Nupr1 mRNA在小鼠早期妊娠子宫中的表达

试验旨在研究核蛋白1(nuclear protein 1,Nupr1)mRNA在小鼠早期妊娠子宫中的表达,探讨Nupr1与小鼠胚胎着床的相关性。通过建立小鼠早期妊娠模型、假孕妊娠模型、延迟着床及激活模型、人工蜕膜化模型和激素处理模型,采用原位杂交的方法检测Nupr1mRNA在小鼠各种模型子宫组织中的定位表达情况,并应用实时荧光定量PCR法检测早期妊娠模型和假孕妊娠模型中Nupr1mRNA的相对表达量。结果显示,Nupr1mRNA在小鼠早期妊娠第1~4天子宫的腔上皮和腺上皮表达,第5~8天表达于蜕膜区域;假孕妊娠第1~5天,Nupr1mRNA主要表达于小鼠子宫腔上皮和腺上皮;延迟着床模型中信号表达于在小鼠子宫的腔上皮和腺上皮,与正常妊娠第4天结果相似;延迟激活模型中信号表达于蜕膜区,与早期妊娠第5天表达结果相似;人工蜕膜化模型中信号表达于蜕膜区,而蜕膜对照组中信号表达于腔上皮和腺上皮;17β-雌二醇(oestrogen,E2)处理组信号表达于腔上皮和腺上皮,信号增强,孕酮(progesterone,P4)和E2共同处理表达无明显变化;实时荧光定量PCR结果显示,正常妊娠第2天Nupr1mRNA相对表达量较高,假孕妊娠第2天Nupr1mRNA相对表达量也较高。本研究结果表明,Nupr1mRNA在小鼠子宫中的表达与小鼠早期妊娠过程相关,Nupr1mRNA在腔上皮和腺上皮的表达可能受激素调节,在子宫基质中的表达与蜕膜化及活化胚泡相关。     

SerpinB6b在大鼠着床期子宫及蜕膜的表达与调控

SerpinB6b是丝氨酸蛋白酶抑制剂亚家族B成员之一。本实验旨在利用荧光定量PCR、原位杂交、免疫组织化学与蛋白质印迹分析等实验技术,研究SerpinB6b mRNA和蛋白在大鼠早期妊娠1~9 d、人工诱导蜕膜化及体外诱导蜕膜化模型子宫中的表达规律。结果显示:SerpinB6b mRNA和蛋白在大鼠早期妊娠1~5 d腔上皮、腺上皮有微弱表达;在6~9 d,表达主要集中在胚胎和蜕膜区,且随蜕膜化程度增加,表达量逐渐增强,且在第8天达到最高;在大鼠人工诱导蜕膜化模型中,SerpinB6b在蜕膜区的表达均显著高于非蜕膜区;在大鼠子宫内膜基质细胞体外诱导蜕膜化模型中,SerpinB6b随着诱导蜕膜化天数的增加呈上升趋势。以上体内和体外实验结果表明,SerpinB6b在大鼠子宫中的表达具有着床相关特异性,SerpinB6b参与大鼠子宫蜕膜化过程。     

Elastin基因在小鼠早期妊娠过程中的表达

弹性蛋白(Elastin)是一种关键的细胞外基质蛋白,对大动脉、肺、韧带、肌腱、皮肤和弹性软骨等许多脊椎动物组织的弹性和复原力至关重要。本实验旨在利用原位杂交、荧光定量PCR方法,研究Elastin mRNA在小鼠早期妊娠、假孕及人工诱导蜕膜化模型子宫中的表达。结果显示:在小鼠早期妊娠1~4d子宫中未检测到ElastinmRNA表达;随着妊娠进行,在妊娠第5天子宫肌层检测到ElastinmRNA微弱表达;在妊娠第6天的子宫壁肌层与第7、8天的肌层及蜕膜区Elastin mRNA表达逐渐增强。在小鼠假孕1~5 d子宫中,ElastinmRNA不表达。在人工诱导蜕膜化模型中,ElastinmRNA在子宫壁肌层及系膜侧蜕膜区均有表达。以上表明Elastin可能参与小鼠早期妊娠子宫壁肌层弹性的调控与蜕膜化过程。     

