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Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions. [ABSTRACT FROM AUTHOR]

To determine the beneficial effects and mechanisms of exogenous glutathione (GSH) during IVC on the embryonic development, bovine IVF zygotes were cultured in medium containing different concentrations of GSH, and the rate of cleavage and blastocyst development, total cell number of blastocysts, the inner cell mass:total cell number ratio, and intracellular GSH and reactive oxygen species concentrations were investigated. Gene expressions associated with embryonic development and GSH metabolism were measured using quantitative real-time polymerase chain reaction. At the concentrations of 1, 3, and 5 mM, GSH significantly increased the blastocyst rate and embryo quality. The highest blastocyst rate (51%) and the best embryo quality appeared in the 3-mM GSH treatment. Intracellular content of GSH of embryos at the two- to four-cell stage significantly increased, and the reactive oxygen species level decreased accordingly with 3-mM GSH treatment, but no significant differences were found in the four- to eight-cell stage and blastocysts. Gene expression analysis of the embryo regulator genes ( OCT4 , SOX2 , and KLF4 ), GSH synthesis genes ( GCLM , GCLC , and GSS ), and GSH utilization genes ( GSTP , GSTM , and GPX ) showed that the GSH had no significant impact on these genes. In conclusion, exogenous GSH during IVC improved developmental potential and quality of bovine IVF embryos, which was probably caused by the ability of GSH to maintain the redox balance. [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10-6 m or 10-8 m, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10-10 m melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10-9 m or 10-12 m melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals. [ABSTRACT FROM AUTHOR]

Abstract: In general, a diet enriched in polyunsaturated fatty acids (PUFAs) inhibits the development of obesity and decreases adipose tissue. The specific impacts of n-3 and n-6 PUFAs on adipogenesis, however, have not been definitively determined. Traditional in vivo and in vitro supplementation studies have yielded inconsistent or even contradictory results, which likely reflect insufficiently controlled experimental systems. Caenorhabditis elegans fat-1 gene encodes an n-3 fatty acid desaturase, and its heterologous expression represents an effective method both for altering the n-6/n-3 PUFA ratio and for evaluating the biological effects of n-3 and n-6 PUFAs. We sought to determine whether a reduced n-6/n-3 ratio could influence adipogenesis in 3T3-L1 cells. Lentivirus-mediated introduction of the fat-1 gene into 3T3-L1 preadipocytes significantly reduced the n-6/n-3 ratio and inhibited preadipocyte proliferation and differentiation. In mature adipocytes, fat-1 expression reduced lipid deposition, as measured by Oil Red O staining, and induced apoptosis. Our results indicate that a reduced n-6/n-3 ratio inhibits adipogenesis through several mechanisms and that n-3 PUFAs more effectively inhibit adipogenesis (but not lipogenesis) than do n-6 PUFAs. [Copyright &y& Elsevier]

Paraquat (PQ), a broad‐spectrum agricultural pesticide, causes cellular toxicity by increasing oxidative stress levels in various biological systems, including the reproductive system. PQ exposure causes embryotoxicity and reduces the developmental abilities of embryos. However, there is little information regarding the toxic effects of PQ on oocyte maturation. In this study, we studied the toxic effects of PQ exposure and the effects of melatonin on PQ‐induced damage in bovine oocytes. PQ exposure disrupted nuclear and cytoplasmic maturation, which was manifested as decreased cumulus cell expansion, reduced first polar body extrusion, and abnormal distribution patterns of cortical granules and mitochondria. In addition, PQ treatment severely disrupted the ability of the resulted in vitro‐produced embryos to develop to the blastocyst stage. Moreover, PQ exposure significantly increased the intracellular reactive oxygen species (ROS) level and early apoptotic rate, and decreased the glutathione (GSH) level, antioxidative CAT and GPx4 mRNA, and apoptotic‐related Bcl-2/Bax mRNA ratio. These results indicated that PQ causes reproductive toxicity in bovine oocytes. Melatonin application resulted in significant protection against the toxic effects of PQ in PQ‐exposed oocytes. The mechanisms underlying the role of melatonin included the inhibition of PQ‐induced p38 mitogen‐activated protein kinase (MAPK) activation, and restoration of abnormal trimethyl‐histone H3 lysine 4 (H3K4me3) and trimethyl‐histone H3 lysine 9 (H3K9me3) levels. These results reveal that melatonin serves as a powerful agent against experimental PQ‐induced toxicity during bovine oocyte maturation and could form a basis for further studies to develop therapeutic strategies against PQ poisoning. [ABSTRACT FROM AUTHOR]

