雌激素受体α在小鼠植入前胚合子型基因激活中的作用机制探讨
发布时间：2018-11-05 14:17
【摘要】：目的:1、探讨雌激素受体α(estrogen receptor alpha,ERα)及其磷酸化激活形式p ERα-S118在小鼠1-细胞胚和2-细胞胚中的阶段特异性表达与小鼠合子型基因激活的关系。2、探讨ERα特异性抑制剂MPP处理对小鼠植入前胚发育的阶段特异性影响,及其对合子型基因激活的作用机制。3、探讨ERα通过micro RNA途径影响小鼠合子型基因激活的机制,并研究其对干细胞关键因子Oct4和Sox2的调控作用。方法:1、采用输卵管冲洗的方式,于hCG注射后15 h~54 h每隔3 h收集小鼠植入前胚,利用细胞免疫荧光技术检测ERα和p ERα-S118的表达定位。2、收集昆明小鼠1-细胞胚、2-细胞胚和4-细胞胚,以空白KSOM培养液(单纯型优化的胚胎培养基)为对照组,以KSOM中添加MPP为实验组;将各时间点收集的植入前胚培养3 h后再移入新KSOM培养液中继续培养至囊胚阶段,并记录囊胚数。于hCG注射后21 h收集小鼠1-细胞胚,置于MPP中培养,于hCG后27 h,提取总RNA,RT-q PCR检测合子型标志基因e IF1A和Mu ERV-L的表达变化;采用细胞免疫荧光技术检测经21~27 h MPP处理后,组蛋白甲基化(H3K4me3和H3K27me3)水平的改变。于hCG注射后21 h收集小鼠1-细胞胚,分别置于KSOM+BrdU和KSOM+MPP+BrdU培养液中培养,于hCG后27 h收集样品,采用抗BrdU抗体免疫染色实验观察DNA复制情况;于hCG注射后27h收集小鼠1-细胞胚,经不同浓度MPP处理后,于hCG后42 h固定细胞,用免疫荧光技术观察2-细胞胚核像的变化;并且用BrdU掺入法检测DNA复制是否受到影响;采用免疫荧光技术检测S期启动相关因子c-MYC和E2F1的表达变化。3、于hCG注射后27 h收集小鼠1-细胞胚,建立27~45 h MPP处理(20μmol/L)实验组和KSOM对照组模型;提取总RNA,经Megaplex逆转录以及预扩增后,进行Taq Man定量PCR芯片检测,收集数据;通过交集分析,筛选出差异表达的micro RNA与已知小鼠精子micro RNA表达谱的一致部分;然后通过micro RNA靶基因注释、信号通路研究等手段,进一步分析ERα通过micro RNA途径影响小鼠植入前胚发育的可能机制;采用细胞免疫荧光技术验证靶基因表达产物(Oct4和Sox2)的变化。结果:1、ERα在小鼠植入前胚中的核内定位首次出现于S期中期,于第一次有丝分裂期核内定位消失,重新出现于2-细胞胚早期(第一次有丝分裂的末期),并保持核内定位和胞质定位至2-细胞胚分裂期。p ERα-S118在小鼠1-细胞胚的早期可见细胞核表面的定位,并且在1-细胞胚和2-细胞胚有丝分裂前期和前中期出现核内定位增强,中期出现胞质表达水平升高,于后期迅速降低;p ERα-S118在2-细胞核内的定位首次出现于S期启动时。2、MPP处理对小鼠植入前胚发育具有显著的抑制作用,其阶段特异性影响与小鼠ZGA发生的时间一致。经21~27 h MPP处理,小鼠合子型基因e IF1A和Mu ERV-L m RNA表达下调;1-细胞胚雄原核H3K27me3表达水平降低。MPP处理对小鼠的1-细胞胚和2-细胞胚的S期进程都有显著的抑制作用。c-MYC和E2F1在小鼠2-细胞胚内的表达不受MPP处理的影响。3、Micro RNA芯片结果显示,表达上调的micro RNA数目(126个)多于表达下调的micro RNA(64个)。受MPP处理影响的37个精源性micro RNA显著参与“应激反应、大分子物质代谢、基因表达和转录后调控、基因表观遗传学调控、负反馈调控(大分子物质)代谢过程、抑制基因表达、抑制m RNA翻译”等多种分子功能。精源性micro RNA靶基因集在“DNA依赖性转录调控”中有显著的富集;而且其中的小规模合子型靶基因更显著地富集于多种生物学过程。以“Akt1 Foxo3 Stat3 ERα Oct4”为主轴的调控网络参与了小鼠小规模合子型基因激活的DNA依赖转录调控。经MPP处理后,小鼠2-细胞胚OCT4表达水平升高,SOX2表达下调;经Akt抑制剂API-2处理后,p ERα-S118表达上调。结论:1.ERα可通过影响小鼠早期植入前胚的S期进程、精源性染色质的组蛋白甲基化修饰、精源性micro RNA表达等途径,参与小鼠小规模合子型基因的DNA依赖性转录。2.ERα在小鼠合子型基因激活中的作用机制还包括对干性维持关键基因Oct4和Sox2的调控。3.而且,ERα在小鼠合子型基因激活中的作用受到Akt的磷酸化激活调控,而ERα又可以通过mi R-125a-5p和mi R-125a-3p途径,影响Akt的功能,形成反馈调控网络。
[Abstract]:Objective: 1. To investigate the relationship between the specific expression of estrogen receptor alpha (ER) and its phosphorylation-activated form (p ER)-S118 in mouse 1-cell embryo and 2-cell embryo and the activation of zygotic gene in mice. Objective: To investigate the specific effect of ER proton-specific inhibitor (MPP) on the stage-specific development of mouse preimplantation embryos and its effect on the activation of zygotes-type genes. The key factors of stem cell factor Oct4 and Sox2 were studied and studied. Methods: 1. The pre-implantation embryos were collected from 15 h ~ 54 h after hCG injection. The expression and localization of ER and p ER-S118 were detected by immunofluorescence technique. The 1-cell embryos, 2-cell embryos and 4-cell embryos of Kunming mice were collected. The blank KSOM culture medium was used as the control group, and MPP was added to KSOM as experimental group. The pre-implantation embryos collected at each time point were cultured for 3 h, then moved into the new KSOM culture medium and then cultured to the blastocyst stage, and the blastocyst count was recorded. 1-cell embryos of mice were collected for 21 h after hCG injection, cultured in MPP, extracted for 27h after hCG, total RNA was extracted, RT-q PCR was used to detect the expression changes of zygotic marker genes e IF1A and Mu ERV-L, and after 21-27h MPP treatment was detected by immunofluorescence technique, Changes in Histone Methylation (H3K4me3 and H3K27me3) levels. The 1-cell embryos of mice were collected at 21h after hCG injection, respectively placed in KSOM + BrdU and KSOM + MPP + BrdU culture solution, samples were collected at 27h after hCG, DNA replication was observed with anti-BrdU antibody immunostaining