甘氨酰tRNA合成酶在小鼠围着床期子宫及蜕膜化过程的时空特异性表达

核编码的甘氨酰-tRNA合成酶(Glycyl-tRNA Synthetase, GlyRS)是细胞质和线粒体蛋白质翻译所必需的,GlyRS除了在蛋白翻译中的作用外,它还参与基因转录、炎症和细胞增殖等其他生物学过程。本研究从mRNA水平和蛋白水平检测了GlyRS在小鼠早期妊娠1～8 d模型、人工诱导蜕膜化模型和体外诱导人子宫内膜基质细胞蜕膜化模型中的表达规律。结果表明:GlyRS mRNA和蛋白在小鼠早期妊娠1～4 d的腔上皮和腺上皮中表达,在小鼠早期妊娠5～8 d主要在胚胎和蜕膜区表达;随着蜕膜化程度增加,表达量逐渐增加;在人工诱导蜕膜化模型中,诱导组GlyRS mRNA和蛋白表达情况类似,表达量均明显高于对照组。在体外诱导的人子宫内膜基质细胞蜕膜化模型中,诱导组GlyRS mRNA表达量随着诱导天数的增加而增加,均显著高于对照组。以上结果表明GlyRS参与小鼠早期妊娠及子宫蜕膜化的过程。     

缝隙连接蛋白43对小鼠子宫基质细胞蜕膜化的影响

缝隙连接蛋白43(connexin 43,Cx43)是缝隙连接蛋白家族成员之一,广泛存在于哺乳动物器官和组织中,参与细胞增殖、分化、凋亡等生理过程。本试验参照Gen Bank中已发表的Cx43基因序列,设计特异性引物,进行PCR扩增。为了提高构建过表达重组质粒的效率,PCR产物先与p GEM-T载体进行连接,经双酶切后与pc DNA3.1载体融合得到过表达重组质粒,经双酶切和测序鉴定后将其转染到体外分离培养的小鼠子宫基质细胞中,利用实时荧光定量PCR(qRT-RCR)方法检测Cx43 mRNA表达变化及Cx43过表达对蜕膜化标志性分子表达的影响。结果表明,与空载体转染组相比,重组质粒pc DNA3.1-Cx43转染子宫基质细胞后可使小鼠子宫基质细胞Cx43 mRNA的表达量显著升高,同时Cx43过表达可使子宫基质细胞蜕膜化标志性分子Prl8a2和Prl3c1 mRNA的表达量均显著升高,表明Cx43可促进小鼠子宫基质细胞发生蜕膜化。     

Dnm1l mRNA在小鼠早期妊娠子宫中的表达研究

为了研究Dnm1l mRNA在小鼠胚胎着床过程中的表达,试验采用原位杂交方法检测早期妊娠、假孕、人工蜕膜化、延迟着床及激活等小鼠模型中Dnm1l mRNA的表达,并应用Real-time PCR检测早期妊娠模型中Dnm1l mRNA的相对表达量。结果表明:Dnm1l mRNA在小鼠早期妊娠的子宫中呈现时空特异性表达,Real-time PCR检测表达的整体趋势变化与原位杂交试验中观测到的结果基本一致。说明Dnm1l mRNA与小鼠早期妊娠过程相关,其表达可能受到着床胚泡的调节。     

BDNF与TrkB蛋白在小鼠子宫和着床前胚胎中表达规律的研究

为了研究BDNF与TrkB蛋白在小鼠发情周期和妊娠早期子宫、输卵管及着床前胚胎中的表达规律,试验采用免疫组织化学方法检测BDNF与TrkB蛋白在子宫和输卵管中的表达,采用免疫荧光技术检测着床前不同发育阶段胚胎中蛋白的表达,并利用图像分析软件对两种蛋白在着床前胚胎中的表达强度进行定量分析。结果表明:BDNF与TrkB蛋白在发情周期及妊娠3.5天和4天子宫腔上皮、子宫腺上皮和血管内皮细胞中表达,在发情周期子宫内膜固有层基质细胞中不表达,但在妊娠3.5天和4天子宫蜕膜细胞中表达,发情前期、发情期和发情后期BDNF蛋白的表达强度高于发情间期;BDNF与TrkB蛋白在妊娠2天输卵管黏膜上皮和血管内皮细胞中表达;两种蛋白在着床前胚胎各发育阶段都有表达,在囊胚期达到最高水平。说明BDNF和TrkB蛋白通过旁分泌或自分泌途径对子宫、输卵管尤其是着床前胚胎发育发挥调控作用。     

