人子宫内膜细胞共培养体系对早期鼠胚体外发育的影响

目的:研究人子宫内膜共培养体系对早期鼠胚体外发育的影响及移植后的妊娠情况。方法:将2-细胞小鼠胚胎与人子宫内膜细胞进行体外共培养,对照组为无营养细胞的单纯培养液,每日在显微镜下观察胚胎的发育情况。将培养到囊胚期的胚胎移植回小鼠的子宫腔,观察着床情况。结果:共培养体系中68.3％的2-细胞胚胎发育至桑椹胚期,50.8％发育至囊胚期,囊胚的孵化率为36.7％,胚胎的着床率为25.0％。而对照组只有24.8％的2-细胞胚胎发育至桑椹胚期,11.4％到达囊胚期,且其中大部分为早期囊胚即停止发育。另外对照组细胞碎片出现早且多,卵裂球不均匀,胚形态差,移植后胚胎的着床率仅为3.1％。结论:人子宫内膜细胞共培养体系可以促进小鼠胚胎的体外发育,改善胚胎的质量,提高着床率。     

人子宫内膜,早孕蜕膜单层细胞及其条件培养液对小鼠胚胎发育的作用

为研究人子宫内膜、早孕蜕单膜单层细胞及其培养液对早期胚胎发育的作用。本地将人增殖晚期－早分泌期子宫内膜及早孕蜕膜消化成单个细胞或细胞团块,分别在体外培养,并冷冻保存。建立单层细胞交制成条件培养液,由于人胚取材较困难,此采用小鼠早期胚胎共培养,结果显示,人子宫内膜及早孕蜕膜在体外发育良好,并可冷冻保存,复苏率〉４０％,小鼠胚胎的卵裂率、桑椹胚及囊胚形成率明显高于对照组。早孕且较子宫内膜组略高,但     

人子宫内膜、早孕蜕膜单层细胞及其条件培养液对小鼠早期胚胎发育的作用

为研究人子宫内膜、早孕蜕膜单层细胞及其条件培养液对早期胚胎发育的作用,本文应用酶消化法将人增殖晚期早分泌期子宫内膜及早孕蜕膜消化成单个细胞或细胞团块,分别在体外培养,并冷冻保存。建立单层细胞并制成条件培养液,由于人胚取材较困难,此文采用小鼠早期胚胎共培养,结果显示,人子宫内膜及早孕蜕膜在体外发育良好,并可冷冻保存,复苏率＞ ４０％ 。小鼠胚胎的卵裂率、桑椹胚及囊胚形成率明显高于对照组（Ｐ＜ ０．００１）,早孕蜕膜组较子宫内膜组略高,但无显著性差异。两种细胞的条件培养液均可刺激胚胎发育,但无显著性差异,而胚泡形成率在早孕蜕膜组明显高于对照组（Ｐ＜ ０．０５）。提示：人子宫内膜及早孕蜕膜细胞能释放某些刺激胚胎发育的物质有利于胚胎发育     

重组人促性腺激素对早期鼠胚发育的影响

目的:探讨重组人促性腺激素对小鼠早期胚胎发育的影响。方法:将35只昆明种小鼠随机分为7组,实验组注射一定剂量的重组人促卵泡激素(rhFSH 20 IU)结合不同剂量的重组人促黄体激素(rhLH)对小鼠进行超促排卵,对照组注射孕马血清(PMSG10 IU)和hCG(10 IU),受精0.5 d处死各组雌鼠,收集2-细胞期的鼠胚进行体外培养,观察并记录鼠胚发育至各细胞期的数目。结果:10 IU和15 IU rhLH 组收集的胚胎数与对照组无差异,20 IU rhLH组则高于对照组,但20 IU rhLH组鼠胚发育至4-细胞、8-细胞、桑椹胚、囊胚、扩张期囊胚和孵出期的比率却较低,与对照组比有统计学差异,其余各组收集的胚胎数和胚胎发育至各细胞期数均低于对照组。结论:合适剂量的rhLH对小鼠早期胚胎的发育有明显的促进作用,但剂量过高则对胚胎发育有抑制作用。     

