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 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.     

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.     

Expression and Hormonal Regulation of E‐Cadherin in Canine Uterus During Early Pregnancy

E‐cadherin, a Ca^{2 +} ‐dependent cell adhesion molecule, is necessary for endometrial receptivity to blastocyst implantation. The aim of this study was to investigate the differential expression of E‐cadherin in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. E‐cadherin mRNA expression was at a low level in the glandular epithelium on days 6, 12 and 17 of pregnancy. On days 20 and 23 of pregnancy, E‐cadherin mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium and declined in villi and placenta on day 28 of pregnancy. During oestrous cycle, a moderate level of E‐cadherin mRNA expression was found in the luminal and glandular epithelium of canine uteri at oestrus stage. The same expression was also found at anoestrus stage. Progesterone slightly induced the expression of E‐cadherin mRNA in the luminal and glandular epithelium of ovariectomized canine uterus. These results suggest that E‐cadherin expression is closely related to canine implantation and can be up‐regulated by progesterone.     

Expression of hedgehog family genes in the rat uterus during early pregnancy

Hedgehog (Hh) plays a pivotal role in various tissues during embryonic development, tissue homeostasis and tumorigenesis. In mammals, Hh exists in three homologs: Desert hedgehog (Dhh), Indian hedgehog (Ihh) and Sonic hedgehog (Shh). In this study, we cloned full-length cDNAs encoding Dhh and Ihh from the rat uterus. Their amino acid sequences have a high homology with those of the mouse and human. In addition, the changes of Hh gene expression in the rat uterus during early pregnancy were analyzed. The results showed that all three hedgehog mRNAs were detected in the rat uterus at the proestrus stage and during early pregnancy (1.5, 3.5, 5.5 and 7.5 days post coitus: dpc). Ihh mRNA expression varied and peaked at 3.5 dpc in the luminal and glandular epithelium. Expression was decreased on 5.5 dpc with the exception of sustained expression in the glandular epithelium. Despite such Ihh variability, the expressions of Dhh and Shh mRNA remained unchanged. This indicated that Ihh was mainly expressed in the rat uterus during early pregnancy. Moreover, the Hh target gene (glioma-associated oncogene homolog 1; Gli1) was also highly expressed at 3.5 dpc in the epithelium and periepithelial stroma in a manner similar to the temporal pattern of Ihh expression. This suggests that Ihh signaling axis play a role in the rat uterus during early pregnancy. In summary, our results elucidate that Ihh is a predominant Hh protein in the rat uterus during early pregnancy and that other Hhs have the potential to be expressed. This observation will help to elucidate the basic molecular mechanism of rat uterus during early pregnancy.     

Expression of Inhibin/Activin Subunits in the Equine Uteri during the Early Pregnancy

The establishment of equine pregnancy is a unique and long process during which a series of physical and possibly biochemical interactions are required between the conceptus and uterus. In this study, we investigated the expression pattern of inhibin/activin subunits in the uterus during early pregnancy. The uteri from four adult mares on cyclic day 13 or pregnancy day 25 were obtained. Immunohistochemical experiments suggested that inhibin/activin subunits were immunolocalized in the luminal and glandular epithelium on pregnancy day 25. In addition, the inhibin α and inhibin/activin β_B subunits were not detected, and inhibin/activin β_A subunit was detected, in the luminal and glandular epithelium on cyclic day 13. Real‐time polymerase chain reaction and Western blotting results for the inhibin/activin subunits suggested a significant increase in the expression of inhibin/activin subunit β_B and a significant decrease in the expression of inhibin/activin subunit β_A on pregnancy day 25 compared with those on cyclic day 13. Enzyme‐linked immunosorbent assays suggested a significant decrease in the concentration of activin A in endometrium extracts from cyclic day 13 to pregnancy day 25. These results suggest that inhibins or activins synthesized in the uterus, as endocrine factors and necessary nutriments, have different expression patterns and may play different, important roles during early embryonic development of the equine.     

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.     

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.     

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。     

Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0 = day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17β (E₂) were detected in day 25 conceptuses. Concentrations of E₂ were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E₂ and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1α and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E₂ and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.     

Expression of Oestrogen Receptor α and Oestrogen Receptor β in the Uterus of the Pregnant Swine

The uterus is a well‐known target of endocrine, paracrine and autocrine acting molecules among which steroid hormones are of special importance. The objective of our work was to localize oestrogen receptors (ERα and ERβ) mRNA and protein in the pig uterus throughout pregnancy (10, 18, 32, 50, 71, 90 days post coitum) using RT‐PCR, Western‐blot and immunohistochemistry. The present study is the first one to demonstrate the presence of ERs protein in the porcine uterus not only at the beginning but also at mid‐ and late pregnancy. In the pregnant swine, ERα was immunolocalized in the luminal epithelium (LE) and glandular epithelium (GE) and the myometrium of the uterus with differences in the intensity of staining at different stages of pregnancy studied. The LE and GE of pregnant swine stained for ERβ regardless of the day of pregnancy examined, whereas only a few cells within the myometrium showed a weak immunoreactivity. Western blot analysis confirmed the presence of ERα and ERβ proteins on all investigated days of gestation. The expression of ERα and ERβ mRNA was detected by RT‐PCR in all examined samples corresponding to each of the consecutive stages of pregnancy. The obtained results show that ERα is more abundant in comparison to ERβ within the porcine pregnant uterus. The presence of ERα and ERβ in all compartments of the pig uterus during pregnancy may indicate direct action of oestrogens on proliferation and differentiation of these cells.     

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.     

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.     

Immunohistochemical localization of beta defensins in the endometrium of rat uterus during the postpartum involution period

β-Defensins are small cationic molecules that have antimicrobial actions against bacteria, fungi and viruses and contribute to mucosal immune responses at epithelial sites. The female reproductive tract is an important site of defensin production. This study was conducted to determine the possible changes in proportions and localization of β-defensin 1-4 in the rat uterus at the 1st, 3th, 5th, 10th and 15th days of postpartum and at the period of diestrus using immunohistochemical techniques. In the present study, it was determined that β-defensin 1-4 were generally found in all structural components of the endometrium (luminal and glandular epithelium, stromal cells and blood vessels) in both the nucleus and the cytoplasm of cells during the involution period and diestrus. Suprisingly, immunoreaction of β-defensin 2 was also observed in the lateral membrane of the luminal and glandular epithelial cells on the 10th day of involution and immunostaining of β-defensin 4 was also localized in the apical membrane of the luminal and glandular epithelial cells. The current study demonstrated β-defensin 1-4 immunoreactivities in the endothelium of blood vessels were stronger throughout the involution period. Although β-defensins 2 and 3 were localized in both the nuclei and the cytoplasm of endothelial cells, β-defensins 1 and 4 were present in only cytoplasm. These results show that the most component of rat endometrium expresses human β-defensin 1-4 in a involution-dependent manner. Therefore it may be asserted that these molecules constitute a organised protection to prevent uterus from probable infections during the involution process.