Physiological roles of FSH and LH in red seabream, Pagrus major

The duality of gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), has been confirmed in most teleost species, but very little is known about their biological functions. To elucidate the physiological roles of FSH and LH in fish reproduction, the expression profiles of GTH subunit genes during gonadal development were analyzed in both male and female red seabream. Furthermore, in vitro studies were carried out to examine the effects of GTHs on steroid hormone production and cytochrome P450 aromatase (P450arom) expression in red seabream gonads. In both sexes, LHβ mRNA was maintained at high levels from the early gametogenesis until spawning season, and declined with gonadal regression. Interestingly, FSHβ mRNA levels in males increased in parallel with testicular development, whereas those in female were remained low throughout oocyte development. From in vitro studies using purified red seabream FSH and LH, both GTHs had a similar potency in stimulating 11-ketotestosterone production by testicular slices, while the biological activity of FSH was much lower than that of LH in stimulating production of estradiol-17β by vitellogenic follicles. Moreover, expression of P450arom mRNA was induced by LH, but not FSH, in ovarian follicles in vitro. FSH was also ineffective in inducing maturational competence and final oocyte maturation. These results suggest that, unlike salmonids, FSH may play an important role during gametogenesis in male, but not female, red seabream, whereas LH may be involved in regulation of both early and late gametogenesis in both sexes.     

Effects of water temperature on the gonadal development and expression of steroidogenic enzymes in the gonad of juvenile red seabream, Pagrus major

The effects of water temperature on the development of hermaphroditic gonads in red seabream (Pagrus major) and on mRNA expression of cytochrome P450 aromatase (P450arom) and 11β-hydroxylase were examined. High water temperature suppressed both expression of P450arom and 11β-hydroxylase and the development of oocytes in ovarian portion of hermaphroditic gonads.     

黄鳝脑芳香化酶基因cDNA的克隆及组织表达特异性分析

根据NCBI数据库报道的黄鳝芳香化酶基因的gDNA序列,设计了一对特异性引物.用Trizol试剂盒提取黄鳝脑总RNA,反转录获得cDNA第一条链,进行PCR扩增.扩增产物经过回收、连接、测序后得到了一条长1514 bp的芳香化酶基因cDNA序列.同源性比较表明该基因属于脑型P450aromB,与其他鱼类脑型P450aromB的同源性较高(>70%),与性腺型P450aromA的同源性较低(<60%),与自身性腺型芳香化酶同源性为59.5%.采用RT-PCR的方法对该基因在雄性黄鳝的各组织表达情况进行了分析,结果表明,该基因的转录本较高的表达于脑和精巢中,较低的在皮肤中表达,而在肝脏、心脏、小肠、肌肉等组织中没有检测到该基因的表达.     

Aromatase (P450arom) and 11β-hydroxylase (P45011β) genes are differentially expressed during the sex change process of the protogynous rice field eel, monopterus albus

Steroids are known to play a crucial role in gonadal sex differentiation in many non-mammalian vertebrates, but also in the gonadal sex change of hermaphroditic teleosts. We investigated the expression of two genes encoding key steroidogenic enzymes, i.e., cytochrome P450 aromatase (P450arom) and cytochrome P45011β-hydroxylase (P45011β), during the sex change of the protogynous rice field eel, Monopterus albus. Using RT-PCR with degenerate primers, we cloned rice field eel homologous fragments for both genes (rcP450arom and rcP45011β) as indicated by the high level of homology with P450arom and P45011β sequences from various vertebrates. Gonadal expression of rcP450arom and rcP45011β mRNA levels were then assessed during the sex change by semi-quantitative RT-PCR and a real-time RT-PCR. rcP450arom was predominantly expressed in ovary, much less in ovotestis, and barely in testis. Conversely, P45011β was markedly up-regulated at the onset of testicular development. These findings underline that regulation of steroidogenesis is an important process in the sex change of protogynous rice field eel, and they clearly indicate that the concomitant down-regulation of P450arom and up-regulation of P45011β are of pivotal importance to the sex change of this species.     