experiment, 1-cell embryos were collected after hCG injection, and treated with MPP treatment at different concentrations, The expression of c-MYC and E2F1 in S phase was detected by immunofluorescence technique, and the expression of c-MYC and E2F1 was detected by immunofluorescence technique. The 1-cell embryos of mice were collected after hCG injection for 27 h, and 27-45h MPP treatment (20 umol/ L) test group and KSOM control group model were established; total RNA was extracted, and after Meaplex reverse transcription and pre-amplification, a TaqMan quantitative PCR chip detection and data collection were carried out; and by intersection analysis, The results showed that the microRNAs of differentially expressed microRNAs were consistent with those of the known mouse sperm micro RNA expression profiles, and then the possible mechanism of ER gene expression was further analyzed by micro RNA target gene annotation, signal path research and so on, and the possible mechanism of the mouse embryo development was further analyzed by micro RNA pathway. The changes of target gene expression products (Oct4 and Sox2) were verified by cell immunofluorescence. Results: 1. The nuclear localization of ER IUD in the embryos of mice was first observed in the mid-stage of S phase, disappeared in the nucleus of the first mitotic phase, and reappeared in the early stage of 2-cell embryo (the end of the first mitosis). and keeping the nuclear localization and cytoplasmic localization to the 2-cell embryo division stage. In the early stage of mitosis of 1-cell embryo and 2-cell embryo, nuclear localization was enhanced in the early stage and metaphase of 1-cell embryo and 2-cell embryo, the level of cytoplasmic expression increased in the medium term, and decreased rapidly in later stage. At the start of S phase, p ER 044-S118 appeared in S phase for the first time. MPP treatment had a significant inhibitory effect on the development of mouse embryos before implantation, and its stage-specific effects were consistent with the time of ZGA in mice. After 21-27h MPP treatment, mouse zygotic gene e IF1A and Mu ERV-L mRNA were down-regulated; 1-cell embryo male pronucleus H3K27me3 expression level decreased. MPP treatment had significant inhibitory effect on the S phase processes of 1-cell and 2-cell embryos in mice. The expression of c-MYC and E2F1 in mouse 2-cell embryos was not affected by MPP treatment. 37 sperm source micro RNAs affected by MPP treatment were significantly involved "Stress response, macromolecular substance metabolism, gene expression and post-transcriptional regulation, gene epigenetic regulation, negative feedback regulation (macromolecular substance) metabolism, inhibition of gene expression, and inhibition of m RNA translation" and the like. The fine-source micro RNA target gene set has significant enrichment in the 鈥淒NA-dependent transcriptional regulation鈥，
本文编号：2312335
[Abstract]:Objective: 1. To investigate the relationship between the specific expression of estrogen receptor alpha (ER) and its phosphorylation-activated form (p ER)-S118 in mouse 1-cell embryo and 2-cell embryo and the activation of zygotic gene in mice. Objective: To investigate the specific effect of ER proton-specific inhibitor (MPP) on the stage-specific development of mouse preimplantation embryos and its effect on the activation of zygotes-type genes. The key factors of stem cell factor Oct4 and Sox2 were studied and studied. Methods: 1. The pre-implantation embryos were collected from 15 h ~ 54 h after hCG injection. The expression and localization of ER and p ER-S118 were detected by immunofluorescence technique. The 1-cell embryos, 2-cell embryos and 4-cell embryos of Kunming mice were collected. The blank KSOM culture medium was used as the control group, and MPP was added to KSOM as experimental group. The pre-implantation embryos collected at each time point were cultured for 3 h, then moved into the new KSOM culture medium and then cultured to the blastocyst stage, and the blastocyst count was recorded. 