AQP7在卵母细胞冷冻保存与成熟中的功能及其机制研究

发布时间：2018-08-27 15:32

【摘要】：1冷冻保护剂调节卵母细胞中AQP7表达而促进冷冻过程中水转运的研究目的：检测卵母细胞中AQP7在卵母细胞冷冻保存中的作用材料与方法： 1.收集行人MⅡ期卵母细胞和C57BL/6J小鼠MⅡ期卵母细胞,检测水通道蛋白AQP3,7,9的mRNA和蛋白水平的表达。 2.比较卵母细胞在汞离子处理和不处理两种情况下,卵母细胞的对低渗溶液膨胀能力和对水的转运速率。 3.分别用8%乙二醇(EG),9.5%DMSO和0.5M蔗糖的HTF溶液处理卵母细胞,检测AQP3,7,9蛋白的表达。 4.转染表达GFP-hAQP7融合蛋白载体到293T细胞中,分别用EG, DMSO和蔗糖的DMEM溶液处理,实时监测细胞中GFP-hAQP7蛋白表达变化。 5.使用显微操作仪固定卵母细胞,实时监测卵母细胞在EG, DMSO溶液中体积的变化。 6.比较使用EG和DMSO作为冷冻保护剂,卵母细胞的存活率。结果： 1.在人和小鼠的MⅡ期的卵母细胞中,都能检测到AQP3,7,9的mRNA和蛋白水平的表达。 2.卵母细胞在汞离子处理后,对低渗溶液膨胀能力减弱,对水的转运速率降低。 3.乙二醇(EG), DMSO和蔗糖可使卵母细胞AQP7蛋白表达水平上调,并且DMSO处理后上调效应最强烈。而AQP3和9表达水平并没有改变。 4. EG, DMSO和蔗糖可以上调293T细胞中GFP-hAQP7蛋白表达。并且同样是DMSO处理组GFP-hAQP7蛋白表达上调效应最明显。 5.相比较EG,卵母细胞在DMSO溶液中体积的变化更快。 6.相比较EG,用DMSO做冷冻保护剂,卵母细胞冷冻保存后的存活率低。结论：DMSO比EG更能刺激卵母细胞中AQP7表达上调。这个上调作用可以促进卵母细胞在冷冻保存过程中对水的渗透,减少卵母细胞达到渗透压平衡时间。 2AQP7在卵母细胞成熟和冷冻中的功能的研究目的：阐明AQP7在卵母细胞成熟和冷冻保存中的功能材料与方法： 1.收集自然周期和控制超促排卵(COH)的小鼠MⅡ期卵母细胞,分别进行体外受精实验。比较两组细胞受精率。用Real-time PCR检测两组细胞AQP7mRNA表达差异。 2.收集GV期和MⅡ期卵母细胞,运用Real-time PCR比较这两种时期卵母细胞中AQP7的mRNA表达差异。运用细胞免疫荧光方法检测AQP7蛋白在这两种细胞中分布差异。 3.采用显微注射技术将AQP7siRNA注射到GV期卵母细胞中,敲减AQP7的表达。与注射Sramble siRNA比较,计算卵细胞体外的成熟率。 4.收集自然周期的MⅡ期卵母细胞,分别用含有insulin, LH和FSH这三种激素的HTF培养细胞1h,采用免疫荧光方法检测AQP7的在细胞内的分布的改变。 5.收集GV,M Ⅰ和MⅡ期卵母细胞,采用免疫荧光方法检测这三种时期的卵母细胞中AQP7与F-actin的共定位情况。 6.分别使用EG和DMSO对卵母细胞玻璃化冷冻,解冻后和新鲜卵母细胞同时进行体外受精,计算受精率。 7.新鲜未冷冻的卵母细胞作为对照组,采用免疫荧光法检测卵母细胞玻璃化冷冻解冻后2h后,AQP7的表达情况。 8.分别注射Sramble RNA和AQP7siRNA到GV卵母细胞,体外培养16-18h后,使用EG进行玻璃化冷冻,解冻统计存活率。结果： 1.COH组组卵母细胞体外受精率明显低于自然周期组,并且COH组MⅡ期卵母细胞AQP7mRNA表达水平显著低于自然周期组。 2.MⅡ期卵母细胞AQP7mRNA表达水平显著低于GV期。在MⅡ期卵母细胞中,相比较GV期而言,AQP7更多分布在细胞膜上,细胞质重分布明显减少。 3.注射靶向AQP7的siRNA到GV期卵母细胞中敲减AQP7表达后,卵母细胞成熟率显著降低。 4. Insulin和LH处理后,AQP7在卵母细胞膜上分布增多,细胞质中分布减少。而使用FSH处理,AQP7分布并没有改变。 5. GV, M I和MⅡ期卵母细胞中都能检测到AQP7与F-actin共定位。