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.     

Sulfation and uptake of the maturation-inducing steroid, 17α,20β-dihydroxy-4-pregnen-3-one by rainbow trout ovarian follicles

Rainbow trout ovarian follicles were incubated in vitro with tritiated 17,20-dihydroxy-4-pregnen-3-one (17,20-P; maturation-inducing steroid). Within 18–24 h, 56–66% had been converted to tritiated 17,20-dihydroxy-4-pregnen-3-one 20-sulfate (identification confirmed by HPLC) and 27% had been taken up (absorbed) by the follicles. Addition of 125 ng of cold (non-tritiated) 17,20-P to the incubations caused a decrease in the percentage of [³H]-17,20-P which was sulfated (56% 10%) and an increase in the percentage that was taken up (27% 57%). Seven steroids were tested for their effectiveness in decreasing the sulfation and increasing the uptake of tritiated [³H]-17,20-P. The order of effectiveness was in both cases the same: 17,20-P > cortisol > 11-deoxycortisol > 17,20,21-trihydroxy-4-pregnen-3-one > 17-hydroxy-4-pregnene-3,20-dione > 17-estradiol > testosterone. This indicated that the processes of sulfation and uptake of [³H]-17,20-P were related to each other and led to the hypothesis that, when cold 17,20-P is added to the medium, it reduces the proportion of [³H]-17,20-P which is sulfated and thus allows more free [³H]-17,20-P to enter the ovarian follicles. This hypothesis was supported by the finding that each ovarian follicle had the capacity in vitro to sulfate only ca. 2 ng of [³H]-17,20-P per 18h but a capacity to take up > 500 ng per 18h.Gonadotropin I, Gonadotropin II, forskolin and phorbol-12-myristate-13-acetate (which all have an affect on steroid biosynthesis) did not affect the amount of 17,20-P which was sulfated. Sulfating activity was localized in the thecal cell layer of the follicle. The yolk fraction was shown to be responsible for absorbing the [³H]-17,20-P.     

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.     

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.     

Circulating levels and in vitro production of two maturation-inducing hormones in teleost: 17α,20β-dihydroxy-4-pregnen-3-one and 17α,20β,21-trihydroxy-4-pregnen-3-one, in a daily spawning wrasse, Pseudolabrus japonicus

The female bambooleaf wrasse, Pseudolabrus japonicus, spawns daily during the spawning season, and exhibits a diurnal rhythm of ovarian development. In the present study, we have investigated: (1) circulating levels of 17a, 20-dihydroxy-4-pregnen- 17,20-P) and 17,20,21-trihydroxy-4-pregnen-3-one (20-S) in females sampled at different times of the day during spawning season in captivity, and (2) in vitro production of 17,20-P and 20-S by follicle-enclosed oocytes at seven different develo tal stages. In addition, we developed a microtiter plate enzyme-linked immunosorbent assay (ELISA) for 17,20-P. Serum levels of 17,20-P and 20-S showed similar diurnal changes; substantial increases in these levels occurred around the time of germinal vesicle breakdown (GVBD). In vitro experiments showed that massive production of 17,20-P and 20-S occurred in follicles collected just before or during GVBD. Further, acute decreases in 17,20-P and 20-S production were found in the ovarian follicles just prior to ovulation, suggesting inactivation of the maturation-inducing hormone (MIH). These results, taken together with our previous data on the occurrence of GVBD in vitro, suggest a role for both 17,20-P and 20b-S as MIHs in the bambooleaf wrasse.     

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.     

Effect of gonadotropin type II and 17-hydroxy-4-pregnene-3,20-dione on 17,20β-dihydroxy-4-pregnen-3-one production by rainbow trout testes at different developmental stages

