Knockdown of KDM5B Leads to DNA Damage and Cell Cycle Arrest in Granulosa Cells via MTF1.

KDM5B is essential for early embryo development, which is under the control of maternal factors in oocytes. Granulosa cells (GCs) play a critical role during oocyte mature. However, the role of KDM5B in GCs remains to be elucidated. In the current study, we found that KDM5B expressed highly in the ovaries and located in goat GCs. Using an RNA sequence, we identified 1353 differentially expressed genes in the KDM5B knockdown GCs, which were mainly enriched in cell cycle, cell division, DNA replication and the cellular oxidative phosphorylation regulation pathway. Moreover, we reported a decrease in the percentage of proliferated cells but an increase in the percentage of apoptotic cells in the KDM5B knockdown GCs. In addition, in the KDM5B knockdown GCs, the percentage of GCs blocked at the S phase was increased compared to the NC group, suggesting a critical role of KDM5B in the cell cycle. Moreover, in the KDM5B knockdown GCs, the reactive oxygen species level, the mitochondrial depolarization ratio, and the expression of intracellular phosphorylated histone H2AX (γH2AX) increased, suggesting that knockdown of KDM5B leads to DNA damage, primarily in the form of DNA double-strand breaks (DSBs). Interestingly, we found a down-regulation of MTF1 in the KDM5B knockdown GCs, and the level of cell proliferation, as well as the cell cycle block in the S phase, was improved. In contrast, in the group with both KDM5B knockdown and MTF1 overexpression, the level of ROS, the expression of γH2AX and the number of DNA DSB sites decreased. Taken together, our results suggest that KDM5B inhibits DNA damage and promotes the cell cycle in GCs, which might occur through the up-regulation of MTF1.

Circular RNA circUSP13 sponges miR-29c to promote differentiation and inhibit apoptosis of goat myoblasts by targeting IGF1.

Circular RNAs (circRNAs) are an indispensable element of post-transcriptional gene regulation, influencing a variety of biological processes including myogenic differentiation; however, little is known about the function of circRNA in goat myogenic differentiation. Using RNA-sequencing data from our laboratory, we explored the influences of circUSP13, as a candidate circRNA, on myoblast differentiation since its expression is higher in myoblasts of lamb (first day of age) than that of the fetus (75th day of pregnancy). In in vitro experiments, circUSP13 significantly promoted differentiation and inhibited apoptosis in goat primary myoblasts. Mechanistically, circUSP13 localized with miR-29c in the cytoplasm of goat myoblasts to regulate IGF1 expression. We further demonstrated that circUSP13 sponges miR-29c, promoting IGF1 expression that upregulated the expression of MyoG and MyHC. Thus, our results identified circUSP13 as a molecular marker for breeding programs of mutton production, as well as the circUSP13-miR-29c-IGF1 axis as a potential therapeutic target for combating muscle wasting.

The regulation of LncRNA GTL2 expression by DNA methylation during sheep skeletal muscle development.

DNA methylation has crucial roles in regulating the expression of genes involved in skeletal muscle development. However, the DNA methylation pattern of lncRNA during sheep skeletal muscle development remains unclear. This study investigated previous WGBS and LncRNA data in skeletal muscle of sheep (fetus and adult). We then focused on LncRNA GTL2, which is differentially expressed in skeletal muscle and has multiple DMRs. We found that the expression level of GTL2 decreased with age. GTL2 DMRs methylation levels were significantly higher in adult muscle than in fetal muscle. After 5AZA treatment, GTL2 expression was significantly increased in a dose-dependent manner.The dCas9-DNMT3A-sgRNA significantly reduced the expression level of GTL2 in cells, but increased GTL2 DMR methylation levels. The above studies indicate that dCas9-DNMT3A can effectively increase the methylation level in the DMR region of GTL2, the expression level of GTL2 is regulated by DNA methylation during muscle development.

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.

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.

LncRNA LOC102176306 plays important roles in goat testicular development.

