Cloning and Sequence Analysis of Major Histocompatibility Complex Ⅰ Gene from Goose F1 of Fast-growth Lines

According to the multiple alignments identified major histocompatibility complex Ⅰ (MHC Ⅰ) gene conserved sequence registered in GenBank from the family ducks (Anatidae) anser waterfowl, a pairs of specific primers for the fragments of MHCⅠgene of goose F1 from fast-growth lines were designed and synthesized by Primer Premier 5.0. Using the genome DNA of goose F1 from fast-growth lines, the target gene fragment was obtained by PCR. To conduct sequencing of the fragments of MHCⅠgene of goose F1 from fast-growth lines and make sequence alignment and analysis of protein structure and function by bioinformatics, and research the characteristics of MHCⅠgene of goose F1 and the physicochemical properties of the protein. Bioinformatics was analyzed the nucleic acid data, deduced amino acid sequence and phylogenetic trees. The result of sequence analysis showed that the fragments of MHCⅠgene of goose F1 from fast-growth lines was 1036 bp in length, which coded 96 amino acids polyprotein. The homology were 93% and 83% with MHC Ⅰ gene and coding sequence of Wulong goose in NCBI respectively. There were 72 different bases sequence and 16 amino acids change. There also was higher homology with other poultry, and existed genetic relationship of Siji goose > chickens > ducks.The homology segment sequences corresponding to the fragments of MHCⅠ gene of goose F1 coded 96 amino acids protein, which molecular weight, PI, positively or negatively charged amino acid, estimated half-life, instability index, aliphatic index and average hydrophobicity were 11.342 ku, 5.32, 14, 17, 2.8 h, 34.92, 42.81, -1.066, respectively, and appeared 9 B cell epitopes, but contained no signal peptide. These results indicated that the protein for hydrophilic non-secreted proteins, had the high immunogenicity. In addition, The protein structure study indicated that alpha-helix, beta-sheet, beta-turn and random coil were 31.25%,16.67%, 14.58% and 37.50%, respectively. There existed amino terminal domain and carboxyl terminal domain in the tertiary structure. Therefore, MHC gene had significant difference between species and populations of individuals by the pathogen pressure in environment, and there were the interaction between polymorphism of MHC molecules and the diversity of antigenic peptide. MHC determined the differences of individual susceptibility to disease, and could be treated as a candidate gene for disease resistance.     

羊绒与羊毛的PCR-RFLP识别方法

羊绒价格昂贵,在销售中时常出现掺假现象,最常见的是用羊毛,因此精确识别羊绒和羊毛纤维的方法非常重要。本试验采用聚合酶链式反应-限制性片段长度多态（PCR-RFLP）的技术方法,利用提取的羊绒和羊毛线粒体基因组,根据细胞色素b（Cytochrome b）基因已知的DNA序列设计引物,通过内切酶SspⅠ进行酶切,得到不同的片段长度,根据酶切图谱鉴别羊绒和羊毛样品,用来进行羊绒掺假试验的定性检测。     

The maturation of the salmon GnRH system and its regulation by gonadal steroids in masu salmon

Pituitary gonadotropin (GTH) secreting cells and brain gonadotropin-releasing hormone (GnRH) secreting neurons are known to be subjected to feedback control by gonadal steroid in teleosts. In masu salmon, Oncorhynchus masou, salmon GnRH (sGnRH) neurons in the ventral telencephalon (VT) and the preoptic area (POA) are involved in the control of GTH cells because sGnRH synthesis in these areas is activated with gonadal maturation. In this study, we attempted to clarify mechanisms of feedback control of sGnRH neurons by gonadal steroids. We examined the effects of 17-methyltestosterone (MT) on sGnRH synthesis in yearling and 2-year-old female fish (which were immature during experimentation in May), and the effects of castration on sGnRH synthesis in underyearling precocious male fish in August. sGnRH synthesis in the POA, but not in the VT, was increased by MT administration in 2-year-old females only, indicating higher sensitivity to MT in the preoptic sGnRH neurons. Castration increased sGnRH synthesis in the VT but not in the POA. These results suggest that sGnRH neurons in the VT and those in the POA are differentially regulated by gonadal steroids.     

