大鼠垂体促性腺激素细胞内cAMP的改变对LHβ mRNA表达的影响

目的 分析大鼠LHβ mRNA表达的促性腺激素释放激素(GnRH)受体后信号转导机制.方法 将体外培养的大鼠腺垂体促性腺激素(GTH)细胞用cAMP的兴奋剂FSK或抑制剂SQ22536处理后,再用高频GnRH脉冲刺激,然后用实时荧光定量PCR法测定细胞LHβ mRNA的Ct值,并与空白组比较.结果 LHβ mRNA的Ct值随着GTH细胞cAMP含量的增高而显著降低,随着cAMP含量的降低而显著增高.结论 cAMP是高频GnRH脉冲刺激所引起的LHβ mRNA表达的受体后的信号转导途径.     

改变大鼠促性腺激素细胞内cAMP含量对卵泡刺激素分泌的影响

目的:分析大鼠卵泡刺激素(FSH)分泌的受体后信号转导机制。方法:将促性腺激素(GTH)细胞用毛喉素(FSK)或腺苷酸环化酶抑制剂SQ22536处理后,用促性腺激素释放激素脉冲刺激,再用酶联免疫吸附法检测其FSH分泌量,并与空白对照组比较。结果:FSK能显著提高GTH细胞中环磷酸腺苷(cAMP)含量,SQ22536能显著降低GTH细胞中的cAMP含量,FSK和SQ22536都不会影响GTH细胞的蛋白激酶C活性,GTH细胞cAMP含量的变化对FSH分泌的影响不显著。结论:cAMP-PKA(蛋白激酶A)不是FSHβ亚基分泌的受体后信号转导途径。     

促黄体素β基因表达中的转导通路及转录因子

促性腺激素释放激素 (GnRH)为下丘脑促垂体激素 ,其脉冲式地释放调节垂体促卵泡素(FSH)和促黄体素 (LH)的合成与释放 ,进而调节动物的生殖活动。LH是由α亚基和 β亚基组成的异二聚体糖蛋白激素 ,其中 β亚基决定激素的特异性。LHβ基因的表达是由GnRH诱发的 ,此过程主要依靠PKC和Ca2 两类信号通路 ,并调节LHβ基因的表达。目前已经发现 ,多种转录因子 ,如早期生长反应基因 (Egr 1)、核受体SF 1基因、Ptx1基因和Sp1基因等 ,通过与LHβ亚基基因的启动子区直接结合 ,而对该基因的表达进行调控。     

大鼠GTH细胞内cAMP的改变对LH分泌的影响

将GTH细胞用FSK(cAMP兴奋剂)或SO22,536(cAMP抑制剂兴奋剂)处理后,用GnRH脉冲刺激,再用ELISA法检测其LH分泌量,并与空白对照组比较。结果表明,FSK能显著提高GTH细胞中cAMP含量,SO22,536能显著降低GTH细胞中cAMP含量,FSK和SO22,536都不会影响GTH细胞的PKC活性,GTH细胞cAMP含量显著影响LH的分泌,LH随着cAMP的升高而升高,随着cAMP的降低而降低。cAMP-PKA是GnRH脉冲刺激所引起LH分泌受体后的信号转导途径。     

促性腺激素抑制激素与生殖轴的关系

下丘脑的促性腺激素释放激素((GnRH)对调控促性腺激素释放十分重要.10年前在鸟类中首先发现了促性腺激素抑制激素(GnIH),它的鉴定提示GnRH不是唯一能直接影响垂体促性腺激素释放的下丘脑神经肤.GnIH及其同系肽广泛存在于鸟类及哺乳动物体内,GnIH在脑部与GnRH神经接触,GnIH可以直接抑制垂体促性腺激素的合成和释放,GnIH及其受体在鸟类和哺乳动物的生殖腺存在.因此GnIH可以在多个水平直接影响生殖轴:脑部、垂体、生殖腺.     

外源激素对雄性黄鳝性类固醇激素分泌的影响

硬骨鱼类促性腺激素(GTH)的分泌受到双重调控,即促性腺激素释放激素(GnRH)的刺激和促性腺激素释放抑制因子(GRIF)的抑制1.       

GnRH及其受体在性成熟前后鲇、黄颡鱼脑、垂体和卵巢中的免疫细胞化学定位

用链霉亲和素 -生物素化过氧化物酶复合物 (StreptAvidinBiotin peroxidaseComplex ,SABC)免疫细胞化学方法 ,使用促性腺激素释放激素 (Gonadotropin releasinghormone ,GnRH)以及促性腺激素释放激素受体 (GnRHR) 2种抗血清对性成熟前后的黄颡鱼 (Pelteobagrusfulvidraco)和鲇鱼 (Silurusasotus)的脑、垂体、卵巢中的免疫活性内分泌细胞进行了免疫细胞化学定位。结果表明GnRH和GnRHR免疫活性在两种鱼的各脑区、垂体、卵巢中均有分布 ;两种鱼在性成熟时它们的下丘脑、垂体和卵巢中的GnRH和GnRHR免疫反应细胞数目和免疫反应强度明显高于性成熟前。本文讨论了GnRH、GnRHR直接或间接参与黄颡鱼和鲇鱼性腺发育成熟调节的可能性及形态学证据。可为下丘脑 垂体 性腺轴、神经 内分泌、GnRH功能的多样性等研究领域提供新的形态学依据。     