Plasma levels of 17,20-dihydroxy-4-pregnen-3-one (17,20OHP), which is involved in the regulation of spermiation in male salmonid fish, increase dramatically at the time of spermiation. To advance the understanding of the regulation of 17,20OHP production during the spermatogenetic cycle in trout, we have studied the in vitro effect of gonadotropin type II (GtH II) and the precursor 17-hydroxy-4-pregnene-3,20-dione (17OHP) on the production of 17,20OHP. The sensitivity with which testes secreted 17,20OHP following stimulation with GtH II was maximum during spermatogenesis. The addition of 17OHP (10 to 1600 ng ml-1) to the culture medium of testes fragments induced a significant and dose-related increase in 17,20OHP secretion. Although the capacity to produce 17,20OHP was not saturated by the 17OHP concentrations used, the conversion rate was highest for tested at an immature stage. As to the regulation of 17OHP, in vivo, a single injection of partially purified salmon gonadotropin (50 ng g-1 body weight) induced a significant increase in the circulating levels of 17OHP of immature males. In conclusion, the maximum sensitivity to GtH II stimulation and the highest conversion rate of 17OHP to 17,20OHP in vitro, occurred before the dramatic increase in the 17,20OHP secretion observed in rainbow trout at the time of spermiation.     

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.     

Survival,growth and sexual maturation of the tiger trout hybrid (Salmo trutta ♀ × Salvelinus fontinalis ♂)

Brown trout ♀ × brook trout ♂ hybrids, together with parental species controls from the same breeders, were tested up to the age of 3 years at four different fish farms. Aside from egg and newborn alevin mortalities, the survival rate of the hybrids was similar to that of brown trout while that of brook trout was much lower. Hybrid growth was about intermediate between those of brown trout (inferior) and brook trout (superior). Sexual maturation was severely impaired in the hybrids, which resulted in high dressing rates: females had practically no ovarian development and male gonadogenesis, although limited, caused some morbidity during the spawning season. The feasibility of hybrid fish culture using genetic improvements is discussed in the light of these results.     

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.     

The modulating effect of vitellogenin on the synthesis of 17β-estradiol by rainbow trout (Oncorhynchus mykiss) ovary

In vivo induction of vitellogenin (VTG) in response to the administration of 17-estradiol (E₂) was achieved and the protein was isolated by gel filtration column chromatography of plasma samples. Adult female trout were injected with the vitellogenic fraction every ten days from July to November and levels were measured by RIA from September to December. The results showed a significant decrease (p<0.01) in plasma E₂ levels in injected females compared with the controls. In December, after finishing the treatment, the plasma E₂ concentration increased, in injected females to reach a level similar to that of control females at vitellogenesis. The in vitro study showed that in early vitellogenic oocyte (from September) the presence of the vitellogenic fraction in the incubation medium causes a significant decrease (p<0.01) in the synthesis of E₂ by the oocytes. These data suggest that the concentration of the VTG into the oocyte can alter VTG production by the liver, moderating the production of E₂ by the ovary.     

Effects of (D-Ala6, Pro9N ethylamide) — LHRH on plasma levels of gonadotropin, 17α,20β-dihydroxy-4-pregnen-3-one and testosterone in male goldeye (hiodon alosoides Rafineque)

Male goldeye were treated with (D-Ala⁶, Pro⁹-N ethylamide) — LHRH (LHRH-A) in saline or a silastic pellet (100 µg.kg^–1 body weight) and changes in plasma gonadotropin (GtH), 17,20-dihydroxy-4-pregnen-3-one (17,20P) and testosterone (T) levels were determined by radioimmunoassay. LHRH-A increased plasma GtH levels, with the response to LHRH-A in saline being of much greater magnitude and duration than the response to silastic pellet implants. Seasonal differences were found in the response to LHRH-A. Spermiated fish were the most responsive, recrudescing fish the least, and regressed fish displayed an intermediate response. Plasma 17,20P levels were elevated in response to LHRHA in fish of all sexual stages although the magnitude of the increase was not related to the magnitude of the increase in GtH levels. Treatment with LHRH-A also resulted in a transitory increase in plasma T levels. The endocrine control of GtH and steroid secretion in goldeye is discussed in relation to studies in cyprinids and salmonids. Address for reprint requests: Department of Zoology, University of Alberta, Edmonton, Alberta T6G 2E9 Canada.     

