Effect of vitrification on protein O-GlcNAcylation in mouse metaphase II oocytes.

In brief: Oocyte vitrification leads to DNA hypomethylation, which results in defect in early embryo development. This study reveals that oocyte vitrification impairs the DNA methylation pattern by influencing protein O-GlcNAcylation. Abstract: Oocyte vitrification leads to decreased DNA methylation levels, which impairs the quality and the developmental potential of oocytes. However, the underlying molecular mechanism still need to be further revealed. In this study, mouse metaphase II (M II) oocytes were frozen by vitrification technology, while fresh oocytes were used as the control group. The effect of oocyte vitrification on protein O-GlcNAcylation and its impact on the developmental potential of oocytes were elucidated. We found that the protein O-GlcNAcylation levels were significantly increased in vitrified oocytes. Increase of protein O-GlcNAcylation levels in control oocytes by PUGNAc (an O-GlcNAcase inhibitor) decreases blastocyst rate after parthenogenetic activation (20.82% in PUGNAc-treated group; 53.82% in control group, P < 0.05). We also discovered that DNA methylation was disrupted in two-cell embryos derived from vitrified oocytes, resulting in decreased 5mC and increased 5hmC, showing similar phenotypes to that derived from PUGNAc-treated oocytes. In vitrified and PUGNAc-treated oocytes, O-GlcNAcylated TET3 was significantly increased. Notably, by inhibiting protein O-GlcNAcylation in vitrified oocytes using OSMI1 (an O-GlcNAc transferase inhibitor) we restored the DNA methylation in two-cell embryos and ameliorated the developmental defects in early embryo. Thus, elevated protein O-GlcNAcylation in vitrified oocytes is an essential contributor to their declining embryonic developmental potential. Modulation of protein O-GlcNAcylation improves the developmental potential of vitrified oocytes.

Enhancement of microbial community dynamics and metabolism in compost through ammonifying cultures inoculation.

The efficient use of livestock and poultry manure waste has become a global challenge, with microorganisms playing an important role. To investigate the impact of novel ammonifying microorganism cultures (NAMC) on microbial community dynamics and carbon and nitrogen metabolism, five treatments [5% (v/w) sterilized distilled water, Amm-1, Amm-2, Amm-3, and Amm-4] were applied to cow manure compost. Inoculation with NAMC improved the structure of bacterial and fungal communities, enriched the populations of the functional microorganisms, enhanced the role of specific microorganisms, and promoted the formation of tight modularity within the microbial network. Further functional predictions indicated a significant increase in both carbon metabolism (CMB) and nitrogen metabolism (NMB). During the thermophilic phase, inoculated NAMC treatments boosted carbon metabolism annotation by 10.55%-33.87% and nitrogen metabolism annotation by 26.69%-63.11. Structural equation modeling supported the NAMC-mediated enhancement of NMB and CMB. In conclusion, NAMC inoculation, particularly with Amm-4, enhanced the synergistic interaction between bacteria and fungi. This collaboration promoted enzymatic catabolic and synthetic processes, resultng in positive feedback loops with the endogenous microbial community. Understanding these mechanisms not only unravels how ammonifying microorganisms influence microbial communities but also paves the way for the development of the composting industry and global waste management practices.

Immunogenicity Analysis of PCV3 Recombinant Capsid Protein Virus-like Particles and Their Application in Antibodies Detection.

Porcine circovirus type 3 is a newly emerging pathogen of porcine circovirus associated disease (PCVAD). Currently, there is no commercially available vaccine, resulting in huge economic losses to the pig industry. Porcine circovirus type 3 capsid protein (Cap) can self-assemble into virus-like particles (VLPs). Therefore, the expression of the recombinant Cap protein is of great significance for the prevention, diagnosis and control of porcine circovirus type 3 associated diseases. In this study, the recombinant Cap protein was successfully expressed in Escherichia coli by deleting the nuclear localization sequence (NLS). The VLPs were observed by transmission electron microscopy. To evaluate the immunogenicity of the recombinant Cap protein, mice were immunized. As a result, the recombinant Cap protein can induce higher levels of humoral and cellular immune responses. A VLP-based ELISA method was developed for the detection of antibodies. The established ELISA method has good sensitivity, specificity, repeatability and clinical applicability. These results demonstrate the successful expression of the PCV3 recombinant Cap protein and the preparation of recombinant Cap protein VLPs, which can be used for the preparation of subunit vaccines. Meanwhile, the established I-ELISA method lays a foundation for the development of the commercial PCV3 serological antibody detection kit.