Pesticide—induced reproductive dysfunction in Indian fishes

Numerous studies are available reporting the effects of pesticides on reproductive activity in Indian fishes. The majority of these reports deals with histopathological changes in gonads and endocrine glands involved in the regulation of reproduction following treatment with different pesticides. Pesticides are reported to cause degenerative changes in gonads and arrest gametogenic processes either by acting directly on the gonads or by interfering with the secretory activity of the hypothalamo-hypophyseal-gonadal/thyroid axis that regulates various reproductive events. Secretion of hormones such as gonadotropin-releasing hormone (GnRH), gonadotropin, growth hormone, adrenocorticotropic hormone (ACTH), testosterone, estrogens, 17,20β-dihydroxyprogesterone and thyroid hormones are in general lowered, leading to cessation of gametogenesis, vitellogenesis, oocyte maturation, ovulation, spermiation, etc. Adverse effects of pesticides have also been demonstrated on fecundity, fertilization, hatching, and postembryonic development. The effects are highly variable and depend on the nature, dose, and mode of application of the pesticides.     

Extrapituitary gonadotropin-releasing hormone (GnRH) binding sites in goldfish

In teleosts, as in other vertebrates, the secretion of pituitary gonadotropin (GTH) is mediated by the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). Recent findings in teleosts indicate that GnRH receptors are not restricted to the pituitary gonadotropes and are also associated with somatotropes as well as being present in a number of other tissues. In the present study, we provide novel information on GnRH binding in a number of extrapituitary tissues in goldfish. However, we do not intend to provide full characterization of GnRH binding sites in various extrapituitary tissues in goldfish as this would clearly be outside the scope of this paper. In this study we examined GnRH binding in a number of extrapituitary tissues in goldfish and observed specific binding in ovary, testis, brain, liver and kidney. No specific GnRH binding was observed in muscle, skin, gut, gill and heart. In general, the present findings together with the results of other studies carried out in our laboratory demonstrate that mature goldfish ovary and testis contain two classes of GnRH binding sites, high affinity/low capacity and low affinity/high capacity sites with binding characteristics similar to those of the pituitary GnRH receptors. The brain of goldfish was also found to contain two classes of GnRH binding sites, a super-high affinity/low capacity and a low affinity/high capacity sites. Furthermore, study of goldfish liver and kidney demonstrated the presence of a single class of GnRH binding sites with characteristics different from those of pituitary, ovary, testis and brain. Overall, it is evident that goldfish contains a family of GnRH binding sites which can be classified into four groups based on binding affinities: 1) A class of high affinity binding sites present in the pituitary, ovary and testis, 2) a class of super high affinity sites so far only detected in the brain, 3) a class of intermediate-affinity GnRH binding sites in the liver and kidney, and 4) a class of low affinity binding sites present in all the tissues containing specific GnRH binding sites except for liver and kidney.     

Distribution of three GnRHs in the brain and pituitary of the wild Japanese flounder Paralichthys olivaceus

ABSTRACT:   Wild adult maturing and immature female Japanese flounder Paralichthys olivaceus were collected in June 2004 and January 2005, respectively, to clarify a possible role of gonadotropin-releasing hormones (GnRHs) in reproduction. Levels of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and sea bream GnRH (sbGnRH) in the brain and pituitary were examined by time-resolved fluoroimmunoassay. Three forms of GnRHs were detected in the discrete brain at various levels. In the pituitary of both maturing and immature fish, sbGnRH was abundant together with a pronounced amount of sGnRH, whereas cGnRH-II was almost below the detectable limit. In maturing fish, levels of sbGnRH were high in the telencephalon, hypothalamus and pituitary, while levels of sbGnRH of immature fish were very low in these regions. These results indicate that sbGnRH is mainly responsible for gonadotropin secretion, and that sbGnRH in the anterior part of the brain is associated with gonadal maturation in the Japanese flounder.     

Characterization of molecular variants of GnRH, induction of spermiation and sex reversal using salmon GnRH-A and domperidone in the protogynous diandric fish, Synbranchus marmoratus Bloch, (Teleostei, Synbranchidae)

