Interrelationships between gill chloride cell morphology and calcium uptake in freshwater teleosts

The involvement of the freshwater fish gill chloride cells (CCs) in trans-branchial calcium uptake (JinCa²⁺) was investigated. This was accomplished by assessing the interspecific relationships between the apical surface area of CCs exposed to the external environment and JinCa²⁺. Three species of freshwater teleosts, the rainbow trout (Oncorhynchus mykiss), the American eel (Anguilla rostrata) and the brown bullhead catfish (Ictalurus nebulosus), were used. Chronic (ten-day) treatment with cortisol in each species was used as a tool to evoke variations in both JinCa²⁺ and gill CC morphology in order to assess intraspecific relationships between CC surface area and JinCa²⁺. The results of quantitative morphometry, based on analysis of scanning electron micrographs, demonstrated that catfish possessed the lowest fractional area of exposed CC (CCFA) on the gill filament epithelium (12,744 ± 2248 m²/mm²) and was followed, in increasing order, by American eel (21,355 ± 981 m²/mm²) and rainbow trout (149,928 ± 26,545 m²/mm²). With the exception of catfish, chronic treatment with cortisol caused significant increases in CCFA owing to proliferation of CCs and/or enlargement of individual CCs (eel only). The rates of JinCa²⁺ closely reflected the CC fractional area in each species. The results of correlation analysis revealed significant correlations between CC fractional area and JinCa²⁺ in trout and eel. Owing to the absence of an effect of cortisol treatment, there was no significant correlation in catfish because of insufficient variation in CC fractional area in this species. CC fractional area was significantly correlated with JinCa²⁺ among the three species examined. These results suggest that CC is involved in calcium uptake in freshwater teleosts and that both intra- and interspecific differences in the rates of calcium uptake can be accounted for by variability in the surface area of exposed CCs on the gill epithelia.     

Ionoregulatory and respiratory responses of brown trout,Salmo trutta,exposed to lethal and sublethal aluminium in acidic soft waters

Soft water acclimated (Ca²⁺ 0.02 mM; Na⁺ 0.03 mM; K⁺ 0.01 mM; pH 7.0), cannulated brown trout (Salmo trutta) were exposed to various pH and aluminium (Al) regimes (pH 7.0, pH 5.0, pH 5.0 plus Al: 50, 25, and 12.5 g l^–1) for up to 5 days in order to determine (i) the sublethal concentration of Al at pH 5.0 for this species (ii) their ionoregulatory and respiratory status. No mortality or physiological disturbances were evident at pH 7.0 or pH 5.0. All trout died within 48 h at pH 5.0 in the presence of Al at 50 g l^–1 and 67% died over the 5 day period at pH 5.0 in the presence of Al at 25 g l^–1. Fish at these lethal Al concentrations showed significant decreases in arterial blood oxygen content (C_aO₂) but no changes in plasma osmolarity or the concentrations of plasma Na⁺, K⁺ and Cl^–. Physiological disturbance was more marked at the 50 g l^–1 Al concentration. The surviving fish at 25 g l^–1 showed few signs of physiological recovery while continually exposed to this regime. No fish died during the exposure to water of pH 5.0 containing 12.5 g l^–1 Al, but physiological disturbance was still apparent. These sublethally-stressed trout showed a transient decline in the plasma concentrations of Na⁺ and Cl^–1. Although C_aO₂ decreased, recovery was evident. The data suggest that in the brown trout, environmental Al concentration is as important as pH and calcium concentration in determining the physiological status of the fish.     

Gill Na+-K+ ATPase, carbonic anhydrase activities and plasma osmotic and ionic variations during smoltification of Atlantic salmon in the north of Portugal

Gill Na⁺-K⁺ ATPase and carbonic anhydrase activities were measured, on a fortnightly basis, from February to July, in 0₊ age Atlantic salmon (Salmo solar), hatched and reared in a freshwater experimental station in Covas, northern Portugal. Plasma osmolarity and ionic composition were also measured. Gill Na⁺-K⁺ ATPase activity increased slowly until April (15–19 moles Pi mg prot^–1 h^–1). From April to late May there was a great increase in activity (19–32 moles Pi mg prot^–1 h^–1) followed by a sharp decline in June (15 moles Pi mg prot^–1 h^–1). In contrast, carbonic anhydrase activity decreased significantly from early April to early June (170-70 moles p-nitrophenol mg prot^–1 h^–1) and increased in late June, suggesting the existence of a compensatory mechanism for the changes in Na⁺-K⁺ ATPase activity. Plasma osmolarity and sodium concentration showed lower levels during the period of high ATPase activity. On the other hand, plasma calcium concentrations showed an increase during the same period (3.47–5.98 mm1^–1 of plasma). A transitory decrease in osmolarity and plasma sodium and chlorine concentrations occurred in March, prior to the surge in Na⁺-K⁺ ATPase activity, suggesting that the physiological changes, characteristic of parr-smolt transformation can be a consequence of this loss of freshwater osmoregulatory capacity.     

