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1.
Although gonadotrophins are major regulators of ovarian function in teleosts and other vertebrates, accumulating evidence indicates that the growth hormone (GH)-insulin-like growth factor (IGF) axis also plays an important role in fish reproduction. As a first step to understand the physiological role of the GH-IGF system in the ovarian development of starry flounder (Platichthys stellatus), the expression profiles of GH and IGF messenger RNAs (mRNAs) and plasma GH, IGF-I, estradiol-17β (E2), and testosterone (T) levels during the ovarian development were investigated. The developmental stages of ovaries were divided into five stages (II, III, IV, V, and VI) by histological analysis. The hepatosomatic index (HSI) and gonadosomatic index (GSI) values increased and peaked at stage IV and stage V, respectively, and then declined at stage VI. Pituitary GH mRNA levels decreased sharply at stage III and raised to top level at stage VI. The hepatic IGF-I mRNA levels ascended to maximum value at stage V and then declined significantly at stage VI. However, the hepatic IGF-II mRNA levels remained stable and increased significantly at stage VI. In contrast, the ovarian IGF-I mRNA levels increased gradually and peaked at stage VI. The ovarian IGF-II mRNA levels were initially stable and increased significantly at stage V until the top level at stage VI. Consistent with the pituitary GH mRNA levels, plasma GH levels reduced sharply at stage III and remained depressed until stage V and then raised remarkably at stage VI. Plasma IGF-I level peaked at stage V and then declined to initial level. Plasma E2 level peaked at stage IV and then dramatically descended to the basal level. Plasma T level peaked at stage V and then declined significantly back to the basal level. Based on statistical analysis, significant positive correlations between hepatic IGF-I mRNA and GSI, ovarian IGF-II mRNA and hepatic IGF-II mRNA, ovarian IGF-I mRNA and ovarian IGF-II mRNA, and plasma IGF-I and plasma T were observed, respectively. These results suggest that the GH-IGF system may be involved in the ovarian development of starry flounder; GH and IGFs appear to play distinct roles in the regulation of the ovarian development in paracrine/autocrine manners. These findings extend our knowledge of the roles of the GH-IGF axis on reproduction regulation in fish.  相似文献   

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为探究Smoothened (Smo)信号在精巢不同细胞增殖与存活中的作用,实验分离鉴定了尼罗罗非鱼smo (命名为Onsmo),检测了其在不同组织中的表达分布及在精巢中的细胞表达模式,在尼罗罗非鱼精巢组织的体外培养体系中,用Smo特异性激动剂SAG或抑制剂环巴胺分别进行处理,EdU掺入法及TUNEL法检测了处理后生殖细胞(Vasa+)、Sertoli细胞(Amh+)与Leydig细胞(Cyp17a1+)增殖或凋亡情况。结果显示,Onsmo开放阅读框全长2 478 bp,编码825个氨基酸,含有7次跨膜结构域,与人SMO氨基酸一致性达77%;Onsmo表达于包括精巢在内的多个组织;在精巢中,Onsmo在多种不同类型细胞表达,包括精原细胞、精母细胞、Sertoli细胞以及Leydig细胞;在精巢组织的体外培养体系中,SAG处理对精原细胞增殖具有显著促进作用,而环巴胺处理对Sertoli细胞、Leydig细胞凋亡具有显著促进作用。研究表明,On Smo信号在尼罗罗非鱼精巢精原细胞增殖与体细胞存活中具有重要作用。该研究首次证实...  相似文献   

