Immunocytological evidence for the presence of vertebrate FSH- and LH-like substances in the brain and thoracic ganglion of the swimming crab，Portunus trituberculatus

Investigating the distribution of follicle stimulating hormone （FSH） and luteinizing hormone （LH） in the swimming crab, Portunus trituberculatus, helps in the understanding of its reproductive regulation mechanism. This study has detected the location of vertebrate FSH- and LH-like substances in the brain and the thoracic ganglion mass with immunocytochemical techniques. Their immunoreaction is very weak or negative during the immature period and the post-spawning period, while it is stronger in the mature period. Localization of FSH-like immunoreactive cells resembles that of LH-like cells. These immunoreactive cells were observed chiefly in the anterior median cell-duster of the protocerebrum with stronger immunostaining. There were only a few in the inboard cell-cluster of the olfactory lobe of the deutocerebrum and the posterior cell-cluster of the tritocerebrum. Immunoreactive cells were located in all the three ganglia in the thoracic ganglion mass. These results indicated that substances resembling the vertebrate FSH and LH exist in P. trituberculatus, and their expression has some correlation with the developmental stage of ovaries, thus they might engage in the ovary development and ovulation.     

锯缘青蟹视神经节FSH和LH的免疫识别

甲壳动物的视神经节含有性腺抑制激素(Gonad inhibiting hormone, GIH),长期以来视神经节被理解为生殖抑制效应.本研究应用脊椎动物卵泡刺激素(Fllicle stimulating hormone,FSH)抗体和黄体生成素(Luteinizing hormone,LH)抗体,从锯缘青蟹(Scylla serrata)视神经节的视外髓、视内髓和视端髓检测到FSH和LH免疫细胞化学阳性反应.锯缘青蟹视神经节中的FSH和LH免疫阳性物质,可能参与了生殖神经内分泌调节.     

雄性三疣梭子蟹神经器官GnRH受体的免疫组织化学研究

采用MaxVisionTM免疫组织化学方法,以兔抗人GnRH受体的多克隆抗体,对雄性三疣梭子蟹(Portunus trituberculatus)的神经器官(视神经节、脑神经节和胸神经团)进行免疫组织化学定位.结果显示:GnRH受体的免疫阳性物质在雄性三疣梭子蟹视神经节、脑神经节和胸神经团的多个部位都有较为广泛的存在,定位在神经细胞的细胞质和神经髓质中,不同神经器官GnRH受体的免疫阳性强度有一定的差异.三疣梭子蟹神经器官存在GnRH受体的免疫阳性物质,为GnRH参与其神经调节作用提供了形态学依据.     

Immunorecognition of estrogen and androgen receptors in the brain and thoracic ganglion mass of mud crab, Scylla paramamosain

The brain and the thoracic ganglion of a crustacean can synthesize and secrete gonad-stimulating hormone (GSH) which stimulates the maturation of gonad. In the previous experiments, sex steroid hormones (estradiol, testosterone, progesterone, etc.) have been detected from the crustacean. However, the feedback regulation of sex steroid hormones on the brain and the thoracic ganglion of the crustacean has not been reported so far. In the present experiment, monoclonal antibodies were applied to investigate the immunorecognition of estrogen receptor (ER) and androgen receptor (AR) in the brain and the thoracic ganglion mass of Scylla paramamosain. The results showed that the distribution of the immunopositive substances of ER and AR was extremely similar. They distributed in the protocerebrum, deutocerebrum and tritocerebrum of the brain, and mainly in protocerebrum. In the thoracic ganglion mass, immunopositive substances distributed in the subesophageal ganglion, thoracic ganglion and abdominal ganglion, and mostly in subesophageal ganglion. Immunopositive substances of ER and AR mostly existed in the cytoplasm of neurons. The present study will provide morphological evidence for the origin and the evolution of ER and AR. In addition, the immunoreactivities of ER and AR suggested that the estrogen and androgen may be involved in the feedback regulation of crustacean neuroendocrine.     

