Effects Of In Vitro Maturation On Mouse Oocytes, Embryos And Offspring

Partâ… Effects of in vitro maturation on mouse oocytes, embryos and the development and neuroethology of offspringObjective:To reveal the effects and degree of IVM on oocytes, embryos and the development and neuroethology of offspring by establishing model of IVM, and to illuminate the short-term and long-term effects of IVM.Materials and methods:1. To establish model of IVM, the VVM (oocytes in vivo maturation) group was used as the control group; the effects of IVM on oocytes and embryos development were detected by comparing the fertilization, cleavage, and embryo development of preimplantation.2. Effects of IVM on the first filial generation1) The rates of pregnancy, birth, survival, and the sex ratio of postnatal mice were detected between IVM and VVM group.2) The effects of IVM on development were detected by checking the growth weight from newborn to the sex matured stages, and the specific gravity of the organs in 10d,3w and 1Ow. All the researches were done in the male and in the female mice.3) The reproductive capability was detected by checking the sperm vigor of 10w male mice using the SCA(Sperm Class Analyzer) system, checking the estrous cycle of female mice from 6w to 9w stages, and checking their procreation ability by mating each other during 6-8w.4) The spatial learning and memory capability was detected by using the water maze, and the incubation period and the times across the platform were recorded in 8w stages.3. Effects of IVM on the second filial generation1) The rates of birth, survival, and the sex ratio were detected in the second filial generation after mating each other, and the natural pregnant mice were used as the negative control.2) The effects of of IVM on growth, reproductive capability and spatial learning and memory capability were detected as the first filial generation.Result(s):1. The model of IVM was established successfully.2. The rates of fertilization, cleavage and development capability were significantly lower in IVM group than in VVM group.3. Effects of IVM on the first filial generation.1) The rates of birth and suvival of offspring were significantly lower in IVM group than in VVM group.2) Weights from 1d to 1Ow stages were significantly higher in IVM group than in the natural group; however, no differences were shown between IVM and VVM group. The specific gravity of brain in 1Od and 3w stages from IVM and VVM group were shown significantly lower than the natural group, no differences were shown between IVM and VVM group. No differences from other organs were detected.3) The quantities of the procreation were shown higher in IVM groups than in the natural groups, while no differences were shown between IVM groups and VVM groups. No differences were detected in the sperm activity and estrous cycle between IVM and the other groups4) No differences were detected in the learning and memory ability including incubation period and the times of crossing by the water maze testing between IVM and the other groups.4. Effects of IVM on the second filial generation1) The survival rates of the second filial generation from IVM group were shown significantly lower than the other groups, which mean the existance of the risk of the mate among IVM groups.2) No differences were detected in weights, specific gravity of organs, sperm activity and estrous cycle, incubation period and the times of crossing among the second filial generation.Conclusion(s):1. IVM could affect the fertilization, cleavage, and development of preimplantation and post-implantation, which indicated the existence of the potential risk of IVM on the development.2. The changes of weight, specific gravities of organs and the procreation capibility illustrated the multifold effects of IVM on the offspring. The degree of the effects should be further investigated.3. IVM could affect the survival rate of the second filial generation, which revealed the risk of the marriage and the procreation for the offspring.4. The effects of IVM covered many sides around oocytes, embryos and offspring (first and second filial generation), and the exact mechanism should be under investigation. Partâ…¡Expression profile of global genome in postnatal mouse brain conceived through in vitro maturationObjective:To investigate the effects of IVM on the expression profile of the whole genome, and to reveal the dynamic changes of the differential genes during development and the effects of inheritance.Materials and methods:1. The gene chip array (mouse genome 430 2.0) was used for the analysis of the gene expression in the brains of newborn mice between IVM and VVM group (3 male mice per group). The differentially expressed genes were