Long ncRNAs regulate a complex array of fundamental biological processes, while its molecular regulatory mechanism in Leydig cells (LCs) remains unclear. In the present study, we established the lncRNA LOC102176306/miR-1197-3p/peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) regulatory network by bioinformatic prediction, and investigated its roles in goat LCs. We found that lncRNA LOC102176306 could efficiently bind to miR-1197-3p and regulate PPARGC1A expression in goat LCs. Downregulation of lncRNA LOC102176306 significantly supressed testosterone (T) synthesis and ATP production, decreased the activities of antioxidant enzymes and mitochondrial complex I and complex III, caused the loss of mitochondrial membrane potential, and inhibited the proliferation of goat LCs by decreasing PPARGC1A expression, while these effects could be restored by miR-1197-3p inhibitor treatment. In addition, miR-1197-3p mimics treatment significantly alleviated the positive effects of lncRNA LOC102176306 overexpression on T and ATP production, antioxidant capacity and proliferation of goat LCs. Taken together, lncRNA LOC102176306 functioned as a sponge for miR-1197-3p to maintain PPARGC1A expression, thereby affecting the steroidogenesis, cell proliferation and oxidative stress of goat LCs. These findings extend our understanding of the molecular mechanisms of T synthesis, cell proliferation and oxidative stress of LCs.

Reinterpreting sheep muscle strand-specific RNA sequencing data showing extensive 3'UTR extensions.

The more complex 3' UTR in higher organisms may have the function of increasing post-transcriptional gene regulation. Recent RNA sequencing technologies have provided us with the possibility to capture the complete 3' UTR landscape of different species and cells. However, no systematic analysis of sheep-related 3' UTR has been performed. Here, we conducted a detailed analysis of the 3' UTR with the primary goal of identifying intact 3' UTR landscapes in the sheep muscles of the three developmental stages. Based on strand-specific RNA sequencing (ssRNA-seq) data, we found that thousands of genes in sheep muscle are continuously transcribed after the UTR of the reference genome (Oar_v4.0). More than 66% of the 3' UTR extensions exhibit similar expression trends to their upstream gene exons. These 3' UTR extensions strongly enrich thousands of conserved microRNA binding sites. The 3' UTR extension-associated RNA of PFKM (PuaRNA) was predicted to be derived from the 3' UTR of PFKM. In sheep myocytes, myotubes and various tissues, the expression pattern of PuaRNA is positively correlated with that of PFKM. Taken together, these new 3' UTR annotations greatly extend the range of mammalian post-transcriptional regulatory networks, which have a particular impact on the regulation of sheep muscle development.

Exchanges of histone methylation and variants during mouse zygotic genome activation.

Minor and major zygotic genome activation (ZGA) are crucial for preimplantation development. During this process, histone variants and methylation influence chromatin accessibility and consequently regulated the expression of zygotic genes. However, the detailed exchanges of these modifications during ZGA remain to be determined. In the present study, the epigenetic modifications of histone 3 on lysine 9 (H3K9), 27 (H3K27) and 36 (H3K36), as well as four histone variants were determined during minor and major ZGA and in post-ZGA stages of mouse embryos. Firstly, microH2A1, H3K27me3 and H3K36me3 were asymmetrically stained in the female pronucleus during minor ZGA but lost staining in major ZGA. Secondly, H3K9me2 and H3K9me3 were strongly stained in the female pronucleus, but weakly stained in the male pronucleus and disappeared after ZGA. Thirdly, H2A.Z and H3.3 were symmetrically stained in male and female pronuclei during minor ZGA. Moreover, H3K27me2 was not statistically changed during mouse early development, while H3K36me2 was only detected in 2- and 4-cell embryos. In conclusion, our data revealed dynamics of histone methylation and variants during mice ZGA and provided details of their exchange in mice embryogenesis. Moreover, we further inferred that macroH2A1, H2A.Z, H3K9me2/3 and H3K27me2/3 may play crucial roles during mouse ZGA.