Immunorecognition of estrogen and androgen receptors in the brain and thoracic ganglion mass of mud crab, Scylla paramamosain

The brain and the thoracic ganglion of crustacean can synthesize and secrete gonad-stimulating hormone (GSH) which stimulates the maturation of gonad. In the previous experiments, sex steroid hormones (estradiol, testosterone, progesterone, etc.) have been detected from crustacean. However, the feedback regulation of sex steroid hormones on the brain and thoracic ganglion of crustacean has not been reported so far. In the present experiment, monoclonal antibodies were applied to investigate the immunorecognition of estrogen receptor (ER) and androgen receptor (AR) in the brain and thoracic ganglion mass of Scylla paramamosain. The results showed that the distribution of the immunopositive substances of ER and AR was extremely similar. They distributed in the protocerebrum, deutocerebrum and tritocerebrum of the brain, and mainly in protocerebrum. In the thoracic ganglion mass, immunopositive substances distributed in the subesophageal ganglion, thoracic ganglion and abdomen ganglion, and mostly in subesophageal ganglion. Immunopositive substances of ER and AR mostly existed in the cytoplasm of neurons. The present study will provide morphological evidence for the origin and evolution of ER and AR. In addition, the immunoreactivities of ER and AR suggested that the estrogen and androgen may be involved in the feedback regulation of crustacean neuroendocrine.     

Immunorecognition of estrogen and androgen receptors in the brain and thoracic ganglion mass of mud crab, Scylla paramamosain

The brain and the thoracic ganglion of crustacean can synthesize and secrete gonad-stimulating hormone (GSH) which stimulates the maturation of gonad. In the previous experiments, sex steroid hormones (estradiol, testosterone, progesterone, etc.) have been detected from crustacean. However, the feedback regulation of sex steroid hormones on the brain and thoracic ganglion of crustacean has not been reported so far. In the present experiment, monoclonal antibodies were applied to investigate the immunorecognition of estrogen receptor (ER) and androgen receptor (AR) in the brain and thoracic ganglion mass of Scylla paramamosain. The results showed that the distribution of the immunopositive substances of ER and AR was extremely similar. They distributed in the protocerebrum, deutocerebrum and tritocerebrum of the brain, and mainly in protocerebrum. In the thoracic ganglion mass, immunopositive substances distributed in the subesophageal ganglion, thoracic ganglion and abdomen ganglion, and mostly in subesophageal ganglion. Immunopositive substances of ER and AR mostly existed in the cytoplasm of neurons. The present study will provide morphological evidence for the origin and evolution of ER and AR. In addition, the immunoreactivities of ER and AR suggested that the estrogen and androgen may be involved in the feedback regulation of crustacean neuroendocrine.     

Distribution of α-MSH-like immunoreactive cells in the nervous system, Hatschek''''s pit and other tissues of amphioxus, Branchiostoma belcheri

Immunohistochemical localization of a-melanocyte-stimulating hormone (α-MSH) in the nervous system, Hatschek's pit and other tissues of amphioxus (Branchiostoma belcheri) was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of brain vesicle, the dorsal side and the surrounding of nerve tube, and in the epithelial cells of Hatschek's pit, the zone 1, 3, and 6 of endostyle and gut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek's pit in amphioxus does not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of the present study provided a new evidence for the endocrinology of Hatschek's pit and for the origin and evolution of vertebrate adenohypophysis.     

虎纹蛙促性腺激素及其释放激素含量的日变化

利用具有高度特异性的放射免疫测定法,研究了10月份虎纹蛙成蛙脑和脑垂体GnRH、以及脑垂体和血浆GtH含量的日变化,结果表明：雌蛙脑和脑垂体mGnRH含量、以及脑垂体cGnRH-Ⅱ含量都存在有明显的日变化;雄蛙脑垂体和血浆LH水平,以及雌、雄蛙血浆FSH水平也都存在明显的日变化;对各种激素日变化产生的原因进行了探讨.     