重组人白血病抑制因子对体外移植前鼠胚发育的影响

目的：观察重组人白血病抑制因子（rhLIF)对体外移植前鼠胚发育的影响。方法：将36只小鼠随机分成3组,每组12只。组I（体内对照）小鼠注射人绒毛膜促性腺激素（hCG)116-120h后处死,组Ⅱ及组Ⅲ（体外对照）小鼠注射hCG44-48h后处死,收集组Ⅱ及组Ⅲ的2细胞期鼠胚,组Ⅱ的鼠胚用人输卵管液（HTF）＋10％人血清培养,组Ⅲ的鼠胚用HTF＋10％人血清＋rhLIF(1000U/ml）培养,观察并记录各细胞期鼠胚发育的数目。结果：（1）组Ⅱ和组Ⅲ发育到4细胞期、8细胞期、桑椹胚阶段的鼠胚百分率（分别为93．4％、87．7％、75．0％和94．5％、91．2％、85．4％）相似,差异无显著性（P＞0．05）。（2）组Ⅱ发育到囊胚期、扩张囊胚期和孵出期的鼠胚百分率低于组Ⅲ（分别为48．1％、32．1％、18．4％和82．3％、59．7％、36．3％）,差异有显著性（P＜0．05）。（3）组I和组Ⅲ发育到囊胚期的鼠胚百分率（分别为86．0％和82．3％）相比,差异无显著性（P＞0．05）。结论：rhLIF对鼠胚的早期发育没有显著影响,但能促进移植前晚期鼠胚的生长、分化和孵出。     

人卵泡液对小鼠胚胎生长发育影响的研究

目的：探讨人卵泡液对小鼠胚胎生长发育的影响。方法：在培养液中分别加入人卵泡液（卵泡液组）和人血清（血清组）,观察和检测2细胞期鼠胚在体外发育至8细胞期、囊胚期以及鼠胚孵出的比率;用放射免疫方法测定人卵泡液和血清中表皮生长因子（EGF）含量。结果：卵泡液组有72.9%的2细胞期鼠胚能发育到8细胞期,血清组仅有48.0%到达8细胞期;继续培养显示,卵泡液组细胞分裂较快,有50.9%的鼠胚可发育至囊胚期,并有26.3%孵出,明显高于血清组（24.5%,6.9%),两组比较,差异有显著性（P＜0.05)。卵泡液中EGF的含量为0.50μg/L,血清中为0.26μg/L,两组比较,差异有显著性（P＜0.05）。结论：卵泡液对早期胚胎发育有促进作用,在早期胚胎培养中,添加人卵泡液比添加人血清对提高早期胚胎培养质量更为有效。     

小鼠及人胚胎分割技术的研究

植入前小鼠胚胎（ｐｒｅｉｍｐｌａｎｔａｔｉｏｎ ｅｍｂｒｙｏ）的透明带经软化后,用固定在显微操作臂上的微玻璃针行胚胎分割,２－、４－、８－细胞胚胎及桑椹胚分割成功率分别为９０．６％、８５．０％、７３．６％、７１．４％。所获半胚体外培养后,胚泡发育率分别为６２．１％、８０．１％、８３．０％、８９．２％。随胚胎细胞数目增多,分割成功率降低,而发育率则提高。将８－细胞胚组及桑椹胚组的半胚移植到假孕母鼠后,幼仔出生率分别为１４．６％、１５．１％。同法分割４－细胞人胚胎２个,获半胚４个,分割成功率为１００％。     

