Sulforaphane suppresses paraquat-induced oxidative damage in bovine in vitro-matured oocytes through Nrf2 transduction pathway.

Sulforaphane (SFN), a bioactive phytocompound extracted from cruciferous plants, has received increasing attention due to its vital cytoprotective role in eliminating oxidative free radical through activation of nuclear factor erythroid 2-related factor (Nrf2)-mediated signal transduction pathway. This study aims at a better insight into the protective benefit of SFN in attenuating paraquat (PQ)-caused impairment in bovine in vitro-matured oocytes and the possible mechanisms involved therein. Results showed that addition of 1 µM SFN during oocyte maturation obtained higher proportions of matured oocytes and in vitro-fertilized embryos. SFN application attenuated the toxicological effects of PQ on bovine oocytes, as manifested by enhanced extending capability of cumulus cell and increased extrusion proportion of first polar body. Following incubation with SFN, oocytes exposed to PQ exhibited reduced intracellular ROS and lipid accumulation levels, and elevated T-SOD and GSH contents. SFN also effectively inhibited PQ-mediated increase in BAX and CASPASE-3 protein expressions. Besides, SFN promoted the transcription of NRF2 and its downstream antioxidative-related genes GCLC, GCLM, HO-1, NQO-1, and TXN1 in a PQ-exposed environment, indicating that SFN prevents PQ-caused cytotoxicity through activation of Nrf2 signal transduction pathway. The mechanisms underlying the role of SFN against PQ-induced injury included the inhibition of TXNIP protein and restoration of the global O-GlcNAc level. Collectively, these findings provide novel evidence for the protective role of SFN in alleviating PQ-caused injury, and suggest that SFN application may be an efficacious intervention strategy against PQ cytotoxicity.

Knockdown of CLAUDIN-6 Inhibited Apoptosis and Induced Proliferation of Bovine Cumulus Cells.

This study aims to investigate the effects of CLAUDIN-6 (CLDN6) on cell apoptosis and proliferation of bovine cumulus cells (CCs). Immunofluorescence staining was used to localize CLDN6 protein in CCs. Three pairs of siRNA targeting CLDN6 and one pair of siRNA universal negative sequence as control were transfected into bovine CCs. Then, the effective siRNA was screened by real-time quantitative PCR (RT-qPCR) and Western blotting. The mRNA expression levels of apoptosis related genes (CASPASE-3, BAX and BCL-2) and proliferation related genes (PCNA, CDC42 and CCND2) were evaluated by RT-qPCR in CCs with CLDN6 knockdown. Cell proliferation, apoptosis and cell cycle were detected by flow cytometry with CCK-8 staining, Annexin V-FITC staining and propidium iodide staining, respectively. Results showed that the CLDN6 gene was expressed in bovine CCs and the protein was localized in cell membranes and cytoplasms. After CLDN6 was knocked down in CCs, the cell apoptosis rate significantly decreased and the pro-apoptotic genes BAX and CASPASE-3 were down-regulated significantly, whereas the anti-apoptotic gene BCL-2 was markedly up-regulated (p < 0.05). Additionally, CLDN6 knockdown significantly enhanced cell proliferation of CCs at 72 h after siRNA transfection. The mRNA levels of proliferation-related genes PCNA, CCND2 and CDC42 increased obviously in CCs with CLDN6 knockdown (p < 0.05). After CLDN6 was down-regulated, the percentage of CCs at S phase was significantly increased (p < 0.05). However, there was no remarkable difference in the percentages of cells at the G0/G1 phase and G2/M phase between CCs with or without CLDN6 knockdown (p > 0.05). Therefore, the expression of CLDN6 and its effects on cell proliferation, apoptosis and cell cycle of bovine CCs were first studied. CLDN6 low expression inhibited cell apoptosis, induced cell proliferation and cell cycle arrest of bovine CCs.

Identification of the porcine IG-DMR and abnormal imprinting of DLK1-DIO3 in cloned pigs.

