The Expression And Localization Of GCN5, HDAC1 And DNMT1 In Early Diploid Parthenogenesic Embryo Of Mouse

BackgroundThere is no doubt that the reproductive assisted techniques have stood out as one of the most rapidly evolving areas in reproductive medicine during the recent 20 years. However, so far the majority of mechanisms of fertilization, development and differentiation of oocytes or embryos have still not been identified. The functions of male or female genome in the process of sperm integrating with oocyte and zygote developing to an individual mostly remain mystery. Parthenogenesis (PA) is a procedure that an oocyte is activated into mitosis by some chemical or physical stimulation other than a sperm and the embryo is formed in the absence of any contribution from a male gamete. PA provides a suitable model for the study of the functions of the genomes from different gamets and a way to avoids social, religion and medical ethic problems caused by using the embryo from the reproductive assisted techniques for study.PA can result from the oocytes which fail to maintain at metaphase â…¡ stage of meiosis (Mâ…¡) before fertilized with sperm. It has been described in many species,including human, that eggs in ovarian and oviduct are able to cleave spontaneously. However, in most cases, PA is artificially induced by various kinds of physical or (and) chemical methods. A combination of calcium ionophore A23187 (Ca-A23187 ) and 6-dimethylaminopurine (6-DMAP) can achieve favorable result. It is generally thought that Ca-A23187 can elevate intracellular calcium levels in the cytoplasm of oocytes through the influx of calcium from extracellular spaces. Therefore, the treatedment of Ca-A23187 can raise the concentration of intracellular Ca2+, increase the electricity digit of inner membrane of oocyte and at last lead to exceed polarization and oocyte activation. As one kind of puromycin, 6-DMAP belongs to phosphorylation inhibitor, which inhibits the activation of maturation promoting factor and other protein factors for maintenance of oocyte in MII stage, induces to chromosome decondensation and nuclear membrance formation. 6-DMAP can lead to the formation of PA bipronucleus embryo by disturbing the configuration of actin filament and spindle, preventing spindle from circumrotation and inhibiting the extrusion of the second polar body.The formation and development of offspring are dependent on both the genomes from parents and epigenetic reprogramming. Epigenetic reprogramming is a process in which gametes transfer to totipotent zygote and then develop to a differentiated embryo or individual without the alteration of nucleotide sequence. Molecular mechanisms that mediate epigenetic regulation include DNA methylation, histone methylation or acetylation and gene imprinting.The levels of histone acetylation are controlled by the interations of two classes of enzymes, histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs). General control of nucleoditde acid synthesis 5 (GCN5) and histone deacetylase 1 (HDAC1) are the representative enzymes of HATs and HDACs respectively. As one of transcription related HATs, GCN5 usually expresses in the nucleolus and catalyze acetylation of core histone and dissociative histone in thecytoplasm. Normally GCN5 is considered as transcriptional coactivator to promote gene transcription. HDAC1 is a kind of RPD3 related HDACs and also mainly expresses in the nucleus. It can deacetylate 4 kinds of core histone to maintain the dynamic balances of acetylation and deacetylation levels in cell cycles. Absolutely contrary to GCN5 in regulation of gene expression, HDAC1 is transcriptional repressor of genes. DNA methyltransferases (DNMTs) catalyze the transfer of methyl donor from S-adenosyl-L-methionine (AdoMet) to 5 position of cytosine in DNA strand, which usually act as transcriptional repressors in expression regulation of a number of genes. DNA methyltransferasel (DNMT1) is one of the first discovered DNA methyltransferases and its level can indirectly reflect the level of DNA methylation in whole genome. Apparently, GCN5, HDAC1 and DNMT1 play important roles in the epigenetic reprogramming and the regulation of the gene expression in the development and differentiation of embryo. However, the functions of female or male genome in epigenetic reprogramming have not been identified so far. The investigation of the expression of GCN5, HDAC1 and DNMT1 in PA embryo would offer us a window to study the functions of the genome from sperm or oocyte in embryo epigenetic modification and gene expression regulation.Therefore, a mouse mode of diploid PA embryos resulted from the activation with combination of Ca-A23187 and 6-DMAP in oocytes was introduced to the present study. Fluorescent immunocytochemistry were applied to investigate the expression changes of GCN5, HDAC1 and DNMT1 in the different stages of early PA embryos by compareing to those of embryos from in-vitro fertilization (IVF).ObjectiveThe aims of the present study are to observe the development of PA embryos and compare the expression of GCN5, HDAC1, DNMT1 protein in PA and IVF mouse early diploid embryos, investigate the change of those key enzymes related to histone acetylation and DNA methylation in the embryo with only female genome,and analyze the influences and possible mechanisms of the genome from female or male gamete on epigenetic reprogramming.Material & methods1. Animals: ICR mice were utilized in the present study.2. PA group: ICR female mice were induced to