弓形虫急性感染与小鼠妊娠失败相关性研究

研究弓形虫感染对小鼠妊娠的影响。用免疫组织化学方法测定子宫部分免疫细胞,用酶联免疫吸附法测定子宫匀浆中肿瘤坏死因子 α(TNF α)和白介素 10(IL 10)的含量。发现孕0天和孕6天接种弓形虫,小鼠全部流产。感染组子宫内膜中仅见少量CD8+T淋巴细胞。对照组未见阳性细胞。F4/80+巨噬细胞在感染组小鼠子宫内膜中大量存在。对照组仅见少量F4/80+细胞。细胞因子测定结果表明,TNF α弓形虫感染组子宫匀浆中含量高达76 862±30 354pg/mg蛋白,显著高于正常妊娠7天时小鼠子宫中TNF α的含量(P<0 01)。结果提示,已妊娠母鼠感染弓形虫可引发流产,并与子宫胎儿界面的TNF α分泌增多有关。孕0天感染,孕鼠子宫匀浆上清液中IL 10含量平均值为19 696±9 811pg/mg蛋白,显著低于对照组(48 856±16 518pg/mg,P<0 05)。孕6天感染,子宫匀浆IL 10含量与对照组比较差异不显著(P>0 05)。     

中药"球康"对E.tenella感染鸡肠道CD4+和CD8+T细胞的影响

本试验采用免疫组化方法检测球虫感染鸡肠道组织中CD4^＋、CD8^＋T细胞数量,探讨中药“球康”抗球虫的作用机理。试验共设不感染不给药组、不感染给“球康”组（2％拌料）、感染给“球康”组和感染不给药组共4组。感染球虫后第2、4、5、7和9天,每组随机捕杀4只鸡,采取盲肠中段进行检测。结果,雏鸡感染球虫后,盲肠组织中CD4^＋、CD8^＋T细胞数量显著高于不感染组,感染给中药“球康”组鸡CD4＋T细胞数量在第2,4,5和9天均高于感染不给药组,在第4天和第5天差异显著（P＜0．05）;CD8＋T细胞数量在第2,5,7和9天高于感染不给药组,第5天差异显著（P＜0．05）,第7天达到最高峰。说明中药“球康”通过增加球虫感染鸡肠道黏膜CD4^＋、CD8^＋T细胞数量,提高细胞免疫水平,以增强机体对球虫的抑杀作用。     

猪瘟免疫猪接种PRRSV Nsp2Δ1882-2241弱毒疫苗后免疫应答及T细胞表型变化特征

选择17头28日龄的CSFV和PRRSV抗体均为阴性的仔猪,于试验的第1天和第14天分别对其进行猪瘟耐热保护剂活疫苗（兔源）和高致病性猪繁殖与呼吸综合征Nsp2A1882—2241弱毒疫苗免疫。在免疫后的第28、42天采集外周血液,分析特异性抗体表达量和外周血T淋巴细胞表型的变化,评估猪瘟免疫对猪繁殖与呼吸综合征免疫的影响。结果显示,在CSF免疫后第28、42天,CSFV高抗组中的CD4^＋、CD4^＋／CD8^＋、CD4^＋CD8^＋和CD4-CD8数目均比CSFV低抗组高,CD3^＋和CD8^＋细胞数量比CSFV低抗组低;PRRS高抗组中,CD4CD8-细胞含量高于PRRS低抗组;在CSF免疫后第28天,CSFV抗体产生较高（阳性比率为73．33％）,PRRSV抗体产生较低（阳性比率仅为6．67％）。在CSF免疫后的第42天,CSF高抗组中PRRSV抗体阳性比率较CSF低抗组高8．33％。结果表明,CSFV特异性抗体产生高时能增加PRRSV特异性细胞免疫应答,增加CD4^＋细胞、CD4^＋CD8^＋细胞数量,提高机体免疫水平。CD3＋和CD4CD8-细胞应答作用值得重视。     

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。     

高温下清凉冲剂对鸡免疫器官中CD4+、CD8+ T细胞数量的影响

采用免疫组化方法检测鸡免疫器官中CD4^＋、CD8^＋T细胞数量，探讨高温下清凉冲剂对机体细胞免疫机能的影响及其药理作用。108只农大3号35目龄公雏，随机分为3组，每纽36只鸡，即常温对照组、高温对照组和高温用药组。用药组饲喂中药煎荆（浓度为1g／ml），用药量与饲料量比为1：1000。在试验的第1、4、8、10天各组捕杀5只。分别采取胸腺、脾脏、法氏囊检测。结果高温下。用药组免疫器官中CD4^＋、CD8^＋T细胞数量均高于高温对照组。第8、10天差异极显著（P〈0．01）。表明高温下清凉冲剂可以增加鸡免疫器官中CD4^＋、CD8^＋T细胞数量，有效地提高鸡细胞免疫水平。     