Genetic improvement of farmed tilapias: Biochemical characterization of strain differences in Nile tilapia

Four African wild strains (Egypt, Ghana, Senegal and Kenya) and four established Asian farmed strains of Nile tilapia, Oreochromis niloticus (popularly known in the Philippines as Taiwan, Thailand, Singapore and Israel) were analysed electrophoretically at 30 protein loci to estimate genetic differences among the strains. All strains shared alleles at 14 monomorphic and 16 variable loci. Among the African strains, characteristic allele frequency differences were observed at AAT-1 ^* 46 for Ghana and Senegal, ADH ^* 83 for Kenya, ADH ^* 120 for Senegal, G3PDH-2 ^* 300 for Egypt, IDDH ^* 67 for Senegal, sMDH-1 ^* 120 for Kenya and SOD ^* 150 for Senegal. Genetic distance values among the strains revealed a clustering of the farmed strains with Egypt and Ghana O. niloticus, a slight separation of the Senegal strain and a larger separation of the Kenya strain. This profile may reflect the origins of the few founder populations of this species previously introduced to Asia. It also confirms the wider genetic divergence of the Kenya strain (O. niloticus vulcani) from the others studied here, which are all O. n. niloticus. Observed heterozygosities of the strains ranged from 0.026 to 0.071, with the African wild strains the lower values (mean Ho = 0.036) and the farmed strains the higher ones (mean Ho = 0.056). The implications of these results to the ongoing tilapia genetic improvement programme in the Philippines are discussed.     

Seasonal changes in reproductive condition and plasma levels of sex steroids in the blue cod,Parapercis colias (Bloch and Schneider) (Mugiloididae)

Gonad and plasma samples were taken from blue cod captured throughout the reproductive cycle, gonad condition was assessed, and plasma levels of 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20P), testosterone (T), 17-estradiol (E₂) and estrone (E₁) were measured by radioimmunoassay. It was confirmed that spawning occurred over an extended period in late winter and spring, with individual fish being involved in multiple spawning events. Plasma levels of T were bimodal in both sexes with peaks (maximum of 6.0 ng.ml^–1) occurring 2 months prior to, and also during the early part of the spawning period. 17,20P was elevated in males (2.1 ng.ml^–1) in mid-spermatogenesis coinciding with the first T peak (4.9 ng.m.^–1). 17,20P was detectable but not significantly elevated (0.6–1.2 ng.ml^–1) at any sample time in females. E₂ was elevated in mature females (1.0 ng.ml^–1) early in the spawning period but remained at assay detection limits (0.3 ng.ml^–1) at all other sample times. Neither 17OHP nor E₁ were detectable in the plasma of either sex. It is suggested that bimodal increases in sex steroids prior to spawning may be a feature of species with rapid recrudescence.     

Dehydration is more effective for the control of embryonic development and larval hatching of Diplectanum aequans (Monogenea, Diplectanidae) than formalin and trichlorphon

Embryonic development and larval hatching of the monogenean Diplectanum aequans, gill parasite of sea bass Dicentrarchus labrax, was studied in relation to different prophylactic treatments. Groups of eggs of D. aequans were submitted to different in vitro treatments: formalin (300 and 100 L L^–1 per 1 hour), Neguvon^® (trichlorphon 0.2 mg L^–1 per 48 hours) and dehydration for 4 and 8 hours. Percentages of hatched larvae, aborted larvae and undeveloped embryos were estimated in comparison with the control group. Results showed that 300 L L^–1 formalin and dehydration treatments were able to reduce larval hatching significantly, while Neguvon^® and 100 L L^–1 formalin treatments had no effect.     

