人类卵母细胞及植入前胚胎中母性效应基因Mater mRNA的表达

目的:了解母性效应基因Mater表达与人类卵母细胞及早期胚胎发育不同阶段的关系。方法:用巢式逆转录多聚酶链式反应(single cell nested RT-PCR)方法检测人类卵子和植入前各阶段胚胎的母性效应基因Mater mRNA的表达。结果:Mater在人类卵母细胞GV、MⅠ、MⅡ都有表达,植入前胚胎1、2、4、6细胞期表达量逐渐下降,到8细胞期、囊胚、孵出囊胚期均未检测到Mater mRNA表达。结论:人类卵母细胞和植入前胚胎Mater基因的表达量随发育时间的改变而变化,对卵子生长和胚胎发育有重要意义,与母性效应基因在小鼠等其他哺乳动物中的表达模式相似。     

小鼠植入前胚胎对输卵管上皮细胞DNA甲基转移酶-1 mRNA的降调节作用

目的:探讨小鼠植入前胚胎对输卵管上皮细胞DNA甲基转移酶I(Dnmt1)mRNA表达的影响。方法:采用免疫组织化学方法检测Dnmt1在小鼠输卵管的组织学定位;Real- timeRT- PCR检测正常妊娠和假孕小鼠在胚胎2细胞期、4细胞期和8细胞期时,输卵管上皮细胞Dnmt1mRNA表达水平的变化。结果:Dnmt1主要表达在小鼠输卵管的上皮细胞层;妊娠组小鼠各期输卵管上皮细胞Dnmt1mRNA表达水平均显著低于同期假孕组(P<0 05)。结论:小鼠植入前期的胚胎对母体输卵管上皮Dnmt1mRNA表达具有降调节作用,胚胎可能通过此作用间接调控输卵管上皮某些基因的表达。     

Expression of estrogen receptor alpha in preimplantation mice embryos

Estrogen,an ovarian steroid,is known to play im-portant roles in early embryonic development,prepara-tion of the uterus for implantation and the establishmentand maintenance of pregnancy[1].Diminishing of thehormone from pregnant animals will cause the delayedentry of embryos into uterus,excluding of embryos inthe uterus and the retarded development of embryos[2].Estrogen action is thought to be mediated by itsreceptors,including the estrogen receptorα(ERα)and estrogen receptorβ(ERβ),whic…     

肿瘤转移抑制因子CD82/KAI1对植入前小鼠胚胎体外发育的影响

目的:研究CD82/KAI1mRNA及蛋白质在小鼠胚胎中的表达规律及其对小鼠胚胎体外发育的影响。方法:①应用RT-PCR及免疫荧光技术观察CD82/KAI1mRNA及蛋白质在小鼠不同发育时期胚胎中的表达规律。②在不同CD82/KAI1抗体浓度的培养液中,培养小鼠8-细胞胚胎,观察囊胚发育率、孵化率和胚胎细胞数的变化。结果:①CD82/KAI1mRNA在小鼠不同时期胚胎中均有表达,于8-细胞期及桑葚胚表达较丰富,CD82/KAI1蛋白表达于各期胚胎细胞的胞膜和胞浆,在桑葚胚期CD82蛋白还强表达于胚胎细胞的胞核。②一定浓度的CD82/KAI1抗体可明显抑制胚胎的发育速度(1∶800,P<0.05),降低囊胚的形成率(1∶400,P<0.05)和孵出率(1∶800,P<0.05),减少囊胚细胞数(1∶800,P<0.05)。结论:CD82/KAI1在小鼠胚胎的发育过程中可能发挥重要作用。     

人类白细胞抗原G及其异构体mRNA在人类着床前胚胎的表达

目的研究人类白细胞抗原G（human leukocyte antigen，HLA-G）及其异构体mRNA在人类着床前胚胎的表达，探讨HLA-G在人类胚胎早期发育中的作用和意义。方法第四军医大学唐都医院妇产科生殖医学中心2003-01-2005-12期间，以体外受精-胚胎移植技术助孕过程中剩余的患者自愿捐赠的人类早期胚胎作为研究对象，采用巢式RT-PCR检测早期胚胎HLA-G及其异构体mRNA的表达。结果检测20个受精后2—3d的人类卵裂期胚胎，2、4、6、8细胞期胚胎各5个，有7个胚胎表达HLA-G及其部分的异构体mRNA。检测5个受精后4d的人类桑葚胚，全部表达HLA-G及其部分的异构体mRNA。检测25个受精后6d的扩张期囊胚，全部受检囊胚均表达HLA-G mRNA，但异构体表达不同。在受检的25个扩张期囊胚中，20个表达HLA-G1（表达率80．0％），4个表达G2（表达率16．0％），25个表达G3（表达率100％）、24个表达G4（表达率96．0％），5个表达G5（表达率20．0％），8个表达G6（表达率32．0％）。结论着床前的人类胚胎表达HLA-G及其异构体mRNA，其阳性表达胚胎的比例，随着胚胎发育的进程而增加。HLA-G异构体在人类着床前胚胎差异表达。     