白血病抑制因子对体外培养小鼠早期胚胎发育的影响

目的：研究白血病抑制因子（ＬＩＦ）对胚胎发育的影响。方法：用小鼠的单细胞胚胎体外培养１２０ｈ,观察不同浓度的ＬＩＦ对早期胚胎发育的影响。结果：①０．１ｎｇ／ｍｌ、１ｎｇ／ｍｌ和１０ｎｇ／ｍｌ浓度的ＬＩＦ对小鼠早期胚胎的发育和胚泡的形成有促进作用,０．０１ｎｇ／ｍｌ和１００ｎｇ／ｍｌ浓度的ＬＩＦ对胚胎的发育无明显影响;②ＬＩＦ对小鼠早期胚胎发育的影响主要发生于桑椹胚和胚泡期,对１～８细胞期胚胎无明显影响。结论：适当浓度的ＬＩＦ可促进胚胎发育和胚泡形成。     

体外着床模型中人孵化后早胚细胞角蛋白和肌动蛋白的表达

目的:建立理想的体外胚胎着床模型,并检测模型中人孵化后早胚细胞角蛋白、肌动蛋白和hCG。方法:孵化后早胚与人子宫内膜蜕膜化的基质细胞共培养,观察胚泡在基质细胞层上的定位、黏附、铺展和侵入过程;用免疫荧光染色技术,测定共培养系统中的细胞角蛋白和肌动蛋白;用免疫荧光分析技术,测定培养液中的hCG水平。结果:胚泡和基质细胞共培养5h起,胚泡开黏附在基质细胞层上,最终侵入蜕膜化的基质细胞间。共培养48h后,细胞角蛋白仅仅在滋养层细胞中表达;肌动蛋白在人蜕膜化的基质细胞和滋养层细胞中均有表达。囊胚与子宫内膜基质细胞共培养的培养液中的hCG水平明显高于囊胚单独培养的(P<0.01)。结论:成功建立了一个能反映人胚泡黏附、铺展及侵入到人子宫基质细胞的体外着床模型,细胞角蛋白、肌动蛋白和hCG在着床早胚细胞中起相应变化。     

盐酸克仑特罗对小鼠胚胎体外发育的影响

目的:探讨盐酸克仑特罗对小鼠1-细胞胚胎和2-细胞胚胎体外发育的影响。方法:获取小鼠1-细胞和2-细胞胚胎,分别与盐酸克仑特罗10 ng/mL,3 ng/mL和1 ng/mL的3个剂量组共培养,观察胚胎各阶段的发育情况并计算胚胎的发育率。结果:1ng/mL和3ng/mL组的1-细胞小鼠胚胎,从4-细胞期到囊胚阶段的发育率与对照组相比差异显著(P<0.05),3 ng/mL组显现出比1ng/mL组更强的抑制作用(P<0.01),10 ng/mL组,2-细胞期就与对照组有差异(P<0.05,其中 4-细胞至囊胚阶段P<0.01),囊胚率只有2.4％。10 g/mL组及3 ng/mL组的2-细胞小鼠胚胎,分别从4-细胞期和8-细胞期与对照组相比差异显著(P<0.05),1 ng/mL组的各个阶段胚胎发育率与对照组相比未见统计学差异(P>0.05)。培养液中含有盐酸克仑特罗使胚胎的粗颗粒增多,部分印裂球碎裂、退化。结论:盐酸克仑特罗对小鼠胚胎的体外发育有毒性作用并呈一定的剂量效应。盐酸克仑特罗使1-细胞小鼠胚胎被抑制在2-细胞期,对处于晚2-细胞期胚胎的影响明显降低。     

Effect of cryotop vitrification on preimplantation developmental competence of murine morula and blastocyst stage embryos

