SRIF及CSH对斜带石斑鱼脑垂体生长激素合成和分泌的调控

斜带石斑鱼 (Epinepheluscoioides)属于雌性先成熟、具有性转变的雌雄同体鱼类。生长激素释放抑制因子 (SRIF)是鱼类生长激素 (GH)分泌的主要抑制性调节剂 ,半胱胺 (CSH)可抑制SRIF的作用。本文采用静态孵育系统 ,应用RPA及RIA研究SRIF及CSH对斜带石斑鱼GHmRNA表达及GH分泌的调节。结果显示 ,SRIF能以剂量依存方式抑制斜带石斑鱼脑垂体释放GH ,时间越长作用越强。但SRIF作用 2 4h对GHmR NA水平的影响不显著 ,表明SRIF是斜带石斑鱼GH释放的抑制性调节剂 ,对GHmRNA的表达没有明显影响。较低剂量的CSH (10 -4- 10 -2 mol/L)使斜带石斑鱼的GH释放量增加 ,较高剂量 (10 -1mol/L)的CSH引起的GH增加趋势减缓 ,这种现象可能与较高剂量的CSH不仅抑制下丘脑SRIF的释放 ,同时影响GHRH的释放 ,使得GH的分泌量增幅下降有关 ;无论是较高剂量还是较低剂量的CSH都不能使GHmRNA的水平增加 ,表明CSH只能引起GH的释放量增加 ,不影响GH的合成。GnRH与CSH共同作用引起的GH释放量明显高于CSH单独作用的效应 ,其主要原因是由于GnRH促进GHmRNA的表达所致     

几种神经内分泌因子对鲤促性腺激素释放激素(GnRH)释放的调节作用:离体研究

用离体静态培育系统进行的初步研究表明 ,在幼鲤 ,多巴胺 (DA)显著刺激下丘脑片段和脑垂体碎片释放GnRH ,并且是剂量依存的 ;促甲状腺素释放激素 (TRH)和γ -氨基酸丁酸 (GABA)对GnRH的释放没有影响。在成鲤 ,DA抑制下丘脑片段和脑垂体碎片释放GnRH ,而TRH和GABA刺激GnRH的释放 ;DA对GABA刺激的GnRH释放也具有抑制作用 ;TRH和GABA的协同作用对下丘脑和脑垂体GnRH释放活动的影响明显低于TRH和GABA的单独作用 ,表明TRH和GABA之间可能存在着某种GnRH释放的相互消竭作用。     

重组斜带石斑鱼生长激素及其抗血清在放射免疫测定中的应用

将斜带石斑鱼(Epinephelus coioides)生长激素成熟多肽cDNA序列克隆到质粒pRSET,与6x组氨酸等原核编码序列融合获得重组质粒pRGH6,转入大肠杆菌BL21(DE3),获得高效表达,表达量占细菌总蛋白的43％。免疫印迹证明表达产物为含斜带石斑鱼生长激素的融合蛋白,Ni^2 亲合层析柱纯化融合蛋白,以此为抗原免疫家兔制备特异性的抗血清。以纯化的重组生长激素和特异性的抗血清建立斜带石斑鱼生长激素的放射免疫测定法,该方法的灵敏度、特异性和重复性均达到测定血液生长激素的水平。研究了多巴胺的受体激动剂阿扑吗啡对静态孵育斜带石斑鱼脑垂体碎片释放生长激素的影响,结果表明,阿扑吗啡能以剂量依存方式促进斜带石斑鱼垂体释放生长激素。     

半胱胺对草鱼下丘脑-脑垂体组织共孵育中生长激素分泌的影响

采用静态孵育和放射免疫测定技术,研究了生长抑素抑制剂半胱胺盐酸盐对草鱼脑垂体组织单独孵育或下丘脑脑垂体组织共孵育中生长激素分泌的影响。结果表明：脑垂体组织单独孵育时,半胱胺盐酸盐(0．1、1和10mmol／L)对基础生长激素分泌无影响;而下丘脑脑垂体组织共孵育时,半胱胺盐酸盐(0．1、1和10mmol／L)对基础生长激素分泌有明显影响,且是剂量依存的。神经肽hGHRH、sGnRH—A和LHRH—A对CSH影响的下丘脑脑垂体组织共孵育中生长激素分泌均无协同作用。我们认为,半胱胺盐酸盐可在下丘脑水平调节生长激素释放,半胱胺盐酸盐调节草鱼离体生长激素分泌是由下丘脑途径介导的。     