The expression and function of bone morphogenetic protein 4 (BMP4) gene in bovine cumulus cells (CCs) was investigated to reveal the mechanisms by which it regulated cell apoptosis and proliferation. The mRNA and protein expression of BMP4 were detected using quantitative PCR (qPCR) and immunofluorescence staining in CCs. The effective siRNAs against BMP4 gene were screened using qPCR and western blotting. The mRNA expression levels of apoptosis-related genes and proliferation-related genes were estimated by qPCR after knocking-down the BMP4 gene in bovine CCs. Cell apoptosis, proliferation and cell cycle were measured with Annexin V-FITC, CCK-8 and propidium iodide staining by flow cytometry. Results showed that the BMP4 gene was expressed and its protein was in the cytoplasm and nuclei of bovine CCs. The BMP4 knockdown increased the cell apoptosis rate and upregulated the mRNA levels of apoptosis genes CASPASE-3 and BAX with downregulation of the anti-apoptosis gene BCL-2 (P < 0.05). The proliferation rate declined and the mRNA expression levels of proliferation-related genes PCNA , CDC42 and CCND2 were downregulated in the bovine CCs with BMP4 low expression (P < 0.05). The BMP4 knockdown significantly increased the percentage of G0/G1 phase cells while decreased that of S phase cells. Therefore, the expression of BMP4 and its biological functions on the cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. BMP4 knockdown induced cell apoptosis, cell cycle arrest and inhibited proliferation of bovine CCs. • Expression and function of BMP4 in cumulus cells cultured in vitro were first studied. • BMP4 knockdown induced the apoptosis of bovine cumulus cells. • BMP4 knockdown inhibited the proliferation of bovine cumulus cells. • BMP4 knockdown induced cell cycle arrest of bovine cumulus cells. [ABSTRACT FROM AUTHOR]

O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a ubiquitous, reversible, and highly dynamic post-translational modification, which takes charge of almost all biological processes examined. However, little information is available regarding the molecular regulation of O-GlcNAcylation in granulosa cell function and glucose metabolism. This study focused on the impact of disrupted O-GlcNAc cycling on the proliferation and apoptosis of bovine granulosa cells, and further aimed to determine how this influenced glucose metabolism. Pharmacological inhibition of OGT with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside (BADGP) led to decreased cellular O-GlcNAc levels, as well as OGT and OGA protein expressions, whereas increasing O-GlcNAc levels with the OGA inhibitor, O-(2-acetamido-2-deoxy- D -gluco-pyranosylidene) (PUGNAc), resulted in elevated OGA protein expression and decreased OGT protein expression in granulosa cells. Dysregulated O-GlcNAc cycling reduced cell viability, downregulated the proliferation-related genes of CDC42 and PCNA transcripts, upregulated the pro-apoptotic genes of BAX and CASPASE-3 mRNA and the ratio of BAX/BCL-2, and increased the apoptotic rate. Glycolytic enzyme activities of hexokinase and pyruvate kinase, metabolite contents of pyruvate and lactate, mitochondrial membrane potential, ATP levels, and intermediate metabolic enzyme activities of succinate dehydrogenase and malate dehydrogenase involved in the tricarboxylic acid cycle, were significantly impaired in response to altered O-GlcNAc levels. Moreover, inhibition of OGT significantly increased the expression level of thioredoxin-interacting protein (TXNIP), but repression of OGA had no effect. Collectively, our results suggest that perturbation of O-GlcNAc cycling has a profound effect on granulosa cell function and glucose metabolism. [ABSTRACT FROM AUTHOR]