Control of phosphorus homeostasis of Atlantic salmon (Salmo salar) in fresh water

Studies were conducted to determine the absorption, excretion and requirement of dietary phosphorus (P) by Atlantic salmon (Salmo salar). Triplicate groups of salmon parr, initial weight 15 ± 0.5±g , were fed, diets containing 4, 5, 6, 7, 8, 9, 11 and 13 mg P and 20 KJ of digestible energy (DE) per±g of diet (dry matter basis, DMB) to satiation for 16 weeks. The basal diet containing 4 mg P g^-1 (0.15 mg available P per KJ DE) was supplemented with graded levels of calcium phosphate, Ca(H₂PO₄)²H₂O to formulate the eight experimental diets. The fish were reared in fresh water at a temperature of 15 °C on a 12 h photoperiod. Vertebrae ash increased from 316 to 516 mg g^-1 fat-free dry matter with an increase in dietary P content. P requirement was estimated by using a four-parameter sigmoidal equation. The data suggests that a diet of 0.28 mg available P per KJ DE is needed for Atlantic salmon. Phosphate and calcium levels in plasma and bone increased, whereas levels of magnesium and liver cholecalciferol decreased, with an increase in dietary P.Phosphate excretion in urine and apparent availability of P were determined in deficient and replete fish. In deficient fish, the urine phosphate concentration was 0.10 mmol L^-1 before feeding and 0.25 mmol l^-1 after feeding, whereas in replete fish these concentrations were 1.09 and 5.11 mmol l^-1, respectively. The increase in urine phosphate concentration was higher in replete fish than in deficient fish, however, the apparent absorption of P was found to be significantly lower in replete fish than in deficient fish. These results suggest that similarly to terrestrial vertebrates, P homeostasis in Atlantic salmon is controlled by absorption in the intestine, conservation in the kidney and storage in the bones.     

Evaluation of vitamin C-enriched Artemia nauplii for larvae of the giant freshwater prawn

The effect of high levels of ascorbic acid (AA) delivered through enriched live food has been verified through the successful culture of larval giant freshwater prawn, Macrobrachium rosenbergii. Two successive feeding trials were set up using a control (550 g AA g^–1 DW) and two different AA-enrichment levels in Artemia (1300 and 2750 g AA g^–1 DW). Under standard culture conditions, no differences in growth nor survival could be observed demonstrating that the nutritional requirements are below 550 g AA g^–1 DW, which is the normal level occurring in freshly-hatched Artemia. However, a significantly positive effect could be demonstrated on the physiological condition of the postlarvae, measured by means of a salinity stress test, when vitamin C-boosted live food was administered. Since the AA levels in the predator larvae are linked with the enrichment levels in the live prey, it may be assumed that a positive influence on stress resistance was caused by feeding vitamin C-enriched Artemia. It is expected that under suboptimal conditions, supplementation of high vitamin C levels might also enhance production characteristics.     

Effects of Varying Dietary Fatty Acid Composition on Growth and Survival of Seahorse, Hippocampus Sp., Juveniles

Three commercially available fatty acid enrichment emulsions (DC Selco, DC DHA Selco and DC Super Selco) were used to enrich Artemia nauplii fed to seahorse, Hippocampus sp. fry. The emulsions varied in their n-3 highly unsaturated fatty acid (HUFA) composition. Total n-3 HUFA content ranged from 200 to 450mgg^-1 between the three emulsions while levels of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) ranged between 47–220 and 80–190mgg^-1, respectively. Survival and growth of seahorses at the end of the 30 day growth trial were greater in treatments receiving enriched Artemia. Seahorses receiving Artemia enriched with DC DHA Selco and DC Super Selco showed significantly (p < 0.05) greater mean survival (71.6 ± 6.0% and 78.3 ± 6.0%, respectively) than those receiving unenriched Artemia (48.3 ± 6.0%). Mean standard length was also significantly greater (p < 0.05) in fry fed DC DHA Selco and DC Super Selco enriched Artemia (20.2 ± 0.3 and 19.7 ± 0.3mm, respectively) compared to those fed unenriched Artemia (18.1 ± 0.3mm). The results show that dietary n-3 HUFA are essential for optimal growth and survival of Hippocampus sp. and, based on the fatty acid compositions of the enriched Artemia used in this study, indicate that the level of dietary DHA supporting optimal growth and survival is greater than 9.3mgDHAg^-1 dry weight.     

