石鲽脑垂体组织生理学研究

石鲽(Kareius bicoloratus)脑垂体呈鸡心形,由神经垂体和腺垂体两部分组成。腺垂体又包括前外侧部(RPD)、中外侧部(PPD)和垂体中间部(PI)。神经纤维束延伸至腺垂体的各个部分。采用HE、Jafri、HA、Mallory和PAS染色法,结合免疫组织化学方法,对石鲽脑垂体的6种内分泌细胞进行鉴别和定位。RPD部位主要含有PRL和ACTH细胞,PI部位主要含有MSH细胞。PPD部位有4类细胞,分别为GH、Gt H、TSH和嫌色细胞。GH、Gt H和TSH细胞数量随鱼体生长发育而变化,Gt H在性腺成熟期达到高峰,GH细胞和TSH细胞在生长早期数量多。     

Immunocytochemical and ultrastructural identification of adenohypophyseal cells in Ctenopharyngodon idella (Cypriniformes: Cyprinidae) during gonadal differentiation

The adenohypophysis was studied by immunocytochemical and ultrastructural methods in juvenile grass carp (Ctenopharyngodon idella) from natural reproduction in Northern Italian rivers. The adenohypophysis included the rostral pars distalis (RPD), the proximal pars distalis (PPD) and the pars intermedia (PI), all deeply penetrated by branches of the neurohypophysis (Nh). The prolactin (PRL), adrenocorticotropic (ACTH), somatotropic (GH), thyrotropic (TSH), gonadotropic type I (GtH I) and type II (GtH II), somatolactin (SL), melanotropic (MSH) and endorphin (END) cells were identified with antisera raised against piscine and human pituitary hormones. In juveniles of 51–69 mm of total body length (TL) with undifferentiated gonads, the PRL cells, arranged in thick strands, occupied most of the RPD. The ACTH and GH cells organized in cords bordering Nh were, respectively, confined to RPD and PPD. The TSH cells were scattered among ACTH cells in RPD and among GH cells in PPD. Cells simultaneously immunoreactive to anti-follicle stimulating hormone and to anti-croaker gonadotropin were intermingled among GH and TSH cells, which were mostly in the dorsal PPD. The SL cells were detected in PI layers bordering the Nh. The MSH and END cells were intermingled in PI and, unlike what observed in other teleosts, their respective antisera did not cross-react. In individuals of 78–112 mm TL with gonads at the beginning of differentiation, the GtH II cells were detected in PPD; all other cell types increased in number. These results, supported by ultrastructural investigations, suggest that SL and GtH II cells are directly involved in gonadal differentiation in C. idella.     

Involvement of protein kinase C in the modulation of gonadotropin and growth hormone secretion from dispersed goldfish pituitary cells

It has been established that secretion of gonadotropin (GtH) and growth hormone (GH) release in goldfish are both stimulated by GtH-releasing hormone (GnRH); in addition GtH secretion is inhibited by dopamine D₂ mechanisms. In the present study, depletion of protein kinase C (PKC) in goldfish pituitary cells reduced the GtH and GH responses to GnRH and an activator of PKC in static culture. In perifusion studies, GtH released in response to sGnRH analog was greatly attenuated in PKC-depleted cells, however, hormone responses to forskolin were enhanced. Stimulation of dopamine D₂ receptors reduced the GtH, but not the GH, responses elicited by PKC activators. These results indicate that PKC participates in the GtH and GH responses to natural neuroendocrine regulators in the goldfish.

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Résumé Il a été établi que chez le poisson rouge, les sécrétions de gonadotropine (GtH) et d'hormone de croissance (GH) sont toutes les deux stimulées par la gonadolibérine (GnRH); de plus, la sécrétion de GtH est inhibée par des mécanismes dopaminergiques de type D₂. Dans le présent travail, la déplétion de la teneur en protéine kinase C (PKC) dans des cellules hypophysaires de poisson rouge réduit les résponses en GtH et GH au GnRH et à un activateur de la PKC de cellules maintenues en incubation statique. Dans des cellules maintenues en périfusion et soumises à une déplétion en PKC, la GtH libérée en réponse à un analogue du sGnRH est fortement diminuée, cependent les réponses hormonales à la forskoline sont augmentées. La stimulation des récepteurs dopaminergiques D₂ réduit, dans le cas d'action d'activateur de la PKC, la réponse en GtH mais pas en GH. Ces résultats indiquent que la PKC est impliquée dans les mécanismes de régulation de GtH et GH par des facteurs neuroendocriniens naturels.

