Heat-Stress Impacts on Developing Bovine Oocytes: Unraveling Epigenetic Changes, Oxidative Stress, and Developmental Resilience.

Extreme temperature during summer may lead to heat stress in cattle and compromise their productivity. It also poses detrimental impacts on the developmental capacity of bovine budding oocytes, which halt their fertility. To mitigate the adverse effects of heat stress, it is necessary to investigate the mechanisms through which it affects the developmental capacity of oocytes. The primary goal of this study was to investigate the impact of heat stress on the epigenetic modifications in bovine oocytes and embryos, as well as on oocyte developmental capacity, reactive oxygen species, mitochondrial membrane potential, apoptosis, transzonal projections, and gene expression levels. Our results showed that heat stress significantly reduced the expression levels of the epigenetic modifications from histone H1, histone H2A, histone H2B, histone H4, DNA methylation, and DNA hydroxymethylation at all stages of the oocyte and embryo. Similarly, heat stress significantly reduced cleavage rate, blastocyst rate, oocyte mitochondrial-membrane potential level, adenosine-triphosphate (ATP) level, mitochondrial DNA copy number, and transzonal projection level. It was also found that heat stress affected mitochondrial distribution in oocytes and significantly increased reactive oxygen species, apoptosis levels and mitochondrial autophagy levels. Our findings suggest that heat stress significantly impacts the expression levels of genes related to oocyte developmental ability, the cytoskeleton, mitochondrial function, and epigenetic modification, lowering their competence during the summer season.

Effects of Regulating Hippo and Wnt on the Development and Fate Differentiation of Bovine Embryo.

The improvement of in vitro embryo development is a gateway to enhance the output of assisted reproductive technologies. The Wnt and Hippo signaling pathways are crucial for the early development of bovine embryos. This study investigated the development of bovine embryos under the influence of a Hippo signaling agonist (LPA) and a Wnt signaling inhibitor (DKK1). In this current study, embryos produced in vitro were cultured in media supplemented with LPA and DKK1. We comprehensively analyzed the impact of LPA and DKK1 on various developmental parameters of the bovine embryo, such as blastocyst formation, differential cell counts, YAP fluorescence intensity and apoptosis rate. Furthermore, single-cell RNA sequencing (scRNA-seq) was employed to elucidate the in vitro embryonic development. Our results revealed that LPA and DKK1 improved the blastocyst developmental potential, total cells, trophectoderm (TE) cells and YAP fluorescence intensity and decreased the apoptosis rate of bovine embryos. A total of 1203 genes exhibited differential expression between the control and LPA/DKK1-treated (LD) groups, with 577 genes upregulated and 626 genes downregulated. KEGG pathway analysis revealed significant enrichment of differentially expressed genes (DEGs) associated with TGF-beta signaling, Wnt signaling, apoptosis, Hippo signaling and other critical developmental pathways. Our study shows the role of LPA and DKK1 in embryonic differentiation and embryo establishment of pregnancy. These findings should be helpful for further unraveling the precise contributions of the Hippo and Wnt pathways in bovine trophoblast formation, thus advancing our comprehension of early bovine embryo development.

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.

Study on the expression patterns and function of JUNO and CD9 in bovine oocytes during in vitro maturation.

Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 µg/ml) or (ii) CD9 antibody (1, 5 and 25 µg/ml) or (iii) CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.

Vitrification of bovine germinal vesicle oocytes significantly decreased the methylation level of their in vitro derived MII oocytes.

CONTEXT: The vitrification of oocytes is important for the conservation of animals, and the effect of vitrification on methylation patterns of bovine oocytes remains unclear. AIMS: This article aims to investigate the effect of vitrification on the DNA methylation patterns on vitrified GV oocytes and their in vitro derived MII oocytes. METHODS: 5-MeC staining and single-cell whole genome bisulphite sequencing (SC-WGBS) were utilised to analyse fresh GV oocytes (F_GV group), MII oocytes (F_MII group), vitrified GV oocytes (V_GV group) and their in vitro derived MII oocytes (V_MII group). KEY RESULTS: Results of both 5-MeC staining and SC-WGBS showed that no significant difference was found between the F_GV group and the V_GV group, while the methylation level of the V_MII group was significantly lower than that of the F_MII group. Moreover, supplementation of 2µM resveratrol (Res) in IVM medium significantly improved maturation and development ability of vitrified GV oocytes by restoring their DNA methylation levels. CONCLUSION: In conclusion, vitrification of bovine GV oocytes significantly decreased the DNA methylation level of their in vitro derived MII oocytes, and 2µM Res improved their development ability by restoring DNA methylation level. IMPLICATIONS: Our results provide an efficient approach to improve the maturation and fertilisation ability of vitrified GV oocytes.

