PTEN在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨存检测肿瘤抑制基因PTEN(phosphatase andtensinhomologdeletedonchromosometen)在早孕小鼠子宫内膜中的表达规律,探讨PTEN在小鼠胚胎着床过程中的作用.采用实时荧光定量聚合酶联反应(real.time fluorescent quantitative PCR.FQ.PCR)和免疫组织化学方法分别检测未孕及孕1、3、4、5、7 d小鼠子宫内膜PTEN mRNA和蛋白的表达;子宫角注射PTEN反义寡核苷酸观察胚泡着床数.FQ-PCR结果显示,妊娠小鼠子宫内膜组织PTENmRNA的表达高于未妊娠小鼠,且随着妊娠天数的增加表达逐渐增强,到妊娠第5天达最高.免疫组织化学分析显示,PTEN蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射PTEN反义寡核苷酸后胚泡着床数明显减少.结果提示,PTEN在妊娠早期子宫内膜持续表达,可能参与了胚泡着床.     

p16INK4a在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨在检测肿瘤抑制基因p16INK4a(inhibitor of cyclin-dependent kinase 4a)在早孕小鼠子宫内膜中的表达规律,探讨p16INK4a在小鼠胚胎着床过程中的作用.采用荧光定量PCR(FQ-PCR)和免疫组织化学方法分别检测未孕小鼠及孕小鼠第2、3、4、5、7天子宫内膜p16INK4a mRNA和蛋白的表达;子宫角注射p16INK4a抗体观察胚泡着床数.FQ-PCR结果显示孕小鼠子宫内膜组织p16INK4amRNA的表达高于未孕小鼠,且随着妊娠天数的增加呈现表达逐渐增强的趋势,到妊娠第5天达到最高,后渐降.免疫组织化学分析显示p16INK4a蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射p16INK4a抗体后胚泡着床数明显减少.以上结果提示,P161INK4a在妊娠早期子宫内膜持续表达,可能参与胚泡着床.     

层粘连蛋白、纤粘连蛋白及Ⅳ型胶原抗血清对小鼠胚泡着床的影响

文中报道了子宫角单次注射层粘连蛋白（ＬＮ）抗血清、纤粘连蛋白（ＦＮ）抗血清以及Ⅳ型胶原（ＣｏｌⅣ）抗血清对小鼠胚胎植入的作用。选用６０只３０ｇ左右的昆明品系雌性成年小鼠,与有生殖能力的雄性小鼠按１：１合笼,翌晨检查外阴,以出现阴栓为妊娠第１天。于妊娠第３天下午,给每个雌性小鼠 侧子宫角单次注射５μｌ ＬＮ抗血清ＮＦ抗血清或Ｃｏｌ Ⅳ抗血清作为实验组;给同一小鼠右侧子宫角注射５μｌ正常兔血清（Ｎｏｒ     

ConA的抗着床效应

本文用凝集素为探针,探索糖复合物在胚泡着床中的作用,报道了与甘露糖苷有专一结合的伴刀豆凝集素(ConA)有明显的抗小鼠胚泡着床作用。妊娠4d的小鼠,每只子宫角中注入Con A 25μg,22只子宫角中只有4只子宫角有胚泡着床,着床率为18.2％,与生理盐水对照组的着床率87.5％相比有明显差异。将相同剂量的Con A先与0.4mol/L α-甲基-D-甘露糖苷温育1—2h后再注入子宫,20只子宫角中有15只子宫角有胚泡着床,着床率提高到75％。用辣根过氧化物酶直接标记法证明,着床前子宫内膜细胞表面有Con A受体存在,并随着妊娠天数而增加,尤其是间质细胞,发情期时时为阴性反应,到着床期蜕膜细胞膜表面呈现出大量Con A受体。提示精复合物在着床中的重要作用。与甘露糖苷同样专一结合的,但寡糖结构专一性与Con A不同的豌豆凝集素注入子宫则无抗着床效应,着床率为85.7％。由此可以推测,N-连接的包含二个未被取代的或只在C-2位被取代的α-甘露糖苷的寡糖参于胚泡与子宫内膜相互作用的着床过程。     

