日粮能量水平对五指山猪初情期性腺轴KISS-1/GPR54 mRNA水平表达影响

本研究采用实时荧光定量PCR技术检测不同日粮能量水平对五指山猪初情期性腺轴Kiss-1、GPR54基因mRNA水平表达的影响。结果显示,在NRC和0.7 NRC两组实验猪的下丘脑、垂体、卵巢组织中均检测到Kiss-1、GPR54基因mRNA水平的表达;NRC组在下丘脑和卵巢kiss-1 mRNA表达量显著高于0.7NRC组(p0.05),在垂体中的表达差异不显著(p0.05);日粮能量水平对初情期性腺轴GPR54 mRNA表达差异不显著(p0.05)。说明KISS-1/GPR54系统对动物初情表达的调节作用可能是通过调节下丘脑KISS-1基因的表达来实现。结果表明kiss-1 mRNA的表达规律在NRC组和0.7NRC组间保持一致,表达量依次为:下丘脑垂体卵巢;GPR54 mRNA的表达规律在NRC组和0.7NRC组间也保持一致,表达量依次为:卵巢垂体下丘脑,表明不同日粮水平的摄入并不影响Kiss-1、GPR54基因在各个组织中的表达规律。     

不同日粮水平对初情期五指山猪性腺轴Kisspeptin/GPR54蛋白表达作用

为了阐明不同日粮水平对初情期五指山猪母猪性腺轴组织中Kisspeptin/GPR54蛋白表达作用影响,本研究采用免疫组织化学法DAB显色技术对其蛋白表达进行检测。结果表明,NRC和0.7 NRC两组实验中猪性腺轴(下丘脑,垂体,卵巢)组织中均检测到Kisspeptin和GPR54 2种蛋白的表达,并确定2种蛋白表达部位为细胞核;其中NRC组Kisspeptin蛋白下丘脑、垂体和卵巢中表达量显著比0.7 NRC组高(p0.05),2组实验中GPR54蛋白在性腺轴中表达无显著差异(p0.05),说明Kisspeptin/GPR54系统在动物初情期中的调控作用是通过个体下丘脑组织中Kiss1蛋白的表达情况得以实现的;Kisspeptin蛋白在2组实验组猪个体性腺轴中的表达规律一致,表达量从高到低排列依次为丘脑、垂体、卵巢;GPR54蛋白则在2组实验组猪个体性腺轴中表达量从高到低排列依次为卵巢、垂体、下丘脑。说明控制日粮能量的摄入并不能影响以上2种基因在猪个体性腺轴各组织中的表达规律。     

Kisspeptin调控鱼类生殖内分泌的研究进展

Kisspeptin是近10年新发现的调控动物生殖内分泌的关键因子,是kiss基因所编码的多肽产物,属神经内分泌肽类激素,为G蛋白偶联受体54(GPR54)的内源性配体。Kisspeptin具多个功能性的分子结构形态,在鱼类中,主要结构形式为Kisspeptin-10肽。Kisspeptin/GPR54系统在生殖中具重要功能。该文综合现有研究资料,就Kisspeptin在鱼类生殖内分泌调控中的概况、Kisspeptin神经元在鱼脑中的分布和定位、鱼类Kisspeptin分子的多态性、Kisspeptin调控鱼生殖内分泌的功能多样性、Kisspeptin调控鱼类生殖内分泌的机制、Kisspeptin的分子进化以及Kisspeptin和其他功能分子对鱼类生殖内分泌的协同调控进行了初步阐述,同时对Kisspeptin在鱼类生殖内分泌调控中的研究进行了展望。     

达氏鲟生长激素基因cDNA克隆、表达及免疫荧光定位研究

为研究达氏鲟(Acipenser dabryanus)生长激素(Growth Hormone, GH)基因的功能, 合成了达氏鲟垂体SMART cDNA, 克隆得到GH全长cDNA序列。达氏鲟GH全长cDNA序列为1008 bp, 由52 bp的5'端非编码区(Untranslated region, UTR)、编码214个氨基酸的645 bp开放阅读框(Open reading frame, ORF)和311 bp的3'UTR构成。运用GH氨基酸序列构建进化树分析发现, 达氏鲟与两栖类、爬行类和哺乳类的一致性要高于真骨鱼类。实时荧光定量PCR结果表明, 达氏鲟GH mRNA主要在垂体和下丘脑中表达, 且垂体中GH的表达量约为下丘脑的110倍; Western-blot研究结果与qRT-PCR一致, 仅在垂体和下丘脑中检测到生长激素蛋白, 且垂体中GH的表达量远高于下丘脑。免疫荧光定位结果显示, GH主要定位于垂体中部, 下丘脑中也有少量荧光信号; 苏木精-伊红组织切片染色研究表明, GH主要是由嗜酸性的生长激素分泌细胞分泌。研究为深入研究脊椎动物生长激素基因的进化和人工养殖达氏鲟的生长调控提供了基础。       

