Xiaorong An

Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation

BackgroundPrevious studies indicated that, unlike mouse zygotes, sheep zygotes lacked the paternal DNA demethylation event. Another epigenetic mark, histone modification, especially at lysine 9 of histone 3 (H3K9), has been suggested to be mechanically linked to DNA methylation. In mouse zygotes, the absence of methylated H3K9 from the paternal pronucleus has been thought to attribute to the paternal DNA demethylation.ResultsBy using the immunofluorescence staining approach, we show that, despite the difference in DNA methylation, modification of H3K9 is similar between the sheep and mouse zygotes. In both species, H3K9 is hyperacetylated or hypomethylated in paternal pronucleus relative to maternal pronucleus. In fact, sheep zygotes can also undergo paternal DNA demethylation, although to a less extent than the mouse. Further examinations of individual zygotes by double immunostaining revealed that, the paternal levels of DNA methylation were not closely associated with that of H3K9 acetylation or tri-methylation. Treatment of either 5-azacytidine or Trichostatin A did not induce a significant decrease of paternal DNA methylation levels.ConclusionOur results suggest that in sheep lower DNA demethylation of paternal genomes is not due to the H3K9 modification and the methylated DNA sustaining in paternal pronucleus does not come from DNA de novo methylation.

Dexamethasone-induced skeletal muscle atrophy was associated with upregulation of myostatin promoter activity

Some recent studies showed that the glucocorticoid-induced muscle atrophy was associated with myostatin, a negative regulator of skeletal muscle. In this study, two experiments were performed to investigate the relationship between the glucocorticoid-induced ultrastructural changes in skeletal muscle and the myostatin gene expression, and to examine in vivo whether the glucocorticoid-induced upregulation of myostatin gene expression is associated with the myostatin promoter activity. In the first experiment, the Kun-Ming mice with similar body weights were treated with high-dose dexamethasone. The results showed that high-dose dexamethasone caused myofibrillar disorganization or degradation and mitochondrial swelling or vacuolization, which were accompanied with the upregulation of myostatin expression. In the second experiment, the mice were treated with the wild-type or GRE (glucocorticoid response elements)-mutant myostatin promoter vector and high-dose dexamethasone alone or together with RU486. The results showed that the mutation of GRE motif resulted in the obvious decrease of the myostatin promoter activity, the high-dose dexamethasone promoted significantly the activity of the wild-type myostatin promoter but did not affect the activity of the GRE-mutant myostatin promoter, and RU486 inhibited the effect of dexamethasone on the wild-type myostatin promoter activity. Taken together, these results suggested that the dexamethasone-induced changes in ultrastructure of skeletal muscle were associated with the upregulation of myostatin gene expression and the upregulation was partly attributed to the binding of glucocorticoid receptor to GRE motifs along myostatin promoter.

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2–3 h, was significantly lower than that of the 3–6 h groups (31.0%), while not significantly different among 3–4 h (P < 0.05), 4–5 h, and 5–6 h groups (P≥0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.

Production of transgenic rabbit embryos through intracytoplasmic sperm injection.

The objective of this study was to test if intracytoplasmic sperm injection (ICSI)-mediated gene transfer was an effective method in the production of transgenic rabbit embryos. Rabbit sperm diluted in different media with various pH were treated by freezing without cryoprotectant, and their ability for DNA uptake was determined. In these experiments using production of transgenic rabbit embryos by ICSI, exogenous genes at three concentrations and of two conformation types were used. The rate of DNA association to the sperm seen by rhodamine-tagged DNA encoding green fluorescent protein (GFP) was 90.0%, 92.7%, 91.0%, 91.7%, and 92.3%, respectively in TCM199, DM, DPBS, CZB, and HCZB media. The DNA attachment to sperm was not affected by media pH within the range of 5.4-9.4 (p > 0.05). Expression of GFP first occurred at the 2-cell stage and continued to blastocyst formation. DNA concentration (between 5, 10, and 20 ng/μl) or conformation (linear and circular) had no effect on the production rate of transgenic embryos. These results indicated that genetically modified rabbit blastocysts can be efficiently produced by ICSI technique.