     

Immunohistochemical investigation of the pituitary of the sturgeon (Acipenser baeri,Chondrostei)

An immunohistochemical study of the sturgeon (Acipenser baeri) pituitary was undertaken using antisera directed against hormones from various classes of vertebrates, including the only pituitary hormone available from sturgeon, gonadotrophin. A positive reaction was obtained after application of antisera towards the following hormones 1–24 synthetic ACTH (1-24 ACTH), melanophore stimulating hormone (MSH), ovine prolactin (oPRL), ovine growth hormone (oGH), salmon growth hormone (sGH), carp gonadotrophin (cGTH) and its beta subunit (cGTH), sturgeon gonadotrophin (aciGTH), carp thyrotrophin (cTSH) and subunit of the human thyrotrophin (hTSH). The results demonstrate that, in general, the sturgeon pituitary resembles that of teleosts as regards the distribution of the different cell types: ACTH and PRL cells in the rostral pars distalis, GTH, TSH and GH cells in the proximal pars distalis and MSH and PAS-cells in pars intermedia. In addition to the topographical organization of the sturgeon pituitary, this study provides data on the immunological relationships between sturgeon pituitary hormones and those of other vertebrates.     

Immunohistochemical localization of three GnRH systems in brain and pituitary of Japanese flounder

ABSTRACT:   To clarify the possible roles of gonadotropin-releasing hormone (GnRH) in the reproduction of Japanese flounder Paralichthys olivaceus , localization of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and sea bream GnRH (sbGnRH) immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined together with follicle stimulating hormone (FSH) and luteinizing hormone (LH)-ir cells in the pituitary by immunohistochemistry. sGnRH-ir cell bodies were localized in the ventromedial part of the rostral olfactory bulb and cGnRH-II-ir cell bodies were restricted to the midbrain tegmentum, while sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were also scattered in many areas of the brain with abundance in the midbrain, but sbGnRH-ir fibers were observed in the preoptic–hypothalamic area and innervated the pituitary. In the pituitary, neither sGnRH-ir fibers nor cGnRH-II-ir fibers were found, but sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis, targeting FSH and LH cells. These results suggest that three GnRH systems can play different physiological roles in the brain of Japanese flounder. Among them, sbGnRH is considered to be involved in reproduction by stimulating gonadotropin secretion, while sGnRH and cGnRH-II can function as a neurotransmitter and/or neuromodulator within the brain in this species.     

The regulatory effects of thyrotropin-releasing hormone on growth hormone secretion from the pituitary of common carp in vitro

The effects of thyrotropin-releasing hormone (TRH) on growth hormone (GH) and gonadotropin (GtH) release, and the influences of somatostatin (SRIF), the dopamine agonist apomorphine (APO) and extracellular calcium on basal and TRH-induced GH release were examined using an in vitro perifusion system for pituitary fragments of common carp (Cyprinus carpio). Five minute pulses of different dosages of TRH stimulated a rapid and dose-dependent increase in GH release from the perifused pituitary fragments with an ED₅₀ of 9.7 ± 2.3 nM. TRH was ineffective on GtH release. SRIF significantly inhibited basal and TRH-induced GH release from the perifused pituitary fragments, and the effects of SRIF were dose-dependent. APO induced a dose-dependent increase in basal and TRH-stimulated GH release from the perifused pituitary fragments. Increasing the concentrations of extracellular calcium from 0 mM to 1.25 mM resulted in an increase in basal and TRH-induced GH release. The high dose of calcium (6.25 mM) caused a slight decrease in basal and TRH-induced GH release compared with those at a concentration of 1.25 mM.