KISS-1/GPR54基因及其在生殖中的作用

KISS-1及其受体GPR54基因对青春期的正常启动具有重要作用。青春期开始前后, 动物下丘脑中KISS-1和GPR54 mRNA水平很高, Kisspeptins(KISS-1基因产物)通过激活GPR54增加促性腺激素的释放, KISS-1基因的表达受性腺类固醇激素的调控。GPR54基因突变可以导致人和鼠的特发性促性腺激素分泌不足性腺机能减退症和促性腺激素依赖性性早熟。文章还介绍了KISS-1、GPR54基因的结构、表达、多态性以及和其它生殖调控因子之间的相互关系。     

KISS-1/GPR54基因在猪繁育性能中的作用概述

KISS-1和GPR54(Kiss-1受体)基因共同作用组成KISS-1/GPR54系统,参与HPGA启动青春期,调节下丘脑分泌Gn RH,并刺激垂体门脉系统分泌LH、FSH对性腺产生作用,刺激雌激素、睾酮等激素合成,进而促使卵子和精子的产生,使个体具备生殖能力,在生殖中起关键调控作用。该文章对这两个基因在猪的繁育性能中的作用进行简述,旨在探讨和挖掘影响猪繁育的遗传机理及作用,为更有效地提高猪场产量及优良繁育猪种的选育提供参考依据。     

Kiss-1及MVD在非小细胞肺癌中检测的临床意义及其相关性研究

目的：测定NSCLC组织中kiss-1基因表达、MVD分布,探讨其临床意义及其相关性。方法：应用免疫组化SP法检测56例NSCLC组织中Kiss-1表达及MVD分布情况,分析其与NSCLC的临床病理参数的关系及其相关性。结果：kiss-1在NSCLC表达低于对照组（P〈0.05）,MVD在NSCLC分布明显高于对照组（P〈0.05）。Kiss-1与NSCLC的临床分期、淋巴结转移、肿瘤大小有关（P〈0.05）。MVD与NSCLC分化程度、临床分期及淋巴结转移、肿瘤大小有关（P〈0.05）.Kiss-1阳性组MVD分布明显低于Kiss-1阴性表达组,二者呈负相关（P〈0.05,r=-0.363）。结论：Kiss-1及MVD在非小细胞肺癌发生、发展中发挥重要作用,二者存在相关性,联合检测可帮助预测NSCLC的侵袭、转移,判断预后。     

达氏鲟Elovl4、ELovl5和Elovl7克隆、组织分布及饥饿对其表达的影响

为研究长链脂肪酸延长酶(Elovls)作用机制, 实验根据前期达氏鲟转录组测序获得的unigene序列为基础, 得到Elovl4、Elovl5和Elovl7 CDS区序列, 并分析了3个基因在各组织及饥饿胁迫下的表达情况。结果显示, 达氏鲟Elovl4、Elovl5和Elovl7编码区为753、885和846 bp, 分别编码250、294和281个氨基酸。达氏鲟Elovl4、Elovl5和Elovl7氨基酸序列与斑点雀鳝Elovls具有较高的同源性; 组织分布结果显示, 达氏鲟Elovl4在卵巢和眼中表达最高; Elovl5在肌肉中的表达显著高于其他各个组织; Elovl7在卵巢中表达最高(P<0.05), 鳃、精巢和肌肉中表达较多。在饥饿条件下, Elovl4、Elovl5和Elovl7在肝脏中的表达量均显著下调(P<0.05), 肠道中的趋势各有不同; 饥饿3d时, Elovl4、Elovl5和Elovl7在胃中的表达量显著降低, 随后上调(P<0.05); 饥饿7d时, 脑和肌肉中Elovl4、Elovl5和Elovl7的表达量显著高于其他各组, 随后显著下降(P<0.05)。这说明Elovl4、Elovl5和Elovl7在达氏鲟营养调控过程中发挥的作用可能存在差异, 其具体作用机制需要进一步研究。     