双炔失碳酯对兔移植胚泡着床的影响

本文用胚泡移植技术验证了双炔失碳酯对子宫内膜的影响是胚泡不能着床的重要原因。将 29只胚龄4d的兔胚泡移植到给药后假孕4d的子宫,结果没有一只胚泡着床;而对照组的5只受卵兔,移植了41只胚泡,有26只着床成功。 为了了解双炔失碳酯对黄体的影响,在5只妊娠兔上,从交配后1—7d与对照组比较血清孕酮浓度的变化,结果表明:对照组的血清孕酮浓度随着妊娠天数显著升高,妊娠第7天时达 17.35±2.12ng/ml,而给药组升高不明显,第7天仅 1.83±1.03ng/ml,为对照组的1/9。已知足够的孕酮浓度对妊娠的维持和胚泡的成功着床具有十分重要的作用。由此推测,双炔失碳酯抑制孕酮的分泌势必影响子宫内膜的发育,从而阻碍胚泡的着床。     

兔卵巢对着床前胚泡的识别

在17只母兔上,用放射免疫学方法对妊娠、假孕、假孕子宫中移植胚泡以及从妊娠子宫中冲去胚泡等情况下的周边血清孕酮浓度进行了测定。比较排卵后每日浓度的变化,借以探讨卵巢黄体能否对着床前胚泡进行识别。结果显示:排卵后第5天开始,妊娠兔的血孕酮水平与假孕兔就有了明显的差异,到第7天,前者血孕酮浓度增至第1天的6倍,而后者仅增3倍左右;将4天胚泡41只同步移植到5只假孕兔子宫,其中26只胚泡着床,血孕酮浓度于移植后第2天起即显著升高,以后逐日上升的趋势近似于妊娠兔所呈现的曲线类型;另从5只妊娠4天的子宫中冲去所有胚泡后,第2天孕酮上升趋势立刻减缓,曲线类型与假孕兔相似。上述结果说明:母体卵巢对已排出的卵是否受精和发育,早在排卵后第5天已能区分;对子宫里有无胚泡存在的反应也显然不同。为了观察卵龄对促黄体作用的影响,分别将2天受精卵和6天胚泡非同步地移植到子宫内膜和卵龄均相差2天的假孕4天子宫中,以了解不同卵龄卵的促黄体效应。结果显示:6天胚泡48只,移植后有24只着床,第2天血孕酮即明显上升;而40只2天受精卵,移植后没有1只着床,对孕酮的变化也未产生影响。由此可见,受精卵必须发育到一定阶段后才出现促黄体的作用。     

细胞周期蛋白D3在兔妊娠早期子宫和胚胎中的表达与调节

利用免疫组织化学及RT—PCR的方法,研究了细胞周期蛋白D3(Cyclin D3)在兔早期妊娠子宫和着床前胚胎中的表达情况,以揭示CyclinD3在兔胚胎着床过程中的可能作用。结果显示：(1)在兔妊娠第3～8天的子宫近肌层的腺上皮中有CyclinD3免疫染色,并且其表达强度在第7天以后呈现下降的趋势,着床的胚胎中未见CyclinD3免疫染色;(2)在第2～5天的假孕兔子宫的腺上皮中有较强的CyclinD3免疫染色;(3)在发情周期的兔子宫中未见其有表达;(4)在切除卵巢的兔中,注射雌激素后子宫中未见CyclinD3免疫染色,注射孕酮后在子宫腺上皮中有CyclinD3免疫染色,在孕酮和雌激素共同处理后的子宫腺上皮中有较强的CyclinD3免疫染色;(5)利用RT—PCR的方法在早期胚胎中均能检测到CyclinD3,但从胚泡期开始表达上升,到扩展胚泡时其表达最强。上述结果表明,CyclinD3的表达可能对兔胚泡的着床具有一定的调节作用。     

绒毛膜促性腺激素和前列腺素F2α抗血清对小鼠胚胎着床效应的研究

本实验用hCG和PGF_(2σ)抗血清对妊娠4天的小鼠胚胎进行体外处理,经过移植后,二个实验组的胚胎着床率(hCG组为25.2±0.37％和PGF_(2σ)组为28.6±0.19％)均显著低于对照组(hCG组为74.9±0.48％,PGF_(2σ)组为73.2±0.17％)P<0.001。研究结果证明;着床前小鼠胚泡能分泌和合成hCG样糖蛋白物质和PGF_(2σ),而hCG和PGF_(2σ)抗血清对小鼠胚泡着床有显著的抑制效应。     