Effect of PPARGC1A on the development and metabolism of early rabbit embryos in vitro.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is a central regulator of mitochondrial biogenesis and metabolism, and its expression is closely related to embryo development. To gain insights into the possible mechanisms of PPARGC1A during early embryogenesis, the development potential, mitochondrial biogenesis, and the culture medium metabolomics of embryos were evaluated when PPARGC1A overexpressed or suppressed in rabbit zygotes. Results showed that different PPARGC1A levels in rabbit zygotes could affect blastocyst percentage, and the expressions of mitochondrial biogenesis and metabolic-related genes, as well as the glutathione and adenosine triphosphate levels during early embryo development. In addition, compared with the controls, 12 and 10 different metabolites involved in carbohydrate, amino acid, and fatty acid metabolism were screened in the 5 day's spent culture medium of PPARGC1A overexpressed and suppressed embryos by gas chromatography-mass spectrometer, respectively. Consistent with these metabolite changes, the transcriptions of genes encoding glucose transporters and fatty acid biosynthetic proteins in the embryos from different groups were regulated by PPARGC1A during rabbit embryo development. Taken together, these data provide evidence that PPARGC1A may regulate early rabbit embryo development through mitochondrial biogenesis and metabolism.

Highly methylated Xist in SCNT embryos was retained in deceased cloned female goats.

X (inactive)-specific transcript (Xist) is crucial in murine cloned embryo development, but its role in cloned goats remains unknown. Therefore, in this study we examined the expression and methylation status of Xist in somatic cell nuclear transfer (SCNT) embryos, as well as in ear, lung, and brain tissue of deceased cloned goats. First, the Xist sequence was amplified and a differentially methylated region was identified in oocytes and spermatozoa. Xist methylation levels were greater in SCNT- than intracytoplasmic sperm injection-generated female 8-cell embryos. In addition, compared with naturally bred controls, Xist methylation levels were significantly increased in the ear, lung, and brain tissue of 3-day-old female deceased cloned goats, but were unchanged in the ear tissue of female live cloned goats and in the lung and brain of male deceased cloned goats. Xist expression was significantly increased in the ear tissue of female live cloned goats, but decreased in the lung and brain of female deceased cloned goats. In conclusion, hypermethylation of Xist may have resulted from incomplete reprogramming and may be retained in 3-day-old female deceased cloned goats, subsequently leading to dysregulation of Xist.

Efficient generation of CLPG1-edited rabbits using the CRISPR/Cas9 system.

The sheep callipyge (CLPG) phenotype, a well-known muscular hypertrophy syndrome, is caused by an A-to-G transition in the CLPG1 locus. The mechanisms of CLPG phenotype are very complicated and remain to be further studied. Lacking suitable animal models containing CLPG mutations may partially contribute to these unanswered mechanisms. In this study, we confirmed that the CLPG1 locus, especially the 12-bp CLPG1 motif, is conserved in mammalian animals including rabbit. Then, we generated seven CLPG1-edited rabbits with 100% efficiency using CRISPR/Cas9 system combined with cytoplasm injection technology. All the newborn rabbits were mosaicism with numerous kinds of mutations around the target sites. Among the nine screened potential off-target sites (POTs) for the two sgRNAs used in this study, none off-target effect was detected. This indicated that we efficiently and precisely generated CLPG1-edited rabbits, and we believe that these newly generated rabbits will do help to unravel the mechanisms of the CLPG phenotype in the future.

Long noncoding RNAs exchange during zygotic genome activation in goat.

In mammals, their proper development during the early cleavage stages strongly relies on the gene products newly transcribed by zygotic genome activation (ZGA). Long noncoding RNAs (lncRNAs) have been characterized as key regulators of the ZGA process in mice and human. However, the ZGA stage has not yet been identified and epigenetic regulations of the ZGA process remain largely unknown in goats. Here, we show that ZGA occurred at the 8-cell stage in goats. During ZGA, trimethylation of H3K9 was dynamically changed but maintained strong staining in development arrested embryos. Using single-cell RNA sequencing, we identified 800 mRNAs and 250 lncRNAs as candidates of key molecules in goat preimplantation embryos. These mRNAs and lncRNAs were differentially expressed from 4- to 8-cell stage embryos and were strongly enriched in terms of retinoic acid receptor signaling pathway as well as signaling pathway regulating pluripotency of stem cells. In particular, we found that microinjection of siRNA against lnc_137 caused development arrest. Our results are consistent with the notion that lncRNAs play vital roles during ZGA, and the data presented here provide an excellent source for further ZGA lncRNA studies.