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Résumé Les effets de la thyrotropine (TRH) sur la sécrétion d'hormone de croissance (GH) et de gonadotropine (GTH), et de la somatostatine (SRIF), de l'apomorphine (APO), antagoniste dopaminergique, et du calcium extracellulaire sur les sécrétions basale et stimulée de GH ont été étudiées in vitro par périfusion, de fragments d'hypophyses de carpe (Cyprinus carpio). Des applications de 5 minutes de TRH à différentes concentrations induisent une stimulation rapide et dose dépendante de la sécrétion de GH (ED₅₀ = 9.7 ± 2.3 nM). Le TRH est sans effet sur la sécrétion de GTH. Le SRIF inhibe la sécrétion basale de GH ainsi que la résponse hypophysaire à l'action du TRH. Son action est dose dépendante. L'apomorphine induit une augmentation dose dépendante de la sécrétion basale de GH et potentialise l'action du TRH sur la stimulation de la sécrétion de GH. Des effets équivalents sont induits par des concentrations croissantes de calcium extra cellulaire de 0 à 1.2 mM, alors qu'à une concentration de 6.25 mM des effets opposés sont obtenus.

     

Immunocytochemical identification of adenohypophyseal cells in the pirarucu (<Emphasis Type="Italic">Arapaima gigas</Emphasis>), an Amazonian basal teleost

The adenohypophysis (AH) of juvenile pirarucu (Arapaima gigas), a representative species of the Osteoglossomorpha (bonytongue fishes, one of the oldest living groups of the teleosts), was studied using histochemical and immunocytochemical methods. The AH is comprised of the pars distalis (PD), without a clear distinction between rostral pars distalis (RPD) and proximal pars distalis (PPD), and the pars intermedia (PI). The neurohypophysis (NH) is positioned on top of the PD and penetrates and branches into the PI. In the most rostral dorsal portion of the PD, adrenocorticotropic cells and fusiform gonadotropic cells were found. In the central PD, scarce prolactin-producing cells and growth-hormone-producing cells were located mainly in the dorsal part, whereas round gonadotropic cells were abundant in the ventral portion of this region. Human thyrotropin immunoreactive cells were not found in the entire AH. In the PI, melanotropic, some adrenocorticotropic, and somatolactin-producing cells were located intermingled surrounding the neurohypophyseal branches. Our results showed that the A. gigas pituitary has some basal characteristics between the ancient Actinopterygii and the more derived teleosts.     

Development of a homologous radioimmunoassay for Mediterranean yellowtail (Seriola dumerilii, Risso 1810) LH

Purified Mediterranean (M.) yellowtail luteinizing hormone-like gonadotropin (MyLH) and its β-subunit (MyLHβ) served to develop a radioimmunoassay (RIA) for MyLH. The rabbit antisera against MyLH and MyLHβ used for this purpose were tested on pituitary sections by immunocytochemistry. Anti-MyLHβ specifically detected a single type of cells, which were located at the periphery of the proximal pars distalis (PPD) and surrounding the pars intermedia (PI). Anti-MyLH, however, also recognized two other cell types, thyrotropin β-immunoreactive (ir) cells and putative follicle-stimulating hormone-like (MyFSH)-producing cells. Labeling of the two latter cell types was prevented by preabsorption of anti-MyLH with M. yellowtail pituitary glycoprotein -subunit. The standard curve for the RIA was generated using purified MyLH, ¹²⁵I-labeled MyLHβ and anti-MyLHβ at a dilution of 1:70,000, which resulted in the binding of 30% of the tracer added. The standard curve ranged from 0.25 to 50 ng/ml. The midrange of the assay (ED50) was obtained with 5.48–7.87 ng LH/ml. The variation between assays was less than 15%. An average cross-reactivity of FSH in the LH RIA of 8.4% was found. Serial dilutions of M. yellowtail pituitary extracts displaced radiolabelled MyLHβ parallel to the MyLH standard. Application of the LH RIA to blood samples and pituitary cell culture medium provided physiological validation of the assay. Significant increases in LH levels were recorded after salmon GnRH treatments in vivo and in vitro. Serum LH levels from wild fish sampled at the spawning season were significantly higher than those from captive fish sampled in the same period.     