Morphometrics and ultrastructure of myocardial tissue in Notothenioid fishes

Antarctic fish of the family Channichthyidae (Icefishes) lack the respiratory pigments haemoglobin and myoglobin. The morphometrics and ultrastructure of the ventricular myocardium of a benthic icefish,Chaenocephalus aceratus has been compared with that of a red-blooded Notothenioid fish,Notothenia neglecta, of similar habit.The mass of ventricular muscle as a percentage of bodyweight is 3 times greater in adultC. aceratus (0.32%) thanN. neglecta (0.11%). Myoglobin concentration in the ventricle ofN. neglecta, 20 nmoles/g, is comparable to that of temperate teleosts with similar activity patterns. The volume and surface densities of mitochondria are 41.5% and 0.32 m^–1 for Icefish and 25% and 0.15 m^–1 forN. neglecta, Cytochrome oxidase activities are similar in the two tissues whilst the volume density of myofibrils is higher forN. neglecta (47%) thanC. aceratus (29.9%).The proliferation of mitochondria in the myocardium of Icefish will reduce the diffusion path-length for oxygen between ventricular lumen and the outer mitochondrial membrane and may compensate for the absence of myoglobin.     

Utilization of carotenoids from various sources by rainbow trout: muscle colour,carotenoid digestibility and retention

Rainbow trout (Oncorhynchus mykiss) with a mean (sd) weight of 120 (2) g were fed diets supplemented with astaxanthin extracted from the yeast Phaffia rhodozyma (OY1 = 50 mg carotenoids kg^–1 feed, OY2 = 100 mg carotenoids kg^–1 feed), astaxanthin (AX = 100 mg astaxanthin kg^–1 feed) and canthaxanthin (CX = 100 mg canthaxanthin kg^–1 feed) for 4 weeks. Muscle analyses at the end of the experiment indicated a significantly higher carotenoid concentration in the AX group, while CX and OY1 groups were similar in spite of the differences in dietary concentration. The measure of total muscle colour difference (E^* _ab) between initial samples and 4 week ones was higher for the AX fish group but showed no significant difference between OY1, OY2, and CX. The hue and the reflectance ratio (R₆₅₀:R₅₁₀) of fish muscle increased in proportion to carotenoid intake. Digestibility (ADC) of yeast astaxanthin in OY1 and OY2 groups was significantly higher than that in the AX group. Canthaxanthin ADC was about one sixth of that of astaxanthin (AX group). Carotenoid retention in the muscle, expressed as a percentage of carotenoid intake, was higher for the AX group than that recorded for OY1 and OY2. According to ADC, carotenoid retention showed a marked lower value for the CX group. Muscle retentions were similar for astaxanthins from both sources.     

Motility of spermatozoa ofAlburnus alburnus (Cyprinidae) and its relationship to seminal plasma composition and sperm metabolism

The composition of seminal plasma and metabolism of sperm of the cyprinid fishAlburnus alburnus were investigated. Statistically significant correlations were found between motility parameters and seminal fluid osmolality, pH, Na⁺, K⁺ and protein levels (negative correlations: % immotile spermatozoa-Na⁺, K⁺; positive correlations: % motile spermatozoa-osmolality, pH, Na⁺, K⁺, protein; % linear motile spermatozoa-pH protein; swimming velocity of spermatozoa-pH, Na⁺, protein). Spermatozoan motility and ATP metabolism and glycolysis were correlated as indicated by measurement of ATPase, pyruvate kinase, adenylate kinase and lactate dehydrogenase activity. The physiological meanings of these correlations and their possible significance for quality control of semen are discussed.Abbreviations used ACP acid phosphatase - ADP adenosine diphosphate - AK adenylate kinase - ALP alkaline phosphatase - ASPAT aspartate aminotransferase - ATP adenosine triphosphate - ATPase magnesium dependent adenosine triphosphatase - CRPO creatine phosphate - -GLU \-D-glucuronidase - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PK pyruvate kinase     

Oxygen consumption and responses to hypoxia of ammocoetes of the southern hemisphere lampreyGeotria australis

