早孕小鼠子宫内膜钙网蛋白的表达规律

采用RT-PCR、间接免疫荧光组织化学、Western 印迹及原位杂交技术分别检测未孕(d0)和妊娠d1、d2、d3、d4、d5、d6、d7天小鼠子宫内膜中钙网蛋白(calreticulin, CRT)的表达规律, 探讨CRT在胚胎着床中的作用.结果显示CRT mRNA在妊娠小鼠子宫内膜中的表达明显高于未孕小鼠(P <0.05), 且随着妊娠天数的增加呈逐渐增强的趋势.间接免役荧光组织化学结果显示CRT表达于子宫内膜基质细胞、腺上皮以及腔上皮, 并在妊娠第4、5天基质细胞的胞浆中呈现高峰.实验结果提示, CRT在妊娠早期子宫内膜的持续表达, 可能通过调节整合素介导的细胞信号通路而调节胚胎滋养层细胞的黏附、侵袭, 参与胚胎着床.     

前蛋白转换酶Furin和PC7在小鼠母胎界面的动态表达及其在胚胎粘附和扩展中的作用

通过免疫组化、免疫荧光和小鼠胚胎-子宫内膜上皮细胞共培养体系,研究了前蛋白转换酶Proprotein Convertases (PCs)家族中的Furin和PC7在小鼠妊娠早期子宫和胚胎中的表达及对胚胎植入的影响.结果显示:Furin和PC7在妊娠D1-D4小鼠子宫的腺上皮和D5-D7小鼠子宫的蜕膜、腺上皮高表达;PC7在植入前胚胎的2-细胞和4-细胞期表达很低,8-细胞期表达开始增加,囊胚期滋养外胚层有显著的高表达.Furin的抑制剂Dec-RVKR-CMK可显著抑制共培养体系中胚胎的粘附和扩展.以上结果表明,Furin在植入期胚胎的粘附和扩展中发挥重要作用.此外,Furin和PC7可能参与子宫内膜蜕膜化和早期胚胎发育.     

Caveolin-1在围植入期小鼠子宫内膜组织中的动态变化

目的检测caveolin-1在胚胎植入过程中小鼠子宫内膜组织中的表达,探讨其在胚胎植入过程中的作用。方法选择成年雌性昆明小白鼠42只,随机均分为7组(处于动情期的未孕组、妊娠3.5天组、妊娠4.5天组、妊娠5.5天组、妊娠6.5天组、妊娠7.5天组、妊娠9天组),采用免疫组织化学和RT-PCR方法检测子宫内膜组织中caveolin-1蛋白及mRNA水平在围植入期的变化。结果 (1)caveolin-1在胚胎植入前期(0d、3.5d)小鼠子宫内膜组织中的表达高于胚胎植入期(4.5d、5.5d、6.5d),差异有显著性(P<0.05)。(2)caveolin-1在胚胎植入后期(7.5d、9d)小鼠子宫内膜组织中的表达高于胚胎植入期(4.5d、5.5d、6.5d),差异有显著性(P<0.05)。(3)caveolin-1在胚胎植入后期小鼠子宫内膜组织中的表达略高于胚胎植入前期,但差异无显著性(P>0.05)。结论 Caveolin-1在胚胎植入前期和后期均高表达,植入期低表达。这种变化提示caveolin-1是影响胚胎植入的重要因素之一。     

斯钙素与哺乳动物生殖的研究进展

斯钙素(stanniocalcin,STC)是一类首先在硬骨鱼类发现的糖蛋白激素,其生理作用在于调节钙磷稳态。近年来在入和哺乳动物组织中也发现存在有STC,并且以旁分泌方式参与机体的多种生理功能。本文综述了近年来哺乳动物胚胎发育、妊娠及哺乳期间STC基因在卵巢内的表达,早期胚胎植入过程中STC基因在子宫内膜的表达,以及人绒毛膜促性腺激素(hCG)对STC分泌的调节作用及其作用机制。研究表明,新生个体的STC基因表达首先出现在卵巢的膜细胞内,随后出现于卵巢基质的间质细胞和内膜细胞;但STC基因的表达产物STC却聚积于卵母细胞和黄体细胞内,提示STC参与卵母细胞的成熟过程。胚胎植入、妊娠、分娩与哺乳期间卵巢内STC基因表达量的动态变化行为,提示STC在哺乳动物生殖过程的各个环节均发挥一定的作用。     

早孕小鼠子宫内膜mTOR基因表达增高

利用实时荧光定量PCR、原位杂交法、免疫组化(SP法)和Western印迹法分别检测未孕、假孕及孕d3、d4、d5、d6、d7小鼠子宫内膜哺乳动物雷帕霉素靶蛋白(mammalian tar get of rapamycin,mTOR)mRNA和蛋白质的表达,研究mTOR基因在早孕小鼠子宫内膜的表达规律.结果显示妊娠子宫内膜组织mTOR的表达较未妊娠的子宫内膜组织显著增加(P＜0.05);着床窗期表达最高(d4,d5),且与其他妊娠各期比较差异有统计学意义(P＜0.05);原位杂交和免疫组化分析显示mTOR mRNA和蛋白质表达主要在子宫内膜基质细胞,与上皮细胞各组比较差异有统计学意义(P＜0.05).研究表明mTOR在子宫内膜基质细胞的规律性表达所调控的细胞生长、增殖和分化可能是胚胎正常着床的分子机制之一.     

