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.     

Thyroxine-induced alterations in the testis and seminal vesicles of air-breathing catfish, Clarias gariepinus

Effect of experimentally induced thyroxine overdose on the testis and seminal vesicles was studied in the air-breathing catfish, Clarias gariepinus during the preparatory and the pre-spawning phase. The present study revealed a marked reduction in testosterone level in serum, testis and seminal vesicles (SV). Histological examination showed a considerable reduction in the number of spermatozoa/spermatids in the seminiferous tubular lumen as well as depletion of fluid in the loculi of SV. SDS-PAGE analysis of SV fluid proteins demonstrated a significant decrease in the level of a ~27 kDa protein in thyroxine treated fishes. Evidences are presented here to indicate that thyroid hormone plays a role in regulating testis and SV function in catfish. T.N. Jacob and J.P. Pandey contributed equally     

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.     

Mismatch between patterns of circulating and testicular androgens in African catfish, Clarias gariepinus

11-Ketotestosterone (11-KT) is an important plasma androgen in male African catfish. The quantitatively predominating androgen produced by the testis, however, is 11-hydroxyandrostenedione (OHA). Our working hypothesis to explain this mismatch assumed that OHA is converted to 11-KT at extratesticular sites. First, we examined the in vivo metabolism of [³H]-OHA in mature males after sham-operation or removal of either the testes (TC), the seminal vesicles (SVC), or both (TSVC) by analysing the pattern of circulating [³H]-androgens two hours after intravenous injection of [³H]-OHA. [³H]-OHA was converted to [³H]-11-KT to the same extent in all groups, indicating that neither ablation of testes nor of seminal vesicles, or both attenuates this conversion. We then examined the in vitro metabolism of [³H]-OHA by several types of tissues. Liver and seminal vesicle tissue were found to produce significant amounts of [³H]-11-KT. The conversion capacity in vivo was assessed by injecting TSVC-castrated males with increasing doses of radioinert OHA, followed by the quantification of OHA and 11-KT plasma levels. Saturation of the conversion capacity was not reached but the 11-KT production capacity is at least 80 ng per ml of plasma per hour. Moreover, liver fragments were tested for their OHA to 11-KT conversion capacity in vitro using increasing concentrations of radioinert OHA. The 11-KT producing increased with time and OHA concentration. The production rate was 90 pg 11-KT mg^-1 liver h^-1. Considering the results of the surgical experiments and the fact that the total hepatic mass by far exceeds that of the seminal vesicles, we conclude that the hepatic conversion is of primary relevance in vivo.     

Catecholestrogens inhibit dopamine methylation in the gonadotrops of the African catfish,Clarias gariepinus

Isolated gonadotrops of the African catfish,Clarias gariepinus, were incubated with dopamine (DA) and/or catecholestrone and the activity of the enzyme catechol-O-methyltransferase (COMT) was determined by measuring the methylated products. From the apparent Km values for DA and catecholestrone of 0.4–1.3 M and 17.9–25.2 M respectively, it was concluded that catecholestrone is a better substrate for the enzyme COMT, compared to DA. Moreover, the methylation of DA is inhibited by comparatively low concentrations of catecholestrone.     

In vitro induction of vitellogenin by estradiol 17 β in isolated hepatocytes of catfish, Clarias gariepinus

Vitellogenin is a female-specific calcium-binding glycolipophosphoprotein synthesized in the hepatocytes of fishes. Its synthesis can be induced in fishes of either sex by estradiol or by xenoestrogens. To study the in vitro synthesis of vitellogenin, different culture conditions were set up using the hepatocytes of Clarias gariepinus. The present study reports on a non-enzymatic procedure for isolation and culture of hepatocytes from the liver of the catfish Clarias gariepinus, in order to study the effects of estradiol on vitellogenin synthesis in vitro. The procedure employs chelating properties of ethylenediamine tetracetic acid to achieve cell viability in excess of 95%. Equal numbers of isolated cells were incubated in different culture media viz. RPMI F1640, Medium-199, and Williams’ Medium E. At 36 h, cell attachment and monolayer formation is faster in M-199 and Williams’ Medium E than in RPMI. In order to study the effects of estradiol on vitellogenin synthesis, the isolated hepatocytes were seeded in Williams’ Medium E in 24-well cell culture plates. 17 β-estradiol (E₂) was introduced in the culture plates at different concentrations and for different time periods. The media were assayed for vitellogenin using competitive ELISA. Vitellogenin appeared in the medium after 48 h of incubation with 10⁻⁵ M estradiol whereas after 72 h of incubation 5×10⁻⁷ M E₂ could elicit the synthesis.     