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SUMMARY: We examined the distribution of two rainbow trout androgen receptors (rtAR: rtAR-α and rtAR-β) in the testis immunohistochemically using a specific antibody to clarify the target cells of androgen in spermatogenesis. Positive rtAR immunoreactivity in paraffin-embedded sections was revealed using microwave treatment, and was detected in the nuclei of Sertoli cells, Leydig cells, and other interstitial cells. The presence of rtAR in Leydig cells suggested that fish androgens regulate Leydig cell activity in an autocrine fashion similar to mammalian androgens. In addition, we found that not all Leydig cells exhibited rtAR immunoreactivity in the mature testis by double staining using anti-3β-hydroxysteroid dehydrogenase (3β-HSD) antibody. Furthermore, rtAR immunoreactivity was also detected in the nuclei of spermatogonia, spermatocytes, and spermatids. The intensity of rtAR immunoreactivity in the nuclei of spermatogonia seemed to be weaker than those of spermatocytes and spermatids. These results suggested that androgens act directly on both germ cells and somatic cells in the regulation of spermatogenesis in the rainbow trout.  相似文献   

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Juvenile rainbow trout (Oncorhynchus mykiss) were passively immunized by intraperitoneal immunization against somatostatin-14 (SS-14) using an antibody originating from egg-laying chicken (Gallus domesticus). Fish were immunized weekly (0, 7, 14, 21, 28, 35 days) with chicken egg yolk-derived immunoglobulin (IgY) against SS-14 (1:25 IgY, 5 mg mL?1), and growth performance, feed utilization as well as plasma concentrations and mRNA levels of growth hormone (GH) and insulin-like growth factor I (IGF-I) were compared to the control group that received placebo immunization with PBS. Passive immunization significantly increased weight gain of treated fish (67.7 ± 7.4 g) compared to the control group (40.1 ± 2.0 g) after 35 days (p < 0.05). Feed conversion ratio (FCR) was significantly improved in the immunized fish (0.7 ± 0.08) compared to control group (1.2 ± 0.06) (p < 0.05). The concentrations of GH and IGF-I in the blood plasma showed no significant differences between the fish treated with anti-SS-14 and those of control during the treatment (p > 0.05). In both groups, GH levels decreased over the 35 days of the experiment (p < 0.05). However, IGF-I level during the period of treatment remained unchanged in both control and immunized fish with the anti-SS-14. Similarly, no changes were observed in pituitary GH and liver IGF-I mRNA levels between treatment and control at each sampling time (p > 0.05). There was no indication of a cumulative, long-lasting effect of repeated immunization on GH or IGF-I plasma concentrations or mRNA expression. The present study shows that a passive immunization of rainbow trout against SS-14 using a chicken egg yolk-derived SS-14 antibody could increase growth rate and improved FCR.  相似文献   

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Using rainbow trout plasma protein (IGF-BP) which specifically binds human insulin-like growth factor (IGF) (Niu and Le Bail 1993), we have developed an assay to measure plasma IGF-like levels in different teleost species. Before the assay and to prevent interference by IGF-BP, IGF-like was extracted from all samples, using SP Sephadex C-25 in acidic conditions. After this treatment, contamination of the IGF fraction by IGF-BP which was estimated by binding assay, was approximately 5%, and was not detectable by western ligand blot. Human IGF-I was used as standard and labelled hormone. Sensitivity of the assay was 0.15–0.40 ng/ml (ED90) and ED50 was 1–3 ng/ml. hIGF-II crossreaction was partial and no significant displacement was observed with Insulin from different species or with other hormones. Inhibition curves were obtained with plasma IGF fractions (but not with tissue extracts) from teleost and mammals and are parallel to the standard curve. These results suggest that the protein binding assay can quantify an IGF-like factor in the plasma of teleost and that the binding sites of IGF are well conserved during vertebrate evolution. Using this IGF binding assay, IGF-like was measured in parallel with growth hormone (GH) in plasma from young rainbow trout killed every 1.5h throughout one day. The daily profiles for both hormones, which appear pulsatile, are similar. A significant correlation was observed between GH levels and IGF-like levels with a 1.5h delay. Analogous observations were obtained in individual catheterized adult rainbow trout. Although plasma GH levels differ greatly between fish, less variability is found with IGF-like. In a third experiment, rainbow trout were starved or submitted to bovine GH treatment for four weeks. Starved fish, in which plasma GH levels increased, had plasma IGF-like level significantly lower than in fed fish. In bGH injected fish, plasma IGF-like level was significantly higher than in non-injected fish. These results suggest that, as in mammals, IGF-like secretion depends on plasma GH level and could be modulated by the nutritional status of fish.  相似文献   