动物季节性繁殖分子调控机理研究进展

动物季节性发情繁殖涉及下丘脑-垂体-性腺轴系统复杂的神经内分泌过程,并受光照周期等环境因素的影响。褪黑激素则作为光周期信号分子调控动物季节性繁殖活动。近年来研究发现,对GnRH分泌有重要影响的Kiss1/GPR54系统既受褪黑激素的调控又受到性腺类固醇激素反馈调节,Kiss1/GPR54系统很可能是调控动物季节性繁殖的关键因子;同时动物季节性繁殖很可能还存在一条涉及TSH-DIO2/DIO3系统的逆向调控通路,该系统同样显著影响GnRH合成释放并受褪黑激素调控。文章就褪黑激素中心信号,特别是Kiss1/GPR54和TSH-DIO2/DIO3系统对繁殖季节性调控的最新研究进展进行综述。     

Kiss1和GPR54基因多态性与多囊卵巢综合征相关性分析

本研究旨在探讨Kiss1和GPR54基因多态性与多囊卵巢综合征的相关性。利用超声检查卵巢体积、血清睾酮、游离雄激素指数情况;临床评估患者身高(cm)和体重(kg)、BMI、静息血压、痤疮和黑棘皮病的分布;ELISA酶联免疫法检测血清中的kisspeptin和睾酮水平,使用Next generation sequencing方法(LGC group, Germany)对基因(Kiss1, GPR54)进行测序。结果显示,PCOS患者比对照组女性具有更高的BMI和mFG评分,PCOS患者血清Kisspeptin和睾酮浓度显著提高,且LH浓度也显著高于对照组(p<0.05)。GPR54和Kiss1 2个基因在患者体内存在多态性;测序分析结果显示GPR54基因存在的2个新的SNP位点(chr19:918686, A→G和chr19:918735, A→G),这2个新的多态性位于内含子区域(内含子2),Kiss1基因也存在两个SNP,位于非翻译变体5的末端(rs5780218)和外显子3 (rs4889),即GPR54基因存在A→G多态性,Kiss1基因为CTT→CT/G→C多态性,且相关性关联分析结果表明,GPR54基因型多态性(Chr19:918735)与PCOS风险增加相关(p<0.05);而Kiss1 SNP的基因型与PCOS风险之间没有关联。此外,PCOS与GPR54和Kiss1基因的单倍型没有显著关联。本研究推论对PCOS发生风险的遗传影响可能不仅是通过直接改变Kiss1/GPR54相互作用,而且还可能通过改变个体与环境因素的相互作用。     

Differential and gonad stage-dependent roles of kisspeptin1 and kisspeptin2 in reproduction in the modern teleosts, morone species

Kisspeptin is an important regulator of reproduction in many vertebrates. The involvement of the two kisspeptins, Kiss1 and Kiss2, and their receptors, Gpr54-1 and Gpr54-2, in controlling reproduction was studied in the brains of the modern teleosts, striped and hybrid basses. In situ hybridization and laser capture microdissection followed by quantitative RT (QRT)-PCR detected coexpression of kiss1 and kiss2 in the hypothalamic nucleus of the lateral recess. Neurons expressing gpr54-1 and gpr54-2 were detected in several brain regions. In the preoptic area, gpr54-2 was colocalized in GnRH1 neurons while gpr54-1 was expressed in cells attached to GnRH1 fibers, indicating two different modes of GnRH1 regulation. The expression of all four genes was measured in the brains of males and females at different life stages using QRT-PCR. The levels of kiss1 and gpr54-1 mRNA, the latter being expressed in minute levels, were consistently lower than those of kiss2 and gpr54-2. While neither gene's expression increased at prepuberty, all were dramatically elevated in mature females. The levels of kiss2 mRNA increased also in mature males. Kiss1 peptide was less potent than Kiss2 in elevating plasma luteinizing hormone levels and in up-regulating gnrh1 and gpr54-2 expression in prepubertal hybrid bass in vivo. In contrast, during recrudescence, Kiss1 was more potent than Kiss2 in inducing luteinizing hormone release, and Kiss2 down-regulated gnrh1 and gpr54-2 expression. This is the first report in fish to demonstrate the alternating actions and the importance of both neuropeptides for reproduction. The organization of the kisspeptin system suggests a transitional evolutionary state between early to late evolving vertebrates.     

