饒金鵬 邱 楓 金 敏
解凍液I溫度設(shè)定對(duì)小鼠卵母細(xì)胞玻璃化凍融效果的影響
饒金鵬 邱 楓 金 敏
目的探討預(yù)熱至37℃的解凍液I和預(yù)熱至室溫(22℃)的解凍液I對(duì)小鼠卵母細(xì)胞玻璃化凍融效果的影響。方法對(duì)6周齡昆明系雌鼠進(jìn)行促排卵,脫去顆粒細(xì)胞后選取形態(tài)良好的MII期卵母細(xì)胞,用含不同濃度DMSO+EG的磷酸鹽緩沖液(5%DMSO+5%EG,10%DMSO+10%EG,20% DMSO+20%EG+30%蔗糖)對(duì)其進(jìn)行玻璃化冷凍,儲(chǔ)存2天后對(duì)小鼠卵子進(jìn)行解凍復(fù)溫。將卵子隨機(jī)分為兩組,一組投入預(yù)熱至37℃的解凍液I(含30%蔗糖的磷酸鹽溶液),另一組投入預(yù)熱至室溫(22℃)的相同濃度的解凍液I中,再分別移至預(yù)熱至室溫(22℃)的解凍液II、III和IV(分別含20%、10%、5%蔗糖的磷酸鹽溶液),隨后對(duì)兩組卵母細(xì)胞的復(fù)蘇率進(jìn)行統(tǒng)計(jì)比較。結(jié)果共計(jì)對(duì)119枚小鼠卵子進(jìn)行玻璃化凍融,其中61枚置于預(yù)熱至37℃的解凍液I,有52枚存活良好,復(fù)蘇率為82.25%(52/61),其余58枚置于預(yù)熱至室溫(22℃)的解凍液I進(jìn)行解凍,有23枚存活良好,復(fù)蘇率為39.66%(23/58),37℃組的復(fù)蘇率顯著高于室溫(22℃)組(P<0.01)。結(jié)論雖然降低平衡時(shí)的溫度可以減少高濃度玻璃化冷凍保護(hù)劑對(duì)卵子的毒性作用,但較快的升溫速度可以幫助卵子快速通過(guò)-120℃~-35℃的危險(xiǎn)階段,而37℃的解凍液I環(huán)境較之室溫(22℃)能更好地加速這一過(guò)程,避免重結(jié)晶對(duì)細(xì)胞的致命損傷。
解凍液I;溫度;小鼠;卵母細(xì)胞;玻璃化冷凍;復(fù)蘇率
人類卵子冷凍作為生育力保存的一種方式,在應(yīng)對(duì)因腫瘤需放療、化療,卵巢早衰等緊急狀況下起著關(guān)鍵性作用。但卵母細(xì)胞因體積大、膜的滲透性低、紡錘體對(duì)溫度敏感,在凍融時(shí)極易產(chǎn)生冰晶,而使得凍融復(fù)蘇率不高。近期研究表明,玻璃化冷凍技術(shù)比傳統(tǒng)的慢速冷凍法更有效[1-3],但溶液要實(shí)現(xiàn)玻璃化狀態(tài),需要更高的冷凍保護(hù)劑(CPA)濃度,而濃度的增加意味著對(duì)細(xì)胞造成更大的毒性及滲透性損傷。降低平衡時(shí)的溫度可減少高濃度CPA的毒性作用,降溫也會(huì)使得復(fù)蘇過(guò)程中因升溫速度不夠而導(dǎo)致重結(jié)晶的產(chǎn)生[4]。凍融液的溫度選擇生理溫度(37℃)亦或是室溫(22℃)還存在爭(zhēng)議[5-7]。本研究以成熟小鼠卵母細(xì)胞(MII期)作為研究對(duì)象,將解凍液I的溫度分別設(shè)定為37℃和室溫(22℃),通過(guò)比較兩組間的復(fù)蘇率,評(píng)估溫度對(duì)重結(jié)晶/毒性兩因素的影響,并為今后人類卵母細(xì)胞凍融技術(shù)的開展積累經(jīng)驗(yàn)。
1.1 動(dòng) 物 6周齡雌性昆明系小白鼠(體質(zhì)量30~35g),購(gòu)自浙江中醫(yī)藥大學(xué)實(shí)驗(yàn)動(dòng)物中心[(動(dòng)物合格證號(hào):SYXK(浙)2013-0184)]。