Vitrification is an effective method for the cryopreservation of mammalian embryos. Nevertheless, it is unclear which embryonic developmental stage is the most suited for vitrification and would ensure maximal developmental competence upon subsequent warming. This study, therefore, compared the effects of cryotop vitrification on the developmental competence of murine morula and blastocyst stage embryos. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in two hatched blastocyst groups derived from vitrified morulae and blastocysts, respectively. The post-vitrification survival rates for mouse embryos at the morula and blastocyst stage were 95.4% (186/195) and 96.5% (195/202), respectively. The blastocyst formation rate was significantly lower for vitrified morulae (90.3%) compared with the non-vitrified control group (98.4%) (P < 0.05). The hatching rates were similar between the vitrified morula (79.6%) and the vitrified blastocyst (81.0%) groups. When further development to the fully hatched blastocyst stage was compared, fully hatched blastocysts derived from vitrified morulae had significantly higher cell counts for both the ICM and TE lineage, as compared with hatched blastocysts derived from vitrified blastocysts (P < 0.001). Cryotop vitrification of mouse embryos at the morula stage rather than blastocyst stage would thus ensure a higher degree of post-warming developmental competence.     

Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching

Purpose

To determine the pattern of expression of parathyroid hormone-related protein (PTHrP) and its receptor, parathyroid hormone receptor 1 (PTHR1), in mouse embryos in different stages of preimplantation development.

Methods

Embryos were cultured from the pronuclear zygote stage and harvested as 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts. RT-PCR was carried out on mRNAs of these and of trophoblast outgrowths for detection of PTHrP and PTHR1. Whole mounted embryos intact or stripped of zonae pellucidae were immunofluorescently stained for PTHrP and PTH receptor and observed with confocal microscopy.

Results

PTHrP mRNA was present in the pronuclear zygote, not present in 2-cell, 4-cell and uncompacted 8-cell embryos, present in the 8-cell compacting embryo, and not detected in 16-cell morulae or blastocysts. The mRNA was present in trophoblasts growing on fibronectin beds. mRNA for PTHR1 was detected in the pronuclear zygote, then undetected until the compacted 8-cell stage and thereafter. PTH receptor protein was observed in 2-cell embryos, morulae and in the inner cell mass and trophectoderm of blastocysts. PTHrP was observed dispersed in the cytoplasm of 2-cell, 4-cell and uncompacted 8-cell embryos, and in distinct foci near the nuclei of morulae. In blastocysts, PTHrP appeared on the apical surface of only trophoblast cells which had extruded from the zona pellucida. Fully hatched blastocysts expressed the protein on the apical side of all trophoblasts. When morulae were prematurely stripped of their zonae, PTHrP was observed on the embryos’ outer surface.

Conclusions

PTHrP protein is expressed throughout early embryo development, and its receptor PTHR1 is expressed from the morula stage. Embryo hatching is associated with translocation of PTHrP to the apical plasma membrane of trophoblasts. PTHrP may thus have autocrine effects on the developing blastocyst.     

The mechanism of action of coculture on embryo development in the mouse model: direct embryo-to-cell contact and the removal of deleterious components

OBJECTIVE: To elucidate the mechanism for the mode of action of coculture by the use of a coculture system for mouse one-cell embryos with human oviductal epithelial cells. DESIGN: Prospective, controlled in vitro experimental study. SETTING: Academic research laboratory. ANIMAL(S): Female ICR strain mice aged between 6 and 8 weeks. INTERVENTION(S): Flushed one-cell embryos were cultured in human tubal fluid medium alone (control), in coculture system with human oviductal cells, in five kinds of conditioned media, and in a contactless coculture system using a cell-culture insert. MAIN OUTCOME MEASURE(S): The percentage of the embryos developed to hatching blastocyst stage and the level of superoxide anion in the supernatant from each culture condition. RESULT(S): The rates of embryo development to the hatching blastocyst stage were significantly higher in the coculture group (43%) than in the control group (none) (P <.05). The embryo development rate in the control group was similar to that of the embryos in the five kinds of conditioned media. The effects of coculture on embryo development disappeared in the contactless coculture group. The level of superoxide anion was significantly reduced in the coculture group compared to the control group. CONCLUSION(S): The present coculture system overcomes the two-cell block in vitro and improves the embryo development. The beneficial effect may be a result of direct cell-to-cell contact between the embryo and helper cells and the removal of deleterious components from medium, rather than a result of embryotrophic factors.     

