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.     

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.     

Purification of vitellogenin from the air breathing catfish, Clarias gariepinus

Vitellogenin (Vtg) is a female specific glycophospholipoprotein which can be induced both in male and female with estradiol and xeno-estrogens. The basic theme behind the purification of vitellogenin from the fish is to understand the evolutionary relationship and for the purification and characterization of the Vtg receptor. The male catfish, Clarias gariepinus was administrated with estradiol over a period of time for the synthesis of Vtg and the serum was collected. The Vtg was purified from the serum using a two step chromatographic technique. The serum was passed on to DEAE-ion exchange column and the protein was eluted using a salt gradient. The fractions containing the Vtg were pooled and passed onto a gel permeation chromatography column and the pure protein was obtained. The molecular weight is around 200 kDa on the SDS-PAGE and around 520 kDa on the native gel electrophoresis.     

Seasonal dynamics in gonadotropin secretion and E2-binding in the catfish Heteropneustes fossilis

In the present investigation, significant annual/seasonal variations were noticed in plasma and pituitary gonadotropin (GTH) which were correlated with gonado-somatic index, plasma estradiol-17β, and nuclear E₂ receptor (NE₂R) in the pituitary, hypothalamus and telencephalon. The NE₂R concentrations and dissociation constant (k _d) values showed significant seasonal variations with high values in the late preparatory phase and low values in the postspawning phase. The NE₂R levels were the highest in the pituitary, followed by the hypothalamus and telencephalon in all the seasons. In the prespawning phase, ovariectomy (OVX) elicited a strong negative feedback on GTH secretion with a bimodal pattern of release and elevated the NE₂R levels and k _d values, without producing any significant change in the resting phase suggesting that E₂ appears to exert differential feedbacks on GTH secretion.     

Molecular endocrinology of oocyte growth and maturation in fish

Pituitary gonadotropins (GTHs) are of primary importance in triggering oocyte growth and maturation. However, the actions of GTHs are not direct, but are mediated by the ovarian production of steroidal mediators of oocyte growth (estradiol-17β) and maturation (maturation-inducing hormone, MIH; 17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP in salmonid fishes; 17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S in sciaenid fishes). It is established that production of estradiol-17β and 17α,20β-DP by salmonid ovarian follicles occurs via the interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). A distinct shift in the salmonid steroidogenesis from estradiol-17β to 17α,20β-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. It is possible that this shift is a consequence of dramatic changes in the expression of the genes encoding various steroidogenic enzymes. As an initial step to address this question, we have isolated and characterized the cDNAs encoding a number of ovarian steroidogenic enzymes including the rainbow trout cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase (HSD), 17α-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAS as well as the pig 20β-HSD cDNA. Estradiol-17β stimulates the hepatic synthesis and secretion of a yolk precursor, vitellogenin. Vitellogenin is then transported to the ovary where it is selectively taken up into the oocyte by a receptor-mediated process involving specific cell-surface receptors. Estradiol-17β was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17α,20β-DP and 20β-S is through the binding to the oocyte plasma membrane. This initial MIH-surface interaction is followed by the formation of the major mediator of MIH, maturation-promoting factor (MPF). We have purified MPF from mature oocytes of carp. Carp MPF consists of two components: the homolog of the cdc2⁺ gene product of fission yeast (p34^cdc2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17α,20β-DP treatment, while cyclin B proteins can be detected only in mature oocytes. Addition of bacterially expressed goldfish cyclin B to the extracts of immature goldfish oocytes induced MPF activation. These results suggest that the appearance of cyclin B protein is a crucial step for 17α,20β-DP-induced oocyte maturation in fish.     

Thyroid hormone modulation of ovarian recrudescence of air-breathing catfish Clarias gariepinus

In the present study, thiourea-induced thyroid hormone depletion and thyroxine (T₄) ‘overdose’ were used as a strategy to understand the influence of thyroid hormones on ovarian recrudescence of juvenile (3-months-old), immature (8-months-old) and adult (1-year-old) air-breathing catfish, Clarias gariepinus. Thiourea-induced thyroid hormone depletion in juvenile catfish impaired ovarian development, but no significant effect was observed in immature catfish and during late stage of ovarian recrudescence of mature catfish. T₄ treatment in females undergoing late stages of ovarian recrudescence induced rapid oocyte growth by promoting its early entry into maturational phase as evident from the presence of more number of vitellogenic and post-vitellogenic follicles, decrease in aromatse immunoreactivity and reduced estradiol–17β levels. Hence, thyroid hormones have an important role to play during early stages of ovarian development and vitellogenesis of catfish and also indicating that thyroid has a stage dependent effect on ovary.     

