Expression of AMH in female fetal intersex gonads in the bovine

Anti Müllerian Hormone, AMH, is believed to be the main agent in the freemartin syndrome. Supposing an active role of freemartin gonads in AMH secretion, in the present study, we aimed at investigating the presence and the localization of AMH producing cells either in fetal or in adult freemartin gonads. Our finding of positive AMH cells in a 26-week-old freemartin fetus indicates an active role of masculinized freemartin gonads in AMH secretion. However, the positive reaction, limited to few cells grouped in 'nests' in proximity to testis cord-like structures, supports a chimeric origin of such cells, migrated from the male co-twin. No adult freemartin, irrespective from the degree of masculinization, showed any AMH positive cell.     

Steroidogenesis in fetal bovine gonads.

Gonadal steroidogenesis in bovine fetuses of 40 to 125 days gestation was examined using histochemical procedures and radioimmunoassay on gonadal cultures to determine the physiological correlates of gonadal morphogenesis in cattle. Gonadal morphology and the in vitro secretion patterns were distinct between the sexes by 45 days when testes secreted significantly higher levels of testosterone and androstenedione and lower levels of estrone and 17 beta-estradiol that the ovaries (p less than 0.0001). It would appear that the main steroid route in the ovaries of 45 to 70 day old fetuses is the androstenedione to estrone to 17 beta-estradiol pathway. The high estrone secretion and the decreasing levels of 17 beta-estradiol and testosterone in the ovaries of 70 to 125 day fetuses suggest an inhibition of 17 beta-hydroxysteroid dehydrogenase activity. It is postulated that this shift in steroid biosynthetic pathways may be related to the change in cellular events from mitosis to meiosis in fetal ovaries.     

雌激素受体在哺乳动物胎儿性腺中的分布

雌激素对胎儿性腺的发育起着重要作用,其作用是通过位于性腺的特异性受体,即雌激素受体(ER)来实现的。ER包括α(ERα)和β(ERβ)两种亚型。本文总结了此前关于两种雌激素受体亚型在胎儿性腺分布的研究,发现对于不同物种,胎儿性腺雌激素受体的分布明显不同,而且即使是在同种动物,在不同年龄段、不同生理状态、不同部位其受体的表达强度也有变化。     

Angiostatin and integrin alphavbeta3 in the feline, bovine, canine, equine, porcine and murine retina and cornea

PURPOSE: Angiogenesis is tightly controlled in the ocular tissues of domestic animals but its mechanisms are not fully understood. This is largely because of insufficient data on the expression of molecules that impact angiogenesis. Because angiostatin and one of its receptors integrin alphavbeta3 inhibit and promote angiogenesis, respectively, we hypothesized that the normal retina and cornea of domestic animals would express angiostatin but not integrin alphavbeta3. PROCEDURE: Normal eyes of the cat, cow, dog, horse, pig and rat were evaluated for angiostatin and integrin alphavbeta3 by light and electron immunocytochemistry and estern blots. RESULTS: Angiostatin was detected in the corneal epithelium of the cat, dog, horse, pig and rat, but was not found in cow corneal epithelium. Angiostatin was localized in the nerve fiber layer, ganglion cell layer, inner and outer plexiform layers, and the photoreceptor layer of the cat, cow, dog and rat. Horse and pig retinas showed additional staining in the matrix of the inner nuclear layer. Immunogold electron microscopy further confirmed angiostatin in cat retina. Western blots showed angiostatin in corneal and retinal homogenates. Integrin alphavbeta3 was absent in cornea and retina of all the species studied. CONCLUSION: These data show that angiostatin, an inhibitor of angiogenesis, is present while integrin alphavbeta3, which promotes angiogenesis, is absent in normal cornea and retina of the domestic animals in this study with the exception being angiostatin absence in cow corneal epithelium. Therefore, angiostatin may contribute to the anti-angiogenic environment in the normal domestic animal eye while its absence in the cow may contribute to greater propensity for corneal vascularization. Because integrin alphavbeta3 is one of the receptors for angiostatin, its absence may prevent angiostatin from killing normal retinal and corneal cells.     

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.     

