Oocyte maturation inClarias batrachus. III. Purification and characterization of maturation-inducing steroid

Maturation-inducing steroid (MIS) in the Indian female catfish,Clarias batrachus, was purified and characterized from the incubation medium in which fully grown but immature folliculated oocytes were incubated with salmon gonadotropin (SG-G100) for 36 h. Maturation-inducing (MI) activity of residues obtained at various steps of extraction and purification was assessed byin vitro germinal vesicle breakdown (GVBD) assay using folliculated oocytes ofC. batrachus. The post incubation medium was extracted with diethyl ether. The ether phase was partitioned using 50% methanol plus n-hexane. The methanol phase which had MI activity was fractionated into 7 fractions using reverse-phase high-performance liquid chromatography. Of these 7 fractions, fraction 3 was found to be active in having MI ability and identified as 17 ,20-dihydroxy-4-pregnen-3-one (17,20-diOHprog). The authenticity of 17,20-diOHprog as the major follicular mediator of gonadotropin-induced oocyte maturation was further confirmed by thin-layer chromatography (TLC) in which fraction 3 was run along with authentic 17,20-diOHprog standard. This investigation gives a direct evidence that 17,20-diOHprog is the major naturally occurring MIS in Indian female catfish,C. batrachus.     

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.     

Presence of maturation-promoting factor in 17α,20β-dihydroxy-4-pregnen-3-one-induced oocytes of catfish,Clarias batrachus

Full-grown immature Clarias batrachus oocytes respond in vitro to exogenous 17,20-dihydroxy-4-preg-nen-3-one ( 17,20-DP) by undergoing germinal vesicle breakdown (GVBD). Cytosolic extract (CE) prepared from 17,20-DP-induced oocytes has been shown to produce similar effect when microinjected into unstimulated immature oocytes of the same fish. A dose of 50 nl is enough to cause 100% GVBD after 4 h. Maturation-promoting factor was investigated from 17,20-DP-induced, immature and cycloheximide treated oocytes incubated in presence of [³⁵S] methionine. When the proteins were extracted and analyzed on SDS-PAGE, two prominent bands corresponding to molecular weight 34- and 46-kDa were detected in the CE of mature oocytes. However, labelling of [³⁵S] methionine was observed mainly in the region of 46 kDa protein band indicating de novo synthesis of this particular protein during l7,20-DP-induction. Further, immunoblotting study by using rabbit anti-cyclin B₁ antibody has clearly demonstrated that the protein which is newly synthesized is highly homologous to Xenopus cyclin B₁ and goldfish cyclin B.     

Effects of acute temperature decreases on turbot fry and juveniles

Turbot fry (10–20 mm) and juveniles (85–110 mm) were transferred directly from 16.0–16.5 C to 1.0 C, 2.5 C, 5.5 C or 8.0 C seawater. The fry were more sensitive to cold water than juveniles. The fry survived for 1 week at 8.0 C but not at 5.5 C, whereas juveniles survived at 5.5 C but not at 2.5 C. Transfer of juveniles to 1.0 C and 2.5 C seawater caused a high mortality, a marked increase in plasma Cl^- concentration, decrease in muscle water content, and hyperglycaemia. Acclimation to 5.5 C (juveniles) or 8.0 C (fry and juveniles) markedly reduced the sensitivity to 1.0 C exposure.     

Steroidogenesis by ovaries and testes of the European catfish,the wels (Silurus glanis), in vitro

Testosterone, 3,17-dihydroxy-5-pregnen-20-one, 17,20-dihydroxy-4-pregnen-3-one (17,20P) and 5-pregnane-3,17,20-triol were identified as the major metabolites of [³H] 17-hydroxyprogesterone in ovarian incubations of the European catfish Silurus glanis. 17,20P and the reduced triol were present only in ovaries from fish primed with carp hypophysial homogenate (chh) while testosterone yields were significantly higher in controls than in treated fish. 11-Ketotestosterone, 11-hydroxytestosterone and 17,20-dihydroxy-4-pregnen-3-one (17,20P) were identified as the major metabolites of [³H]17-hydroxyprogesterone in in vitro incubations of testes of a spermiating catfish. There was no significant production of conjugates or other water soluble metabolites by either sex. The stimulation of plasma 17,20P, 17,20P and 11-hydroxytestosterone by chh in primed but not control males suggests that the role of these steroids in spermiation should be further examined.     

