Estrogen inhibits interleukin-18 mRNA expression in the mouse uterus

Interleukin-18 (IL-18) is a proinflammatory cytokine expressed in female reproductive organs in humans, rats and mice. The physiological roles of uterine IL-18 and the regulatory mechanisms of IL-18 gene expression are unclear. The present study aimed to clarify the effects of estradiol-17beta (E2) and progesterone (P4) on IL-18 mRNA expression in the mouse uterus. Distribution and expression levels of IL-18 mRNA were studied using an RNase protection assay. Expression of IL-18 mRNA was observed in all organs studied, including testes, ovaries and uteri. The uterine IL-18 mRNA level of estrous mice was higher than that of diestrous mice. E2 treatment (1, 5, 25 or 250 ng/mouse) decreased uterine IL-18 mRNA levels in ovariectomized mice dose-dependently. E2 treatment acutely decreased IL-18 mRNA levels 3 h after injection, but these levels returned to the initial level after 48 h. P4 treatment (1 mg/mouse) decreased uterine IL-18 mRNA levels after 12 h, but levels returned to the initial level after 48 h. Both uterine IL-18 and IL-18Ralpha mRNAs were detected in cultured endometrial epithelial and stromal cells. These results suggest that uterine IL-18 expression is reduced by sex steroid hormones and that IL-18 acts on endometrial cells in a paracrine or autocrine manner.     

Analyses of circRNA profiling during the development from pre-receptive to receptive phases in the goat endometrium

Background: Recent studies have revealed that noncoding RNAs play important regulatory roles in the formation of endometrial receptivity. Circular RNAs(circRNAs) are a universally expressed noncoding RNA species that have been recently proposed to act as miRNA sponges that directly regulate expression of target genes or parental genes.Results: We used Illumina Solexa technology to analyze the expression profiles of circRNAs in the endometrium from three goats at gestational day 5(pre-receptive endometrium, PE) and three goats at gestational day 15(receptive endometrium, RE). Overall, 21,813 circRNAs were identified, of which 5,925 circRNAs were specific to the RE and 9,078 were specific to the PE, which suggested high stage-specificity. Further analysis found 334 differentially expressed circRNAs in the RE compared with PE(P 0.05). The analysis of the circRNA-miRNA interaction network further supported the idea that circRNAs act as miRNA sponges to regulate gene expression.Moreover, some circRNAs were regulated by estrogen(E2)/progesterone(P4) in endometrial epithelium cell lines(EECs) and endometrial stromal cell line(ESCs), and each circRNA molecule exhibited unique regulation characteristics with respect to E2 and P4.Conclusions: These data provide an endometrium circRNA expression atlas corresponding to the biology of the goat receptive endometrium during embryo implantation.     

玉米赤霉烯酮诱导山羊子宫内膜基质细胞凋亡的研究

玉米赤霉烯酮(zearalenone,ZEN)是由镰刀菌产生的一种霉菌毒素,具有细胞毒性和雌激素活性,会对生殖系统、泌尿系统和消化系统等产生严重毒性影响.目前,ZEN对山羊子宫内膜基质细胞(endometrial stromal cells,ESCs)凋亡的潜在作用仍不清楚.本研究通过体外培养山羊永生化ESCs,采用C...     

山羊子宫内膜上皮细胞转染pCI—neo—hTERT质粒后的永生化

为获得大量的、具有高度同一性和表型功能正常的山羊子宫内膜上皮细胞（EEC）,本试验将含人端粒酶逆转录酶（hTERT）基因的真核表达质粒pCI—neo—hTERT导入原代山羊EEC中,筛选稳定转染的细胞克隆,进行表型鉴定,利用免疫细胞化学方法检测hTERT导入后细胞端粒酶活性的表达情况,探讨转染后细胞的生长特性。结果显示,转染后获得的阳性克隆细胞及其传代细胞呈明显铺路石状。与原代细胞形态一致,具有接触抑制性;角蛋白检测阳性;细胞具有较高的端粒酶活性,目前已传至50代仍稳定增殖;100nmol／L的雌二醇（E2）可促进该细胞的增殖;50,100nmol／L的孕酮（P4）可明显抑制该细胞的增殖。这表明hTERT导入山羊EEC后,可使其重建端粒酶活性,从而获得大量的、具有高度同一性和表型正常的细胞。     

