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.

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.

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.

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.

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.

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 apoptosis and improves the developmental potential of vitrified bovine oocytes.

Vitrification of oocytes has been shown to be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. However, little information is available the effect of melatonin on the ROS levels and apoptotic events in vitrified oocytes. Therefore, we studied the effect of melatonin on ROS and apoptotic events in vitrified bovine oocytes by supplementing vitrification solution or in vitro maturation (IVM) and vitrification solution with 10(-9) m melatonin. We analyzed the ROS, mitochondrial Ca(2+) (mCa(2+) ) and membrane potential (ΔΨm), externalization of phosphatidylserine (PS), caspase-3 activation, DNA fragmentation, mRNA expression levels of Bax and Bcl2 l1, and developmental potential of vitrified bovine oocytes. Vitrified bovine oocytes exhibited increased levels of ROS, mCa(2+) , Bax mRNA, and caspase-3 protein and higher rates of PS externalization and DNA fragmentation, and decreased ΔΨm and Bcl2 l1 mRNA expression level. However, melatonin supplementation in vitrification solution or IVM and vitrification solution significantly decreased the levels of ROS, mCa(2+) , Bax mRNA expression, and caspase-3 protein, and PS externalization and DNA fragmentation rates, and increased the ΔΨm and Bcl2 l1 mRNA expression level in vitrified oocytes, resulting in an increased developmental ability of vitrified bovine oocytes after parthenogenetic activation. The developmental ability of vitrified oocytes with melatonin supplementation in IVM and vitrification solution was similar to that of fresh ones. This study showed that supplementing the IVM and vitrification medium or vitrification medium with 10(-9) m melatonin significantly decreased the ROS level and inhibited apoptotic events of vitrified bovine oocytes, consequently increasing their developmental potential.

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.

Melatonin enhances the in vitro maturation and developmental potential of bovine oocytes denuded of the cumulus oophorus.

This study was designed to determine the effect of melatonin on the in vitro maturation (IVM) and developmental potential of bovine oocytes denuded of the cumulus oophorus (DOs). DOs were cultured alone (DOs) or with 10-9 M melatonin (DOs + MT), cumulus-oocyte complexes (COCs) were cultured without melatonin as the control. After IVM, meiosis II (MII) rates of DOs, and reactive oxygen species (ROS) levels, apoptotic rates and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression of ATP synthase F0 Subunit 6 and 8 (ATP6 and ATP8), bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) mRNA in MII oocytes and IFN-tau (IFN-τ), Na+/K+-ATPase, catenin-beta like 1 (CTNNBL1) and AQP3 mRNA in parthenogenetic blastocysts were quantified using real-time polymerase chain reaction (PCR). The results showed that: (1) melatonin significantly increased the MII rate of DOs (65.67 ± 3.59 % vs. 82.29 ± 3.92%; P < 0.05), decreased the ROS level (4.83 ± 0.42 counts per second (c.p.s) vs. 3.78 ± 0.29 c.p.s; P < 0.05) and apoptotic rate (36.99 ± 3.62 % vs. 21.88 ± 2.08 %; P < 0.05) and moderated the reduction of relative mRNA levels of ATP6, ATP8, BMP-15 and GDF-9 caused by oocyte denudation; (2) melatonin significantly increased the developmental rate (24.17 ± 3.54 % vs. 35.26 ± 4.87%; P < 0.05), and expression levels of IFN-τ, Na+/K+-ATPase, CTNNBL1 and AQP3 mRNA of blastocyst. These results indicated that melatonin significantly improved the IVM quality of DOs, leading to an increased parthenogenetic blastocyst formation rate and quality.

Effect of mouse cumulus cells on the in vitro maturation and developmental potential of bovine denuded germinal vesicle oocytes.

