Hsp27在卵母细胞成熟和植入前胚胎发育过程中的功能研究

发布时间：2018-02-16 08:24

本文关键词： Hsp27 多囊卵巢综合征细胞凋亡卵母细胞成熟胚胎发育　出处：《南京医科大学》2014年博士论文　论文类型：学位论文

【摘要】：研究背景多囊卵巢综合征(Polycystic Ovary Syndrome, PCOS)是育龄期妇女最常见的内分泌和代谢紊乱性疾病,育龄妇女中发病率为5%-10%,主要特征为排卵障碍、高雄激素血症、胰岛素抵抗以及卵巢多囊样改变。PCOS的病理生理变化涉及多种神经内分泌及代谢系统通路和多种卵巢局部的蛋白质调控因子等,某种调节机制的失常可以导致多种反馈失衡和连锁反应,产生临床表型的多样化和分类治疗的复杂性。因此,更好的了解PCOS与卵巢外部和内部的因子异常的关系,以及这些异常对颗粒细胞与卵母细胞之间的联系,卵子成熟和胚胎发育潜力的影响等,对于需要进行IVF治疗的PCOS女性提高生育力、优化临床卵巢刺激方案和提高妊娠结局都是至关重要的。虽然PCOS患者的特点是在IVF促排卵过程中产生大量的卵子,但是通常卵子质量较差,从而随后的受精率、卵裂率和着床率都较低,流产率高。有证据表明卵子和胚胎质量差可能是由于非整倍体率增加引起的,但最近的数据提示PCOS患者行体外受精(In Vitro Fertilization, ⅣF)治疗产生大量的卵子,整倍体率卵子也较多,但总体妊娠率仍较低,流产率增高,提示这与胚胎非整倍体无关。因此,染色体之外还有其它因素导致PCOS患者妊娠丢失率增高。PCOS女性卵子成熟受损和胚胎发育潜能降低可能与患者的内分泌／旁分因子异常、代谢异常和卵泡发育成熟过程中卵泡内环境的改变都有关。 PCOS的病因和病理生理机制尚有很多未知的环节。大量研究提示,在PCOS患者卵巢中存在着凋亡／抗凋亡的失衡。本实验室在前期的研究工作中构建了正常人与PCOS患者卵巢中差异蛋白质表达谱系,其中54种蛋白质在PCOS患者卵巢中表达上调,15种蛋白质表达下调。热休克蛋白27(Heat Shock Protein27, Hsp27)是该差异表达谱系表达下调的蛋白质之一。Hsp27是小分子量热休克蛋白亚家族中的重要成员之一,Hsp27作为调控凋亡因子,通过与细胞色素c结合,防止Caspase9激活,阻碍Fas、TNF介导的外源性凋亡途径产生抗凋亡作用。Hsp27可能通过抗凋亡机制的紊乱导致卵泡发育的停滞而不能正常周期性凋亡。我们前期研究了Hsp27对小鼠卵母细胞成熟的影响,研究证实Hsp27可以通过调节卵母细胞的早期凋亡从而调控其成熟,保证卵母细胞的正常生长发育和成熟。Hsp27通过活化Caspase8介导的外源性凋亡信号通路调节卵母细胞的凋亡进而调控卵母细胞的成熟；通过调节PKAc、PKCδ、PKOλ的表达影响卵母细胞的成熟。有研究提示Hsp27在受精和早期胚胎发育过程中可能也起着重要作用。而对于Hsp27对卵母细胞的成熟受精和随后胚胎发育的影响以及Hsp27在一系列过程中所起到的作用,尚未见报道。PCOS患者卵巢中存在着凋亡与抗凋亡的失衡,卵泡发育成熟障碍。而我们前期研究证实,PCOS卵泡中Hsp27低表达,可能引起抗凋亡机制的紊乱导致卵泡发育的停滞而不能正常周期性凋亡。卵泡发育障碍是否与此相关还不明确。而在临床治疗中发现,PCOS患者进行IVF促排卵治疗时,可以获得大量的卵母细胞,但获得的胚胎发育潜力和着床率较低。这是否与卵母细胞Hsp27的低表达有关,还是胚胎发育过程中Hsp27低表达所致。这些问题都值得深入探讨。因此,我们期望能研究PCOS患者小卵泡中HSP27的低表达对于卵母细胞成熟和随后受精和胚胎发育的影响,进一步探讨凋亡在卵母细胞成熟和早期胚胎发育过程中起到的作用。本研究以小鼠原核期胚胎和人PCOS患者未成熟的卵母细胞为对象,研究Hsp27在小鼠及PCOS患者卵母细胞成熟、受精、胚胎早期发育中的作用,观察Hsp27对卵子成熟及胚胎体外发育能力的影响,进一步探讨PCOS患者卵母细胞Hsp27低表达对随后胚胎发育潜力的影响。研究内容和结果 1.Hsp27在小鼠早期胚胎体外发育过程中的表达与定位 6-8周龄ICR小鼠超排后,合笼。从输卵管获取受精卵。置于培养液中体外培养,在培养的24h,48h,72h,96h后,受精卵分别发育到2细胞期,4-细胞期,8-16细胞期和囊胚期。收集后采用免疫荧光法对胚胎发育的各个时期Hsp27表达进行定位。结果显示,Hsp27蛋白定位于小鼠早期胚胎发育各时期的卵裂球除核仁外的细胞质和细胞核中。 2.Hsp27在小鼠早期胚胎发育过程中的功能应用显微注射方法分别将Hsp27抗体和已构建成功的重组腺病毒Ad-H1-siRNA注射入小鼠原核期受精卵,分别从蛋白和RNA水平上阻断受精卵的Hsp27的活性,观察Hsp27下调对胚胎体外发育的影响。同时重组腺病毒Ad-CMV-Hsp27显微注入受精卵胞浆中,上调受精卵的Hsp27表达,观察Hsp27上调后对胚胎发育的影响,将胚胎继续体外培养,观察各组囊胚形成率和囊胚细胞总数的差异,通过TUNEL法检测囊胚细胞的凋亡情况。