Alkaline Dilution Alters Sperm Motility in Dairy Goat by Affecting sAC/cAMP/PKA Pathway Activity.

In dairy goat farming, increasing the female kid rate is beneficial to milk production and is, therefore, economically beneficial to farms. Our previous study demonstrated that alkaline incubation enriched the concentration of X-chromosome-bearing sperm; however, the mechanism by which pH affects the motility of X-chromosome-bearing sperm remains unclear. In this study, we explored this mechanism by incubating dairy goat sperm in alkaline dilutions, examining the pattern of changes in sperm internal pH and Ca2+ concentrations and investigating the role of the sAC/cAMP/PKA pathway in influencing sperm motility. The results showed that adding a calcium channel inhibitor during incubation resulted in a concentration-dependent decrease in the proportion of spermatozoa with forward motility, and the sperm sAC protein activity was positively correlated with the calcium ion concentration (r = 0.9972). The total motility activity, proportion of forward motility, and proportion of X-chromosome-bearing sperm decreased (p < 0.05) when cAMP/PKA protease activity was inhibited. Meanwhile, the enrichment of X-chromosome-bearing sperm by pH did not affect the sperm capacitation state. These results indicate that alkaline dilution incubation reduces Ca2+ entry into X-sperm and the motility was slowed down through the sAC/cAMP/PKA signaling pathway, providing a theoretical foundation for further optimization of the sex control method.

Asymmetric expression of maternal mRNA governs first cell-fate decision.

The goal of preimplantation development is to establish the fates of the embryonic and extra-embryonic cells. However, when and how cell fates are determined during early mammalian embryonic development remains unclear. We report that the high mobility group (HMG) protein family member HMGA1 was distributed differentially in mouse two-cell blastomeres. Knockdown of Hmga1 expression in one of the two cells reduced the number of cells contributing to the inner cell mass (ICM), suggesting that differential distribution of HMGA1 in the blastomeres in two-cell mouse embryos affected the selection of embryonic cell lineages. Mechanistically, HMGA1 promotes the expression of the ICM-specific gene Sox2. The results of this study show that mouse embryos demonstrate heterogeneity as early as the two-cell stage, and that these differences are related to cell-fate differentiation in early mouse embryos.

Advancements in mammalian X and Y sperm differences and sex control technology.

Mammal sex determination depends on whether the X sperm or Y sperm binds to the oocyte during fertilization. If the X sperm joins in oocyte, the offspring will be female, if the Y sperm fertilizes, the offspring will be male. Livestock sex control technology has tremendous value for livestock breeding as it can increase the proportion of female offspring and improve the efficiency of livestock production. This review discusses the detailed differences between mammalian X and Y sperm with respect to their morphology, size, and motility in the reproductive tract and in in vitro conditions, as well as 'omics analysis results. Moreover, research progress in mammalian sex control technology has been summarized.

Effects of MICU1-Mediated Mitochondrial Calcium Uptake on Energy Metabolism and Quality of Vitrified-Thawed Mouse Metaphase II Oocytes.