Synthesis and regulation of 17α-hydroxy-20β-dihydroprogesterone in immature males of Oncorhynchus mykiss

Three experimental approaches were chosen to study the question if the progestin 17-hydroxy-20-dihydroprogesterone (1720OHP) is synthesised in testes of young Oncorhynchus mykiss, in which the absence of spermatozoa was verified histologically: first, in order to detect 20-hydroxysteroid dehydrogenase activity (20HSD), testes homogenates were incubated with ³H-labeled 17OHP.Metabolites were analysed by TLC, HPLC, and repeated crystallization to constant isotope ratios. One of the metabolites was identified as 1720OHP-³H, indicating that already immature testes contain 20HSD activity and are able to produce 20-reduced steroids. Second, 1720OHP was quantified by radioimmunoassay in incubates of testes fragments. The sensitivity of the gonads to gonadotropin II (GtH II) became evident when comparing incubations in the absence and presence of GtH II. Third, plasma levels of 1720OHP were significantly higher in animals injected with partially purified salmon gonadotropin, compared to controls. Thus, for the first time, it could be shown that 20HSD is present in testicular cells other than spermatozoa. Furthermore, 1720OHP is indeed secreted at a very early stage of testicular development; 1720OHP secretion is also responsive to GtH II. Future studies will have to show if the functions of this progestin include the stimulation of spermatogenesis.     

The nature of hematological response in fish

Hematological status was examined in rainbow trout,Oncorhynchus mykiss, held for 3–4 weeks under temperature, photoperiod and P_{O 2} conditions approximating those of their winter, spring and summer habitats. The most striking change observed was in red cell population composition. In winter fish mature cells were predominant; juvenile and developing erythrocytes characterized spring and summer animals. Hemoglobin, hematocrit and both mean erythrocytic volume and hemoglobin were modestly lower in spring and summer than in winter fish. Red cell numbers were not significantly affected. These observations suggest that avoidance of viscosity-based increases in circulatory work cost is more advantageous than elevation of blood O₂-carrying capacity. Although hemoglobin isomorph profiles were significantly altered, there is little evidence that such changes are of critical adaptive importance. Given presumed age-based reduction in gas transport effectiveness, the replacement of mature and senescent cells by more metabolically-competent juvenile cells appears to be the pivotal event in hematological response. Leucocyte counts were significantly elevated in spring and summer as compared to winter fish. Lymphocyte/heterophil ratios declined from 8.27 in winter fish to 3.13 in summer trout. Thrombocyte, monocyte, eosinophil and basophil abundances were little changed.     

Transport of 17α,20β-dihydroxy-4-pregnen-3-one in brook troutSalvelinus fontinalis ovarian follicles

Extremely low levels of the maturation inducing steroid (MIS) 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP) were found in the ooplasm and ovarian follicle membranes of Atlantic salmonSalmo salar ouananiche, a finding that is at variance with the elevated blood levels of the steroid. The uptake of MIS at physiological concentrations into brook trout follicles occurred by passive diffusion. Uptake of the steroid into the ovarian follicle membrane, consisting of zona radiata and the attached follicle cells, deviated from linearity in a double reciprocal plot. These results suggest that 17,20-DHP is binding to a receptor-like protein in the ovarian follicle or the zona radiata membrane surrounding the oocyte, and extend our previous demonstration of 17,20-DHP receptor-like activity in the zona radiata membrane of the late stage brook trout oocytes. An oocyte cytoplasmic receptor gave subunits on SDS PAGE that were similar to the membrane and cytosol receptors previously described.     