superovulate by injection pregnant mare serum gonadotropin (PMSG), followed 48h later by injection human chorionic gonadotropin (HCG). Then the superovulated female mice were killed by neck broken after injection of HCG (pHCG) 16~18h and the oviductal ampullae were broken to release oocyte-conora-cumulus complexes (OCCs) and washed by modified human tubal fluid (MHTF). Oocytes were freed of cumulus cells by a brief exposure hyaluronidase, followed by gentle washings before activation treatment. The nude oocytes at Mil stage were treated with 5umol/L Ca-A23187 for 5 minutes and washed thrice by MHTF. Then the oocytes were cultured in 25 u mol/L 6-DMAP for 4 hours. After washing thrice, the treated oocytes were cultured in human tubal fluid (HTF) medium and observed at different time points. PA embryos at the stages of bipronucleus, two-cell and four-cell were picked up from culture at 4~6h, 24~26h and 48~50h after parthenogenetic activation respectively.3. IVF group: ICR female mice were induced to superovulate as the same as PA group. Male mice were killed by neck broken after female mice injected HCG 13h and the cauda epididymises were dissected out to achieve the sperms. Diluted sperm was placed on a culture dish for 1 ~2h capacitation time. The superovulated females were killed pHCG 14h to retrieve the OCCs. The sperm was added to the culture of oocytes at MII stage and observed at different time points. The mouse embryos at the stages of pronucleus, two-cell and four-cell were picked up through the culture at 4~6h, 24~26h and 48~50h after IVF.4. Fluorescent immunocytochemistry was used to determine the localizations and intensities of the expressions of GCN5, HDAC1, DNMT1 and proliferating cellnuclear antigen (PCNA) which was used as the criteria of proliferating potential in PA and IVF embryos at the different stages respectively.Results1. The development of PA embryoWith treatment of Ca-A23187 followed by 6-DMAP, PA activation rate in mouse oocyte is 87.07%, and cleavage rate is 73.28%, while blastocyst formation rate is 3.45%. There is no obvious morphologic abnormality in PA embryos picked up in present study and the embryos developed to the stages of pronucleus, two-cell, four-cell, eight-cell, morula, early blastocyst, blastocyst at pHCG 22~30h, 43~ 49h, 49~68h, 68~96h, 96~115h, 138~140h, 140~160h, which were similar with those in IVF embryos.2. Immunocytochemistry(1) No matter the embryos from in IVF group or PA group, or in the different cell stages, the expression level of PCNA remained almost the same in the tested embryos, which suggested that there is no significant differences of cell proliferation capacity between the IVF and PA embryos in the study and the expression alteration of the investigated proteins caused by the change of proliferation capacity of PA embryos might be excluded.(2) In IVF group, the expression of GCN5 was observed in the cytoplasm and nuclei of pronucleus, two-cell and four-cell embryos. The strongest fluorescent intensity was seen in four-cell embryos. Meantime the distribution of GCN5 fluorescence in blastomeres of four-cell embryo was found to be uneven and there is more intensive fluorescences in the side of blastomere near to the center of embryo than that to the peripheral. In PA group, the expression of GCN5 was observed in the cytoplasm and nuclei of pronucleus, two-cell and four-cell embryos but the intensities were significantly reduced and uneven distribution of GCN5 fluorescence in blastomeres disappeared.(3) In IVF group, the obvious expression of HDAC1 was also observed in the cytoplasm of pronucleus, two-cell and four-cell embryos. The most intensive HDAC1 fluorescence was seen in the cytoplasm of four-cell embryos. However, HDAC1 fluorescent staining in the nuclei was only seen in two-cell and four-cell embryos. There is no uneven distribution of HDAC1 fluorescence. In PA group, the expression of HDAC1 was observed in pronucleus, two-cell and four-cell embryos while their fluorescent intensities were significantly decreased.(4) In IVF embryos, the expression of DNMTl protein was seen in cytoplasm at the stages of pronucleus, two-cell and four-cell and the expression intensities was increased with the development of embryos. Comparing to IVF, the expression model of DNMTl protein in PA embryos was the same but the intensities were significantly decreased.Conclusions1. Mouse diploid PA embryos activated by the combination of Ca-A23187 with 6-DMAP possess the similar proliferation capacity with IVF embryos, which is not only able to provide an useful model for study on the mechanisms of fertilization, development and differentiation but also possible to be used for the establishment of PA stem cell line, which will be very useful in transplant medicine.2. GCN5 protein distributed in mouse four-cell embryo is uneven, which might play important roles in the differentiation of embryos. The absence of a male genome in mouse early embryo could decrease of expression of GCN5 as well as disturb the uneven distributions of GCN5 in the cytoplasma of blastomere.3. There is obvious expression of HDAC1 in the cytoplasm and nuclei of mouse early embryos. Male genome is necessary for the normal expression of HDAC1.4. The expression of DNMTl protein in PA embryos is significantly reduced compared to IVF embryo.5. The remarkable changes of expression levels of the GCN5, HDAC1 and DNMT1 in PA embryos imply that epigenetic reprogramming procedure depends on the co-operation of the genomes from both male gamete and female gamete.