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.     

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.     

MicroRNA调控胚胎植入研究进展

植入是胚胎与子宫内膜识别、接触并依附于子宫内膜上或进一步侵入、包埋于子宫内膜中的过程,是哺乳动物生殖生理的重要环节,这一过程对于胚胎能够在子宫持续的发育并最终获得成功的妊娠是必需的。成功的植入需要同步发育的正常胚胎、容受性的子宫内膜及母胎的和谐对话来实现。胚胎植入过程复杂而短暂,参与调控的因素有很多,包括microRNA。论文介绍了与植入相关microRNA的最新研究进展,包括microRNA对胚胎发育、子宫内膜的容受性等方面的作用,将为我们理解植入机制提供新的视角。     

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.     

Two concepts on the immunological aspect of blastocyst implantation

The process of blastocyst implantation is a series of interactions between the blastocyst and maternal tissues. The purpose of this process is (1) to provide nourishment to the embryo for developmental growth in appropriate physiological and endocrinological environment until a placenta is established, and (2) to protect the (semi-)allogeneic embryo from any attacks from the maternal immune system. To facilitate successful implantation, therefore, these two aspects of the embryonic demand must be satisfied in the embryo-maternal interface throughout the entire process of implantation. The first concept I present in this paper is that blastocyst implantation essential factors (BIEFs) have dual functions: one, for structural and functional modification of the endometrium to accommodate the developing embryo and provide nourishment and suitable environment for its development, and the other, for modulation, directly or indirectly, of the maternal immune system to prevent attacks by the maternal immune system. The second concept is that BIEFs convert the endometrium (or uterus) from an immunologically non-privileged site to a privileged site. This endometrial (uterine) conversion is the immunological aspect of the blastocyst implantation process. When the endometrium has become receptive for blastocyst implantation, it signifies that the immunological conversion of the endometrium by BIEFs has been sufficiently attained to let the embryo start contacting maternal tissues. During the early stages of placentation, as the trophoblast cells differentiate and make their way to the maternal blood vessels to establish the placenta, BIEFs continuously provide nourishment and immunological protection to the developing embryo. The immunological protection of the embryo/fetus from potential attacks by the maternal immune system appears to reach a peak at the time of establishment of the placenta. Thus, clarification of the roles of BIEFs in both the physiological/endocrinological aspect as well as the immunological aspect is essential for understanding the biological process of implantation.     

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.     

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.     

猪胚胎附植部分因子研究进展

产仔数性状是养猪业中一个重要的经济性状,而胚胎附植对猪产仔数的高低有重要的影响。胚胎附植的调控涉及到多个生物学过程,在其中发挥作用的物质（附植因子）很多。作者针对孕酮、孕酮受体、雌二醇、雌激素受体α（ESR1）和促红细胞生成素产生肝细胞受体配体（Eph-ephrin）系统这几种附植因子的相关研究进行了归纳总结,重点阐述了这些因子在母胎对话过程中的时空表达、功能、突变、调控等方面的研究进展。其中,孕酮及其受体在猪胚胎附植活动中全程发挥着重要作用,黄体分泌的孕酮与母猪子宫内膜腔上皮、腺上皮、基质和肌细胞中的孕酮受体结合发挥作用,使这些组织细胞分泌多种附植因子,这些附植因子进一步参与胚胎附植过程。雌二醇是雌激素中含量最多、活性最强的一种激素,主要由卵巢颗粒细胞分泌,雌激素受体α是子宫内雌二醇的主要受体,二者结合可使母猪发情;此外,附植中处于游离状态的胚泡也分泌雌二醇,其是一个让母猪子宫内膜得以识别的信号,允许胚泡附植。Eph-ephrin系统作用广泛,其在人、小鼠和猪的胚胎附植过程中发挥着重要作用。系统中的EphA1、ephrin A1、EphA4等基因在猪的胚胎附植期表达量显著高于空怀猪,它们在子宫内膜附植点的表达量显著高于附植点间的部位,且ephrin A1的抑制表达会降低子宫内膜上皮细胞的迁移和黏附能力。这些附植因子都在猪胚胎附植活动中发挥着重要作用,对它们的深入研究将有利于明确猪胚胎附植调控机理,进一步揭示猪高繁机理。     

Expression and regulation of Foxa2 in the rat uterus during early pregnancy

The forkhead box a (Foxa) protein family has been found to play important roles in mammals. Recently, the expression of Foxa2 was reported in the mouse uterus, and it was reported to be involved in regulation of implantation. However, the regulation of Foxa2 expression in the uterus is still poorly understood. Therefore, the present study was conducted to investigate the expressional profiles of Foxa2 in the rat uterus during the estrus cycle and pregnancy. Furthermore, the effect of steroid hormones and Hedgehog protein on the expression of Foxa2 was analyzed in vivo and in vitro. In this study, the level of expression of Foxa2 was low in the rat uterus during the different stages of the estrus cycle. However, the expression increased transiently during early pregnancy at 3.5 days post coitus (dpc) and decreased at 5.5 dpc. In ovariectomized rats, P4 treatment had no effect on the expression of Foxa2 compared with the expression in control animals. Moreover, the expression of Foxa2 in cultured epithelial cells was not increased by P4 treatment in vitro. However, Foxa2 expression was significantly decreased in the rat uterus after 24 h of E2 treatment. Treatment of cells with a recombinant Hedgehog protein significantly increased the expression of Foxa2. These results suggest that the expression of Foxa2 may transiently increase just before the implantation and it may be regulated by E2 and Hedgehog protein.     

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.     