结果发现,Hsp27抗体组的囊胚形成比例为84.8%,对照组分别为81.8%和82.2%,P0.05。囊胚细胞数分别为：67.5、67.1和71.0,p0.05,没有统计学差异。将重组腺病毒AdSiRNA-Hsp27采用显微注射方法注射入小鼠原核期受精卵,注射AdSiRNA-GFP作为对照,空白对照不注射。结果发现,三组之间囊胚形成率分别为,83.1%,86.3%87.7%,P0.05,没有统计学差异。Hsp27表达上调结果显示,显微注射重组腺病毒Ad-CMV-Hsp27和Ad-CMV-GFP以及Control组,三组的囊胚形成率分别为：84.5%、83.8%和86.4%,P0.05,没有统计学差异。以上试验结果表明,小鼠受精卵中Hsp27下调和上调后,对胚胎发育潜力均没有明显影响。通过总细胞数(total cell number,TCN)和细胞死亡率(cell death index,CDI)用来评估囊胚质量,结果发现超标达和干涉Hsp27表达后各组的囊胚质量没有差异。 3.Hsp27对人PCOS卵母细胞体外成熟的影响 3.1Hsp27表达上调对卵母细胞成熟的影响通过小卵泡穿刺,获得人PCOS患者的未成熟卵。将获得的GV期卵母细胞随机分为三组,利用显微注射技术,将重组的Hsp27质粒载体(pAdTrack-CMV-Hsp27)注射到GV期卵子的生发泡中,从基因水平上调Hsp27的表达。实验组注射pAdTrack-CMV-Hsp27质粒,一个对照组只注射空质粒载体(pAdTrack-CMV),另一对照组不行注射。注射完成后,将GV期卵子转移到体外成熟培养液中进行成熟培养,48h后观察卵母细胞体外成熟情况。结果显示,注射Hsp27超表达载体组卵母细胞的发育成熟率为33.5%,对照组分别为55.9%和61.4%,P0.05。表明,PCOS患者获得的未成熟卵Hsp27表达上调后,抑制了卵母细胞的体外成熟。 3.2Real time RT-PCR验证卵母细胞特异的分泌因子的表达 Hsp27超表达载体(pAdTrack-CMV-hHsp27)显微注射到人PCOS卵母细胞后,体外培养48h后,收集卵母细胞,提取mRNA,检测卵母细胞特异性的分泌因子Bmp15口Gdf9的表达变化,结果发现,Hsp27表达增加后,Bmp15和Gdf9的mRNA表达下降,从另一方面验证Hsp27对卵母细胞成熟的影响。 3.3Hsp27上调后凋亡调控因子的表达为了检测Hsp27上调后激活的凋亡通路,显微注射pAdTrack-CMV-hHsp27后,卵母细胞继续体外成熟培养,培养48h后,收集各组卵母细胞,提取mRNA,RNA反转录为cDNA,运用Real time RT-PCR检测Caspase3、Caspase8、Caspase9、Cytc的表达。结果显示,Track-CMV-hHsp27显微注射组中Caspase8、Caspase9和Caspase3的表达明显低于pAdTrack-CMV注射组(P0.05),而Cyt c的表达未有明显的差异。说明,HSP27在人PCOS卵母细胞中通过capase-8介导的外源性及capase-9介导的内源性凋亡通路发挥作用。 4.Hsp27表达上调对人PCOS卵母细胞体外受精胚胎发育能力的影响。获取的PCOS患者的GV期卵母细胞,显微注射Hsp27超表达载体后,继续体外成熟培养,将培养成熟的MII期卵母细胞行卵胞浆内单精子注射(ICSI)法受精。注射完成后,将卵子移入卵裂期胚胎培养液中培养,ICSI后16～-18h观察卵子受精情况。培养的第三天,将胚胎移入囊胚培养液中继续培养。在第五天和第六天观察囊胚形成情况,统计囊胚形成率。结果提示,Hsp27超表达后,卵子成熟率显著低于对照组(33.5%vs55.9%和61.4%,P0.05),Hsp27上调后成熟卵子的受精率、day3优质胚胎率与对照组没有显著差异,而Hsp27上调组的囊胚形成率显著高于对照组(41.30%vs23.53%,P0.05)。说明HSP27表达上调导致PCOS卵母细胞成熟发生不同程度的阻滞,然而HSP27却显著提高卵母细胞发育潜能,说明HSP27在卵母细胞进一步发育潜能起着重要的调节作用。数据分析运用SPSS17.0进行数据统计分析,采用单向方差分析和对数线性模型来比较mRNA和蛋白水平,而对于卵母细胞成熟率,受精率和胚胎发育数据,采用卡方分析进行比较,P0.05认为是具有统计学差异。结论 1.在小鼠早期胚胎发育过程中,Hsp27蛋白定位于早期胚胎发育各时期的卵裂球除核仁外的细胞质和细胞核中。Hsp27的上调或下调对体外培养胚胎发育潜能没有显著影响。 2.Hsp27通过Caspase8介导的外源性及capase-9介导的内源性凋亡信号通路调节卵母细胞的凋亡而影响其成熟,Hsp27的表达上调,使得caspase8凋亡相关因子表达降低,降低卵母细胞体外成熟率。 3.Hsp27作为抗凋亡活性因子,参与了PCOS卵母细胞凋亡的失衡。由于PCOS的卵巢凋亡的失衡状态导致了Hsp27的代偿性下降。
