【作者】 梁琳琳；

【导师】 朱桂金；

【作者基本信息】 华中科技大学 ， 妇产科学， 2009， 博士

【摘要】 第一部分研究玻璃化冷冻对小鼠胚胎体内外发育潜能的影响目的：观察新鲜6～8-细胞胚胎和玻璃化冷冻复苏6～8-细胞胚胎在囊胚形成率、囊胚孵出率及妊娠率、小鼠出生率的差异，初步探讨玻璃化冷冻对胚胎体内外发育潜能的影响。方法：共取性成熟期雌鼠(大于6～7周龄)200只。同笼雌鼠于见阴栓后20小时脱颈处死，取2-细胞胚胎。取胚后36小时左右观察胚胎，将6～8细胞胚胎分为两组，一组新鲜胚胎进行移植(新鲜移植组)，一组进行玻璃化冷冻(玻璃化冷冻组)2周后复苏移植。两组各取50枚移植前胚胎，体外培养48～72小时，观察两组胚胎体外培养发育至囊胚、及囊胚孵出的情况。复苏后的6～8-细胞胚胎经1～3 h培养，选择外形正常的胚胎移植于假孕63～67 h的受体母鼠的两侧子宫中，每侧8枚。新鲜移植组为新鲜6～8-细胞胚胎移植。妊娠17～20 d自然分娩后记录产仔数。对两组妊娠率、小鼠出生率进行比较。结果：1．新鲜移植组和玻璃化冷冻组胚胎一般情况观察：新鲜移植组共获823枚6～8细胞优质胚胎。玻璃化冷冻组共获1156枚6～8细胞优质胚胎，冷冻1143枚胚胎，复苏后存活胚胎为864枚。复苏成活率为75.59％。2．新鲜移植组和玻璃化冷冻组胚胎囊胚形成率、囊胚孵出率的比较：两组各取50枚移植前胚胎，体外培养观察，新鲜移植组形成囊胚46枚，囊胚形成率为92％；孵出囊胚42枚，囊胚孵出率为84％。玻璃化冷冻组形成囊胚45枚，囊胚形成率为90％；孵出囊胚43枚，囊胚孵出率为86％。两组比较差异无统计学意义(P＞0.05)。3．新鲜移植组和玻璃化冷冻组妊娠率的比较：新鲜移植组移植假孕鼠47只，妊娠23只，妊娠率为48.93％；玻璃化冷冻组移植假孕鼠51只，妊娠24只，妊娠率为47.06％。两组比较差异无统计学意义(P＞0.05)。4．新鲜移植组和玻璃化冷冻组仔鼠出生率的比较：新鲜移植组共移植胚胎752枚，分娩子代99只，出生率为13.16％；玻璃化冷冻组共移植胚胎816枚，分娩子代103只，出生率为12.62％。两组比较差异无统计学意义(P＞0.05)。结论：玻璃化冷冻复苏后，成活胚胎囊胚形成率、囊胚孵出率及移植后的妊娠率，仔鼠出生率，与新鲜胚胎相比无明显差异。说明玻璃化冷冻对胚胎体内外发育潜能无明显影响。第二部分研究玻璃化冷冻对子代一般发育情况的影响目的：对新鲜移植组和玻璃化冷冻组子代小鼠的生理发育、运动协调功能、器官畸形发病率进行观察，进一步探讨胚胎玻璃化冷冻对子代发育的影响。方法：1．仔鼠出生当天定为出生后0d。记录新生小鼠体重，仔鼠耳廓分离、上下牙萌出、张耳、睁眼、睾丸下降、阴道开口、全身绒毛、全身白毛等生理发育指标；平面翻正、前肢悬挂、爬行、空中翻正等神经反射和运动协调功能指标。2．两组均于出生后第3天，第7天，第14天，第21天，第28天，第60天分别取雌雄小鼠各6只，脱颈处死，解剖后观察各器官发育情况。3．两组均于上述时间段分别取小鼠子宫、卵巢、睾丸，4％多聚甲醛固定，石蜡包埋，行HE染色。进一步观察小鼠生殖器官发育过程中的形态学变化，每个时间段雌雄小鼠各6只。结果：1．生长发育：两组新生仔鼠平均体重分别为1.42±0.26g，1.39±0.38g。两组小鼠生理发育和运动协调指标测试结果提示两组小鼠发育无明显异常。2．大体解剖：玻璃化冷冻组共出生103只仔鼠，其中雄鼠58只。有一只子代解剖后可见雄性生殖器官发育正常，但副中肾管无完全退化，呈白色羊角样。该鼠染色体分析为40，XY。故认为是雄性畸形，畸变原因未知。余器官未见明显畸形。新鲜移植组共出生99只仔鼠，其中雄鼠56只，各器官均未见明显畸形。3．生殖器官形态学观察：两组小鼠在上述相应时期，子宫、卵巢、睾丸发育中均未见明显形态学异常。结论：玻璃化冷冻不影响小鼠的生理及运动协调能力的发育。但对出现的小鼠生殖管道畸形的原因，需要进一步研究。第三部分研究玻璃化冷冻对雌性子代子宫发育的影响目的：通过比较两组子代子宫发育过程中，Wnt4、Wnt5a、Wnt7a、β-catinin、Hoxa10基因的表达，进一步研究玻璃化冷冻复苏过程对雌性小鼠子宫形态和功能的影响。方法：1．两组均于生后第3天，第7天，第14天，第21天，第28天，第60天取雌性小鼠各6只，分别取其子宫。取其中3只，左侧子宫固定行免疫组化，右侧行PCR，另3只欲行Western-blot。2．用于免疫组化的子宫固定，石蜡包埋，水平横切面切成4μm切片，行Wnt4、Wnt5a、Wnt7a、β-catinin、Hoxa10基因蛋白免疫组化染色。3．两组上述不同时间段的小鼠子宫，PCR及Western-blot进一步研究Wnt4、Wnt5a、Wnt7a、β-catinin、Hoxa10基因、蛋白在子代子宫发育过程中的表达变化。结果：1．免疫组化结果：Wnt4表达仅限于腔上皮和邻近子宫腺体之间的基质。Wnt5a在基质内呈强表达，在腔上皮和腺上皮内弱表达。Wnt7a在腔上皮和腺上皮内表达。β-catenin在子宫内膜上皮及基质内均有表达。Hoxa10主要在基质及子宫肌层内表达。对Wnt4、Wnt5a、Wnt7a、β-catenin和Hoxa10在两组相应时期的免疫反应性进行统计学分析，两组相应时期的平均密度值无明显差异(P＞0.05)。2．RT-PCR结果RT-PCR结果显示：Wnt4、Wnt5a、Wnt7a、Hoxa10、β-catenin在两组子代子宫发育过程中均有表达，在上述相应时期表达无明显差异(P＞0.05)。3．Western-blot结果Western-blot结果显示：Wnt4、Wnt5a、Wnt7a、Hoxa10、β-catenin在两组子代子宫发育过程中均有表达，在上述相应时期表达无明显差异(P＞0.05)。结论：玻璃化冷冻不影响出生后小鼠子代子宫的发育和功能。更多还原

【Abstract】 PartⅠThe study of embryos developmental capacity of vitrifiedmouse 8-cell embryos in vivo or in vitroObjective:To observe blastocyst formation rate, blastocyst hatching rate and pregnancy rate, birthrate in mice of vitrified 8-cell embryos, compared with fresh 8-cell embryos. And toevaluate the impact of vitrification on embryos developmental ability in vitro and in vivoMethods:A total of 200 females mice (more than 6～7 weeks) were used in this experiment. Thefemale were sacrified by cervical dislocation after 20h of the presence of vaginal plug. 2-cell embryos were obtained, and to observe embryonic development after 36h of culture. 