FZD5在山羊妊娠早期的表达及激素调控

实验旨在研究雌性山羊早期妊娠和发情周期中 Frizzled-5(FZD5)蛋白在子宫中的表达,以及类固醇激素对 FZD5 的表达调控。选取发情周期、早期妊娠及雌激素(50 μg/mL)与孕酮(50 ng/mL)处理山羊的子宫组织,采用免疫组织化学染色、荧光定量 PCR和 Western blot检测 FZD5在山羊子宫中的表达规律。结果表明:FZD5 蛋白在山羊妊娠早期的子宫腔上皮和腺上皮中表达;FZD5 mRNA 和蛋白在胚胎与子宫的黏附前(D6)和黏附中(D16)表达较高,在黏附后(D19)和胎盘形成早期(D25)呈下降趋势;孕酮处理导致FZD5表达降低,表明山羊子宫中孕酮下调 FZD5的表达。研究提示FZD5可能在山羊胚胎着床过程中发挥作用。     

Hmgn3和Hoxa10对小鼠子宫基质细胞蜕膜化过程中Foxo1表达的调控

利用小鼠子宫基质细胞体外诱导蜕膜化模型转染Hmgn3或Hoxa10过表达载体及siRNA片段,通过荧光定量PCR方法检测Hmgn3和Hoxa10对Foxo1表达的调控。结果显示:转染Hmgn3或Hoxa10过表达载体可促进Foxo1在子宫基质细胞蜕膜化过程中的表达,而转染Hmgn3或Hoxa10siRNA则可抑制Foxo1的表达。在小鼠子宫基质细胞中转染Hmgn3或Hoxa10siRNA后再添加孕酮,Foxo1的表达显著下降。同样,干扰Hmgn3或Hoxa10也可减低cAMP对Foxo1表达的调控。结果表明:Hmgn3和Hoxa10可通过Foxo1来影响小鼠子宫基质细胞的蜕膜化,孕酮和cAMP可通过Hmgn3和Hoxa10来调控Foxo1在子宫基质细胞中的表达。     

妊娠早期蒙古绵羊子宫内膜enJSRV及IFN-τ基因表达水平的分析

本研究旨在检测内源性绵羊肺腺瘤反转录病毒(enJSRV)与干扰素-τ(IFN-τ)在妊娠早期蒙古绵羊子宫内膜组织的表达以及enJSRV的表达与外周血孕酮水平变化的关系。运用TaqMan实时荧光定量PCR技术和电化学发光法对enJSRV和IFN-τ在妊娠早期蒙古绵羊子宫内膜组织相对表达及孕酮水平进行了测定。实时荧光定量PCR结果显示,enJSRV和IFN-τmRNA在妊娠早期绵羊子宫内膜组织有不同程度的表达。SAS统计学软件分析得出,子宫内膜组织中enJSRV mRNA在妊娠12～14d(交配日为0d)表达较高,2～10、16～30d的表达均低于前者。IFN-τmRNA仅在妊娠12～25d表达,14d达其峰值,30d就不能检出,且差异都极显著(P<0.01)。电化学发光法结果显示,孕酮水平在妊娠2d为0.4ng·mL-1,以后升高,妊娠8～16d维持在8.3ng·mL-1左右,在妊娠19～30d孕酮水平有所下降,30d为2.5ng·mL-1。以上结果提示,子宫内膜组织中enJSRV mRNA的表达与外周血孕酮含量及IFN-τ的变化高度相关,且enJSRV在胎盘的形态发生及生殖生物学方面发挥重要作用。     