胚泡对子宫的局部影响:雌、孕激素及其受体量的变化

前文指出,大鼠在妊娠第六天(D_6)血液和子宫组织中雌二醇含量明显上升;子宫胞质受体此时也达高峰;特别值得注意的是在D_6细胞核雌、孕激素受体皆比D_5显著升高(刘以训、贾晓池1981;刘以训、贾晓池1982)。Dickmann等用组织化学的方法在大鼠、小鼠、地鼠和家兔等动物胚泡中发现有△~5-3β-羟脱氢酶及17-β-羟甾体脱氢酶的活性,作者认为胚泡能合成孕酮和雌激素(Dey et al., 1976;Dickmann et al., 1976、1977:Sen Gupta,1977)。Psychoyos(1973)给妊娠大鼠子宫中注射3—5毫微克雌二醇,动物去卵巢后只要每天注射孕酮仍能着床。Dickmann等(1977)进而指出,胚泡雌激素能引     

Inhibition of vascular endothelial growth factor/vascular permeability factor action blocks estrogen-induced uterine edema and implantation in rodents

Estrogen induces a rapid increase in microvascular permeability in the rodent uterus, leading to stromal edema and a marked increase in uterine wet weight. This edema is believed to create an environment optimal for the growth and remodeling of the endometrium in preparation for implantation and pregnancy. Increased endometrial microvascular permeability also occurs in conjunction with implantation. Estrogen-induced uterine edema is immediately preceded by an increase in the expression of vascular endothelial growth factor (VEGF), a potent stimulator of microvascular permeability. The objective of this study was to determine to what degree immunoneutralization of VEGF would interfere with a) estradiol-induced uterine edema and b) pregnancy. In the first set of experiments, immature female rats were injected with either VEGF antiserum or normal rabbit serum (NRS) prior to 17beta-estradiol treatment. Rats treated with estradiol alone showed a 57% increase in uterine wet weight at 6 h compared with controls. Injection of 200 or 300 micro l of VEGF antiserum reduced the response to only 20% and 10% above controls, respectively. In the second set of experiments, young adult female mice were treated with 100 micro l of either VEGF antiserum or NRS at 1200 h on the fourth day after mating. NRS-treated mice had normal pregnancies. VEGF antiserum, however, completely blocked pregnancy. When VEGF antiserum-treated females were examined on Day 5 for the presence of implantation sites, none were found. These results show that a) VEGF is the major mediator of estrogen-induced increase in uterine vascular permeability and b) VEGF-induced edema is absolutely essential for implantation to take place.     

Effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation

Mouse embryo implantation depends on the complex interaction between the embryo trophoblast cells and the uterine environment, which deposits an extracellular matrix with abundant amounts of laminin. Intrauterine injection and blastocyst or ectoplacental cone culture models were used to study the effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation in vivo and in vitro. Intrauterine injection of 32/67 kDa laminin-binding protein antibody (0.4 mg in 1 ml Ham's F-10 medium, 5 microl per mouse) into the left uterine horns of mice (n = 22) on day 3 of pregnancy inhibited embryo implantation significantly (P < 0.001) compared with the contralateral horns that had been injected with normal rabbit IgG. A continuous section study on day 5 after injection showed that the embryos in the control uteri implanted normally and developed healthily, but there were no embryos or the remaining embryos had disintegrated in the uteri injected with 32/67 kDa laminin-binding protein antibody. Blastocysts or ectoplacental cones were cultured in media containing 32/67 kDa laminin-binding protein antibody (0.2 mg ml(-1)) on laminin-coated dishes with normal rabbit IgG at the same concentration as in the controls. The 32/67 kDa laminin-binding protein had no effect on blastocyst or ectoplacental cone attachment, but prohibited the blastocyst or ectoplacental cone outgrowth and primary or secondary trophoblast giant cell migration. These results indicate that 32/67 kDa laminin-binding protein antibody blocked mouse embryo implantation by preventing embryo trophoblast cell invasion and migration through the uterine decidual basement membrane-like extracellular matrix which has a high laminin content.     

Cytoskeletal control of the apical surface transformation of rat uterine epithelium

Summary— During early pregnancy, in the lead up to blastocyst implantation, the apical cell surface of luminal epithelial cells of the rat uterus undergo a dramatic shape transformation. This study aims to investigate the role of the cytoskeleton in this apical transformation by considering the effects of the drugs cytochalasin D and colchicine on the uterine luminal cell surface. The results are determined using transmission and scanning electron microscopy. In vivo exposure to cytochalasin D during oestrus, as well as on day 1 of pregnancy, did not affect the long, regular surface microvilli. This drug, however, did disrupt the terminal web within the apical cytoplasm of these cells. Disruption of microfilament (MF) polymerization by cytochalasin D on day 4 of pregnancy induced a cell surface transformation, resulting in the appearance of numerous irregular projections normally present during blastocyst implantation on day 6 of pregnancy. Colchicine did not alter the uterine microvilli of oestrus or day 1 pregnant tissue. Unlike the effect of cytochalasin D, colchicine-induced microtubule (MT) disruption on day 4 of pregnancy did not increase irregular projections and hence this treatment did not result in the cell surface appearance associated with blastocyst implantation. These results indicate that the disruption of MF, rather than MT, contributes to the transformation of the uterine luminal cell surface during the lead up to blastocyst attachment.     