Expression of fibronectin and its integrin receptor α5β1 in canine mammary tumours

Fibronectin and its integrin receptor α5β1 were studied by immunohistochemical methods in five normal canine mammary glands, four dysplastic glands and 18 mammary tumours. The aim of the study was to evaluate the possible changes in the α5β1 integrin receptor and its ligand fibronectin in relation to the metastatic capacity of canine mammary neoplasms. The immunostaining of α5β1 was very uniform in the hyperplastic glands but uneven in the mammary tumours. The expression of α5 and β1 was diminished in metastatic tumours but there were some α5-positive cells with pronounced features of malignancy and immaturity. Stromal fibronectin was increased in most cases and cytoplasmic staining of fibronectin was observed in epithelial and myoepithelial cells in mammary neoplasms but not in normal or dysplastic mammary tissue. There was no relationship between the content of α5β1 and the expression of fibronectin in canine mammary tumours.     

CDK5对羊驼皮肤黑色素细胞TYR和MITF mRNA表达的调节

为了证实CDK5是否参与羊驼毛色的形成,本研究主要对CDK5在羊驼黑色素细胞中调节TYR和MI TF的表达进行了研究.本研究首先采用免疫组织化学方法检测CDK5在羊驼皮肤黑色素细胞中的定位,再通过脂质体将CDK5转染于羊驼皮肤黑色素细胞,之后通过Western blot和qRT-PCR的方法检测转染后黑色素细胞中CDK5蛋白、TYR和MITF mRNA的表达.免疫组化结果显示CDK5位于黑色素细胞的胞质和细胞核内;Western blot结果显示转染组黑色素细胞中CDK5蛋白表达量明显高于对照组;qRT-PCR结果显示CDK5可下调MITF的表达,同时上调TYR的表达,转染组黑色素细胞中MITF和TYR mRNA的表达水平分别是对照组细胞的0.264 9和3.931 3倍.结果揭示CDK5可能通过调节黑色素细胞核中TYR和MITF mRNA的表达,从而参与调控羊驼毛色形成.     

斑马鱼E3泛素连接酶基因的生物信息学分析和mRNA表达

目的对斑马鱼E3泛素连接酶基因进行生物信息学分析,并研究其mRNA表达情况。方法利用生物信息学方法分析斑马鱼E3泛素连接酶基因的cDNA序列、蛋白质二级结构、结构域、氨基酸序列同源性,并构建进化树;利用整胚原位杂交技术研究目的基因在斑马鱼体内的mRNA表达模式。结果在NCBI基因库中找出TRIM54、TRIM55a、TRIM55b、TRIM63a、TRIM63b和TRIM101 6个斑马鱼E3泛素连接酶基因。这些基因之间有较高的同源性,二级结构以α-螺旋和无规卷曲为主,编码的蛋白质均含有3个保守结构域：环锌指、B-box锌指和卷曲螺旋。进化树分析显示,所有基因聚为4组：各物种TRIM54基因聚为一组;各物种TRIM55基因聚为一组,但靠近于斑马鱼TRIM101基因分支;TRIM63基因分为一组。整胚原位杂交结果显示,这些TRIM基因的mRNA均在受精后24 h的斑马鱼的肌节中表达,且TRIM55a、TRIM63a和TRIM101基因表达量较高。这些TRIM基因在肌肉发育和肌肉功能中可能发挥作用。结论斑马鱼的6个E3泛素连接酶基因有较高的保守性,其mRNA特异性表达于肌节部位。结果对深入研究这些基因在鱼类肌肉中的作用和指导鱼类生产具有一定的意义。     

围产期酮病牛脂联素mRNA和甘油三酯脂肪酶mRNA的表达

选择自然发生酮病牛10头、正常对照组奶牛（健康牛）10头。分别采取酮病牛和产后不同时期的奶牛尾部皮下脂肪组织,应用荧光定量PCR法检测脂联素（ADPN）mRNA与甘油三酯脂肪酶（HSL）mRNA表达的状况。酮病牛与对照组（健康）牛分别颈静脉采血,测定葡萄糖（GLU）、游离脂肪酸（NEFA）、β羟丁酸（BHBA）、甘油三酯（TG）以及胰岛素（INS）、胰高血糖素（GN）等相关指标。结果表明：酮病奶牛血糖显著降低,血浆NEFA和BHBA、血清INS降低和INS/GN变小;酮病奶牛HSLmRNA的表达低于围产期健康奶牛产后14d和产后28d,但ADPN mRNA却极显著地高于围产期健康奶牛产后的各生理时期,表明酮病牛因能量代谢紊乱脂肪动员产物NEFA和BHBA的增多,激发导致ADPN mRNA的表达,但ADPN mRNA的表达增加到一定量时反而会降低HSL mRNA的表达,说明ADPN参与了脂肪代谢的调节。     

