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.     

Hormonal changes during meiotic maturation and ovulation in the brook trout (Salvelinus fontinalis)

The plasma levels of estradiol-17 (E2), 17, 20-dihydroxy-4-pregnen-3-one (17,20-P) and gonadotropin (GTH) were measured in brook trout (Salvelinus fontinalis) during the period from the end of vitellogenesis to postovulation. Blood samples were taken according to specific stages of maturation, including germinal vesicle breakdown (GVBD) and ovulation. E2 levels were quite high (45 ng/ml) at the end of vitellogenesis (and prior to GVBD) and dropped precipitously by GVBD (2 ng/ml). They remained low through ovulation and postovulation. 17,20-P levels were low prior to GVBD (0.7 ng/ml) and increased dramatically at GVBD (148 ng/ml). The levels of 17,20-P remained high at ovulation (142 ng/ml) and then dropped significantly within 24 h to approximately half of the ovulatory values. They decreased even further by 7 days postovulation. GTH levels rose gradually through GVBD and ovulation from a postvitellogenic level of approximately 3 ng/ml to a 7 day postovulatory value of approximately 10 ng/ml. The overall results; 1) decrease in estradiol prior to GVBD, 2) increase in 17,20-P at GVBD and 3) gradual GTH rise through GVBD and ovulation, are similar to those reported for other salmonids.     

Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon (Oncorhynchus kisutch) during vitellogenesis

17-estradiol, 17-20-dihydroxy-4-pregnen-3-one (17-20-P), and testosterone levels were measured in plasma samples obtained from vitellogenic coho salmon (Oncorhynchus kisutch) before and 32 days after injection of the aromatase inhibitor Fadrozole (AI). Plasma 17-estradiol levels decreased significantly 6 h after injection in all AI treated fish. The higher the dose the longer the maintenance of low plasma 17-estradiol levels. Inversely, plasma 17-20-P increased significantly 6 h after injection in all AI treated fish, and the higher the dose the longer the maintenance of high plasma 17-20-P levels. At 48 h after injection plasma testosterone levels were significantly higher in the AI treated groups. The oocyte maturation index showed that multiple injections with AI retarded oocyte development. Besides, oocyte diameter and GSI were lower in the same group, which presented high incidence of atresia of vitellogenic oocytes. The ovarian follicles and brain of the fish which received multiple injections secreted less 17-estradiol, in vitro. These findings suggest that aromatase inhibitors such as Fadrozole may have a potential as a tool to regulate sexual development in salmon.     

Synthesis of 17,20α- and 17,20β-dihydroxy-4-pregnen-3-ones, 11-ketotestosterone and their conjugates by gills of teleost fish

Goldfish, carp and trout gills were incubated with ³H-17-hydroxyprogesterone (17P). With goldfish gills, the metabolites were 17,20-dihydroxy-4-pregnen-3-one (17,20P; 82%), 17,20-dihydroxy-4-pregnen-3-one (17,20P; 8%), 11-ketotestosterone (KT) glucuronide (5.4%) and 17,20P glucuronide (0.2%). Sulfates were not detected. Carp gills converted 17P into 17,20P (11.2%), 17,20P (9.6%), KT (8.4%), glucuronides of 17,20P (1.3%) and 17,20P (1.6%) and sulfates of 17,20P (5.1%) and 17,20P (7.2%). 17,20P (38% free, 1.8% glucuronide and 21.1% sulfate) was the sole metabolite of ³H-17P in trout gill incubations. In the presence of high (10; µg ml^-1) substrate concentration, cyprinid gills gave predominantly free 17,20P, while trout gills yielded only free 17,20P. Production of 17,20P, predominantly as its sulfate, from endogenous precursors was demonstrated in trout gills but was not stimulated by trout primary extract. Our results demonstrate for the first time the steroidogenic potential of teleost gills and suggest that they may play a role in secretion of pheromones in some species.     