BACKGROUND: Oocyte vitrification has been widely used in the treatment of infertility and fertility preservation. However, vitrification-induced mitochondrial damage adversely affects oocyte development. Several studies have reported that mitochondrial calcium uptake protein 1 (MICU1) regulates the uptake of mitochondrial calcium by the mitochondrial calcium uniporter (MCU) and subsequently controls aerobic metabolism and oxidative stress in mitochondria, but research considering oocytes remains unreported. We evaluated whether the addition of MICU1 modulators enhances mitochondrial activity, pyruvate metabolism, and developmental competence after warming of MII oocytes. METHODS: Retrieved MII oocytes of mice were classified as vitrified or control groups. After thawing, oocytes of vitrified group were cultured with or without DS16570511 (MICU1 inhibitor) and MCU-i4 (MICU1 activator) for 2 h. RESULTS: Mitochondrial Ca2+ concentration, pyruvate dephosphorylation level, and MICU1 expression of MII oocytes were significantly increased after vitrification. These phenomena were further exacerbated by the addition of MCU-i4 and reversed by the addition of DS16570511 after warming. However, the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) in vitrified-warmed MII oocytes drop significantly after vitrification, which was improved after MCU-i4 treatment and decreased significantly after DS16570511 treatment. The vitrification process was able to elicit a development competence reduction. After parthenogenetic activation, incubation of the thawed oocytes with MCU-i4 did not alter the cleavage and blastocyst rates. Moreover, incubation of the thawed oocytes with DS16570511 reduced the cleavage and blastocyst rates. CONCLUSIONS: MICU1-mediated increasing mitochondrial calcium uptake after vitrification of the MII oocytes promoted the pyruvate oxidation, and this process may maintain oocyte development competence by compensating for the consumption of ATP under stress state.

Effect of inoculation with newly isolated thermotolerant ammonia-oxidizing bacteria on nitrogen conversion and microbial community during cattle manure composting.

Nitrogen loss during composting is closely related to NH4+-N conversion, and ammonia-oxidizing bacteria (AOB) are important microorganisms that promote NH4+-N conversion. Since the biological activity of conventional AOB agents used for compost inoculation declines rapidly during the thermophilic phase of composting, new compound inoculants should be developed that are active during that phase. In the current study, the effects of inoculating cattle manure compost with newly isolated AOB (5%, v/w) [thermotolerant AOB X-2 strain (T-AOB-2), mesophilic AOB X-4 strain (M-AOB-4), and AOB X-2 combined with AOB X-4 (MT-AOB-2-4)] on the conversion of nitrogen, compost maturity, and the resident microbial community were studied. During 35 days of composting, compared with the control, AOB inoculation reduced NH3 emissions by 29.98-46.94%, accelerated the conversion of NH4+-N to NO2--N, increased seed germination values by 13.00-25.90%, and increased the abundance of the microbial community at the thermophilic phase (16.38-68.81%). Network analysis revealed that Bacillaceae play a crucial role in the composting process, with the correlation coefficients: 0.83 (p < 0.05) with NH3, 0.64 (p < 0.05) with NH4+-N, and 0.81 (p < 0.05) with NO2--N. In addition, inoculation with MT-AOB-2-4 notably increased the total nitrogen content of compost, prolonged the sanitation stage, and promoted compost maturity. Hence, MT-AOB-2-4 may be used to increase the microbial community abundance and improve the efficiency of cattle manure composting.

Myostatin site-directed mutation and simultaneous PPARγ site-directed knockin in bovine genome.

Most studies on the acquisition of advantageous traits in transgenic animals only focus on monogenic traits. In practical applications, transgenic animals need to possess multiple advantages. Therefore, multiple genes need to be edited simultaneously. CRISPR/Cas9 technology has been widely used in many research fields. However, few studies on endogenous gene mutation and simultaneous exogenous gene insertion performed via CRISPR/Cas9 technology are available. In this study, the CRISPR/Cas9 technology was used to achieve myostatin (MSTN) point mutation and simultaneous peroxisome proliferator-activated receptor-γ (PPARγ) site-directed knockin in the bovine genome. The feasibility of this gene editing strategy was verified on a myoblast model. The same gene editing strategy was used to construct a mutant myoblast model with MSTN mutation and simultaneous PPARγ knockin. Quantitative reverse-transcription polymerase chain reaction, immunofluorescence staining, and western blot analyses were used to detect the expression levels of MSTN and PPARγ in the mutant myoblast. Results showed that this strategy can inhibit the expression of MSTN and promote the expression of PPARγ. The cell counting kit-8 cell proliferation analysis, 5-ethynyl-2'-deoxyuridine cell proliferation analysis, myotube fusion index statistics, oil red O staining, and triglyceride content detection revealed that the proliferation, myogenic differentiation, and adipogenic transdifferentiation abilities of the mutant myoblasts were higher than those of the wild myoblasts. Finally, transgenic bovine embryos were obtained via somatic cell nuclear transfer. This study provides a breeding material and technical strategy to breed high-quality bovine and a gene editing method to realize the mutation of endogenous genes and simultaneous insertion of exogenous genes in genomes.