chosen by significance analysis of microarray (SAM)2. The differentially expressed genes were analyzed by hierarchical cluster, gene ontology classification and pathway analysis.3. Real-time RT-PCR was used to verify the results of the gene chip.4. IVM,VVM and the natural pregnant mice were used to detect the dynamic changes of these differentially expressed genes during the development from newborn to adult in male and female mice.5. The altered genes in the first filial generation were detected about their conditions in 10w of the second filial generation by real-time RT-PCR.Results:1. Gene chip microarray analysis showed that there were 231 differentially expressed probes between the brain tissues of IVM and VVM group, representing 196 differentially expressed genes with 158 up-regulations and 38 down-regulations. The IVM group and VVM group could be distinguished by analysis of hierarchical cluster.2. GO analysis showed that differentially expressed genes were involved in many functions such as neural development, neural differentiation and the regulation of neural factor. 3. For the pathway analysis, in general, differential gene expression followed specific molecular pathways, and the most significantly altered one was the pathway named neuroactive ligand-receptor interaction4. Eight genes in neuroactive ligand-receptor interaction pathway and five important genes involved in the development of brain and nerves were found to be up-regulated in IVM group, and were confirmed by real-time RT-PCR.5. Some of the differential expressions of those genes disappeared during the development. Until lOw stage, most of the thirtheen differential gene recovered except that Neurodl and Pax6 were up-regulated in male mice, while Agtr2 were up-regulated and Pax6 were down-regulated in female mice.6. Expressions of Agtr2 were kept up-regulated in IVM male mice of the second filial generation, and no differences were shown among all the groups on the expressions of Neurodl and Pax6.Conclusions:1. Evidence presented here using a mouse model suggested that IVM could affect the brain gene expressions of the newborn mice, especially the neuroactive ligand-receptor interaction pathway.2. During the development, some of the genes recovered to normal expression while the others expressed abnormally.3. Some of the differentially expressed genes could last to the second filial generation, and show the sex difference. The relationship between the risk of nervousness disorders and the effects of IVM on offspring should be further evaluated. Partâ…¢Effects of IVM on the expression and the regulation of the imprinted genesObjective:To investigate the effects of IVM on the expression and regulation of the imprinted genes of the offspring.Materials and methods:1. The imprinted genes were chosen according to the gene chip, and were verified by the real-time RT-PCR.2. The expressions of the key regulating enzymes (Dnmtl, Dnmt3a, Dnmt3b, Dnmt31) were detected by real-time RT-PCR3. The DNA methylation status of CpG islands of the imprinted genes were detected by Bisufite Sequence PCR.Results:1. Eight imprinting genes were chosen according to the gene chip, including H19, Igf2, Kcnq1ot1, Mest, Peg3, Ube3a, Snrpn and Peg12.2. H19 were detected down-regulated and Kcnqlotl were detected up-regulated by real-time RT-PCR in IVM offspring.3. The expressions of Dnmtl were shown the higher in IVM group than the other groups, and the expressions of Dnmt3a were shown higher in IVM and VVM group than the Natural group. No differences were detected in the expressions of the other genes among the three groups.4. All the CpGs of H19 were moderately methylated, and the levels of methylation in IVM group were shown higher than the other groups, however, no statistical differences were shown. Some sites of maternal expression were methylated in IVM group, but were not detected in the other groups. All the CpGs of Kcnq1ot1 were moderately methylated. The methylations of Kcnqlotl were lower in IVM group; however, no stastical differences were shown. Conclusion:1. IVM could affect the expressions of H19 and Kcnq 1ot1 of the offspring.2. No differences in the status of the whole methylation were detected; however, the abnormalities of some important methylation sites might be involved in the differential expression of H19, which might be related to the abnormal expressions of Dnmtl and Dnmt3a, and lead to the abnormal expression of H19.3. No differences in the status of the whole methylation were detected in Kcnqlotl, and other regulating ways might be involved in the abnormal expression.4. The exact effects of IVM on the establishment, maintenance and regulation of the imprinting genes of the offspring should be under investigation.