Pituitary gonadotropin-releasing hormone (GnRH) receptor activity in goldfish and catfish: seasonal and gonadal effects

The goldfish pituitary contains two classes of gonadotropin-releasing hormone (GnRH) binding sites, a high affinity/low capacity site and a low affinity/high capacity site (Habibiet al. 1987a), whereas the catfish pituitary contains a single class of high affinity GnRH binding sites (De Leeuwet al. 1988a). Seasonal variations in pituitary GnRH receptor binding parameters, and the effect of castration on pituitary GnRH receptor binding were investigated in goldfish and catfish, respectively. In goldfish, GnRH receptors undergo seasonal variation with the highest pituitary content of both high and low affinity sites occurring during the late stages of gonadal recrudescence. The observed changes in pituitary GnRH receptor content correlate closely with responsiveness to a GnRH agonistin vivo in terms of serum gonadotropin (GTH) levels. In catfish, castration results in a two-fold increase in pituitary GnRH receptor content, which can be reversed by concomitant treatment with androstenedione, but not by the non-aromatizable androgen 11β-hydroxyandrostenedione; changes observed in GnRH receptor content correlate with variations in serum GTH levels and responsiveness to a GnRH agonist. In summary, the present study provides a clear evidence for seasonal variation in pituitary GnRH receptor activity in goldfish, and demonstrates a gonadal feedback mechanism regulating GnRH receptor activity in the catfish pituitary.     

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.     

Characterization of growth hormone binding sites in the goldfish,Carassius auratus: effects of hypophysectomy and hormone injection

A recombinant carp growth hormone (rcGH) was used to develop for a GH radioreceptor binding assay in the goldfish (Carassius auratus). Specific binding of¹²⁵I-rcGH to goldfish liver membranes was a pH, time, temperature, and membrane protein dependent process. Scatchard and LIGAND analysis indicated a single class of high affinity and low capacity binding site, with an association constant (Ka) of 1.9×10¹⁰ M^–1 and a maximum binding capacity (B_max) of 9 fmol mg^–1 protein. Liver tissue displayed the highest¹²⁵I-rcGH binding of all the tissues examined. Displacement of¹²⁵I-rcGH with various unlabeled teleost and mammalian GHs and prolactins revealed that the goldfish hepatic binding site was highly specific for teleost GH. Intraperitoneal administration of 0.1, 1.0, and 10 g rcGH g^–1 body weight to hypophysectomized goldfish resulted in a 27, 52, and 68% decrease in total binding sites, respectively. Injection of a high dose of rat prolactin (rPRL) (5 g rPRL g^–1 body weight) also resulted in a 32% decrease in total binding sites. These results suggest that endogenous GH may have a role in the regulation of its own receptors in the goldfish.     

Molecular characterization and expression pattern of insulin-like growth factor binding protein-3 (IGFBP-3) in common carp, Cyprinus carpio

A full-length cDNA encoding the insulin-like growth factor binding protein-3 (IGFBP-3) was cloned from the liver of common carp (Cyprinus carpio) by RT-PCR. The IGFBP-3 cDNA sequence is 1,680 bp long and has an open reading frame of 882 bp encoding a predicted polypeptide of 293 amino acid residues. The deduced amino acid sequence contains a putative signal peptide of 25 amino acid residues resulting in a mature protein of 268 amino acids. A single band of approximate 1.9 kb was found in liver by Northern blot analysis. IGFBP-3 mRNA was observed in all regions of brain with high levels. In peripheral tissues, high levels of IGFBP-3 mRNA were found in retina, red muscle, liver, heart, posterior intestine, spleen, and testis. Relatively lower levels were found in white muscle, kidney, thymus gland, and ovary, while in head kidney, blood, skin, gill, middle intestine, and anterior intestine, the IGFBP-3 mRNA levels were much lower. IGFBP-3 mRNA was first detected in the blastula stage with significantly high level. The level sharply decreased in gastrula stage, and it became to increase in the following stages. During the reproductive cycle, the abundance of IGFBP-3 mRNA significantly decreased between the recrudescing stage and the matured stage in ovary, although in testis, IGFBP-3 mRNA expression level did not exhibit a significant change. The mRNA expression profiles in the present study imply that the IGFBP-3 may play important physiological functions in common carp development and reproduction.     