Identification of small extracellular vesicle subtypes in follicular fluid: Insights into the function and miRNA profiles.

The study of small extracellular vesicles (sEVs) heterogeneity is one of the main problems that must be solved, and the different sEV subtypes in follicular fluid are still unclear, limiting our understanding of their function. This study first separated sEV subtypes from follicular fluid using differential ultracentrifugation combined with iodixanol density gradient flotation and then evaluated their miRNA profile and effects on the proliferation and apoptosis of granulosa cells (GCs). We also performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of potential target genes of differentially expressed miRNAs (DEMs) and KEGG analysis of potential target genes of non-DEMs. Low-density sEVs (sEV_F6) were enriched in TSG101, while high-density sEVs (sEV_F8) were enriched in CD63. The miRNA profiles of sEV_F6 and sEV_F8 were heterogeneous, and the differential signaling pathways were mainly related to the adhesion and hypoxic stress pathways, while the same signaling pathways were mainly related to cell proliferation, apoptosis, cell cycle, and autophagy pathways. In addition, the highly expressed miRNAs in both subtypes were mainly related to cell proliferation and apoptosis. Both subtypes transferred their miRNAs into GCs and promoted the proliferation ability of the GCs and inhibited their apoptosis. The results showed for the first time that there are different subtypes of sEVs in follicular fluid and that the miRNA profiles of subtypes are heterogeneous.

Effect of oocyte vitrification on glucose transport in mouse metaphase II oocytes.

Oocyte vitrification has significantly improved the survival rate and become the mainstream method for cryopreserving oocytes. Previous studies have demonstrated that the ultrastructure, mitochondrial function, DNA methylation, and histone modification exhibit an irreversible effect after oocyte vitrification. However, little is known about the effects of oocyte vitrification on glucose transport and metabolism. This study aims to determine whether mouse oocyte vitrification causes abnormal glucose metabolism and identify a strategy to correct abnormal glucose metabolism. Furthermore, this study further investigates the effects of oocyte vitrification on glucose uptake, and glucose metabolism, and energy levels. The results indicated that vitrification significantly reduced the glucose transport activity, NADPH, glutathione, and ATP levels, and increased reactive oxygen species levels in oocytes (P < 0.01). Vitrification also reduced the expression of glucose transporter isoform 1 (GLUT1) (P < 0.01). Adding a GLUT1 inhibitor reduced the glucose uptake capacity of oocytes. Furthermore, the inclusion of vitamin C into thawing and culture solutions restored abnormal glucose transportation and metabolism and improved the survival, two-cell embryo, and blastocyst rates of the vitrified groups via parthenogenesis (P < 0.05). Overall, this method may improve the quality and efficiency of oocyte vitrification.

Alpha-lipoic acid improves the maturation and the developmental potential of goat oocytes in vitro.