睾酮对日本鳗鲡离体下丘脑-脑垂体复合物GtH合成与分泌的影响

用离体孵育的方法研究了睾酮（T）对日本鳗鲡（Anguilla japonica)完整的正丘脑-脑垂体复合物（hypothalamus-pituiary complex,HPC)、分离的下丘脑（hypothalamus,H)加脑垂体（pituitary,P)以及单独的脑垂体（pituitary,P)促性腺激素（GtH)合成与释放的影响,在不加T的孵育液中孵育,HPC组的孵育液及其脑垂体中的GtH含量最高,H P组次之,而P组最低,表明下丘脑通过GnRH直接刺激脑垂体GtH的合成与释放,在加入T的孵育液中孵育,P组的孵育液及脑垂体中的GtH含量显著增加,并且和孵育液中的T呈剂量依存的正反馈作用,当T和H与P一起孵育时,低浓度的T（0.1和1μmol/L)刺激HPC组和H P组的GtH释放,呈现正反馈作用,而高浓度的T（10μmol/L)则抑制HPC组和H P组的GtH释放,表现负反馈作用。这些结果直接证明日本鳗鲡下丘脑对脑垂体GtH分泌的调控以及性类固醇激素（如雄鳗的睾酮）对脑垂体GtH分泌的反馈作用。     

瘦素对GH3细胞分泌和凋亡的影响

本文旨在探讨瘦素(leptin)对垂体瘤GH3细胞的生长激素(growth hormone,GH)分泌的作用及可能机制。我们观察了leptin对GH3细胞生长激素的分泌、细胞的增殖和凋亡的影响,结果显示:leptin(1、10和100 nmol/L)对GH3细胞的基础GH分泌有抑制作用(P<0.05),并存在剂量依赖效应。用10 nmol/L的leptin作用30 min、1和3 h对GH分泌无明显影响,而作用1、2和3 d则可抑制GH分泌(P<0.05)。应用噻唑蓝(MTT)比色分析法和流式细胞仪研究leptin对GH3细胞增殖和凋亡的影响,我们发现leptin对GH3细胞的增殖有抑制作用,并存在剂量依赖效应;同时leptin可减低GH3细胞的S期细胞比例,而G1期的细胞比例明显增加,进入2相和4相的凋亡细胞比例增加。上述结果表明,leptin可抑制GH3 细胞的基础GH分泌,其作用可能是通过抑制GH3细胞的DNA合成,促进GH3细胞的凋亡,从而影响GH的分泌。     

野生鲇鱼生长激素分泌的季节变化及其神经内分泌调控

采用离体垂体碎片灌流孵育系统 ,将处于性腺退化期野生鲇鱼垂体切成约 1mm3 的碎片 ,用M 199冲洗之后放入灌流柱的两层Cytodex -Ⅲ微载体之间 (温度为 19± 1℃ )。每 5分钟收集一管灌流液 ,- 2 5℃贮存待测GH。采用鲤鱼GH放射免疫测定方法 (cGHRIA)测定鲇鱼垂体碎片灌流液以及血清和垂体中的GH含量。结果表明 :促黄体素释放激素类似物 [desGly10 (D Ala6)LHRHethylamide ,LHRH A]不能显著刺激离体垂体碎片基础GH分泌 ,注射LHRH A后不能显著提高血清基础GH水平 ;注射DA能显著提高鲇鱼血清基础GH水平 ,APO能以剂量依赖方式显著刺激垂体碎片基础GH分泌。雌、雄鲇鱼血清GH水平在 6月达到峰值 ,垂体GH水平在 3月和 7月份各出现一个峰值 ,各个季节雌鱼垂体和血清GH水平均显著高于雄鱼。鲇鱼血清和垂体GH水平与生殖周期有密切联系。     