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.     

Regional distribution of microsomal xenobiotic and steroid metabolism in kidney microsomes from rainbow trout

The regional distribution of microsomal cytochrome P-450-mediated reactions with exogenous and endogenous substrates in the kidney of rainbow trout was studied. The cytochrome P-450-dependent 7-ethoxyresorufin- and 7-ethoxycoumarin-0-deethylase activities were significantly higher (3–4 and 10–14 fold, respectively) in the trunk kidney than in the head kidney, whereas ethylmorphine-N-demethylase activity was evenly distributed along the kidney. The microsomal cytochrome P-450-dependent steroid hydroxylases and steroid reducing enzymes when using androstenedione as substrate also exhibited a regional distribution in trout kidney. The 6β- and 16-hydroxylase activities as well as the 5α-reductase and 17 hydroxysteroid oxidoreductase activities were higher in the anterior part of the trunk kidney than in the head kidney and posterior trunk kidney.     

Changes of plasma steroid concentrations associated with spontaneous or induced ovulation in yellow perch Perca flavescens

This study examined the changes in plasma steroids during natural (Experiment 1) and induced (Experiment 2) final maturation in yellow perch Perca flavescens. In experiment 1, ovulating yellow perch were stripped of eggs and blood samples collected to determine the concentrations of testosterone (T), estradiol-17β (E2), and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). Eggs from individual females were weighed and fertilized. Fertilization rate was determined at the embryo eyed stage. In experiment 2, females were randomly assigned to one of the following treatment groups: (1) saline (0.7% NaCl), (2) des-Gly¹⁰[D-Ala⁶] LHRH-ethylamide (100 μg LHRHa/kg), and (3) LHRHa plus 17,20βP (100 μg LHRHa/kg + 2 mg 17,20βP/kg). Fish were injected intraperitoneally with two doses at a two-day interval. Blood was collected prior to injections and at the time of ovulation/spawning and concentrations of T, E2, and 17,20βP (free and conjugated) were determined. In experiment 1, low concentrations of 17,20βP were recorded at spawning. In experiment 2, all surviving fish injected with LHRHa (5 of 5) released their eggs spontaneously during the week following injections. None of the surviving control fish (0 of 5) ovulated during this period, whereas only 1 of 3 surviving fish injected with LHRHa + 17,20βP released eggs. In the control group, concentrations of E2 and 17,20βP did not show significant differences over the experimental period, whereas plasma T concentrations increased significantly. In fish injected with LHRHa, the concentrations of T and 17,20βP increased significantly after the first injection but then declined at ovulation/spawning. It also appears that 17,20βP was conjugated to its sulfated form. Mortality reached 62.5% in the group injected with LHRHa + 17,20βP indicating that this treatment was severe. Thus, LHRHa alone appears highly effective in inducing ovulation in yellow perch. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production, release and olfactory detection of sex steroids by the tench (Tinca tinca L.)

The present study is concerned with pheromone communication in tench (Tinca tinca L.), establishing firstly whether males have a high olfactory sensitivity to some typical teleost sex steroids and prostaglandins; and secondly whether males and females might be able to synthesise and release some of these steroids into the water. The C₂₁ steroid, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was found to give large electro-olfactogram responses with an estimated threshold of detection of 10⁻¹² M. The male tench were equally sensitive to glucuronidated 17,20β-P (10^−11.6 M) but 100 times less sensitive to sulphated 17,20β-P (11^−9.7 M). Preliminary data from cross-adaptation studies suggest that both the free and conjugated forms are detected by the same olfactory receptor(s). Male tench also had high olfactory sensitivity to prostaglandin F_2α (PGF_2α) and 15-keto PGF_2α (11^−11.5 and 10^−11.4 M). They were relatively insensitive, however, to testosterone (T), androstenedione (AD), 11-ketotestosterone (11-KT), 17β-oestradiol (E₂), 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) and 17,20α-dihydroxy-4-pregnen-3-one (17,20α-P). Radioimmunoassays were used to measure the steroids in plasma and water and all samples were processed for the measurement of free, sulphated and glucuronidated fractions. In females, free 17,20β-P, 17,20α-P, free and glucuronidated T, and AD in plasma showed the largest increases in response to injection with mammalian gonadotropin-releasing hormone analogue (GnRHa) or Ovaprim (a mixture of GnRHa and a dopamine inhibitor). Free 17,20β-P was released into the water at the greatest rate. Plasma concentrations of the two conjugated forms of 17,20β-P were also elevated 18 h after the administration of GnRHa, but not by as much as the free steroid. In males, AD and 11-KT showed the greatest increase in response to GnRHa and were moreover released into the water at a higher rate in the treated group than in the control. The data support a possible pheromonal role for free and glucuronidated 17,20β-P. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular cloning and sequencing of hemoglobin-β gene of channel catfish, Ictalurus punctatus Rafinesque