[Abstract]:Research background
Polycystic ovary syndrome (Polycystic Ovary, Syndrome, PCOS) is a disease of endocrine and metabolic disorders are most common in women of childbearing age, the incidence rate of 5%-10% in women of childbearing age, the main features of anovulation, Kaohsiung hormones, insulin resistance and polycystic ovary.PCOS involves a variety of pathophysiological changes of neuroendocrine and metabolic pathways and systems a variety of ovarian local regulatory protein factors, some regulatory mechanisms of arrhythmias can lead to a variety of complex feedback imbalance and chain reaction, clinical phenotype and diverse classification treatment. Therefore, a better understanding of the relationship between abnormal factor PCOS and ovarian internal and external, and these abnormalities of cumulus cells and oocytes contact oocyte maturation and embryo development potential, and the effects of the need for IVF treatment PCOS women improve fertility, optimize clinical Ovarian stimulation protocols and improve the outcome of pregnancy is essential. Although the characteristics of PCOS patients in IVF induced ovulation process produces a large number of eggs, but eggs usually of poor quality, and then the fertilization rate, cleavage rate and implantation rate were lower, the abortion rate is high. There is evidence that the quality of oocytes and embryos may be poor the aneuploidy rate increased, but recent data suggest that patients with PCOS in vitro fertilization (In Vitro Fertilization F, IV) treatment produced a large number of eggs, aneuploid rate of eggs is more, but the overall pregnancy rate is still low, the abortion rate increased, suggesting that the embryo aneuploidy has nothing to do therefore, other factors lead to the loss of chromosome.PCOS increased female egg maturation and impaired embryo development potential decrease may be associated with endocrine / paracrine factor PCOS in patients with abnormal pregnancy, abnormal metabolism and follicle development The changes in the follicular environment in the process of ripening are all related.