虎纹蛙促性腺激素释放激素分泌调节的离体研究

利用离体静态孵育系统和放射免疫测定法,研究了性成熟的虎纹蛙雌蛙离体的视前－下丘脑－正中隆起（P－H－ME）片段促性腺激素释放激素（GnRH)的分泌调节。结果表明：γ－氨基丁酸（GABA）对成熟前期蛙离体P－H－ME片段的哺乳类GnRH（mGnRH)的释放有显著的刺激作用;随着GABA作用浓度的增加,刺激作用逐渐增强。100μmol/L的多巴胺（DA）及1μmol/L和10μmol/L的雌二醇(E2）则显著抑制鸡ⅡGnRH(cGnRH-Ⅱ)的释放。10μmol/L和100μmol/L的睾酮（T）以及10μmol/L的E2显著刺激冬眠期蛙P－H－ME片段mGnRH的释放。这些结果表明,GABA,DA及E2和T对虎蚊蛙GnRH的释放有直接的调节作用。     

斜带石斑鱼生长激素/催乳素家族基因cDNAs的分子克隆及其进化意义

从斜带石斑鱼垂体提取总。RNA,再取其50ng合成SMART cDNA。从所构建的垂体SMART cDNA质粒文库中筛选到生长激素／催乳素基因家族的2个成员的全长cDNA片段：生长激素(GH)基因全长为938bp,编码204个氨基酸;催乳素基因(PRI．)全长为1429bp,编码212个氨基酸。采用计算机软件Mega 2和CLUSTAL W1．64b对9种鱼的生长激素／催乳素基因家族的3个成员(GH、PRL和生长催乳素SL)的氨基酸序列进行系统分析,构建NJ分支系统树,对于序列中的插入／缺失位点则采用Pairaise Deletion,1000次自展(Bootstrap)分析计算各节点支持率。根据3个基因的氨基酸序列构建的系统树表明,石斑鱼与金头鲷、金鲈和牙鲆聚成一类,虹鳟与大马哈鱼聚成一类,鲫鱼与鲶鱼聚成一类,鳗鲡成另外一类。根据石斑鱼全长cDNA推断的氨基酸序列比较表明,SL相对GH和PRL有较高的保守性。石斑鱼的GH、PRL和SL的氨基酸同源性在24％～31％,但其C-端的氨基酸同源性较高,尤其是C-端的3个Cys是严格保守的。其中SL与GH的同源性(30．8％)高于与PRL的同源性(25．6％),GH和PRL的同源性最低(24．1％)。     

垂体腺苷酸环化酶激活多肽对鲤鱼脑垂体细胞内cAMP和Ca^2＋的影响

采用环腺苷酸 (cAMP)放射免疫测定法和活细胞内Ca2 荧光探针Indo 1,研究绵羊垂体腺苷酸环化酶激活多肽 (oPACAP)对原代培养的鲤鱼脑垂体细胞内cAMP和游离Ca2 ([Ca2 ]i)的影响 ,以期探讨PACAP调节脑垂体生长激素 (GH)分泌的机制受体后。oPACAP 38和oPACAP 2 7以剂量依存方式促进脑垂体细胞内cAMP释放和合成。oPACAP 38和oPACAP 2 7也能升高脑垂体细胞内 [Ca2 ]i 水平 ,该作用会因用EGTA消竭细胞外Ca2 ([Ca2 ]e)而迅速消失 ;L型电位敏感性Ca2 通道 (VSCC)阻断剂硝苯吡啶可抑制oPACAP 38诱导的 [Ca2 ]i 水平的升高 ,而当用硝苯吡啶预处理脑垂体细胞 ,oPACAP 38诱导 [Ca2 ]i 水平升高作用完全被抑制。可见 ,PACAP刺激鲤鱼脑垂体GH分泌机制包括依赖于cAMP和依赖于通过L型VSCC内流的 [Ca2 ]e 的机制。     

Effects of gonadotropin-releasing hormone on growth hormone secretion and gene expression in common carp pituitary

Using radioimmuno- and ribonuclease protection assays, we examined the effects of gonadotropin-releasing hormone and its analogs on the growth hormone mRNA level and growth hormone secretion in common carp (Cyprinus carpio) pituitary fragments with static incubation. After a 24 h treatment, sGnRH ([Trp(7),Leu(8)]-LHRH) and sGnRH-A ([D-Arg(6),Pro(9)]-LHRH) (0.1 nM-1 microM) elevated the GH mRNA level and stimulated the GH secretion in a dose-dependent manner, with a higher potency for sGnRH-A. In a time-course experiment, the function of sGnRH and sGnRH-A (10 nM) on GH secretion was observed after 6 h incubation, while no action on the GH mRNA level were noted until 12 h after treatment. Comparing mammalian GnRH, avian GnRH and piscine GnRH, sGnRH and sGnRH-A showed the highest potency in increasing GH mRNA level and GH-release, followed by cGnRH-II ([His(5),Tyr(8)]-LHRH), and finally LHRH and LHRH-A([D-Trp(6), Pro(9)]-LHRH). These findings, taken together, suggest that GnRH not only can influence GH release, but also play a role in the regulation of GH synthesis.     