Isolation, cloning, sequencing of brain type aromatase and its expression in male and female Wrasse, Pseudolabrus sieboldi

Pseudolabrus sieboldi, wrasse being a diurnal spawner provides a good opportunity to study the endocrine mechanism of estrogen formation in brain and gonads. Moreover, an extremely large amount of E2 was produced in serum and testis of wrasse. It is assumed that the presence of E2 may play a major role in diurnal gametogenesis in male fish. In this study brain type aromatase have been isolated, cloned and sequenced from the brain of wrasse. Further, the expression pattern of brain type aromatase in gonads and adult tissue of male and female fish have been analyzed. In addition, the diurnal expression pattern of brain type aromatase in both male and female fish brain during spawning season have been analyzed. The P450arom (br) was isolated, cloned and sequenced from both male and female bamboleaf wrasse. The P450arom (br) gene (1877 sequenced nucleotide) contains an ORF of 1470 bp, a 5′-UTR of 18 bp and at least 407 bp in 3′-UTR. The amino acid sequence homology in the coding region of wrasse P450arom (br) is high compared to that of medaka, Oryzias latipes (80%), rainbow trout type 2, Oncorhynchu mykiss (78.2%), fugu, Takifugu ribripes (78%) rainbow trout type 1, (76%), goldfish, Carassius auratus (66.8%) and zebrafish, Danio rerio (66.2%). Expression study reveals that P450arom (br) mRNA were most abundant in brains of both male and female fish throughout the day during the spawning season. RT-PCR study revealed that P450arom (br) was expressed in skin, anal fin and tail fin of both male and female wrasse. P450arom (br) was not detected at any time of the spawning day in either ovary or testis of wrasse.     

Gonadal development, aromatase activity and P450 aromatase gene expression during sex inversion of protogynous red-spotted grouper Epinephelus akaara (Temminck and Schlegel) after implantation of the aromatase inhibitor, fadrozole

In the present study, we implanted 2‐year‐old female red‐spotted grouper, Epinephelus akaara, with a non‐steroidal aromatase inhibitor (AI), fadrozole, in the breeding season and examined changes in gonadal histology, serum sex steroids, aromatase activities and P450 aromatase (P450arom) gene expression in gonads after AI implantation. Aromatase inhibitor at doses from 0.1 to 10.0 mg kg^?1 BW induced a sex inversion and completion of spermatogenesis up to the functional male phase, but doses of 1.0 and 10.0 mg kg^?1 BW AI produced more males than 0.1 mg kg^?1 BW AI. Serum estradiol‐17β (E₂) levels decreased, but 11‐ketotestosterone (11‐KT) levels increased significantly in all the AI‐implanted groups, whereas testosterone (T) levels increased significantly only in the 1.0 mg kg^?1 BW AI‐implanted group. Aromatase activities and P450arom gene expression in gonads were inhibited significantly in the AI‐implanted groups, which was in accordance with the decrease in serum E₂ levels. These results suggested the optimal dose of AI to induce sex inversion to be 1.0 mg kg^?1 BW. Furthermore, the sex inversion induced by AI may be attributed to the inhibition of P450arom gene expression and aromatase activity and the resultant decrease in the biosynthesis of endogenous E₂. Meanwhile, the elevated 11‐KT levels were also associated closely with the occurrence of sex inversion in protogynous red‐spotted grouper.     

Immunolocalization of steroidogenic enzymes (P450scc, P450c17, P450arom) in gonad of Japanese eel

Antibodies against P450scc, P450c17 and P450arom were generated using recombinant proteins. In eel testis, P450scc and P450c17 were immunolocalized as clusters in Leydig cells. In vitellogenic eel ovary, P450scc and P450c17 immunoreactive cells were localized as clusters in the outer layer of the ovarian follicle. In contrast, P450arom seemed to be immunolocalized in the innermost follicle layer.     

