促性腺激素释放激素受体在雌性山羊颈胸神经节中的分布

目的探讨颈胸神经节是否具备接受促性腺激素释放激素(GnRH)作用的条件。方法实验采用免疫组织化学SP法,观察GnRH受体在雌性山羊颈胸神经节中的分布特点。结果在颈胸神经节中,神经元胞体均为GnRH受体免疫反应阳性,其细胞膜以及膜下胞质中的颗粒状物质呈棕黄色,GnRH受体为强阳性表达,而胞核呈阴性表达。部分神经元可见其突起,呈黄色,GnRH受体为中等阳性表达。卫星细胞也呈黄色,GnRH受体为中等阳性表达。图像分析表明,呈阳性反应的神经元胞体与节内其他阳性成分相比,其GnRH受体相对表达量差异性极显著(P0.01)。结论在雌性山羊颈胸神经节中,神经元胞体是GnRH发挥作用的主要靶细胞,颈胸神经节具备接受GnRH作用的条件。提示GnRH有可能通过作用于颈胸神经节交感节后神经元上的GnRH受体来影响心肺器官的功能活动,即颈胸神经节可能是心肺功能活动中协调GnRH内分泌调节和该神经节自主神经调节两途径之间的中继站。     

孕激素受体在雌性山羊颈胸神经节的表达

目的观察雌性山羊颈胸神经节内神经元是否具备接受孕激素作用的条件。方法采用免疫组织化学SP法观察孕激素受体(PR)在成年雌性山羊颈胸神经节的表达。结果雌性山羊颈胸神经节内神经元胞体均呈孕激素受体免疫反应阳性;卫星细胞、施万细胞、神经突起和神经纤维中也有较弱的着色。神经元的细胞膜和细胞质呈棕褐色为强阳性,而神经元的细胞核着色出现异质性:85.10%的神经元细胞核为强阳性,其中31.91%的核仁呈清亮的空泡,为阴性;而14.90%的神经元细胞核呈空泡状不着色,其中7.45%的核仁为强阳性。卫星细胞、施万细胞和神经纤维均呈淡黄色,为弱阳性。图像分析表明,神经元胞体PR的相对表达量与其他非神经成分相比差异性极显著(P0.01)。结论在颈胸神经节内神经元是孕激素作用的主要靶细胞之一,提示孕激素有可能通过影响雌性山羊颈胸神经节内神经元的活动参与心脏功能的神经调节,而颈胸神经节上的PR可能作为一个网络节点协调孕激素对心脏功能的内分泌调节和自主神经对心脏活动的神经调节。     

催产素受体在雌性山羊肠系膜后神经节的分布

目的:研究雌性山羊肠系膜后神经节的神经元是否具备接受催产素(OT)调节的条件,探讨是否可以通过其节后纤维这一途径实现催产素参与生殖器官活动的调节.方法:采用免疫组织化学SP法研究雌性成年山羊肠系膜后神经节中催产素受体(OTR)的分布特点.结果:OTR在雌性山羊肠系膜后神经节的神经元、支持细胞、神经纤维及血管内皮细胞均有不同程度的阳性染色.神经元中OTR强阳性产物主要分布于胞质,多数呈棕褐色,为强阳性,少部分呈黄褐色,为中等阳性;神经纤维、血管内皮细胞和支持细胞等其他结构呈淡黄色,为弱阳性;核仁呈空泡状,着色极浅或不着色,为阴性.图像分析表明神经元胞体的相对表达量与其他非神经结构相比差异有统计学意义.结论:肠系膜后神经节的神经元具备接受催产素作用的条件,提示肠系膜后神经节的交感节后神经元可能是催产素对生殖系统的内分泌调节和经肠系膜后神经节的神经调节两种途径相互协调作用的节点.     