This paper studies the molecular variants of gonadotropin-releasing hormone (GnRH) present in the brain of the protogynous swamp eel, Synbranchus marmoratus, and the effects of the administration of salmon GnRH analogue (sGnRH-A) and the dopamine receptor antagonist, domperidone (DOM) on final maturation and gamete release in this species. Evidence for the presence of two GnRH variants, sGnRH and cIIGnRH were obtained by reverse phase high-pressure liquid chromatography (RP-HPLC) and radioimmunoassay with different antisera. The effects of treatment with sGnRH-A+DOM were checked by three ways: oocyte and milt release by stripping, histological analysis of the gonadal tissue, and androgen serum levels at different times throughout the experiment. In males, spermiation was induced after three weeks of treatment. In the female group, sGnRH-A+DOM did not induce ovulation at the end of the experiment. Histological analysis of the gonads from the female group showed evidence of sex reversal. All the treated fish had elevated androgen serum levels from the third week, with respect to control fish. In all cases, serum estradiol levels were undetectable. These results suggest that treatment with sGnRH analog and DOM induce sex reversal in female and spermiation in males. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vivo activity of native GnRHs and their analogues on ovulation in the African catfish, Clarias gariepinus (Burchell)

Four distinct forms of native gonadotropin‐releasing hormone (GnRH) and two newly designed analogues were tested for their in vivo activity to induce ovulation in African catfish. The effects of these peptides on ovulatory parameters were compared with those of carp pituitary and [d ‐Ala⁶, Pro⁹‐NEt]‐mammalian GnRH analogue (mGnRHa), two tested ovulation‐inducing agents in African catfish. Assessment of ovulation was carried out by determining the ovulation ratio and the relative quantity of egg produced. From the results of the experiments, the order of potency of the native GnRH peptides is summarized as chicken GnRH‐II (cGnRH‐II) >salmon GnRH (sGnRH) >mammalian GnRH >chicken GnRH‐I (cGnRH‐I). Chicken GnRH‐II was as potent as mGnRHa while cGnRH‐I was totally ineffective. The new d ‐Orn⁶‐cGnRH‐II and d ‐Orn⁶‐sGnRH with a substitution at position 6 with d ‐isomer residue were as potent as the most extensively used mGnRHa, indicating that the position 6 modification might be more crucial than the substitution at the C‐terminal. On the basis of our results, the potential use and incorporation of cGnRH‐II and sGnRH for the development of more generic spawning induction therapies are suggested.     