The effect of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the pituitary of goldfish, Carassius auratus

The goldfish brain contains at least two forms of gonadotropin-releasing hormone (GnRH): sGnRH and cGnRH-II. In goldfish sGnRH and cGnRH-II are present both in the brain and pituitary, and exert direct effects via specific GnRH receptors stimulating growth hormone (GH) and gonadotropin hormone (GtH) synthesis and secretion. In this study, we investigated the effects of sGnRH and cGnRH-II on GtH subunit (alpha, FSH-beta and LH-beta) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 microg/fish) stimulated GtH-alpha, FSH-beta and LH-beta mRNA levels after 24 h. For in vitro studies, goldfish pituitary fragments were treated continuously for 12 h with 10(-7) M sGnRH or cGnRH-II. Both sGnRH and cGnRH-II stimulated GtH-alpha, FSH-beta, LH-beta and GH mRNA levels, however, cGnRH-II appeared to have a more pronounced effect. Similar experiments were carried out using cultured dispersed goldfish pituitary cells. In this study, treatments for 12 h with 10(-7) M sGnRH or cGnRH-II also stimulated GtH and GH gene expression. The present results provide a basis for the investigation of the signal transduction pathways that mediate GnRH-induced changes in GtH subunit and GH mRNA levels in the goldfish pituitary.     

Lack of gonadotropin releasing-hormone action on in vivo and in vitro growth hormone release,in rainbow trout (Oncorhynchus mykiss)

In order to understand the mechanisms implicated at the hypothalamo-pituitary level in growth-reproduction interaction in salmonids, the gonadotropin-releasing hormone (GnRH) action on growth hormone (GH) release was studied, in rainbow trout (Oncorhynchus mykiss). In vivo, acute treatment with salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and an sGnRH analogue [(DArg6Pro9)sGnRH] was performed on catheterized fish. The different forms of GnRH have no effect on plasma GH levels of immature and mature fish, but induce a stimulation of gonadotropin (GtH) release in mature fish. In the present work we have adapted and validated a culture system for GH regulation studies. In this system, increasing doses of sGnRH, (DArg6Pro9)sGnRH and cGnRH-II are inactive on GH release (24 hr incubation) in immature or mature fish, but stimulate GtH release in a dose-dependent manner. sGnRH (10⁻⁶ M) has no action on GH release, whatever the incubation time (15 min–24 hr). In a perifusion system, sGnRH also has no action on GH release but stimulates GtH release. The present results obtained using in vivo and in vitro techniques adapted for GH regulation studies, show that GnRH does not function as a growth hormone-releasing factor in rainbow trout as it does in goldfish.     

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary.

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Time- and dose-related effects of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the goldfish pituitary

The goldfish brain contains two molecular forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). In a preliminary report, we demonstrated the stimulation of gonadotropin hormone (GtH) subunit and growth hormone (GH) mRNA levels by a single dose of GnRH at a single time point in the goldfish pituitary. Here we extend the work and demonstrate time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH gene expression in vivo and in vitro. The present study demonstrates important differences between the time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels. Using primary cultures of dispersed pituitary cells, the minimal effective dose of cGnRH-II required to stimulate GtH subunit mRNA levels was found to be 10-fold lower than that of sGnRH. In addition, the magnitudes of the increases in GtH subunit and GH mRNA levels stimulated by cGnRH-II were found to be higher than the sGnRH-induced responses. However, no significant difference was observed between sGnRH and cGnRH-II-induced responses in vivo. Time-related studies also revealed significant differences between sGnRH- and cGnRH-II-induced production of GtH subunit and GH mRNA in the goldfish pituitary. In general, the present study provides novel information on time- and dose-related effects of sGnRH and cGnRH-II on GtH subunit and GH mRNA levels and provides a framework for further investigation of GnRH mechanisms of action in the goldfish pituitary.     