Cloning and expression of goat interleukin-18 gene

We isolated and sequenced a 480 bp cDNA encoding mature goat interleukin-18 (gIL-18) from alveolar macrophages and splenocytes activated with LPS by RT-PCR. The gIL-18 gene was cloned into pET32a (+) vectors and sequenced. Nucleotide sequence of gIL-18 shares high homology with cattle. Fusional expression with pET32a (+) of gIL-18 of about 38kD was obtained by SDS-PAGE analysis after induction by IPTG in the E. Coli BL21 expression system. The recombinant protein can induce IFN-gamma production in PBMC. The IL-18 mRNA was constitutively detected in goat alveolar macrophages with or without LPS, While, enhanced expression was detected in splenocytes and liver cells if treated by LPS, and can be weakly detected in Peripheral blood mononuclear cells (PBMCs) treated by activators. Significant deference of IL-18 mRNA level may reflect the capacity to produce mature IL-18 in such tissues.     

Regulation of endometrial granulocyte macrophage-colony stimulating factor (GM-CSF) in the ewe

Granulocyte macrophage-colony stimulating factor (GM-CSF) increases ovine interferon-tau (oIFNtau) secretion by ovine conceptuses, but endometrial production of GM-CSF has not been characterized. Endometrial GM-CSF expression was evaluated in ovariectomized ewes implanted with estradiol-17beta (E(2)) and/or progesterone (P(4)) for 14 days, in day 14 cyclic and day 14 pregnant ewes. Relative levels of endometrial GM-CSF mRNA were 3-fold higher in E(2)- and E(2)/P(4)-treated ewes than that of control or P(4)-treated ovariectomized ewes. Levels of endometrial GM-CSF mRNA for cyclic ewes were similar to E(2)- and E(2)/P(4)-treated ewes, but amounts of GM-CSF mRNA in pregnant ewes were 2-fold higher. GM-CSF concentrations in endometrial culture media, determined by GM-CSF bioassay, for cyclic and E(2)/P(4)-treated ovariectomized ewes were 3-fold higher than those of control, E(2)- and P(4)-treated ovariectomized ewes; however, amounts of GM-CSF in pregnant ewes were 2-fold higher. Immunoreactive GM-CSF, examined by western blot, was detected in the culture medium from E(2)/P(4)-treated ovariectomized, cyclic and pregnant ewes. Luminal and glandular epithelia and stromal regions were determined to be sites of GM-CSF expression by immunohistochemistry and in situ hybridization techniques. Data indicate that combined E(2) and P(4) treatment of ovariectomized ewes is sufficient to restore GM-CSF expression to the level found in cyclic ewes; however, GM-CSF mRNA and protein in pregnant ewes is 2-fold greater than in ovariectomized or cyclic ewes. These data suggest that the conceptus, in addition to steroids, may play a role in the regulation of endometrial production of GM-CSF.     

Regulation by gonadal steroids of estrogen and progesterone receptors along the reproductive tract in female lambs.

The regulation of estrogen and progesterone receptor (ER, PR) expression by estradiol (E2) and progesterone (P4) in the oviduct, uterus and cervix of female lambs was studied. The animals received three intramuscular injections of E2, P4 or vehicle with an interval of 24 h and they were slaugthered 24 h after the third injection. Determinations of ER and PR were performed by binding assays and mRNAs of ER alpha and PR by solution hybridization. High levels of ER and PR in both cervix and oviduct were found in the female lamb, differing from other mammalian species. No significant effects by either E2 or P4 treatment on ER and PR levels in the cervix and oviduct could be observed. E2 treatment increased the mRNA levels of ERa and PR more than 3-fold in the cervix, while P4 treatment increased the mRNA levels of ERa and PR in the uterus. The results show differential effects of gonadal steroids on sex steroid receptor expression along the reproductive tract in female lambs, suggesting that steroid target tissues can modulate responses to the same circulating levels of steroid hormones.     

山羊子宫内膜基质细胞对上皮细胞IL-18分泌活性的调节作用

为了探究性腺激素作用下山羊子宫内膜上皮细胞（EEC）中白细胞介素-18（IL-18）的分泌活性,以及子宫内膜基质细胞（ESC）对其分泌活性的调节作用。基于套皿共培养永生化山羊EEC和ESC为体外研究模型,通过ELISA和Western blot方法检测E2和/或P4对EEC中IL-18分泌水平的作用以及ESC对该作用的影响。ELISA结果显示,E2较无激素对照组可显著增强单独培养EEC中IL-18的分泌水平（P〈0.05）,P4单独或与E2联合作用则进一步增强其分泌活性（P〈0.01）;EEC与ESC共培养模式下,ESC细胞对性激素作用下EEC中IL-18分泌活性具有显著的抑制作用。Western blot结果显示,在单独培养或与ESC共培养模式下,EEC培养上清液中IL-18均以相对分子质量大小18 300的具有生物活性的蛋白形式存在。本研究结果表明,山羊ESC对性腺激素作用下EEC中IL-18的分泌活性具有重要调节作用。     