牙鲆脑中促性腺激素释放激素受体的克隆和鉴定

GnRH（gonadotropin-releasing hormone）是下丘脑-垂体-性腺轴中重要的十肽信息分子，在所有的脊椎动物的生殖神经内分泌调控中具有重要的作用。GnRH成熟的十肽沿着轴突被运送到下丘脑正中隆起（median eminence）末端，然后进入下丘脑垂体门脉循环（hypothalamo hypophyseal portal circulation）（四足动物）或直接通过轴突末梢（大多数硬骨鱼）把信号传递给刺激性腺的细胞，与特异性受体结合，刺激促性腺激素（GtHs，gonadotropins）的合成与释放，参与脊椎动物繁殖的起始和维持正常生殖功能。伴随着GnRH的研究，研究者对鱼类GnRH受体的研究也越来越感兴趣，牙鲆（Paralichthys olivaceus）是中国重要的海水养殖鱼类，本研究从牙鲆脑组织中克隆得到全长的Type Ⅰ GnRH受体，并对其组织特异性表达作了分析，为牙鲆的神经生殖内分泌研究奠定了一定的基础。 在本研究中，以海水养殖牙鲆为材料，从牙鲆脑组织中采用巢式PCR、5′-和3′-RACE技术克隆得到了1671bp牙鲆促性腺激素释放激素受体（GnRH-R）全长cDNA序列，包括147bp的5′-UTR、1248bp的开放阅读框和276bp的3′-UTR，GenBank登录号是DQ011872。牙鲆GnRH-R和其他物种GnRH-R的氨基酸序列的多序列比对结果显示，其存在许多保守的特征，牙鲆GnRH-R显示有3个主要的功能区域：1个N端胞外结构域（1～44个氨基酸残基）、1个大的跨膜结构域（45～324个氨基酸残基）和1个C端细胞质区域（325～415个氨基酸残基）。空间结构预测显示牙鲆GnRH-R跨膜区域包含7个高度保守的跨膜α螺旋（45—64、76—95、114—135、156—176、207—224、267—286、305—324），这些α螺旋是受体锚定到细胞膜上必需的。 氨基酸序列系统进化分析表明，牙鲆GnRH-R与琥珀鱼GnRH-R1具有高度同源性（85％同源性）。已知的硬骨鱼GnRH—R基因的系统进化分析表明，存在两种类型的GnRH-R基因：Type Ⅰ GnRH—R和Tpye Ⅱ GnRH-R基因。在牙鲆GnRH—R的氨基酸序列中，具有Type Ⅰ GnRH—R共有的典型基序：CAFVT（TMⅢ）和DlLEGKVSHSLTH（TMⅦ开始的序列处），表明克隆得到的牙鲆GnRH-R属于Type Ⅰ GnRH—R。 GnRH-R跨膜α螺旋之间的区域形成了胞内或胞外loops，这些区域参与受体信号转导和肽选择性功能上。牙鲆GnRH-R在TMⅢ的细胞质边界处具有一个保守的DRQSA／（DRXXXI／V）基序，这个基序被认为参与G蛋白偶联信号转导和GnRH肽诱导受体的激活；另一个保守的区域是位于TMⅦ内的NPXXY或DPXXY基序同样存在于牙鲆中（DPVIY），这个基序参与到一些GPCRs（包括GnRH-R）的内在化（internalization），同时这个基序特别是基序中的Tyr残基对于受体激活和信号转导起到关键作用。在第三个胞内loop中，鱼类GnRH—R有个保守的Ala残基（在牙鲆中为Ala^250），其也在G蛋白偶联和受体内在化方面具有重要作用。 在哺乳动物或非哺乳动物GnRH-R中，在第一个和第二个胞外loop之间由2个Cys形成1个保守二硫桥，是受体的正确折叠必需的。和其他的GnRH-R一样，牙鲆GnRH-R在第一个和第二个胞外loop之间存在由2个Cys（Cys^112和Cys^190）可以形成的潜在的二硫桥。这个二硫桥的完全缺失将会影响受体的结构完整性和降低鱼类GnRH—R对GnRH肽的选择性。 GnRH-Rs的NH2-末端区域不是很保守，但含有糖基化位点，是GnRH—Rs表达、GnRH—Rs穿梭到细胞胞表面或受体稳定必需的。将mouse的GnRH—R糖基化位点引进到人GnRH-R中，可以增加受体的数量，但不影响受体结合力或肽选择性。牙鲆Type Ⅰ GnRH—R中在NH2-末端含有3个潜在的糖基化位点（N^11SSW、N^15GSS和N^22WTA），此外，在第一个胞外loop和第二个胞外loop中分别存在1个糖基化位点（N^100ITV和N^178VTI），牙鲆Type Ⅰ GnRH—R含有的糖基化位点比哺乳动物要多，牙鲆糖基化位点的数目是否／怎样影响牙鲆GnRH-R的表达、降解率或亲和力，应该进一步研究。 在牙鲆GnRH-R的第一个胞内Ioop中，存在1个和tGnRH-R Ⅲ基序（^80KRKSH^84）一致的基序（^69KRKSH^74），这个基序是Gs识别基序（BBXXB，B代表碱性氨基酸，X代表任何一种氨基酸）。已经证明这个基序和cAMP产生相关，cGnRH-Ⅱ能通过cAMP-PKA途径提高stbGnRH-R在COS7细胞中的活性。 和其他非哺乳动物GnRH-Rs相似，牙鲆GnRH-R含有1个C-末端细胞内尾巴。这种胞内尾巴是鱼类GnRH-Rs功能必需的，在第三个胞内loop中，牙鲆GnRH-R含有2个PKC磷酸化位点（^233SKR^235和^262TLK^264）；在C端尾巴中，存在1个PKC磷酸化位点（^402TAR^404）。牙鲆GnRH-RC-末端尾巴中含有鱼类GnRH-R具有的Src同源区3（SH3）结合区域（PxxP序列）（^340PPAP），能够潜在地传送偶联的能力到丝裂原活化蛋白激酶（MAPKs），GnRH通过PKC和PKA调控GtHα和LHβ转录，二者都集中于MAPK水平。 RT—PCR分析表明，GnRH-R在牙鲆脑和垂体有表达，暗示牙鲆GnRH-R参与到生殖行为如产卵行为；RT-PCR也检测到牙鲆GnRH-R在卵巢和睾丸中有表达，其能与牙鲆cGnRH-Ⅱ和sbGnRH在卵巢中共表达，GnRH肽在卵巢中具有自分泌／旁分泌功能，对牙鲆卵巢中的GnRH受体可能起到调控作用。[中国水产科学，2006，13（4）：536—546]     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.