Effect of gonadotropin-releasing hormone on phagocytic leucocytes of rainbow trout

To clarify the role of gonadotropin-releasing hormone (GnRH) in the fish immune system, in vitro effect of GnRH was examined in phagocytic leucocytes of rainbow trout (Oncorhynchus mykiss). Gene expression of GnRH-receptor was detected by RT-PCR in leucocytes from head kidney. Administration of sGnRH increased proliferation and mRNA levels of a proinflammatory cytokine, tumor necrosis factor (TNF)-α, in trout leucocytes. Superoxide production in zymosan-stimulated phagocytic leucocytes was also increased by sGnRH in a dose-related manner from 0.01 to 100 nM. There was no significant effect of sGnRH on mRNA levels of growth hormone (GH) expressed in trout phagocytic leucocytes. Immunoneutralization of GH by addition of anti-salmon GH serum into the medium could not block the stimulatory effect of sGnRH on superoxide production. These results indicate that GnRH stimulates phagocytosis in fish leucocytes through a GnRH-receptor-dependent pathway, and that the effect of GnRH is not mediated through paracrine GH in leucocytes.     

Molecular characterization of the GHRH/GHRH-R and its effect on GH synthesis and release in orange-spotted grouper (Epinephelus coioides)

Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide that stimulates growth hormone (GH) synthesis and secretion in the pituitary gland. In this paper, the full-length cDNAs of orange-spotted grouper GHRH and its receptor (GHRH-R) were cloned. The grouper GHRH cDNA is 713 bp in length and encodes a 141-aa precursor that includes an 18-aa signal peptide, a 27-aa mature GHRH mature peptide and a 47-aa carboxyl terminus. The grouper GHRH-R cDNA sequence is 1495 bp in length, encoding a 422-aa receptor with seven transmembrane domains. Tissue distribution analyses showed that both GHRH and GHRH-R mRNAs were predominantly expressed in the brain, while the GHRH-R mRNA was also abundantly detected in the pituitary gland. Both GHRH and GHRH-R mRNAs were expressed throughout embryonic development from the multi-cell stage to the newly hatched larvae stage, and the highest GHRH and GHRH-R expressions appeared at the brain vesicle stage and the heart stage, respectively. In vitro studies performed on the grouper pituitary primary cells showed that a synthetic grouper GHRH-NH(2) increased both GH mRNA expression and GH protein release in a dose-dependent manner. Together, these results suggest that the newly obtained grouper GHRH was able to stimulate GH synthesis and release, similar to its mammalian counterparts.     

In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.     

Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in the goldfish pituitary: effects of native GnRH peptides and a synthetic GnRH antagonist.

Gonadotropin-releasing hormone (GnRH) stimulates release of gonadotropin hormone (GTH) through interaction with high affinity receptors in the goldfish pituitary. In the present study, we investigated desensitization of two native GnRH peptides, [Trp7, Leu8]-GnRH (sGnRH) and [His5, Trp7, Tyr8]-GnRH (cGnRH-II), using superfused fragments of goldfish pituitary in vitro. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses given every 60 min) resulted in dose-dependent secretion of GTH from the goldfish pituitary; cGnRH-II had a greater GTH release potency and displayed a greater receptor binding affinity than sGnRH. Both sGnRH and cGnRH-II-induced GTH release were partially inhibited by concomitant treatment with either [D-Phe2, Pro3, D-Phe6]-GnRH or [D-pGlu1, D-Phe2, D-Trp3.6]-GnRH. These antagonists had greater receptor binding affinities than the native peptides, with no stimulatory action on GTH release in the absence of the GnRH agonists. Continuous treatment with either sGnRH or cGnRH-II (10(-7) M), rapidly desensitized pituitary GTH release in a biphasic fashion; initially there was a rapid increase in GTH release of approximately 10-20-fold (phase 1), followed by a sharp decline in GTH release, reaching a stable concentration 2-3-fold above the basal level (phase 2). Further stimulation of the pituitaries with sGnRH or cGnRH-II (10(-7) M) (second treatment) after 60 min recovery resulted in a significantly lower sGnRH or cGnRH-II-induced GTH release compared to that observed during the initial treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)