Positive feedback control by the gonads on gonadotropin (GTH) and gonadoliberin (GnRH) levels in experimentally matured female silver eels,Anguilla anguilla

Treatment of sham-operated female silver eels with carp pituitary extract stimulated ovarian development and induced increases in pituitary gonadotropin (GTH) and gonadoliberin (GnRH) contents. Both effects of carp pituitary extract were abolished in ovariectomized eels, indicating the involvement of the gonads. Endogenous sexual steroids, the secretion of which was increased during sexual maturation, should be responsible for the stimulation of GTH and GnRH levels. Ovariectomy itself had no significant effect on pituitary GTH and GnRH contents, reflecting the fact that, at the silver stage, sexual steroid levels are too low to exert any significant effect on pituitary GTH and GnRH. The positive feedback control exerted by the gonads on GTH and GnRH levels during sexual maturation, in the eel as well as in some other teleosts, would produce an amplification of the pubertal stimulation of the hypothalamo-pituitary-gonadal axis.     

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.     

Cloning,molecular characterization and expression analysis of insulin-like growth factor binding protein-2 (IGFBP-2) cDNA in goldfish,Carassius auratus

In the present study, a full-length cDNA encoding the insulin-like growth factor binding protein-2 (IGFBP-2) was cloned from the liver of goldfish (Carassius auratus) by rapid amplification of cDNA ends technique. The goldfish IGFBP-2 cDNA sequence was 1,513 bp long and had an open reading frame of 825 bp encoding a predicted polypeptide of 274 amino acid residues. Semi-quantitative RT-PCR results revealed that goldfish IGFBP-2 mRNA was expressed in all detected tissues. In liver, central nervous system and pituitary gland, goldfish IGFBP-2 expressed at high levels, followed by anterior intestine, middle intestine and kidney. In posterior intestine, ovary, skin, fat, spleen, muscle and gill, the goldfish IGFBP-2 expression levels were very low. Fasting and refeeding experiment showed that the mRNA expression of goldfish IGFBP-2 was up-regulated significantly in liver compared to the fed group and restored rapidly to normal level after refed. However, the mRNA expressions of IGFBP-2 in hypothalamus and pituitary of goldfish were insensitive to fasting. Furthermore, the mRNA expressions of IGFBP-2 in hypothalamus, pituitary and liver were varied in periprandial changes and significantly down-regulated at 2 and 4 h after meal. These results imply that the IGFBP-2 mRNA expression may be associated with anabolic and catabolic metabolism and regulated by metabolic factors in goldfish.     

Activin and its receptors in the goldfish

Activin (_AA, _AB and _BB) is a dimeric protein that belongs to the transforming growth factor- (TGF-) superfamily of growth factors and is involved in the regulation of many physiological and developmental processes. Recently, we have demonstrated that porcine activin stimulated goldfish gonadotropin-II (GTH-II) and growth hormone (GH) secretion from dispersed pituitary cells in static culture and pituitary fragments in perifusion. The action of activin in the goldfish is unique in that it has an acute stimulatory effect on the secretion of GTH-II and GH, whereas in mammals activin usually exhibits long-term stimulatory actions on FSH secretion. The action mechanism of activin is different from that of gonadotropin-releasing hormone (GnRH). Using domain-specific antibodies against mammalian activin subunits, we subsequently demonstrated the existence of immunoreactive activin subunits (_A and _B) in the goldfish ovary, testis, pituitary and brain, suggesting endocrine, paracrine and autocrine roles for activin in the regulation of goldfish reproduction. Both activin _A and _B subunits have been cloned from goldfish genome by polymerase chain reaction (PCR). Using the PCR fragments as probes, we have cloned a full length cDNA coding for activin _B subunit from the goldfish ovary. Both activin _A and _B subunits show high homology to those of other vertebrates with the _B subunit much more conserved (93 and 98% identity with human and zebrafish _B subunit, respectively). The identity of the cloned _B subunit was further confirmed by expression in the Chinese hamster ovary (CHO) cells and detection of the specific activity of activin in the culture medium. The messenger RNA of activin _B subunit is expressed in a variety of goldfish tissues including ovary, testis, brain, pituitary, kidney and liver, suggesting a wide range of physiological roles for activin in the goldfish. We have also cloned a full length cDNA coding for the activin Type IIB receptor from the goldfish ovary, suggesting that activin may have paracrine or autocrine actions on the ovarian functions. The identity of the cloned receptor was confirmed by specific binding of¹²⁵ I-activin on COS-1 cells transfected with the cloned Type IIB receptor.     