1-cell embryos of mice were collected for 21 h after hCG injection, cultured in MPP, extracted for 27h after hCG, total RNA was extracted, RT-q PCR was used to detect the expression changes of zygotic marker genes e IF1A and Mu ERV-L, and after 21-27h MPP treatment was detected by immunofluorescence technique, Changes in Histone Methylation (H3K4me3 and H3K27me3) levels. The 1-cell embryos of mice were collected at 21h after hCG injection, respectively placed in KSOM + BrdU and KSOM + MPP + BrdU culture solution, samples were collected at 27h after hCG, DNA replication was observed with anti-BrdU antibody immunostaining experiment, 1-cell embryos were collected after hCG injection, and treated with MPP treatment at different concentrations, The expression of c-MYC and E2F1 in S phase was detected by immunofluorescence technique, and the expression of c-MYC and E2F1 was detected by immunofluorescence technique. The 1-cell embryos of mice were collected after hCG injection for 27 h, and 27-45h MPP treatment (20 umol/ L) test group and KSOM control group model were established; total RNA was extracted, and after Meaplex reverse transcription and pre-amplification, a TaqMan quantitative PCR chip detection and data collection were carried out; and by intersection analysis, The results showed that the microRNAs of differentially expressed microRNAs were consistent with those of the known mouse sperm micro RNA expression profiles, and then the possible mechanism of ER gene expression was further analyzed by micro RNA target gene annotation, signal path research and so on, and the possible mechanism of the mouse embryo development was further analyzed by micro RNA pathway. The changes of target gene expression products (Oct4 and Sox2) were verified by cell immunofluorescence. Results: 1. The nuclear localization of ER IUD in the embryos of mice was first observed in the mid-stage of S phase, disappeared in the nucleus of the first mitotic phase, and reappeared in the early stage of 2-cell embryo (the end of the first mitosis). and keeping the nuclear localization and cytoplasmic localization to the 2-cell embryo division stage. In the early stage of mitosis of 1-cell embryo and 2-cell embryo, nuclear localization was enhanced in the early stage and metaphase of 1-cell embryo and 2-cell embryo, the level of cytoplasmic expression increased in the medium term, and decreased rapidly in later stage. At the start of S phase, p ER 044-S118 appeared in S phase for the first time. MPP treatment had a significant inhibitory effect on the development of mouse embryos before implantation, and its stage-specific effects were consistent with the time of ZGA in mice. After 21-27h MPP treatment, mouse zygotic gene e IF1A and Mu ERV-L mRNA were down-regulated; 1-cell embryo male pronucleus H3K27me3 expression level decreased. MPP treatment had significant inhibitory effect on the S phase processes of 1-cell and 2-cell embryos in mice. The expression of c-MYC and E2F1 in mouse 2-cell embryos was not affected by MPP treatment. 37 sperm source micro RNAs affected by MPP treatment were significantly involved "Stress response, macromolecular substance metabolism, gene expression and post-transcriptional regulation, gene epigenetic regulation, negative feedback regulation (macromolecular substance) metabolism, inhibition of gene expression, and inhibition of m RNA translation" and the like. The fine-source micro RNA target gene set has significant enrichment in the 鈥淒NA-dependent transcriptional regulation鈥，
本文编号：2312335
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