Structural and functional effects of early exposure to estradiol-17β and 17α-ethynylestradiol on the gonads of the gonochoristic teleost Dicentrarchus labrax

This study investigated the effects of estrogens on sexual differentiation in sea bass (Dicentrarchus labrax L.), a gonochoristic marine teleost that under culture conditions has a histologically sexual undifferentiated period that covers most of the first year of life, after which most individuals develop as males. Sea bass that had no noticeable histological sign of sex differentiation were fed estrogens at two doses (5 or 10 mg kg^-1 food) and for different periods ranging from 48 to 426 days post fertilization (DPF). Exposure to the synthetic estrogen 17-ethynylestradiol (EE₂) at 10 mg kg^-1 food from 60 to 260 DPF, including the sensitive period to equivalent doses of synthetic androgens previously determined for this species (126-226 DPF), significantly (p < 0.05) more than doubled the number of juvenile females to 80%, compared to the control value of 33%, and completely suppressed gonadal development in the remaining 20% of the population. This suggests that the period during which sea bass gonads exhibit high sensitivity to androgens is also very sensitive to estrogens. A comparable exposure to the natural estrogen estradiol-17 (E₂) resulted in 13% of the fish having suppressed gonadal development, but induced 57% of the fish to develop gonads with germinal tissue of both sexes, suggesting a pivotal role for E₂ during this sensitive period. Earlier exposure to EE₂ at 10 mg kg^-1 food from 48-88 DPF, significantly (p < 0.05) increased the number of females to 62% from 36% in the control group, allowing for the normal testicular development in the remaining fish. In contrast, a later chronic exposure (226-426 DPF) to E₂, at either 5 or 10 mg kg^-1 food, starting when the gonads showed no sign of sexual differentiation but past the critical sensitive period, had no effect on the resulting overall sex ratios, indicating that after this period responsiveness of the gonads to estrogens decreases as gonadal sexual differentiation progresses. However, the consequences of this apparently innocuous exposure were later manifested in adults, exemplified by a significant dose-dependent reduction in the number of mature males at 626 DPF, coinciding with the second reproductive season, the time when males normally reach sexual maturation in cultured sea bass. This suggests that chronic exposure to E₂ past the critical sensitive period may not affect the sex ratio, but could result in alterations in the male reproductive organs. This was later verified by histological analysis which revealed a significant (p < 0.05) dose-dependent reduction of the surface of the testicular lobules in the remaining males that did not mature. Together, these experiments illustrate both readily observable and subtle effects of estrogens on sex proportions, gonadal morphology and maturation rates, providing evidence that estrogen exposure can have delayed action in a teleost in a manner similar to the effects described for mammalian species. The possible existence of effects of this latter type in adult fish could be considered when evaluating the consequences of deliberate or accidental exposure to estrogens or putative estrogenic chemicals, particularly if such exposure had taken place during sex differentiation.     

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.     

17α,20β,21-trihydroxy-4-pregnen-3-one and 17α,20β-dihydroxy-4-pregnen-3-one stimulated spermiation in protandrous black porgy, Acanthopagrus schlegeli

The objective of this study was to investigate the effects of sex steroids on spermiation in protandrous male black porgy, Acanthopagrus schlegeli. Experiments on common carp (Cyprinus carpio) were also conducted for comparison. Fifty male black porgy were divided into 5 groups and injected with a superactive analogue of mammalian luteinizing hormone releasing hormone (LHRH-A), 17,20,21-trihydroxy-4-pregnen-3-one (20-S), 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP), 11-ketotestosterone (11-KT) or saline. The dosage of the sex steroids given on days 0, 2, 4 and 6 was 330, 330, 990 and 1980 µg kg^-1 body weight, respectively. Milt volume and sperm concentrations were measured on days 0, 2, 4, 6, 8 and 10. Similar treatments were also conducted in 45 male common carp. Milt volume was significantly increased in black porgy after treatment with 20-S and 17,20-DHP; 17,20-DHP had stimulatory effects on spermiation at a lower dose (900 µg kg^-1 body weight, p < 0.05) as compared to 20-S (1980 µg kg^-1 body weight, p < 0.01). In the common carp, milt volume was also increased after treatment with LHRH-A and 17,20-DHP but not with 20-S. 17,20-DHP stimulated spermiation at a lower dose in common carp (330 µg kg^-1 body weight) than in black porgy (990 µg kg^-1 body weight). However, 11-KT did not stimulate spermiation in black porgy or common carp. The concentrations of plasma 11-KT could immediately reflect to the administration of exogenous 11-KT in black porgy.     