Effects of NRF1 on steroidogenesis and apoptosis in goat luteinized granulosa cells.

During goat follicular development, abnormal expression of nuclear respiratory factor 1 (NRF1) in granulosa cells may drive follicular atresia with unknown regulatory mechanisms. In this study, we investigated the effects of NRF1 on steroidogenesis and cell apoptosis by overexpressing or silencing it in goat luteinized granulosa cells (LGCs). Results showed that knockdown of NRF1 expression significantly inhibited the expression of STAR and CYP19A1, which are involved in sex steroid hormones synthesis, and led to lower estrogen levels. Knockdown of NRF1 resulted in an increased percentage of apoptosis, probably due to the release of cytochrome c from mitochondria, accompanied by upregulating mRNA and protein levels of apoptosis-related markers BAX, caspase 3 and caspase 9. These data indicate that NRF1 might be related with steroidogenesis and cell apoptosis. Furthermore, NRF1 silence reduced mitochondrial transcription factor A (TFAM) transcription activity, mtDNA copy number and ATP level. Simultaneously, knockdown of NRF1 suppressed the transcription and translation levels of SOD, GPx and CAT, decreased glutathione level and increased 8-OHdG level. However, the overexpression of NRF1 in LGCs or gain of TFAM in NRF1 silenced LGCs increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the antioxidant stress system and steroids synthesis. Taken together, aberrant expression of NRF1 could induce mitochondrial dysfunction and disturb the cellular redox balance, which lead to disturbance of steroid hormone synthesis, and trigger LGC apoptosis through the mitochondria-dependent pathway. These findings will be helpful for understanding the role of NRF1 in goat ovarian follicular development and atresia.

Effect of PGC-1α overexpression or silencing on mitochondrial apoptosis of goat luteinized granulosa cells.

During goat follicular development, abnormal expression of peroxisome proliferator- activated receptor gamma coactivator-1 alpha (PGC-1α) in granulosa cells (GCs) may contribute to follicular atresia with unknown regulatory mechanisms. In this study, we investigate the effect of ectopic expression or interference of PGC-1α on cell apoptosis of goat first passage granulosa cells (FGCs) in vitro. The results indicate that PGC-1α silencing by short hairpin RNA (shRNA) in goat FGCs significantly reduced mitochondrial DNA (mtDNA) copy number (P < 0.05), changed mitochondria ultrastructure, and induced cell apoptosis (P < 0.05). The transcription and translation levels of the apoptosis-related genes BCL-2-associated X protein (BAX), caspase 3, and caspase 9 were significantly up-regulated (P < 0.05, respectively). Moreover, the ratio of BAX/B-cell lymphoma 2 (BCL-2) was reduced (P < 0.05), and the release of cytochrome c (cyt c) and lactate dehydrogenase (LDH) was significantly enhanced (P < 0.05, respectively) in PGC-1α interference goat FGCs. Furthermore, the expression of anti-oxidative related genes superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx) and catalase (CAT) was down-regulated (P < 0.05, respectively) and the activity of glutathione/glutathione disulfide (GSH/GSSG) was inhibited (P < 0.05). While enforced expression of PGC-1α increased the levels of genes involved in the regulation of mitochondrial function and biogenesis, and enhanced the anti-oxidative and anti-apoptosis capacity. Taken together, our results reveal that lack of PGC-1α may lead to mitochondrial dysfunction and disrupt the cellular redox balance, thus resulting in goat GCs apoptosis through the mitochondria-dependent apoptotic pathway.