Metabolic enzyme activities in larvae of the African catfish,Clarias gariepinus: changes in relation to age and nutrition

The influence of ontogeny and nutrition on metabolic enzyme activities in larvae of the African catfish, Clarias gariepinus, was studied. After start of exogenous feeding, the larvae were reared for 10 days under three different nutritional conditions: Artemia nauplii, a dry starter diet, and starvation. The live feed gave the best growth (96 mg within 10 days) whereas the dry diet resulted in low growth (33 mg). This growth difference was reflected in larval RNA and DNA concentrations, but not in the levels of soluble protein. Enzymes representing the following aspects of metabolism have been analysed: NADPH generation (G6PDH, ME), glycolysis (PFK, PK), gluconeogenesis (FDPase), amino acid catabolism (GOT, GPT) and oxidative catabolism (CS). All enzymes were present from the start of exogenous feeding onwards, but their maximum specific activities displayed different developmental patterns. In catfish larvae fed on Artemia, G6PDH and ME activities steadily increased with age and weight of the larvae. CS levels remained, after an immediate enhancement upon onset of exogenous feeding, on a rather stable plateau. The amino acid-degrading enzymes GOT and GPT showed maximum levels at days 3–5 of feeding or at a body weight of 10–20 mg, but decreased thereafter. Activities of PFK, PK and FDPase showed low initial levels, and increased significantly with age and size. Based on the ontogenetic patterns of metabolic enzymes, in C. gariepinus larvae an early and a late developmental phase can be distinguished. During the early phase, the glycolytic and gluconeogenetic capacities are low, whereas they are enforced during the later phase. The oxidative capacity is high both during the early and the late phase. The metabolic changes in catfish development coincide with other major ontogenetic events, e.g., alterations of muscle organization, gill morphology, respiration and stomach structure and function. Rearing catfish larvae on a dry diet instead of Artemia partly altered the developmental pattern described: The ontogenetic elevation of CS, PFK and FDPase was delayed and the early peak in GOT and GPT activities was not realized. Particularly during the early developmental phase, the enzyme behaviour of the larvae fed on dry food was similar to that of starved larvae.Abbreviations CS citrate synthase - FDPase fructose-1,6-diphosphatase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - G6PDH glucose-6-phosphate dehydrogenase - ME malic enzyme - PFK phosphofructokinase - PK pyruvate kinase     

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.     

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.     

黄颡鱼促黄体激素受体基因克隆及在雌鱼繁殖周期中的表达

通过简并引物扩增及SmartTM Race技术,首次克隆了黄颡鱼促黄体激素受体(LHR)基因cDNA全长序列.该基因全长2 524 bp,编码701aa,含有属于糖蛋白激素受体(GpHR)家族的典型跨膜螺旋结构区域(TM helix).9个富含亮氨酸的重复性序列(LRRs);4个潜在的N-端糖基化位点:29NFTC,82 NVSR,203 NGSR,548NLTV;24个Ser,6个Thr和5个Tyr磷酸化位点.同时400S为潜在的PKC位点.通过RT-PCR分析组织表达,卵巢繁殖周期中卵巢和脑的表达水平并结合血浆中E2含量的变化,发现LHR在卵巢中大量表达,其次是脑、肾脏、心脏、肝脏和肠存在少量或微量的表达;在卵巢繁殖周期中,卵巢LHR表达从Ⅲ期-V期处于较高水平,V期达到最高峰,随后下降到最低值;而脑的表达高峰出现在Ⅳ期,与血浆中E2变化相一致.推测卵巢和脑中LHR基因参与卵黄的生成,调控卵母细胞的成熟和排卵.     

黄颡鱼促卵泡激素受体基因的克隆及其在雌鱼生殖周期中的表达

为研究黄颡鱼促卵泡激素受体(FSHR)基因的克隆以及该基因在卵巢发育周期中的表达情况,实验首先采用SmartTMRace技术,获得了黄颡鱼促卵泡激素受体基因cDNA全长序列,该基因全长2 340 bp,编码661aa,具有G蛋白偶联受体超家族(G protein-coupled receptor,GPCR)的7个跨膜螺旋区(transmembrane domain)。经分析发现,得到的FSHR基因序列具有9个富含亮氨酸的重复性序列(LRRs);5个潜在的N-端糖基化位点;蛋白激酶C(proteinkinase C,PKC)磷酸化位点(403Y)位于第一个包外环,C-末端(C-terminal domain)G蛋白偶联区域,存在3个蛋白激酶C磷酸化位点(634S,643S,632T)。这些区域可能与FSHR的功能有密切关系。RT-PCR发现,FSHR基因在卵巢和脑中大量表达,在其他组织中均存在少量或微量的表达;在繁殖周期中,卵巢FSHR基因表达从Ⅲ期～Ⅳ期处于较高水平,在Ⅵ期时下降。研究推测,卵巢中FSHR基因参与黄颡鱼卵母细胞成熟及卵黄蛋白的积累过程。     

