The Preliminary Research Of The Mice Development After The Transfer Of Vitrified 8-Embryo Obtained

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.