1.2 藥物與試劑 孕馬血清(PMSG)(批號(hào)151029)與絨促性素(HCG)(批號(hào)150709)均購(gòu)自寧波第二激素廠,配子處理液G-GAMETE(批號(hào)505768)和GIVF(批號(hào)505784)購(gòu)自Vitrolife公司,二甲基亞砜(DMSO)(批號(hào)112K2389)購(gòu)自SIGMA公司,磷酸鹽緩沖液(PBS)(批號(hào)C0221A)購(gòu)自碧云天生物技術(shù)公司,蔗糖(sucrose)(批號(hào)78-01-25)購(gòu)自中國(guó)上海試劑一廠,透明質(zhì)酸酶(hyaluronidas)(批號(hào)F078A),人血清白蛋白(HSA)(批號(hào)F084A)以及石蠟油(批號(hào)E147A)購(gòu)自SAGE公司。
1.3 耗材與設(shè)備 四孔皿購(gòu)自NUNC公司,60mm培養(yǎng)皿及巴斯德管購(gòu)自FALCON公司,裝載工具CRYOTOP購(gòu)自KITAZATO公司。超凈工作臺(tái)為丹麥IVFTECH,解剖顯微鏡為日本OLYMPUS,培養(yǎng)箱為美國(guó)THEMRO,液氮罐為美國(guó)MVE。
2.1 玻璃化冷凍解凍液的制備 冷凍液分為濃度由低到高的4個(gè)梯度:預(yù)平衡液(95%DPBS+5%HSA);冷凍液Ⅰ(85%DPBS+5%DMSO+5%EG+5%HSA);冷凍液Ⅱ(75%DPBS+10%DMSO+10%EG+5%HSA);冷凍液Ⅲ(25%DPBS+20%DMSO+20%EG+30%蔗糖+5%HSA)。解凍液則分為濃度由高到低的4個(gè)梯度:解凍液I(65% DPBS+30%蔗糖+5%HSA);解凍液Ⅱ(75%DPBS+20%蔗糖+5%HSA);解凍液Ⅲ(85%DPBS+10%蔗糖+5%HSA);解凍液Ⅳ(90%DPBS+5%蔗糖+5%HSA)。
2.2 凍融前小鼠卵母細(xì)胞的準(zhǔn)備 雌鼠腹腔注射10IU的PMSG,48h后再注射10IU的HCG,15h后將小鼠頸椎脫臼處死,用注射器于覆油的GGAMETE配子處理液中刺破輸卵管膨大部,擠出成簇卵冠丘復(fù)合物(COCs)。置于37℃培養(yǎng)箱4h后,將COCs放入含透明質(zhì)酸酶(hyaluronidas)的四孔皿內(nèi)1min,再移入剩余3孔的GAMETE中反復(fù)的吹打直至顆粒細(xì)胞剝離干凈,此時(shí)可選取形態(tài)良好的MII期卵母細(xì)胞放入培養(yǎng)箱內(nèi)培養(yǎng)1h待用。
2.3 卵母細(xì)胞玻璃化凍融 將預(yù)平衡液及冷凍液在室溫下平衡40min,把卵母細(xì)胞依次放入預(yù)平衡液(2min),冷凍液Ⅰ(2min),冷凍液Ⅱ(3min)以及冷凍液Ⅲ(40s),在1min內(nèi)將卵母細(xì)胞轉(zhuǎn)移到冷凍載桿上并投入液氮,此即為冷凍過(guò)程。2天后,將解凍液分為兩組,1組的解凍液Ⅰ預(yù)先放到37℃培養(yǎng)箱預(yù)熱1h,另1組則將解凍液Ⅰ置于室溫環(huán)境下平衡,而兩組其余解凍液Ⅱ、Ⅲ、Ⅳ均置于室溫下平衡40min。解凍時(shí),迅速?gòu)囊旱腥〕鲚d桿,將前端載片浸入解凍液Ⅰ中,輕晃以使得卵子脫落,2min內(nèi)將卵子轉(zhuǎn)移到解凍液Ⅱ,再分別將卵母細(xì)胞放入解凍液Ⅱ、Ⅲ、Ⅳ各3min,最后轉(zhuǎn)移到G-IVF培養(yǎng)液,放于37℃、6% CO2飽和濕度培養(yǎng)箱后繼續(xù)培養(yǎng)。
2.4 卵母細(xì)胞存活判斷 經(jīng)解凍的卵母細(xì)胞放于培養(yǎng)箱內(nèi)培養(yǎng),次日早間觀察依然可見(jiàn)透明帶與細(xì)胞膜完整,卵周間隙清晰,大小正常,折光度好,且沒(méi)有明顯的胞漿溢出與皺縮,則判定其存活良好。