This study aims to investigate the expression and function of absent, small, or homeotic 1-like (ASH1L) methyltransferase in bovine cumulus cells in order to reveal by which mechanisms ASH1L regulates epigenetic modification and apoptosis in cumulus cells. The location of ASH1L and the methylation pattern of H3K36 were detected using immunofluorescence staining in cumulus cells. Quantitative PCR (qPCR) and western blotting were used to screen for effective siRNA targeting the ASH1L gene. Also, the mRNA expression levels of apoptosis-related genes and polycomb inhibitory complex genes were estimated by qPCR after knocking down the ASH1L gene in bovine cumulus cells. Cell proliferation and apoptosis were measured with the CCK-8 method and Annexin V-FITC by flow cytometry, respectively. The results of immunofluorescence analysis showed that ASH1L is located in the nucleus of bovine cumulus cells and is distributed in a dotted pattern. ASH1L knockdown in cumulus cells induced a decrease in the levels of H3K36me1/2/3 methylation (P < 0.05). Additionally, ASH1L knockdown inhibited cell proliferation, increased the apoptosis rate, and upregulated the expression of apoptosis genes CASPASE-3 , BAX and BAX/BCL-2 ratio (P < 0.05). Meanwhile, the mRNA expression levels of EZH2 and SUZ12 , two subunits of PRC2 protein, were increased in cells with ASH1L knockdown (P < 0.05). Therefore, the expression of ASH1L methyltransferase and its function in on the apoptosis of bovine cumulus cells were first studied. The mechanism by which ASH1L regulates the histone methylation and apoptosis in cumulus cells was also revealed. • Expression and function of ASH1L in livestock cells were first studied. • ASH1L knockdown significantly declined H3K36me1/2/3 methylation in bovine cumulus cells. • ASH1L knockdown inhibited the proliferation of bovine cumulus cells. • ASH1L knockdown induced the apoptosis of bovine cumulus cells. [ABSTRACT FROM AUTHOR]

The fertilization capacity of sex‐sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex‐sorted bull sperm. In this study, the washing medium and fertilization medium of sex‐sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10–3, 1 × 10–4, 1 × 10–5, 1 × 10–6 M) or Lyc (0, 1 × 10–4, 1 × 10–5, 1 × 10–6, 1 × 10–7). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex‐sorted sperm were investigated. For the sex‐sorted sperm of bulls A, B and C, 1 × 10–3 M VC or 1 × 10–4 M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10–4 M Lyc significantly improved the blastocyst rates and their IFN‐tau expression of bulls A and C. In conclusion, supplementation of 1 × 10–3 M VC or 1 × 10–4 M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex‐sorted bull sperm during IVF procedure. [ABSTRACT FROM AUTHOR]

To explore the mechanisms leading to the poor quality of IVF blastocysts, the single-cell whole-genome methylation sequencing technique was used in this study to analyse the methylation patterns of bovine blastocysts derived from in vivo , fresh (IVF) or vitrified (V_IVF) oocytes. Genome methylation levels of blastocysts in the IVF and V_IVF groups were significantly lower than those of the in vivo group (P < 0.05). In all, 1149 differentially methylated regions (DMRs) were identified between the IVF and in vivo groups, 1578 DMRs were identified between the V_IVF and in vivo groups and 151 DMRs were identified between the V_IVF and IVF groups. For imprinted genes, methylation levels of insulin-like growth factor 2 receptor (IGF2R) and protein phosphatase 1 regulatory subunit 9A (PPP1R9A) were lower in the IVF and V_IVF groups than in the in vivo group, and the methylation level of paternally expressed 3 (PEG3) was lower in the V_IVF group than in the IVF and in vivo groups. Genes with DMRs between the IVF and in vivo and the V_IVF and IVF groups were primarily enriched in oocyte maturation pathways, whereas DMRs between the V_IVF and in vivo groups were enriched in fertilisation and vitrification-vulnerable pathways. The results of this study indicate that differences in the methylation of critical DMRs may contribute to the differences in quality between in vitro- and in vivo -derived embryos. Exploration of methylation characteristics of in vitro -produced (IVP) and in vivo -derived embryos can help improve the quality of IVP embryos. In this study, methylation patterns of IVP and in vivo- derived blastocysts were investigated by single-cell genome methylation sequencing, and the differentially methylated regions identified were primarily involved in oocyte maturation and activation. More attention should be paid to oocyte factors to improve the quality of IVP embryos. [ABSTRACT FROM AUTHOR]