成年猕猴背根神经节nNOS免疫阳性神经元的分布

目的:观察正常成年猕猴背根神经节神经元型一氧化氮合酶(nNOS)免疫阳性神经元的分布。方法:ABC免疫细胞化学方法显示nNOS免疫阳性神经元,并用体视方法进行定量分析。结果:猕猴颈、胸、腰各段背根神经节nNOS免疫阳性神经元的分布相似,数量较多,阳性神经元的大小不等,多呈圆形或椭圆形;胞浆着色较深,胞核位于细胞中央,不着色,细胞被神经纤维束分隔成群。nNOS免疫阳性神经元以中型神经元为主,其次为小型神经元,其胞浆呈强阳性染色,细胞直径<50μm,大型神经元较少。颈、胸、腰各段背根神经节nNOS免疫阳性神经元的密度以及阳性细胞与总细胞数的比值均无明显差异。结论:猕猴背根神经节nNOS主要表达在中、小型神经元,提示NO可能主要参与痛觉等浅感觉的传导和调制。     

成年弥猴背根神经节nNOS免疫阳性神经元的分布

目的：观察正常成年称猴背根神经节神经元型一氧化氮合酶(nNOS)免疫阳性神经元的分布。方法：ABC免疫细胞化学方法显示nNOS免疫阳性神经元，并用体视方法进行定量分析。结果：猕猴颈、胸、腰各段背根神经节nNOS免疫阳性神经元的分布相似，数量较多，阳性神经元的大小不等，多呈圆形或椭圆形；胞浆着色较深，胞核位于细胞中央，不着色，细胞被神经纤维束分隔成群。nNOS免疫阳性神经元以中型神经元为主，其次为小型神经元，其胞浆呈强阳性染色，细胞直径＜50μm，大型神经元较少。颈、胸、腰各段背根神经节nNOS免疫阳性神经元的密度以及阳性细胞与总细胞数的比值均无明显差异。结论：称猴背根神经节nNOS主要表达在中、小型神经元，提示NO可能主要参与痛觉等浅感觉的传导和调制。     

大鼠心内神经节细胞雄激素受体的研究

有关雄激素受体在动物心肌组织的分布已有报道,但该受体在心内神经节细胞的配布尚未见报道.本实验取成年SD雄性大鼠的心房做冰冻切片,微波抗原修复后进行免疫组织化学染色(SP法,一抗为鼠抗雄激素受体1:200,博士德公司.二抗、三抗均为1:200,中山公司).光镜下观察,在心房的心内神经节发现雄激素受体阳性神经元,棕色的阳性反应颗粒定位于细胞核区,胞浆空白,亦见部分阳性神经元胞浆呈棕色淡染现象;阳性细胞数约占神经节细胞总数的60%;该类神经元胞体多数呈圆形或椭圆形,大、中、小型细胞均有发现.     

儿茶酚胺能神经元在雏鸡脊髓内的分布

用ＰＡＰ免疫组织化学方法对１０只白菜享雏鸡脊髓酷氨羟化酶阳性神经元的分布进行了研究。结果表明，脊髓内儿茶酚胺类，除来自脑的儿茶酚胺类神经元的下行纤维外；并有ＴＨ免疫反应阳性的神经元分布于脊髓的颈，胸和腰段。根据细胞形态和分布区域可将神经元分为３种：１．无绕中央管腹侧呈放射状分布的神经元；在腰髓的胞体为双极的圆形，其轴突细长；在胸髓的胞体为梭形，其突起粗短，两处细胞的突起均行于室管膜细胞之间，末端呈     

大鼠纹状体边缘区P物质受体的原位杂交和免疫组织化学研究

为了观察 P物质受体 m RNA在大鼠纹状体边缘区内的表达 ,本实验采用原位杂交和免疫组织化学方法从基因分子水平和细胞水平探索了大鼠纹状体边缘区能否合成 P物质受体。结果证明 ,P物质受体 m RNA阳性杂交信号不均匀地分布于大鼠中枢神经系统 ,在纹状体内的分布也不均匀 ,尾壳核内有少量中等大小的阳性胞体 ,苍白球内也有少量大的阳性胞体 ;而在尾壳核和苍白球之间的边缘区内则有许多梭形强阳性神经元胞体呈带状分布。 P物质受体单克隆抗体的免疫组织化学阳性神经元细胞体的分布与原位杂交组织化学结果一致。本研究结果提示 ,大鼠纹状体边缘区内可以合成 P物质受体 ,具有接受和整合 P物质的功能。     

