体外成熟对小鼠卵母细胞、胚胎及出生后子代的影响
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摘要
第一部分体外成熟对小鼠卵母细胞、胚胎及出生后子代生长发育行为学的影响
目的：本研究拟通过建立小鼠卵母细胞体外成熟(in vitro maturation, IVM)技术平台,揭示IVM对小鼠卵母细胞,胚胎及出生后子代生长发育及行为学的影响,阐明IVM的短期及中长期效应。
材料和方法：
1.采用卵母细胞体外成熟技术结合胞浆内单精子注射技术(intracytoplasmic sperm injection, ICSI)建立小鼠IVM技术平台。比较IVM卵母细胞与体内成熟(in vivo maturation, VVM)卵母细胞受精率,卵裂率,胚胎发育率。
2.检测IVM对出生后子一代生长发育及相关行为学影响
1)比较IVM和VVM组移植后妊娠率,妊娠周期,子代出生率,子代存活率及雌雄比的差异。
2)生长曲线及器官发育检测：以IVM子代为研究对象,以VVM子代为对照组,同时设立自然妊娠组为阴性对照,自出生第1天起至第10周性成熟期测量不同性别各组小鼠体重变化；于10d,3w,10w取小鼠各器官,测量各组器官重量所占体重百分比。
3)发育期生殖能力检测：利用SCA (Sperm Class Analyzer)动物精子分析统检测10w雄鼠精子活力；雌鼠自6w起连续3周做阴道图片,观测动情周期；性成熟期各组雌雄小鼠两两1：1自然交配,比较其生育能力。
4)学习认知能力检测：水迷宫实验比较各组小鼠潜伏期及穿台次数。
2.检测IVM对出生后子二代生长发育及相关行为学影响
1)性成熟期各组子一代雌雄小鼠两两1：1自然交配,比较各组妊娠率,子二代存活率及雌雄比的差异。
2)生长曲线及器官发育,发育期生殖能力检测,学习认知能力检测同子一代。
结果：
1.通过优化IVM条件,成功建立小鼠IVM技术平台。
2.IVM组卵母细胞受精率、卵裂率及胚胎发育率均较VVM组显著性降低。
3.IVM对出生后子一代生长发育及相关行为学影响
1)IVM子一代出生率及存活率均较VVM组显著性降低。
2)IVM组子一代体重较自然妊娠组显著性升高,其脑组织器官比重较自然妊娠组显著性降低,但与VVM组比较无显著性差异。
3)IVM组子一代产仔数量显著性高于自然妊娠组,但与VVM组比较没有显著性差异。各组精子活力相关指数、动情周期无显著性差异。
4)水迷宫实验示各组潜伏期及穿台次数无显著性差异。
3.IVM对出生后子二代生长发育及相关行为学影响
1)IVM组子二代存活率较其余各组显著性降低。
2)IVM组子二代体重及器官比重与其余各组比较均无显著性差异。
3)子二代各组精子活力相关指数、动情周期无显著性差异。
4)水迷宫实验示各组潜伏期及穿台次数无显著性差异。
结论：
1.IVM影响卵母细胞受精、卵裂及胚胎发育。
2.IVM子一代出生率及存活率降低,体重增大,器官比重降低,表明IVM可影响子一代生长发育。
3.IVM影响子一代生育能力及子二代存活率,提示IVM存在婚配风险性。
4.IVM的影响涵盖卵母细胞、胚胎直至出生后子一代甚至子二代,是多方面多层次的,具体效应还有待进一步考证。
第二部分IVM对子代脑组织全基因组表达谱的影响
目的：通过研究IVM对子代脑组织全基因组表达谱的影响,阐明其对基因表达影响的差异程度及涉及的主要方面,并分析差异基因在生长发育过程中的动态变化及子二代效应,揭示IVM对子代神经系统及脑发育的影响机制。
材料和方法：
1.应用Affymetrix公司的Affymetrix mouse 430小鼠基因组表达芯片,对IVM子代新生雄性小鼠及VVM子代新生雄性小鼠各三只进行检测,利用芯片显著性分析(significance analysis of microarray, SAM),比较两组标本基因表达差异。
2.通过分层聚类,GO (gene ontology)分类和信号通路(Pathway)分析等方法对差异基因进行分析。
3.选择对芯片分析表达显著性差异的重要基因,经实时荧光定量RT-PCR验证
4.以验证证实表达显著性差异的基因为目标基因,以IVM、WM和自然妊娠出生(子一代)的10d龄(新生期),3w龄(生长期)和10w龄(性成熟期)小鼠脑组织为对象,RT-PCR分析表达差异基因在不同性别小鼠的不同生长发育时期的变化。
5.选取IVM子一代10w龄仍差异显著的基因,利用RT-PCR检测其在子二代小鼠脑组织的表达变化。
结果：
1.基因芯片结果显示共196个差异基因,其中IVM组较VVM组基因表达上调158个,下调38个,对差异基因进行无监督分层聚类,可完全区分IVM组和VVM组子代。
2.GO分析显示差异基因包括神经系统发育和神经分化相关基因。
3. Pathway分析发现10条主要的差异信号通路,差异最显著的是神经活动配体-受体相互作用(neuroactive ligand-receptor interaction)通路。
4.荧光定量RT-PCR检测神经活动配体-受体相互作用通路中8个基因及与脑发育相关的5个基因表达水平,与芯片结果一致。
