The Mechanism And Functions Of AQP7in Oocyte Cryopreservation And Maturation

Part1Cryoprotectants upregulate the expression of mouse oocyte AQP7that facilitates water diffusion during cryopreservationObjective: To investigate the effects of cryoprotectants on the expression of AQP7in oocytes.Materials and Methods:1. Collecting metaphase Ⅱ (MII) oocytes donated by fertile women, and, from adult female C57BL/6J mice.2. Comparing the water permeability coefficient of oocytes in hypotonic medium between with and without mercury.3. Treating mouse oocytes with human tubal fluid medium containing8%ethylene glycol (EG),9.5%dimethylsulfoxide (DMSO), and0.5M sucrose, respectively, and, examing the expression of AQP3,7,9in oocytes.4. Transfecting293T cells with GFP-hAQP7expression vector.5. Examing the mouse oocyte volume change in the presence of8%ethylene glycol (EG),9.5%dimethylsulfoxide (DMSO), respectively.6. Comparing the survival rate of oocytes after vitrification between EG and DMSO treatments..Result(s):1. AQPs3,7,9were expressed in human and mouse oocytes.2. Cryoprotectants, including EG, DMSO and sucrose, up-regulated AQP7 3. Pretreatment of oocytes with mercury partly blocked the increase in cell volume and permeability coefficient.4. Immunofluorescence intensities of AQP7were significantly increased in the oocytes cultured with human tubal fluid medium containing8%ethylene glycol (EG),9.5%dimethylsulfoxide (DMSO) and0.5M sucrose, respectively. However, AQP3and9were not changed.5. AQP7expression level in293T cells transfected with GFP-hAQP7expression vector was significantly increased in the human tubal fluid medium containing8%ethylene glycol (EG),9.5%dimethylsulfoxide (DMSO) and0.5M sucrose, respectively. Compared with sucrose solution, DMSO, not EG, induced a significantly higher expression level of AQP7.6. DMSO treatment facilitates water transport more than EG treatment.7. The survival rate of the DMSO-treated oocytes was significantly lower than that of the EG-treated oocytes..Conclusion(s): DMSO up-regulates AQP7expression inmouse oocytesmore than EG. This may facilitate water diffusion and reduce the time for oocytes to reach osmotic balance in the cryoprotectant solution during cryopreservation. Part2. AQP7functions in oocyte maturation and cryopreservationObjective: To investigate the functions of AQP7in oocyte maturation and cryopreservation.Materials and Methods:1. Collecting the oocytes from mice with natural cycle or controlled ovarian stimulation (COH), fertilizing them with sperms, and, comparing their fertilization rate and expression levels of AQP7mRNA.2. Collecting mouse oocytes of GV stage and MII stage were collected, and, examining the expression level of AQP7mRNA and location of AQP7protein.3. Injecting AQP7siRNA into the GV stage oocytes with the microinjection technique,. Using real-time PCR and immunofluorescence to detect the expression of AQP7. Examing in vitro oocyte maturation rate in sramble siRNA and AQP7siRNA groups.4. Collecting MII oocytes from natural cycle, and, treating them with the HTF medium containing insulin, LH and FSH, respectively, for one hour. Using immunofluorescence to examine the location change of AQP7in oocytes.5. Collecting of GV, MI and MII oocytes, and, observing co-localization of AQP7and F-actin with immunofluorescence analysis.6. Comaring the in vitro fertilization rate between fresh and thawed oocytes vitrified with EG or DMSO.7. Comaring AQP7expression levels between fresh and thawed oocytes vitrified with EG or DMSO, using immunofluorescence assay.8. Comparing the survival rate of the GV stage oocytes vitrified with EG between sramble siRNA and AQP7siRNA injection groups..Results:1. Fertilization rate of oocytes in COH group was significantly lower than that in the natural cycle group. Real-time PCR results showed that AQP7mRNA expression level in COH oocytes was significantly lower than that in the natural cycle group.2. Real-time PCR results showed that, AQP7mRNA expression level of MII oocytes was significantly lower than that of GV stage oocytes. Immunofluorescence showed that AQP7in GV oocytes were distributed in the cytoplasm and cell membrane. In MII oocytes, more AQP7was distributed in the cell membrane.3. Injections of siRNA targeting AQP7to GV oocytes significantly knockdown AQP7expression.4. Insulin and LH treatments significantly upregulated the distribution of AQP7in the oocyte membrane. However, FSH treatment did not change AQP7distribution.5. Immunofluorescence showed that colocalization of AQP7with F-actin in GV, MI and MII of oocytes. As oocyte development, reduced distribution of AQP7in cytoplasm, and, increased distribution of AQP7in cell membrane were observed. However, increased distribution of F-actin was observed in the cytoplasm as oocyte development. 