MK基因对小鼠子宫基质细胞蜕膜化进程的影响

为探索肿瘤坏死因子相关凋亡诱导配体(TNF related apoptosis inducing ligand,TRAIL)的死亡受体(mouse killer,MK)对小鼠子宫基质细胞蜕膜化进程的影响,构建MK基因过表达和siRNA干扰重组腺病毒.原代培养的小鼠子宫基质细胞感染MK过表达或者干扰重组腺病毒并诱导蜕膜化,72 h后用免疫细胞化学与流式细胞术分别检测蜕膜细胞的标志物催乳素(prolactin,PRL)与蜕膜细胞凋亡率的变化情况.妊娠d4小鼠子宫角注射MK重组腺病毒,观察胚胎植入点的数量变化.实验结果表明,与对照组相比,在诱导的蜕膜细胞中过表达MK使得催乳素的含量显著降低(P<0.05),同时,蜕膜细胞的凋亡率明显升高(P<0.05),而siRNA干扰之后催乳素的含量显著升高,凋亡率明显下降(P<0.05),但是,宫角注射MK基因过表达和siRNA干扰重组腺病毒之后,胚胎植入数量均显著减少(P<0.01).提示MK基因通过参与小鼠子宫内膜基质细胞的蜕膜化进程,调节蜕膜细胞增殖与凋亡之间的平衡从而影响胚胎的植入.     

CT感染大鼠子宫内膜植入窗期基质金属蛋白酶的表达

目的 通过大鼠生殖道沙眼衣原体（chlamydia trachomatis,CT）感染模型研究CT初次感染后妊娠大鼠在子宫内膜植入窗期基质金属蛋白酶-9（MMP-9）及金属蛋白酶组织抑制物-1（TIMP-1）的表达对胚胎着床的影响。方法选择成年雌性SD大鼠30只,随机均分为对照组和感染组,感染组通过阴道接种CTD型株,而后在妊娠大鼠的植入窗期（即妊娠第5、6、7天）分别处死大鼠,采用链菌生物素蛋白一过氧化酶连接法和免疫组化测定两组子宫内膜种植窗期MMP-9及TIMP-1。采用HPIAS-1000高清晰度彩色病理图文小报告管理系统测定感染组和对照组中MMP-9＼T1MP-1在单位面积中各项免疫组织化学反应阳性颗粒的平均光密度值,并通过SPSS软件对数据差异显著性进行分析。结果在两组大鼠的子宫内膜中MMP-9均呈阳性表达。腺上皮细胞、腔上皮细胞和基质细胞胞浆内均可见棕黄色颗粒沉着,上皮细胞染色强度大于基质;感染组的表达较对照组弱,差异有显著性（P〈0．05）。TIMP-1表达特点和MMP-9相似,感染组的表达也较对照组弱,差异有显著性（P〈0．05）。结论生殖道感染CT后,妊娠大鼠子宫内膜植入窗期MMP-9及TIMP-1的低表达,可能是影响胚胎着床、导致不孕的重要因素。     

G0101C03-3和L0254H10-3 基因在小鼠动情周期子宫中的差异表达

正常动情周期的维持是小鼠子宫多功能的必要条件 ,但对于其分子基础至今尚不十分明了。我们曾通过基因芯片技术分析了小鼠动情期与间情期子宫的基因表达谱 ,发现了许多差异表达的基因或表达序列标签(ESTs)。本实验选取了G0 1 0 1C0 3 3及L0 2 5 4H1 0 3两个差异表达的EST ,通过Northern印迹与原位杂交方法分析了它们在小鼠动情周期子宫中的时空表达模式。结果表明 :这两个基因的表达水平都发生周期性变化 ,在动情期表达量较少 ,而在间情期表达量较高 ;G0 1 0 1C0 3 3在动情期的表达量是间情期的 2 7% ,L0 2 5 4H1 0 3在动情期检测不到有表达 ,提示它们的表达受卵巢类固醇激素的调控 ;G0 1 0 1C0 3 3基因主要在子宫的腔上皮与腺上皮中表达 ,可能与子宫细胞的程序性死亡有关 ;而L0 2 5 4H1 0 3基因则主要位于基质细胞中     