miR‐182 selectively targets HOXA10 in goat endometrial epithelium cells in vitro

Proper HOXA10 expression was essential for endometrial receptivity what was crucial for successful embryo implantation in mammalian. This study confirmed that miR‐182 regulated the expression levels of HOXA10 by binding to its 3′ UTR, selectively downregulated HOXA10 in goat endometrial epithelium cells (gEECs) but not stromal cell (gESCs) in vitro. However, HOXA10 and miR‐182 both up‐expressed in the goat endometrium at gestational day 15 (D15) compared with gestational day 5 (D5), suggesting that there were some other factors regulated the expression of HOXA10 during the development of goat endometrium in vivo. What's more, HOXA10 gene silencing (HOXA10‐siRNA) resulted in gEECs apoptosis in vitro, and it regulated the protein levels of oestrogen receptor a (ERa), progesterone receptor B (PRb), insulin‐like growth factor 1 receptor (IGF1R), BCL‐2, pleiotrophin (PTN), AKT and p‐JNK in gEECs. Furthermore, HOXA10 might regulate the protein levels of endometrial receptivity biomarker genes, including vascular endothelial growth factor (VEGF), osteopontin (OPN), cyclooxygenase‐2 (COX‐2) and prolactin receptor (PRLR) in gEECs. In conclusion, miR‐182 targeted HOXA10 selectively in EECs in vitro, and HOXA10 played an important role in maintaining the function of EECs in dairy goats.     

山羊子宫内膜上皮细胞转染pCI-neo-hTERT质粒后的永生化

为获得大量的、具有高度同一性和表型功能正常的山羊子宫内膜上皮细胞(EEC),本试验将含人端粒酶逆转录酶(hTERT)基因的真核表达质粒pCI-neo-hT ERT导入原代山羊EEC中,筛选稳定转染的细胞克隆,进行表型鉴定,利用免疫细胞化学方法检测hTERT导入后细胞端粒酶活性的表达情况,探讨转染后细胞的生长特性。结果显示,转染后获得的阳性克隆细胞及其传代细胞呈明显铺路石状,与原代细胞形态一致,具有接触抑制性;角蛋白检测阳性;细胞具有较高的端粒酶活性,目前已传至50代仍稳定增殖;100nmol/L的雌二醇(E2)可促进该细胞的增殖;50,100nmol/L的孕酮(P4)可明显抑制该细胞的增殖。这表明hTERT导入山羊EEC后,可使其重建端粒酶活性,从而获得大量的、具有高度同一性和表型正常的细胞。     

Expression of interleukin-18 receptor mRNA in the mouse endometrium

Interleukin-18 (IL-18) is a proinflammatory cytokine involved in chronic inflammation, autoimmune diseases, and a variety of cancers, and is expressed in mouse uteri. Our previous study suggested that IL-18 acts as a paracrine factor, regulating endometrial function. To elucidate the physiological roles of IL-18 in the mouse endometrium, the expression of the IL-18 receptor (IL-18R) alpha subunit was analyzed. IL-18Ralpha mRNA was expressed in several mouse organs in addition to the endometrium. In situ hybridization analysis using a biotin-labeled mouse IL-18Ralpha riboprobe demonstrated that IL-18Ralpha mRNA expression was detected in glandular epithelial cells, stromal cells around uterine glands, and myometrial cells in the mouse uterus, suggesting that these cells are targets for IL-18. The uterine IL-18Ralpha mRNA expression level changed with the estrous cycle. The uterine IL-18Ralpha mRNA levels of estrous mice were higher than those of diestrous mice. In addition, the IL-18Ralpha mRNA levels in uteri at 3 and 14 days after ovariectomy were higher than those at diestrus and decreased following treatment with estradiol-17beta or progesterone. These findings suggest that IL-18Ralpha gene expression is regulated by estrogen and progesterone and that the uterine IL-18 system is involved in the regulation of uterine functions in a paracrine manner.     