2.5 統(tǒng)計(jì)學(xué)方法 應(yīng)用SPSS19.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。計(jì)數(shù)資料采用χ2檢驗(yàn)。P<0.01表示差異有統(tǒng)計(jì)學(xué)意義。
共計(jì)對(duì)119枚小鼠卵子進(jìn)行玻璃化凍融,其中61枚置于預(yù)熱至37℃的解凍液Ⅰ,有52枚存活良好,復(fù)蘇率82.25%(52/61),其余58枚置于預(yù)熱至室溫(22℃)的解凍液I進(jìn)行解凍,有23枚存活良好,復(fù)蘇率39.66%(23/ 58),37℃組的復(fù)蘇率顯著高于室溫(22℃)組(P<0.01)。
本研究比較了不同解凍液Ⅰ溫度環(huán)境下,成熟小鼠卵母細(xì)胞(MⅡ期)的復(fù)蘇率,發(fā)現(xiàn)復(fù)蘇解凍液Ⅰ設(shè)定為生理溫度(37℃)較之室溫(22℃)能獲得更高的復(fù)蘇率(P<0.01)。Larman等[6]發(fā)現(xiàn)人MⅡ卵子在37℃解凍液環(huán)境下進(jìn)行復(fù)蘇紡錘體完好而在室溫的條件下(21~22℃)解凍則紡錘體發(fā)生解聚。這一結(jié)果支持了卵母細(xì)胞應(yīng)該在生理溫度(37℃)環(huán)境下進(jìn)行解凍,其不僅能克服重結(jié)晶對(duì)細(xì)胞造成的致命損傷獲得較高的復(fù)蘇率,而且紡錘體結(jié)構(gòu)完整性的維持有利于卵母細(xì)胞受精及受精后胚胎發(fā)育。但龍曉林等[7]通過(guò)玻璃化法凍融未成熟人卵母細(xì)胞(GV期)的實(shí)驗(yàn),給出了不同觀點(diǎn)。在其試驗(yàn)中,37℃環(huán)境下GV期人卵母細(xì)胞的復(fù)蘇率為68.75%(11/16),要低于室溫環(huán)境下的復(fù)蘇率81.25%(13/16),而后期通過(guò)對(duì)卵母細(xì)胞紡錘體和染色體形態(tài)的觀察,也是常溫解凍要優(yōu)于37℃解凍。考慮到37℃較之22℃確實(shí)會(huì)增加細(xì)胞膜的通透性,而過(guò)快的復(fù)溫,也可能因體積與溫度的急劇變化而導(dǎo)致透明帶破裂,且過(guò)高溫度下接觸EG、DMSO等高濃度滲透性保護(hù)劑增加了其化學(xué)毒性[4],所以本研究?jī)H將解凍液I的溫度設(shè)計(jì)為37℃,以獲得較快的升溫速度幫助卵子加速通過(guò)-120℃~-35℃這一重結(jié)晶易發(fā)階段,而其后的解凍液II、III、IV的溫度均設(shè)為室溫則又可以減小殘留的高濃度滲透性保護(hù)劑對(duì)其產(chǎn)生的滲透毒性。由于選擇的實(shí)驗(yàn)對(duì)象為不同的物種的卵母細(xì)胞(小鼠/人),或同一物種但處于不同發(fā)育階段的卵母細(xì)胞(成熟MII期/未成熟GV期),它們的生物特性(例如透明帶通透性)各有差異,加之不同實(shí)驗(yàn)所選擇的玻璃化試劑組成上有所差別,得到不同的實(shí)驗(yàn)結(jié)果在所難免。
綜上所述,影響卵母細(xì)胞玻璃化凍融的因素很多,結(jié)晶的形成、冷凍保護(hù)劑的滲透毒性、溫度和滲透壓的改變均會(huì)影響其凍融效果。本研究通過(guò)將解凍液I設(shè)定為37℃與22℃,通過(guò)比對(duì)MII期小鼠卵母細(xì)胞的復(fù)蘇率,提示解凍第一步采用生理溫度(37℃)更為合適。
[1]Noyes N,Boldt J,Nagy ZP.Oocyte cryopreservation:is it time to remove its experimental label[J].J Assist Reprod Genet,2010,27(5):69-74.