Expression of Nupr1 mRNA in Mouse Uterus During Early Pregnancy

The test was aimed to study the expression of nuclear protein 1 (Nupr1) mRNA in mouse uterus during early pregnancy.The method of in situ hybridization was used to investigate Nupr1 mRNA expression in animal models that included early pregnancy,pseudopregnancy,delayed implantation and activation,artificial decidualization and hormonal treatments.The relative expression level of Nupr1 mRNA was detected in early pregnancy and pseudopregnancy using Real-time PCR.During mouse early pregnancy,the signal of Nupr1 mRNA was detected in luminal epithelium and glandular epithelium during the 1st to 4th day and in the decidua area during the 5th to 8th day.Nupr1 mRNA was mainly expressed in the luminal epithelium and glandular epithelium of mose uterus on the 1st to 5th day of pseudopregnancy.The signal was detected in luminal epithelium and glandular epithelium of the mouse uterus in the delayed implantation,which was similar to the results of early pregnancy on the 4th day.The signal was detected in decidua in the model of delayed activation,which was similar to the results of early pregnancy on the 5th day.The expression of Nupr1 mRNA in the model of artificial decidualization was detected in decidua area.In the control of artificial decidualization the slight signal appeared in luminal epithelium and glandular epithelium of the mouse uterus.After treated with oestrogen (E₂) the signal appeared in luminal epithelium and glandular epithelium of the mouse uterus,and the signal was enhanced.After treated with both of E₂ and progesterone (P₄), the expression of the signal was not changed significantly.Real-time PCR result showed that the relative expression on the 2nd day was higher than other days in early pregnancy and pseudopregnancy.The results indicated that the expression of Nupr1 mRNA in mouse uterus was related to the process of mouse early pregnancy.The expression of signal in luminal epithelium and glandular epithelium of the mouse uterus might be regulated by hormones.Nupr1 mRNA expression in uterine stroma was associated with decidualization and active blastocysts.     

Expression and Hormonal Regulation of IL-11Rα in Canine Uterus During Early Pregnancy

Embryo implantation is critical for the successful establishment of pregnancy. Interleukin-11 (IL-11) is essential for adequate decidualization in the mouse and human via binding to the specific IL-11 receptor α (IL-11Rα). But the expression and regulation of IL-11 and IL-11Rα in the canine endometrium remain unknown. The aim of this study was to investigate the differential expression of IL-11Rα in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. Interleukin-11Rα mRNA was mainly localized in glandular epithelium in canine uterus. There was a low level of IL-11Rα expression in the glandular epithelium on days 6, 12 and 17 of pregnancy. On day 20 of pregnancy when embryo implanted, IL-11Rα mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium and stroma. On day 23 of pregnancy, the expression of IL-11Rα mRNA maintained a constant level compared with the expression of day 20 and increased on day 28 of pregnancy. During the oestrous cycle, a high level of IL-11Rα mRNA expression was seen in the oestrous uterus. Progesterone slightly induced the expression of IL-11Rα mRNA in the ovariectomized canine uterus. These results suggest that IL-11Rα expression is closely related to canine implantation and up-regulated by progesterone.     

Involvement of stathmin in proliferation and differentiation of immortalized human endometrial stromal cells

Uterine endometrial stromal cells differentiate into decidual cells during the late secretory phase of the menstrual cycle and pregnancy. However, the biochemical mechanisms of decidualization have yet to be definitively elucidated. In the present study, we transfected primary human endometrial stromal cell with a temperature-sensitive mutant of simian virus 40 large T antigen and thereby established an immortalized stromal cell line (EtsT) in order to examine the role of stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics, in stromal cell differentiation. When treated with the decidual stimulus dibutyryl-cAMP (db-cAMP) or forskolin, the fibroblastic cell-shaped EtsT cells transformed into large- and round-shaped cells and secreted large amounts of the decidual markers prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1). Analysis of the stathmin protein levels in the db-cAMP- and forskolin-treated EtsT cells revealed that the total and phosphorylated protein levels dropped as decidualization progressed. Suppression of stathmin expression by transfection with small interfering RNA (siRNA) suppressed EtsT cell proliferation. It also abolished db-cAMP-induced PRL and IGFBP-1 mRNA expression and protein secretion. Thus, stathmin expression can be considered an integral factor regulating the initial stage of the process of human endometrial stromal cell differentiation.     

Expression and Hormonal Regulation of Hoxa10 in Canine Uterus During the Peri-implantation Period

Hoxa10, a homeobox gene, is necessary for endometrial receptivity to blastocyst implantation. The aim of this study was to investigate the differential expression of Hoxa10 in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. Hoxa10 mRNA was mainly localized in glandular epithelium and myometrium in canine uterus. There was a low level of Hoxa10 expression in the glandular epithelium on days 6, 12 and 17 of pregnancy. On day 20 of pregnancy when embryo implanted, Hoxa10 mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium. The expression of Hoxa10 mRNA gradually declined from day 23 and reached a low level on day 28. In the myometrium, a low level of Hoxa10 mRNA signal was seen on days 6, 12 and 17 of pregnancy and reached a high level on day 20 of pregnancy. During the estrous cycle, a high level of Hoxa10 mRNA expression was seen in the estrous uterus. Either estrogen or progesterone significantly induced the expression of Hoxa10 mRNA in the ovariectomized canine uterus. These results suggest that Hoxa10 expression is closely related to canine embryo implantation and upregulated by estrogen and progesterone.     