Melatonin, but not somatostatin-14 influences in vitro steroidogenesis by ovarian follicles of rainbow trout

Ovarian follicles taken from sexually maturing rainbow trout at the mid-vitellogenic stage of ovarian development were incubated in vitro in the presence or absence of melatonin or somatostatin-14 (SRIF-14) to determine whether there is evidence of a direct action of these factors on gonadal steroidogenesis in fishes. The steroidogenic capacity of the ovarian follicles was assessed by measuring testosterone (T) and 17-estradiol (E₂) release into the incubation medium, and by examining the steroid metabolites produced following incubation of follicles with radiolabelled steroid precursors.Melatonin appears to elicit a biphasic effect on steroidogenesis by in vitro rainbow trout ovarian follicles; at a concentration of 1 × 10^–3 M, melatonin stimulated basal T and E₂ production, but at a concentration of 1 × 10^–2 M there was an inhibition of basal and sGtH-stimulated T and E₂ Melatonin may act to reduce the activity of specific steroidogenic enzymes, since there was evidence of melatonin at 1 × 10^–2 M enhancing the accumulation of [³H]17-hydroxyprogesterone in the medium following incubation with [³H]pregnenolone, possibly suggesting the inhibition of C_17,20-lyase activity. In contrast, SRIF-14, used at concentrations of 1 × 10^–8 M and 1 × 10^–6 M, had no effect on basal or sGtH-stimulated E₂ or T production by ovarian follicles, incubated in vitro.     

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.     

Plasma levels of Gonadotropin-II and gonadal sex steroids in triploid catfish, Heteropneustes fossilis (Bloch)

Triploid fish have under-developed gonads due to altered reproductive endocrinology. Triploids of Indian catfish (H. fossilis) showed significantly reduced plasma levels of gonadotropin (GtH-II), testosterone (T) and estradiol-17 (E₂) than that of diploids throughout the year, except for the resting phase, irrespective of sex. Plasma levels of GtH-II were significantly different (p<0.001) between diploid and triploid fish during preparatory, prespawning and spawning phase. The plasma testosterone contents in triploids were significantly less (p<0.001) than that of diploids, except during the resting phase. Triploid females showed very low titres of estradiol-17 (<1 ng ml^–1) throughout the annual reproductive cycle in contrast to highly fluctuating levels in diploid females. Thus, this study for the first time provides information on reduced levels of GtH-II and sex steroids in plasma of male triploid fish and additional information on species-specific alteration of sex hormones in female triploids.     

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.     

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.     

Inhibition of HPI axis response to stress in gilthead sea bream (Sparus aurata) with physiological plasma levels of cortisol

This study investigates the effect of corticosteroid (cortisol) administration on the stress response of the gilthead sea bream Sparus aurata subjected to a 48 h confinement. The effect of (in-vitro and in-vivo ) cortisol administration on the in-vitro ACTH sensitivity of the interrenal tissue; the plasma levels and tissue concentration of cortisol; and the plasma levels of ACTH, -MSH, -endorphin and glucose were determined. Confinement caused a transient and concomitant increase in plasma cortisol and ACTH levels. However, in cortisol-fed fish the plasma ACTH levels were lower, indicating a suppresion of the ACTH release from the corticotropes by cortisol. In contrast to the activation of the corticotropes, the levels of plasma melanotrope derived peptides were not affected. In spite of the fact that interrenal cells of cortisol-fed gilthead sea bream released less cortisol than controls, the interrenal sensitivity to ACTH was not affected by in-vivo and in-vitro cortisol administration. This suggests that the interrenal sensitivity to ACTH in stressed (confinement) sea bream is probably not regulated by -MSH, N-ac--END, or by cortisol. Thus, in gilthead sea bream the interrenal sensitivity to ACTH could be regulated at the hypothalamus and/or pituitary and communicated via circulating ACTH levels.     