Steroid hormone-regulated let-7b mediates cell proliferation and basigin expression in the mouse endometrium

Let-7b, one of the let-7 family members, was studied for its regulative role in endometrial cells during early pregnancy in mice. According to real-time RT-PCR analysis, the expression of let-7b in epithelial cells increased gradually from day 1 to day 4 of preimplantation stages and reached the highest level on day 4. On the other hand, the highest level of let-7b in stromal cells was observed on day 1, although the expression was decreased on day 2 and increased significantly on day 4. By in situ hybridization, let-7b was also found to express in uteri during days 6-8 of pregnancy. Endometrial cells isolated from prepubertal mice were treated with steroid hormones, progesterone (P4), estradiol (E2) and P4 plus E2. After 96 h of culture in the presence of steroid hormones, the expression levels of let-7b were increased in the endometrial cells, although significant differences were only observed after P4 treatment in stromal cells and after individual E2 and P4 treatments in the epithelial cells. In association with the increased let-7b expression, the cell proliferation slope, measured by a MTT assay, significantly decreased in the presence of P4 and P4 plus E2 compared with the nonhormone and E2 treatment groups during 72-108 h of culture. Furthermore, results from transfection of let-7b into stromal cells isolated from day 4 pregnant mice or prepubertal mice demonstrated that let-7b attenuated the proliferation during the periods of time examined. After transfection of let-7b into mouse stromal cells isolated from day 7 of pregnancy, the expression of Basigin (Bsg), a matrix metalloproteinase (MMP) inducer, was suppressed, as well as that of MMP-9. In conclusion, this study clarifies the expression pattern of let-7b in uterine epithelial and stromal cells during preimplantation stages in mice, as well as the inhibitory effect of let-7b associated with steroid hormones on stromal cell proliferation and on the expression of MMP-9.     

大肠杆菌与金黄色葡萄球菌对奶牛子宫内膜组织细胞因子表达及损伤程度的影响

本试验旨在研究体外感染金黄色葡萄球菌与大肠杆菌对奶牛子宫内膜组织中细胞因子白介素-6（IL-6）、IL-1β和IL-8的表达及损伤程度的影响。以体外培养的奶牛子宫内膜组织作为研究对象,采用1×10⁵~1×10⁹ CFU/mL大肠杆菌与金黄色葡萄球菌对奶牛子宫内膜组织进行体外感染,通过实时荧光定量PCR和ELISA方法检测两种细菌刺激后奶牛子宫内膜组织中IL-6、IL-1β及IL-8 mRNA与蛋白的表达量,并用HE染色法观察两种细菌感染后奶牛子宫内膜组织病理学切片。结果显示,1×10⁵~1×10⁹ CFU/mL大肠杆菌体外感染后,奶牛子宫内膜组织中IL-6、IL-1β和IL-8 mRNA表达量均极显著高于空白对照组（P<0.01）;金黄色葡萄球菌感染浓度为1×10⁵、1×10⁶ CFU/mL时,IL-6、IL-1β mRNA表达量极显著高于空白对照组（P<0.01）,感染浓度为1×10⁷ CFU/mL时,IL-6、IL-1β mRNA表达量显著高于空白对照组（P<0.05）,感染浓度为1×10⁶ CFU/mL时,IL-8 mRNA表达量显著高于空白对照组（P<0.05）,其他感染浓度均与空白对照组无显著差异（P>0.05）。奶牛子宫内膜组织感染1×10⁵~1×10⁹ CFU/mL金黄色葡萄球菌和大肠杆菌时,IL-6、IL-1β及IL-8蛋白表达量均极显著高于空白对照组（P<0.01）。相同浓度的大肠杆菌感染奶牛子宫内膜组织后,IL-6、IL-1β及IL-8 mRNA与蛋白表达量均极显著高于金黄色葡萄球菌感染组（P<0.01）。HE切片染色结果显示,大肠杆菌感染后仍有部分上皮细胞保留,而金黄色葡萄球菌感染后上皮细胞全部脱落。本试验结果表明,大肠杆菌与金黄色葡萄球菌感染奶牛子宫内膜组织后,引起的炎症反应不同。大肠杆菌感染后,促炎性细胞因子被显著上调,而金黄色葡萄球菌感染后破坏子宫内膜上皮细胞程度更加严重。     

miR-495-3p对山羊卵巢颗粒细胞功能的影响

旨在探究miR-495-3p对山羊卵巢颗粒细胞功能的影响及作用机制.本研究选取健康的3～4月龄大足黑山羊母羊,收集卵巢颗粒细胞,利用miR-495-3p模拟物(mimics)和抑制物(inhibitor)构建过表达和抑制模型,通过流式细胞术检测细胞凋亡和周期,ELISA分析颗粒细胞的雌二醇(E2)和孕酮(P4)分泌,采...