多巴胺能药物对虎纹蛙GnRH和LH分泌活动的影响

利用在体注射实验和放射免疫测定法,研究了多巴胺能药物对性腺处于再发育期虎纹蛙的促性腺激素释放激素（GnRH)及促黄体激素（LH)分泌活动的影响。结果是：多巴胺（DA）及其激素剂阿扑吗啡（APO）可显著降低血浆LH水平;而多巴胺的拮抗剂－地欧酮（DOM）可显著增加垂体LH含量。DA对脑中cGnRH-Ⅱ的合成有抑制作用,而OM对其mGnRH的释放有一定的刺激作用。结果表明：DA可在脑及垂体水平分别抑制虎纹蛙GnRH和LH的释放,DA对LH释放的抑制作用很可能是通过D2受体实现的。     

促性腺激素释放激素类似物对长臀wei脑垂体促性腺激素分泌的影响

采用离体灌流孵育技术和促性腺激素的放射免疫测定方法,对长臀wei(Cranoglanis bouderius)脑垂体碎片促性腺激素的分泌进行了研究。结果表明：持续的促性腺激素释放激素类似物(GnRH-A)能显著刺激退化期的长臀wei离体脑垂体碎片促性腺激素(GTH)的分泌,并且长臀wei脑垂体碎片对持续的GnRH-A刺激未表现出脱敏性,该结果与胡子鲇和鲇鱼相似,而与金鱼和鲤科鱼类不同;重复脉冲GnRH-A刺激对长臀wei脑垂体碎片GTH分泌具有促进作用,而且存在剂量依存关系,与鲇鱼和鲤科鱼类相类似。上述结果表明在长臀wei的人工繁殖中可以用持续高浓度GnRH-A刺激对长臀wei进行催熟和催产。     

促性腺激素释放激素类似物对长臀(鱼危)脑垂体促性腺激素分泌的影响

采用离体灌流孵育技术和促性腺激素的放射免疫测定方法,对长臀(鱼危)(Cranoglanis bouderius)脑垂体碎片促性腺激素的分泌进行了研究.结果表明:持续的促性腺激素释放激素类似物(GnRH-A)能显著刺激退化期的长臀(鱼危)离体脑垂体碎片促性腺激素(GTH)的分泌,并且长臀(鱼危)脑垂体碎片对持续的GnRH-A刺激未表现出脱敏性,该结果与胡子鲇和鲇鱼相似,而与金鱼和鲤科鱼类不同;重复脉冲GnRH-A刺激对长臀(鱼危)脑垂体碎片GTH分泌具有促进作用,而且存在剂量依存关系,与鲇鱼和鲤科鱼类相类似.上述结果表明在长臀(鱼危)的人工繁殖中可以用持续高浓度GnRH-A刺激对长臀(鱼危)进行催熟和催产.     

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.     

Modulation of LH release by estradiol. Involvement of protein kinase C in the action mechanism of GnRH

The involvement of PKC in GnRH action is still controversial. Discrepancies between different results could be due to the endocrine status of cells used for the studies. In order to determine a putative role for PKC in GnRH action and if gonadal steroids could be implicated in the PKC contribution to GnRH action, we have conducted a study of LH release in response to GnRH and to PMA, an activator of PKC, using an anterior pituitary cell culture system. The direct effects of E2 were considered coupled or not with the effect of PKC depletion. GnRH and PMA induced LH releases in a dose-dependent manner. Both are increased by E2. The PKC depletion had no effect on GnRH stimulated LH release in cells deprived of gonadal steroid influence but induced a significant decrease in cells which had been treated by E2. These results indicate that E2 alters cell sensitivity to GnRH by affecting post-receptor intracellular pathways such as PKC activation.     

GnRH-A免疫与母兔生殖激素浓度的变化

目的探讨促性腺激素释放激素类似物（GnRH-A）对动物生殖功能调节的效果和作用机制。方法 24只日本大耳白兔分为四组,分别在实验Ⅰ组（EG-I）、实验Ⅱ组（EG-II）和实验III（EG-III）组兔的颈背侧注射1.0 mL（100、100和50μg/mL）GnRH-A抗原,实验II组和实验III组于第3周以原剂量加强注射一次,用ELISA法测定血清GnRH抗体效价、促卵泡刺激素（FSH）和促黄体生成素（LH）含量。结果注射GnRH-A后10 d实验组兔均出现GnRH抗体,而对照组未检测到;EG-I在第30天达到高峰,而EG-II和EG-III于40～50 d至峰值,但在实验结束时（70 d）实验组均高于对照组,40～70 d时EG-II显著高于EG-I和EG-III。30～50 d时EG-II的LH明显高于EG-I和EG-III及对照组。EG-II和EG-III的FSH浓度在40 d达到峰值,但EG-II高于EG-I、对照组及EG-III,EG-I和对照组无显著差异。结论兔体内注射GnRH-A可以明显提高GnRH抗体效价,增强LH和FSH的合成与分泌,加强注射效果更明显,且与注射剂量相关,持续时间为40 d左右。     

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of G_q/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.     

