Disruption of O-GlcNAcylation Homeostasis Induced Ovarian Granulosa Cell Injury in Bovine.

O-linked ß-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.

Knockdown of bone morphogenetic protein 4 gene induces apoptosis and inhibits proliferation of bovine cumulus cells.

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.

Knockdown of ASH1L methyltransferase induced apoptosis inhibiting proliferation and H3K36 methylation in bovine cumulus cells.

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.

Protective effect of vitamin C and lycopene on the in vitro fertilization capacity of sex-sorted bull sperm by inhibiting the oxidative stress.

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.

Oocyte IVM or vitrification significantly impairs DNA methylation patterns in blastocysts as analysed by single-cell whole-genome methylation sequencing.

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 invivo, 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 invivo group (P<0.05). In all, 1149 differentially methylated regions (DMRs) were identified between the IVF and invivo groups, 1578 DMRs were identified between the V_IVF and invivo 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 invivo group, and the methylation level of paternally expressed 3 (PEG3) was lower in the V_IVF group than in the IVF and invivo groups. Genes with DMRs between the IVF and invivo and the V_IVF and IVF groups were primarily enriched in oocyte maturation pathways, whereas DMRs between the V_IVF and invivo 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 invitro- and invivo-derived embryos.

Melatonin Improves the Fertilization Capacity of Sex-Sorted Bull Sperm by Inhibiting Apoptosis and Increasing Fertilization Capacitation via MT1.

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.

Melatonin protects against paraquat-induced damage during in vitro maturation of bovine oocytes.

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.

Resveratrol significantly improves the fertilisation capacity of bovine sex-sorted semen by inhibiting apoptosis and lipid peroxidation.

The aim of this study was to test the effects of five different concentrations (0, 10-3, 10-4, 10-5, and 10-6 M) of resveratrol (Res) supplementation in bull sperm washing and fertilisation medium on levels of reactive oxygen species (ROS), phosphatidylserine (PS) externalisation, mitochondrial membrane potential (Δψm), ATP and malondialdehyde (MDA), acrosomal integrity, blastocyst rate, and blastocyst quality after in vitro fertilisation (IVF). The results for sex-sorted sperm from three bulls showed: (1) ROS and MDA levels in 10-3 M and 10-4 M Res groups were significantly lower than those of controls (P < 0.05); (2) the percentage of viable sperm, percentage of sperm with high Δψm, and the ATP content in 10-3 M and 10-4 M Res groups were significantly higher than those of controls (P < 0.05); (3) the percentage of viable sperm with acrosomal integrity, and the blastocyst percentage and quality of the 10-4 M Res group were significantly higher than those of controls (P < 0.05). In conclusion, 10-4 M Res supplementation in washing and fertilisation medium of sex-sorted bull sperm significantly decreased ROS, PS externalisation, and MDA, and protected mitochondrial function and acrosomal integrity, thereby increasing blastocyst percentage and quality following IVF.

Melatonin improves the fertilization capacity and developmental ability of bovine oocytes by regulating cytoplasmic maturation events.

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.

Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes.

Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca2+]i). However, little information about the mechanism by which vitrification increases [Ca2+]i levels or a procedure to regulate [Ca2+]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca2+]i, endoplasmic reticulum Ca2+ (ER Ca2+), and mitochondrial Ca2+ (mCa2+) levels. Our results showed that vitrification, especially with dimethyl sulfoxide (DMSO), can induce ER Ca2+ release into the cytoplasm, consequently increasing the [Ca2+]i and mCa2+ levels. Supplementing the cells with 10 µM 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM or BAPTA) significantly decreased the [Ca2+]i level and maintained the normal distribution of cortical granules in the vitrified bovine oocytes, increasing their fertilisation ability and cleavage rate after in vitro fertilisation (IVF). Treating vitrified bovine oocytes with 1 µM ruthenium red (RR) significantly inhibited the Ca2+ flux from the cytoplasm into mitochondria; maintained normal mCa2+ levels, mitochondrial membrane potential, and ATP content; and inhibited apoptosis. Treating vitrified oocytes with a combination of BAPTA and RR significantly improved embryo development and quality after IVF.

Effects of lipopolysaccharide on maturation of bovine oocyte in vitro and its possible mechanisms.

Lipopolysaccharide disturbs the secretion of gonadotropin, endometrial function and implantation efficiency. However, there is little information regarding the effects of lipopolysaccharide on cyclic ovary activity, especially oocyte maturation. Therefore, we aimed to investigate the effects of lipopolysaccharide on the maturation potential of bovine oocytes. We found that lipopolysaccharide exposure significantly decreased the first polar body extrusion rate and delayed the cell cycle progression. The abnormal spindle rate was significantly increased in lipopolysaccharide treatment group, accompanied by disrupted localization and level of phosphorylated mitogen-activated protein kinase (p-MAPK). Moreover, lipopolysaccharide treatment significantly increased intracellular reactive oxygen species (ROS) levels and the early apoptotic rate in oocytes. The pro-apoptotic caspase-3 and Bax mRNA levels and caspase-3 protein level were significantly increased, whereas the anti-apoptotic Bcl-2 and XIAP transcript abundance were significantly decreased in lipopolysaccharide exposure group. Furthermore, the dimethyl-histone H3 lysine 4 (H3K4me2) level was significantly increased, while the DNA methylation (5-mC) and dimethyl-histone H3 lysine 9 (H3K9me2) levels were markedly decreased in oocytes treated with lipopolysaccharide. In conclusion, lipopolysaccharide exposure inhibits the maturation potential of bovine oocytes by affecting cell cycle, cytoskeletal dynamics, oxidative stress, and epigenetic modifications.

Protective effects of melatonin on bovine sperm characteristics and subsequent in vitro embryo development.

We aimed to investigate the effect of melatonin on bovine frozen-thawed semen and its impact on fertilization outcome. Plasma membrane integrity, mitochondrial activity, acrosome integrity, and levels of intracellular reactive oxygen species (ROS) were measured in spermatozoa treated with different concentrations of melatonin. Melatonin-treated spermatozoa were then used for in vitro fertilization, followed by analysis of subsequent embryo development and the expression of apoptosis- and antioxidant-related genes. The results revealed that 10-5 and 10-3 M melatonin led to higher plasma membrane integrity, mitochondrial activity, and acrosome integrity, and significantly decreased intracellular ROS levels (P < 0.05). The blastocyst development rate of in vitro-produced bovine embryos originating from 10-3 M melatonin-treated spermatozoa was significantly higher, while the incidence of apoptotic nuclei in blastocysts was markedly lower than for embryos from any other group (P < 0.05). CASP3 and BAX mRNA abundance were significantly reduced whereas BCL2, XIAP, and CAT transcript abundance were significantly increased in embryos produced from spermatozoa treated with 10-3 M melatonin; GPX4 expression, however, was comparable in all treatment groups. Thus, 10-3 M melatonin can improve the quality of bovine frozen-thawed semen. These beneficial effects appear to influence preimplantation embryos, given the correlation with its anti-apoptotic and anti-oxidative properties. Mol. Reprod. Dev. 83: 993-1002, 2016 © 2016 Wiley Periodicals, Inc.

Melatonin inhibits paraquat-induced cell death in bovine preimplantation embryos.

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.

Exogenous glutathione supplementation in culture medium improves the bovine embryo development after in vitro fertilization.

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.

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

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.

Heterologous expression of C. elegans fat-1 decreases the n-6/n-3 fatty acid ratio and inhibits adipogenesis in 3T3-L1 cells.

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. Caenorhabditiselegans 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.