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 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 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.     

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。     

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。     

CD58对妊娠早期山羊子宫局部细胞因子的调节

采用IL-2的生物学测定方法，IL-4、TGF-β1、TGF-β2试剂盒等方法，对正常妊娠组山羊妊娠早期（15-19d)子宫局部细胞因子IL-2、IL-4、TGF-β、TGF-β2水平（活性）的变化规律进行了研究，结果发现，妊娠早期山羊子宫两侧局部细胞因子水平（活性）无显著性差异，IL-2活性先上升，后出现下降趋势；IL-4水平一直呈上升趋势；TGF-β1水平较高，但呈不规则曲线变化；TGF-β2先上升，后趋于平稳，并保持在较高水平。证明妊娠早期山羊子宫局部细胞因子分泌及活性有一动态变化规律。对CD58处理后山羊妊娠16-18d子宫局部上述细胞因子水平（活性）与同期正常妊娠相比的变化情况的研究发现，CD58极显著抑制IL-2的产生，而明显促进IL-4的产生，对TGF-β2的产生具有一定促进作用，而对TGF-β1无影响，证明CD58对妊娠早期山羊子宫局部细胞因子的产生具有调节作用。     

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.     

Expression of Genes in the Canine Pre‐implantation Uterus and Embryo: Implications for an Active Role of the Embryo Before and During Invasion

The aim of the present study was to assess genes expressed in maternal uterine tissue and pre‐implantation embryos which are presumably involved in maternal recognition and establishment of canine pregnancy. For this purpose, 10 pregnant bitches were ovariohysterectomized between days 10 and 12 after mating. Four non‐pregnant bitches served as controls. Early pregnancy was verified by flushing the uterine horns with PBS solution. The collected embryos (n = 60) were stored deep‐frozen (?80°C). Uterine tissue was excised, snaps frozen in liquid nitrogen and homogenized using TRI Reagent. All embryos from one litter were thawed together and also homogenized in TRI Reagent. RT‐PCR was performed to prove mRNA expression of progesterone receptor, key enzymes of the prostaglandin synthesis pathway, selected growth factors, cytokines, immune cell receptors, major histocompatibility complex (MHC) and matrix‐metalloproteinases (MMP). Only pregnant uteri revealed the presence of mRNA for interferon (IFN)‐γ, IL‐4 and CD‐8, which resembles the milieu in humans and other mammalians. Similarly, in day 10 embryos, mRNA for transforming growth factor‐β, insulin‐like growth factor‐1,‐2, hepatocyte growth factor, leukaemia inhibitor factor, tumour necrosis factor‐α, interleukin‐1β,‐6,‐8, cyclooxygenase‐2, CD4⁺ cells, and MMP‐2 and ‐9 were detected, but not MHC‐I or ‐II. We therefore suppose that the canine embryo, like its human counterpart, actively initiates measures to prevent attacks from the maternal immune system to prepare its own adhesion, nidation, growth and further development.     

Pulmonary Maturation in Canine Foetuses From Early Pregnancy to Parturition

Development of the foetal respiratory system includes both pulmonary growth and maturation. In human medicine, a higher incidence of respiratory distress is reported in newborn males. This study aimed to identify different phases of canine foetal lung maturation throughout pregnancy, to determine the stage of pregnancy in which surfactant production begins and to compare pulmonary development of male and female foetuses. Pregnant bitches (34) were subjected to elective ovariohysterectomy and allocated into four groups, according to the stage of pregnancy: 30–40 days of pregnancy (n = 10), 41–50 days (n = 10), 51–60 days (n = 10) and bitches in the first stage of parturition (n = 4). Foetal lungs were histologically processed and evaluated by optical microscopy. The pseudoglandular phase was identified between the 35th day and 46th day of gestation; the onset of canalicular and saccular periods was observed, respectively, from the 48th day and 60th day of pregnancy. Lungs from foetuses at term were in the saccular phase; thus, the development into the alveolar period occurs in the neonatal period. The histological analyses revealed that respiratory tract development is centrifugal, from upper to lower airways. Therefore, it is possible to identify distinct development periods in different portions of the same organ. In conclusion, the saccular phase of lung development begins around 57 and 60 days of pregnancy, the period in which surfactant production is believed to occur. Male and female foetuses present similar pulmonary development from early pregnancy until parturition.     

Leptin Gene and Protein Expression in the Ovary During the Oestrous Cycle and Early Pregnancy in Pigs

Leptin, the product of the obese gene, is the hormone originally identified in adipocytes. It is involved in the control of satiety and energy metabolism. More recent observations suggest that leptin plays an important role in reproduction. Leptin mRNA and protein have been found in the human and the murine ovary. However, the expression of leptin in the porcine ovary has not been examined. Therefore, the aim of the present work was to compare the expression levels of porcine leptin mRNA by semiquantitative RT‐PCR and in situ hybridization, as well as leptin protein by Western blotting in the corpus luteum (CL) and ovarian stroma (OS) during mid‐ and late‐luteal phase of the oestrous cycle as well as during days 14–16 and 30–32 of pregnancy. Leptin gene and protein expression in CL was increased on days 14–16 of the cycle compared with pregnant animals. Leptin gene expression in OS was higher during the late‐luteal phase of the cycle than on days 30–32 after conception. However, comparison of leptin protein expression in OS between days 14–16 of the cycle and days 30–32 of pregnancy indicates a higher protein expression during pregnancy. Moreover, leptin gene expression was higher in porcine CL and OS on days 14–16 of pregnancy in comparison to days 30–32. Contrary to leptin mRNA expression, a higher leptin protein expression was observed on days 30–32 compared with days 14–16 after conception. In summary, the present study provides the first evidence that leptin mRNA and protein occur in porcine ovary and vary during the oestrous cycle and pregnancy. Moreover, the obtained results indicate that also locally synthesized leptin may participate in the control of pig reproduction by exercising its action at the ovarian level.     