NO在小鼠早期胚胎吸收过程中的作用

给妊娠 7d小鼠尾静脉注射细菌脂多糖 (L PS)诱导早期胚胎吸收。注射 L PS后 12、2 4、36 h,以 EL ISA、比色法检测血清和子宫匀浆中 Th1型细胞因子 IFN- γ、IL- 12与 NO的含量变化 ;免疫组织化学法观察 3种一氧化氮合酶(n NOS,e NOS,i NOS)和 Th2型细胞在小鼠子宫表达的变化。此外 ,还观察了 NO供体硝普钠 (SNP)诱导孕鼠流产效果及氨基胍 (AG)对抗 L PS诱导孕鼠流产的效果。结果显示 ,相对于正常妊娠组 ,L PS处理组孕鼠子宫匀浆 NO含量及 IFN- γ、IL- 12水平极显著升高 (P<0 .0 1) ,血清 NO含量也极显著升高 (P<0 .0 1) ;n NOS、i NOS在 L PS处理组小鼠子宫可见阳性标记 ,而 e NOS未见阳性标记 ;大量 Th2型阳性细胞标记仅在正常妊娠组小鼠子宫内膜基质可见 ,L PS处理组未见阳性细胞。腹腔注射 SNP致使孕鼠早期胚胎吸收 ,然而妊娠 6～ 9d腹腔注射 AG却未能降低 L PS诱导的孕鼠早期胚胎吸收。上述结果提示 ,L PS处理后 ,Th1型免疫反应增强 ;源自升高表达的 i NOS的子宫局部高浓度 NO可能作为一种效应分子介导小鼠早期胚胎吸收。     

母猪妊娠诊断的方法

1雌激素测定 猪妊娠早期的一个最显著的特点是雌激素的浓度明显增加。硫酸雌酮的浓度从妊娠第16天的浓度迅速增加到25-30天的2纳克/毫升,然后在妊娠40天时又降低到很低的水平（0.2纳克/毫升）。在未孕猪,硫酸雌酮的浓度很低,胚胎可能是这种雌激素的主要来源,而且子宫中胚胎的数量和母体血液中硫酸雌酮的浓度之间有直接关系。因此,在配种后适当时间测定硫酸雌酮浓度可以作为灵敏的诊断早期妊娠的方法。进行妊娠诊断时,可从猪的耳静脉采集少量血液,硫酸雌酮在室温下可以稳定一星期,因此将样品送实验室测定不会存在任何问题。据报道,如果在配种后22-28天采样测定硫酸雌酮,不仅可以准确鉴别妊娠和未孕猪,而且可以比较准确判断子宫中胎儿的数目。妊娠30天时硫酸雌酮的含量与产仔时的窝产仔数高度相关,妊娠22-29天硫酸雌酮的含量与窝产仔数呈高度相关,因此可以预测产仔数量。通过测定硫酸雌酮含量预测产仔数,其准确率为73%。另据报道,猪妊娠25-30天时子宫内的胎儿数由于可能发生胚胎死亡,因此并不能完全代表分娩时的产仔数。注意窝产仔数少的母猪,由于硫酸雌酮含量低,因此有时错误地诊断为未孕。     

交感神经阻断小鼠妊娠早期子宫内肥大细胞的分布

为了研究交感神经对哺乳动物早期胚胎发育的影响机制,通过腹腔注射6-羟多巴胺使交感神经阻断后,观察了小鼠妊娠前期胚胎早期发育和肥大细胞数量和型别的变化。结果显示,交感神经阻断后,不仅对胚胎早期发育有影响,使胚胎着床数降低64.4%,而且影响子宫内肥大细胞的数量及其型别分布。妊娠4d(E4)时肥大细胞数量明显升高(P<0.01),同时肥大细胞分型也有所变化,即黏膜型肥大细胞主要存在于胚胎着床前和着床后,结缔组织型肥大细胞没有明显差异,混合型肥大细胞主要存在于着床期间。这一结果表明,妊娠早期子宫肥大细胞数量与型别的变化可能是交感神经影响早期胚胎发育的途径之一。     

