【摘要】：1冷凍保護(hù)劑調(diào)節(jié)卵母細(xì)胞中AQP7表達(dá)而促進(jìn)冷凍過程中水轉(zhuǎn)運的研究 目的：檢測卵母細(xì)胞中AQP7在卵母細(xì)胞冷凍保存中的作用 材料與方法： 1.收集行人MⅡ期卵母細(xì)胞和C57BL/6J小鼠MⅡ期卵母細(xì)胞,檢測水通道蛋白AQP3,7,9的mRNA和蛋白水平的表達(dá)。 2.比較卵母細(xì)胞在汞離子處理和不處理兩種情況下,卵母細(xì)胞的對低滲溶液膨脹能力和對水的轉(zhuǎn)運速率。 3.分別用8%乙二醇(EG),9.5%DMSO和0.5M蔗糖的HTF溶液處理卵母細(xì)胞,檢測AQP3,7,9蛋白的表達(dá)。 4.轉(zhuǎn)染表達(dá)GFP-hAQP7融合蛋白載體到293T細(xì)胞中,分別用EG, DMSO和蔗糖的DMEM溶液處理,實時監(jiān)測細(xì)胞中GFP-hAQP7蛋白表達(dá)變化。 5.使用顯微操作儀固定卵母細(xì)胞,實時監(jiān)測卵母細(xì)胞在EG, DMSO溶液中體積的變化。 6.比較使用EG和DMSO作為冷凍保護(hù)劑,卵母細(xì)胞的存活率。 結(jié)果： 1.在人和小鼠的MⅡ期的卵母細(xì)胞中,都能檢測到AQP3,7,9的mRNA和蛋白水平的表達(dá)。 2.卵母細(xì)胞在汞離子處理后,對低滲溶液膨脹能力減弱,對水的轉(zhuǎn)運速率降低。 3.乙二醇(EG), DMSO和蔗糖可使卵母細(xì)胞AQP7蛋白表達(dá)水平上調(diào),并且DMSO處理后上調(diào)效應(yīng)最強(qiáng)烈。而AQP3和9表達(dá)水平并沒有改變。 4. EG, DMSO和蔗糖可以上調(diào)293T細(xì)胞中GFP-hAQP7蛋白表達(dá)。并且同樣是DMSO處理組GFP-hAQP7蛋白表達(dá)上調(diào)效應(yīng)最明顯。 5.相比較EG,卵母細(xì)胞在DMSO溶液中體積的變化更快。 6.相比較EG,用DMSO做冷凍保護(hù)劑,卵母細(xì)胞冷凍保存后的存活率低。 結(jié)論：DMSO比EG更能刺激卵母細(xì)胞中AQP7表達(dá)上調(diào)。這個上調(diào)作用可以促進(jìn)卵母細(xì)胞在冷凍保存過程中對水的滲透,減少卵母細(xì)胞達(dá)到滲透壓平衡時間。 2AQP7在卵母細(xì)胞成熟和冷凍中的功能的研究 目的：闡明AQP7在卵母細(xì)胞成熟和冷凍保存中的功能 材料與方法： 1.收集自然周期和控制超促排卵(COH)的小鼠MⅡ期卵母細(xì)胞,分別進(jìn)行體外受精實驗。比較兩組細(xì)胞受精率。用Real-time PCR檢測兩組細(xì)胞AQP7mRNA表達(dá)差異。 2.收集GV期和MⅡ期卵母細(xì)胞,運用Real-time PCR比較這兩種時期卵母細(xì)胞中AQP7的mRNA表達(dá)差異。運用細(xì)胞免疫熒光方法檢測AQP7蛋白在這兩種細(xì)胞中分布差異。 3.采用顯微注射技術(shù)將AQP7siRNA注射到GV期卵母細(xì)胞中,敲減AQP7的表達(dá)。與注射Sramble siRNA比較,計算卵細(xì)胞體外的成熟率。 4.收集自然周期的MⅡ期卵母細(xì)胞,分別用含有insulin, LH和FSH這三種激素的HTF培養(yǎng)細(xì)胞1h,采用免疫熒光方法檢測AQP7的在細(xì)胞內(nèi)的分布的改變。 5.收集GV,M Ⅰ和MⅡ期卵母細(xì)胞,采用免疫熒光方法檢測這三種時期的卵母細(xì)胞中AQP7與F-actin的共定位情況。 6.分別使用EG和DMSO對卵母細(xì)胞玻璃化冷凍,解凍后和新鮮卵母細(xì)胞同時進(jìn)行體外受精,計算受精率。 7.新鮮未冷凍的卵母細(xì)胞作為對照組,采用免疫熒光法檢測卵母細(xì)胞玻璃化冷凍解凍后2h后,AQP7的表達(dá)情況。 8.分別注射Sramble RNA和AQP7siRNA到GV卵母細(xì)胞,體外培養(yǎng)16-18h后,使用EG進(jìn)行玻璃化冷凍,解凍統(tǒng)計存活率。 結(jié)果： 1.COH組組卵母細(xì)胞體外受精率明顯低于自然周期組,并且COH組MⅡ期卵母細(xì)胞AQP7mRNA表達(dá)水平顯著低于自然周期組。 2.MⅡ期卵母細(xì)胞AQP7mRNA表達(dá)水平顯著低于GV期。在MⅡ期卵母細(xì)胞中,相比較GV期而言,AQP7更多分布在細(xì)胞膜上,細(xì)胞質(zhì)重分布明顯減少。 3.注射靶向AQP7的siRNA到GV期卵母細(xì)胞中敲減AQP7表達(dá)后,卵母細(xì)胞成熟率顯著降低。 4. Insulin和LH處理后,AQP7在卵母細(xì)胞膜上分布增多,細(xì)胞質(zhì)中分布減少。而使用FSH處理,AQP7分布并沒有改變。 5. GV, M I和MⅡ期卵母細(xì)胞中都能檢測到AQP7與F-actin共定位。