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The role of growth hormone (GH) in regulating hepatic mRNA expression of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) in yellowtail Seriola quinqueradiata was examined using in vivo and in vitro assays. Yellowtail hepatic IGF-I, IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-5 mRNAs were measured by real-time quantitative RT-PCR. Intraperitoneal injection of recombinant GH of chum salmon Oncorhynchus keta (rsGH) at a dose of 1 μg/g body weight resulted in a significant increases in hepatic IGF-I, IGFBP-3, and IGFBP-5 mRNA levels, whereas significant reductions in hepatic IGFBP-1 and IGFBP-2 mRNA levels were observed. For in vitro assays, liver slices were incubated with rsGH at different concentrations (doses: 0, 1, 10, 100, 500, and 1,000 ng/ml). Liver slices incubated with 100 ng/ml rsGH elicited a significant increase in IGF-I mRNA level. Similarly, a slight increase in IGFBP-3 and IGFBP-5 mRNA levels were also observed in liver explants incubated with rsGH. In contrast, a significant decline in IGFBP-1 mRNA levels was observed in liver slices incubated with 1,000 ng/ml rsGH. A slight decline in the level of IGFBP-2 mRNA was noted in liver explants with rsGH treatment. This study demonstrates the modulating effect of GH on the IGF system.  相似文献   

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Fish testis is equipped with different isoforms of nitric oxide synthase (NOSs) and is capable of producing nitric oxide (NO). Cellular sources of NO in the catfish testis are germ cells, Leydig cells, and macrophages. Production of testicular NO is under endocrine inhibitory control. Expression of NOSs exhibits seasonality and that depends on the reproductive status of fish. Leydig cells are highly sensitive to chemical as well as biological NO. NO inhibits testosterone production by the testis in vivo as well as by the isolated Leydig cells in vitro.  相似文献   

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The tilapia, Oreochromis mossambicus, exhibits a sexually dimorphic pattern of growth, males growing larger than females. We examined the effects of E2 and DHT on the GH/IGF-I axis and on VTG production in the tilapia. Sexually mature tilapia were injected with 5 μg g body weight of E2 (males) or DHT (females) every 5 days for a total of 3 injections. Female tilapia had significantly higher plasma GH levels than males. However, plasma and liver mRNA levels of IGF-I were significantly lower in females than in males, whereas VTG levels in both the plasma and liver mRNA were significantly higher in females than in males. Although significant amounts of VTG were detected in control males (8 ± 0.3 μg ml), the levels in control females (3000 ± 500 μg ml) were about 400 times higher than in males. Males treated with E2 exhibited a female-like GH/IGF-I profile. That is, they had significantly elevated levels of plasma GH with lower plasma IGF-I and liver IGF-I mRNA levels. Estradiol treatment significantly elevated both plasma and liver mRNA VTG levels. Dihydrotestosterone treatment in females induced a male-like GH/IGF-I profile: plasma GH levels were significantly reduced, whereas plasma and liver IGF-I mRNA levels were significantly elevated. Both plasma and liver mRNA levels of VTG were not altered by DHT treatment. Pituitary GH mRNA levels were similar in all treatment groups. These results clearly indicate that estrogens and androgens feminize and masculinize the GH/IGF-I axis, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