Histological changes in insulin-immunoreactive pancreatic β-cells,and suppression of insulin secretion and somatotrope activity in brook trout (Salvelinus fontinalis) maintained on reduced food intake or exposed to acidic environment

Immature brook trout (Salvelinus fontinalis) were randomly divided into a pH control, a pH and food control and an acid-stressed group. Fish in the first two groups were held at neutral pH and those in the last group were maintained at pH 4.2 for up to two months. The food supply to the pH and food control group was restricted to simulate the reduction in food intake demonstrated for acid-stressed trout. Plasma insulin levels were significantly decreased from 5–20 ng/ml to 1–2 ng/ml and plasma cortisol levels were significantly increased from 5–10 ng/ml to as high as 70 ng/ml in the acid-stressed brook trout. Concomitantly, a significant decrease of 21–39% in the proportion (volume density) of insulin immunoreactive -cells was observed within the principal pancreatic islets. Somatic growth was stunted and ultrastructural morphometry revealed the suppression of somatotrope secretory activity in the acid-stressed fish. Restriction of food supply induced a smaller but still significant decrease in circulating levels of insulin which was however not accompanied by a reduction in insulin immunoreactive -cells. The rise in plasma cortisol levels was not significant, and the plasma levels of glucose and protein were unaffected. Nevertheless, somatotrope secretory activity was suppressed and somatic growth was stunted. This study demonstrates for the first time the complexity of the endocrine response to acid stress and that some of the response to acid stress can be attributed to the lowering of food intake.     

Binding characteristics and regulation of the 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) receptor on testicular and sperm plasma membranes of spotted seatrout (Cynoscion nebulosus)

The binding characteristics of 17,20,21-trihydroxy-4-pregnen-3-one (20-S) to plasma membranes prepared from the testes and sperm of spotted seatrout (Cynoscion nebulosus) were investigated using a filtration method to retain the bound 20-S. A single class of high affinity (K_d = 17.9 nM), low capacity (B_max = 0.072 nM g^-1 testes) binding sites was identified by saturation and Scatchard analyses on testicular membranes of spermiating spotted seatrout. A corresponding receptor (K_d = 22.17 nM, B_max = 0.00261 nM ml^-1 milt) was also detected in spermatozoan membrane preparations. The rates of 20-S association and dissociation were rapid, both had T_halfs of less than 1 min. Competition studies indicated that the receptor was highly specific for 20-S. 17,20-dihydroxy-4-pregnen-3-one, which had the highest affinity of the other steroids tested, had a relative binding affinity (RBA) of 14.3%. Progesterone, 11-deoxycortisol and testosterone competed with an order of magnitude less affinity (RBA's of 7.4, 1.8 and 1.1%, respectively). Estradiol displayed low affinity for the receptor (RBA = 0.4%) and cortisol did not cause any displacement at 1000-fold excess concentration. Specific 20-S receptor binding was detected in plasma membranes from testes of both spermiating and non-spermiating seatrout and on spermatozoa. Prolonged incubation of testicular fragments from a spermiating fish with gonadotropin (15 IU ml^-1 human chorionic gonadotropin) or forskolin (10 µM) caused a 2–3 fold increase in membrane receptor binding. Previous studies have shown that gonadotropin-induced upregulation of the 20-S plasma membrane receptor in seatrout ovaries is required for the oocytes to become responsive to 20-S and undergo final maturation. The existence of a 20-S membrane receptor on sperm and its upregulation in the testes by gonadotropin raises the possibility that final maturation of spermatozoa in male seatrout may be regulated by a similar mechanism.     

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.     

An enzyme linked immunosorbant assay (ELISA) for testosterone, estradiol, and 17,20β-dihydroxy-4-pregenen-3-one using acetylcholinesterase as tracer: application to measurement of diel patterns in rainbow trout (Oncorhynchus mykiss)

A simple and rapid Enzyme Linked ImmunoSorbant Assay (ELISA) is described and validated for testosterone, estradiol, and 17,20-dihydroxy-4-pregnen-3-one (17,20P). A general procedure for preparation of the acetylcholinesterase labeled steroid is described which is applicable to any steroid. Use of acetylcholinesterase tracer increased the sensitivity of assay so that reliable measurements of each steroid could be achieved with only 10 l of plasma. The ELISA was applied to measurement of all three steroids every hour for over 24 hours in a female trout using cannulation of the dorsal aorta. This high sampling frequency revealed several short-term (<2 h) episodic pulses of testosterone and estradiol.