Improvement in blastocyst hatching of mouse embryos cocultured with human placental cells

Purpose Although the hatching of embryos is an important phenomenon, the mechanism of hatching remains controversial. Therefore, we attempted to develop a new coculture system with human placental cells to investigate the hatching of mouse embryos.Results In our new system there was no difference in development from the two-cell stage to blastocysts between embryos cultured with a T6 medium and embryos cocultured with human placental cells at 1 × 10⁵, 5 × 10⁵, and 1 × 10⁶ cells/ml. However, the hatching-rate cell number increased significantly in embryos cocultured with placental cells compared to embryos cultured without placental cells. [³H]Thymidine uptake did not show any significant difference from the beginning of in vitro culture to the hatching stage between the coculture group and the control group. Nevertheless, the [³H]uridine uptake was significantly different in the two groups, measuring 2167 ± 532 cpm/10 embryos in the coculture group and 804 ± 86 cpm/10 embryos in the control group at 114 hr after human chorionic gonadotropin injection (P < 0.01).Conclusion These results therefore seem to indicate that the hatching of blastocysts depends on the protein synthesis of the embryos and not on DNA duplications.     

小鼠体外培养囊胚辅助孵化的初步研究

目的 :探索体外培养和辅助孵化技术对体外发育小鼠囊胚孵出率的影响。方法 :将昆明白种小鼠的 2～ 4-细胞胚于体外培养到囊胚 ,观察比较 :(1 )体外培养囊胚与体内发育囊胚的自然孵化率 ;(2 )体外培养囊胚经辅助孵化后与未行辅助孵化 (对照组 )的体外孵化率。结果 :(1 )体外培养囊胚与体内发育囊胚的自然孵化率分别为 75.4%和 96 .2 % ;有显著差异 (P<0 .0 0 1 )。 (2 )辅助孵化与对照组的体外孵化率分别为 97.7%和 74.0 % ,有明显差异 (P<0 .0 5)。结论 :体外培养对囊胚的孵化有不利影响 ;辅助孵化则能明显提高体外培养囊胚的孵出效率     

Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo.

OBJECTIVE: To investigate whether the deleterious effect of E(2) on embryonic implantation is due to a direct effect on the endometrium, on the embryo, or both. DESIGN: Prospective, controlled in vitro study. SETTING: Tertiary infertility center. PATIENT(S): Fertile patients in the luteal phase with histologically normal endometrium who were attending the infertility clinic as oocyte donors (n = 14). INTERVENTION(S): E(2) dose-response (0, 10(-8), 10(-7), 10(-6), 10(-5), and 10(-4) M) and time course (day 2 vs. day 5) experiments were performed in an in vitro embryo adhesion assay composed of human polarized endometrial epithelial cells obtained from fertile patients and mouse embryos. MAIN OUTCOME MEASURE(S): Blastocyst formation rate and embryo adhesion rate. RESULTS: Monolayers of polarized endometrial epithelial cells expressed ERalpha at the mRNA level. The E(2) dose response of blastocysts with polarized endometrial epithelial cells (n = 235) demonstrated a progressive reduction in embryonic adhesion that was statistically significant at 10(-6) M. When polarized endometrial epithelial cells were treated alone with increasing doses of E(2) for 3 days and E(2) was then removed and blastocysts added (n = 410), embryonic adhesion was not significantly reduced, except at 10(-4) M. When 2-day mouse embryos (n = 609) were treated with increasing E(2) concentrations until day 5, the rate of blastocyst formation significantly decreased at a concentration >or= 10(-6) M, and embryonic adhesion decreased when blastocysts (n = 400) were obtained at a concentration >or= 10(-7) M. Time course experiments of embryos cultured for 2 days with polarized endometrial epithelial cells (n = 426) showed that the adhesion rate was higher at E(2) levels of 10(-7), 10(-6) and 10(-5) M compared with embryos cultured for 5 days (n = 495). CONCLUSION(S): High E(2) levels are deleterious to embryo adhesion in vitro, mainly because they have a direct toxic effect on the embryo that may occur at the cleavage stage.     

