Expression And Roles Of Aquaporin3in Mouse Early Embryonic Development And Vitrification

BackgroundAquaporins (AQP) channels are widely distributed in various epithelium andendothelium both animals and plants involving fluid seeretion and basoprtion, theymediate the transmembrane movement of water to maintain cellular fluid and osmoticbalance. Since the diseove r yof first water chnnael(AQP1) from the human red bloodmembrnae by Preston, Agre et al.in1988，rapid and serial porgresses have been mdae incharacterization of AQP sturcture and function. So far, at least12homologousmembers(AQPO~AQP12) hvae been moleeularly identified in mmamals, they are nowsubdivided into two major groups: orthodox AQPs and aquaglyceroporins.Aquaglyceroporins includes five members: AQP3, AQP7, AQP8, AQP9and AQP10. Theyare permeable to glycerol, urea and other small non-electrolytes as well as to water. Aprevious study reported that AQPs are highly expressed in the mammalian reproductivesystem and actively participate in multiple reproductive processes, including folliclegrowth, spermatogenesis, embryo formation, embryonic development, and implantation.Previous studies show that AQPs are expressed in mouse early embryos of eachdevelopmental stage, and the distribution within the embryo are developmentally regulated. Among those studies, AQP3and AQP7mRNAs were highly expressed in allstage and AQP8only expressed in blastocys are uniformity.Since the first frozen-thawed embryo transfer(F-ET) had succesfully acquired clinicalpregnancy in1983by Trounson, F-ET had been becomed a significance component ofAssisted Reproductive Technology(ART). Accorrding to24th ESHRE, entire world,25%live birth babies who applied ART came from F-ET, which componented about42%frozen–thawed embryos. So, frozen–thawed embryo is an important modern assistedreproductive technology.Previous animal studies concluded that cellular freezing is a dehydrant procedureindeed, while thawing meaning the reabsorption of water into cell. Therefore, thetransmembrane flux of water and cryoprotectant is a key factor affecting the viability ofembryos following freeze–thawing, or could course some damage to embyo.The majordamage to embryos induced by freeze–thawing was due to intracellular and extracellularice formation, thus reducing embryonic reproductive potential. A previous study revealedthat that changes in the expression of AQP and shifts in protein location were major stressresponses to hypertonic solution treatment before embryonic implantation, besides, exceptfor AQPs, not any other substance which could mediate water transmembrane movementhad been found yet. Then, what is the pattern of AQPs expression in early embryos? Whatis the role of AQPs in early embryonic development? If aquaglyceroporins couldcontribute the protective response during early embryos vitrification? And which themolecular mehcnaism of that? These are still to be detennined.This study was designed to detect changes in the expression and distribution of AQP3,which is a member of aquaglyceroporins and several independent investigationsdemonstrated it is highly expressed in mouse embryos of all early developmental stages,in early stage mouse embryos before and after vitrification, to examine the role of thisAQP isoform in early embryonic development and in protecting embryos subjected tofreeze–thawing for assisted reproduction. The study aiming to elucidat the mechanism ofearly embryonic development and embryo vitrification process, and could lead to moreeffective methods for enhancing the viability of freeze–thawed embryos. ContentPartⅠ Expression and function of aquaporin3in mouse earlyembryonic development processObjectiveTo investigated the expression and distribution of aquaporin3(AQP3) in mouse earlyembryos at different developmental stages.MethodsControlled ovarian hyperstimulation (COH) was used to collect four-cell embryos,eight-cell embryos, morulae, and early blastocysts from mice. qRT-PCR,Immunofluorescence, laser confocal microscopy were used to measure the expression anddistribution of AQP3channels in these early embryonic stages. Using2-△CTmethodaccount the relative expression of AQP3mRNA. Using SPSS16.0statistical software forstatistical analysis, P <0.05think that the difference was statistically significantResults1. qRT-PCR show that AQP3mRNA was expressed