The standard rate of oxygen consumption of ammocoetes (larvae) ofGeotria australis with a mean weight of c. 0.5 g was 9.6, 31.4 and 59.4l g^–1 h^–1 at 4.5, 15.5 and 25.0°C respectively, which gives an overall Q₁₀ of 2.4. The regression coefficient for the logarithmic relationship between oxygen consumption and body weight at 15.5°C was 0.704. The ammocoetes ofG. australis have a much lower rate of oxygen consumption at 15.5 and 25.0°C than those of holarctic lampreys. This presumably reflects the lower oxygen delivery pressure to their tissues and helps account for their slow growth rate. At 15.5°C, ammocoetes ofG. australis emerged from the substrate at 21–25 mm Hg and, unlike those of the Northern HemisphereIchthyomyzon greeleyi, died at 14–17 mm Hg. Thus, despite having a thinner water/blood barrier in the gills and blood with a higher oxygen affinity and capacity than holarctic ammocoetes, the larvae ofG. australis cannot survive very low dissolved oxygen tensions. This is apparently related to an inability of larvalG. australis to meet the high oxygen requirements of the respiratory pump at these oxygen tensions. During metamorphosis, oxygen consumption at 15.5°C rose from approximately 27l g^–1 h^–1 at the beginning of transformation to 33.2l g^–1 h^–1 by Stage 3 and then rapidly to 66l g^–1 h^–1 at Stage 6. It remained near this level in Stage 7 and the downstream migrant.     

The role of Ca2+ and Na+ membrane transport in brook trout (Salvelinus fontinalis) spermatozoa motility

The role of environmental ion composition and osmolality in Ca²⁺ signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris–HCl pH 8.5. For investigation of spermatozoa reaction to external Ca²⁺ concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na⁺ concentration in conditions of low external Ca²⁺, 100 µM amiloride was added to the EGTA-containing solutions as a Na⁺ transport blocker. Low motility was observed in sucrose (Na⁺ free) solutions containing 2 mM EGTA but not in Na⁺ solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na⁺ free) solutions containing 2 mM EGTA. We conclude that Na⁺ transport in Ca²⁺-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na⁺ and Ca²⁺ transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.     

Quantitation of inert feed ingestion in larval silver sea bream (Sparus sarba) using auto-fluorescence of alginate-based microparticulate diets

The ingestion of an inert feed as a sole food source was investigated in larval silver sea bream (Sparus sarba) fed an alginate-based microparticulate diet. Using the auto-fluorescent properties of pigments associated with the alginate base, ingestion and gut content were investigated over a 6 h experimental period in fed and unfed larvae. By extracting and measuring chlorophyll a (Chl a) and phaeopigment content of feeding larval fish and relating this to standardized Chl a and phaeopigment content of the diet, relative to diet weight, it was determined that individual fed 7-day old larvae had a maximum gut content of 1.05±0.09 g diet while 14-day old fed fish had a maximum gut content of 3.17±0.90 g diet. On average, the gut content of 14-day old fish was 2.89 times greater than the gut content of 7-day old fish. The dry weight of larval sea bream increased from 43±4.2 g at day 7 to 134.3±20.4 g at day 14 indicating that growth of fish fed this inert feed was substantial. Gut pigment dynamics suggested that Chl a was degraded to phaeopigments by 7-day but not 14-day old larvae and the individual gut dietary content varied considerably in 14-day old fish. The maximum Chl a and phaeopigment content in larval sea bream was 0.4 ng ind^–1 and 0.55 ng ind^–1 for 7-day old fish and 1.54 ng ind^–1 and 2.81 ng ind^–1 for 14-day old fish respectively. The present method may potentially allow simple and direct assessment of larval fish feed ingestion in both an experimental and commercial setting.     

Replacement of Artemia with Moina micrura in the rearing of freshwater shrimp larvae

The effect of an abrupt change in the live diet of shrimp larvae was investigated by replacing Artemia with Moina micrura. The control treatment consisted of feeding Artemia throughout the rearing period (regime A), while in the other treatments the onset of Moina feeding was arbitrarily chosen at larval stages iv (A3M), vi (A5M), viii (A7M) and x (A9M). No significant differences ( = 0.05) were observed among the treatments during larval production, mean stage development (MSD) and growth of postlarvae. The mean (SD) yields of postlarvae (PL) were 11.97 (1.98), 15.10 (2.92), 14.72(1.56), 13.51 (1.74) and 12.70 (1.40) PL l^–1 respectively for the feeding regimes A3M, A5M, A7M, A9M and A. Up to stage v, the ingestion rate in the Moina treatment was as low as 0.01–0.47 larva^–1 h^–1 compared with that in the Artemia treatment (0.29–1.77 larva^–1 h^–1). However, the ingestion of Moina increased from stage vi–vii onwards.     