Oxidative stress inevitably occurs during oocyte maturation in vitro. α-lipoic acid (α-LA) has a strong antioxidant capacity, but the effect of α-LA on parthenogenetic activation of oocytes was rarely reported. This study aims to investigate the effect of supplementing α-LA to in vitro maturation medium on the subsequent developmental ability of goat parthenogenetic embryos during oocytes maturation. In the study, the goat cumulus-oocyte complex was divided into the experimental (with 25 µmol/L α-LA) and the control (without α-LA) groups. Oxidase expression was measured using RT-qPCR. After 18-22 hr of maturation, the oocytes were then parthenogenetic activated. The total antioxidant capacity of embryos was measured after 0, 24, 48, 72 and 96 hr of culture. Rates of oocyte maturation and the rates of development for parthenogenetic embryos in the α-LA group were significantly improved by 7.88% (p < .05) and 5.41% (p < .05) compared with those in the control group, respectively. After 24 hr, the difference in total antioxidant capacity was extremely significant in both groups. An evident decrease in the control group and a minor decrease in the α-LA group were observed (p < .01). The ratio of inner cell mass cells to the total cell number of blastocysts in the α-LA group increased compared with that in the control group (p < .05) on day 8. α-LA significantly promoted the expression of SOD and GPX4 of parthenogenetic blastocysts and maturated oocytes. α-LA (25 µmol/L) improved the maturation rate and the developmental competence of the parthenogenetic activation of oocytes, which might be mediated by maintaining the total antioxidant ability of oocytes during the culture period.

Genome-wide analysis of alternative splicing differences between oocyte and zygote†.

Alternative splicing (AS) of mRNA precursors allows the synthesis of multiple mRNAs from a single primary transcript, significantly expanding the information content and regulatory possibilities of higher eukaryotic genomes. During mammalian development, AS drives certain decisive changes in different physiological processes. As development progresses, the maternal-to-zygotic transition (MZT) will trigger two processes: elimination of a subset of maternal mRNA and transcription of the zygote genome begins. Recent high-throughput technological advancements have facilitated genome-wide AS, whereas its analysis in mouse oocyte transition to the zygote stage has not been reported. We present a high-resolution global analysis of AS transitions and discovered extensive AS transitions between mouse oocyte and zygote. The difference of AS patterns was further confirmed using reverse transcription-polymerase chain reaction analysis. Many genes with specific AS events in mouse oocytes are differentially expressed between oocyte and zygote, but only a few genes with specific AS events in zygote are differentially expressed between oocyte and zygote. We provide a landscape of AS events in mouse oocyte and zygote. Our results advance the understanding of AS transitions during mouse fertilization and its potential functions for MZT and further development.

Comparison of Bovine Small Antral Follicle Development in Two- and Three-Dimensional Culture Systems.

To compare the effects of two-(2D, microplate) and three-dimensional (3D, alginate) culture systems on the in vitro growth of small antral follicles in cattle, individual follicles were separately cultured in the two culture systems for 8 days. Half of the culture medium was replaced by fresh medium every 2 days; the former medium was used to assess the amount of follicular hormone secretion using ELISA. Individual follicle morphology, diameter, and survival rate were recorded every alternate day. The results showed that in 4 days, there was no significant difference between the two systems, except that the growth rate of follicles in 2D system was relatively faster. After 4 days, estradiol concentration in 3D system was higher than that in 2D system. However, progesterone concentration was lower than that in the 2D system. The survival rate and oocyte quality of follicles in 2D system were significantly lower than those in 3D system on day 8. The follicle diameter slightly increased (30-60 µm) in the entire process. Taken together, for in vitro culture of follicles within 4 days, the 2D culture system is more suitable. However, when the culture duration is >4 days, the 3D culture system is more suitable.

The Wilms tumor gene (WT1) (+/-KTS) isoforms regulate steroidogenesis by modulating the PI3K/AKT and ERK1/2 pathways in bovine granulosa cells†.