Characterization of a population of unique granular lymphocytes in a bitch deciduoma, using a panel of histo- and immunohistochemical markers

The ovaries and uterus were collected after ovariohysterectomy from a 16-month-old Labrador bitch in diestrus that never mated. Discrete swellings were found in the uterine horns, with the macroscopic appearance of normal early pregnancy. At histologic examination, the endometrium, devoid of any conceptus and chorion, showed a marked proliferation, on the basis of which a diagnosis of deciduoma was made. A remarkable population of stromal eosinophilic granular lymphocytes was present, especially in the axis of the endometrial folds. Periodic acid-Schiff and Dolichos biflorus-lectin histochemical reaction and a panel of 10 immunohistochemical markers were used to characterize eosinophilic granular cells. Our findings allowed us to compare these granular cells with the granulated decidual cells, whose presence was until now described only in primates, rodents, or a few other epitheliochorial species. On the basis of our results, the importance of eosinophilic granular cells in a decidualization process is hypothesized to occur also in the bitch.     

Distribution of androgen receptor in the porcine uterus throughout pregnancy.

The uterus is a well-known target of endocrine, paracrine and autocrine acting molecules among which steroid hormones (oestrogens, androgens and progesterone) are of special importance. The uterine tissues (endometrium and myometrium) undergo morphological and physiological changes which are associated with changes in expression of steroid hormone receptors. Androgen receptors (AR) that mediate the action of androgens have already been detected in porcine uteri during the oestrous cycle and early pregnancy. To evaluate the role of AR in uterine physiology, the presence of ARmRNA and AR protein localization in the porcine uterus from day 10 to day 90 of pregnancy and in the uterus from the foetus of day 90 postcoitum (p.c.) and from the neonatal 1-day-old piglet was studied. ARmRNA was detected in the porcine endometrium up to day 18 p.c., while AR protein was detectable in glandular epithelium and stromal cells as through day 90 of pregnancy. AR was also detected in the myometrium on all investigated days of pregnancy; however, on day 90, the immunostaining was present only in a limited number of cells. AR immunostaining was clearly demonstrated in the uterus of the female foetuses on day 90 as well as in the uterus of 1-day-old piglets. The physiological relevance of this finding needs further elucidation.     

Expression of uterine sensitization-associated gene-1 (USAG-1) in the mouse uterus during the peri-implantation period

Rat uterine sensitization-associated gene-1 (USAG-1) mRNA is expressed in the uterus during the peri-implantation period, and its mRNA expression in uterine epithelial cells is highest on day 5 of pregnancy. On the other hand, since changes in USAG-1 mRNA expression in the mouse uterus are not seen during the estrous cycle, USAG-1 expression might be specifically regulated by embryonic factors rather than by the maternal environment. However, the expression pattern and function of USAG-1 in the mouse uterus have not been determined. Thus, we examined the tissue-specific USAG-1 mRNA expression in the uteri of ICR mice during peri-implantation using real-time quantitative PCR. Uterine tissues, such as the myometrium, luminal epithelium, and stroma, were collected by laser capture microdissection at 3.5-6.5 dpc. USAG-1 mRNA was expressed in the uteri of pregnant mice from 3.5 dpc to 6.5 dpc, and the highest level of expression was seen at 4.5 dpc (P<0.01). Significantly high USAG-1 mRNA expression was detected in the luminal epithelium at 4.5 dpc (P<0.05). The stroma and myometrium exhibited unchanged expression levels of USAG-1 mRNA at 3.5-5.5 dpc. USAG-1 mRNA was undetectable in blastocysts and implanting embryos. Expression of USAG-1 mRNA appears to be associated with blastocyst implantation to the luminal epithelium, suggesting that physiological or biochemical contact of the blastocyst to the uterus is required for USAG-1 expression.