二甲双胍对多囊卵巢综合征肥胖型患者LH和FSH的影响

目的：探讨二甲双胍对多囊卵巢综合征肥胖型患者血清中胰岛素、LH和FSH水平的影响。方法：将84例PCOS肥胖型患者随机分成44例对照组（克罗米芬）和40例观察组（二甲双胍）,采用放射免疫法测定黄体生成素（LH）和卵泡刺激素（FSH）的水平,分别于服药前（0分钟）和服后60、120分钟经前臂静脉采血,测血糖浓度及血清胰岛素水平。结果：对照组患者治疗前0min、60min及120min的血糖OGTT分别为（4．57±0．25）mmol／L、（8．38±7．05）mmol/L（7.21±0．12）mmol／L。治疗后0min、60min及120min的血糖OGTT无明显变化。观察组患者治疗前0min、60min及120min的血糖OGTT分别为（4．11±0．31）mmol／L、（8．23±6．57）mmol／L及（7．25±0．13）mmol／L,治疗后0min、60min及120min的血糖0GTT明显降低。对照组患者治疗前血清中胰岛素为（47．32±9．52）U／ml,治疗后为（42．25±7．65）U／ml,治疗前后无明显差异。观察组患者治疗前血清中胰岛素为（46．41±6．11）U／ml,治疗后血清胰岛素水平明显降低。对照组患者治疗后血清中LH为（17．22±2．14）mU／ml,FSH为（1．24±0．33）mU／ml,而与对照组相比,观察组患者血清中的LH明显降低,而FSH水平升高。结论：二甲双胍导致多囊卵巢综合征患者血清中胰岛素水平降低,从而减轻了胰岛素对LH的刺激作用,使LH水平下降,FSH升高,进而改善机体的激素紊乱,最终达到治疗PCOS的目的。     

Secretoneurin stimulates the production and release of luteinizing hormone in mouse L{beta}T2 gonadotropin cells

Secretoneurin (SN) is a functional secretogranin II (SgII)-derived peptide that stimulates luteinizing hormone (LH) production and its release in the goldfish. However, the effects of SN on the pituitary of mammalian species and the underlying mechanisms remain poorly understood. To study SN in mammals, we adopted the mouse LβT2 gonadotropin cell line that has characteristics consistent with normal pituitary gonadotrophs. Using radioimmunoassay and real-time RT-PCR, we demonstrated that static treatment with SN induced a significant increment of LH release and production in LβT2 cells in vitro. We found that GnRH increased cellular SgII mRNA level and total SN-immunoreactive protein release into the culture medium. We also report that SN activated the extracellular signal-regulated kinases (ERK) in either 10-min acute stimulation or 3-h chronic treatment. The SN-induced ERK activation was significantly blocked by pharmacological inhibition of MAPK kinase (MEK) with PD-98059 and protein kinase C (PKC) with bisindolylmaleimide. SN also increased the total cyclic adenosine monophosphate (cAMP) levels similarly to GnRH. However, SN did not activate the GnRH receptor. These data indicate that SN activates the protein kinase A (PKA) and cAMP-induced ERK signaling pathways in the LH-secreting mouse LβT2 pituitary cell line.     

Actions of 17 beta-estradiol on gonadotropin release induced by drugs that activate intracellular signal transduction mechanisms in rat anterior pituitary cells

We studied the effects of 17 beta-estradiol (E2) on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release induced by drugs that activate different intracellular signal transduction mechanisms in rat anterior pituitary cells. Cells were pretreated with E2 (6 x 10(-10) M) or diluent for 24 h. Then, both E2- and diluent-pretreated cells were incubated for 4 h with E2 or diluent, respectively, with or without drugs, and in the presence or absence of gonadotropin-releasing hormone (GnRH). Media were assayed for LH and FSH by radioimmunoassays. E2 treatment had no effect on basal FSH release, but occasionally stimulated basal LH release. Phospholipase C (PLC), L-alpha-1,2-dioctanoyl glycerol (C8), veratridine, 8-bromo-cyclic adenosine 3',5'-monophosphate (8-Br-cAMP), melittin (a phospholipase A2 [PLA2] activator), arachidonic acid, PLA2, and GnRH all stimulated LH and FSH release in both E2- and diluent-treated cells. E2 treatment increased both LH and FSH release induced by GnRH, PLC, C8, veratridine, and 8-Br-cAMP, but not by melittin, arachidonic acid, and PLA2. Neither C8, PLA2, nor arachidonic acid in combination with a maximal dose of GnRH had additive effects on either LH or FSH release, whereas melittin increased the maximal response to GnRH in both E2- and diluent-treated cells. The effects of veratridine and 8-Br-cAMP depended on dose of GnRH and presence or absence of E2. These results suggest that E2 augments stimulus-coupled gonadotropin release by interacting with the Ca2+-, and/or diacylglycerol-, and cAMP-activated pathways, but not with the arachidonic acid-activated pathway.