5.IVM小鼠不同生长发育时期脑组织基因表达动态变化研究显示：上述13个基因在新生小鼠表达上调；3w龄时部分基因表达归于正常；到10w龄,除雄鼠Neurod1和Pax6表达上调,雌鼠Agtr2基因表达上调,Pax6基因表达下调外,其余基因表达均趋于正常。
6. RT-PCR分析IVM子二代小鼠脑组织基因表达,结果显示,Agtr2在IVM组子二代雄性小鼠仍表现为表达上升,而Neurod1和Pax6在IVM组子二代表达趋于正常。
结论：
1.IVM可影响子代脑组织基因表达,涉及神经系统发育和神经分化相关基因。
2.IVM子代脑组织中大部分差异表达基因会随生长发育逐步纠正,至性成熟期仍有部分未得到纠正而继续异常表达。
3.部分基因差异表达延续到子二代,并出现性别差异,提示IVM子代可能会有神经行为学改变风险。这些改变可能与某些神经系统疾病相关,仍需大量研究评估IVM远期安全性及潜在发病风险。
第三部分IVM对子代脑组织印记基因差异表达及调节的影响
目的：研究IVM对子代脑组织印记基因表达的影响及调控机制,评估IVM对子代印记基因甲基化调控改变的风险。
材料和方法：
1.本研究以IVM子代为研究对象,以VVM子代为对照,同时设自然妊娠子代为阴性对照,结合基因芯片,筛选可能存在差异的印记基因进行荧光定量RT-PCR检测。
2.荧光定量RT-PCR检测印记基因表达关键调节酶(Dnmt1, Dnmt3a, Dnmt3b, Dnmt31)的表达变化。
3.通过亚硫酸氢盐测序PCR (bisulfite-sequencing PCR, BSP)检测差异印记基因CpG岛的甲基化状态。
结果：
1.芯片共包含了70个印记基因,放宽条件,降低Fold值至1.4,筛选到8个印记基因,分别是H19, Igf2, Kcnq1ot1, Mest, Peg3, Ube3a, Snrpn和Peg12.
2.对IVM, VVM和自然妊娠三组子代进行上述8个基因的实时荧光定量PCR验证,结果显示,IVM子代中,H19表达下调,Kcnq1ot1表达上调,其余6个基因表达水平三组间无显著性差异。
3. Dnmt1表达水平IVM组较VVM及自然妊娠组显著性升高；Dnmt3a表达水平IVM及VVM组均较自然妊娠组显著性升高,而Dnmt3b及Dnmt31表达水平三组间无显著性差异。
4.采用BSP法分析H19基因启动子区CpG岛的12个CpG位点,结果发现,H19三组均呈中等程度甲基化,三组间甲基化状况基本相似,IVM组甲基化程度较其他两组高,但无显著性差异,部分母源性表达位点出现甲基化现象,而在其他两组均未发现；BSP法分析了Kcnq1ot1基因启动子区CpG岛的20个CpG位点,结果发现,三组均呈中等程度甲基化,三组间甲基化状况基本相似,IVM组甲基化程度较其他两组低,但无显著性差异。
结论：
1.IVM可影响子代脑组织印记基因H19及Kcnq1ot1表达水平。
2.IVM子代H19差异甲基化区CpG岛甲基化水平未见明显差异,但有部分母源性甲基化,可能与Dnmt1及Dnmt3a表达异常有一定关系,其相互作用从而影响基因表达。
3.IVM子代Kcnq1ot1差异甲基化区CpG岛甲基化水平未见明显差异,可能有其他调节方式影响该印记基因的表达。
4.IVM子代脑组织中的印记基因表达差异,表明IVM过程可能通过不同方式干扰印记的建立与维持,提示IVM子代可能存在一定的表遗传风险。在IVM乃至ART子代中印记破坏是否对胚胎发育和出生后患病高风险的确切影响尚需进一步研究。
PartⅠEffects of in vitro maturation on mouse oocytes, embryos and the development and neuroethology of offspring
Objective: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 generation
1) 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 generation
1) 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 groups
4) 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 generation
1) 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.
PartⅡExpression profile of global genome in postnatal mouse brain conceived through in vitro maturation
Objective: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 interaction
4. 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.