Metabolic Profile of Broodmares During Late Pregnancy and Early Post‐Partum

Pregnancy represents a specific physiological status characterized by continuous adjustments that affect maternal metabolism of all nutrients. In the last trimester of pregnancy, mare's nutrient requirements greatly increase and most pregnancy‐associated diseases are likely to occur. Therefore, we aimed to assess the metabolic profile of broodmares focusing on the last 3 months of pregnancy and the early post‐partum. Fifteen pregnant mares (Group A) were monitored from 263 ± 3 days of pregnancy until 21 days after foaling. Seven non‐pregnant mares (Group B) were used as the control group. Blood samples were collected weekly by jugular venipuncture throughout the experimental period, and additional blood samples were collected within 24 ± 12 h of foaling. Obtained sera were analyzed for urea (Ur), creatinine (Cre), total protein (TP), total, direct and indirect bilirubin (tB, dB, iB), triglycerides (TG), total cholesterol (tChol) and β‐hydroxybutyrate (BHB). During pregnancy, Ur (p = 0.015) and dB (p = 0.028) were higher in Group A than Group B. Serum Cre, tB, iB and TG (p < 0.001) decreased after foaling; furthermore, Group A exhibited lower tChol (p < 0.001) and higher BHB (p < 0.001) than Group B during the study. There were significant interactions of time × peripartum on Ur (p = 0.007), Cre (p < 0.001), tB (p = 0.018), TG (p < 0.001) and tChol (p < 0.001). These results can be used in the interpretation of biochemical studies in late gestation mares which develop systemic disease. The improved understanding of the metabolic profile during the peripartum might assist in monitoring the health status of the broodmares to promote foetal growth and well‐being.     

营养水平对母猪妊娠早期类胰岛素生长因子-I及其mRNA表达的调控

类胰岛素生长因子-I(IGF-I)是促合成代谢、促有丝分裂和抗细胞凋亡的因子。妊娠早期母猪体内有大量IGF-I表达,对胚胎发育有重要作用。营养水平影响IGF-I及其mRNA的表达。本文介绍了母猪卵巢、输卵管和子宫内IGF-I及其mRNA的表达特点,重点阐述了营养水平对母猪IGF-I及其mRNA表达的调控与可能机制。     

早期妊娠、发情周期及假孕小鼠子宫与荆豆素-Ⅰ的结合特性及激素调节

应用免疫荧光技术以生物素化的荆豆素-Ⅰ(UEA -Ⅰ)为探针,检测其结合位点α-L-岩藻糖在小鼠妊娠1～8 d、假孕1～6 d及发情周期子宫中的表达情况,并以摘除卵巢的小鼠为模型观察雌激素、孕酮对小鼠子宫α-L-岩藻糖表达的影响.结果,UEA-Ⅰ的结合位点主要位于妊娠1～4 d的子宫腔上皮及腺上皮中.在假孕小鼠,UEA-Ⅰ的结合位点主要位于假孕1～2 d的子宫腔上皮及腺上皮中,假孕3～4 d时在这些部位的结合逐渐减少或消失.另外,在发情周期各期的子宫腔上皮和腺上皮中也发现很多UEA-Ⅰ的结合部位,在发情期明显增多,当用雌激素处理时在这些部位的结合明显增加.这些表明,UEA-Ⅰ在子宫腔上皮和腺上皮上的结合位点可能与胚胎的存在与否无关, 而是受母体雌激素的调节.但胚胎着床后UEA-Ⅰ的结合位点消失,以后又在初级蜕膜中出现,表明UEA-Ⅰ的结合位点可能与胚胎着床后的蜕膜化反应有关.     

Sialomucin Complex (Muc4) Expression in Porcine Endometrium During the Oestrous Cycle and Early Pregnancy

The non‐invasive type of implantation in the pig is characterized by the maintenance of a thick glycocalyx coating on the uterine epithelial surface microvilli. Present study investigated the alteration in the sialomucin complex (Muc4) expression during the oestrous cycle and early pregnancy in the pig. Endometrial tissue samples were immunostained with the primary antibody to the Muc4 transmembrane subunit ASGP‐2. Muc4 immunostaining increased in the surface and glandular epithelia between days 5 and 10 of oestrous cycle. Immunostaining continued to increase on day 12 with the greatest intensity of uterine Muc4 immunostaining detected on day 15 of the oestrous cycle and early pregnancy. Endometrial Muc4 expression in cyclic gilts decreased dramatically during early proestrous but continued to remain abundant in the surface and glandular epithelium of pregnant gilts during the period of conceptus attachment to the uterine surface.     

Expression of Aquaporin Water Channels in Equine Endometrium is Differentially Regulated During the Oestrous Cycle and Early Pregnancy

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non‐pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time‐dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non‐pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non‐pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up‐regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine‐specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.     

犬子宫蓄脓症的综合诊断与治疗

犬子宫蓄脓是母犬生殖系统的一种常见病。根据临床症状结合影像学诊断可确诊本病。卵巢-子宫全切除术是目前最有效彻底的治疗方法。