Molecular cloning of the bullfrog kisspeptin receptor GPR54 with high sensitivity to Xenopus kisspeptin

Kisspeptin and its receptor, GPR54, play important roles in mammalian reproduction and cancer development. However, little is known about their function in nonmammalian species. In the present study, we have isolated the cDNA encoding the kisspeptin receptor, GPR54, from the bullfrog, Rana catesbeiana. The bullfrog GPR54 (bfGPR54) cDNA encodes a 379-amino acid heptahelical G protein-coupled receptor. bfGPR54 exhibits 45-46% amino acid identity with mammalian GPR54s and 70-74% identity with fish GPR54s. RT-PCR analysis showed that bfGPR54 mRNA is highly expressed in the forebrain, hypothalamus and pituitary. Upon stimulation by synthetic human kisspeptin-10 with Phe-amide residue at the C-terminus (h-Kiss-10F), bfGPR54 induces SRE-luc activity, a PKC-specific reporter, evidencing the PKC-linked signaling pathway of bfGPR54. Using a blast search, we found a gene encoding a kisspeptin-like peptide in Xenopus. The C-terminal decapeptide of Xenopus kisspeptin shows higher amino acid sequence identity to fish Kiss-10s than mammalian Kiss-10s. A synthetic Xenopus kisspeptin peptide (x-Kiss-12Y) showed a higher potency than mammalian Kiss-10s in the activation of bfGPR54. This study expands our understanding of the physiological roles and molecular evolution of kisspeptins and their receptors.     

The kisspeptin/gonadotropin-releasing hormone pathway and molecular signaling of puberty in fish

The mechanisms underlying the initiation of puberty in fish are poorly understood, and whether the Kiss1 receptor (Kiss1r; previously designated G protein-coupled receptor 54; GPR54) and its ligands, kisspeptins, play a significant role, as has been established in mammals, is not yet known. We determined (via real-time PCR) temporal patterns of expression in the brain of kiss1r, gnrh2, and gnrh3 and a suite of related genes in the hypothalamo-pituitary-gonadal (HPG) axis and analyzed them against the timing of gonadal germ cell development in male and female fathead minnow (Pimephales promelas). Full- or partial-length cDNAs for kiss1r (736 bp), gnrh2 (698 bp), and gnrh3 (804 bp) cloned from fathead minnow were found to be expressed only in the brain, testis, and ovary of adult fish. Localization of kiss1r, gnrh2, and gnrh3 within the brain provided evidence for their physiological roles and a likely hypophysiotropic role for GnRH3 in this species (which, like other cyprinids, does not appear to express gnrh1). In both sexes, kiss1r expression in the brain increased at the onset of puberty and reached maximal expression in males when spermatagonia type B appeared in the testis and in females when cortical alveolus-stage oocytes first appeared in the ovary, the timings of which differed for the two sexes. However, kiss1r expression was considerably lower during more advanced stages of spermatogenesis and oogenesis. The expression of kiss1r closely aligned with that of the gnrh genes (gnrh3 in particular), suggesting the Kiss1r/kisspeptin system in fish has a similar role in puberty to that occurring in mammals, and this hypothesis was supported by the induction of gnrh3 (2.25-fold) and kiss1r (1.5-fold) in early-mid pubertal fish injected with mammalian kisspeptin-10 (2 nmol/g wet weight). An intriguing finding, and contrasting that in mammals, was an elevated expression of esr1, ar, and cyp19a2 (genes involved in sex steroid signaling) in the brain at the onset of puberty, and in females slightly in advance of the elevation in the expression of kiss1r.     

Differential ovarian expression of KiSS-1 and GPR-54 during the estrous cycle and photoperiod induced recrudescence in Siberian hamsters (Phodopus sungorus)

Kisspeptins, coded by the KiSS-1 gene, regulate aspects of the reproductive axis by stimulating GnRH release via the G protein coupled receptor, GPR54. Recent reports show that KiSS/GPR54 may be key mediators in photoperiod-controlled reproduction in seasonal breeders, and that KiSS-1/GPR54 are expressed in the hypothalamus, ovaries, placenta, and pancreas. This study examined the expression of KiSS-1/GPR54 mRNA and protein in ovaries of Siberian hamsters (Phodopus sungorus). Ovaries from cycling hamsters were collected during proestrus (P), estrus (E), diestrus I (DI), and diestrus II (DII). To examine KiSS-1/GPR54 during stimulated recrudescence, additional hamsters were maintained either in long day (LD 16L:8D, control) or short day (SD 8L:16D) for 14 weeks and then transferred to LD for 0-8 weeks. Staining of KiSS-1/GPR54 protein was detected by immunohistochemistry in steroidogenic cells of pre-antral and antral follicles, and corpora lutea. Immunostaining peaked in P and E, but decreased in the diestrus stages (P < 0.05). In recrudescing ovaries, KiSS-1/GPR54 immunostaining was low after 14 weeks of SD exposure (post-transfer [PT] week 0), and increased during the early weeks of recrudescence. Expression of KiSS-1/GPR54 mRNA was low with short day exposure, but increased during recrudescence and was higher at PT week 8 as compared to PT weeks 0 and 2 (P < 0.05). The elevated KiSS-1/GPR54 expression during P and E suggests a potential role in ovulation in Siberian hamsters. Transient increases in KiSS-1/GPR54 expression following LD stimulation are also suggestive of possible involvement in ovulation and/or restoration of ovarian function.     