Central regulation of reproduction in teleosts

As in other vertebrates, reproduction in teleosts depends upon interactions taking place along the brain-pituitary-gonads axis. At the central level, these interactions involve at least three types of factors:A gonadotrophin-releasing factor which has recently been isolated from chum salmon brain extracts. This decapeptide, whose structure is (Trp⁷-Leu⁸)-LHRH, appears to have a widespread distribution among teleosts, and is less active that LHRH or LHRH analogues in releasing gonadotrophin from the teleost pituitary. Immunohistochemical and quantitative studies have demonstrated that Gn-RH neurons are mainly located in the ventral telencephalon and the preoptic area, while projections are found in the entire brain and the pituitary gland.A gonadotrophin release-inhibiting factor has been demonstrated in the anterior preoptic region of the goldfish and a large set of data suggests that dopamine has GRIF activity in goldfish, and in other teleost species, by direct action on the gonadotrophs. Accordingly, a dopaminergic preoptico-hypophyseal pathway could be demonstrated in the goldfish brain.Sex steroids exert, depending on the dosages, either a negative feedback in sexually mature fish or a positive feedback in immature fish. Such a positive feedback is caused by estrogens and aromatizable androgens. Accordingly, the brain of teleosts contains high levels of aromatase activity in particular in the telencephalon and anterior hypothalamus. The distribution of estrogens concentrating cells within the brain is consistent with possible interactions with Gn-RH or catecholaminergic neurons at the level of certain brain territories.These data are discussed in relation with the functional significance of different brain areas where interactions between these different factors possibly take place, in particular the terminal nerve, the ventral telencephalon, the preoptic area and nucleus lateralis tuberis.     

Nucleotide sequence and mRNA expression analysis of <Emphasis Type="Italic">β</Emphasis>-actin gene in the orange-spotted grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The full-length cDNA, encoding the orange-spotted grouper β-actin and spanning 1920 bp including a poly (A) tail, was cloned from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate β-actin than any other members of the actin family. The partial genomic sequence indicated that the organization of the β-actin gene in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low level in the kidney. The expression of β-actin gene in the skeletal muscle was barely detectable. These results indicated that the expression of the orange-spotted grouper β-actin gene showed significant variation in different tissues. Therefore, caution should be taken when using β-actin gene as an internal control in the normalization of gene expression among tissues. Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17α–methyltestosterone (MT) had little effect on the mRNA expression of β-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting β-actin can be used as an internal control for RT-PCR analysis of MT effects on gene expression in these tissues.     

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.     

The GnRH systems in the brain and pituitary of normal and hCG treated European silver eels

The distribution of immunoreactive GnRH was studied in the brain and pituitary gland of normal and human chorionic gonadotrophin (hCG) injected silver eels. It was found that the general organization of GnRH systems in this species is similar to that reported in other teleosts. Cell bodies were present in the olfactory bulbs, ventral telencephalon, periventricular hypothalamus and dorsal tegmentum. No positive perikarya could be detected in the preoptic region. Only scarce fibers were observed in the proximal neurohypophysis. Treatment with hCG does not modify the distribution of GnRH but it increases the density of positive structures, in particular at the level of the pituitary. The results are discussed in relation with the present status of knowledge of the mechanisms underlying the blockage of sexual maturation in the European eel at the silver stage.     