6. There was no difference in in vitro fertilization capacity between oocytes vitrified with DMSO and EG. Howerver, in vitro fertilization capacity of oocytes vitrified with DMSO and EG was lower than that of control group. On ther other hand, AQP7expression levels were not significantly different between DMSO and EG groups, but they were higher than that in control group.7. The survival rate of the oocytes with AQP7knockdown was significantly lower than that of control oocytes.Conclusion:AQP7was co-localization with F-actin. AQP7may be transported from cytoplasm to the cell membrane by F-actin. AQP7plays a role in the oocyte maturation from the GV stage to MII stage. The survival rate after oocyte cryopreservation was significantly reduced after AQP7knockdown. Part3PI3K/PKC pathway mediates the alteration of AQP7expression and location in mouse oocyte induced by cryoprotectants and hyperosmolar stimulationObjective: To explore molecular mechanisms of cryoprotectants and high osmolarity stimulating oocytes AQP7expression and localization changes.Materials and Methods1. Treating the mouse oocytes with human tubal fluid medium containing8%ethylene glycol (EG),9.5%dimethylsulfoxide (DMSO), and0.5M sucrose, respectively, and, detecting the expression of phosphorylation of CPEB and Aurora A.2. Dividing oocytes into five groups randomly, and, treating them with staurosporine/HTF, LY294002/HTF, U0126/HTF, SP600125/HTF and HTF alone, respectively. Then, treating these oocytes with8%EG/HTF solution for20minutes. Examing AQP7, CPEB, phosphorylation of CPEB and Aurora A and phosphorylation of Aurora A, using immunofluorescence3. Transfecting293FT cells with GFP-hAQP7vector. Treating the cells with five different media as described above, and, examing expression levels of GFP-hAQP7 using confocal microscope and western blotting.4. Treating mouse oocytes with medium at different hyper-osmotic pressure using0.25M,0.5M,0.7M and1M sucrose/HTF, respectively, and, examing the expression levels of AQP7.5. Transfecting293FT cells with GFP-hAQP7vector and pEGFP-Cl vector, respectively. Examing the expression levels of GFP-hAQP7and GFP in the cells treated with solution containing8%ethylene glycol (EG),9.5%dimethylsulfoxide (DMSO), and0.5M sucrose, respectively.6. Treating Transfecting293FT cells transfected with GFP-hAQP7for20minutes, and, examing the F-actin and AQP7binding using immunoprecipitation.Results:1. Immunofluorescence intensities of phosphorylated CPEB and phosphorylated Aurora A were significantly increased in mouse oocytes treated with HTF medium containing8%ethylene glycol (EG),9.5%dimethylsulfoxide (DMSO), and0.5M sucrose, respectively. The total protein levels of CPEB and Aurora A were not changed.2. PKC pathway inhibitor staurosporine and PI3K pathway inhibitor LY294002significantly inhibited the effects of cryoprotectant EG on AQP7expression in mouse oocytes. Erkl/2pathway and JNK pathway inhibitors had no effect on AQP7expression induced by cryoprotectant EG. On the other hand, the same results were also observed in293FT cells transfected with GFP-hAQP7293FT.3. PKC and PI3K pathway inhibitors inhibited phosphorylation level of CPEB and expression level of Aurora A.4. Increased osmotic pressure induced an increased AQP7expression level and an increased distribution of AQP7in cell membrane of mouse oocytes. On the other hand, in the293FT cells transfected with the GFP-hAQP7, increased osmotic pressure also induced an increased AQP7expression level and an increased distribution of AQP7in cell membrane.5. Co-immunoprecipitation analysis showed that AQP7and F-actin bound together.Conclusion: PI3K/PKC pathway mediates the upregulation of mRNA translation of CPEB and Aurora A, upregulation of AQP7expression level, and, altered location in mouse oocyte induced by cryoprotectants. Hyperosmolar stimulation could increase AQP7expression, alter the distribution of AQP7, and, increase the binding of AQP7and F-actin that may facilitate AQP7re-distribution from cytoplasm to membrane.