The Wilms tumor (WT) gene WT1 encodes the splicing variants WT1(+KTS) and WT1(-KTS). Recent data suggest that WT1 plays an important role in the development of mice follicles. However, the mechanism through which WT1 influences ovarian steroidogenesis remains unknown. This study identified WT1 and evaluated the impact of splicing variants WT1(+KTS) and WT1(-KTS) on steroidogenesis using adult bovine granulosa cells (GCs). Using RT-qPCR and western blotting, we found that the ratio between WT1(+KTS) and WT1(-KTS) was stabilized. WT1 expression, however, decreased gradually in bovine GCs in response to follicle enlargement or atresia. The downregulation of WT1 increased the secretion of basal and follicle-stimulating-hormone-induced progesterone (P4), but decreased the secretion of basal-induced estradiol (E2). This was associated with an increase in the expression of 3ß-HSD, and a decrease in the expression of CYP19A1. In addition, WT1(-KTS) overexpression suppresses the secretion of E2 and P4 compared with WT1(+KTS) overexpression. This was associated with a decrease in the expression of CYP19A1, CYP11A1, and 3ß-HSD in cultured bovine GCs. Of note, the downregulation of WT1 suppresses the phosphorylation levels of AKT and p-ERK1/2. However, WT1(-KTS) overexpression promotes the phosphorylation levels of AKT and suppresses p-ERK1/2 levels. LY294002 (AKT inhibitor) increases MKP3 mRNA expression levels but decreases the level of p-AKT and p-ERK1/2. Collectively, WT1 significantly suppresses the mRNA expression of CYP11A1 and 3ß-HSD and the secretion of P4 in bovine GCs. Moreover, it regulates CYP19A1 mRNA expression and E2 secretion with complex networks, at least in part, by modulating AKT and ERK1/2 signaling. The effect of WT1(-KTS) was more pronounced than that exerted by WT1(+KTS).

Evidence that downregulation of Wilms' tumor 1 (WT1) is involved in cortical stromal cell differentiation into theca cells in adult bovine ovaries.

Bovine theca cells are thought to differentiate from cortical stromal cells, and ovary-derived Wilms' tumor 1+ (WT1+ ) cells are the primary source of mouse theca cells. However, it is not known whether the differentiation of cortical stromal cells is regulated by WT1. Here, we identified WT1 in the cortical stroma and theca layer of the bovine ovary and analyzed the theca cell functional markers in cortical stromal cells and theca cells; in addition, we determined the effects of this gene on the secretion of androstenedione and progesterone by cortical stromal cells and the responsiveness of cortical stromal cells to luteinizing hormone (LH) in vitro. We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR), western blot analysis, and immunohistochemistry to discover that the cortical stroma had higher WT1 expression than the theca layer. We used RT-qPCR and ELISA analyses to determine that the cortical stromal cells had lower levels of androstenedione and progesterone secretion and LHR messenger RNA expression than the levels of the theca cells. In cultured bovine cortical stromal cells, we found that WT1 downregulation increased androstenedione and progesterone secretion but had no effect on the LH responsiveness. Notably, the increase in androstenedione and progesterone secretion was associated with an increase in 3-ß-hydroxysteroid dehydrogenase expression. In conclusion, the results suggest that WT1 is involved in the differentiation of cortical stromal cells into cells with characteristics similar to theca cells of antral follicles in adult bovine ovaries.

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos.

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification-induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ-H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage-related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ-H2AX foci in zygotes and two-cell embryos. Trp53bp1 was upregulated in zygotes, two-cell embryos and four-cell embryos in the vitrified group, and Brca1 was increased in two-cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two-cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4'-trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ-H2AX foci in zygotes and two-cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification-induced abnormal ROS generation, γ-H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ-H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification-associated γ-H2AX accumulation is at least partially due to abnormal ROS generation.

Resveratrol improved the developmental potential of oocytes after vitrification by modifying the epigenetics.