Correct reprogramming of the DLK1-DIO3 imprinted region is critical for the development of cloned animals. However, in pigs, the imprinting and regulation of the DLK1-DIO3 region has not been systematically analyzed. The objective of this study was to investigate the imprinting status and methylation regulation of the DLK1-DIO3 region in wild-type and cloned neonatal pigs. We mapped the imprinting control region, IG-DMR, by homologous alignment and validated it in sperm, oocytes, fibroblasts, and parthenogenetic embryos. Subsequently, single nucleotide polymorphism-based sequencing and bisulfite sequencing polymerase chain reaction were conducted to analyze imprinting and methylation in different types of fibroblasts, as well as wild-type and cloned neonatal pigs. The results showed that Somatic cell nuclear transfer (SCNT) resulted in hypermethylation of the IG-DMR and aberrant gene expression in the DLK1-DIO3 region. Similar to wild-type pigs, imprinted expression and methylation were observed in the surviving cloned pigs, whereas in dead cloned pigs, the IG-DMR was hypermethylated and the expression of GTL2 was nearly undetectable. Our study reveals that abnormal imprinting of the DLK1-DIO3 region occurs in cloned pigs, which provides a theoretical basis for improving the cloning efficiency by gene editing to correct abnormal imprinting.

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.

Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System.

BACKGROUND: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of IGF2-which disrupts the ZBED6-binding site-affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-binding site is also conserved in cattle. METHODS: In the present study, we introduced mutations in the ZBED6-binding site in intron3 of IGF2 in bovine fetal fibroblasts using the CRISPR/Cas9 system, and investigated the effect of disruption of ZBED6 binding on IGF2 expression. RESULTS: Eleven biallelic-mutant single-cell clones were established, three of which contained no foreign DNA residues. Single-cell clones 93 and 135 were used to produce cloned embryos. Dual-luciferase reporter assay in C2C12 cells demonstrated that the mutation in the ZBED6-binding site increases the promoter 3 activity of bovine IGF2. A total of 49 mutant cloned embryos were transplanted into surrogate cows. Unfortunately, all cloned embryos died before birth. IGF2 was found to be hypomethylated in the only fetus born (stillborn), which may have been due to the incomplete reprogramming. CONCLUSIONS: We efficiently constructed IGF2-edited cell lines and cloned embryos, which provided a theoretical basis and experimental materials for beef cattle breeding.

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.

Supplementation of EGF, IGF-1, and Connexin 37 in IVM Medium Significantly Improved the Maturation of Bovine Oocytes and Vitrification of Their IVF Blastocysts.

The quality and developmental capacity of oocytes derived from in vitro maturation (IVM) remain unsatisfactory, which greatly impairs the efficiency and application of embryo technologies. The present experiment was designed to investigate the effect of the supplementation of EGF, IGF-1, and Cx37 in an IVM medium on the maturation quality and development ability of bovine oocytes. The cytoplasmic maturation events of oocytes and the quality of in vitro fertilization (IVF) blastocysts were examined to investigate the relative mechanisms. Our results showed that the nuclear maturation and blastocyst development after the IVF of oocytes treated with 25 µg/mL Cx37 or the combination of 50 ng/mL EGF and 100 ng/mL IGF-1 were significantly increased compared to those of the control group (p < 0.05). Furthermore, the blastocyst rate, and blastocyst total cell number and survival rate after vitrification of the EGF+IGF-1+Cx37 group, were significantly higher than those of the control group (p < 0.05), but lower than those of the FSH+LH+EGF+IGF-1+Cx37 group (p < 0.05). The transzonal projection (TZP) intensity, glutathione (GSH) level, and mitochondrial function of the EGF+IGF-1+Cx37 group were significantly higher than that of the control group, and lower than those of the FSH+LH+EGF+IGF-1+Cx37 group, in contrast to the results of the reactive oxygen species (ROS) levels. In conclusion, our results showed that the supplementation of 50 ng/mL EGF, 100 ng/mL IGF-1, and 25 µg/mL Cx37 in the IVM of bovine oocytes significantly improved their quality and developmental ability by increasing the TZP, mitochondrial function, and GSH level.

Melatonin improves the maturation and developmental ability of bovine oocytes by up-regulating GJA4 to enhance gap junction intercellular communication.

Melatonin (MT) increases oocyte maturation by reducing reactive oxygen species level and enhancing oocyte antioxidant capacity. However, the mechanisms via which MT works are still poorly understood. In the present study, the effects of MT on the maturation rate and development ability of bovine oocytes were investigated. Then, the transcriptome of oocytes treated by MT was sequenced. Finally, the expression of gap junction protein alpha 4 (GJA4) protein and cAMP level were detected in bovine oocytes, and isoprenaline (enhancer of gap junctional intercellular communication (GJIC)) and heptanol (inhibitor of GJIC) were used to investigate the effect of MT on GJIC activity in bovine oocytes. Our results showed that MT significantly improved the maturation, developmental ability and mRNA expression of GJA4 of bovine oocytes. Meanwhile, MT significantly increased GJA4 protein level and cAMP level in bovine oocytes. In contrast to heptanol, both isoproterenol and MT significantly increased GJIC activity, nuclear maturation and the development ability of bovine oocytes. However, MT significantly restored the nuclear maturation and developmental ability of oocytes treated by heptanol. In conclusion, our results showed that MT improves the maturation and developmental ability of bovine oocytes by enhancing GJIC activity via up-regulating GJA4 protein expression in IVM progress.