Promoter analysis of two aromatase genes in the serial-sex changing gobiid fish, Trimma okinawae

Despite numerous endocrine studies on sex change in teleost, no general mechanism that mediates sex change has emerged. The gobiid fish, Trimma okinawae, can change sex in both directions repeatedly. This phenomenon of sex change in goby assigns it as an excellent animal model to elucidate the understanding mechanisms of sex change. In hermaphrodite fishes, estrogen plays a particularly important role in natural and experimentally induced sex change. To investigate the role of estrogen in the serial-sex changing fish T. okinawae, we cloned and analyzed the 5′-flanking regions of P450arom genes from goby genome DNA. Both regions have consensus sequences of TATA, CRE and ERE. Ad4 binding site was restricted in the region of P450aromA. These findings indicate that different regulators control the expression of the two P450arom genes.     

Expression of cytochrome P-450aromatases in the sex-reversed Nile tilapia

Present study revealed the expression of ovarian (oP450arom) and brain (bP450arom) type cytochrome P-450 aromatases in the gonads and brains of sex reversed Nile tilapia.     

雄性黄颡鱼芳香化酶基因mRNA在性成熟期组织中表达分析

在水温分别为20(常温对照组)、25、30℃条件下,采用RT-PCR方法,研究性腺成熟期雄性黄颡鱼组织中P450芳香化酶(P450aromA和P450aromB)的mRNA表达水平,同时测定性腺成熟系数(GSI)、肝重指数(HSI)、肥满度(CF)和血浆睾酮(T),雌二醇(E2)含量。结果表明,P450aromA在性腺发育成熟期雄性黄颡鱼心脏、肝、脾、胃、肾脏、精巢、鳃、脑、头肾、肠管组织中均无表达,P450aromB只在脑和肠中有表达,在脑中表达量高于肠,表达量随着水温升高而显著下降;各组实验鱼血浆T和E2含量与芳香化酶基因表达量存在相关关系。该实验结果表明,P450aromA可能与精子发生相关,可能不参与黄颡鱼精子排放过程;P450aromB在雄鱼脑中高度表达,可能参与性腺成熟期精子活力保持与排放过程,且应激温度越高,对性腺成熟期精子活力保持与排放过程影响越大。     

条斑星鲽CYP19a基因克隆及其在雄鱼生殖周期中的表达

细胞色素P450芳香化酶(P450arom)作为P450细胞色素酶超家族中的一员,是性类固醇生成途径中的末端酶,能够将雄激素转化为雌激素。在大多数脊椎动物中,P450arom由CYP19单基因编码。但是在鱼类中存在两种P450芳香化酶,分为性腺型芳香化酶(P450aromA)和脑型芳香化酶(P450aromB),它们由不同的基因(CYP19a和CYP19b)编码,分布在不同的组织。通过简并引物扩增及RACE cDNA扩增克隆,在国内外首次获得全长为2167bp的条斑星鲽CYP19a cDNA序列,并将推测的氨基酸序列与其它物种P450arom氨基酸序列进行多重比较,发现存在跨膜螺旋区、I-螺旋区、Ozol肽区、芳香化酶特异保守区以及血红素结合区。通过RT-PCR分析了P450aromA mRNA在条斑星鲽不同组织中的表达情况,结果表明,CYP19a基因主要在脑、卵巢和精巢中表达,其次在肠、肝脏、肾脏也有少量表达。同时也分析了P450aromA mRNA在处于不同发育期的精巢中的表达情况,发现在Ⅱ期精巢中表达量最高,在Ⅴ期精巢中表达量最低。     