N-carbamylglutamate and L-arginine improved maternal and placental development in underfed ewes.

The objectives of this study were to determine how dietary supplementation of N-carbamylglutamate (NCG) and rumen-protected L-arginine (RP-Arg) in nutrient-restricted pregnant Hu sheep would affect (1) maternal endocrine status; (2) maternal, fetal, and placental antioxidation capability; and (3) placental development. From day 35 to day 110 of gestation, 32 Hu ewes carrying twin fetuses were allocated randomly into four groups: 100% of NRC-recommended nutrient requirements, 50% of NRC recommendations, 50% of NRC recommendations supplemented with 20g/day RP-Arg, and 50% of NRC recommendations supplemented with 5g/day NCG product. The results showed that in maternal and fetal plasma and placentomes, the activities of total antioxidant capacity and superoxide dismutase were increased (P<0.05); however, the activity of glutathione peroxidase and the concentration of maleic dialdehyde were decreased (P<0.05) in both NCG- and RP-Arg-treated underfed ewes. The mRNA expression of vascular endothelial growth factor and Fms-like tyrosine kinase 1 was increased (P<0.05) in 50% NRC ewes than in 100% NRC ewes, and had no effect (P>0.05) in both NCG- and RP-Arg-treated underfed ewes. A supplement of RP-Arg and NCG reduced (P<0.05) the concentrations of progesterone, cortisol, and estradiol-17ß; had no effect on T4/T3; and improved (P<0.05) the concentrations of leptin, insulin-like growth factor 1, tri-iodothyronine (T3), and thyroxine (T4) in serum from underfed ewes. These results indicate that dietary supplementation of NCG and RP-Arg in underfed ewes could influence maternal endocrine status, improve the maternal-fetal-placental antioxidation capability, and promote fetal and placental development during early-to-late gestation.

Abnormal expression of DNA methyltransferases and genomic imprinting in cloned goat fibroblasts.

Somatic cell nuclear transfer (SCNT) is a useful way to produce cloned animals. However, SCNT animals exhibit DNA methylation and genomic imprinting abnormalities. These abnormalities may be due to the faulty epigenetic reprogramming of donor cells. To investigate the consequence of SCNT on the genomic imprinting and global methylation in the donor cells, growth patterns and apoptosis of cloned goat fibroblast cells (CGFCs) at passage 7 were determined. Growth patterns in CGFCs were similar to the controls; however, the growth rate in log phase was lower and apoptosis in CGFCs were significantly higher (P < 0.01). In addition, quantitative expression analysis of three DNA methyltransferases (Dnmt) and two imprinted genes (H19, IGF2R) was conducted in CGFCs: Dnmt1 and Dnmt3b expression was significantly reduced (P < 0.01), and H19 expression was decreased sixfold (P < 0.01); however, the expression of Dnmt3a was unaltered and IGF2R expression was significantly increased (P < 0.05). Finally, we used bisulfite sequencing PCR to compare the DNA methylation patterns in differentially methylated regions (DMRs) of H19 and IGF2R. The DMRs of H19 (P < 0.01) and IGF2R (P < 0.01) were both highly methylated in CGFCs. These results indicate that the global genome might be hypomethylated. Moreover, there is an aberrant expression of imprinted genes and DMR methylation in CGFCs.

Ythdf2-mediated STK11 mRNA decay supports myogenesis by inhibiting the AMPK/mTOR pathway.