增免散对鸡脾T淋巴细胞CD4^＋和CD8^＋亚群的影响

将300只1日龄雏鸡随机分为中药增免散组、环磷酰胺组、环磷酰胺＋左旋咪唑组、环磷酰胺＋增免散组和正常对照组，6日龄时用鸡新城疫LaSota疫苗点眼滴鼻免疫，14、21、28、35日龄时测定鸡血液NDV抗体效价，脾器官指数，脾组织中CD4^＋、CD8^＋T淋巴细胞浓度和脾细胞凋亡率，观察脾的组织结构变化。结果显示，14、21日龄增免散组抗体水平高于其他各组（P〈0．01）；14、21、28日龄增免散组脾指数高于其他各组，增免散组脾CD4^＋细胞浓度、CD4^＋／CD8^＋比例高于其他各组，差异极显著（P〈0．01）；14、21、28日龄增免散组脾细胞凋亡率低于其他各组，差异极显著（P〈0．01）；14、21日龄时，增免散组脾白髓和红髓内网状细胞和中淋巴细胞较多，脾小体比对照组略有增加。表明，中药增免散不仅能促进鸡脾的发育，增高脾组织中CD4^＋／CD8^＋比例，而且对环磷酰胺的免疫抑制有一定拮抗作用。     

川楝素对昆明小鼠的胚胎毒性研究

取体重18~25 g雌鼠,超数排卵后与公鼠合笼。获得的孕鼠随机分为 6 组,分别用于研究川楝素对孕鼠2 细胞胚、桑椹胚、囊胚3个时期胚胎的毒性。每个时期均设对照组,以小鼠腹腔注射1/30半数致死量(LD50)的川楝素剂量对以上3个时期试验组孕鼠染毒2次,对照组孕鼠用等量PBS注射。研究其着床时期胚胎毒性时不对小鼠进行超数排卵,但试验组孕鼠染毒3次,对照组孕鼠用等量PBS注射。最后计数、观察4个时期孕鼠子宫内的胚胎数量和形态。结果表明:2 细胞胚、桑椹胚、囊胚时期试验组胚胎总数与对照组相比,没有统计学上差异(P>0 05); 2 细胞期试验组正常胚胎、异常胚胎数量与对照组相比,也无统计学上的差异(P> 0 05);桑椹胚、囊胚时期试验组正常胚胎、异常胚胎数量与对照组相比,具有极显著差异(P<0 01)。3 次染毒对着床后的胚胎毒性最大,子宫内胚胎几乎全部溶解,无法计数。母体组织病理切片观察发现川楝素对主要器官心、肝、肺、肾、子宫仅有轻微损害,提示腹腔注射小剂量川楝素对怀孕小鼠具有特定的胚胎毒性。     

Immunohistochemical Studies on Oestrogen Receptor Alpha (ERα) and the Proliferative Marker Ki-67 in the Sow Uterus at Oestrus and Early Pregnancy

Oestrogen receptor alpha (ERalpha), the main subtype in the uterus, is involved in the regulation of uterine growth/proliferation. A relationship between ERalpha and proliferative activity has been shown in the cyclic sow uterus, but to our knowledge, no study has been carried out on early pregnant sows. Therefore, by means of immunohistochemistry and use of mouse monoclonal antibodies to ERalpha and a proliferative marker, Ki-67, the localization of these proteins was investigated in the sow uterus during early pregnancy. Eighteen crossbred multiparous sows were artificially inseminated once at 20-15 h before expected ovulation. After artificial insemination (AI), they were slaughtered at five different times: at oestrus, 5-6 h after AI (n = 4), 20-25 h after ovulation (n =4), 70 h after ovulation (n = 4), on day 11 (the first day of standing oestrus = day 1, n = 3) and on day 19 (n = 3). Immediately after slaughter, uterine samples were collected at the mesometrial side of the uteri, fixed in 10% formaldehyde and embedded in paraffin. Immunohistochemistry was performed by using mouse monoclonal antibodies to ERalpha (C-311) and Ki-67 (MM1). All sows slaughtered after ovulation were pregnant. In general, positive immunostaining for ERalpha and Ki-67 was found in the nuclei. Variations in staining intensity and proportion of positive nuclei were observed in different uterine compartments and stages of early pregnancy. The highest level of ERalpha presence in the surface epithelium and myometrium was found at oestrus (5-6 h after AI), and low levels of ERalpha in these compartments were observed as early as 20-25 h after ovulation. In the glandular epithelia, presence of ERalpha was highest at 70 h after ovulation. The largest number of ERalpha-positive cells in the stroma was observed at oestrus and early after ovulation. Low proliferation was observed, and with no significant difference in tissue compartments except in the glandular epithelium. High proliferative activity in the glandular epithelium at 70 h after ovulation indicated involvement in preparation for secretory activity and growth during pregnancy establishment. Significant positive correlation was found between the number of ERalpha-positive cells in the stroma and Ki-67-positive cells in the surface epithelium. In conclusion, the present study showed differences in immunolocalization of ERalpha and the proliferative marker Ki-67 in different tissue compartments of the sow uterus at oestrus and early pregnancy. In some uterine compartments, the patterns of ERalpha and Ki-67 immunostaining seemed to be influenced by insemination and the presence of embryos, in addition to the effects of steroid hormones.     