随着卵母细胞的发育,AQP7在这三种时期细胞内细胞质中的分布减少,细胞膜上分布增多。而F-actin正好相反,在胞质中的分布增多,细胞膜上分布减少。 6.使用DMSO或者EG作为冷冻保护剂冻存的卵母细胞解冻后体外受精能力没有差异,均低于新鲜对照组。AQP7表达量在DMSO和EG组之间并没有显著差异,但均高于未冷冻的对照组。 7.AQP7表达敲减后,卵母细胞冷冻保存后存活率是显著降低。结论：AQP7通过与F-actin共定位在一起,通过F-actin运输作用,在卵母细胞从GV期发育到MⅡ期过程中从胞质向胞膜上转运,参与卵母细胞的成熟。敲减AQP7表达后,卵母细胞冷冻保存后的存活率显著降低。 3冷冻保护剂和高渗透压刺激卵母细胞中AQP7表达和定位的改变是通过PI3K/PKC通路调节的机制研究目的：明确冷冻保护剂和高渗透压刺激卵母细胞中AQP7表达和定位改变的分子机制。材料与方法： 1.采用细胞免疫荧光和蛋白免疫印(Western blotting)检测卵母细胞上蛋白CPEB和Aurora A磷酸化和总蛋白在冷冻保护剂EQDMSO和蔗糖溶液处理后的表达水平。 2.分别Staurosporine/HTF, LY294002/HTF, U0126/HTF, SP600125/HTF预处理MⅡ期的卵母细胞,第五组为对照。再用8%EG/HTF溶液处理20min后采用免疫荧光方法检测AQP7, CPEB,磷酸化CPEB, Aurora A和磷酸化Aurora A蛋白表达水平。 3.293FT细胞中表达GFP-hAQP7,同2使用方法处理,激光共聚焦显微镜下检测GFP-hAQP7的表达量。用Western blotting检测GFP-hAQP7蛋白水平。 4.用浓度分别为0.25M,0.5M,0.7M,1M蔗糖的高渗透压溶液分别处理卵母细胞20min, HTF为对照组,使用免疫荧光方法检测各组AQP7的表达。 5.293FT细胞转染GFP-hAQP7载体,48h后分别用DMSO,EG和蔗糖溶液处理20min, HTF组为对照。使用激光共聚焦显微镜下检测各组GFP-hAQP7的表达。转入pEGFP-Cl载体作为对照。 6.对表达GFP-hAQP7的293FT细胞,裂解细胞,使用免疫共沉淀的方法检测AQP7和F-ACTIN在细胞中绑定情况。结果： 1. EG, DMSO和蔗糖均可以上调卵母细胞中磷酸化的CPEB蛋白表达水平。DMSO组与EG组比较起来,DMSO上调磷酸化的CPEB蛋白表达更多。CPEB磷酸化的上游激酶Aurora A的磷酸化蛋白水平也被上调,DMSO上调效应最明显。总蛋白表达水平不变。 2.PKC通路抑制剂Staurosporine和PI3K通路抑制剂LY294002可以显著抑制冷冻保护剂EG对AQP7表达上调的效应,而Erkl/2通路和JNK通路抑制剂对上调效应并没抑制作用。在表达GFP-hAQP7的293FT细胞上发现同样的结果。 3.在卵母细胞水平上,PKC和P13K通路抑制剂可以抑制CPEB和Aurora A磷酸化水平表达增高的效应。 4.AQP7表达水平随着渗透压增大而上升。并且随着渗透压增大,AQP7在细胞膜上分布增多。在表达GFP-hAQP7的293FT细胞上,冷冻保护剂所形成的高渗透压溶液同样也能刺激GFP-hAQP7在细胞膜上表达增多。 5.免疫共沉淀实验结果显示细胞内AQP7和F-ACTIN绑定在一起。结论：冷冻保护剂通过PI3K/PKC通路激活卵母细胞内调控mRNA翻译的蛋白CPEB和Aurora磷酸化活性来上调AQP7表达。高渗透压刺激卵母细胞中AQP7在细胞膜上分布增加。在细胞内,AQP7和F-ACTIN绑定在一起。很可能通过F-ACTIN运动作用将AQP7由胞质中运输到胞膜上。
[Abstract]:1 cryoprotectant regulates AQP7 expression in oocytes and promotes water transport during cryopreservation.