Hypothalamic GPR40 Signaling Activated by Free Long Chain Fatty Acids Suppresses CFA-Induced Inflammatory Chronic Pain

GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund''s adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty acids might have an important role in this pain control system.     

人孤儿受体GPR81分子克隆、组织分布及表达工程细胞株的建立

采用PCR技术分别从人全血基因组DNA及引产胚胎肾组织cDNA中扩增得到gpr81的全长cDNA序列(1041bp),运用生物信息学手段绘制该基因的分子进化树,显示该基因的氨基酸序列与烟酸受体同源性最高;然后,采用RT-PCR法分析该基因表达的组织分布,组织表达谱显示该基因在多种组织均有表达,以心脏及肝脏组织为最高;利用分子克隆手段构建含6×组氨酸(His)标签蛋白的真核表达载体pcDNA3·1(-)/his-myc-A-gpr81,通过脂质体介导,将该重组质粒转染CHO-K1细胞,RT-PCR证实该基因已整合入CHO-K1细胞的基因组中,Western-blot表明GPR81在CHO-GPR81工程细胞株中有表达。组织表达谱的检测和工程细胞株的建立为进一步研究该受体的生物学功能奠定了基础。     

Kiss-1 及MVD 在非小细胞肺癌中检测的临床意义及其相关性研究

目的:测定NSCLC组织中kiss-1基因表达、MVD分布,探讨其临床意义及其相关性.方法:应用免疫组化SP法检测56例NSCLC组织中Kiss-1表达及MVD分布情况,分析其与NSCLC的临床病理参数的关系及其相关性.结果:Kiss-1在NSCLC表达低于对照组(P＜0.05),MVD在NSCLC分布明显高于对照组(P＜0.05).Kiss-1与NSCLC的临床分期、淋巴结转移、肿瘤大小有关(P＜0.05).MVD与NSCLC分化程度、临床分期及淋巴结转移、肿瘤大小有关(P＜0.05).Kiss-1阳性组MVD分布明显低于Kiss-1阴性表达组,二者呈负相关(P＜0.05,r=-0.363).结论:Kiss-1及MVD在非小细胞肺癌发生、发展中发挥重要作用,二者存在相关性,联合检测可帮助预测NSCLC的侵袭、转移,判断预后.     

Neuroanatomical Evidence That Kisspeptin Directly Regulates Isotocin and Vasotocin Neurons

Neuropeptide kisspeptin has been suggested to be an essential central regulator of reproduction in response to changes in serum gonadal steroid concentrations. However, in spite of wide kisspeptin receptor distribution in the brain, especially in the preoptic area and hypothalamus, the research focus has mostly been confined to the kisspeptin regulation on GnRH neurons. Here, by using medaka whose kisspeptin (kiss1) neurons have been clearly demonstrated to be regulated by sex steroids, we analyzed the anatomical distribution of kisspeptin receptors Gpr54-1 and Gpr54-2. Because the both receptors were shown to be activated by kisspeptins (Kiss1 and Kiss2), we analyzed the anatomical distribution of the both receptors by in situ hybridization. They were mainly expressed in the ventral telencephalon, preoptic area, and hypothalamus, which have been suggested to be involved in homeostatic functions including reproduction. First, we found gpr54-2 mRNA expression in nucleus preopticus pars magnocellularis and demonstrated that vasotocin and isotocin (Vasopressin and Oxytocin ortholog, respectively) neurons express gpr54-2 by dual in situ hybridization. Given that kisspeptin administration increases serum oxytocin and vasopressin concentration in mammals, the present finding are likely to be vertebrate-wide phenomenon, although direct regulation has not yet been demonstrated in mammals. We then analyzed co-expression of kisspeptin receptors in three types of GnRH neurons. It was clearly demonstrated that gpr54-expressing cells were located adjacent to GnRH1 neurons, although they were not GnRH1 neurons themselves. In contrast, there was no gpr54-expressing cell in the vicinities of neuromodulatory GnRH2 or GnRH3 neurons. From these results, we suggest that medaka kisspeptin neurons directly regulate some behavioral and neuroendocrine functions via vasotocin/isotocin neurons, whereas they do not regulate hypophysiotropic GnRH1 neurons at least in a direct manner. Thus, direct kisspeptin regulation of GnRH1 neurons proposed in mammals may not be the universal feature of vertebrate kisspeptin system in general.