Qinggang Meng

Changes in acetylation on lysine 12 of histone H4 (acH4K12) of murine oocytes during maternal aging may affect fertilization and subsequent embryo development

OBJECTIVE To compare acH4K12 levels in oocytes during mouse aging and then assess how such changes might affect the developmental potential of oocytes. DESIGN Experimental animal study. SETTING State key laboratory and university research laboratory. ANIMAL(S) Kunming white strain mice. INTERVENTION(S) Oocytes obtained from TSA treated group or aging mouse group were fertilized and the formation of pronuclei and subsequently developmental potential in vitro or in vivo were assessed. MAIN OUTCOME MEASURE(S) AcH4K12 levels in oocytes were assessed using fluorescence staining, and confocal microscopy and oocyte developmental potentials were determined by in vitro or in vivo methods. RESULT(S) The AcH4K12 levels in oocytes statistically significantly increased during mouse aging. When histone acetylation of oocytes of young mice was artificially increased by trichostatin A (TSA) treatment, the acH4K12 levels in male and female pronuclei in fertilized oocytes showed statistically significant changes. About 38.9% of TSA-treated oocytes failed to form pronuclei or formed morphologically abnormal pronuclei 6 hours after fertilization, which statistically significantly decreased the blastocyst rate of TSA-treated oocytes when compared with the control group (41.5% vs. 60.5%). A similar reduction in blastocyst development was also observed when oocytes collected in older mice were compared with younger mice (17.3% vs. 69.4%). CONCLUSION(S) The AcH4K12 levels in oocytes statistically significantly increased during the aging process in mice, and such changes may affect the acetylation patterns and morphology of pronuclei during fertilization and lead to a reduction in oocyte developmental potential.

Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.

The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)—three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C—and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.

Increased Susceptibility to Atrial Fibrillation Secondary to Atrial Fibrosis in Transgenic Goats Expressing Transforming Growth Factor-β1.

Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF‐β1 and investigated the changes in the cardiac structure and function leading to AF.

Decreased expression of CD9 in bovine oocytes after cryopreservation and the relationship to fertilization capacity.

This study was conducted to investigate the effect of vitrification of bovine metaphase‐II (MII) oocytes on CD9 expression and fertilization capacity. Surviving vitrified/warmed oocytes were used to detect CD9 distribution (fluorescence microscopy), CD9 mRNA (qRT‐PCR), and CD9 protein expression (Western blot), and to analyze in vitro fertilization rates (number of sperm bound to or that penetrated the oocytes) after removing the zona pellucida. Fresh oocytes acted as control. The experimental results showed that the vitrification/warming procedures significantly decreased CD9 expression at the mRNA and protein levels, and changed the CD9 distribution pattern in bovine oocytes. After fertilization in vitro, the average number of sperm binding and penetration of vitrified oocytes were significantly lower than those of the non‐vitrified oocytes. In conclusion, vitrification of bovine oocytes caused a decrease in CD9 expression at the mRNA and protein levels, and an alteration of CD9 distribution pattern, which may have resulted in lowered fertilization capacity. Mol. Reprod. Dev. 80: 451–459, 2013.

Enucleation of Demecolcine-Treated Bovine Oocytes in Cytochalasin-Free Medium: Mechanism Investigation and Practical Improvement

Demecolcine-assisted/induced enucleation has been used in nuclear transfer cloning procedures for many species, yet its mechanism of action remains unclear. Primarily because oocytoplasm protrusion induced by demecolcine is inhibited by the presence of cytochalasin, its use has had limited application. In this experiment, we investigated the microtubule and microfilament alterations in bovine oocytes after demecolcine and/or cytochalasin B (CB) treatments by immunocytochemical staining. We also examined mechanical enucleation of demecolcine-treated oocytes in cytochalasin-free medium. The results showed that demecolcine-treatment disrupts the balance between microtubule/microfilament interactions primarily by deleting microtubules and with little effect on the microfilaments that we believe accounts for the membrane protrusion. The CB treatment reduced the amount of microfilaments but had little effect on the microtubules. Most demecolcine-induced membrane protrusions disappeared when exposed to CB. Western blotting showed that CB treatment increases G-actin, which indicates a decrease in the microfilaments. High oocyte enucleation, survival, and developmental rates occurred when demecolcine-assisted enucleation was carried out in a CB-free solution. Higher blastocyst development rates and blastocyst cell numbers were achieved compared to control, indicating that CB is not necessary in the enucleation procedure of bovine oocytes. This study provides a clearer understanding of the mechanism for the demecolcine-induced oocyte membrane protrusion, and substantiates the practical use of demecolcine-assisted enucleation in a CB-free environment.