Antioxidant effect of dietary tocopherol and ascorbic acid on growth and survival of Litopenaeus vannamei postlarvae

The nutritional effect of vitamin E in dietsfor Litopenaeus vannamei postlarve (PL19)was investigated. Four formulated diets withdifferent combinations of -tocopherylacetate (-TA), ascorbic acid (AA) andhighly unsaturated fatty acids (HUFA) weretested, using four replicates.No significant differences in survival wereobserved among treatments after 34 days offeeding. However, shrimp fed with a dietcontaining 2% fish oil (low n-3 HUFA content),200 mg.kg^–1 -TA and100 mg.kg^–1 AA (diet H/E/C) showedsignificantly better growth than those fed adiet supplemented with 5% fish oil (high n-3HUFA content), 200 mg.kg^–1 -TA and100 mg.kg^–1 AA (diet H+/E/C). Shrimp fedwith a diet containing 5% fish oil,900 mg.kg^–1 -TA and100 mg.kg^–1 AA (diet H+/E+/C) showed a significantly higher tissue level of n-6 PUFAthan postlarvae fed diet H+/E/C. No definiteconclusion could be drawn about a possibleinteraction between -TA and AA, since acomparison of the diet containing 5% fish oil,200 mg.kg^–1 -TA and700 mg.kg^–1 AA (H+/E+/C+) and the dietH+/E/C did not show any significant differencesin any of the measured parameters. Theantioxidative status of the shrimp tissue(measured by means of the thiobarbituric acid(TBA) assay and expressed as nM malonaldehyde(MA) per gramme dry weight) was equal for alltreatments. Nevertheless, there was a slightlylower MA value with the diet H+/E/C+,indicating that AA may be an effectiveantioxidant in the aqueous phase and at thewater/lipid interface of the tissue. The tissuelevels of -T and AA were highlydependent on the amounts in diets and nocorrelation between -T and AAincorporation could be observed.     

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.     

The presence of 17α,20β-dihydroxy-4-pregnen-3-one receptor activity in the ovary of the brook trout,Salvelinus fontinalis,during terminal stages of oocyte maturation

The presence of 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP) oocyte receptor activity has been demonstrated in brook troutSalvelinus fontinalis. Scatchard analyses of the cytosol fraction during various terminal stages of oocyte maturation gave a high equilibrium association constant (K_a) value of 1.394±0.669 10⁸M^–1 (n=7) and low maximum binding capacities (N_max). The association kinetics of the receptor was second order k₊₁=2.292×10⁶M^–1 sec^–1. The dissociation rate constant k_a was 1.502×10^–2 sec^–1 for the first order dissociation reaction. The K_a=1.526×10⁸M^–1, when it was determined from k₊₁/k_–1 a value close to that found from the Scatchard analysis. Competition studies showed the following binding affinities testosterone > 17-HP > 17,20-DHP > Promegestone > progesterone > estradiol > pregnenolone; cortisol showed no competitive inhibition. Cytosolic extracts when pre-equilibrated with various labelled steroids and eluted from a Sephacryl S-300 column gave multiple specific binding peaks. On sucrose density gradient centrifugation specific binding was observed at 3.05 S in cytosol containing 0.15M sodium chloride buffer. The receptor lost binding activity when incubated with various proteases, but DNase and RNase had no effect. Blood plasma without heparin at (110) dilution also bound [³H]17,20-DHP, Ka was 8.04×10⁷ M^–1.The nuclear pellet extract (750×g) gave very little specific binding activity even at high radiolabelled steroid concentrations and a linear Scatchard plot was not obtained. Nevertheless the nuclear extract, after dextran-charcoal treatment, pre-equilibrated with [³H]17,20-DHP, bound specifically to DNA cellulose, and cytosol from the same oocytes also bound to DNA cellulose under similar conditions. Although specific binding to DNA cellulose was obtained the salt concentrations at which the steroid-receptor complex elution took place was not reproducible in both nuclear extracts and cytosol samples. Also binding activity was extremely small compared to the total cytosolic binding. The nuclear extract when pre-equilibrated with high concentrations (20 nM) of the labelled steroid and then chromatographed on Sephacryl S-300 column gave a specific binding peak which was similar to that of the cytosolic preparation.The receptor levels in cytosol decreased progressively during final maturation (Stages 1–7). There is preliminary evidence for the presence of 17,20-DHP receptor activity in cytosol of landlocked Atlantic salmonSalmo salar ouananiche, and rainbow troutSalmo gairdneri. The zona radiata fraction from late stages oocyes 5, 6, and 7 in brook and rainbow trout oocytes were isolated by ultracentrifugation; from this fraction a protein was characterized which covalently bound [³H]R5020 after photoaffinity labelling. The same protein also bound [³H]17,20-DHP after solubilization in Brig 35 buffer. The SDS gel electrophoresis subunit composition of the above protein was similar to the cytosol counterpart binding [³H]17,20-DHP, although the molecular weights were different. The blood sample [³H]R5020 binding component subunit composition was different from that of the membrane extracted protein. These results demonstrate the presence of 17,20-DHP receptor activity in the cytosol and zona radiata membranes of the oocytes during final maturation.A. Maneckjee is presently NSERC postgraduate scholar at MSRL and Ph.D. candidate at Department of Biochemistry, Memorial University of Newfoundland.     

