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.     

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.     

Diurnal rhythm of steroid biosynthesis in the testis of terminal phase male of protogynous wrasse, Pseudolabrus sieboldi, a daily spawner

The wrasse, Pseudolabrus sieboldi, is a diandric protogynous labrid fish. Spawning is performed by a terminal phase (TP) male and an initial phase (IP) female between 6:00 and 9:00 h daily during two-month-long spawning season. In the present study, to investigate the roles of steroid hormones in the diurnal spermatogenesis of the P. sieboldi TP male, all steroid hormones produced in the testis were identified and the synthetic pathways of these steroids were determined. Furthermore, the circulating levels of the major steroids produced were analyzed throughout a day at 3-hour intervals during spawning season. In the testis, 11-ketotestosterone (11-KT), estradiol-17β (E2), 17,20β-dihydoxy-4-pregnane-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) were synthesized as the major metabolites. In vitro steroid biosynthesis experiments showed similar results to the circulation profiles of the major steroids. This study is the first to clarify the complete steroidogenic pathways in the gonads of a diandric protogynous species throughout its life, when combined with the results of the steroidogenesis in the ovarian follicles. This is also the first report of a clear diurnal rhythm of the steroid production corresponding to the spermatogenic process in the testis of a male teleost.     

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.     

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.     

Embryonic development in corkwing wrasse,Symphodus melops

Corkwing wrasse, Symphodus melops, is one of the main species used as cleaner fish to combat sea lice infestation in salmon aquaculture; however, there is little knowledge about its biology. Here, we describe the embryonic development of this species and examine the viability of the eggs under three temperature regimes. The experiments were conducted at three water temperature regimes, 12, 15, and 18°C, which resemble common sea water temperatures registered during the spawning season of corkwing wrasse at different latitudes along the Norwegian coast. Corkwing wrasse spawn small spherical eggs of 0.75–0.80 mm in diameter (mean 0.78, CV = 3.6%) with several oil droplets and go through eight developmental stages until hatching. The shortest hatching time was registered after 144 hr at 18°C and after 222 and 372 hr at 15 and 12°C, respectively. These observations provide important baseline biological information to advance the establishment of commercial rearing techniques and sustainable fishing management practices for this heavily exploited species.     

食蚊鱼CYP19a基因的克隆与序列分析

硬骨鱼类CYP19基因与生物的性别分化和激素调节相关,因此可开发用来探究环境激素污染与基因表达的关系。本研究首次克隆和分析了食蚊鱼CYP19a cDNA的全系列,为将CYP19基因作为监测环境激素生物标志物的研究提供了全面的实验数据。根据CYP19a基因c DNA保守区域设计引物,扩增保守区域并测序。采用RACE法扩增食蚊鱼CYP19a基因c DNA序列全长,对其蛋白序列进行同源性分析,并将序列应用于CYP19a mRNA转录水平的RT-PCR法检测中。成功克隆食蚊鱼CYP19a基因全长,获得CYP19a基因总长为2020 bp,ORF为238~1791 bp,共编码518个氨基酸,对其编码的蛋白质进行有关信号肽、跨膜螺旋、亲水性/疏水性、一级结构、二级结构和三级结构分析,与其他硬骨鱼类底鰆、青鰆、平鲷、鲫、鲤和斑马鱼的性腺CYP19a基因作同源性比较,其基因相似度分别为93%、84%、84%、71%、71%和66%。用MEGA6.0软件对19个物种的CYP19a基因进行聚类分析,食蚊鱼CYP19a基因与底鰆、青鰆同源性最高,说明芳香化酶在进化上相对保守。确定从食蚊鱼性腺所克隆的CYP19a基因是芳香化酶基因,证明食蚊鱼的芳香化酶是由CYP19a和CYP19b两种基因编码的。食蚊鱼卵巢芳香化酶具有3个高度保守的片段,并具有催化活性。     

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。     

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.     

Spawning site fidelity and apparent annual survival of walleye (Sander vitreus) differ between a Lake Huron and Lake Erie tributary

Fidelity to spawning habitats can maximise reproductive success of fish by synchronising movements to sites of previous recruitment. To determine the role of reproductive fidelity in structuring walleye Sander vitreus populations in the Laurentian Great Lakes, we used acoustic telemetry combined with Cormack–Jolly–Seber capture–recapture models to estimate spawning site fidelity and apparent annual survival for the Tittabawassee River in Lake Huron and Maumee River in Lake Erie. Walleye in spawning condition were tagged from the Tittabawassee River in Lake Huron and Maumee River in Lake Erie in 2011–2012. Site fidelity and apparent annual survival were estimated from return of individuals to the stream where tagged. Site fidelity estimates were higher in the Tittabawassee River (95%) than the Maumee River (70%) and were not related to sex or fish length at tagging. Apparent annual survival of walleye tagged in the Tittabawassee did not differ among spawning seasons but was higher for female than male walleye and decreased linearly as fish length increased. Apparent annual survival of walleye tagged in the Maumee River did not differ among spawning seasons but was higher for female walleye than male walleye and increased linearly as fish length increased. Greater fidelity of walleye tagged in the Tittabawassee River than walleye tagged in the Maumee River may be related to the close proximity to the Maumee River of other spawning aggregations and multiple spawning sites in Lake Erie. As spawning site fidelity increases, management actions to conserve population structure require an increasing focus on individual stocks.     

Frequency and distribution patterns of Saprolegnia infection in wild and hatchery-reared brown trout Salmo trutta L. and char Salvelinus alpinus (L.)

Abstract. Fungal infection of sexually mature brown trout and char was associated with a particular type of Saprolegnia exhibiting a low degree of homothallic sexuality. Hatchery-reared brown trout were more severely infected (in terms of the % area of the body covered with the fungus) than were wild fish. The fins of hatchery-reared fish were particularly prone to Saprolegnia infection regardless of sex. In wild brown trout, a sexual difference in the pattern of infection was demonstrated. The flanks of the male fish appeared to be more prone to infection when compared with the female and there was a greater susceptibility of the caudal and ventral fin of the female when compared with the male. Evidence is presented which suggests that the incidence of infection in mature male salmonid fish prior to spawning is significantly greater than in the females. This difference may not be apparent in spent fish after spawning. These findings are discussed in relation to the background concentration of fungal spores in the water, the behavioural characteristics of spawning fish and differences in epidermal structure.     

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。     

Use of an acoustic telemetry array for fine scale fish behaviour assessment of captive Paiche,Arapaima gigas,breeders

As Arapaima gigas is one of the most valuable species for the growing production of Amazonian aquaculture, knowledge of its reproductive behaviour and its application to increase reproduction success in captivity is of great importance as no hormonal spawning induction technique exists for this species. An acoustic positioning system (LOTEK Inc.) was used to observe the interactions of adult fish to better understand the formation of mating pairs. Fish were placed in a 4,500 m² aquaculture pond over a 6‐month period in the IIAP field station of Pucallpa, Perú. This paper describes the methodological protocols used to set up and test the hydrophone array and presents the methodology used for the analysis of the huge amount of collected data. This methodology is illustrated by the analysis of a 6‐day period for a mating pair that showed a spawning event. The results indicated that male and female occupied mostly one preferential area in one pond edge where the nesting area is located. Different activity patterns were observed during the spawning event, with male and female being closer during the spawning day. The results also showed that male travelled less distance than female during the studied period. Finally these results demonstrated the suitability of such equipment to monitor fish interactions at fine spatial (sub meter) and temporal (5 s) scales in confined environments like aquaculture ponds.