Cytokine gene expression at the maternal-fetal interface after somatic cell nuclear transfer pregnancies in small ruminants

The present retrospective study investigated pregnancy rates, the incidence of pregnancy loss and large offspring syndrome (LOS) and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesised that significantly higher pregnancy losses observed in sheep compared with goat SCNT pregnancies are due to the increased amounts of T-helper 1 cytokines and proinflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days of gestation and the incidence of LOS were significantly greater in sheep than in goats. At term, the expression of proinflammatory genes in sheep SCNT placentas was increased, whereas that in goats was similar to that in control animals. Genes with altered expression in sheep SCNT placentas included cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 2 receptor alpha (IL2RA), cluster of differentiation 28 (CD28), interferon gamma (IFNG), interleukin 6 (IL6), interleukin 10 (IL10), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-α), interleukin 1 alpha (IL1A) and chemokine (C-X-C motif) ligand 8 (CXCL8). Major histocompatibility complex-I protein expression was greater in sheep and goat SCNT placentas at term than in control pregnancies. An unfavourable immune environment is present at the maternal-fetal interface in sheep SCNT pregnancies.The present retrospective study investigated pregnancy rates, the incidence of pregnancy loss and large offspring syndrome (LOS) and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesised that significantly higher pregnancy losses observed in sheep compared with goat SCNT pregnancies are due to the increased amounts of T-helper 1 cytokines and proinflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days of gestation and the incidence of LOS were significantly greater in sheep than in goats. At term, the expression of proinflammatory genes in sheep SCNT placentas was increased, whereas that in goats was similar to that in control animals. Genes with altered expression in sheep SCNT placentas included cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 2 receptor alpha (IL2RA), cluster of differentiation 28 (CD28), interferon gamma (IFNG), interleukin 6 (IL6), interleukin 10 (IL10), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-α), interleukin 1 alpha (IL1A) and chemokine (C-X-C motif) ligand 8 (CXCL8). Major histocompatibility complex-I protein expression was greater in sheep and goat SCNT placentas at term than in control pregnancies. An unfavourable immune environment is present at the maternal-fetal interface in sheep SCNT pregnancies.

In Vitro Development and Chromosomal Configuration of Bovine Somatic Cloned Embryos with Nonenucleated Metaphase II Oocytes

This study was designed to examine the effects of the presence of oocyte nuclei on the donor cell nuclear remodeling, including premature chromosome condensation (PCC) and DNA configuration, and subsequent embryo development. The results showed that: (1) the presence of oocyte MII spindles was more likely to induce donor cell PCC. (2) The positional relationship between the fused donor cell and the oocyte metaphase spindle had an effect on oocyte PB2 extrusion. When the fused donor cell was widely separated from the MII spindle, 94.4% of the reconstructed oocytes expelled a PB2. When the donor cell was fused adjacently to the MII spindle, almost all of the reconstructed oocytes did not expel the PB2; the majority (67.9%) formed a very large M-phase spindle in which the oocyte and the donor cell chromosomes merged. (3) After activation, the oocyte and donor nuclei exhibited a variety of pronuclear patterns and asynchronous development. (4) The embryos reconstituted with nonenucleated oocytes resulted in a similar cleavage rate as observed in the control embryos reconstituted with enucleated oocytes. Blastocyst developmental rates were no different between nonenucleated and enucleated cloned embryos; however, the development rates from early to hatching blastocysts significantly decreased in the nonenucleation group compared to enucleation controls (0 vs. 23.1%; 27.5 vs. 67.8%), regardless with either cumulus cells or fibroblasts as donor cells. (5) All nonenucleated oocyte-derived blastocysts contained mixed polyploidy with a variety of compositions that included 2n/4n, 2n/6n, 2n/8n, and 2n/4n/8n. (6) Nuclear transfer preceding the oocyte enucleation experiment indicated that prolonged presence of oocyte nuclei induced abnormal DNA configuration and reduced in vitro development of transferred somatic nuclei, but short time presence of oocyte nuclei did not affect the in vitro development of cloned embryos. We conclude that oocyte MII spindles induce donor cell PCC, the developmental capacity of cloned embryos reconstituted with nonenucleated oocytes is inferior to those with enucleated oocytes, and that all such derived blastocysts are polyploidy.