[2]Cao YX,Chian RC.Fertility preservation with immature and in vitro matured oocytes[J].Semin Reprod Med,2009,27 (6):456-464.
[3]Forman EJ,Li X,F(xiàn)erry KM,et al.Oocyte vitrification does not increase the risk of embryonic aneuploidy or diminish the implantation potential of blastocysts created after intracytoplasmicsperm injection:a novel,paired randomized controlled trial using DNA fingerprinting[J].Fertil Steril,2012,98(3):644-649.
[4]黃國(guó)寧,孫海翔.體外受精-胚胎移植實(shí)驗(yàn)室技術(shù)[M].北京:人民衛(wèi)生出版社,2012:265-271.
[5]Grifo JA,Noyes N.Delivery rate using cryopreserved oocytes is comparable to conventional in vitro fertilization using fresh oocytes:potential fertility preservation for female cancer patients[J].Fertil Steril,2010,93(2):391-396.
[6]Larman M,Minasi M,Rienzi L,et al.Maintenance of the meiotic spindle during vitrification in human and mouse oocytes[J].Reprod Biomed Online,2007,15(6):692-700.
[7]龍曉林,黃玉玲,孫筱放,等.凍融液的溫度對(duì)人卵玻璃化凍融效果的影響[J].實(shí)用婦產(chǎn)科雜志,2009,25(10):603-605.
(收稿:2016-06-10 修回:2016-06-17)
Effects of Setting Temperature of Thawing Medium I on Mouse Oocytes Vitrification
RAO Jinpeng,QIU Feng,JIN Min. ReproductiveMedicineCenter,theSecond Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou(310052),China
ObjectiveTo investigate the effect of thawing medium I preheating to 37℃ and 22℃(room temperature)on mouse oocytes vitrification.MethodsSix-week old Kunming female mice were induced to superovulate.After granulosa cells of cumulus-oocyte-comlexes(COC)were taken off in hyaluronic acid,the mature oocytes were frozen by using phosphate buffer containing different concentrations of DMSO+EG (5%DMSO+5% EG,10%DMSO+10%EG,20%DMSO+20%EG+30%sucrose).After 2 d,the frozen oocytes were randomly allocated to two thawing groups:thawing medium I(phosphate buffer containing 30%of sucrose)preheating to 37℃group and room temperature(22℃)group,respectively,then both to thawing mediumⅡ,Ⅲ andⅣ(phosphate buffer containing 20%,10%,and 5%of sucrose,respectively).The survival rates of two groups were calculated.ResultsA total of 119 oocytes were frozen,61 out of which were allocated to thawing medium I preheating to 37℃,and the survival rate was 82.25%(52/61);the rest 58 oocytes were in thawing medium I preheating to room temperature group,and 23 of 58(39.66%)were survived.A significant difference was found between two groups (P<0.01).ConclusionThough balance in lower temperature can reduce the toxic effects of high concentrations of vitrification cryoprotectants on the oocytes,a faster heating rate can help the oocytes quickly pass the dangerous phase of-120℃ ~-35℃.37℃ thawing medium I environment could speed up this process better than the room temperature(22℃)one,and could avoid the recrystallization which cause fatal damage to the cells.
thawing medium I;temperature;mice;oocytes;vitrification;survival rate
浙江大學(xué)醫(yī)學(xué)院附屬第二醫(yī)院生殖醫(yī)學(xué)中心(杭州 310052)
金敏,Tel:13588803459;E-mail:Jinmin76@hotmail.com