Direct evidence for the involvement of prostaglandin F2 alpha in the first step of estrone-induced blastocyst implantation in the spayed rat.

The effect of prostaglandin F2 alpha (PGF2 alpha) on blastocyst implantation in spayed rats has been studied. In preliminary experiments, the first implantation sites were observed 8 - 12 hours after a single injection of estrone in ovariectomized and progesterone-conditioned rats. Intraluminal instillation of PGF2 alpha into the right uterine horn 8 - 10 h after the estrone injection increased the number of implantation sites. Even treatment with PGF2 alpha without previous estrone injection induced the first step of blastocyst implantation as shown by uterine dye site reaction (Niagara-blue test). The results are discussed with regard to the possible role of PGF2 alpha in the regulation of the blastocyst implantation processes in the rat.     

Heparin-binding EGF-like growth factor is seen on the extracellular surface of uterine epithelial cells only after the initial stages of blastocyst attachment

The presence and distribution of heparin-binding epidermal growth factor in rat uterine epithelial cells was determined immunohistochemically and localized ultrastructurally. Rat uterine tissue was examined on days 1, 3, 6 and 8 of pregnancy and it was found that while presence of this growth factor was evident from day 1, spatial reorganization occurred by the time of blastocyst implantation. Strong apical staining was evident from day 6 to day 8, day 6 being the approximate time of blastocyst implantation. Electron microscopy further revealed that this growth factor while shown to be expressed very strongly apically from day 6, actually localized on the plasma membrane only after attachment of the blastocyst. This suggests that heparin-binding epidermal growth factor is not involved in the initial stages of implantation but is more likely involved in the post attachment stages of pregnancy.     

The effects of heparin-binding epidermal growth factor-like growth factor on preimplantation-embryo development and implantation in the rat.

This study examined the effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on preimplantation-embryo development and initiation of implantation in the rat. In vitro studies showed that HB-EGF improved the development of 8-cell embryos to the blastocyst stage in a concentration-dependent manner, and the growth factor had no effect on the cell number of the blastocyst developed. Intraluminal injection of an anti-HB-EGF antiserum into the uterine horns at 0600 h on day 5 of pregnancy decreased the number of implantation sites (blue dye reaction) at 0200 h on day 6. Intraluminal injection of 20 microl of HB-EGF solution (10 or 100 ng/ml) into each uterine horn induced implantation in about half of the ovariectomized progesterone-treated delayed implanting rats, and the number of implantation sites per rat increased dose-dependently. These results suggest that HB-EGF is involved in the preimplantation-embryo development and initiation of implantation in the rat.     

Dysregulated cannabinoid signaling disrupts uterine receptivity for embryo implantation

The mechanisms by which synchronized embryonic development to the blastocyst stage, preparation of the uterus for the receptive state, and reciprocal embryo-uterine interactions for implantation are coordinated are still unclear. We show in this study that preimplantation embryo development became asynchronous in mice that are deficient in brain-type (CB1) and/or spleen-type (CB2) cannabinoid receptor genes. Furthermore, whereas the levels of uterine anandamide (endocannabinoid) and blastocyst CB1 are coordinately down-regulated with the onset of uterine receptivity and blastocyst activation prior to implantation, these levels remained high in the nonreceptive uterus and in dormant blastocysts during delayed implantation and in pregnant, leukemia inhibitory factor (LIF)-deficient mice with implantation failure. These results suggest that a tight regulation of endocannabinoid signaling is important for synchronizing embryo development with uterine receptivity for implantation. Indeed this is consistent with our finding that while an experimentally induced, sustained level of an exogenously administered, natural cannabinoid inhibited implantation in wild-type mice, it failed to do so in CB1(-/-)/CB2(-/-) double mutant mice. The present study is clinically important because of the widely debated medicinal use of cannabinoids and their reported adverse effects on pregnancy.