Genetic and epigenetic regulation of major histocompatibility complex class I gene expression in bovine trophoblast cells

The regulatory mechanisms governing differential expression of classical major histocompatibility complex (MHC) class I (MHC‐Ia) and non‐classical MHC class I (MHC‐Ib) genes are poorly understood.

A sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR gene

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The major cause of limited life span in CF patients is progressive lung disease. CF models have been generated in 4 species (mice, rats, ferrets, and pigs) to enhance our understanding of the CF pathogenesis. Sheep may be a particularly relevant animal to model CF in humans due to the similarities in lung anatomy and development in the two species. Here, we describe the generation of a sheep model for CF using CRISPR/Cas9 genome editing and somatic cell nuclear transfer (SCNT) techniques. We generated cells with CFTR gene disruption and used them for production of CFTR-/- and CFTR+/- lambs. The newborn CFTR-/- sheep developed severe disease consistent with CF pathology in humans. Of particular relevance were pancreatic fibrosis, intestinal obstruction, and absence of the vas deferens. Also, substantial liver and gallbladder disease may reflect CF liver disease that is evident in humans. The phenotype of CFTR-/- sheep suggests this large animal model will be a useful resource to advance the development of new CF therapeutics. Moreover, the generation of specific human CF disease-associated mutations in sheep may advance personalized medicine for this common genetic disorder.

36 SERIAL SOMATIC CELL NUCLEAR TRANSFER INCREASES PREGNANCY LOSSES IN GOATS

Serial cloning by somatic cell nuclear transfer (SCNT) has been successful in several mammalian species. This method can be beneficial for transgenic line expansion or resetting the lifespan of transgenic cells. Previous studies in bovine and porcine have shown a decrease in efficiency over multiple iterations of serial cloning. However, the contradictory data has been reported in mice where no decrease in cloning efficiency was observed after 25 generations of recloning. To our knowledge, no data have been reported investigating the efficiency of serial cloning in goats. The aim of this study was to evaluate whether there is an effect of recloning on goat SCNT efficiency. αMHC-TGF-β1 fetal fibroblast cells (containing transforming growth factor-β under control of a cardiac-specific promoter) were produced by electroporation and used for the first round of SCNT. For serial cloning, we used neonatal fibroblast cells obtained from skin biopsies used as nuclear donors. These cells were collected from the transgenic cloned goats generated by the first round of SCNT. Cumulus-oocyte complexes recovered from abattoir-derived ovaries using slicing technique were matured in vitro for 20 to 24h. The first polar body and metaphase plate were removed from a cumulus cell-free oocyte, and a donor fibroblast cell was subsequently transferred into the enucleated oocyte. Fused embryos were then activated for 5min in 5mM ionomycin followed by 4h in 2mM DMAP with 5mgmL-1 cycloheximide. Activated embryos were cultured in G1 medium with 5mgmL-1 BSA for 12h, followed by surgical transfer into the oviducts of recipients synchronized to show oestrus within 12h of SCNT. In total, 592 and 395 embryos were transferred to 37 and 25 recipient goats, respectively, for the first and second round of SCNT. Pregnancy rate, rate of pregnancy loss, and term rate were analysed by Chi-squared with a 2-tailed P-value. No significant difference was observed in Day 40 pregnancy rates (32.4v. 36%) and term rates (32.4v. 20%) between the first round of cloning and the successive recloning. However, the rate of pregnancy losses was significantly greater in recloning group (P<0.05), with 4 out of 9 pregnancies lost between Day 40 of gestation and term, whereas no pregnancy losses were observed after Day 40 of gestation in the first-round cloning group. The greater pregnancy loss in the recloning procedure might be caused by accumulation of epigenetic errors resulting from incomplete reprogramming. We are assessing the DNA methylation pattern of differentially methylated regions (DMR) of 2 paternally imprinted genes (H19 and IGF2R) in the cloned and recloned goats and expect to see a difference in their imprinted gene DNA methylation pattern, which could explain the greater rate of pregnancy loss in recloned goats.