Fel d 1-airway inflammation prevention and treatment by co-immunization vaccine via induction of CD4+CD25-Foxp3+ Treg cells

Pet allergens are major causes for asthma and allergic rhinitis. Fel d 1 protein, a key pet allergen from domestic cat, can sensitize host and trigger asthma attack. In this study, we report that co-immunization with recombinant Fel d 1 protein (rFel d 1) plus plasmid DNA that contains Fe1 d 1 gene was effective in preventing and treating the natural Fel d 1 (nFel d 1) induced allergic airway inflammation in mice. A population of T regulatory cells (iTreg) exhibiting a CD4+CD25-Foxp3+ phenotype and expressing IL-10 and TGF-β was induced by this co-immunization strategy. Furthermore, after adoptive transfers of the iTreg cells, mice that were pre-sensitized and challenged with nFel d 1 exhibited less signs of allergic inflammation, AHR and a reduced allergic immune response. These data indicate that co-immunization with DNA and protein mixture vaccine may be an effective treatment for cat allergy.

Production of transgenic blastocyst of sheep by somatic cell cloning

Five samples from primary cultures of five sheep ovarian granulosa cells were transfected by pEGFP-N1 DNA. Five transgenic positive cell lines, each from one of the five samples above, were used as donor nuclei for somatic nucleus transfer. A total of 352in vitro matured and enucleated sheep oocytes were fused electrically with transgenic granulosa cells and 329 reconstructed embryos were obtained after activation by Ionomycin/6-DMAP, and these embryos were cultured in SOFaaBSA medium for 7 d. The result shows that 312 embryos (94.8%) had gone through cleavage and among them 63 (19.1%) had developed to the blastocyst stage. Expression of GFP gene was detected in various stages of early embryonic development by sampling randomly. Blastocyst rates given by the four cells treated with 0.5% FCS starvation was 19.6% (55/280) and it had not shown difference significantly (P>0.05) with the result obtained with another cell line that had not gone through serum starvation (16.3%, 8/49). This experiment indicates that sheep transgenic embryos up to the blastocyst stage can be produced effectively by the combination of gene transfection in somatic cells in culture and somatic cell cloning.

Viable rabbits derived from oocytes by intracytoplasmic injection of spermatozoa from an infertile male.

Intracytoplasmic sperm injection (ICSI) is an assisted fertilization technique and has been widely applied in human medicine to overcome some obstacles of infertility. However, this technology has not yet been used as a mainstream technique for animal production, including the rabbit, due to its limited success. The aim of this study was to improve ICSI techniques and establish an efficient ICSI method for rabbits. Spermatozoa used for ICSI were collected from mature New Zealand white male rabbits. They were washed two to three times with HEPES-buffered TCM199 containing 10% FBS and then mixed with 10% polyvinylpyrrolidone (PVP) prior to microinjection. Oocytes were harvested from superovulated donor rabbits after 14-15 h hCG treatment and were fertilized by microinjection of a single living spermatozoon into the ooplasm of each oocyte without additional activation treatment. A total of 317 injected oocytes resulted in the high survival rate of 86.1%. Among the surviving oocytes, 273 were placed into culture dishes for in vitro development. The fertilization, cleavage and blastocyst rates were 59.0%, 88.2% and 45.3% respectively. Furthermore, ICSI embryos were produced with spermatozoa from an infertile male rabbit, and 21 early-stage embryos (2-cell and 4-cell) were surgically transferred into the oviducts of two adult female rabbits. On day 31 after transfer, one out of the two recipients gave birth to two normal and healthy young rabbits. These results demonstrate that rabbit oocytes can be successfully activated and fertilized by the new ICSI protocol. Spermatozoa derived from infertile rabbits can successful fertilize oocytes and produce offspring by the simple ICSI technique.

Extraction of sp18 family membrane proteins on posterior head of bull sperm and the effect of anti-sp18 IgG on sperm motility and murine in vitro fertilization.

Bovine sperm heads were separated via ultrasonic treatment and centrifugation. Anti-bull sperm IgG was produced by immunizing rabbits with acrosome-reacted bull sperm heads. SDS PAGE patterns revealed that the main membrane proteins on acrosome-reacted bull sperm head were sp18 family, including 18, 16, and 14 kD, which represented about 64% of the total membrane proteins in bull sperm. Indirect immunofluorescence shown sp18 antigens primarily distributed in postacrosomal and proximal tail regions. Western blot analysis revealed that the anti-bull sperm IgG reacted with sp18 antigens in acrosome-reacted bull sperm head and bull seminal plasma. Anti-bull sperm IgG also reacted with 14, 16, 18, 42, 57 and 60 kD proteins in fresh bull, mouse and rabbit sperm. Anti-sp18 IgG caused agglutination of bull and rabbit sperm, but had no effect on murine sperm. In murine in vitro fertilization trials, preincubating capacitated sperm with 0.364 mg/ml of anti-sp18 IgG resulted in a decrease in the fertilization rate from 75.6% in the controls to 50.8% in the experimental groups (p < 0.001).