The Expression Pattern Of Neuronatin (NNAT) In Pig Parthenogenetic Fetuses And Mouse Preimplantation Embryos

In nature, the parthenogenesis is common in invertebrates and lower vertebrates,but there isn’t natural parthenogenesis in mammals. Scientists found theparthenogenetic embryos can be implanted and develop to the somite stage byactivating MetaphaseⅡ(MⅡ) oocytes in vitro and transplanted into receptor. It isgenerally believed that aberrant expression of imprinted genes participates in growthretardation of mammalian parthenogenesis. Many studies have determined themethylation dynamics of some imprinted genes in gamete and preimplantationembryos of mouse, human and bovine. Neuronatin (NNAT), amaternally imprintedgene, firstly found in the brains of newborn rats, plays important roles in neuronalgrowth and metabolic regulation. In order to investigate the role of NNAT on theparthenogenetic development and early embryonic development, the pigparthenogenetic (PA) fetus and mouse preimplantation embryos were selected tostudy the expression patterns and methylation status of NNAT.Firstly, pig PA and normally fertilizated fetuses (control) were collected on Day28of gestation by the PA activation technique and by in vitro fertilization. Here wehave compared the gene expression and promoter methylation pattern of NNATbetween pig control and PA samples using Q-PCR (quantitative real-time PCR) andBSP (bisulfite sequencing PCR). The results showed loss of NNAT expression (p<0.001) and hypermethylation of NNAT promoter in PA samples. Additionally, partialmethylation was observed in control fetuses, while almost full methylation andunmethylation of NNAT promoter were apparent in Metaphase Ⅱ (MⅡ) oocytes andmature sperms, respectively, which identified the CpG promoter region as a putativedifferentially methylated region (DMR) of NNAT. The data demonstrate thatpromoter hypermethylation is associated with the silencing of NNAT in pig PA fetuses, which may be related to developmental failure of pig parthenogenesis atearly stages.The mRNA of NNAT was observed in mouse brain but lost in liver by Q-PCR(P<0.001). Therefore, the brain and liver were selected as positive and negativecontrol to determine the methylation patterns off our predicted CpG islands. Data ofBSP showed there were no differences in the four CpGs with hypermethylationexcept the CpG3with obvious different methylation levels between brain and liver.So we speculate that the CpG3is the putative DMR region of mouse NNAT. Inaddition, the methylation level of CpG3was also detected in mouse gamete,inconsistent with hypermethylation in pig MⅡ oocytes, the hypomethylation wasfound in mouse MⅡ oocytes. Therefore, we hypothesize that there may bedifferences of the expression patterns of NNAT between species.The mouse fertilized embryos obtained by superovulation were cultured in vitrousing KSOM to harvest preimplantation embryos. BSP results showed that, with the4cell stage and the morula stage as the turning points, the methylation level of CpG3was firstly increased then decreased and then elevated again in blastocyst stage. Andthe methylation levels of1,2,4-6,8,10,12-14sites of CpG3were obviouslydifferent in mouse embryos and tissues. Therefore, we speculate that these sites maybe the key sites of mouse NNAT regulatory region.This study reveals that promoter CpG hypermethylation mediated the silence ofNNAT gene may be related to developmental failure of pig parthenogenesis at earlystages. Additionally, methylation dynamics of NNAT in mouse preimplantationembryos were determined for the first time, which may contribute to the functionstudy of NNAT in mammalian development.