Endocrine stress response and abnormal development in carp (Cyprinus carpio) larvae after exposure of the embryos to PCB 126

To investigate whether PCB 126 exposure duringembryonic development induces an endocrine stressresponse in larval carp, eggs were exposed,containing 0.01% ethanol (vehicle-control), 10^-11,immediately after fertilization, for 48 h to water10^-10 or 10^-9 mol l^-1 PCB in 0.01% ethanol. Eggsincubated in water served as controls. After transferto PCB-free water, mortality, the incidence ofyolk-sac and pe-ricardial oedema, wet and dry weight,rate of skin pigmentation, and whole-body contents ofthe stress hormones ACTH, -MSH and cortisol weredetermined at 48, 96, 144, 168, 192 and 216 hpost-fertilization. Except for the dry weight, allparameters of animals exposed to 10^-10 and 10^-9 moll^-1 PCB increased in a concentration-related manner.However, these changes became evident only at 144 hpost-fertilization, i.e. after resorption of theyolk-sac. Swelling of the yolk sac and pericardiumoccurred, and whole-body ACTH, -MSH and cortisollevels increased. Although animals exposed to 10^-10and 10^-9 mol l^-1 PCB displayed stable but elevatedwhole-body ACTH and -MSH levels until 216 h,whole-body cortisol concentrations gradually decreasedfrom 168 h post-fertilization, and were significantlybelow control values at 216 h post-fertilization.Exposure of the carp embryos to 10^-11 mol l^-1 PCB only increased whole-body -MSH levels. Increased whole-body ACTH and cortisol levels indicate that PCBinduces a stress response in carp larvae, possiblymediated by a disturbed hydromineral balance (oedema).We further suggest that the PCB-stimulated bodypigmentation is mediated by a stimulation of -MSHsecretion.     

Progestogen metabolism by ovaries of the roach (Rutilus rutilus L.) and the rudd (Scardinius erythrophthalmus L.)

Roach ovaries converted 17-hydroxyprogesterone to 17,20-dihydroxy-4-pregnen-3-one (17,20P) and to glucuronides of testosterone and 17,20P. Small amounts of 5-pregnane-3- and -3, 17, 20-triols, 7-hydroxy-5-reduced metabolites and 17,20-dihydroxy-4-pregnen-3-one (17,20P) were also formed. Rudd ovaries converted this substrate mainly to 17,20P, 5-pregnane-3- and -3,17,20-triols, 17,20-dihydroxy-5-pregnan-3-one and testosterone glucuronide. The main metabolites of progesterone with both species were 17,20P, 5-pregnane-3,17,20-triol and 7-hydroxy-5-reduced steroids. Rudd ovaries formed, in addition, 17,20-dihydroxy-5-pregnan-3-one from progesterone. The pattern of metabolites was markedly altered when the concentration of substrate was increased from 42ng to 1 µg or 100 µg. At the highest concentration, glucuronides and polar steroids were not detectable, while at low concentrations they accounted for over 50% of the metabolites. 20-Hydroxysteroid dehydrogenase was shown to have a very high capacity, producing 21–47 µg 17,20P from 100 µg 17-hydroxyprogesterone substrate with 200 mg ovarian tissue in 5h.     