猕猴胸腰段脊神经节nNOS免疫阳性神经元的分布

目的　观察正常猕猴胸腰段脊神经节神经元型一氧化氮合酶 (nNOS)免疫阳性神经元的分布。方法　用ABC免疫细胞化学方法显示nNOS免疫阳性神经元 ,并用体视学方法进行定量分析。结果　胸段和腰段脊神经节nNOS免疫阳性神经元的分布相似 ,均可见较丰富的nNOS免疫阳性神经元分布 ,神经元的大小不等 ,多呈圆形或椭圆形。胞浆着色较深 ,胞核位于细胞中央 ,不着色 ,细胞被神经纤维束分隔成群。nNOS免疫阳性神经元以中型神经元为主 ,其次为小型神经元 ,其胞浆呈强阳性染色 ,细胞直径 <50 μm ,大型神经元较少。胸、腰段脊神经节nNOS免疫阳性神经元的密度无明显差异 (P >0 0 5)。结论　在灵长类动物中 ,NO可能在感觉的传导和调节中发挥重要作用 ,但由于nNOS主要在中、小型神经元中表达 ,提示NO可能主要参与痛觉的调制     

BDNF及其受体TrkB在成年恒河猴脊髓的表达

目的观察脑源性神经营养因子BDNF及其高亲合力受体TrkB在成年恒河猴胸髓的表达分布,为成年恒河猴脊髓BDNF、TrkB的功能研究提供形态学依据。方法灌注固定成年雄性恒河猴,取其胸段脊髓制作冰冻切片,用BDNF、TrkB特异性抗体行免疫组织化学SP法染色,观察BDNF、TrkB免疫阳性物质在胸段脊髓的分布。结果成年恒河猴胸髓内均可见BDNF、TrkB免疫反应阳性神经元,反应产物呈棕黄色颗粒。BDNF和TrkB免疫反应阳性细胞遍及胸髓灰质各板层,在腹角、侧角以及中央管邻近区域均可见阳性神经元,它们的阳性产物主要定位于神经元胞浆中,胞浆着色深,胞浆与胞核的界线清楚。BDNF免疫反应细胞的胞核未见染色,而不同的TrkB阳性细胞其胞核染色不一。结论成年恒河猴胸髓存在BDNF、TrkB的表达,其作用可能与猴脊髓的正常生理功能和损伤后的修复有关。     

卵泡刺激素受体在大鼠下颌下腺及胃的定位和分布研究

目的研究卵泡刺激素受体（FSHR）在大鼠下颌下腺和胃的定位、分布.方法采用免疫组织化学ABC法.结果大鼠下颌下腺浆液性腺上皮细胞,颗粒曲管及大于颗粒曲管的导管上皮细胞以及胃底腺壁细胞均呈FSHR免疫反应阳性,阳性物质主要位于细胞质内,细胞核呈阴性反应.结论大鼠下颌下腺、胃底腺壁细胞存在FSHR,卵泡刺激素可能通过FSHR的介导对上述两个器官功能进行调控.     

Differential regulation of leucine-rich primary response gene 1 (LRPR1) mRNA expression in rat testis and ovary

In immature rat Sertoli cells, leucine-rich primary response gene 1 (LRPR1) represents a follicle stimulating hormone (FSH)-responsive gene; the function of the encoded protein is not yet known. LRPR1 mRNA expression is up-regulated very rapidly and specifically by FSH, both in cultured Sertoli cells and in vivo in regulation in more detail, in testis and ovary of fetal, immature, and adult rats. In addition, we have studied the expression of FSH receptor (FSHR) mRNA in relation to LRPR1 mRNA expression. In rat testis, LRPR1 mRNA and FSHR mRNA followed a similar expression pattern, during postnatal development and also at different stages of the spermatogenic cycle in the adult rat. Furthermore, after short-term challenge of the FSH signal transduction pathway in intact immature rats by injection with a relatively high dose of FSH, an inverse relationship between LRPR1 mRNA (up-regulation) and FSHR mRNA expression (down-regulation) was observed. Similar studies in the ovary provided completely different results. LRPR1 mRNA in the postnatal ovary is present well before expression of FSHR mRNA can be first detected. In addition, incubation of ovaries of immature rats with FSH or dibutyryl cyclic AMP (dbcAMP) did not result in up- regulation of LRPR1 mRNA expression. During fetal development, the LRPR1 mRNA expression pattern involved many more tissues, in contrast to the relatively tissue-specific expression of LRPR1 mRNA in gonads of 21 day old and adult rats. Moreover, LRPR1 mRNA expression could be detected as early as 12.5 days post-coitum, whereas FSHR mRNA is absent at this stage of fetal development. We concluded that the pronounced regulation of LRPR1 by FSH observed in the immature rat testis does not occur in the ovary. Furthermore, in the ovary LRPR1 mRNA expression does not appear to be dependent on FSH action. Finally, the LRPR1 gene product may play a general role during fetal development.      