PartⅢEffects of IVM on the expression and the regulation of the imprinted genes
Objective: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-PCR
3. 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.
目的：本研究拟通过建立小鼠卵母细胞体外成熟(in vitro maturation, IVM)技术平台,揭示IVM对小鼠卵母细胞,胚胎及出生后子代生长发育及行为学的影响,阐明IVM的短期及中长期效应。
材料和方法：
1.采用卵母细胞体外成熟技术结合胞浆内单精子注射技术(intracytoplasmic sperm injection, ICSI)建立小鼠IVM技术平台。比较IVM卵母细胞与体内成熟(in vivo maturation, VVM)卵母细胞受精率,卵裂率,胚胎发育率。
2.检测IVM对出生后子一代生长发育及相关行为学影响
1)比较IVM和VVM组移植后妊娠率,妊娠周期,子代出生率,子代存活率及雌雄比的差异。
2)生长曲线及器官发育检测：以IVM子代为研究对象,以VVM子代为对照组,同时设立自然妊娠组为阴性对照,自出生第1天起至第10周性成熟期测量不同性别各组小鼠体重变化；于10d,3w,10w取小鼠各器官,测量各组器官重量所占体重百分比。
3)发育期生殖能力检测：利用SCA (Sperm Class Analyzer)动物精子分析统检测10w雄鼠精子活力；雌鼠自6w起连续3周做阴道图片,观测动情周期；性成熟期各组雌雄小鼠两两1：1自然交配,比较其生育能力。
4)学习认知能力检测：水迷宫实验比较各组小鼠潜伏期及穿台次数。
2.检测IVM对出生后子二代生长发育及相关行为学影响
1)性成熟期各组子一代雌雄小鼠两两1：1自然交配,比较各组妊娠率,子二代存活率及雌雄比的差异。
2)生长曲线及器官发育,发育期生殖能力检测,学习认知能力检测同子一代。
结果：
1.通过优化IVM条件,成功建立小鼠IVM技术平台。
2.IVM组卵母细胞受精率、卵裂率及胚胎发育率均较VVM组显著性降低。
3.IVM对出生后子一代生长发育及相关行为学影响
1)IVM子一代出生率及存活率均较VVM组显著性降低。
2)IVM组子一代体重较自然妊娠组显著性升高,其脑组织器官比重较自然妊娠组显著性降低,但与VVM组比较无显著性差异。
3)IVM组子一代产仔数量显著性高于自然妊娠组,但与VVM组比较没有显著性差异。各组精子活力相关指数、动情周期无显著性差异。
4)水迷宫实验示各组潜伏期及穿台次数无显著性差异。
3.IVM对出生后子二代生长发育及相关行为学影响
1)IVM组子二代存活率较其余各组显著性降低。
2)IVM组子二代体重及器官比重与其余各组比较均无显著性差异。
3)子二代各组精子活力相关指数、动情周期无显著性差异。
4)水迷宫实验示各组潜伏期及穿台次数无显著性差异。
结论：
1.IVM影响卵母细胞受精、卵裂及胚胎发育。
2.IVM子一代出生率及存活率降低,体重增大,器官比重降低,表明IVM可影响子一代生长发育。
3.IVM影响子一代生育能力及子二代存活率,提示IVM存在婚配风险性。
4.IVM的影响涵盖卵母细胞、胚胎直至出生后子一代甚至子二代,是多方面多层次的,具体效应还有待进一步考证。
第二部分IVM对子代脑组织全基因组表达谱的影响
目的：通过研究IVM对子代脑组织全基因组表达谱的影响,阐明其对基因表达影响的差异程度及涉及的主要方面,并分析差异基因在生长发育过程中的动态变化及子二代效应,揭示IVM对子代神经系统及脑发育的影响机制。
材料和方法：
1.应用Affymetrix公司的Affymetrix mouse 430小鼠基因组表达芯片,对IVM子代新生雄性小鼠及VVM子代新生雄性小鼠各三只进行检测,利用芯片显著性分析(significance analysis of microarray, SAM),比较两组标本基因表达差异。
2.通过分层聚类,GO (gene ontology)分类和信号通路(Pathway)分析等方法对差异基因进行分析。
3.选择对芯片分析表达显著性差异的重要基因,经实时荧光定量RT-PCR验证
4.以验证证实表达显著性差异的基因为目标基因,以IVM、WM和自然妊娠出生(子一代)的10d龄(新生期),3w龄(生长期)和10w龄(性成熟期)小鼠脑组织为对象,RT-PCR分析表达差异基因在不同性别小鼠的不同生长发育时期的变化。
5.选取IVM子一代10w龄仍差异显著的基因,利用RT-PCR检测其在子二代小鼠脑组织的表达变化。
结果：
1.基因芯片结果显示共196个差异基因,其中IVM组较VVM组基因表达上调158个,下调38个,对差异基因进行无监督分层聚类,可完全区分IVM组和VVM组子代。
2.GO分析显示差异基因包括神经系统发育和神经分化相关基因。
3. Pathway分析发现10条主要的差异信号通路,差异最显著的是神经活动配体-受体相互作用(neuroactive ligand-receptor interaction)通路。
4.荧光定量RT-PCR检测神经活动配体-受体相互作用通路中8个基因及与脑发育相关的5个基因表达水平,与芯片结果一致。
5.IVM小鼠不同生长发育时期脑组织基因表达动态变化研究显示：上述13个基因在新生小鼠表达上调；3w龄时部分基因表达归于正常；到10w龄,除雄鼠Neurod1和Pax6表达上调,雌鼠Agtr2基因表达上调,Pax6基因表达下调外,其余基因表达均趋于正常。