The etiology and pathophysiology of PCOS still has many unknown links. A large number of studies suggest that, in patients with ovarian PCOS exist in apoptosis / anti apoptosis imbalance. The previous study was constructed between normal persons and patients with ovarian PCOS in protein expression spectrum, upregulation of the expression of 54 proteins in patients with ovarian PCOS in 15 kinds of protein expression. Heat shock protein 27 (Heat Shock, Protein27, Hsp27) is one of the pedigree of the down regulated protein.Hsp27 is an important member of the small heat shock protein subfamily of the Hsp27 as a differential expression, regulation of apoptosis factor by binding to cytochrome c, preventing Caspase9 activation block Fas, exogenous apoptosis mediated by TNF may have anti apoptosis effect of.Hsp27 through anti apoptosis mechanism of follicular development disorder leads to stagnation and not the normal cycle of apoptosis. We Preliminary study of the effects of Hsp27 on mouse oocyte maturation, the study confirmed that Hsp27 can regulate the apoptosis of oocytes to regulate its mature through the development and maturation of.Hsp27, ensure the apoptosis of activated extrinsic apoptosis pathway of Caspase8 mediated regulation of oocyte and control oocytes through normal growth of oocytes. Mature; by adjusting the PKAc, PKC 8, PKO lambda expression affects oocyte maturation.
Studies have shown that Hsp27 may also play an important role in fertilization and early embryonic development process. But for Hsp27 on oocyte maturation and fertilization affect subsequent embryo development and Hsp27 in a series of process function, has not been reported there and anti apoptotic ovarian imbalance in.PCOS patients. Follicular maturation disorder. Our preliminary studies confirmed that PCOS follicles in the low expression of Hsp27, may cause the anti apoptosis mechanism of follicular development disorder leads to stagnation and not the normal cycle. The apoptosis of follicular development disorder is associated with this is not clear. But in clinical treatment, PCOS patients with IVF in ovulation induction treatment. You can get a lot of oocytes, but the embryo developmental potential and implantation rate is relatively low. This is the low expression of Hsp27 on oocytes, or during embryonic development of Hsp27 low expression . these problems are worth further discussion. Therefore, we can expect the low expression of HSP27 on PCOS patients with small follicle in effect on oocyte maturation and subsequent fertilization and embryo development, further discussion to apoptosis in oocyte maturation and early embryonic development process.
In this study, mouse pronuclear embryos and PCOS were immature oocytes as the object of study of Hsp27 in mice and PCOS patients with oocyte maturation, fertilization and early development of embryos, the effects of Hsp27 on oocyte maturation and developmental ability of embryos in vitro, to further explore the oocytes with PCOS low expression of Hsp27 influence on subsequent embryonic development potential.
Research content and results
Expression and localization of 1.Hsp27 in the development of early mouse embryos in vitro
6-8 week old ICR mice after superovulation, cage. Obtaining fertilized eggs from the fallopian tube. In cultured medium, in cultured 24h, 48h, 72h, 96h, respectively. The fertilized eggs develop into 2 cell stage, 4- cell stage, 8-16 cell and blastocyst stage. After collected by immunofluorescence the embryonic development of each period the expression of Hsp27 positioning. The results showed that Hsp27 protein was located in the blastomeres of mouse early embryo development in the nucleolus in the cytoplasm and the nucleus.
Function of 2.Hsp27 in the development of early mouse embryos
Application of micro injection methods respectively, Hsp27 antibody and has successfully constructed the recombinant adenovirus Ad-H1-siRNA was injected into mouse pronuclear zygote, blocking the activity of Hsp27 from the fertilized egg protein and the level of RNA, observe the Hsp27 downward effect on embryo development in vitro. The recombinant adenovirus Ad-CMV-Hsp27 were injected into a fertilized egg in the cytoplasm. Expression of Hsp27 of zygotes, influence on embryonic development observation of Hsp27 increases, will continue to observe the in vitro embryo, blastocyst formation rate and blastocyst cell number differences, the apoptosis of blastocyst cells was detected by TUNEL. The results showed that Hsp27 antibody group of the blastocyst formation ratio was 84.8%, the control group were 81.8% and 82.2%, P0.05. cell number of blastocyst were 67.5,67.1 and 71, P0.05, the difference was not statistically significant. The recombinant adenovirus AdSiRNA-Hsp27 by microinjection method was injected into mouse primary The nuclear stage fertilized eggs, AdSiRNA-GFP injection as control, blank control without injection. The results showed that the blastocyst formation rates between the three groups respectively, 83.1%, 86.3%87.7%, P0.05, no statistical difference between the.Hsp27 expression results showed that microinjection of recombinant adenovirus Ad-CMV-Hsp27 and Ad-CMV-GFP and Control group, three groups of blastocyst formation rates were: 84.5% 83.8%, and 86.4%, P0.05, the difference was not statistically significant. The above results show that the downregulation of Hsp27 and upregulation of mouse fertilized eggs, had no obvious effect on embryonic development potential. The total cell number (total cell, number, TCN) and cell death rate (cell death index, CDI) were used to assess the quality of blastocysts, the results found to exceed the standard of quality and interference blastocysts of Hsp27 expression after no difference.