Effects of salinity, dietary level of protein and 17α-methyltestosterone on growth hormone (GH) and prolactin (tPRL177 and tPRL188) levels in the tilapia, Oreochromis mossambicus

In the present study, we examined the long-term effects of environmental salinity, diet (35% and 25% crude protein) and 17-methyltestosterone (MT) on corresponding levels of pituitary and serum growth hormone (GH) and prolactins (tPRL₁₇₇ and tPRL₁₈₈) in the tilapia (Oreochromis mossambicus). We observed no discernible patterns in serum GH that would suggest an effect of salinity, diet or MT. However, serum GH levels in all treatments declined at 1 and 3h after first feeding. Serum tPRL₁₇₇ and tPRL₁₈₈ were significantly higher in freshwater (FW) than in seawater (SW) and levels were significantly affected by dietary protein. tPRL₁₇₇ levels were higher in all groups fed a 35% protein diet, but tPRL₁₈₈ levels were higher only in the groups fed the MT-treated 35% protein diet; only serum tPRL₁₈₈ levels were affected by MT. Moreover, serum tPRL₁₇₇ and tPRL₁₈₈ increased throughout the sampling time-course. Subsequent work using fasted tilapia suggests that first feeding is likely to initiate the post-prandial suppression of serum GH levels. In contrast with the picture observed in blood, pituitary glands of SW animals showed higher levels of GH than FW fish. Pituitary GH was elevated by MT in both FW and SW. We also observed that pituitary tPRL₁₇₇ and tPRL₁₈₈ levels were higher in FW fish than in SW fish; tPRL₁₇₇ and tPRL₁₈₈ levels were elevated by MT only in FW animals. To assess the somatomedin activity of plasma from FW- and SW-reared tilapia, we measured [35S]-sulfate incorporation into ceratobranchial cartilage explants in vitro. Plasma from SW-adapted tilapia showed greater activity in this assay than plasma from FW-reared tilapia, suggesting that the GH-dependent IGF bioactivity of plasma is higher in SW-reared tilapia. Collectively, these studies suggest that the growth-promoting actions of SW rearing and of MT administration in tilapia may be linked to elevations in GH and/or prolactin (tPRL₁₇₇ and tPRL₁₈₈)levels.     

In vitro steroidogenesis by testicular fragments and ovarian follicles in a hybrid sturgeon, Bester

In this study, developmental changes in the steroidogenic capacity of testicular fragments and isolated ovarian follicles of a hybrid sturgeon, Bester, at a variety stage of developments were examined. Testicular fragments or isolated ovarian follicles were incubated in L-15 medium in the presence or absence of different concentrations of five preparations; forskolin, human chorionic gonadotropin (HCG), pregnenolone (P₅), 17-hydroxyprogesterone (17OHP) and testosterone (T) for 18 h at 15 °C. After incubation, concentrations of 11-ketotestosterone (11 KT) (testis) and, 17-estradiol (E₂) (ovarian follicles) and 17,20-dihydroxy-4-pregnen-3-one (DHP) (testis and ovarian follicles) were measured. 11KT was detected in the media following incubation with P₅, 17OHP and T. Its concentration was higher during late spermatogenesis and prespermiation and lower at the degeneration stage. Both P₅ and 17OHP were converted to DHP during the prespermiation stage. Forskolin had little stimulatory effect on the synthesis of 11KT and DHP and HCG did not induce the production of these steroids.E₂ was detected in the medium following incubation of follicles with P₅, 17OHP and T at all stages of oocyte development. The concentration of E₂ in the medium increased during vitellogenesis with the peak production occurring at the tertiary yolk stage. In contrast, the potencies of follicles to produce steroids shifted to the production of DHP during migratory nucleus stage. Forskolin and HCG had little effect on the synthesis of E₂ and DHP. These results demonstrated that the failure of spontaneous spermiation or ovulation is not due to the insufficient synthesis of DHP, but may due to the lack of availability of precursors.     