Hypothalamic peptides influencing growth hormone secretion in the goldfish,Carassius auratus

In vivo andin vitro techniques were used to examine the influence of various vertebrate peptides on growth hormone (GH) secretion in the goldfish. Tetradecapeptide somatostatin (SRIF-14) was found to inhibit GH secretionin vitro from perifused pituitary fragments, whereas similar concentrations of a salmonid SRIF peptide (sSRIF-25) did not affect GH secretion from the goldfish pituitary fragments. This indicates that SRIF receptors on the goldfish pituitary are very specific for SRIF-14-like peptides. Salmon gonadotropin (GTH)-releasing hormone (sGnRH) was found to elevate serum GH levels in male goldfish. The dopamine antagonist pimozide alone or injected in combination with sGnRH did not influence serum GH levels, although injection of pimozide alone significantly elevated serum GTH levels, in addition to potentiating the effects of sGnRH on GTH secretion. sGnRH stimulated GH secretion from goldfish pituitary fragmentsin vitro, indicating that sGnRH acts directly at the level of the pituitary to stimulate GH secretion in the goldfish. These results suggest that GnRH may also function as a GH-releasing factor in the goldfish, although the release-inhibitory factors for GH and GTH secretion do appear to be separate and distinct. Two human GH-releasing hormone (hGHRH) peptides were found to be ineffective in altering GH secretionin vitro from the perifused pituitary fragments. Consequently, a role for a mammalian GHRH-like peptide in the hypothalamic regulation of GH secretion in the goldfish remains questionable.     

GnRH_A和紫萁抑制大黄鱼性腺早熟的机制

应用脑垂体组织生理学和免疫组织化学方法，对GnRH-A和紫萁抑制大黄鱼性早熟的作用机制进行了研究。结果表明，性早熟大黄鱼脑垂体许多促性腺激素分泌细胞的胞质出现空泡，提示性早熟的原因可能是由于GtH细胞提早进入分泌活动所致。长期服用促性腺激素释放激素类似物的养殖大黄鱼（没有性早熟），它的脑垂体GtH细胞对GnRH抗独特型发生弱的免疫阳性反应，而对照组性早熟鱼仍出现强的反应，表明实验组大黄鱼脑垂体GtH细胞对GnRH-A的应答能力下降，出现脱敏效应，这可能与GnRH-A抑制大黄鱼性早熟有关。养殖大黄鱼长期服用紫萁，它的脑垂体GtH细胞膜上GnRH受体则没有出现脱敏现象，提示紫萁抑制大黄鱼性早熟的机制不同于GnRH类似物，确切机制有待进一步研究。     

LHRH-A、DA对施氏鲟脑垂体碎片释放GtH的影响

为了探讨在古老的软骨硬鳞鱼中促性腺激素(GtH)的双重内分泌调节作用,本实验设计用离体灌流的方法研究促黄体素释放激素类似物(LHRH-A)和多巴胺(DA)对施氏鲟脑垂体碎片分泌GtH的影响。引入10、100和1 000 nmol/L 3个浓度的LHRH-A对施氏鲟脑垂体碎片3次脉冲式刺激实验;每次间隔1 h,持续5 min,研究不同剂量LHRH-A对鲟鱼脑垂体释放GtH的作用;用200 nmol/L DA对施氏鲟脑垂体碎片持续2 h灌流后引入5 min的1 000nmol/L LHRH-A刺激实验,研究DA如何抑制鲟鱼脑垂体释放GtH。每5 min收集一管灌流液,用放射免疫测定法(RIA)检测灌流液中GtH的含量。结果显示,低剂量LHRH-A随着刺激引入脑垂体释放GtH出现波浪式的增加,中、高剂量出现释放延后现象。LHRH-A在10nmol/L到1 000 nmol/L范围内对刺激脑垂体释放GtH没有剂量依存关系。DA对施氏鲟脑垂体碎片GtH的分泌没有显著影响,但是可以抑制LHRH-A引起的GtH分泌,即DA不能抑制施氏鲟GtH的基础分泌,而只能抑制LHRH-A诱导的GtH分泌。研究结果证明,在高等硬骨鱼类中存在的双重神经内分泌调节在古老的鲟鱼中也存在。     