6. RT-PCR分析IVM子二代小鼠脑组织基因表达,结果显示,Agtr2在IVM组子二代雄性小鼠仍表现为表达上升,而Neurod1和Pax6在IVM组子二代表达趋于正常。
结论：
1.IVM可影响子代脑组织基因表达,涉及神经系统发育和神经分化相关基因。
2.IVM子代脑组织中大部分差异表达基因会随生长发育逐步纠正,至性成熟期仍有部分未得到纠正而继续异常表达。
3.部分基因差异表达延续到子二代,并出现性别差异,提示IVM子代可能会有神经行为学改变风险。这些改变可能与某些神经系统疾病相关,仍需大量研究评估IVM远期安全性及潜在发病风险。
第三部分IVM对子代脑组织印记基因差异表达及调节的影响
目的：研究IVM对子代脑组织印记基因表达的影响及调控机制,评估IVM对子代印记基因甲基化调控改变的风险。
材料和方法：
1.本研究以IVM子代为研究对象,以VVM子代为对照,同时设自然妊娠子代为阴性对照,结合基因芯片,筛选可能存在差异的印记基因进行荧光定量RT-PCR检测。
2.荧光定量RT-PCR检测印记基因表达关键调节酶(Dnmt1, Dnmt3a, Dnmt3b, Dnmt31)的表达变化。
3.通过亚硫酸氢盐测序PCR (bisulfite-sequencing PCR, BSP)检测差异印记基因CpG岛的甲基化状态。
结果：
1.芯片共包含了70个印记基因,放宽条件,降低Fold值至1.4,筛选到8个印记基因,分别是H19, Igf2, Kcnq1ot1, Mest, Peg3, Ube3a, Snrpn和Peg12.
2.对IVM, VVM和自然妊娠三组子代进行上述8个基因的实时荧光定量PCR验证,结果显示,IVM子代中,H19表达下调,Kcnq1ot1表达上调,其余6个基因表达水平三组间无显著性差异。
3. Dnmt1表达水平IVM组较VVM及自然妊娠组显著性升高；Dnmt3a表达水平IVM及VVM组均较自然妊娠组显著性升高,而Dnmt3b及Dnmt31表达水平三组间无显著性差异。
4.采用BSP法分析H19基因启动子区CpG岛的12个CpG位点,结果发现,H19三组均呈中等程度甲基化,三组间甲基化状况基本相似,IVM组甲基化程度较其他两组高,但无显著性差异,部分母源性表达位点出现甲基化现象,而在其他两组均未发现；BSP法分析了Kcnq1ot1基因启动子区CpG岛的20个CpG位点,结果发现,三组均呈中等程度甲基化,三组间甲基化状况基本相似,IVM组甲基化程度较其他两组低,但无显著性差异。
结论：
1.IVM可影响子代脑组织印记基因H19及Kcnq1ot1表达水平。
2.IVM子代H19差异甲基化区CpG岛甲基化水平未见明显差异,但有部分母源性甲基化,可能与Dnmt1及Dnmt3a表达异常有一定关系,其相互作用从而影响基因表达。
3.IVM子代Kcnq1ot1差异甲基化区CpG岛甲基化水平未见明显差异,可能有其他调节方式影响该印记基因的表达。
4.IVM子代脑组织中的印记基因表达差异,表明IVM过程可能通过不同方式干扰印记的建立与维持,提示IVM子代可能存在一定的表遗传风险。在IVM乃至ART子代中印记破坏是否对胚胎发育和出生后患病高风险的确切影响尚需进一步研究。
PartⅠEffects of in vitro maturation on mouse oocytes, embryos and the development and neuroethology of offspring
Objective: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 generation
1) 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 generation
1) 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 groups
4) 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 generation
1) 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.
PartⅡExpression profile of global genome in postnatal mouse brain conceived through in vitro maturation
Objective: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 interaction
4. 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.
PartⅢEffects of IVM on the expression and the regulation of the imprinted genes
Objective: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-PCR
3. 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.
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