDB1, SETD2, KDM5B, IGF2BP1, and YTHDF2) were differentially expressed in SCNT embryos of all species. Finally, we identified three modules correlated with the development of SCNT embryos in mice and screened 288 genes (such as BTG4, WEE1, KLF3, and USP21) that are likely critical for SCNT reprogramming using weighted gene correlation network analysis. Our data will broaden the current understanding of transcriptome activity during stochastic reprogramming events and provide an excellent source for future studies.

Comprehensive Transcriptome Analysis of mRNA Expression Patterns of Early Embryo Development in Goat under Hypoxic and Normoxic Conditions.

It has been reported that hypoxic environments were more suitable for the in vitro development of mammalian embryos, but the underlying mechanisms were still unclear. In the present study, RNA-seq was performed to compare 8-cell-stage and blastocyst-stage goat embryos under hypoxic and normoxic conditions; zygotes were checked at 72 and 168 h to 8-cell stage (L8C) and blastocyst stage (LM) in hypoxic conditions and 8-cell stage (H8C) and blastocyst stage (HM) in normoxic conditions. In the H8C and L8C groups, 399 DEGs were identified, including 348 up- and 51 down-regulated DEGs. In the HM and LM groups, 1710 DEGs were identified, including 1516 up- and 194 down-regulated DEGs. The expression levels of zygotic genes, transcription factors, and maternal genes, such as WEE2, GDF9, HSP70.1, BTG4, and UBE2S showed significant changes. Functional enrichment analysis indicated that these DEGs were mainly related to biological processes and function regulation. In addition, combined with the pathway-gene interaction network and protein-protein interaction network, twenty-two of the hub genes were identified and they are mainly involved in energy metabolism, immune stress response, cell cycle, receptor binding, and signal transduction pathways. The present study provides comprehensive insights into the effects of oxidative stress on early embryo development in goats.

Long non-coding RNA lnc_3712 impedes nuclear reprogramming via repressing Kdm5b.

Long non-coding RNAs (lncRNAs) are involved in shaping chromosome conformation and regulation of preimplantation development. However, the role of lncRNA during somatic cell nuclear transfer (SCNT) reprogramming remains largely unknown. In the present study, we identified 114 upregulated lncRNAs in the 8-cell SCNT embryos as candidate key molecules involved in nuclear reprogramming in goat. We found that H3K4me3 was an epigenetic barrier in goat nuclear reprogramming that and injection of Kdm5b mRNA greatly improved SCNT embryos development through removal of H3K4me3. We further reported that knockdown of lnc_3712 increased the expression of Kdm5b, which led to H3K4me3 demethylation. Of note, the development of goat SCNT embryos was improved when lnc_3712 was knocked down, whereas the blastocyst rate showed no difference in lnc_3712 and Kdm5b double knockdown SCNT embryos compared with the negative control SCNT embryos. Specifically, in lnc_3712 knockdown SCNT embryos, partial of the transcriptional activity and the expression of critical embryonic genes (Wee1, Ctsb, and Ybx1) were similar with that of in vitro fertilization embryos. Therefore, our results elucidate the critical role of lnc_3712 in regulating the development of goat SCNT embryos via repressing Kdm5b, which advances our current understanding of the role of lncRNAs during nuclear reprogramming.

YTHDF2 Regulates Maternal Transcriptome Degradation and Embryo Development in Goat.

Maternal mRNA clearance is critical for the early embryo development, which is under the tight control of RNA N6-methyladenosine (m6A). However, little information is known regarding the maternal mRNA clearance and mechanisms behind it in farm animals. In the present study, 3362 differentially expressed genes (DEGs) were found during the maternal-to-zygotic transition (MZT) and determined as maternal mRNAs in goat. Of which, 1961 was decreased at the 4-cell stage embryos, while 1401 was trigged down-regulation at the 8-cell stage embryos, which were termed as maternally encoded mRNA decay genes and zygotic genome activation (ZGA)-dependent maternal mRNAs, respectively. The expression of m6A reader YTHDF2 was increased during goat ZGA, and knockdown of YTHDF2 resulted in decreased blastocyst rate. In the 8-cell stage YTHDF2 knockdown embryos, the M-decay and Z-decay maternal mRNA clearance were impaired. Specifically, the expression of deadenylase (CNOT1 and CNOT11) and decapping enzymes (DCP1A and DCP2) was decreased. In conclusion, we ascertained maternal mRNAs and inferred that maternal mRNA clearance is also ZGA-dependent in goat. We reported that YTHDF2 is vital for goat early embryogenesis as it advances maternal mRNA clearance, which might through the recruitment of deadenylases and mRNA decapping enzymes. This work will be of great value for understanding the stochastic reprogramming events during MZT and achieving better development of goat embryos in vitro.

DNA methylation dynamics during zygotic genome activation in goat.

DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, the subtle changes in DNA methylation differ in species, and, little information is known regarding the dynamics of DNA methylation at the single-base resolution in goat. In the present study, we studied the DNA methylation dynamics during goat zygotic genome activation (ZGA) at global and single-base resolution using immunostaining and reduced representation bisulfite sequencing, respectively. We showed that DNA methylation was decreased both at global and single-base resolution, and the expression of TET1 was increased while DNMT1 was decreased during ZGA in goat. We identified 51058 tiles of differential methylation regions (DMRs), which were enriched in the developmental process, the regulation of developmental process, AMPK signaling pathway, mTOR signaling pathway, autophagy, and lysosome, as revealed by GO and KEGG enrichment analysis. Furthermore, we found an association between the methylation level and the expression of imprinted genes (IGF2R, PEG3, and ZFP64), maternal genes (TRIM28, SETD1A, SIN3A, and NPM2), and zygotic genes (DUXA, IGF2BP1, WT1, and ZIM3), suggesting that DNA methylation is in the tight control of ZGA in goat by regulating the expression of the critical genes. Our data will help to understand the stochastic ZGA events to achieve better development of goat embryos in vitro and provide an excellent source for further ZGA studies.

Facile preparation of iron oxide doped Fe-MOFs-MW as robust peroxydisulfate catalyst for emerging pollutants degradation.

A novel catalyst (Fe-MOFs-MW) was facilely synthesized under microwave-assisted with NaOH as modulator for activating peroxydisulfate (PDS). The accelerated nucleation process was confirmed by Johnson-Mehl-Avrami (JMA) model. There were abundant reactive sites on prepared Fe-MOFs-MW while maintaining high Space-Time-Yield value up to 2300 kg/m3·d. Degradation performance of Fe-MOFs-MW as PDS catalyst on sulfamethoxazole (SMX) removal was evaluated. Results indicated that Fe-MOFs-MW with more Fe element anchored (10%) exhibited excellent catalytic capacity for PDS. Besides, the fantastic stability and reusability were confirmed through recycle experiment. After recycled for 4 times, the removal efficiency of SMX and TOC was 88% and 31.3% compared to 98% and 38% without recycling, respectively. An accurate prediction model on the degradation effect with water matrices coexisted was established by response surface methodology (RSM) method. Moreover, SO4·-, O2·- and ·OH were confirmed as the main reactive species through chemical quenching and EPR tests. The mechanism of Fe-MOFs-MW/PDS process mainly based on electron circulation theory was proposed. As the robust PDS catalyst, facile prepared Fe-MOFs-MW was promising in the treatment of emerging pollutants.

Aberrant DNA and histone methylation during zygotic genome activation in goat cloned embryos.

In somatic cell nuclear transfer (SCNT) embryos, developmental defects first appear at the time of zygotic genome activation (ZGA), a process that is under the control of DNA and histone methylation. However, dynamics of 5-mC and 5-hmC during ZGA differ between porcine and bovine SCNT embryos, and histone methylation during ZGA in goat SCNT embryos remains poorly understood. Therefore, in the present study, we investigated the dynamic changes of 5-mC, 5-hmC, H3K4me2/3, and H3K9me3, as well as the expression of key genes related to these epigenetic modifications, during ZGA in goat cloned embryos. Compared with the IVF embryos, the 5-mC signal intensity was significantly increased at the 2- and 4-cell stage SCNT embryos, and the H3K4me3 and H3K9me3 signal intensity was significantly increased at 2- to 8-cell stage SCNT embryos, while the 5-hmC and H3K4me2 signal intensity was significantly lower at the 4- and 8-cell stage SCNT embryos. Of note, the H3K9me3 level was also significantly higher, whereas H3K4me3 signal intensity showed no statistical difference in the pronuclear stage SCNT embryos. Moreover, the expression of TET2, DNMT3B, KDM4A, SUV39H1, G9A, and SETDB1 was significantly increased, while the expression of UHRF1, PCNA, KDM4B, KDM4D, KDM5A, KDM5B, and KDM5C was significantly decreased at the 8-cell stage SCNT embryos. Our data revealed aberrant DNA and histone methylation during ZGA in goat cloned embryos. We further inferred that the abnormally higher level of 5-mC, H3K4me3, and H3K9me3 might serve as epigenetic barriers of the reprogramming and modifying these aberrant modifications might be a promising strategy to improve cloning efficiency in goat.

Locus-Specific Regulation of Xist Expression Using the CRISPR-Cas9-Based System.

DNA methylation inhibitor or loss and gain of function of DNA methylation key players were widely used to investigate the regulation of X inactive-specific transcript (Xist) expression by DNA methylation, which results in global change of DNA methylation. Here, we reported a novel method for regulation of Xist using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. First, Xist expression was increased in 5-aza-2'-deoxycytidine-treated female goat fibroblast cells. Second, three single-guide RNAs (sgRNAs) that target the Xist differential methylation region (DMR) were inserted to deactivated Cas9 (dCas9) nuclease and the catalytic domain of the DNA methyltransferase Dnmt3a coexpression plasmid. Bisulfite PCR analysis and quantitative real-time PCR revealed that the methylation level of the DMR was significantly increased, while the expression of Xist was downregulated in all three sgRNAs, compared with the mock-transfected cells. Third, the methylation activity at the sites of 37 bp from the protospacer-adjacent motif sequence showed the strong change relative to the mock-transfected cells. Furthermore, genome-wide DNA methylation and expression of the DNA methylation key players were not statistically changed in all three sgRNAs. Therefore, we confirmed that Xist expression was regulated by DNA methylation, and directed DNA methylation of Xist DMR at locus-specific solution decreased Xist expression.