Aberrant Gene Expressions Of Neuroactive Ligand-receptor Interaction Pathway In Brain Of Male-line-derived F2 Generation Conceived By In Vitro Maturation

Objective:To investigate the expression of relative genes of neuroactive ligand-receptor interaction pathway (Gabra6, Glra1, Gria4, Grid2, Agtr2, Slpr5, Galr1, Grm4) in IVM F2 mice, and to explore the transgenerational effect of the differential expression of these genes in newborn F1 IVM mice. Moreover, to illuminate the epigenetic modification of Glral and Glarl protein expression in brains of F2 IVM mice.Materials and methods:1. To obtained immature oocytes (GV oocytes) after the injection of pregnant mare serum gonadotropin (PMSG) in C57BL/6J mice. Then we cultured the GV oocytes to MII stage oocytes (IVM oocytes) in vitro. Meanwhile, we got in vivo mature oocytes (VVM oocytes) after the injection of PMSG and human chorionic gonadotrophin (HCG) in C57BL/6J mice. Both IVM oocytes and VVM oocytes were fertilized by ICSI and transferred to pseudopregnant ICR mice, respectively. First filial generation mice model of IVM was established. Then gene chip array (mouse genome 430 2.0) was used for the analysis of the gene expression in the brains of newborn F1 mice. The further Pathway analysis reveal that the expression of genes (Gabra6, Glral, Gria4, Grid2, Agtr2, Slpr5, Galr1, Grm4) in neuroactive ligand-receptor interaction pathway were the most significantly altered. The real-time PCR was used to detect the expression of these genes. These genes were all up-regulated in newborn F1 mice, but most of these differential genes recovered until lOw stage except Agtr2 were still up-regulated in female mice.2. After mating each other among different group of F1 mice, the F2 mice model was founded, classified as IVM, VVM and NC group. IVM group include:IVM(?)+natural pregnant(?) (MN), IVM(?)+IVM(?)(MM), natural pregnant(?)+IVM(?)(NM); VVM group include:VVM(?)+NC(?)(IN), VVM(?)+VVM(?)(â…¡), NC(?)+VVM(?)(NI); NC group: NC(?)+NC(?). Each group has 8 samples. VVM group was used as the control group and the natural pregnant mice were used as the negative control.3. The spatial learning and memory capability of second filial generation was detected by the water maze test. The incubation period and the times across the platform were recorded in 10week stages (the data was finished by our research group and the data have not been published. The result indicated that there was no different between the spatial learning and memory capability of these groups). Then the each group mice were sacrificed at 10w respectively.4. The expressions of relative genes of neuroactive ligand-receptor interaction pathway (Gabra6, Glra1, Gria4, Grid2, Agtr2, Slpr5, Galr1, Grm4) were detected by using real-time quantitative PCR in each 10w F2 mice group.5. The methylation status of neural Glral gene was meassured by pyrosequencing in 10w F2 mice.6. The Glral protein in lOw mice brains of the above groups were determined by Western blot.Result (s):1.Effects of IVM on the expression levels of relative genes of neuroactive ligand-receptor interaction pathway in F2 mice.When compared to WM and NC second filial generation at lOw period, Glra1 showed significantly down-regulated in IVM group and no significant differences were found in Gabra6, Gria4, Grid2, Agtr2, Slpr5, Galr1, Grm4. The single paternal transgenerational effects is remarkable therein (the Glral expressions of MN offspring is significantly down-regulated compared with IN offspring).2.Effects of IVM on the DNA methylation status of neural Glral in F2 mice.(1)When compared to VVM second filial generation at lOw period, neural Glra1 of IVM F2 offspring showed higher methylation rate in CpG6 site. In CpG2 site, IVM was significanly higher methylated than NC, but no statistical differences compared to VVM. No another differences were found in other CpG sites of Glral among above three groups.(2) The single paternal factor affected the methylation status of neural Glra1 F2 mice significantly. In this study, the methylation status of single IVM paternal second filial generational mice(MN) were significantly increased compare to single VVM paternal second filial generational mice(IN) in CpG1, CpG2 sites.3. Effects of IVM on the neural Glral protein in 10w F2 miceWhen compared with NC F2 offspring, the Glral protein level was expressed at lower level in IVM mice brain tissue at age of 10w. However, there were no statistically significant differences between IVM and VVM. Furthermore, through the independent-t test between MN and IN, single IVM paternal second filial generational mice(MN) have significant lower Glral protein expressions than single VVM paternal second filial generational mice(IN).Conclusion:1. The alterated expression of relative genes of neuroactive ligand-receptor interaction pathway in brain of IVM F1 newborn mice could change the cerebral gene expression of the male-line F2 mice.2.The differential expression of Glra1 gene in IVM F2 mice brain might be caused by the epimutation inheritance from male-line IVM F1 mice.3.The neural phenotype of IVM second filial generation mice have no differences compare to VVM or NC second filial generation mice, but they may possess higher risks of neural disease morbidity. Especially, the IVM paternal factor may cause the harmful effects for IVM second filial generation offspring.