Characterization,cerebral distribution and gonadotropin release activity of neuropeptide Y (NPY) in the goldfish

The presence of a peptide closely related to porcine NPY has been demonstrated in the goldfish brain and pituitary by means of radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). The RIA data demonstrate that displacement curves of brain extracts are parallel to a porcine NPY standard and that in HPLC a compound present in brain extracts is co-eluted with porcine NPY. The distribution of this NPY-like factor within the central nervous system was studied by radioimmunoassay and immunohistochemistry. The results indicated that NPY has a widespread distribution with the highest concentrations being found in the telencephalon and diencephalon. In the pituitary gland, NPY immunoreactive terminals characterized at the electron microscope level were found in the different lobes and, in particular, in close association with the gonadotrophin (GTH) secreting cells. Using anin vitro perifusion system, it was shown that NPY causes a dose dependent increase of GTH release from anterior lobe fragments. These data indicate for the first time in teleosts that NPY is present and widely distributed in the brain and pituitary, and that among other putative functions, could be implicated in the multihormonal release of GTH from the pituitary.     

中国大鲵生长激素受体的克隆与表达分析

利用中国大鲵(Andrias davidianus)性腺转录组测序获得的生长激素受体(GHR)基因部分序列,克隆获得基因全长2992 bp,开放阅读框1812 bp,编码604个氨基酸,该蛋白具有高度保守的FGEFS基序与BOX框。系统进化分析结果显示,中国大鲵GHR氨基酸序列与两栖类非洲爪蟾(Xenopus laevis)同源性最高,蜥形纲锦龟(Chrysemys picta bellii)次之,哺乳类水牛(Bubalus bubalis)和鱼类半滑舌鳎(Cynoglossus semilaevis))最低。实时定量PCR结果表明,GHR基因在肝中表达最高,肌肉、垂体、肾、性腺中的表达量次之,其他各组织表达量较低。在精巢发育早期GHR表达较高,随后表达量逐渐降低,在卵巢中表达量随时间增长而增加;17α-甲基睾丸酮(MT)与芳香化酶抑制剂来曲唑(LE)短暂处理后GHR基因在脑与卵巢中表达量发生变化,MT处理后,脑与卵巢中GHR表达量增加,LE处理后脑与卵巢中表达量降低。研究表明,GHR基因在大鲵性腺发育中可能发挥作用,且MT与LE可能通过不同的途径调节GHR基因的表达。     

孕酮受体膜组分1基因在性成熟雌性半滑舌鳎(Cynoglossus semilaevis)的组织学定位定量分析

运用Western blotting、免疫组化和原位杂交方法检测性成熟半滑舌鳎(Cynoglossus semilaevis)孕酮受体膜组分1(Progesterone receptor membrane component 1,PGRMC1)蛋白和mRNA在不同组织的分布和表达特征.原位杂交结果发现,PGRMC1 mRNA主要分布在成熟的卵母细胞膜上,在脑组织神经元和分散的垂体细胞中也有表达.利用制备的半滑舌鳎PGRMC1多克隆抗体,对不同组织中的PGRMC1蛋白表达量进行Western blotting检测,发现在半滑舌鳎卵巢、脑、肝脏中PGRMC1蛋白表达量相对较高,在垂体、头肾、肾也有表达,但表达量相对较少.免疫组化结果表明,半滑舌鳎PGRMC1蛋白在成熟的卵母细胞膜上显著表达,进一步证明PGRMC1为卵膜上的受体基因,推测其主要在卵膜上行使相关的生理功能.研究结果为探究PGRMC1在半滑舌鳎卵母细胞成熟过程中的生理功能提供了重要参考.     

Estrogen binding sites in the ovary of the European sea bass (Dicentrarchus labrax L.)§

The binding characteristics of the European sea bass estrogen receptor (ER) were determined in ovarian cytosolic and nuclear extracts. Results were consistent with the presence of a single class of high affinity and low capacity ER, the first to be characterized in teleost ovary. This will contribute to our understanding of changes in ER affinity and abundance during sex differentiation in this economically important species.