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.     

Effects of feeding (ω-3) HUFA-enriched Artemia during a progressively increasing period on the larviculture of freshwater prawns

Larvae of the freshwater prawn (Macrobrachium rosenbergii) were reared to post-larvae in a closed recirculation system. Six treatments were designed where freshly-hatched Artemia nauplii were first given to the larvae for 3, 7, 11, 15, 17 and 25 days according to the treatment. After that (-3) HUFA-enriched Artemia were given until the 28th day at which time the test was terminated and evaluated. The requirement for (-3) Highly Unsaturated Fatty Acids (HUFA) for the larval stages of M. rosenbergii was confirmed by this experiment. Moreover, the longer the period of feeding on (-3) HUFA-enriched Artemia nauplii, the better the results in terms of growth, metamorphosis rate, survival and stress resistance.     

Steroidogenesis in rainbow trout (Salmo gairdneri) at various preovulatory stages: changes in plasma hormone levels andin vivo andin vitro responses of the ovary to salmon gonadotropin

In order to specify the timing of some changes in ovarian steroid production during the transition from vitellogenesis to ovulation, plasma hormones levels andin vivo andin vitro responses of the ovary to salmon gonadotropin (s-GtH) or dibutyryl-cyclic adenosine mono-phosphate (db-cAMP) were recorded in relationship with the state of germinal vesicle migration in the oocyte.In vivo, a small, but significant, increase of plasma 17-hydroxy-20-dihydroprogesterone (17, 20-OH-P) level was detected earlier (at the subperipheral germinal vesicle stage) than the increase of GtH level (detectable at the peripheral germinal vesicle stage) and the decline of oestradiol-17 (E2–17) (also detectable at the peripheral germinal vesicle stage). Negative correlations were established between E2–17 levels and GtH (=–0.53) or 17,20-OH-P (=–0,43) levels while a positive correlation occurred between 17,20-OH-P and GtH levels (=+0,54).In vivo no action of GtH on the decline of E2–17 levels was detected GtH did not stimulate 17,20-OH-P production, within 72h, in females at the end of vitellogenesis stage. It had significant effect in females at other stages closer to ovulation, but the pattern of responses changed according to the stage.In vitro db-cAMP like GtH was able to stimulate 17,20-OH-P output from ovarian follicles. The greatest response was observed at the later stage. (GVBD). Testosterone output was also increased by GtH, but the lowest response was observed at the later stage (GVBD). Androstenedione output was lower than testosterone output.In vitro, a small but significant decline of E2–17 output was induced by GtH. We conclude that substantial changes occur during the very last stages prior to ovulation, both in the steroidogenic potential of the ovary and in the ovarian sensitivity to GtH. 20-oxydoreductase is probably progressively induced during GV migration when GtH basal levels are increasing but still relatively low. Without minimizing the role of discrete pulses of GtH on this induction, we could expect synergic actions of other hormones. Thus a high testosterone/oestradiol ratio in the follicle environment favours 17,20-OH-P secretion.     