The combination treatment of cholesterol-loaded methyl-ß-cyclodextrin and methyl-ß-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.

Many experiments show that vitrification significantly reduces the fertilization capacity of mammalian oocytes, restricting the application of vitrified oocytes. It has been proven that the JUNO protein plays a vital role in mammalian oocytes fertilization. However, little information is available about the effects of vitrification on the JUNO protein and the procedure to protect it in bovine oocytes. Here, the present study was designed to investigate the effect of vitrification on the JUNO protein level in bovine oocytes. In this study, MII oocytes were treated with cholesterol-loaded methyl-ß-cyclodextrin (CLC; 0, 10, 15, 20 mM) for 45 min before vitrification and methyl-ß-cyclodextrin (MßCD; 0, 2.25, 4.25, 6.25 mM) for 45 min after thawing (38-39°C). Then, the expression level and function of JUNO protein, cholesterol level in the membrane, the externalization of phosphatidylserine, sperm binding capacity and the developmental ability of vitrified bovine oocytes were examined. Our results showed that vitrification significantly decreased the JUNO protein level, cholesterol level, sperm binding capacity, development ability, and increased the promoter methylation level of the JUNO gene and apoptosis level of bovine oocytes. Furthermore, 15 mM CLC + 4.25 mM MßCD treatment significantly improved the cholesterol level and increased sperm binding and development ability of vitrified bovine oocytes. In conclusion, the combination treatment of cholesterol-loaded methyl-ß-cyclodextrin and methyl-ß-cyclodextrin significantly improves the fertilization capacity of vitrified bovine oocytes by protecting fertilization protein JUNO.

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.

Effects of Inhibin A on Apoptosis and Proliferation of Bovine Granulosa Cells.

Inhibin A is well known for its inhibitory properties against follicle-stimulating hormone (FSH), released through a pituitary-gonadal negative feedback loop to regulate follicular development. Ovarian folliculogenesis, hormonal biosynthesis, and gametogenesis are dependent on inhibins, playing vital roles in promoting or inhibiting cell proliferation. The present study explored the physiological and molecular response of bovine granulosa cells (GCs) to different concentrations of inhibin A in vitro. We treated the primary GCs isolated from ovarian follicles (3-6 mm) with different levels of inhibin A (20, 50, and 100 ng/mL) along with the control (0 ng/mL) for 24 h. To evaluate the impact of inhibin A on GCs, several in vitro cellular parameters, including cell apoptosis, viability, cell cycle, and mitochondrial membrane potential (MMP) were detected. Besides, the transcriptional regulation of pro-apoptotic (BAX, Caspase-3) and cell proliferation (PCNA, CyclinB1) genes were also quantified. The results indicated a significant (p < 0.05) increase in the cell viability in a dose-dependent manner of inhibin A. Likewise, MMP was significantly (p < 0.05) enhanced when GCs were treated with high doses (50, 100 ng/mL) of inhibin A. Furthermore, inhibin A dose (100 ng/mL) markedly improved the progression of the G1 phase of the cell cycle and increased the cell number in the S phase, which was supported by the up-regulation of the proliferating cell nuclear antigen PCNA (20, 50, and 100ng/mL) and CyclinB (100 ng/mL) genes. In addition, higher doses of inhibin A (50 and 100 ng/mL) significantly (p < 0.05) decreased the apoptotic rate in GCs, which was manifested by down regulating BAX and Caspase-3 genes. Conclusively, our study presented a worthy strategy for the first time to characterize the cellular adaptation of bovine GCs under different concentrations of inhibin A. Our results conclude that inhibin A is a broad regulatory marker in GCs by regulating apoptosis and cellular progression.

Thioredoxin-interacting protein regulates glucose metabolism and improves the intracellular redox state in bovine oocytes during in vitro maturation.