We investigated the effect mouse cumulus cells (mCCs) on the in vitro maturation (IVM) and developmental potential of bovine denuded germinal vesicle oocytes (DOs). Cumulus-oocyte complexes (COCs), DOs and DOs cocultured with either mCCs (DOs + mCCs) or bovine cumulus cells (bCCs; DOs + bCCs) were subjected to IVM. The meiosis II (MII) rates of DOs, glutathione (GSH) contents, zona pellucida (ZP) hardening and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression levels of bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) in MII oocytes were measured using quantitative real-time polymerase chain reaction (PCR). mCCs significantly increased the MII rate of DOs from 53.5 ± 3.58% to 69.67 ± 4.72% (p < 0.05) but had no effect on the GSH content (2.17 ± 0.31 pmol/oocyte with mCCs, 2.14 ± 0.53 pmol/oocyte without mCCs). For the DOs + mCCs group, the BMP-15 and GDF-9 expression levels were significantly higher and the ZP dissolution time was significantly lower (162.49 ± 12.51 s) than that of the DOs group (213.95 ± 18.87 s; p < 0.05). The blastocyst rate of the DOs + mCCs group (32.56 ± 4.94%) was similar to that of the DOs group (31.75 ± 3.65%) but was significantly lower than that of the COCs group (43.52 ± 5.37%; p < 0.05). In conclusion, mCCs increased the MII rate of DOs and expression of certain genes in MII oocytes, and decreased the ZP hardening of MII oocytes, but could not improve their GSH content or developmental potential.

[Somatic cells reprogramming using cell extracts].

The process of differentiated somatic cells to a pluripotent state or to another unrelated cell type is referred to as cellular reprogramming. This can be achieved through somatic cell nuclear transfer, cell fusion, specific transcription-factor transduction, and exposure to cell extracts. Cellular reprogramming using cell extracts has gained increasing attention in recent years and has been applied to acquire the target cell types, recognize the functional factors, explore the reprogramming mechanism. In this review, we described different methods of reprogramming somatic cells, which mainly focused on the advances in cellular reprogramming using cell extracts.

Effect of vitrification on promoter methylation and the expression of pluripotency and differentiation genes in mouse blastocysts.

The present study was designed to determine the effects of vitrification on promoter methylation and the expression levels of pluripotency and differentiation genes in mouse blastocysts. Promoter region CpG methylation patterns and the expression levels of octamer-binding transcription factor (Oct4), Nanog homeobox (Nanog), caudal-type homeobox 2 (Cdx2), and heart and neural crest derivatives-expressed transcript 1 (Hand1) were analyzed in fresh and vitrified mouse blastocysts. Methylation was measured by bisulphate mutagenesis and sequencing; gene expression was determined by real-time reverse transcription-PCR. The results showed that vitrification significantly reduced the methylation levels of the Oct4 (85% vs. 62.5%), Nanog (77.5% vs. 55%), and Cdx2 promoters (4.6% vs. 1.4%; P < 0.05) in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog in vitrified blastocysts. Hand1 promoter methylation was not significantly different in the fresh (17.9%) versus vitrification group (21.4%; P > 0.05). The expression levels of Cdx2 and Hand1 were not significantly different in fresh and vitrified blastocysts. In conclusion, vitrification significantly decreased Oct4, Nanog, and Cdx2 promoter methylation in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog.

[Relationship of epidermal growth factor receptor in lung development].

The epidermal growth factor receptor (EGFR), a transmembrane protein receptor, is a member of ErbB family with signal-transducing tyrosine kinase activity. After combined with the ligand, EGFR homologous or heterologous dimers are formed to induce intracellular signal transduction, activate downstream signal transduction pathways, and then produce a series of biological effects. RAF/MEK/RAS/ERK pathway is relevant to cell proliferation, differentiation and apoptosis; while PDK1/AKT /PI3K pathway is involved in cell migration and adhesion. EGFR can promote the maturity of pulmonary type II epithelial cells and the synthesis and secretion of pulmonary surfactant. EGFR shows the effect on mammal lungs in a time-space and dose-dependent manner. The down-regulated expression of it will lead to immature lung development, while the over-expression can promote the cell proliferation, invasion and metastasis of the lung cancer cells. This paper reviewed advances in the study for EGFR and its signal pathway, as well as the relationship among EGFR, atelectasis and lung cancer.