Resveratrol (Res) has been reported to be able to improve oocyte vitrification because of its antioxidative properties. The objective of this study was to further assess the positive effect of Res addition on the developmental potential of vitrified mouse oocytes from the perspective of epigenetic alterations. First, 2 µM Res was chosen as the optimal concentration on the basis of its effects on survival and its antioxidative properties. We found that Res addition significantly promoted fertilization (63.8% vs. 42.9%) and blastocyst formation (68.3% vs. 50.2%) after oocyte vitrification. The quality of the derived blastocysts was also higher after Res treatment. Regarding epigenetic aspects, the expression of the important deacetylase SIRT1 was found to decrease significantly upon vitrification, but it was rescued by Res. The abnormal levels of H3K9 acetylation and DNA methylation in vitrified oocytes were restored by Res addition. Moreover, the expression of several imprinted genes was affected by oocyte vitrification. Among them, abnormal Gtl2 and Peg3 expression levels were restored by Res addition. Therefore, the methylation of their imprinted control regions (ICRs) was examined. Surprisingly, the abnormal patterns of Gtl2 and Peg3 methylation in blastocysts developed from vitrified oocytes were both restored by Res addition. Finally, the full-term embryonic development showed that the birth rate was improved significantly by Res addition (56.2% vs. 38.1%). Collectively, Res was beneficial for the pre- and postimplantation embryonic development. Except for the antioxidative activity, Res also played a role in the correction of some abnormal epigenetic modifications caused by oocyte vitrification.

The effects of TETs on DNA methylation and hydroxymethylation of mouse oocytes after vitrification and warming.

Oocyte vitrification has extensively been applied in the field of embryo engineering and in the preservation of genetic resources of fine livestock. Following our previous work in oocyte vitrification and the level change of DNA methylation, here we further explored the dynamic change of three active demethylation proteins: Ten-Eleven-Translocation 1/2/3(TET1/2/3), 5-methylcytosine (5 mC) and 5-hydroxymethycytosine (5hmC) after vitrification and warming. In order to observe the active demethylation in vitrified oocytes, two small molecular regulators, i.e. Vitamin C (VC) and dimethyloxaloylglycine (DMOG) were used to adjust activity and level of the TET 3 protein. The results showed that the levels of 5 mC and 5hmC were significantly decreased after 2 h of vitrification (P < 0.01). Moreover, the level of TET3 protein was significantly increased after 2 h warming (P < 0.01). And the relative gene expression of TET2/3 did not change in the first 2 h, but significantly increased after 2 h (P < 0.01). When VC was added to vitrification and recovery medium, it could not significantly improve the level of TET3 gene expression, and affect 5 mC and 5hmC expression (P > 0.05). When the DMOG was added to the solutions of vitrification, the level of 5hmC showed significantly increase (P < 0.01). In conclusion, the oocyte vitrification procedure reduced DNA methylation and hydroxymethylation in MII oocytes, but adding VC and DMOG to vitrification medium can prevent the reduction of DNA hydroxymethylation by increasing activity of TET3 methylation protein after vitrification and warming.

The growth and reproduction performance of TALEN-mediated ß-lactoglobulin-knockout bucks.

With the technological development of several engineered endonucleases (EENs), such as zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9, gene targeting by homologous recombination has been efficiently improved to generate site-specifically genetically modified livestock. However, few studies have been done to investigate the health and fertility of these animals. The purpose of the present study is to investigate if gene targeting events and a recloning procedure would affect the production traits of EEN-mediated gene targeted bucks. TALEN-mediated ß-lactoglobulin (BLG) gene mono-allelic knockout (BLG (+/-)) goats and bi-allelic knockout (BLG (-/-)) buck produced by using sequential gene targeting combined with recloning in fibroblasts from BLG (+/-) buck were used to evaluate their health and fertility. Birth weight and postnatal growth of BLG (+/-) bucks were similar to the wild-type goats. None of the parameters for both fresh and frozen-thawed semen quality were significantly different in BLG (+/-) or BLG (-/-) bucks compared to their corresponding comparators. In vitro fertilization (IVF) test revealed that the proportion of IVF oocytes developing to the blastocyst stage was identical among BLG (+/-), BLG (-/-) and wild-type bucks. Conception rates of artificial insemination were respectively 42.3, 38.0 and 42.6 % for frozen-thawed semen from the BLG (+/-), BLG (-/-) and wild-type bucks. In addition, germline transmission of the targeted BLG modification was in accordance with Mendelian rules. These results demonstrated that the analyzed growth and reproductive traits were not impacted by targeting BLG gene and recloning, implicating the potential for dairy goat breeding of BLG (+/-) and BLG (-/-) bucks.