An emerging research focus is the role of m6A modifications in mediating the post-transcriptional regulation of mRNA during mammalian development. Recent evidence suggests that m6A methyltransferases and demethylases play critical roles in skeletal muscle development. Ythdf2 is a m6A "reader" protein that mediates mRNA degradation in an m6A-dependent manner. However, the specific function of Ythdf2 in skeletal muscle development and the underlying mechanisms remain unclear. Here, we observed that Ythdf2 expression was significantly upregulated during myogenic differentiation, whereas Ythdf2 knockdown markedly inhibited myoblast proliferation and differentiation. Combined analysis of high-throughput sequencing, Co-IP, and RIP assay revealed that Ythdf2 could bind to m6A sites in STK11 mRNA and form an Ago2 silencing complex to promote its degradation, thereby regulating its expression and consequently, the AMPK/mTOR pathway. Furthermore, STK11 downregulation partially rescued Ythdf2 knockdown-induced impairment of proliferation and myogenic differentiation by inhibiting the AMPK/mTOR pathway. Collectively, our results indicate that Ythdf2 mediates the decay of STK11 mRNA, an AMPK activator, in an Ago2 system-dependent manner, thereby driving skeletal myogenesis by suppressing the AMPK/mTOR pathway. These findings further enhance our understanding of the molecular mechanisms underlying RNA methylation in the regulation of myogenesis and provide valuable insights for conducting in-depth studies on myogenesis.

Lysine-Specific Demethylase 4D Is Critical for the Regulation of the Cell Cycle and Antioxidant Capacity in Goat Fibroblast Cells.

Oxidative damage to skin fibroblast cells is a causative factor in many skin diseases. Previous studies have reported that lysine-specific demethylase 4D (Kdm4d) is involved in DNA replication, but its role on antioxidant capacity remains unclear. In the present study, we used goat fibroblast cells (GFCs) as the research model and identified 504 up-regulated and 1013 down-regulated genes following the knockdown of Kdm4d, respectively. The down-regulated genes of this enzyme were found to be enriched in the cell cycle, DNA replication, mitotic processes, and the oxidative phosphorylation pathway, as previously revealed from gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and gene set enrichment analysis (GSEA), suggesting vital roles of the Kdm4d enzyme in the cell cycle and in antioxidant regulation. To this end, we found the cell proliferation rate was significantly decreased after the knockdown of Kdm4d. Moreover, both the mRNA and protein expression levels of superoxide dismutase 2 (SOD2), one of the major antioxidant enzymes, was decreased, while the reactive oxygen species (ROS) level was significantly increased in Kdm4d knocked-down cells. In addition, the expression of γH2A histone family member X (γH2AX) increased significantly, indicating the presence of DNA double-strand breaks after the knockdown of the Kdm4d enzyme. In conclusion, the knockdown of Kdm4d inhibited DNA replication and the cell cycle, repressed the expression of SOD2, and increased the generation of ROS, which led to the production of DNA damage in GFCs. Our data will be helpful for understanding the mechanism underlying antioxidant capacity regulation in fibroblast cells.

miR-101-5p overexpression suppresses the proliferation of goat spermatogonial stem cells by targeting EZH2.

MicroRNAs (miRNAs), as post-transcriptional gene mediators, regulate the biological characteristics of spermatogonial stem cells (SSCs), including proliferation, differentiation and apoptosis. However, the potential roles and mechanisms by which miR-101-5p affected the biological characters of goat SSCs have not been fully elucidated. Herein, we reported that miR-101-5p overexpression decreased cell viability (P < 0.01), arrested cell cycle in the G1 phase (P < 0.05), and aggravated apoptosis of goat SSCs (P < 0.01) compared with negative control (NC), as determined by CCK-8 assay and flow cytometry analysis. Additionally, PCNA protein expression was attenuated by miR-101-5p overexpression (P < 0.05). Notably, the expression of SSCs specific genes Oct4 (P < 0.05), PLZF (P < 0.01) and DAZL (P < 0.01) were decreased in miR-101-5p overexpressed SSCs. Furthermore, the dual luciferase reporter assay showed that, when co-transfected with miR-101-5p mimics, the relative luciferase activity of EZH2 wide-type (WT) was inhibited (P < 0.05) compared with the transfection of EZH2 mutant (MUT). EZH2 expression was negatively correlated with miR-101-5p expression in goat SSCs. Collectively, our data implicates that miR-101-5p overexpression aggravates cell apoptosis, and suppresses cell proliferation of goat SSCs via targeting EZH2, which may impair spermatogenesis.