Gonadotropins I and II do not stimulate thein vitro secretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate by rainbow trout gonads during final sexual maturation

Thein vitro secretion of 17,20-dihydroxy-4-pregnen-3-one 20-sulphate (17,20-P-sulphate) and the free steroid 17,20-dihydroxy-4-pregnen-3-one (17,20-P), by rainbow trout (Oncorhynchus mykiss) gonads, in response to gonadotropin (GTH) I and GTH II, were studied during the final stages of sexual maturation. Substantial amounts of 17,20-P-sulphate were produced, by both mature ovaries and testes, indicating considerable 20-hydroxysteroid sulphotransferase (20-HST) activity within these tissues. In the post-ovulatory ovary the level of 17,20-P-sulphate (36.6 ng ml^–1) greatly exceeded that of 17,20-P (8.59 ng ml^–1). The amount of 17,20-P-sulphate produced in incubations of both mature ovary and testes was unaffected by either GTH I or GTH II treatment at physiological concentrations up to 100 ng ml^–1. Similarly, incubations of maturing ovary and testes, treated with GTH I or GTH II, in the presence of added 17,20-P at 100 ng ml^–1 of medium, produced levels of 17,20-P-sulphate that were similar to those of the controls. In incubations of mature ovarian follicles at the stages of germinal vesicle breakdown and preovulation, both GTHs significantly stimulated secretion of 17,20-P, although GTH II was always more potent than GTH I. GTH II significantly elevated the levels of 17,20-P in testicular incubations from mature males more than 4-fold relative to GTH I and controls, which did not differ from one another.In conclusion, 20-HST, the enzyme responsible for the sulphate conjugation of 17,20-P, was found to be active in the ovaries and testes of rainbow troutin vitro. However, the levels of this enzyme do not appear to be regulated by either GTH I or GTH II.     

Role of thyroid hormones in tilapia larvae (Oreochromis mossambicus): II. Changes in the hormones and 5′-monodeiodinase activity during development

Thyroid hormone profiles and 5-monodeiodinase activity were determined in tilapia at different stages of early development. The results showed that both T₄ and T₃ were present in significant amounts in fertilized eggs. There was a steady decrease in both T₄ and T₃ levels during embryonic development. The levels continued to decline after hatching until around 7 days later when most of the yolk had been absorbed. The T₄ level started to rise then, suggesting that the larval thyroid had begun to produce T₄ at this time, which coincided with the period of faster growth of the larvae. The T₃ level remained fairly constant until around 20 days after which it rose significantly. In vitro determination of 5-monodeiodinase activity (5-D activity) in the whole-body homogenates of larvae showed that the enzymatic conversion of T₄ to T₃ was not detectable in eggs and 3-day-old larvae but detected in 5-day-old and older larvae. There was a gradual increase in the V_max as development proceeded indicating increasing 5-D activity during larval development. The K_m values did not differ significantly in the different stages of development. These results are discussed in relation to the growth and development of the larvae.     

Oxidative metabolism in a teleost,Anabas testudineus Bloch: effect of testosterone and estradiol-17β on hepatic enzyme activities

Testosterone (T) administration to maleAnabas testudineus significantly stimulated the activities of cytochrome oxidase, -glycerophosphate dehydrogenase (-GPDH) and Mg²⁺ adenosine triphosphatase (Mg²⁺ ATPase) and inhibited lactate dehydrogenase (LDH), cytosolic and mitochondrial malate dehydrogenases (MDH). The activities of succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G-6-PDH) and catalase were unaffected by testosterone treatment. Administration of estradiol-17 (E2) in female fish, significantly stimulated cytochrome oxidase activity, inhibited Mg²⁺ ATPase, SDH, catalase and cytosolic and mitochondrial MDH activity, and was without effect on other enzymes studied.The simultaneous injections of actinomycin D or chloramphenicol and T or E2 prevented the hormonal influence on hepatic enzyme activities. The present study demonstrates that inA. testudineus sex steroids influence hepatic oxidative metabolism by a mechanism sensitive to the action of inhibitors of protein synthesis.