The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that negatively regulates glucose uptake into cells. However, little information is available regarding the function of TXNIP in bovine oocytes. Accordingly, the present study was performed to investigate the influence of TXNIP on glucose metabolism in bovine oocytes during in vitro maturation. Pharmacological inhibition of TXNIP by azaserine enhanced glucose uptake and imparted a specific metabolic effect on glycolysis and pentose phosphate pathway (PPP). RNA interference (RNAi) was adopted to further determine the biological significance of TXNIP in regulating glucose metabolism. The maturation rate and the developmental competence of TXNIP siRNA-treated oocytes were significantly improved. Knockdown of TXNIP in bovine oocytes significantly increased glycolysis by increasing the activities of phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase; pyruvate and lactate production; and intracellular ATP level, as well as mitochondrial activity. Furthermore, glucose metabolism through PPP was also enhanced by TXNIP depletion, as TXNIP siRNA treatment promoted glucose-6-phosphate dehydrogenase (G6PDH) activity and NADPH content, and helped maintain a high level of glutathione and a low level of reactive oxygen species within the oocytes. Further studies revealed that inhibition of TXNIP resulted increases in glucose transporter 1 (GLUT1) expression, as well as PFK1 platelet isoform (PFKP) and G6PDH mRNA levels. These results reveal that TXNIP depletion promotes oocyte maturation by enhancing both glycolysis and the PPP. During in vitro maturation of bovine oocytes, TXNIP serves as a key regulator of glucose uptake by controlling GLUT1 expression.

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.

No imprinted XIST expression in pigs: biallelic XIST expression in early embryos and random X inactivation in placentas.

Dosage compensation, which is achieved by X-chromosome inactivation (XCI) in female mammals, ensures balanced X-linked gene expression levels between the sexes. Although eutherian mammals commonly display random XCI in embryonic and adult tissues, imprinted XCI has also been identified in extraembryonic tissues of mouse, rat, and cow. Little is known about XCI in pigs. Here, we sequenced the porcine XIST gene and identified an insertion/deletion mutation between Asian- and Western-origin pig breeds. Allele-specific analysis revealed biallelic XIST expression in porcine ICSI blastocysts. To investigate the XCI pattern in porcine placentas, we performed allele-specific RNA sequencing analysis on individuals from reciprocal crosses between Duroc and Rongchang pigs. Our results were the first to reveal that random XCI occurs in the placentas of pigs. Next, we investigated the H3K27me3 histone pattern in porcine blastocysts, showing that only 17-31.8% cells have attained XCI. The hypomethylation status of an important XIST DMR (differentially methylated region) in gametes and early embryos demonstrated that no methylation is pre-deposited on XIST in pigs. Our findings reveal that the XCI regulation mechanism in pigs is different from that in mice and highlight the importance of further study of the mechanisms regulating XCI during early porcine embryo development.

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.

Detrimental effects of lipopolysaccharides on maturation of bovine oocytes.

OBJECTIVE: Gram-negative bacteria lipopolysaccharide (LPS) has been reported to be associated with uterine impairment, embryonic resorption, ovarian dysfunction, and follicle retardation. Here, we aimed to investigate the toxic effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. METHODS: First, we developed an in vitro model to study the response of bovine cumulus-oocyte complexes (COCs) to LPS stress. After incubating germinal vesicle COCs in 10 µg/mL of LPS, we analyzed the following three aspects: the expression levels of the LPS receptor toll-like receptor 4 (TLR4) in COCs, activities of intracellular signaling protein p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-κB); and the concentrations of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6. Furthermore, we determined the effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. RESULTS: The results revealed that LPS treatment significantly elevated TLR4 mRNA and protein expression levels in COCs. Exposure of COCs to LPS also resulted in a marked increase in activity of the intracellular signaling protein p-p38 MAPK and NF-κB. Furthermore, oocytes cultured in maturation medium containing LPS had significantly higher concentrations of the proinflammatory cytokines IL-1ß, TNF-α, and IL-6. LPS exposure significantly decreased the first polar body extrusion rate. The cytoplasmic maturation, characterized by polar body extrusion and distribution of peripheral cortical granules, was significantly impaired in LPS-treated oocytes. Moreover, LPS exposure significantly increased intracellular reactive oxygen species levels and the relative mRNA abundance of the antioxidants thioredoxin (Trx), Trx2, and peroxiredoxin 1 in oocytes. Moreover, the early apoptotic rate and the release of cytochrome C were significantly increased in response to LPS. The cleavage, morula, and blastocyst formation rates were significantly lower in parthenogenetically activated oocytes exposed to LPS, while the incidence of apoptotic nuclei in blastocysts was significantly increased. CONCLUSION: Together, these results provide an underlying mechanism by which LPS impairs maturation potential in bovine oocytes.

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos.

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.

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.