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The morphometric study of spermatogenic cysts in sexually mature tilapias, during the evolution of spermatogenesis, showed a dramatic increase in both number of germ cells and cyst volume. However, the opposite trend was observed for germ cell size. Nevertheless, the number of Sertoli cells increased gradually up to leptotene/zygotene cysts, stabilizing thereafter. Based on the number of spermatids supported by each Sertoli cell and compared to mammals, Sertoli cell efficiency in tilapias is remarkably high. Sertoli cell proliferation was frequently observed, mainly in spermatogonial cysts, and probably is the major factor related to the testis growth and the increase in sperm production that normally occurs in adult tilapias. The combined duration of spermatocytes (5 days) and spermiogenic (5–6 days) phases of spermatogenesis in fish kept at 25 °C was 10–11 days. Mainly due to acceleration in meiosis, these two phases lasted a total of 6 days in tilapias kept at 30 °C, in the opposite way, at 20 °C spermatogenesis was arrested at pachytene spermatocytes. To our knowledge, this is the most comprehensive investigation performed up to date on testis morphometry and function in adult tilapias.  相似文献   

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The effect of gonadotropins, forskolin and IGF-I on steroidogenic enzyme gene expression and E2 production by the rainbow trout follicle were measured. The results indicate that 3β-HSD gene expression is regulated by gonadotropins partially through the cAMP/PKA mechanism. The effect of IGF-I on P450arom mRNA levels suggests that IGF-I is a major regulator of P450arom gene expression.  相似文献   

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To examine the hormonal and nutritional regulation of insulin-like growth factor I (IGF-I) mRNA expression, a sequence-specific solution hybridization/RNase protection assay for coho salmon IGF-I mRNA was developed. This assay is both rapid and sensitive and has low inter- (less than 15%) and intra-assay variations (less than 5%). Using this assay, the tissue distribution of IGF-I mRNA and effects of growth hormone (GH), prolactin (PRL) and somatolactin (SL) on hepatic IGF-I mRNA expression in coho salmon were examined in vivo. Liver had the highest IGF-I mRNA level of 16 pg/μg DNA. Significant amounts of IGF-I mRNA were also found in all other tissues examined (intestine 4.1, kidney 3.8, gill arch 2.4, brain 2.4, ovary 2.3, muscle 2.1, spleen 1.7 and fat 1.1 pg/μg DNA). Injection of coho salmon GH at doses of 0.1 and 1 μg/g body weight significantly increased the hepatic IGF-I mRNA levels in a dose-dependent manner. Injection of coho salmon SL, a recently discovered member of the GH/PRL family, stimulated the IGF-I mRNA expression at the higher dose (1 μg/g), whereas coho salmon PRL had no effect at either dose. Concentration-dependent stimulation by coho salmon GH was also obtained in vitro in primary culture of salmon hepatocytes in concentrations ranging from 0.01 to 1 μg/ml. These results indicate that IGF-I mRNA expression occurs in a variety of tissues in coho salmon, and that at least the hepatic expression is under the regulation of GH and possibly other hormones. The sequence-specific assay established in the present study can be used for accurate quantitation of IGF-I mRNA in salmonid species, and can contribute to a better understanding of the physiology of IGF-I in salmonids.
Résumé Afin d'étudier les régulations homronales et nutritionnelles de l'expression des ARNm de l'IGF-I (insulin-like growth factor I), un dosage spécifique par hybridation en solution des ARNm d'IGF-I de saumon coho et protégé des RNases, a été développé. Ce dosage, à la fois rapide et sensible, présente un faible coefficient de variation inter- (< 15%) et intra- (< 5%) dosage. L'étude de la distribution tissulaire des ARNm de l'IGF-I et des effets de l'hormone de croissance (GH), de la prolactine (Prl) et de la somatolactine (SI) sur l'expression hépatique des ARNm de l'IGF-I, a été entreprise in vivo chez le saumon coho en utilisant ce dosage. Le foie présente les plus grandes quantités d'ARNm d'IGF-I (16 pg/μg d'ADN). Des quantités significatives d'ARNm d'IGF-I ont été également détectées dans tous les autres tissus étudiés (intestin 4,1; rein 3,8; branchie 2,4; ovaire 2,3; muscle 2,1; rate 1,7 et graisse 1,1 pg/μg d'ADN). L'injection à des saumons coho, de GH à des doses de 0,1 et 1 μg/g de poids vif, augmente significativement et de manière dose dépendante les niveaux hépatiques d'ARNm d'IGF-I. L'injection de SI de saumon coho, un membre récemment découvert de la famille GH/Prl, stimule avec la plus haute dose utilisée, l'expression des ARNm d'IGF-I alors que la Prl n'a aucun effet. La GH augmente de manière dose dépendante (0,01–1 μg/ml) l'expression in vitro des ARNm d'IGF-I par des ARNm d'IGF-I par des hépatocytes de saumon coho en culture. Ces résultats indiquent que, chez le saumon coho, l'expression des ARNm d'IGF-I est présente dans le nombreaux tissus et que, l'expression hépatique est, au moins en partie, régulée par la GH et peut-être par d'autres hormones. Le dosage par séquence spécifique mise au point dans le présent travail, peut-être utilisé pour la quantification précise des ARNm, d'IGF-I de salmonidés et devrait permettre une meilleure connaissance de la physiologie de L'IGF-I chez les salmonidés.
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瓦氏黄颡鱼生长激素基因克隆及其组织特异性表达分析   总被引:1,自引:1,他引:0  
生长激素(growth hormone,GH)对脊椎动物的生长发育及代谢具有重要作用。采用RT-PCR和RACE技术,克隆了瓦氏黄颡鱼垂体GH cDNA全长序列,应用real-time qPCR法对不同组织中GH mRNA的表达进行检测。序列分析表明,GH cDNA(GenBank登录号:GU395549)序列全长1 203 bp,其5’端非编码区77 bp、3’端非编码区523 bp,开放阅读框(open reading frame,ORF)603 bp,由此推导GH前体蛋白由200个氨基酸组成。同源性比较结果表明,瓦氏黄颡鱼与同目鱼类的GH编码序列同源性较高,与哺乳类和鸟类的同源性较低。Real-time qPCR结果显示,GH mRNA在垂体中的表达量最高,其次是脑、肌肉、肝脏、脂肪组织、胃、脾脏、卵巢或精巢,而在肾脏、心脏、鳃和肠中没有明显的表达。实验结果表明,GH基因在瓦氏黄颡鱼组织中广泛表达,提示GH可能以旁分泌或自分泌的方式对其生长和繁殖发挥重要作用。  相似文献   