Follicle stimulating hormone receptor protein is expressed in ovine uterus during the estrous cycle and utero-placenta during early pregnancy: An immunohistochemical study

Follicle stimulating hormone (FSH) is a well characterized gonadotropin that controls primarily development and functions of ovarian follicles in mammalian species. FSH binds to a specific G protein-coupled receptor (FSHR) belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although the primary location of FSHR is in the gonads (mainly in ovarian follicles), FSHR protein and/or mRNA have also been detected in extragonadal female reproductive tissues including embryo, placenta, endometrium, cervix, ovarian cancer tissues, and/or endometriotic lesions in several species. To determine the pattern of FSHR expression in the uterus and placenta, uterine tissues were collected at the early, mid- and/or late luteal phases of the estrous cycle from non-treated or FSH-treated ewes, and utero-placental tissues were collected during early pregnancy followed by immunohistochemistry and image generation. FSHR was immunolocalized to several uterine and utero-placental compartments including luminal epithelium, endometrial glands and surrounding stroma, myometrium, and endothelium and vascular smooth muscle cells in endometrium, myometrium and mesometrium. Intensity of staining and distribution of FSHR in selected compartments differed and seems to depend on the stage of the estrous cycle or pregnancy, and FSH-treatment. These novel data demonstrate differential expression of FSHR protein indicating that FSH plays a specific role in regulation of uterine and utero-placenta functions in sheep.     

FSH受体在大鼠下颌下腺的定位及其与FSH的共存关系

目的研究大鼠下颌下腺FSH受体的定位及其与FSH的免疫共定位关系,证明下颌下腺是FSH的一个靶器官。方法制备大鼠下颌下腺石蜡连续切片以免疫组化SABC法分别研究FSH和FSHR的免疫定位。结果大鼠下颌下腺浆液性腺泡上皮细胞、颗粒曲管、排泄管及分泌管上皮细胞内有FSHR和FSH免疫反应阳性物质共存,阳性物质分布在胞质内,胞核阴性。结论大鼠下颌下腺存在FSHR,是雌激素的靶器官,而且FSHR和FSH共存于下颌下腺相同的细胞,FSH可能通过FSHR的介导调节下颌下腺的功能。     

Seasonal changes in the hypothalamo‐pituitary‐testes axis of the japanese wood mouse (Apodemus speciosus)

Seasonal changes in the hypothalamo‐pituitary‐testes axis of the Japanese wood mice (Apodemus speciosus) were studied. The testes, epididymis, pituitary and hypothalamus were compared between mice in the breeding season (July) and non‐breeding season (October) using morphological techniques, and the plasma testosterone level was evaluated by enzyme immunoassay. Significant differences in these tissues were observed between the breeding season and the non‐breeding season. Specifically, differences in the non‐breeding season included 1) a decline in testicular and epididymal weights, arrest of spermatogenesis and decrease of serum testosterone concentration; 2) a decrease in the number of luteinizing hormone (LH)‐, follicle stimulating hormone (FSH)‐, prolactin (PRL)‐, and growth hormone (GH)‐immunoreactive cells, and decrease in the size of FSH, PRL, and GH‐immunoreactive cells; and 3) an increase in the size of gonadotropin‐releasing hormone (GnRH)‐immunoreactive neurons. Our findings indicate that the male adult Japanese wood mouse exhibits unique seasonal changes in the hypothalamo‐pituitary‐testes axis which are not found in laboratory mice. Anat Rec 260:366–372, 2000. © 2000 Wiley‐Liss, Inc.     

Intracerebroventricular infusion of neuropeptide Y up-regulates synthesis and accumulation of luteinizing hormone but not follicle stimulating hormone in the pituitary cells of prepubertal female lambs