Objective: to detect the role of AQP7 in oocyte cryopreservation.
Materials and methods:
1. The expression of aquaporin AQP3,7,9 mRNA and protein in MII oocytes and MII oocytes of C57BL/6J mice were detected.
2. Comparing the expansion ability of oocytes to hypotonic solution and water transport rate under mercury ion treatment and non-treatment.
3. The oocytes were treated with 8% ethylene glycol (EG), 9.5% DMSO and 0.5M sucrose respectively. The expression of AQP3,7,9 protein was detected.
4. GFP-hAQP7 fusion protein vector was transfected into 293T cells and treated with EG, DMSO and sucrose DMEM respectively. The expression of GFP-hAQP7 protein in 293T cells was monitored in real time.
5. Fixed oocytes with a micromanipulator, the volume of oocytes in EG and DMSO solution was monitored in real time.
6. compare EG and DMSO as cryoprotectant and oocyte survival rate.
Result:
1. AQP3,7,9 mRNA and protein levels were detected in both human and mouse M2 oocytes.
2. after the treatment of Hg ion, the oocytes of the oocytes decreased and the transport rate of the water decreased.
3. Ethylene glycol (EG), DMSO and sucrose could up-regulate the expression of AQP7 protein in oocytes, and the up-regulation effect was the strongest after DMSO treatment.
4. EG, DMSO and sucrose can up-regulate the expression of GFP-hAQP7 protein in 293T cells, and the up-regulation effect of GFP-hAQP7 protein in DMSO treatment group is the most obvious.
5. compared to EG, the volume of oocytes in DMSO solution changed faster.
6. compared to EG, DMSO was used as cryoprotectant, and the survival rate of oocytes cryopreservation was low.
CONCLUSION: DMSO can stimulate the up-regulation of AQP7 expression in oocytes more than EG. This up-regulation can promote the water permeation of oocytes during cryopreservation and reduce the time for oocytes to reach osmotic equilibrium.
The function of 2AQP7 in oocyte maturation and cryopreservation
Objective: to elucidate the function of AQP7 in oocyte maturation and cryopreservation.
Materials and methods:
1. The natural cycle and controlled superovulation (COH) mouse oocytes were collected and fertilized in vitro. The fertilization rates of the two groups were compared. The expression of AQP7 mRNA was detected by Real-time PCR.
2. The expression of AQP7 mRNA in GV and MII oocytes was compared by Real-time PCR, and the distribution of AQP7 protein was detected by immunofluorescence.
3. AQP7 siRNA was injected into GV oocytes by microinjection and the expression of AQP7 was knocked down.
4. The MII oocytes of natural cycle were collected and cultured in HTF containing insulin, LH and FSH for 1 hour. The distribution of AQP7 in the cells was detected by immunofluorescence assay.
5. The co-localization of AQP7 and F-actin in GV, MI and MII oocytes was detected by immunofluorescence.
6. EG and DMSO were used to vitrify and freeze the oocytes respectively. After thawing and fresh oocytes were fertilized simultaneously in vitro to calculate the fertilization rate.
7. Fresh unfrozen oocytes were used as control group. The expression of AQP 7 was detected by immunofluorescence 2 hours after vitrification.
8. GV oocytes were injected with Sramble RNA and AQP7 siRNA respectively, and cultured in vitro for 16-18 hours, then vitrified with EG. The survival rate was calculated by thawing.
Result:
1. The in vitro fertilization rate of oocytes in COH group was significantly lower than that in natural cycle group, and the expression level of AQP7 mRNA in MII oocytes in COH group was significantly lower than that in natural cycle group.