Influence of temperature on the hatching of eggs of Diplectanum aequans,a parasite of sea bass

This study deals with the influence of temperature on the hatching of eggs of Diplectanum aequans, a branchial ectoparasite of sea bass (Dicentrarchus labrax). At temperatures of 20 °C, 25 C and 30 °C hatching takes place within a few days of laying, whereas at 15 C and 10 C it occurs respectively between the 7th and the 12th day and between the 11th and the 19th day. No hatching has been observed at 5 C. Development is not completely inhibited at 5 C as the eggs can, at least partially, go on maturing and will subsequently hatch as soon as higher temperatures are encountered.     

Biochemical composition of eggsin relation to egg quality in the Japanese eel, Anguilla japonica

This paper presents the relationship between egg quality and egg biochemical composition of cultured and wild Japanese eel, Anguilla japonica. Eggs were obtained by artificialinduction of maturation. Fertilization and hatching rates were used as characteristics of egg quality. Egg quality characteristics showed large variation; fertilization rate, 0–96; hatching rate, 0–84%. The biochemical composition also showed a large variation. There was no marked relationship between egg quality and fatty acid contents of eggs, except for n-6 highly unsaturated fatty acids (HUFA). Both the fertilization and hatching ratesincreased proportionally withincreases of the -tocopherol g(-Toc) contentin eggs. A more significant correlation was found between the amount of -Toc relative to the amount of HUFA and egg quality. The results of this study show that the egg quality of Japanese eel is affected by the –Toc level, andin particular, the ratio of -Toc to HUFAin the eggs. Abbreviations: BHT – butylhydroxytoluene; EFA – essential fatty acids; FAME – fatty acid methyl esters; HPLC – high performance liquid chromatography; HUFA – highly unsaturated fatty acids; NADH – nicotinamide adenine dinucleotide; NADPH - nicotinamide adenine dinucleotide phosphate; ROS – reactive oxygen species; -Toc –-tocopherol.     

Steroid release from separated theca and granulosa layers of Atlantic salmon (Salmo salar) ovarian follicles: the effects of a purified salmon gonadotrophin preparation

Theca and granulosa layers were removed from ovarian follicles of mature Atlantic salmon (Salmo salar) and were separately incubated under sterile conditions with and without a partially purified salmon gonadotrophin preparation (GTH). Aliquots of the incubation media were removed at intervals and analysed for the steroids 17, 20-dihydroxy-4-pregnen-3-one (1720P), 17-hydroxyprogesterone, progesterone, androstenedione, testosterone and oestradiol. The biosynthesis of C19 and C21 steroids was very largely restricted to the thecal tissue and was markedly stimulated in the presence of GTH. Androstenedione (max 65 ng/ml) and testosterone (max 14 ng/ml) were released from the earliest stages of incubation whereas the release of 17-hydroxyprogesterone (max 51 ng/ml) and progesterone (max 5.5 ng/ml) commenced only after a lengthy induction period. A trace (1.0 ng/ml) of 1720P was produced by the theca in the presence of GTH but oestradiol was not detected. The granulosa preparations released levels of 17-hydroxyprogesterone and androstenedione only marginally above the detection limits (ca 0.7 ng/ml) and there was little stimulation of output with GTH. Oestradiol (max 4 ng/ml) was released only in the presence of GTH. 1720P, progesterone and testosterone were not detected as products of this tissue. These results, together with those derived earlier from incubations of complete follicles support the view that the synthesis of 1720P is essentially a two-cell process in which 17-hydroxyprogesterone produced in the theca is subject to the action of steroid 20-hydroxysteroid dehydrogenase in the granulosa. The temporal pattern of release of steroids in these and earlier experiments is considered in relation to mechanisms of steroid biosynthesis and to their possible roles in oocyte final maturation.     