脂肪和蛋白质营养对工业化养殖大西洋鲑相关代谢酶和生长基因表达的影响

为探究主要脂肪和蛋白质水平对工业化养殖大西洋鲑(Salmo salar)成鱼脂肪相关代谢酶和生长相关基因表达的影响,本实验采用3×2双因素随机实验设计,设置3个脂肪水平:18%、21%、24%(分别以F18、F21、F24表示),2个蛋白质水平:38%、48%(分别以P38、P48表示)。组合为6个实验处理组,每组3个重复,每重复40尾鱼。实验在室内封闭循环水养殖系统中进行,实验鱼初始体重(650.0±45.50)g,实验期56 d。结果表明:(1)工业化养殖实验鱼的脂肪需求较国外深海网箱养殖明显降低,蛋白质需求相近。P48F21组增重率显著最佳,较其他各组提高22.23%~125.86%(P0.05);P48F24组饲料系数显著最低,较其他各组降低16.24%~30.00%(P0.05)。(2)单因素高脂肪显著提高肝体比(P0.05),高蛋白极显著降低肥满度(P0.01);P48F24组肝体比较其他各组显著提高10.92%~28.16%(P0.05),P48F18组肥满度较其他各组显著降低10.24%~12.31%(P0.05);并创新提出了600~900 g大西洋鲑形体营养调控初步方案。(3)单因素高、中脂肪显著提高肝脂肪分解酶(HL、LPL和总酯酶)活力,高蛋白显著提高LPL和总酯酶活力;P48F21和P48F24组饲粮显著提高肝脂肪分解酶活力,其中LPL活力比P38F18组分别提高46.51%、48.84%(P0.05)。实验处理主要对肝脂肪分解酶产生作用,显现了两组优良饲粮改善生长性能的脂肪生理代谢机制。(4)单因素中脂肪极显著增加肌肉和肝IGF-I基因表达量(P0.01);高蛋白极显著增加肌肉GH、IGF-I及肝IGF-I基因表达量(P0.01);GHR基因表达量,随脂肪或蛋白水平升高均有显著下降特征(P0.05)。P48F21和P48F24组试鱼肌肉GH、IGF-I及肝IGF-I基因表达量显著提高(P0.05),肌肉和肝GHR基因表达量显著降低(P0.05)。初步的新发现是,GH和IGF-I与GHR存在相互制约的负相关内在调控关系,以自身控制鱼类生长和生殖活动处于相对稳定和可控状态下。本研究初步确定,P48F21和P48F24组是工业化养殖大西洋鲑成鱼的主要营养素优良组合饲粮,其中以显著降低脂肪水平和饲料成本的P48F21组合饲粮更佳。     

Gene expression levels of growth hormone receptor and insulin-like growth factor-I in gonads of maturing coho salmon (Oncorhynchus kisutch)

The expression levels of growth hormone receptor (GHR) and insulin-like growth factor (IGF-I) in gonads of maturing coho salmon were measured by real-time PCR. The GHR and IGF-I mRNAs showed different gene expression pattern between male and female. These results suggest that a sexual dimorphism may exist in the GHR and IGF-I mRNA expressions of coho salmon during gonadal maturation     

Stimulation of insulin-like growth factor-I production by recombinant bovine growth hormone in Mozambique tilapia, Oreochromis mossambicus

We have previously reported growth-promoting effects of recombinant bovine growth hormone (rbGH) in Mozambique tilapia, Oreochromis mossambicus, after 4 weekly injections or a single injection of slow-releasing formulation (Posilac^®) (Leedom et al. 2002). In order to obtain further understanding of the role of the growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis in growth in the tilapia, the effects of rbGH on plasma and mRNA levels of IGF-I were examined. Plasma IGF-I levels were significantly increased after rbGH and Posilac^® injections, and a significant correlation was observed between plasma IGF-I levels, body length and mass in both treatments. IGF-I mRNA levels in the liver and in the skeletal muscle were also significantly increased after rbGH and Posilac^® injections, indicating that IGF-I gene expression in these tissues is under control of circulating GH. IGF-I mRNA levels in the gill were not affected by treatment. Liver IGF-I mRNA levels were significantly correlated with body length and with body mass after rbGH and Posilac^® injections. These results indicate that the growth-promoting effect of rbGH in this species is mediated to a significant extent via its stimulation of hepatic production of IGF-I and the resulting increase in plasma IGF-I, and also possibly through locally produced IGF-I in the skeletal muscle, acting in a paracrine or autocrine fashion.