The hemoglobin-β gene of channel catfish, Ictalurus punctatus, was cloned and sequenced. Total RNA from head kidneys was isolated, reverse transcribed and amplified. The sequence of the channel catfish hemoglobin-β gene consists of 600 nucleotides. Analysis of the nucleotide sequence reveals one open reading frame and 5′- as well as 3′-untranslated regions. The open reading frame of the sequence potentially encodes 148 amino acids with a calculated molecular mass of 16.3 kDa. The pI and charge at pH 7.0 of the deduced hemoglobin-β protein were 7.28 and 0.47, respectively. Overall, 22 amino acid residues were conserved throughout the sequences, including His64 and His93, the sites for heme-binding. Unlike the counterpart of other common cultured fish such as Salmo salar, Oncorhynchus nerka, Oncorhynchus mykiss, Cyprinus carpio and Ctenopharyngodon idella, the hemoglobin-β of channel catfish did not have cysteine. The amino acid sequence of channel catfish hemoglobin-β shows 84% homology with that of Silurus asotus (both are in the order Siluriformes). However, comparison with those of other fish species shows homology ranging from 53 to 68%. Structural analysis by the 3D-PSSM program displays that channel catfish hemoglobin-β has eight α-helices, A–H.     

Diel interactions between sprat and mackerel in a marine lough and their effects upon acoustic measurements of fish abundance

We have investigated how diel changes in fish behaviour may affect acoustic estimates of fish abundance in a marine system. Studies in Lough Hyne (Ireland), a marine lake with only a narrow and shallow connection to the sea, showed the presence of large numbers of sprat (Sprattus sprattus), mackerel (Scomber scombrus), and zooplankton. Fish species were identified from regular rod and line fishing. Zooplankton were identified from net hauls. Acoustic measures of zooplankton were low with little diel variation. The sprat formed dense schools during the day and dispersed into the water column at night. The acoustic estimate of sprat abundance at night, obtained by means of a standard hydroacoustic technique and protocol, was more than double the estimated biomass during the day. We have considered whether the lower fish estimate during daytime resulted from acoustic shadowing due to aggregation of the fish into dense schools. However, no decrease in echo energy was evident from the top to the bottom of the schools, and there was no reduction in the seabed echo beneath the schools. Acoustic shadowing was therefore not responsible for the diel differences in the estimates of abundance. Instead, we suggest that the target strength of individual sprat diminished during the day as a result of attacks from predatory mackerel. We observed echoes from gas released by the sprat as they gathered into dense plumes close to the seabed. Compression of the gas remaining within the swim bladder as the fish were moving deeper would also reduce swim bladder volume. Finally, negative buoyancy due to reduced swim bladder volume may in addition have forced the fish to change tilt angle to compensate for sinking. All these effects will reduce the target strengths of individual fish and lead to underestimation of fish abundance based on daytime surveys.     

Uptake and clearance of exogenous estradiol-17β and testosterone during the early development of coho salmon (Oncorhynchus kisutch), including eggs,alevins and fry

The uptake and clearance of estradiol-17β (E₂) and testosterone (T) were examined during the initial stages of development of coho salmon (Oncorhynchus kisutch), including eyed-eggs, newly hatched alevins and first feeding fry. Radiolabeled steroids were administered through the water in tracer amounts with or without their nonradioactive form at 400 μg l⁻¹. Regardless of developmental stage, saturation levels were invariably attained earlier for T than for E₂, thus resulting in a higher incorporation of E₂. However, both steroids had similar clearance patterns. Uptake and clearance was clearly stage-dependent, being fastest in fry, intermediate in alevins and slowest in eggs. Furthermore, combined uptake and clearance patterns showed that exposure to steroid was also higher for E₂ than for T and stage-dependent, but always markedly highest in alevins. Subsequently, based on the observed elimination of the estrogen, a double immersion in E₂ at 400 μg 1⁻¹, administered 2 days apart to maximize exposure during the alevin stage, was assayed for its effect on sex reversal and found to induce the production of 100% females. We suggest that the yolk, which is present in substantial amounts during the initial stages of development in salmonids, can retain the exogenously administered liposoluble steroids, thus providing developing embryos with an extended supply of, and exposure to, these steroids well after the treatment is finished. Together, these findings help to explain the previously observed high effectiveness of sex steroids administered during early development in regulating gonadal differentiation in salmonids, the higher effectiveness of E₂ compared to T, and clarify the localization of the most sensitive period to the action of exogenous steroids at the alevin stage in the coho salmon.     