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.     

Distinct alpha (α)-subunits of salmon glycoprotein hormones: production sites in the pituitary with sexual maturity

Salmon pituitary glands contain two structurally distinct -subunit proteins (1 and 2) of glycoprotein hormones: the 2-subunit is common to all salmon gonadotropins (GTH I and GTH II), whereas the 1-subunit is present in only some GTH I molecules. GTH I is predominant in the pituitary gland and plasma during gametogenesis of salmon, but the roles of the 2 GTHs in gametogenesis remain unclear. To understand the roles of GTH I, it is important to clarify patterns of 1- and 2-subunit production with sexual maturity. Thus, we produced antisera that recognized the 1- or 2-subunit, and then immunohistochemically examined the production sites of these subunits in the trout pituitary gland during ovarian development. In all pituitary glands examined, the immunoreactivity of both the 1- and 2-subunits was strong in the GTH II-producing cells, although salmon GTH II, both 1- and II-subunits, has not been detected. However, GTH I-producing cells showed a less dense immunoreactivity for 1- and 2-subunits, whereas the I-subunit was abundant. On the other hand, TSH cells, reacted with 2 but not with 1.     

Regulation of oocyte maturation in the rainbow trout,Salmo gairdneri: role of cyclic AMP in the mechanism of action of the maturation inducing steroid (MIS), 17α-hydroxy, 20β-dihydroprogesterone

In fish, oocyte maturation (resumption of meiosis after completion of vitellogenesis and before ovulation) is triggered by maturation inducing steroids (MIS) which generally appear to be secreted in the ovary in response to stimulation by a pituitary maturational gonadotropin. Converging data from different laboratories show that 17-hydroxy, 20-dihydroprogesterone (17, 20-OH-P) is the principal MIS in salmonoids; but clear identification remains to be done in other taxonomic groups.The experiments reported here in the rainbow troutSalmo gairdneri examine the possible involvement of oocyte cAMP on the mechanism of MIS action. The action of 17, 20-OH-P, on germinal vesicle breakdown (GVBD) in oocytes incubatedin vitro within the follicle, was inhibited by various substances expected to elevate the intraoocyte concentrations of cAMP: cAMP ( 1 mM) or dibutyril cAMP ( 2 mM), phosphodiesterase inhibitors such as theophylline ( 0.2 mM) or 3-isobutyl-1 methylxanthine (IBMX 0.1 mM), adenylate cyclase activators such as cholera toxin (> 100 nM) or forskolin ( 0.03 mM). In fact, the combined action of IBMX (1 mM) and forskolin (0.01 or 0.05 mM)in vitro was to promote accumulation of intraoocyte cAMP within 1 to 5 hours. Oocyte cAMP concentrations exhibited a large variability between different females, depending on the stage of oocyte development; a significant positive correlation between oocyte cAMP concentration and the follicular weight, and a significant negative correlation between oocyte cAMP concentration and the median efficient dose of 17, 20-OH-P for induction of GVBD, were observed. Finally, when intrafollicular oocytes were incubatedin vitro, the addition of a maturation-inducing concentration of 17, 20-OH-P (3×10^–6M) induced a significant decrease of oocyte cAMP within the first 10 hours of incubation. These results show that cAMP appears to play a central role in the regulation of oocyte sensitivity to 17, 20-OH-P and in the intraoocyte mechanisms leading to GVBD in trout.These data are discussed together with the few indications available in fish concerning the mechanism of MIS action which can be compared to some extent with the amphibian model.