环氧合酶-1和环氧合酶-2在兔早期妊娠子宫内膜及早期胚胎中的表达

环氧合酶(cyclooxygenase，Cox)是由花生四烯酸合成前列腺素的限速酶，而前列腺素在哺乳动物的生殖过程中起着很重要的作用。本试验采用RT—PCR方法对环氧合酶-1(Cox-1)及环氧舍酶-2(Cox-2)在早期妊娠兔子宫内膜中的表达进行了半定量研究和在早期胚胎中的表达定性研究。结果表明，Cox-1在早期妊娠兔子宫内膜中表达较平稳，呈组成型表达；在早期胚胎中各个时期均可以检测到Cox-1mRNA，表明它在早期胚胎发育过程中起作用。Cox-2在早期妊娠子宫内膜中的表达水平有很大差异，妊娠第l天表达水平较高，从第2天开始表达下降，到第6天表达又明显增加，第8天达到最高。Cox-2在囊胚之前的各个时期胚胎均未检测到，而在扩展胚泡和孵出胚泡中表达，Cox-2的时期特异性表达可能与兔胚泡孵出及胚胎着床等过程相关。     

Proteomic analysis demonstrates that parthenogenetically activated swamp buffalo embryos have dysregulated energy metabolism

The comprehensive understanding of early embryo development is essential to optimize in vitro culture conditions. Protein expression landscape of parthenogenetically produced embryo remains unexplored. This study aimed to investigate the protein expression dynamics with a particular focus on energy metabolism throughout the early developmental stages of parthenogenetic buffalo embryos. For this purpose, we performed iTRAQ-based quantitative mass spectrometry and identified 280 proteins common in all stages. A total of 933 proteins were identified during the proteomics analysis. The data depicted that morula and blastocyst had distinct protein expression dynamics as compared to 2- to 16-cell-stage embryo. KEGG pathway analysis showed 23 proteins belonging to energy metabolism appeared in the data. Study of energy metabolism-related protein's expression pattern demonstrated that there was asynchrony in proteins related to glycolysis throughout the examined developmental stages. The expression pattern of pyruvate kinase mutase (PKM), an essential protein of glycolysis, indicated a slightly decreasing trend from 2-cell-stage embryo to blastocyst, and it was supported by expression of proteins involved in lactate production (LDHA and LDHB) suggesting the decreasing rate of aerobic glycolysis (Warburg Effect) at morula and blastocyst stage. The increased Warburg Effect is considered as the hallmark of proliferating cells or embryo at the blastocyst stage. Furthermore, the proteins involved in the citric acid cycle also showed down-regulation at the blastocyst stage, indicating a lesser role of oxidative phosphorylation at this stage. Therefore, it could be divulged from the study that there may be an irregular pattern of energy metabolism in early parthenogenetic embryos. Further studies are recommended to understand this phenomenon.     

荧光定量PCR分析水牛胚胎致密化相关基因表达方法的建立

胚胎致密化是哺乳动物早期胚胎发育过程中一个非常重要的环节,有研究发现致密化相关基因的表达水平与体外发育胚胎的质量之间存在着相互关联作用。为了分析水牛胚胎早期发育中致密化相关基因在mRNA水平上的表达并确定其定量分析方法,本研究根据黄牛有关基因序列设计了Cx43、Cx31、E-cad以及组蛋白基因(Histone H2A)引物序列,分析了黄牛和水牛致密化基因的同源性,并确定各目的基因在水牛胚胎的SYBR Green I实时定量PCR反应条件。     

囊胚注射转基因ES细胞制作嵌合体的研究

在小鼠胚胎成纤维细胞制作的饲养层上培养并成功的维持了携带LacZ基因的胚胎干细胞系（S8），在此基础上，以S8为供体细胞，以远交系昆明白小鼠3．5d胚胎为受体，通过显微注射法将供体细胞转移到受体的囊胚腔内，经过恢复培养，移植到代孕鼠昆明白雌鼠的子宫中；后代在嵌合体出生一周后进行判定。本试验用8～13代的S8细胞共注射胚胎597枚，经1～3h恢复培养，有585枚胚胎重新具有膨大的囊胚腔，细胞轮廓分明，滋养层细胞间连接也清晰可见，胚胎成活率为97％；胚胎移植后，代孕母鼠共移植胚胎228枚，经17～19d的妊娠期后，产仔37只（2只死胎），产仔率为16％；有35只仔鼠（雄鼠18只，雌鼠17只）存活到可以判断毛色，共获得8只S8细胞毛色嵌合体小鼠，嵌合体的产生率为21．6％。结果表明用S8细胞经囊胚注射后能够获得嵌合体，并且嵌合体明显发生了性偏离现象。本试验为国内利用囊胚注射携带LacZ基因的胚胎干细胞获得嵌合体小鼠的首例报道。     

哺乳动物胚胎附植与中药保胎作用机制研究进展

附植是指哺乳动物囊胚滋养层细胞与母体子宫上皮细胞之间逐步建立组织生理上联系的过程。成功的附植有赖于胚泡与母体相互识别,胚泡发育与母体子宫变化的同步以及抑制母体的免疫排斥反应和母体接受性等一系列事件。这些事件又受到母体和胚泡的激素以及一些因子的调控。传统中药可通过提高孕酮的含量、类雌激素样作用、治疗子宫内膜炎的作用以及影响白血病抑制因子和白细胞介素1的表达量来促进山羊胚胎附植。文章主要论述了哺乳动物胚胎附植的机制以及中药对胚胎附植的影响,这有助于进一步阐明中药保胎的作用机理。     

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.