6to 8- cell embryos were divided into two groups, one group was fresh-embryo group (freshembryo transferred), and the other group was vitrified-embryo group (vitrified embryoswere storage for 2 weeks in liquid nitrogen, then thawed and transferred).50 pre-transferring embryos were cultured 48～72 hours in vitro in each group. Thenumbers of blastocysts and hatching blastocyst was recorded .After 1～3 h of culture fromthawing, the morphological normal 6 to 8 - cell embryo were selected to transfer to uterus of pseudopregnant mouse, 8 for each side uterus. Fresh-embryo group transferred fresh 6 to8 - cell embryos. Recorded the number of live pups after 17～20 d of pregnancy by naturaldelivery. To compare pregnancy rate, birth rate in mice between two groups.Results:1. Embryos observation in fresh-embryo group and vitrified-embryo group: A total of 823high-quality 6 to 8-cell embryos were obtained in fresh-embryo group. And a total of 11566 to 8-cell embryos were obtained in vitrified-embryo group, 1143 embryos underwentvitrification and 864 embryos were survival after thawing. Survival rate was 75.59%.2. The comparison of blastocyst formation rate, blastocyst hatching rate betweenfresh-embryo group and vitrified-embryo group: 50 pre-transferring embryos were culturedin vitro in each group. In fresh-embryo group, the number of blastocyst and hatchingblastocyst were 46, 42. Blastocyst formation rate and blastocyst hatching rate were 92%,84%. In vitrified-embryo group, the number of blastocyst and hatching blastocyst were 45,43. Blastocyst formation rate and blastocyst hatching rate were 90%, 86%. There was nosignificant difference between two groups (P＞0.05).3.The comparison of the pregnancy rate between fresh-embryo group andvitrified-embryo group: In fresh-embryo group, pseudopregnancy mice were 47, 23 ofpregnancy, the pregnancy rate was 48.93%; in vitrified-embryo group, pseudopregnancymice were 51, 24 of pregnancy, the pregnancy rate was 47.06%. There was no significantdifference in two groups (P＞0.05).4. The comparison of the birth rate between fresh-embryo group and vitrified-embryogroup: In fresh-embryo group, a total of 752 embryos were transferred. 99 offspring bybirth, the birth rate was 13.16%; In vitrified-embryo group, a total of 816 embryos weretransferred. 103 offspring birth, the birth rate was 12.62%. There was no significantdifference in two groups (P＞0.05).Conclution:In vitrified-thawing survival embryos, the resulting blastocyst formation rate, blastocyst hatching rate and pregnancy rate, birth rate in mice were no significantdifference comparing with the fresh-embryo groups. Analysis revealed that the process ofvitrification had less impact on the embryos developmental ability in vitro and in vito.PartⅡThe study of the effect of vitrification on the developmentof offspring in miceObjective:The aim of this part was to investigate the indicators of physiological development,movement coordination function, the incidence of organ abnormalities in fresh-embryogroup and vitrified-embryo group, and to provide further evidence for the effect ofvitrification on the development of offspring in mice.Methods:1. The day of mice birth was considered to be 0 postnatal day. Recorded the indicators ofphysiological development such as weight, ear unfolding, upper and lower teeth eruption,eye opening, drop in testis, vaginal opening, pinna detachment; movement coordinationfunction such as flat righting, forelimb hanging test, crawling, air righting of newborn mice.2. The mice were sacrified by cervical dislocation on postnatal days (PNDs) 3, 7, 14, 21,28, 60 in two groups to observe the development of all organs after anatoming. 