Permeability of the plasma membrane to water and cryoprotectants in mammalian oocytes and embryos: Its relevance to vitrification

The permeability of the plasma membrane to water and cryoprotectants is one of the important factors for determining the suitable condition for the vitrification of mammalian oocytes and embryos. Water and cryoprotectants move slowly through oocytes and early embryos, principally by simple diffusion, in the mouse, bovine, pig, and human. In contrast, water, glycerol, and ethylene glycerol move rapidly through morulae and blastocysts, principally by facilitated diffusion via aquaporin 3, in the mouse and bovine; whereas, in the pig, the permeability to water and these cryoprotectants increases not at the morula stage but at the blastocyst stage and further increases at the expanded blastocyst stage. Dimethyl sulfoxide also moves rapidly via channels other than aquaporin 3 in the mouse. In contrast, propylene glycol moves through morulae and blastocysts principally by simple diffusion in the mouse, bovine, and pig, as through oocytes. Therefore, the permeability of mammalian oocytes and embryos at early stages to water and cryoprotectants is low, but that of embryos at later stages to water and some cryoprotectants is markedly high by channel processes, although species specificity exists in some cases.     

The expression and distribution of aquaporin 3 in mouse embryos before and after vitrification

Purpose

To determine the role of aquaporin 3 (AQP3) isoform in early embryonic development and protecting embryos subjected to freeze–thawing for assisted reproduction, we investigated the expression and distribution of AQP3 in mouse embryos at different developmental stages before and after vitrification.

Methods

Eight-cell embryos, morulae, and blastocysts were obtained from female mice that had been superovulated by controlled ovarian hyperstimulation. Immunofluorescence staining, laser confocal microscopy, and Western blot were used to determine the expression and distribution of AQP3 in preimplantation mouse embryos before and after vitrification.

Results

AQP3 was expressed at the 8-cell to blastocyst stage before and after vitrification. The expression and distribution of AQP3 was developmentally regulated at the 8-cell to blastocyst stage. The expression of AQP3 was significantly decreased in 8-cell embryos and early blastocysts after vitrification. However, at the morulae stage, the expression of AQP3 was increased after vitrification.

Conclusions

The developmental and vitrification-dependent changes in AQP3 expression and distribution suggest that this transmembrane channel might regulate mouse embryo development and contribute to the protective response during vitrification.     

Human oviductal cells reduce the incidence of apoptosis in cocultured mouse embryos

Objective: To investigate the effect of human oviductal cell coculture on the incidence of apoptosis in mouse embryos.

Design: Experimental laboratory study.

Setting: University gynecology unit.

Patient(s): Fallopian tubes were obtained from patients undergoing hysterectomy.

Intervention(s): Mouse embryos were cocultured with human oviductal cells.

Main Outcome Measure(s): Blastocyst development, allocation of inner cell mass (ICM) and trophectoderm (TE) in blastocyst, and apoptosis in embryos.

Results: Oviductal cells significantly enhanced the blastulation (38%) and hatching rate (22%) of the cocultured zygotes. The corresponding values in medium alone culture were 21% and 9%, respectively. The cocultured embryos also had higher blastomere count at blastocyst stage (P<0.005). This was due to increase in both the cell count of ICM (P<0.05) and TE (P<0.001). Coculture reduced the incidence of apoptosis in the cultured morula and blastocyst from 38% and 48% to 16% (P<0.001) and 27% (P<0.05), respectively. The number of apoptotic blastomeres per morula (1.5 ± 0.6; P<0.005) and blastocyst (2.3 ± 0.7; P<0.005) after coculture was also significantly lower than that of the corresponding control (morula, 2.1 ± 0.8; blastocyst, 3.5 ± 1.1).

Conclusion(s): Human oviductal cells improved mouse embryo development partly by decreasing the incidence of apoptosis.