Determination of dosage of Azotobacter and organic fertilizer for optimum nutrient release, net primary productivity and fish growth in fresh water fish ponds

Four experiments were conducted in order to determine the optimum dosageof Azotobacter chroococcum vis-a-vis organic fertilizer(cow-dung) required for optimum pond productivity. Hydrobiological parameters ofpond water, Azotobacter survival (viable counts), netprimary productivity (NPP) and fish growth were monitored. Studies have revealedthat irrespective of the treatments, dissolved oxygen (DO) levels weresignificantly (P < 0.05) lowered on inoculating the ponds withAzotobacter. Alkalinity, O-PO₄,NO₃-N, turbidity, NPP, plankton population and fish growth weresignificantly (P < 0.05) enhanced in ponds inoculated withAzotobacter @ 100.0 ml pond^–1w^–2 in combination with cow-dung @ 10000 kgha^–1 y^–1. At higher or lower dosages offertilizers, the values in most of these parameters remained low. On the otherhand, total kjeldahl nitrogen and NH₄-N increased continuously. Ingeneral, viable bacterial counts decreased with increase in pH, however, therate of nitrogen fixation was not affected. Multivariate analysis of the data revealed a significantpositive correlation of nutrients (Total kjeldahl Nitrogen, NO₃-N andO-PO₄), with NPP and plankton populations. NH₄-N, however,showed a significant negative correlation with DO, NPP and plankton populations.Highest fish biomass and SGR also coincided with the highest NPP and planktonpopulations, revealing that a dose of 100.0 ml pond^–1w^–2 (for 25 m³ ponds) ofAzotobacter along with 10000 kg ha^–1y^–1 of cow-dung appears to be optimum for obtainingoptimum pond productivity and fish yield. Nutrients in the sediment(NO₃-N and O-PO₄) also followed similar trend. On theother hand, organic carbon increased continuously with each increase in thedosage of fertilizers. A decline in fish biomass and pond productivity at higherfertilizer dosages has been attributed to low DO, high NH₄-N and BOD.     

Glycosidase and sulfatase activities and their possible role in keratan sulfate degradation in metamorphosing bonefish (Albula sp.) leptocephali

During metamorphosis of leptocephalous larvae of the bonefish (Albula sp.), keratan sulfate, the principal glycosaminoglycan of the extracellular gelatinous body matrix, is degraded. Artificial substrates have been utilized to demonstrate the presence of β-N-acetylglucosaminidase, β-galactosidase and sulfatase activities in whole-body homogenates of early metamorphosing leptocephali. The concerted action of these enzymes has been shown to degrade the keratan sulfate polymer in other tissues. This paper describes the extraction, partial purification and some of the physical and kinetic properties of these enzymes. Additionally, starch gel electrophoresis was used to follow glycosidase activities in early, intermediate and advanced metamorphosing larvae. No differences were observed in electrophoretic migration or banding pattern of either β-N-acetylglucosaminidase or β-galactosidase during metamorphosis.     

绿鳍马面鲀CYP17-I基因克隆及其在繁殖周期中的表达

为探究CYP17-I基因在绿鳍马面鲀繁殖周期中的作用及其与性激素水平变化的相关性,实验首先通过SmartTMRace技术克隆得到了绿鳍马面鲀CYP17-I c DNA全长序列1 580 bp,编码511个氨基酸。通过半定量RT-PCR技术检测了CYP17-I基因的时空表达,并采用放射免疫测定法检测雄鱼和雌鱼血清睾酮(T)和雌二醇(E2)的含量变化。结果发现,CYP17-I在卵巢、精巢和脑中表达量较高,其次为胃、心脏、脾脏、肾脏,在垂体和鳃中也检测到了微弱表达。在周期表达中发现,无论在卵巢还是精巢中,CYP17-I基因的表达水平与血清T含量密切相关,呈明显周期性变化,最高值均出现在5月份的卵细胞或精子成熟期,而与血清E2含量无显著的相关性。研究表明,CYP17-I在鱼类繁殖周期中对雄性激素睾酮的生成起关键的调控作用。