Characterization of the receptor for gonadotropin-releasing hormone in the pituitary of the African catfish,Clarias gariepinus

Receptors for gonadotropin-releasing hormone (GnRH) were characterized using a radioligand prepared from a superactive analog of salmon GnRH (sGnRH), D-Arg⁶-Pro⁹-sGnRH-NEt (sGnRHa). Binding of¹²⁵I-sGnRHa to catfish pituitary membrane fractions reached equilibrium after 2 h incubation at 4°C. Displacement experiments with several GnRH analogs as well as other peptides, demonstrated the specificity of¹²⁵I-sGnRHa binding. Specific binding was enhanced in the presence of the cation chelator ethylene bis (oxyethylenenitrilo) tetra-acetic acid (EGTA), indicating an inhibitory effect of cations on GnRH-receptor binding. The binding of¹²⁵I-sGnRHa to pituitary membranes was found to be saturable at radioligand concentrations of 5 nM and above. A Scatchard analysis of the saturation data suggested the presence of a single class of high-affinity binding sites (Ka=0.901±0.06×10⁹M^–1, Bmax=1678±150 fmol/mg protein). A comparative study on¹²⁵I-sGnRHa binding to pituitary membrane fractions of male and female catfish, indicated that there were no differences in binding affinity and binding capacity between both sexes. The results demonstrate the presence of specific, saturable GnRH receptors in the African catfish pituitary.     

大鳞大马哈鱼生长激素抗体的制备与鉴定

本文用大鳞大马哈鱼生长激素（ｓＧＨ）免疫日本大耳白兔制备了特异抗血清。应用常规酶联免疫吸附测定法（ＥＬＩＳＡ）对抗血清效价和特异性进行了测定，结果表明ｓＧＨ抗血清只与ｓＧＨ特异反应，与草鱼ＧＨ、草鱼催乳素（ＰＲＬ）、牛ＧＨ及大群大马哈鱼促性腺激素（ｓＧｔＨ）等基本上没有交叉反应，该抗血清的效价约为１：７００００左右。应用ｓＧＨ抗血清，初步建立了ｓＧＨＥＬＩＳＡ检测方法。     

Gonadotropin-releasing hormone receptors: neuroendocrine regulators and neuromodulators

Several lines of evidence support the idea that more than one gonadotropin-releasing hormone receptor (GnRH-R) subtype exists to accommodate the presence of multiple GnRH forms seen in vertebrate species. The development of pituitary endocrine cells, GnRH-Rs type IA (GfA), IB (GfB) and type III in the pituitary and their synchronization with the establishment of GnRH1-3 regulatory pathways reflect a neuroendocrine function for the GnRH-Rs. Based on developmental, morphological and biochemical evidence it can be hypothesized that GnRH1 regulates gonadotrophs through GnRH-R type IA, GnRH2 regulates prolactin secretion through GnRH-R type IB and GnRH3 regulates growth hormone secretion through GnRH-R type III. Therefore, the three native GnRH variants in advance teleost like tilapia might have their respective cognate receptors, each being capable of regulating different and, to some degree the same pituitary endocrine cells. In the brain, the expression pattern of GnRH-R type IB and type III parallels the distribution of GnRH2 and GnRH3 fiber network, which suggests their role in neuromodulation and reproductive behavior.     