5-Aza-dC对牛成肌细胞MyoD1启动子甲基化及mRNA表达的影响

旨在探究5-氮杂-2-脱氧胞苷（5-Aza-2’-deoxycytidine,5-Aza-dC）对牛成肌细胞的增殖和肌分化因子（myogenic differentiation 1,MyoD1）启动子甲基化以及mRNA表达的影响。本研究复苏了前期冻存的关岭牛成肌细胞,并进行培养,待其生长到对数生长期,再通过不同浓度的5-Aza-dC对牛成肌细胞进行处理,利用流式细胞仪检测细胞凋亡和周期;结合高通量检测方法检测MyoD1启动子甲基化水平,并利用qRT-PCR检测MyoD1及相关基因的表达水平。研究发现,0.1 μmol·L^-15-Aza-dC为最适浓度,该浓度下细胞抗凋亡因子Bcl的表达极显著降低（P<0.01）,促凋亡因子Bax的表达极显著提高（P<0.01）,促凋亡因子Caspase-9的表达显著提高（P<0.05）;周期因子Cyclin A2的表达显著提高（P<0.05）,而细胞因子Cyclin B1、Cyclin D的表达无显著变化;检测空白组和试验组MyoD1启动子甲基化水平发现,试验组甲基化水平极显著低于空白组（P<0.01）;而mRNA的表达水平极显著高于空白组（P<0.01）。5-Aza-dC能够通过改变Caspase-9、Bcl、Bax、Cyclin A2等基因的表达来调节关岭牛成肌细胞的增殖和凋亡,并能够有效降低MyoD1基因启动子的甲基化水平（P<0.01）;极显著提高其mRNA的表达量（P<0.01）。低浓度的5-Aza-dC能通过促进细胞凋亡及调控关岭牛MyoD1的甲基化水平来调控MyoD1的表达;同时推测,MyoD1基因启动子的甲基化水平能够影响关岭牛成肌细胞的生长发育,可为遗传标记辅助关岭牛的改良提供理论参考。     

猪肠道钠氢交换载体(NHE3)mRNA表达的肠段特异性和发育性变化

旨在探讨猪肠道钠氢交换载体（NHE3）mRNA表达的肠段特异性和发育模式,为NHE在养猪生产中的应用提供理论依据。选取遗传背景相同的1、7、26、30、60、90和150 d蓝塘和长白公猪各5头,测体质量后屠宰,取十二指肠、空肠、回肠和结肠组织样品;以18S rRNA为内标基因,用实时荧光定量PCR法检测NHE3 mRNA在60 d长白猪表达的肠段特异性及其在蓝塘和长白猪肠道表达的发育模式。结果显示：长白猪肠道NHE3 mRNA的表达丰度为结肠、十二指肠、空肠和回肠依次降低,且结肠显著高于空肠和回肠（P〈0.05）。不同猪种NHE3mRNA在十二指肠和空肠的表达模式相似;蓝塘和长白猪NHE3 mRNA的表达丰度分别在7和30 d（十二指肠）、7和26 d（空肠）达最高水平（P〈0.05）。不同猪种结肠NHE3 mRNA的表达模式不同,分别与其在十二指肠和空肠的发育呈现不同的模式;蓝塘猪结肠NHE3 mRNA的表达丰度在26、90和150 d时显著低于长白猪（P〈0.05）。以上结果说明,猪肠道NHE3 mRNA的表达受到发育阶段、品种和肠段的调控,且在十二指肠和空肠间具有品种稳定性。     