6 femaleand 6 male for each phase in each group.3. In the above-mentioned time period, mouse uterus, testis, ovary were taken from 6female and 6 male in each group, fixed and paraffin-embedded, HE staining, and to observethe morphological changes in reproductive organs of mice between two groups. Results:1. Growth and development:The average weight of newborn in two groups was 1.42±0.26g, 1.39±0.38grespectively. The mice physical development and movement coordinationfunction test results were considered that the two groups had no significant differencein mice, which suggest the vitrification had less impact in the physiological development ofmice.2. The development of organ:A total of 103 new-born mice were obtained in vitrified- embryo group, 58 were males.One offspring of mouse can be seen that the normal development of male reproductive tract(MRT) and the non-degraded female reproductive tract (FRT) were both present in thisgroup.The malformed FRT was shown white selenastrum-like. The abnormal mousechromosome was 40, XY. Therefore we thought it was a male malformation. The reasonsof malformation were unknown. There were no obvious abnormalities in other organ ofother mice. A total of 99 pups were born in fresh - embryo group. 56 males, there was noobvious deformity in all organ.3. Morphological observation:No obvious morphological difference in appearance was found in the correspondingperiod in the development of genital organ between vitrification group and fresh group.Conclution:Vitrification does not affect the physiological development and movementcoordination function of mouse. But the reasons of the abnormal reproductive tract of themice, required further study. PartⅢThe study of the impact of vitrification in thedevelopment of the uterus of offspring in miceObjective:In this experiment, we compared the expression of Wnt4, Wnt5a, Wnt7a,β-catinin,Hoxa10 in the developing uterus in two groups. And to further study the impact ofvitrifying preimplantation embryos on morphology and function of uterus of offspring inmice.Methods:1. Uteri were obtained from 6 different female mice on each postnatal days (PND) 3, 7,14, 21, 28, and 60 in two groups. 3 mice were taken from them, the left uterus forimmunohistochemistry, the right uterus for PCR; another 3 mice for Western-blot.2. Tissues for immunohistochemistry were fixed and then processed and embedded inparaffin and sectioned at 4μm, The expression of mouse Wnt4, Wnt5a, Wnt7a, beta-catinin,Hoxa10 in the uterus were evaluated firstly by immunohistochemistry.3. Reverse transcription (RT)-PCR and Western-blot were performed to determinechanges in gene expression in the uterus of above-mentioned time period in two groups.Results:1. Results of immunohistochemistry:Wnt4 expression was restricted to the stroma between the luminal epithelium andadjacent uterine glands. Wnt5a expression was abundant throughout the stroma, in addition,low but detectable levels of Wnt5a expression were observed in luminal and glandularepithelium. Wnt7a was expressed exclusively in luminal epithelium and in glandularepithelium.β-catenin was expressed in luminal epithelium and the stroma. Hoxa10 wasexpressed in the stroma and smooth muscle layer.Statistic analysis of the Wnt4, Wnt5a,Wnt7a,β-catenin and Hoxa10 immunoreactivity was performed on the correspondingstage between two groups, and the average abundance in each group was no significant difference(P＞0.05).2. Results of RT-PCR:To further assess whether vitrification affects expression of genes during development,the different stage expression was determined by RT-PCR. No difference was apparentbetween two groups in the levels of expression of the development of uterus in mice (P＞0.05).3. Results of western-blot:This was confirmed by western-blot on different days of postnatal. Densitometricanalysis was performed on all samples, and the average levels of Wnt4, Wnt5a and Wnt7aHoxa10,β-catenin protein were no difference in statistic between two groups (P＞0.05).Conelution:These results were a first indication that vitrifying preimplantation embryos did notaffect the development and function of uterus of offsprings in mice.更多还原