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.

Recovery of mitochondrial function and endogenous antioxidant systems in vitrified bovine oocytes during extended in vitro culture.

This study was designed to examine the recovery of mitochondrial function and endogenous antioxidant systems in vitrified oocytes during extended incubations. After 16 hr of in vitro maturation, bovine meiosis-II oocytes were vitrified, and then surviving oocytes were cultured an additional 8 hr. ATP content, ATP synthase activity, expression of ATP synthase F0 subunit 6 (ATP6) and 8 (ATP8) genes, and reactive oxygen species (ROS) levels were investigated in the vitrified oocytes during this additional period (4 or 8 hr). The results showed that: (1) the ATP content and ATP synthase activities in vitrified oocytes at 8 hr post-warming (754.6 fmol, 25.9 nmol NADH/min/mg) were significantly higher than in oocytes immediately warmed (568.3 fmol, 8.7 nmol NADH/min/mg), but still lower than in control oocytes (901.5 fmol, 30.7 nmol NADH/min/mg); (2) the relative expression of ATP6 and ATP8 was initially down-regulated in oocytes when they were first warmed, increased by 4 hr post-warming, and were again down-regulated by 8 hr post-warming; (3) ROS levels in oocytes at 0, 4, and 8 hr post-warming were significantly higher than in control oocytes; and (4) after parthenogenetic activation, the blastocyst rate of oocytes at 8 hr post-warming (26.7%) was significantly higher than that of oocytes immediately warmed (16.9%). These results indicated that mitochondrial function and endogenous antioxidant systems recovered significantly better in vitrified-thawed bovine oocytes with 8 hr of additional incubation, but they did not achieve the activity levels found in fresh oocytes.

[Advances of transgenic breeding in livestock].

Transgenic technology represents a revolutionary way to produce elite livestock breeds, allowing introduction of alien gene into livestock genome. Currently, pronuclear microinjection of DNA and somatic cell nuclear transfer are two popular methods used to make transgenic farm animals. Transgenic technology can be used in livestock breeding for improving disease resistance, carcass composition, lactational performance, wool production, growth rate, and reproductive performance, as well as reducing negative environmental impact. In addition to introduction of animal transgenic technologies, this review described the status and the future perspective of transgenic breeding in livestock.

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.

[Somatic cell nuclear transfer and centrosome inheritance].

The developmental competence of embryos cloned from somatic cells depends on the cellular event and molecular process, such as separation of chromosomes and reorganization of spindle after nuclear transfer. Centrosome, the main microtubule organizing centers in a cell, is crucial for reorganization of spindle and normal separation of chromosomes during mitosis. Aberrant of centrosomes will lead to aneuploidy of blastomere and developmental failure of embryo. This paper expounded the situation of animal somatic cell nuclear transfer (SCNT) and biological functions of centrosome and analyzed the inheritance mechanism of centrosome during gametogenesis and fertilization. Additionally, the study condition of centrosome and its associated proteins in SCNT embryos were introduced, which provided a new clue to study the de-velopmental abnormality of cloned embryos and animals.

[Progress on methods for purity assessment of separated chromosome X- or Y-bearing sperm].

In this review of methods for purity assessment of isolated chromosome X- and Y-bearing sperm, we compared the principles, operating procedures, as well as pros and cons for various methods. We conclude that nested PCR of single sperm will become a conventional and popular method with lower costs, and the method will play a very important role in optimizing the X, Y sorting method, if the sensitivity and accuracy of the method can be increased and the testing time decreased, and promote the new progress in other genetic testing techniques on single sperm.