KAI1/CD82 在早孕小鼠子宫内膜组织的表达研究

目的：观察KAI1／CD82 mRNA和蛋白在小鼠妊娠D1-D8子宫内膜组织的表达。方法：以胚胎与肿瘤同源性为理论基础,胚胎植入与肿瘤侵袭转移相似为切入点,采用免疫组化和RT-PCR技术。结果：KAI1／CD82 mRNA和蛋白在早孕子宫中,KAI1／CD82mRNA的表达渐增多,蛋白表达的量和范围也渐增强。结论：KAI1／CD82mRNA和蛋白在早孕子宫组织中的动态表达,提示它在胚胎精确侵袭子宫内膜的调节中发挥作用,是滋养层细胞精确侵袭调控的分子机制之一。     

环状RNA circCapzb在早孕小鼠围植入期子宫内膜中的表达

环状RNA(circular RNA,circRNA)是一类新型内源性非编码RNA,与多种疾病的发生、发展密切相关,但在胚胎着床的过程中罕见报道。该文旨在探讨环状RNA circCapzb在早孕小鼠围植入期子宫内膜中的表达。采用Real-time PCR检测正常妊娠小鼠孕第5天(d5)至第7天(d7)胚胎着床点及胚胎着床旁组织中circCapzb的表达水平;分别构建小鼠体内人工诱导蜕膜化模型和原代小鼠子宫内膜基质细胞体外人工诱导蜕膜化模型,采用Real-time PCR分别检测circCapzb在组织及细胞蜕膜化诱导模型中的表达;通过生物信息学预测circCapzb下游靶miRNA:miR-377-3p和miR-7005-5p,并采用Real-time PCR检测其在蜕膜化诱导模型中的表达。结果表明,circCapzb在小鼠孕第5天至第7天胚胎着床点的表达明显高于着床旁;circCapzb在组织及体内外细胞蜕膜化诱导模型中诱导组的表达明显高于未诱导组(对照组);circCapzb下游靶miR-377-3p和miR-7005-5p在组织及体内外细胞蜕膜化诱导模型中诱导组的表达明显低于未诱导组。该研究初步表明,circCapzb在小鼠早孕期胚胎着床点高表达,在组织及体内外细胞蜕膜化诱导模型中高表达,在小鼠妊娠早期子宫内膜蜕膜化过程中可能发挥作用,但具体机制有待进一步研究。     

富含脯氨酸小蛋白-2在小鼠子宫中的表达及调节

富含脯氨酸小蛋白(Sprrs)参与构建复层扁平上皮的角化细胞壳（CE）,它们在子宫单层上皮中的作用还不清楚.采用RNA印迹和半定量RT-PCR方法,研究了Sprr2在小鼠动情周期和妊娠子宫中的表达及其激素调控.实验结果发现:Sprr2在动情前期和动情期表达上调,而动情后期和间情期表达下调.在妊娠初期表达迅速下调,直至临产期表达重新受到诱导并在产后达到高峰.鉴于其在不同生殖阶段子宫中独特的表达模式和在复层上皮中保护性的功能,推测Sprr2与子宫对交配和分娩所产生的应激反应有关.     

Estrogen receptor mRNA in uteri of normal estrous cycling and ovariectomized rats by in situ hybridization.

Biological effects of estrogen are mediated via its binding to the estrogen receptor (ER), the contents of its protein and mRNA varying during the estrous cycle. In the present study, the ERalpha mRNA expression in different cell components of the uterus was investigated in normal estrous cycling rats using nonisotopic in situ hybridization. Additionally, ovariectomized (OVX) rats treated with 17beta-estradiol (E2: 5 microg/kg, sc injection daily) were also investigated to clarify the effects of exogenous E2. At proestrus and diestrus, and especially the former, the luminal and glandular epithelial (LE and GE) cells were strongly positive, along with stromal cells beneath the luminal epithelium. At estrus, the expression was slightly diminished in LE cells, but almost completely lacking in GE cells. At metestrus, positive signals appeared again in GE cells. In the myometrium, ER mRNA was demonstrated to be constantly positive in all estrous cycle stages. OVX rat uteri underwent marked atrophy, but ER mRNA still remained in all cell types. After 2 consecutive days of E2 treatment, markedly increased intensity was observed, especially in LE and GE cells. The uteri of OVX rats treated with E2 for 14 days, however, showed slightly diminished expression, whereas the serum concentration of E2 was comparable to that in rats after 2 days. These results provide evidence that cell-type specific patterns of ER mRNA expression characterize the uteri of both normal estrous cycling rats and OVX rats after estrogen treatment.     