隨著卵母細(xì)胞的發(fā)育,AQP7在這三種時期細(xì)胞內(nèi)細(xì)胞質(zhì)中的分布減少,細(xì)胞膜上分布增多。而F-actin正好相反,在胞質(zhì)中的分布增多,細(xì)胞膜上分布減少。 6.使用DMSO或者EG作為冷凍保護(hù)劑凍存的卵母細(xì)胞解凍后體外受精能力沒有差異,均低于新鮮對照組。AQP7表達(dá)量在DMSO和EG組之間并沒有顯著差異,但均高于未冷凍的對照組。 7.AQP7表達(dá)敲減后,卵母細(xì)胞冷凍保存后存活率是顯著降低。 結(jié)論：AQP7通過與F-actin共定位在一起,通過F-actin運輸作用,在卵母細(xì)胞從GV期發(fā)育到MⅡ期過程中從胞質(zhì)向胞膜上轉(zhuǎn)運,參與卵母細(xì)胞的成熟。敲減AQP7表達(dá)后,卵母細(xì)胞冷凍保存后的存活率顯著降低。 3冷凍保護(hù)劑和高滲透壓刺激卵母細(xì)胞中AQP7表達(dá)和定位的改變是通過PI3K/PKC通路調(diào)節(jié)的機(jī)制研究 目的：明確冷凍保護(hù)劑和高滲透壓刺激卵母細(xì)胞中AQP7表達(dá)和定位改變的分子機(jī)制。 材料與方法： 1.采用細(xì)胞免疫熒光和蛋白免疫印(Western blotting)檢測卵母細(xì)胞上蛋白CPEB和Aurora A磷酸化和總蛋白在冷凍保護(hù)劑EQDMSO和蔗糖溶液處理后的表達(dá)水平。 2.分別Staurosporine/HTF, LY294002/HTF, U0126/HTF, SP600125/HTF預(yù)處理MⅡ期的卵母細(xì)胞,第五組為對照。再用8%EG/HTF溶液處理20min后采用免疫熒光方法檢測AQP7, CPEB,磷酸化CPEB, Aurora A和磷酸化Aurora A蛋白表達(dá)水平。 3.293FT細(xì)胞中表達(dá)GFP-hAQP7,同2使用方法處理,激光共聚焦顯微鏡下檢測GFP-hAQP7的表達(dá)量。用Western blotting檢測GFP-hAQP7蛋白水平。 4.用濃度分別為0.25M,0.5M,0.7M,1M蔗糖的高滲透壓溶液分別處理卵母細(xì)胞20min, HTF為對照組,使用免疫熒光方法檢測各組AQP7的表達(dá)。 5.293FT細(xì)胞轉(zhuǎn)染GFP-hAQP7載體,48h后分別用DMSO,EG和蔗糖溶液處理20min, HTF組為對照。使用激光共聚焦顯微鏡下檢測各組GFP-hAQP7的表達(dá)。轉(zhuǎn)入pEGFP-Cl載體作為對照。 6.對表達(dá)GFP-hAQP7的293FT細(xì)胞,裂解細(xì)胞,使用免疫共沉淀的方法檢測AQP7和F-ACTIN在細(xì)胞中綁定情況。 結(jié)果： 1. EG, DMSO和蔗糖均可以上調(diào)卵母細(xì)胞中磷酸化的CPEB蛋白表達(dá)水平。DMSO組與EG組比較起來,DMSO上調(diào)磷酸化的CPEB蛋白表達(dá)更多。CPEB磷酸化的上游激酶Aurora A的磷酸化蛋白水平也被上調(diào),DMSO上調(diào)效應(yīng)最明顯�？偟鞍妆磉_(dá)水平不變。 2.PKC通路抑制劑Staurosporine和PI3K通路抑制劑LY294002可以顯著抑制冷凍保護(hù)劑EG對AQP7表達(dá)上調(diào)的效應(yīng),而Erkl/2通路和JNK通路抑制劑對上調(diào)效應(yīng)并沒抑制作用。在表達(dá)GFP-hAQP7的293FT細(xì)胞上發(fā)現(xiàn)同樣的結(jié)果。 3.在卵母細(xì)胞水平上,PKC和P13K通路抑制劑可以抑制CPEB和Aurora A磷酸化水平表達(dá)增高的效應(yīng)。 4.AQP7表達(dá)水平隨著滲透壓增大而上升。并且隨著滲透壓增大,AQP7在細(xì)胞膜上分布增多。在表達(dá)GFP-hAQP7的293FT細(xì)胞上,冷凍保護(hù)劑所形成的高滲透壓溶液同樣也能刺激GFP-hAQP7在細(xì)胞膜上表達(dá)增多。 5.免疫共沉淀實驗結(jié)果顯示細(xì)胞內(nèi)AQP7和F-ACTIN綁定在一起。 結(jié)論：冷凍保護(hù)劑通過PI3K/PKC通路激活卵母細(xì)胞內(nèi)調(diào)控mRNA翻譯的蛋白CPEB和Aurora磷酸化活性來上調(diào)AQP7表達(dá)。高滲透壓刺激卵母細(xì)胞中AQP7在細(xì)胞膜上分布增加。在細(xì)胞內(nèi),AQP7和F-ACTIN綁定在一起。很可能通過F-ACTIN運動作用將AQP7由胞質(zhì)中運輸?shù)桨ど稀?br/>[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R318.52

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