拟穴青蟹GnRH-R的免疫识别

促性腺激素释放激素(GnRH)及其受体(GnRH-R)对生殖具有重要的调控作用.迄今无脊椎动物GnRH及GnRH-R的研究尚少.本研究采用兔抗人GnRH-R、兔抗果蝇GnRH-R和兔抗海鞘GnRH-R的抗体,应用免疫印迹和免疫共沉淀技术对拟穴青蟹(Scyllaparamamosain)GnRH-R进行了免疫识别,所得结果如下:1)经免疫印迹检测,拟穴青蟹脑、胸神经团、视神经节和精巢中共有的免疫阳性条带分子质量为45~55 ku.脑和胸神经团中另有一条36 ku条带;在精巢中,兔抗人GnRH-R和兔抗果蝇GnRH-R抗体只有一条28 ku条带,而兔抗海鞘GnRH-R抗体显示有28,36和30 ku的条带.2)利用免疫共沉淀技术,分离出与GnRH-R抗体相结合的两种物质,分子质量分别为38.1和54.0 ku,分别与哺乳动物和非哺乳动物GnRH-R的分子质量接近.该发现提示拟穴青蟹体内存在GnRH-R,为深入了解甲壳动物生殖调控机理提供了理论依据.     

广西巴马小型猪不同年龄生殖激素水平的分析

目的探讨广西巴马小型猪不同年龄生殖激素水平。方法利用ELISA酶免疫试剂盒和多功能标记免疫测定系统,对不同年龄的雌雄巴马小型猪血清生殖激素浓度进行测定。结果雄性巴马小型猪血清TESTO、FSH浓度从2月龄到6月龄呈上升趋势,在6月龄时达到最大值(分别为12.2 ng±8.9 ng/mL和64.6 ng±6.1 ng/mL),6月龄后浓度呈下降趋势;血清LH浓度从2月龄到4月龄呈上升趋势,在4月龄时达到最大值(46.8 ng±12.5 ng/mL),4月龄后呈下降趋势。雌性巴马小型猪从2月龄到4月龄,血清FSH、LH、E2浓度都呈现出显著升高趋势,到4月龄时达到最大值(分别为51.9 ng±54.0 ng/mL,23.2 ng±31.6 ng/mL,79.3 pg±93.0 pg/mL),4月龄后又都呈现较明显下降趋势。     

补肾活血颗粒对多囊卵巢综合征大鼠血清性激素水平、 血糖及胰岛素的影响

目的 观察补肾活血颗粒对多囊卵巢综合征（PCOS）模型大鼠血清性激素水平、血糖及胰岛素的影响。方法 采用颈背部皮下注射脱氢表雄酮（DHEA）造模法建立PCOS大鼠模型,将72只雌性SD大鼠随机分为正常对照组、模型组、二甲双胍组、中药高剂量组、中药中剂量组、中药低剂量组,每组12只,各组给予相应药物连续灌胃 20 d,用酶联免疫吸附测定法测定血清17-羟孕酮（17-OHP）、促黄体生成素（LH）、睾酮（T）、促卵泡素（FSH）、空腹 血糖（FPG）及胰岛素（INS）水平。结果模型组血清中17-OHP、LH、T、FSH、FPG、INS水平明显高于正常对照组 (P<0.01 ,P<0. 05 ),中药高剂量组降低大鼠血清LH、T、FSH及INS水平最为明显(P<0.01),二甲双胍组、中药中剂量组显著降低FPG（P<0. 01）,中药各治疗组均能降低17-OHP（P<0. 05）。结论 补肾活血颗粒能对PCOS 大鼠有一定的治疗作用,可改善激素水平、血糖及胰岛素的异常     