in all four embryonicstages.AQP3mRNA expression was highest in morulae and lowest in blastocysts,butthere was no significant difference between groups(P＞0.05).2.Immunoflurescence revealed that AQP3was highly expressed inall fourembryonic stages.3. Laser confocal microscopy revealed distinct spatial expression patterns at thesestages: in the eight-cell embryos stage, the AQP3-specific fluorescence signal was evenlydistributed over the cytoplasm and karyotheca of blastomeres, with stronger expression inthe karyotheca relative to the cytoplasm; In the morula stage, AQP3was evenlydistributed over the plasma membrane of each blastomere; in early blastocysts, AQP3waspredominantly distributed over the plasma membrane and cytoplasm of trophoblastic cell,as well as blastocyst cavity.Conclusions 1. qRT-PCR show that AQP3mRNA was expressed in all four embryonic stages,suggest it may regulate mouse embryonic development.2. The subcellular localization of AQP3are different in each embryonic stagessuggest that the expression and function of AQP3could corresponding adjust with theneed of each embryonic development stage. Part Ⅱ Expression and roles of aquaporin3in mouse early embryosbefore and after vitrificationObjectiveTo investigated the expression and distribution of aquaporin3(AQP3) in mouseembryos at different developmental stages before and after vitrification.MethodsControlled ovarian hyperstimulation (COH) was used to collect eight-cell embryos,morulae, and early early blastocysts from mice. Immunofluorescence, laser confocalmicroscopy, and Western blotting were used to measure the expression and distribution ofAQP3channels in these early embryonic stages before and after vitrification. Allexperiments were repeated three times. GAPDH strip as the internal control of proteinsample volume correction in Western Blot, using Image Plus6.0professional imageanalysis software to carry on the analysis, taking the average value calculated.UsingSPSS16.0statistical software for statistical analysis, P <0.05think that the difference wasstatistically significantResults1.Immunoflurescence revealed thataquaporin3was expressed in all threeembryonic stages both prior to and after vitrification.2. Laser confocal microscopy revealed distinct spatial expression patterns at thesestages. Prior to vitrification: in the eight-cell embryos stage, the AQP3-specific fluorescence signal was evenly distributed over the cytoplasm and karyotheca ofblastomeres, with stronger expression in the karyotheca relative to the cytoplasm; In themorula stage, AQP3was evenly distributed over the plasma membrane of each blastomere;In early blastocysts, AQP3was predominantly distributed over the plasma membrane andcytoplasm of trophoblastic cell, as well as blastocyst cavity. After freeze–thawing: in theeight-cell embryos stage, AQP3mainly distributed to the cytoplasm, while much lessAQP3immunofluorescence was detected in karyotheca; In the morula stage, AQP3wasdistributed over the plasma membrane of each blastomere as before vitrification; In earlyblastocysts, also the same as before vitrification, AQP3was distributed over the plasmamembrane and cytoplasm of trophoblastic cell, as well as blastocyst cavityBoth channelexpression and distribution within the embryo were developmentally regulated.3. Western blot result revealed that AQP3expression was obviously detected inembryos of all stages before and after vitrification. The results also indicated that aftervitrification, AQP3expression was highest in8-cell embryos stage, followed by morulae,early blastocysts was lowest（P<0.05）. The expression level of AQP3in8-cell embryosand early blastocysts after vitrification was lower than before vitrification, while theexpression of AQP3in morulae was higher after vitrification（P<0.05）.Conclusions1.These developmental and vitrification-dependent changes in AQP3expression anddistribution suggest that this transmembrane channel may contribute to the protectiveresponse during vitrification.2. AQP3expression is maintained or upregulated in morulae after vitrification,suggesting that AQP3probably plays an important role in fluid exchange during thisparticular stage. Besides, we speculate that other AQP subtypes mayplay a moredominant role in the transport and distribution of fluids during freeze–thawing of8-cellembryos and early blastocysts.