Development of transferred xenogeneic vole embryos in mouse uteri

An experimental model to study interspecific pregnancy using voles, Microtus arvalis, and green fluorescent protein gene‐induced transgenic mice is presented. Xenogeneic blastocysts from the vole were transferred into the uteri of pseudopregnant mice along with allogeneic blastocysts from green fluorescent protein gene‐induced transgenic mice. The uteri containing xeno‐allo combined transfers were examined from day 6 to 13 of gestation. Although the vole embryos implanted, the uteri containing vole embryos were smaller compared with those having allogeneic mouse embryos. On day 8, the uteri containing vole embryos hemorrhaged internally and no vole embryo was found in the pregnant uterus after day 11. Allogeneic mouse embryos developed normally despite the presence and abortion of the vole embryos. In uteri implanted with vole embryos, decidua were formed and numerous blood vessels were distributed around the embryo. Maternal blood cells infiltrated into the celomic cavity of the vole embryo through the discontinuous region of trophoblast. Periodic acid‐Schiff‐positive granulated metrial gland cells were remarkably increased in the decidual sites. These findings suggest that a disorder of embryo–maternal interaction might induce the appearance of numerous granulated metrial gland cells and rejection of the embryos.     

Effects of bisphenol A (BPA) on placentation and survival of the neonates in mice

The effects of bisphenol A (BPA) on placentation have not been fully determined. The aim of this study was to clarify the structural changes of the placenta, abortion rate, and survival of neonates after BPA administration in mice. BPA (10 mg/kg/day) was administered to pregnant mice (BPA mice) subcutaneously from the first day of pregnancy (Day 0) to Day 7 (8 days total). The number of embryos and weights of whole uteri were measured on Days 10 and 12. Morphological changes in the placentae were examined by light microscopy on the corresponding days of pregnancy. The number of neonates was also counted. Survival rates were periodically calculated for neonates from the first day after parturition (P-Day 0) to P-Day 56. The number of embryos and weight of the uterus on Days 10 and 12 were significantly decreased by BPA injection. No notable differences were recognized between the left and right uteri. The proportion of the labyrinthine zone per whole placenta in the BPA mice became lower than that in the controls, and that of the metrial gland was higher in the BPA mice. The intervillous spaces of the placenta were narrower in the BPA mice. Degenerative changes were found in the trophoblastic giant cells and spongiotrophoblast layers of the BPA mice. The number of BPA mouse neonates was drastically decreased within 3 days after birth, and no mice survived after P-Day 56. The results suggest that BPA not only disrupts placental functions and leads to abortion through chronic stimulation of gene expression by binding to DNA but that it also affects the mortality of neonates through indirect exposure of embryos.     

Localization of interleukin-6 receptor mRNA in the pregnant and non-pregnant mouse uterus

To understand roles of interleukin 6 (IL-6) family cytokines for pregnancy in mice, localization of IL-6 receptor (IL-6R) mRNA was investigated in non- and early pregnant uteri by in situ hybridization. IL-6R mRNA was expressed in all non-pregnant uteri and in pregnant uteri from the third day (Day 3) to the sixth day of pregnancy (Day 6; the day of plug = Day 1). IL-6R mRNA signals were detected in non-pregnant mice in the luminal and glandular epithelium. Signal strength varied according to the sexual cycle. There was no correlation between the signal strength of the IL-6R mRNA and the serum concentrations of progesterone and 17beta-estradiol, which show a monophasic rise in the non-pregnant sexual cycle. In pregnant mice, slight signals were detectable in the luminal and glandular epithelium on Day 3. IL-6R mRNA messages increased with progression towards Day 4, however, localization changed drastically on Day 5. Stromal cells abruptly expressed their mRNA on Day 5, and these cells strongly expressed it on Day 6. The function of IL-6R in the luminal and glandular epithelium might be different from that in the stroma during the implantation period. In addition, few signals were identified in the stromal cells adjacent to the luminal epithelium on Day 6. This suggests that there are two types of stromal cells on Day 6 in mice.     