Neuropeptide Y (NPY) is a putative neuroregulator of the reproductive axis in the central nervous system. In this study we evaluated the effects of central infusion of exogenous NPY on the secretory activity of pituitary gonadotrophic cells in prepubertal lambs. Immature female Merino sheep (n=12) were infused of Ringer solution (control) or 50 microg of NPY to the third ventricle for 5 min and then slaughtered 3 h later. Immunoreactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) cells were localised by immunohistochemistry using antibody raised against LHbeta and FSHbeta. Messenger RNA analyses were performed by in situ hybridisation using sense and antisense riboprobes produced from beta subunits of LH and FSH cDNA clones. The results were generated by computer image analysis to determine the area fraction occupied by immunoreactive and/or hybridising cells and optical density for immunostaining and hybridisation signal. LH in the blood plasma was determined by radioimmunoassay. It was found, that in the lambs infused with NPY the area fraction and optical density for immunoreactive LH cells and mRNA LHbeta-expressing cells increased significantly (P<0.001), compared to the vehicle-infused animals. The concentration of LH in the blood plasma did not differ between control and treated groups. The NPY infusions had no effect on the immunoreactivity of FSH cells or on expression of mRNA for FSHbeta. In conclusion we suggest that NPY may be an important component of mechanisms stimulating the synthesis and storage but not the release of LH in the pituitary gonadotrophs from prepubertal female sheep. In addition, this effect is specific for LH, no such effect was apparent on FSH.     

FSHR胞外区基因片段的原核表达及多克隆抗体的制备

目的 构建人卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)胞外区的原核表达载体,并用纯化的重组蛋白免疫小鼠,制备抗血清.方法 采用RT-PCR克隆人FSHR胞外区,将其克降到原核表达载体pET32a(+),并通过IPTG诱导目的 基因在大肠杆菌中表达,所获得的包涵体蛋白通过Ni-NAT离子交换柱层析纯化重组蛋白,并用SDS-PAGE和蛋白印迹法鉴定.结果 PCR扩增出1 047 bp目的 基因片段,测序证实克隆的基因序列与GenBank中的FSHR序列相符.工程菌pET32 a(+)/FSHR胞外区经IPTG诱导表达后,SDS-PAGE显示有新生的蛋白表达条带,Mr约为58 000,与预期的一致.其表达形式为不溶性包涵体,此包涵体蛋白通过Ni-NAT离子交换柱层析纯化,纯化的蛋白质经SDS-PAGE分析可见单一条带.该蛋白免疫小鼠制备抗血清,抗体效价为1:12 800,Western blot检测证实该抗体能与目的 蛋白发生特异性结合.结论 获得了人FSHR胞外区融合蛋白及特异性多克隆抗体,为进一步研究FSHR蛋白的功能奠定了基础.     

三叶因子3在大鼠腺垂体嗜色细胞中的定位

目的探讨三叶因子3(TFF3)在腺垂体远侧部嗜酸性细胞和嗜碱性细胞中的表达,明确TFF3在远侧部的分布。方法采用相邻切片的免疫组织化学染色,在相邻切片上分别显示TFF3/生长激素(GH)、TFF3/催乳素(PRL)、TFF3/促甲状腺激素(TSH)、TFF3/促肾上腺皮质激素(ACTH)、TFF3/卵泡刺激素(FSH)、TFF3/黄体生成素(LH)的表达。结果 TFF3和各嗜色细胞的免疫反应产物为棕黄色,主要位于细胞质,ACTH免疫阳性信号在胞膜也有表达,主要分布在腺垂体的远侧部。邻片显示TFF3存在于部分GH、PRL、TSH、ACTH、FSH、LH细胞,分别占19.4%、22.4%、9.2%、6.5%、35.7%、8.3%,以FSH最多,PRL、GH次之。结论垂体远侧部TFF3可分别表达于GH、PRL、TSH、ACTH、FSH、LH细胞。     

大鼠下颌下腺卵泡刺激素及其受体的原位杂交及核心片段的序列分析

目的探讨大鼠下颌下腺组织中是否表达卵泡刺激素(FSH)及其受体(FSHR),为进一步研究FSH对下颌下腺的功能调节提供理论依据。方法正常SD大鼠20只,体质量(200±20)g,腹腔麻醉后切取下颌下腺,采用原位杂交方法对FSH及FSHR进行细胞定位,探讨大鼠下颌下腺是否存在FSH及其受体mRNA,从下颌下腺组织中分别提取总RNA,运用RT-PCR获得FSH及其受体基因的cDNA核心序列,并对其序列进行分析。结果大鼠下颌下腺浆液性腺泡及颗粒曲管上皮细胞含有FSH和FSHR mRNA杂交信号,信号物质分布于胞质内,胞核呈阴性反应。从大鼠下颌下腺组织中扩增的FSH及FSHR基因的特异性条带分别为193bp和413bp。结论大鼠下颌下腺浆液性腺泡及颗粒曲管上皮细胞能合成卵泡刺激素及其受体,进一步说明下颌下腺是FSH作用的靶器官。