Effects of ambient ion concentrations on gill ATPases in fresh water eel,Anguilla anguilla

Branchial activities of Na⁺,K⁺-ATPase (ouabain sensitive), Mg²⁺ ATPase (ouabain insensitive) and kinetic analysis of high and low affinity Ca²⁺ ATPase were measured inAnguilla anguilla that had been acclimated to demineralized water (DW, Ca < 10 M), freshwater (FW, Ca = 2 mM), and Low calcium freshwater (L-Ca, Ca = 0.9 mM). Na⁺,K⁺-ATPase activity decreased while ouabain insensitive activity increased when ambient Ca²⁺ decreased. Two kinetic forms of Ca²⁺ ATPase could be resolved in each environmental condition. The stimulation coefficients of both sites or enzymes were not affected by ambient Ca²⁺ concentrations. The maximal velocity of both the high and the low affinity Ca²⁺ ATPase was increased when external Ca²⁺ was decreased during acclimation. The low affinity Ca²⁺ ATPase and the Mg²⁺ stimulated enzyme could be a non specific enzyme accepting either Ca²⁺ or Mg²⁺. Results are compared with previous results in the literature and in relation to the branchial morphology and ionic exchanges in fish.     

Effects of steroid hormones on in vitro oocyte maturation in white sturgeon (Acipenser transmontanus)

The in vitro effects of several steroids on the maturation of intact white sturgeon (Acipenser transmontanus) ovarian follicles were investigated. At the highest concentration (1024 ng ml^–1 for the C21 steroids and 1139 ng ml^–1 for the C19 steroids), all of the C21 steroids tested, progesterone (P4), 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,(20,21-trihydroxy-4-pregnen-3-one 20-S), 11-deoxycortisol (S) and cortisol (F), as well as testosterone (T) induced germinal vesicle breakdown (GVBD) at 14 and 22 h. At 6 h, only P4 and 17,20-P induced maturation at the highest concentration (1024 ng ml^–1). At 14 and 22 h, 11-deoxycortisol was the most potent steroid inducer of GVBD followed by P4, 17OHP, 17,20-P, and 20-S. The steroid 11-hydroxytestosterone (11OHT) was completely ineffective at all concentrations and exposure times. The C21 steroids induced oocyte maturation at concentrations ranging from 4 to 1024 ng ml^–1, whereas T induced GVBD at 225 to 1139 ng ml^–1. Calculation of the mean effective concentration that induced 50% GVBD (EC50) from the 22 h incubations revealed the following order of potencies: S > P4 > 17OHP > 17,20-P > 20-S >> F > T. These bioassay results, together with previous findings on the endogenous production of steroids by ovarian follicles from gonadotropin-primed females, indicate that more than one steroid has a biological role in the resumption of meiosis in sturgeon oocytes and provides empirical evidence for P4, 17OHP, S, 20-S, and 17,20-P as maturation-inducing steroids in white sturgeon.     

Demonstration of putative membrane and cytosol steroid receptors for 17α, 20β-dihydroxy-4-pregnen-3-one in brook troutSalvelinus fontinalis oocytes by photoaffinity labelling using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020)

Cytosol from brook trout ovarian follicles (stages 1–3) was photoaffinity (PA) labelled using synthetic progestin 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione ([³H]R5020). The covalently bound cytosol protein had a relative mass of 501,000 M_r following Sephacryl S-300 column chromatography. The zona radiata membrane fraction from brook trout oocytes which had gone through the first phase of meiotic maturation (stages 6–7) was isolated by ultracentrifugation of the whole oocytes. The zona radiata solubilized protein presumably from the oocyte membrane was also PA labelled and found to give a peak at 355,000 M_r. The SDS PAGE of the cytosol and zona radiata PA labelled protein gave very similar subunits indicating that the membrane protein and the cytosol protein, both of which bind the maturation inducing steroid (MIS) 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP), have similar subunit structures. The isolated zona radiata protein showed cooperativity of binding to [³H]17,20-DHP and PA labelling to [³H]R5020. The association constant (K_a) was 2.0×10⁷M^–1 and maximum binding capacity (N_max) 427 fmoles/mg protein with MIS [³H]17,20-DHP.No evidence for nuclear binding of MIS [³H]17,20-DHP or PA labelling of [³H]R5020 to nuclei was observed. The nuclei were isolated from stages 1 and 3 fresh ovarian follicles of brook trout. The experimental evidence presented demonstrates the presence of MIS 17,20-DHP receptor-like protein from the zona radiata membranes by PA labelling in brook trout oocytes during final stages of maturation.