Direct actions of serotonin on gonadotropin-II and growth hormone release from goldfish pituitary cells: interactions with gonadotropin-releasing hormone and dopamine and further evaluation of serotonin receptor specificity

In this study, the direct actions of serotonin (5HT) on gonadotropin (GTH)-II and growth hormone (GH) release in the goldfish were tested at the pituitary cell level. 5HT (10 nM - 10 µM) stimulated GTH-II but inhibited GH release from perifused goldfish pituitary cells in a dose-dependent manner. The minimal effective dose of 5HT tested to suppress basal GH secretion (10 nM) was 10-fold lower than that to stimulate GTH-II release (100 nM). The GTH-II releasing effect of 5HT was abolished by repeated 5HT treatment (10 µM) whereas the corresponding inhibition on GH release was unaffected. These results suggest that 5HT receptors on goldfish gonadotrophs and somatotrophs exhibit intrinsic differences in terms of sensitivity to stimulation and resistance to desensitization. Salmon GTH-releasing hormone (sGnRH, 100 nM) stimulated GTH-II and GH release from goldfish pituitary cells. The GTH-II releasing action of sGnRH was unaffected by simultaneous treatment of 5HT (1 µM). However, the corresponding GH response to sGnRH (100 nM) was inhibited. In the goldfish, dopamine is known to stimulate GH release through activation of pituitary D1 receptors. In the present study, the GH-releasing action of dopamine (1 µM) and the D1 agonist SKF38393 (1 µM) was significantly reduced by 5HT (1 µM). To examine the receptor specificity of 5HT action, the effects of 5HT1 and 5HT2 analogs on GTH-II and GH release were tested in goldfish pituitary cells. The 5HT1 agonist 8OH DPAT (0.1 and 1µM) and 5HT2 agonist methyl 5HT (0.1 1µM) mimicked the GTH-II releasing effect of 5HT. The 5HT1 agonist 8OH DPAT (0.1 and 1µM) also stimulated GH release but the 5HT2 agonist methyl 5HT (0.1 and 1µM) was inhibitory to basal GH secretion. In addition, 5HT (1µM) -stimulated GTH-II release was abolished by the 5HT1 antagonist methiothepin (10µM) and 5HT2 antagonist mianserin (10µM). Similarly, the inhibitory action of 5HT (1µM) on basal GH release was blocked by the 5HT2 antagonist mianserin (10µM). The 5HT1 antagonist methiothepin (10µM) was not effective in this regard. These results, taken together, indicate that 5HT exerts its regulatory actions on GTH-II and GH release in the goldfish directly at the pituitary cell level, probably through interactions with other regulators including sGnRH and dopamine. The GTH-II releasing action of 5HT is mediated through 5HT2 and possibly 5HT1 receptors. The inhibition of 5HT on basal GH release is mediated through 5HT2 receptors only. Apparently, 5HT1 receptors are not involved in this inhibitory action. In this study, a paradoxical stimulatory component of 5HT on GH release by activating 5HT1 receptors is also implicated.     

Immunohistochemical changes in production of pituitary hormones during artificial maturation of female Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

ABSTRACT:   Specific antibodies against follicle-stimulating hormone β subunit (FSHβ), prolactin (PRL), and somatolactin (SL) of the Japanese eel Anguilla japonica were produced. These antibodies, as well as antibodies against luteinizing hormone β subunit (LHβ) and growth hormone (GH) produced previously, were used to examine changes in the production of pituitary hormones in female eels during maturation induced by salmon pituitary homogenate (SPH) injection. Immunohistochemical observations showed a decrease in FSH production after SPH injection, suggesting that SPH inhibits FSH production. In contrast, LH production increased markedly with maturation. The number of GH producing cells decreased gradually during maturation, possibly because of inhibition by exogenous GH present in the SPH and/or endogenous insulin-like growth factor-I produced by the stimulation of salmon GH. Although changes in the number of PRL producing cells with maturation were not evident, the number of SL producing cells showed a peak at the late vitellogenic stages, and thereafter decreased to the migratory nucleus stage. These results suggest that GH and SL are involved in sexual maturation in SPH injected eels, as in other fishes.     

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.     