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.

Improvement of Fertilization Capacity and Developmental Ability of Vitrified Bovine Oocytes by JUNO mRNA Microinjection and Cholesterol-Loaded Methyl-ß-Cyclodextrin Treatment.

Vitrification of oocytes is crucial for embryo biotechnologies, germplasm cryopreservation of endangered and excellent female animals, and the fertility of humans. However, vitrification significantly impairs the fertilization ability of oocytes, which significantly limits its widely used application. JUNO protein, a receptor for Izumo1, is involved in sperm-oocyte fusion and is an indispensable protein for mammalian fertilization, and its abundance is susceptible to vitrification. However, it is still unclear how vitrification reduces the fertilization capacity of bovine oocytes by affecting JUNO protein. This study was designed to investigate the effect of vitrification on the abundance and post-translational modifications of JUNO protein in bovine oocytes. Our results showed that vitrification did not alter the amino acid sequence of JUNO protein in bovine oocytes. Furthermore, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis results showed that vitrification significantly reduced the number and changed the location of disulfide bonds, and increased the number of both phosphorylation and glycosylation sites of JUNO protein in bovine oocytes. Finally, the fertilization capacity and development ability of vitrified oocytes treated with 200 pg JUNO mRNA microinjection and cholesterol-loaded methyl-ß-cyclodextrin (CLC/MßCD) were similar to those of fresh oocytes. In conclusion, our results showed that vitrification of bovine oocytes did not alter the protein sequence of JUNO, but induced post-translational modifications and changed protein abundance. Moreover, the fertilization and development ability of vitrified bovine oocytes were improved by the combination treatment of JUNO mRNA microinjection and CLC/MßCD.

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.

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.

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.

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.

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.

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.

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.

MiR-744-3p regulates keratinocyte proliferation and differentiation via targeting KLLN in psoriasis.

Psoriasis is a chronic inflammatory disease, and microRNAs have been reported to regulate the pathogenesis of psoriasis. Up-regulated miR-744-3p level was identified to associate with psoriasis while the precise functions of miR-744-3p in psoriasis were not well-elucidated. We first confirmed the up-regulation of miR-744-3p in psoriasis by measuring its expression level in psoriatic samples. We explored the roles of miR-744-3p on keratinocytes proliferation and differentiation. We searched the targets of miR-744-3p and evaluated the roles of one target, KLLN on keratinocytes proliferation and differentiation. We confirmed the up-regulation of miR-744-3p in psoriatic samples. MiR-744-3p promoted keratinocytes proliferation while inhibited differentiation. MiR-744-3p targeted KLLN and overexpression of miR-744-3p resulted in decreased expression of KLLN. Overexpression of KLLN prevented the effects of miR-744-3p on keratinocytes proliferation and differentiation. MiR-744-3p regulated the proliferation and differentiation of keratinocytes through targeting KLLN in psoriasis.

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.

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.

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.

Neonatal respiratory distress syndrome and underlying mechanisms in cloned cattle.

Neonatal respiratory distress is a major mortality factor in cloned animals, but the pathogenesis of this disease is rarely investigated. In this study, four neonatal cloned cattle, born after full-term gestation, exhibited symptoms of neonatal respiratory distress syndrome (NRDS), which included symptoms of hyaline membrane disease as well as disordered surfactant homeostasis in their collapsed lungs. No differences in DNA methylation or histone modifications correlated with the suppressed SPB and SPC transcription observed in the cloned cattle group (p > 0.05), whereas TTF-1 occupancy at SPB and SPC promoter regions in cloned cattle was significantly reduced to 24% and 20% that of normal lungs, respectively (SPB, p < 0.05; SPC, p < 0.01). Decreased TTF1 expression, dysregulation of SPB and SPC transcription by TTF-1, and disordered proteolytic processing of Surfactant protein B precursor together potentially contribute to the disruption of surfactant homeostasis and NRDS in bovine clones. Elucidation of the associated mechanisms should facilitate the development of novel preventive or therapeutic strategies to reduce the mortality rate of cloned animals and to improve the efficiency of SCNT technology.