采用染料亲和层析的方法纯化卵泡刺激素

采用染料亲和层析、QAE离子交换层析等纯化步骤，从绝经期妇女尿促性腺激素(HMG)粗品中分离纯化获得高纯度卵泡刺激素(FSH)产品，其中FSH的生物活性达到268.1IU／mg，卵泡刺激素与黄体生长素(LH)的活性比值达到259，产品超过欧洲药典对高纯度卵泡刺激素的要求。此工艺FSH的活性回收率高达65％，而且分离纯化步骤少，简单易行，适合于大规模生产。     

甜菜碱对蛋鸡产蛋期血清激素及环腺苷酸影响的研究

取３００日龄罗曼蛋鸡７６８羽,随机分为２组（每组设４个重复,每个重复９６羽）,在自由采食、饮水和１６ｈ／ｄ光照的条件下,分别饲喂添加０ｍｇ／ｋｇ和７５０ｍｇ／ｋｇ甜菜碱的玉米－豆粕型日粮９１ｄ。结果表明,甜菜碱使蛋鸡血清中促卵泡生成素（ＦＳＨ）、促黄体生成素（ＬＨ）和雌二醇（Ｅ２）含量分别提出了５６．５９％（Ｐ〈０．０１）,２０．７５％（Ｐ〉０．０５）和２２．０９％（Ｐ〈０．０５）;使下丘脑、腺垂     

锯缘青蟹胸神经团的组织学研究

锯缘青蟹胸神经团食道下神经节、胸神经节和腹神经节高度愈合而成，胸神经团具有3种类型的神经分泌细胞（NSC），食道下神经节腹面NSC的数量多于背面，胸神经节背面的NSC靠近步足神经分布；腹面的NSC较多，靠近胸动脉孔发布，腹神经节的NSC聚集成群，主要位于腹面，不同类型的NSC通常参杂分布。     

Position in the evolution of reproductive endocrine of amphioxus,Branchiostoma belcheri

This review summarizes the recent discoveries of many authors who found that in amphioxus Hatschek’s pit is capable of synthesizing vertebrate gonadotropin-like substance, and that the content of gonadotropin-releasing hormone in the amphioxus’ body shows a positive correlation with the reproductive cycle, and that the sex steroid hormone exists in gonads. Exogenous hormones could promote gonadal development, maturation and reproductive activity in amphioxus. A possible implication might be that the reproductive activity in amphioxus is regulated by reproductive hormones like vertebrate, indicating the existence of primitive reproductive endocrine regulatory axis, brain vesicle-Hatschek’s pit-gonads axis, as compared with regulatory axis of vertebrate. It will provide a new line for establishing the position of reproductive endocrine evolution in lancelet.     

Distribution of CRH-and ACTH-like immunoreactive cells in amphioxus,Branchiostoma belcheri

Immunocytochemical studies on the nervous system,Hatschek's pit,digestive tract and gonads tissues of an amphioxus(Branchiostoma belcheri)were performed using polyclonal antibodies against human corticotrophin-releasing hormone(CRH)and human adrenocorticotropin(ACTH).The results showed that many CRH-like immunoreactive neurons were distributed specifically on the dorsal side and ventral side of brain vesicle,while a few CRH-like neurons and their fibers in spinal cord.At the same time,the epithelial cells in the basic region of Hatschek's pit were shown immunopositive to CRH antibody.In gonads(ovary and testis),CRH-immunopositive substance was localized in the cytoplasm near oocyte nucleus and in early spermatogenic cells.ACTH-like immunoreactivities were observed specially in the neurons and their protrusions localized on the ventral side of the brain vesicle and in spinal cord,and also in epithelial cells of Hatschek's pit,enteric neurons of digestive tract,oocytes in ovary and in early spermatogenic cells as well.It was found for the first time that CRH-like neurons existed in the middle region of brain vesicle(corresponding to the hypothalamus of vertebrates)and ACTH-like immunopositive cells existed in Hatschek's pit,implying that a control mechanism between brain vesicle and Hatschek's pit maybe had been already built in amphioxus as that in vertebrates.The present study will provide new morphological evidence for the origin and evolution of ACTH.In addition,the immunoreactivities of CRH and ACTH in the digestive tract and gonads suggested other physiological function of CRH and ACTH in amphioxus.     

Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid

A variety of hypophysiotropic peptides or proteins have been reported to be present in mammalian gonads. Inhibin, a hormone that under most circumstances selectively suppresses the secretion of follicle-stimulating hormone (FSH) but not luteinizing hormone (LH), has been isolated from the gonadal fluids of several species and characterized as a heterodimeric protein consisting of alpha- and beta-polypeptides associated by disulphide bonds. The complete amino-acid sequences of the precursors of porcine and human inhibin alpha-subunits and two distinct porcine inhibin beta-subunits (beta A and beta B) have been deduced from complementary DNA sequences. Gonadotropin releasing peptides have also been found in the gonad and have generally been shown to be active in radioreceptor assays for gonadotropin releasing hormone (GnRH) but to exhibit different chromatographic and immunological characteristics from those of GnRH. During our purification of inhibin from porcine follicular fluid, we noted fractions that could stimulate the secretion of FSH by cultured anterior pituitary cells. We report here the purification of an FSH releasing protein (FRP) and its characterization by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions and by partial sequence analysis. Our results indicate that porcine gonadal FRP is a homodimer consisting of two inhibin beta A-chains linked by disulphide bonds. FRP is highly potent (50% effective concentration (EC50) approximately 25 pM) in stimulating the secretion and biosynthesis of FSH but not of LH or any other pituitary hormone. In contrast to the effects of GnRH and other reported gonadal gonadotropin releasing fractions, the action of FRP is not mediated by GnRH receptors.     

奶牛生殖激素浓度与胎次的相关性研究

为探讨奶牛血液生殖激素浓度随不同胎次的变化规律及相关性和回归关系,用ELISA法测定了48头奶牛促卵泡激素(FSH)、促黄体激素(LH)、绒毛膜促性腺激素(β-HCG)、睾酮(T)、孕酮(P)和雌二醇(E2)的浓度,分析了激素浓度胎次间的相关关系和回归方程.结果表明,HCG浓度以第0、1胎为最高和最低(p<0.05);P、LH和E2浓度均以第三胎最高,各胎次P浓度差异极显著(p<0.01),第3胎与第1、2胎E2浓度差异显著(p<0.05).各胎次间FSH和T浓度差异不显著(p>0.05);胎次与六种激素以及各激素间的相关性均很小,胎次与孕酮(P)和促卵泡激素(FSH)呈负相关,FSH与LH、T和E2为负相关,LH与E2间的相关性具有显著性(p<0.05);六种激素与胎次回归方程的截距A和回归系数均不相同.     

大负荷运动对摔跤运动员内分泌指标影响

为研究大负荷运动刺激下古典式摔跤运动员适应性内分泌指标的变化规律,选择男性优秀古典式摔跤运动员11名,动态观察在大负荷运动前后以及1d后促肾上腺皮质激素(ACTH)、卵泡刺激素(FSH)、生乳素(PRL)、黄体生成素(LH)、皮质醇(C)、血清睾酮(T)指标和红细胞压积(Hct)的变化.结果:ACTH从运动后即刻起至运动后次日浓度有上升的趋势;而FSH,PRL和LH在运动后即刻上升(P<0.01,P<0.05),而在运动后次日就已经有恢复的趋势(P<0.01,P<0.05);血清皮质醇先升后降,血清睾酮在运动后即刻没有明显变化,而在运动后次日上升,表现出来的T/C比值持续升高;而血细胞压积(Hct)变化不明显.结果提示,大负荷运动对古典式摔跤运动员内分泌指标中的ACTH,T的升高有长时效应;而对FSH,PRL,LH和C的变化出现即刻效应.