Effect of 3.Hsp27 on the in vitro maturation of human PCOS oocytes
The effect of up-regulated expression of 3.1Hsp27 on oocyte maturation
Through the small follicle puncture, human immature oocytes retrieved from PCOS patients. GV stage oocytes were randomly divided into three groups obtained, using microinjection technique, the recombinant plasmid vector Hsp27 (pAdTrack-CMV-Hsp27) injection into germinal vesicle GV oocytes in gene expression level from the upregulation of Hsp27. The experimental group was injected with pAdTrack-CMV-Hsp27 plasmid, a control group was injected with empty plasmid vector (pAdTrack-CMV), a control group without injection. After the injection, the GV oocytes transferred to culture medium on in vitro maturation of mature oocytes in vitro, observe the maturation of 48h. The results showed that the injection of Hsp27 over expression vector group of oocytes maturation rate was 33.5%, the control group were 55.9% and 61.4%, P0.05. showed that the expression of PCOS in patients with immature oocytes after Hsp27 inhibited oocyte maturation in vitro.
3.2Real time RT-PCR verifies the expression of specific secretory factors in oocytes
Hsp27 over expression vector (pAdTrack-CMV-hHsp27) microinjection into human PCOS oocytes after 48h cultured in vitro after extraction of mRNA oocytes were collected, and the expression of secreted Bmp15 Gdf9 in detection of oocyte specific results showed that increased expression of Hsp27, Bmp15 and Gdf9 decreased expression of mRNA, from the impact of on the other hand, verification of Hsp27 on oocyte maturation.
Expression of regulation factor of apoptosis after up regulation of 3.3Hsp27
In order to detect the apoptosis pathway upregulation of Hsp27 after activation, micro pAdTrack-CMV-hHsp27 after injection of oocytes to mature in vitro, cultured 48h, collected oocytes, mRNA extraction, RNA reverse transcription into cDNA, detected by Caspase3, Real time RT-PCR Caspase8, Caspase9, Cytc expression. The results showed that Caspase8 Track-CMV-hHsp27 microinjection in the group, the expression of Caspase9 and Caspase3 were significantly lower than that of pAdTrack-CMV group (P0.05), and the expression of Cyt C. There is no obvious explanation, PCOS HSP27 in human oocytes by exogenous and endogenous apoptosis pathway mediated by capase-9 capase-8 mediated by play a role.
The effect of up-regulated expression of 4.Hsp27 on the development ability of human PCOS oocyte in vitro fertilization embryo.
Gets the PCOS of patients with GV stage oocytes microinjected with Hsp27 over expression vector, continue to mature in vitro, cultured mature MII oocytes intracytoplasmic sperm injection (ICSI) method of fertilization. After injection, the eggs in cleavage stage embryos cultured in ICSI 16 ~ -18h observation of fertilization. Third days of culture, the embryo into blastocyst culture medium to culture. Observation on the fifth day and the sixth day of blastocyst formation and blastocyst formation rate. The statistical results suggest that over expression of Hsp27, egg maturation rate was significantly lower than that of control group (33.5%vs55.9% and 61.4%, P0.05), mature the fertilization rate of Hsp27 increases, day3 high-quality embryo rate no significant difference between the two groups, Hsp27 increased group blastocyst formation rate was significantly higher than the control group (41.30%vs23.53%, P0.05). The results showed that HSP27 up-regulated the expression of PCOS in oocyte maturation occurs However, HSP27 significantly improves the developmental potential of oocyte, indicating that HSP27 plays an important role in the further developmental potential of oocyte.
Data analysis
SPSS17.0 was used for data analysis. One-way ANOVA and log linear models were used to compare mRNA and protein levels. For oocyte maturation rate, fertilization rate and embryo development data, chi square analysis was used for comparison. P0.05 thought there was statistical difference.
conclusion
1. in the process of early embryonic development in mice, the Hsp27 protein was located in the period of early embryonic development of blastomere.Hsp27 except outside the nucleolus in the cytoplasm and the nucleus upregulation or downregulation of embryo development potential has no significant effect on in vitro.
2.Hsp27 regulates the apoptosis of oocytes by Caspase8 mediated exogenous and capase-9 mediated endogenous apoptotic signaling pathways, and affects the maturation. Hsp27 expression is upregulated, which reduces the expression of apoptosis related factors and reduces the in vitro maturation rate of oocytes.
As an anti apoptotic active factor, 3.Hsp27 is involved in the imbalance of apoptosis in PCOS oocytes. The imbalance of PCOS's ovarian apoptosis leads to a compensatory decline in Hsp27.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.75

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