Gonadotropin-releasing hormone and gonadotropin in goldfish and masu salmon

Reproductive activities in vertebrates are regulated by an endocrine system, consisting of the brain-pituitary-gonad axis. In teleosts, gonadotropin-releasing hormone (GnRH) in the brain stimulates gonadotropin (GTH) release in the pituitary gland, but because of lack of the portal vessel, it is not known when and how much GnRH is released for the regulation of GTH release. There are multiple molecular types of GnRH in teleosts and several distinct populations of GnRH neurons in the brain. However, we do not know which types and populations of GnRH neurons regulate reproductive activities. Here we summarize our recent studies on GnRH and GTH in masu salmon Oncorhynchus masou and goldfish Carassius auratus. Immunocytochemistry showed the location and molecular types of GnRH neurons. Salmon (sGnRH) and chicken-II GnRH (cGnRH-II) neuronal fibers were widely distributed in the brain of both masu salmon and goldfish. Only sGnRH fibers were observed in the pituitary of masu salmon, whereas both sGnRH and cGnRH-II fibers were observed in the goldfish pituitary, indicating that species specific GnRH profiles are involved in the regulation of pituitary function in teleosts. A series of experiments in masu salmon and goldfish suggest that among GnRH neuron populations GnRH neurons in the ventral telencephalon and the hypothalamus regulate GTH release, and that GnRH of the terminal nerve origin is not essential to gonadal maturation and ovulation. The biological function of other GnRH neurons remains unkown. Two GTHs appear to be characteristic of teleost; however, regulation of reproduction by these GTHs is a question that remains to be elucidated. In salmonid species, it is proposed that GTH I stimulates early gonadal development, whereas GTH II acts in later stages. When GTH expression was examined in goldfish, both GTH I and II mRNA levels in the pituitary gland showed increases in accordance with gonadal development, unlike the sequential expression of GTH subunits in salmonids. The expression of these GTH subunit mRNAs were affected by water temperature, starvation, and steroid hormones in goldfish, but in what manner these two GTHs regulate gonadal development remains to be clarified.     

养殖鱼类生殖内分泌调控相关功能基因的研究和应用

新型的神经内分泌因子kisspeptin与GnIH(gonadotropin-inhibitory hormone)对哺乳动物的生殖轴起着直接而相反的调控作用,kisspeptin刺激脑垂体促性腺激素(GtH,gonadotropin)的合成与释放,而GnIH抑制GtH的合成与释放。然而,在鱼类中,相关研究甚少。本研究室利用比较基因组学技术,通过同线性分析结合分子生物学手段,在斑马鱼(Danio rerio)、金鱼(Carassius auratus)中克隆得到了kisspeptin(kiss1、kiss2)与GnIH以及它们相关受体的基因cDNA序列。氨基酸序列同源比对结果显示:在脊椎动物中,kisspeptin的核心肽相对保守,而GnIH的核心肽在不同物种中则有较大的差异。比较基因组分析显示kisspeptin与GnIH基因在鱼类、两栖类、鸟类和哺乳类都维持着保守的同线性结构;并且,同线性的结果表明,鱼类kiss1与kiss2基因来源于早期的基因组复制,在漫长的进化过程中,哺乳类丢失了kiss2基因。通过配体受体结合实验,证明2种kisspeptins均能激活其相关受体GPR54(GPR54a、GPR54b),启动下游通路,但却存在一定的配体-受体选择性差异。金鱼、斑马鱼组织表达模式研究显示:kisspeptins与GnIH以及它们相关受体在生殖相关组织或区域(下丘脑、垂体、性腺)均有丰富的表达,提示kisspeptins与GnIH可能参与鱼类的生殖调控。通过化学合成金鱼kisspeptins与GnIH的核心肽,在体注射成熟的雌性金鱼,发现kiss1能显著刺激金鱼LH的分泌,并且能诱导金鱼排卵,而kiss2不能;在高剂量的状况下,GnIH亦能有效抑制金鱼LH的分泌。然而,在离体实验中,2种kisspeptin与GnIH对金鱼垂体细胞LH的分泌均没有显著的影响,提示kisspeptins与GnIH对金鱼的LH调控可能发生于下丘脑水平。以上结果表明:与哺乳动物相类似,在鱼类生殖轴中也存在kisspeptins与GnIH的正负调控系统。本文侧重对这项研究以及相关的研究成果进行归纳与分析,旨为深入探讨鱼类生殖内分泌调控相关功能基因提供参考。