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Insulin-like growth factors I and II (IGF-I and IGF-II) are two highly homologous mitogenic peptides that are expressed ubiquitously and show diverse effects on development, growth, and metabolism. The cDNA encoding IGF-I of a teleost, the orange spotted grouper (Epinephelus coioides) was produced from liver by RT-PCR, and rapid amplification of cDNA ends, RACE. Typically, the deduced 186 amino acid protein contains a signal peptide, B, C, A, D and E domains. On the amino acid level, grouper IGF-I shares 97.3% similarity with black seabream (Sparus macrocephalus) with the differences focusing on the B and C domains. The analysis of the E domain showed that grouper IGF-I belonged to Ea-4 type. When mature amino acid sequence was compared with other vertebrates, it revealed higher similarity with black seabream and halibut, while lower similarity with human and mouse. The expression of IGF-I mRNA in adult tissues was studied using RT-PCR. IGF-I mRNA expression level in the liver was significantly higher than those in the brain and muscles. In other tissues, low amount of IGF-I mRNA expression was also detected. The coding region of IGF-I cDNA for mature IGF-I protein was subcloned into an expression plasmid pTRX and fused with E. coli thioredoxin (Trx). Moreover, we have successfully developed an expression system in E. coli to overproduce recombinant grouper IGF-I. Using western blotting, we found that the fusion protein could blot with antiserum to barramundi IGF-I further confirming the immunoactivity of the recombinant IGF-I.  相似文献   

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