2. The expression level of AQP7 mRNA in MII oocytes was significantly lower than that in GV oocytes. Compared with GV oocytes, AQP7 was more distributed on the cell membrane and cytoplasmic redistribution was significantly reduced.
3. After injecting siRNA targeting AQP7 into GV oocytes and knocking down AQP7 expression, the maturation rate of oocytes decreased significantly.
4. After treatment with Insulin and LH, the distribution of AQP7 increased on the oocyte membrane and decreased in the cytoplasm.
5. Co-localization of AQP7 and F-actin was detected in GV, M I and M I I oocytes. With the development of oocytes, the distribution of AQP7 decreased in cytoplasm and increased in cell membrane.
6. There was no significant difference in fertilization ability of cryopreserved oocytes thawed with DMSO or EG as cryoprotectants, which was lower than that of fresh control group. The expression of AQP7 was not significantly different between DMSO and EG groups, but higher than that of unfrozen control group.
After knockdown of 7.AQP7 expression, the survival rate of oocytes cryopreservation was significantly reduced.
CONCLUSION: AQP7 is involved in oocyte maturation through co-localization with F-actin and F-actin transport from cytoplasm to membrane during oocyte development from GV stage to MII stage.
3 Cryoprotectant and hyperosmotic pressure stimulate the expression and localization of AQP7 in oocytes through the PI3K/PKC pathway
Objective: To clarify the molecular mechanism of AQP7 expression and localization in oocytes stimulated by cryoprotectants and hyperosmotic pressure.
Materials and methods:
1. The expression levels of CPEB and Aurora A phosphorylation and total protein in oocytes treated with cryoprotectant EQDMSO and sucrose solution were detected by immunofluorescence and Western blotting.
2. Staurosporine/HTF, LY294002/HTF, U0126/HTF, SP600125/HTF pretreated MII oocytes and control group 5 oocytes were treated with 8% EG/HTF solution for 20 minutes. The expression of AQP7, CPEB, phosphorylated CPEB, Aurora A and phosphorylated Aurora A protein was detected by immunofluorescence assay.
GFP-hAQP7 was expressed in 3.293FT cells. The expression of GFP-hAQP7 was detected by confocal laser microscopy and the level of GFP-hAQP7 protein was detected by Western blotting.
4. Oocytes were treated with 0.25M, 0.5M, 0.7M and 1M sucrose solution for 20 minutes respectively. HTF was used as control group. The expression of AQP7 was detected by immunofluorescence.
5.293 FT cells were transfected with GFP-hAQP7 vector and treated with DMSO, EG and sucrose solution for 20 min respectively. The expression of GFP-hAQP7 in each group was detected by confocal laser microscopy. The expression of GFP-hAQP7 in each group was transfected into pEGFP-Cl vector as control.
6. Immunocoprecipitation was used to detect the binding of AQP7 and F-ACTIN in 293FT cells expressing GFP-hAQP7.
Result:
1. EG, DMSO and sucrose can up-regulate the expression of phosphorylated CPEB protein in oocytes. Compared with EG group, DMSO group has more up-regulated expression of phosphorylated CPEB protein. The up-regulated level of phosphorylated CPEB kinase Aurora A is also up-regulated, and the up-regulated effect of DMSO is most obvious.
2. PKC pathway inhibitor Staurosporine and PI3K pathway inhibitor LY294002 significantly inhibited the up-regulation effect of cryoprotectant EG on AQP7 expression, while Erkl/2 pathway and JNK pathway inhibitor did not inhibit the up-regulation effect. The same result was found in 293FT cells expressing GFP-hAQP7.
3. PKC and P13K pathway inhibitors inhibited the increased expression of CPEB and Aurora A at oocyte level.
4. The expression level of AQP7 increased with the increase of osmotic pressure, and the distribution of AQP7 increased with the increase of osmotic pressure.
5. co immunoprecipitation assay showed that AQP7 and F-ACTIN were bound together.
CONCLUSION: Cryoprotectants up-regulate the expression of AQP7 by activating the protein CPEB and Aurora phosphorylation of mRNA translation in oocytes via PI3K/PKC pathway. High osmotic pressure stimulates the increased distribution of AQP7 on the cell membrane. In cells, AQP7 is bound to F-ACTIN. It is possible that AQP7 is bound to the cytoplasm by F-ACTIN motility. Transport to the cell membrane.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.52

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