Expression of cyclooxygenases 1 and 2 and prostaglandin E synthase in bovine endometrial tissue during the estrous cycle

In ruminants, endometrial prostaglandin F(2alpha) (PGF(2alpha)) is responsible for luteolysis and prostaglandin E(2) (PGE(2)) is thought to be involved in maternal recognition of pregnancy. In the present study, healthy uteri were collected from cows at the abattoir, and days of the estrous cycle were determined macroscopically. The uteri were classified into seven groups as Days 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, and 19-21 of the estrous cycle. Endometrial scrapings were collected. The expression of cyclooxygenase (COX)-1 and COX-2 mRNAs and proteins and PGE synthase (PGES) mRNA was analyzed by Northern and Western blot. There was no expression of COX-1, either mRNA or protein, on any day of the estrous cycle. In contrast, COX-2 mRNA and protein were expressed at low and high levels on Days 1-12 and 13-21 of the estrous cycle, respectively. The level of expression of PGES was moderate, low, and high on Days 1-3, 4-12, and 13-21 of the estrous cycle, respectively. There were significant correlations between COX-2 mRNA and protein levels and between COX-2 and PGES mRNA levels. COX-1 mRNA and protein are not expressed on any day of the estrous cycle, whereas COX-2 mRNA and protein and PGES mRNA are differentially expressed and regulated in bovine endometrium during the estrous cycle. COX-2, rather than COX-1, is the primary isoenzyme involved in the endometrial production of prostaglandins, and the COX-2 and PGES pathway is responsible for the endometrial production of PGE(2) in the bovine endometrium during the estrous cycle.     

Expression and hormonal regulation of calcyclin-binding protein (CacyBP) in the mouse uterus during early pregnancy

Calcyclin-binding protein (Siah-1-Interacting Protein, CacyBP/SIP), is a calcium signaling protein involved in the degradation of beta-catenin, however, little is known about its role in reproductive biology. The present study was to character its temporospatial expression pattern and regulation in mouse uterus and to investigate whether it plays a role in the regulation of normal endometrial events. While prominently expressed in both luminal and glandular epithelia, CacyBP underwent dynamic changes during early pregnancy. CacyBP expression was observed weakly from days 1-4. An intense accumulation in luminal and glandular epithelia as well as decidua surrounding the embryo at later stages (days 5-7) was observed. Most notably, CacyBP accumulation in trophoblast was pronounced at day 7. Using ovariectomized and pseudopregnant mice, we found that progesterone (P(4)) and 17beta-estradiol (E(2)) led to increased expression of CacyBP gene and this could be abolished by Ru486 and tamoxifen, respectively. Antisense oligonucleotides (ODNs) against CacyBP significantly inhibited cultured endometrial stromal cells' (ESCs) apoptosis induced by UV irradiation. Injection of antisense ODNs into mouse uterine horn severely impaired the number of implanted blastocysts. Taken together, our results suggested that CacyBP expression was positively regulated by P(4) and E(2). CacyBP may be involved in the regulation of endometrial cell apoptosis during early pregnancy and play an important role in mouse endometrial events such as pregrancy establishment.     

Regulation of cyclic guanosine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.     

Expression of heparin/heparan sulfate interacting protein/ribosomal protein l29 during the estrous cycle and early pregnancy in the mouse

Using a variety of approaches, we have examined the expression of the heparin/heparan sulfate (Hp/HS) interacting protein/ribosomal protein L29 (HIP/RPL29) in mouse uteri during the estrous cycle and early pregnancy. HIP/RPL29 selectively binds heparin and HS and may promote HS-dependent embryo adhesion. HIP/RPL29 was prominently expressed in both luminal and glandular epithelia under almost all conditions, including the phase of embryo attachment. In contrast, differences were noted in HIP/RPL29 expression in the stromal compartment both during the estrous cycle and during early pregnancy. Most notably, HIP/RPL29 accumulated in decidua, where it displayed a pattern complementary to that of pericellular deposition of the HS proteoglycan, perlecan. HIP/RPL29 protein was detected in implanted embryos at both initial and later stages of implantation; however, embryonic HIP/RPL29 mRNA accumulation was more pronounced at later stages (Day 7.5 postcoitum). In situ hybridization revealed similar spatial changes for HIP/RPL29 mRNA during these different physiological states. Whereas differences in the spatial pattern of HIP/RPL29 protein and mRNA expression were demonstrable, little change was detected in the level of HIP/RPL29 mRNA or protein in total endometrial extracts. Mouse blastocysts attached, but did not outgrow, on surfaces coated with recombinant murine HIP/RPL29. Surprisingly, soluble glycosaminoglycans including heparin, low molecular weight heparin, or chondroitin sulfate were not able to inhibit embryo attachment to HIP/RPL29-coated surfaces. These latter observations indicate that embryonic cell surface components other than HS proteoglycans can promote binding to HIP/RPL29 expressed by uterine cells.