SQ RT-PCR检测不同品种猪脂肪组织SOCS-3基因表达

取4月龄八眉猪和大白猪背部皮下脂肪组织，提取总RNA，根据大鼠细胞因子信号转导抑制因子-3基因（Suppressor Of Cytokine Signaling-3，SOCS-3）设计并合成引物，以猪β-actin基因作为内参，优化反应条件和体系，半定量（Semi Quantitative，SQ）RT-PCR单管扩增猪SOC93基因，经琼脂糖凝胶电泳分离后，Dolphin-DOC凝胶图像分析软件分析，测定各条带的光密度值，检测八眉猪和大白猪脂肪组织中SOCS-3基因mRNA的表达差异。结果表明：八眉猪脂肪组织SOCS-3 mRNA的表达丰度极显著高于大白猪（P〈0．01）。这种差异可能是2种经济类型猪脂肪沉积能力不同的主要原因之一。     

冷应激对猪脂肪、肝脏组织中细胞因子信号转导抑制因子3 mRNA表达的影响

试验选用30头"军牧1号"猪,随机分成5组,包括常温对照组和4个冷应激组。常温组在21 ℃±2 ℃饲养,冷应激组的温度设置分别为:-10 ℃±2 ℃、-5 ℃±2 ℃、0 ℃±2 ℃、5 ℃±2 ℃。冷应激2 h屠宰后采集腹腔脂肪、颈部皮下脂肪、胸部皮下脂肪和肝脏组织样品,通过荧光定量PCR方法检测细胞因子信号转导抑制因子3(suppressor of cytokine signaling 3,SOCS3)的mRNA的表达量,初步探讨冷应激对猪脂肪及肝脏组织中SOCS3 mRNA表达量的影响。试验结果显示,冷应激猪SOCS3在腹腔脂肪、颈部皮下脂肪、胸部皮下脂肪和肝脏组织中,随着温度的降低,表达量逐渐升高,且总体差异显著(P＜0.05)。试验结果表明,SOCS3受到冷应激的影响,在不同脂肪组织部位发生了变化,可能参加了脂肪细胞因子的调控,从而改变脂肪组织分布及脂肪代谢平衡,为研究冷应激对机体的影响及作用机制奠定了试验依据和理论基础。     

牦牛、犏牛睾丸组织中SYCP 3基因mRNA表达水平研究

本文采用RT-PCR和荧光定量PCR对牦牛SYCP3基因的组织表达,以及SYCP3基因在牦牛和犏牛睾丸组织中的表达水平进行分析。结果表明,SYCP3基因在牦牛睾丸组织中特异表达,牦牛与犏牛睾丸组织中SYCP3基因的表达水平差异极显著（P〈0．01）。睾丸和附睾组织切片显示,牦牛睾丸组织中可见各级生精细胞,附睾内可见发育良好的精子,而犏牛睾丸组织中无次级精母细胞和更高级生精细胞、附睾内未观测到精子,与人和小鼠SY-CP3基因缺失或表达水平降低出现的表型一致,可以认为SYCP3基因的表达水平可能与犏牛的雄性不育有一定的关系。     

Expression of CD34 mRNA and protein in cattle

CD34 is a transmembrane glycoprotein expressed by hematopoietic progenitors and endothelial cells. It is used widely in the clinic for purification of human hematopoietic stem cells transplants, and as an endothelial marker for several species. The aim of this study was to produce an anti-bovine CD34 antibody and to characterize the expression of CD34 mRNA and protein in cattle tissues. The bovine CD34 cDNA was cloned by RT-PCR, and the expression of bovine CD34 mRNA investigated by RT-PCR and in situ hybridization. Polyclonal antibodies were raised against CD34 polypeptide fragments expressed in Escherichia coli, and affinity purified. Alternative splicing of bovine CD34 mRNA was observed. Both splice variants were readily observed in endothelium, while the variant encoding a truncated cytoplasmic domain was mostly undetectable in bone marrow mononuclear cells. A polyclonal antibody against an extracellular fragment of the CD34 polypeptide was characterized using Western blots, cytocentrifuge preparates, and paraffin sections. CD34 immunoreactivity was enriched in lineage-depleted bone marrow cells. The antibody labelled most blood vessel endothelia in fetal and adult cattle, with highest intensity in capillaries. Newly forming capillaries in granulation tissue were also stained. Lymphatic vessels and the endothelium of liver sinusoids were negative.