Isolation and expression of rainbow trout (Oncorhynchus mykiss) ovarian cDNA encoding 3β-hydroxysteroid dehydrogenase/Δ5−4-isomerase

A distinct shift in steroidogenesis from testosterone to 17α-hydroxyprogesterone occurs in the salmonid ovarian thecal cell layers immediately prior to oocyte maturation, and is a prerequisite for the production of 17α,20β-dihydroxy-4-pregnen-3-one (maturation-inducing hormone of salmonid fishes) by granulosa cells during oocyte maturation. 17α-Hydroxylase/17,20-lyase cytochrome P-450 (P-450_17α) and 3β-hydroxysteroid dehydrogenase/Δ^5?4-isomerase (3β-HSD) are the two major steroidogenic enzymes involved in the production of 17α-hydroxyprogesterone and testosterone. Using mammalian cDNA probes, we isolated and characterized full-length cDNAs encoding these two enzymes from a rainbow trout (Oncorhynchus mykiss) ovarian thecal cell cDNA library. The cloning of 2.4-kilobase cDNA encoding P-450_17α and transient expression of this clone in nonsteroidogenic monkey kidney tumor COS-1 cells have recently been reported (Sakai et al. 1992). We have isolated a 1.4-kilobase cDNA which is hybridized to the mammalian 3β-HSD cDNAs. Expression of this cDNA in COS-1 cells led to the production of an enzyme which is capable of converting dehydroepiandrosterone to androstenedione. In this study, enzymatic activities and expression of rainbow trout ovarian P-450_17α and 3β-HSD are discussed in relation of the steroidogenic shift occurring in the ovarian follicle layers.     

Androgen metabolism in the skin of the rainbow trout (Oncorhynchus mykiss)

Pieces of skin of male and female rainbow trout (Oncorhynchus mykiss) were incubated with testosterone and 11-ketotestosterone as substrates. In immature fish the conversion rate was low. In non-spawning adult males 11-ketotestosterone was reduced to 5α-11KDHT (up to 5.2%). In the fish in spawning condition the 5α-reduction rate was only about 1 to 2%. In the same specimens incubated with testosterone a high 11β-hydroxylase activity (23.8-25% in the male and 13% in the female skin) was found. Similar sex specific differences were observed for the occurence of 5α-reduced metabolites (about 20% in the male and 13% in the female tissue).

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Résumé Des fragments de peau de truites arc en ciel (Oncorhynchus mykiss, males ou femelles, ont été incubés avec de la testostérone ou de la 11-cétotestostérone, utilisées comme substrats. Chez les poissons immatures, les taux de conversion restent faibles. Chez les males adultes ne donnant pas de sperme, la 11-cétotestostérone est réduite en 5α-androstane-17β-ol-3,11-dione (jusqu'à 5.2%). Chezles poissons en conditions de fraie, le taux de 5α-réduction est seulement de l'ordre de 1 à 2%. Pour ce derniers individus, les incubations de peau en présence de testostérone montrent l'existence d'une forte activité 11β-hydroxylase (23.8-25% pour le male, et 13% pour la femelle). Des différences liées au sexe sont observées de la même manière dans la production de métabolites 5α-réduits (environ 20% avec le tissu male et 13% avect le tissu femelle).

     

The effect of synthetic and natural pigments on the colour of the cichlids (<Emphasis Type="Italic">Cichlasoma severum</Emphasis> sp., Heckel 1840)

In this study, we have investigated the effects of Porphyridium cruentum (Rodophyta) as a natural pigment source and astaxanthin and β-carotene as synthetic pigment sources on the skin colour of cichlid fish (Cichlasoma severum sp., Heckel 1840), which are generally light orange with white patches and becomes shiny orange in the reproductive phase. The fish were fed diets containing 50 mg kg⁻¹ astaxanthin and β-carotene, and P. cruentum powder. The amount of both natural and synthetic pigment sources given as feed was 50 mg kg⁻¹, and the experiment was continued for 50 days. Total carotenoid content of the fish was determined spectrophotometrically at the end of the experiment. As a result, while a visible change of colour in the skin of the fish fed on the feed containing astaxanthin was observed with 0.34 ± 0.2 mg g⁻¹ of pigment accumulation, a relatively small change of colour was observed in the skin of other fish that were fed on the feed containing P. cruentum and β-carotene with 0.22 ± 0.2 mg g⁻¹ and 0.26 ± 0.1 mg g⁻¹ of pigment accumulations, respectively. Therefore, it was determined that these pigment sources have an effect on the colour of cichlid fish.     

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。