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.     

膜联蛋白A8在小鼠早期妊娠子宫中表达研究

膜联蛋白A8(Annexin A8,ANXA8)是一种磷脂结合蛋白,与炎症反应、癌症的发生以及血管生成有密切联系。本实验旨在利用实时荧光定量PCR、原位杂交与免疫组织化学的方法研究ANXA8 mRNA与蛋白在小鼠早期妊娠和人工蜕膜子宫中的表达。原位杂交结果表明:ANXA8 mRNA在小鼠早期妊娠第1～4天子宫腔上皮和腺上皮有微弱表达,ANXA8 mRNA在妊娠第5、6天的初级蜕膜区与第7、8天的次级蜕膜区表达,并随妊娠进行逐渐增强;人工蜕膜化模型中ANXA8 mRNA表达在蜕膜区。实时荧光定量PCR证明:ANXA8 mRNA的表达量在早期妊娠模型中的第7、8天显著提高,人工蜕膜侧子宫与对照侧相比也显著提高。免疫组织化学结果表明:ANXA8蛋白与ANXA8 mRNA表达规律相似。体外分离培养小鼠子宫基质细胞,并诱导蜕膜化,实时荧光定量PCR结果表明ANXA8随着基质细胞的蜕膜化表达升高。以上体内和体外实验表明,ANXA8在小鼠子宫中的表达具有着床相关特异性,ANXA8参与小鼠子宫蜕膜化过程。     

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.     

Influence of post-ovulatory insemination on sperm distribution,pregnancy and the infiltration by cells of the immune system,and the distribution of CD2, CD4, CD8 and MHC class II expressing cells in the sow endometrium

This study investigates the distribution of leucocytes, CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following post-ovulatory insemination in relation to clinical findings and pregnancy outcome. Crossbred multiparous sows were inseminated once either at 15-20 h after ovulation [experiment 1, slaughtered at 20-25 h (5-6 h after artificial insemination (AI), group 1-A, n = 4), at 70 h after ovulation (group 1-B, n = 4), on day 11 (group 1-C, n = 4, first day of standing oestrus = day 1) or on day 19 (group 1-D, n = 4)] or 30 h after ovulation [experiment 2, slaughtered at 5-6 h after AI (group 2-A, n = 4) or on day 19 (group 2-D, n = 3)]. The uterine horns were flushed to control for the presence of spermatozoa and neutrophils and/or for recovery of oocytes and/or embryos. Mesometrial uterine samples were plastic embedded and stained. Cryofixed uterine samples were analysed by immunohistochemistry using mAbs to lymphocyte subpopulations and MHC class II molecules. Light microscopy was used to examine surface (SE) and glandular epithelia (GE), and connective tissue layers, both subepithelially (SL) and glandular (GL). In experiment 1, group 1-A, only one sow had spermatozoa in the utero-tubal junction (UTJ). Marked/moderated numbers of neutrophils and spermatozoa were observed in the flushings of two sows. In group 1-B, altogether 23 of 48 oocytes were cleaved. Day 11 (1-C), embryos with small diameter were observed. Day 19 (1-D), no embryos were found but small pieces of foetal membrane were observed in one of the sows. In group 1-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. For T lymphocyte subpopulations, in the SE, most CD2+ cells were found in group 1-A. For both SE and GE in all groups, the number of CD8+ cells was significantly larger than that of CD4+ cells. In experiment 2, group 2-A, no sow had spermatozoa in the UTJ or in the uterine flushings. At day 19, no sow was pregnant. In group 2-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. At day 19, high E2 levels showed a hormonal prooestrous stage but the endometrial neutrophil infiltration normally expected at pro-oestrus was absent. In conclusion, post-ovulatory insemination (about 18 h after ovulation) resulted in impaired spermatozoa transport within the uterus and embryonic degeneration. In sows post-ovulatory inseminated at a later stage (30 h after ovulation), no sow was pregnant. In both experiments, disturbed immune cell patterns were observed in some individuals.     

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.