Syngeneic antiserum to Nulli SCC1 embryonal carcinoma cells recognizing surface antigens of embryonic cells

A syngeneic antiserum was prepared in mice against the nullipotent embryonal carcinoma cell line Nulli SCC1 to determine whether cell surface determinants are expressed commonly by this cell line and early embryos. After antibodies to calf serum components were removed from the antiserum by affinity purification, the antiserum was tested on preimplantation and $\text{6}\text{1}\text{2}$-day postimplantation mouse embryos, embryonal carcinoma cells, tumor cells, and somatic cells. It recognizes antigens which are stage and tissue specific on early embryos and have a restricted distribution in adults. These antigens are first detected on 8-cell embryos and display a polarized distribution on blastomeres corresponding to the distribution of microvilli.     

14-3-3蛋白在小鼠受精卵及2-细胞期胚胎中的表达与定位

目的:研究小鼠受精卵及2-细胞期胚胎中14-3-3蛋白的表达及亚细胞定位。方法:采用Western blot方法鉴定小鼠受精卵和2-细胞期胚胎14-3-3蛋白的表达,利用间接免疫荧光技术观察其在细胞中的定位。结果:在小鼠受精卵和2-细胞期胚胎中,全部表达14-3-3蛋白,并且为同一亚型;14-3-3蛋白主要定位于受精卵的细胞质及2-细胞期胚胎中的细胞质和细胞核。结论:14-3-3蛋白正常表达于小鼠受精卵及2-细胞期胚胎中,其定位可能影响胚胎的早期发育。     

14-3-3蛋白在小鼠受精卯及2-细胞期胚胎中的表达与定位

目的：研究小鼠受精卵及2-细胞期胚胎中14-3-3蛋白的表达及亚细胞定位。方法：采用Western blot方法鉴定小鼠受精卵和2-细胞期胚胎14-3-3蛋白的表达，利用间接免疫荧光技术观察其在细胞中的定位。结果：在小鼠受精卵和2-细胞期胚胎中，全部表达14-3-3蛋白，并且为同一亚型：14-3-3蛋白主要定位于受精卵的细胞质及2-细胞期胚胎中的细胞质和细胞核。结论：14-3-3蛋白正常表达于小鼠受精卵及2-细胞期胚胎中，其定位可能影响胚胎的早期发育。     

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.     

Sex determination in single mouse blastomeres by polymerase chain reaction

In sex determination of mammalian preimplantation embryos, viability of biopsied embryos and accuracy of sexing are together important. In consideration of this point, single blastomeres were mechanically isolated from mouse embryos using a micromanipulator and then sexed by polymerase chain reaction (PCR) using mouse Y chromosome-specific primers. All of 260 embryos biopsied at the four-cell and morula stage survived. Developmental rate of the embryos to normal blastocysts was 93 and 94%, respectively. Sex determination of single blastomeres was performed by amplification of a mouse Y chromosome-specific DNA sequence using PCR technique. The ratio of male to female embryos was 53 and 47%, respectively. The sex-determined embryos were transferred to the uteri of pseudopregnant recipients to test the consistency of the assay system. The sex of 27 of 29 mice developed from male and female embryos agreed with the predicted sex. The method developed for embryo biopsy and sexing could be used for diagnosis of defective genes at the stage of the preimplantation embryos of human and other domestic animals.     

五个抑癌印迹基因在人类卵子及种植前胚胎的表达

目的:检测5个抑癌印迹基因在人类卵子和种植前胚胎的正常表达,完善印迹基因在人类种植前胚胎的表达图谱。方法:应用巢式RT-PCR技术检测印迹基因ARHI、LOT1、P57KIP2、Peg3、TSSC3在人类卵子和种植前胚胎中的表达。结果:卵子和各期种植前胚胎、囊胚中均存在LOT1、P57kip2转录;除4-细胞胚胎外,卵子和其余各期胚胎均可测得ARHI表达; Peg3表达于4-细胞、8-细胞胚胎和囊胚;TSSC3于卵子及各期种植前胚胎中均未表达。结论:除TSSC3外的4个抑癌印迹基因均表达于种植前胚胎,为探讨印迹基因与ART及肿瘤的关系提供理论依据。     

Initial differentiation of blastomeres in 4-cell human embryos and its significance for early embryogenesis and implantation

This brief review is devoted to the nature of early blastomere differentiation in human 4-cell embryos and its consequences for embryonic development. Precursor cells of inner cell mass, germline, and trophectoderm may be formed at this stage, the clearest evidence being available for trophectoderm. The sites of these precursor cells in the embryo could be ascertained using markers for animal and vegetal poles, observing specific cleavage planes, and assessing gene and protein expression. This opens new opportunities for studying 4-cell embryos and removing or replacing specific cells. Knowledge of the properties of individual blastomeres should help in improving assisted human reproduction, performing preimplantation genetic diagnosis, and perhaps establishing specific stem cell lines. Special attention is paid to well-characterized trophectoderm, the trophectoderm stem cell, and possible new forms of clinical application.