Melatonin is a well-characterized antioxidant that has been successfully used to protect oocytes from reactive oxygen species during in vitro maturation (IVM), resulting in improved fertilization capacity and development ability. However, the mechanism via which melatonin improves oocyte fertilization capacity and development ability remains to be determined. Here, we studied the effects of melatonin on cytoplasmic maturation of bovine oocytes. In the present study, bovine oocytes were cultured in IVM medium supplemented with 0, 10-7, 10-9, and 10−11 mol/L melatonin, and the cytoplasmic maturation parameters of MII oocytes after IVM were investigated, including redistribution of organelles (mitochondria, cortical granules [ CGs], and endoplasmic reticulum [ ER]), intracellular glutathione ( GSH) and ATP levels, expression of endogenous antioxidant genes ( Cat, Sod1, and GPx), and fertilization-related events ( IP3R1 distribution and expression of CD9 and Juno). Our results showed that melatonin significantly improved the cytoplasmic maturation of bovine oocytes by improving the normal distribution of organelles, increasing intracellular GSH and ATP levels, enhancing antioxidant gene expression levels, and modulating fertilization-related events, all of which resulted in increased fertilization capacity and developmental ability. Meanwhile, melatonin also increased the mRNA and protein expression levels of the Tet1 gene and decreased the Dnmt1 gene mRNA and protein levels in bovine oocytes, indicating that melatonin regulates the expression of the detected genes via demethylation. These findings shed insights into the potential mechanisms by which melatonin improves oocyte quality during IVM. [ABSTRACT FROM AUTHOR]

Little information is available regarding the effect of melatonin on the quality and fertilization capability of sex-sorted bull sperm, and even less about the associated mechanism. Sex-sorted sperm from three individual bulls were washed twice in wash medium and incubated in a fertilization medium for 1.5 h, and each was supplemented with melatonin (0, 10−3 M, 10−5 M, 10−7 M, and 10−9 M). The reactive oxygen species (ROS) and endogenous antioxidant activity (glutathione peroxidase (GPx); superoxide dismutase (SOD); catalase (CAT)), apoptosis (phosphatidylserine [PS] externalization; mitochondrial membrane potential (Δψm)), acrosomal integrity events (malondialdehyde (MDA) level; acrosomal integrity), capacitation (calcium ion [Ca2+]i level; cyclic adenosine monophosphate (cAMP); capacitation level), and fertilization ability of the sperm were assessed. Melatonin receptor 1 (MT1) and 2 (MT2) expression were examined to investigate the involvement of melatonin receptors on sex-sorted bull sperm capacitation. Our results show that treatment with 10−5 M melatonin significantly decreased the ROS level and increased the GPx, SOD, and CAT activities of sex-sorted bull sperm, which inhibited PS externalization and MDA levels, and improved Δψm, acrosomal integrity, and fertilization ability. Further experiments showed that melatonin regulates sperm capacitation via MT1. These findings contribute to improving the fertilization capacity of sex-sorted bull sperm and exploring the associated mechanism. [ABSTRACT FROM AUTHOR]