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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

17α-羟基-20β-双羟孕酮诱导泥鳅排卵的效应

有关17α-羟基-20β双羟孕酮[17α-Hydroxy-20β-dihydroprogesterone(4 Pregnen-17α-20B diol-3one)](以下简称17α-20βP)对鱼类排卵效应及其生殖机理作用的研究,自七十年代以来,国外已开展了这方面的工作。Schuetz, A.(1974),Jalabert, B.(1978)和Jalabert, B.et al.(1978)曾论述过,用脑垂体促性腺激素(GTH)诱导鱼类卵细胞的成熟是通过中介物质性类固醇激素在起作用的。Jababert,B.(1976)叙述了用类固醇激素(17α-20βP)能诱发虹鳟鱼的卵细胞达到最终成熟。Montalembert.     

用17α—羟基—20β—双羟基黄体酮促使泥鳅在低温下排卵效应的研究

国外已有不少文献记载17α-羟基-20β-双羟基黄体酮(以下简称17α-20βP)对鱼类生殖生理方面的机理作用,在国内尚未见有关这方面的报道。Jalabert(1976)曾报道17α-20βP能诱导鲤鱼产卵。Jalabert(1978)报道对银鲑、北美狗鱼进行的试验情况,注射17α-20βP能诱导这几种鱼类卵细胞成熟。Breton(1983)采取多次注射少量的LH-RH或LH-RH-A,然后再注射17α-20βP,其结果同样能诱导鱼类卵细胞成熟。     

栉孔扇贝17β-HSD4基因的克隆和表达分析

为了研究17β-hsd4基因在无脊椎动物生殖过程中的作用,实验从栉孔扇贝转录组数据库中获得一个表达序列标签,采用cDNA末端快速扩增技术克隆得到1条全长为2 417 bp的cDNA序列,其开放阅读框为2 223 bp,编码740个氨基酸,推测的氨基酸序列含有17β-HSDs特有的4个保守区和17β3-HSD4的3个酶结构域,且与人等脊椎动物的序列相似性均在58％以上.半定量RT-PCR显示,该基因在栉孔扇贝精巢、卵巢、肌肉、外套膜、鳃、肝胰腺和肾脏等各组织中均有表达,其中在肝胰腺和肾脏中表达量明显高于其他组织.对不同发育时期性腺中该基因表达的qRT-PCR检测发现,其在精卵巢发育周期的表达模式不同,且在生长期和成熟期的精巢中表达量显著性高于同时期的卵巢.由此推测,栉孔扇贝多种组织中均可以合成17β-HSD4,在精巢的发育和成熟的作用明显高于卵巢.     

17α, 20β 双羟孕酮诱导鲤卵母细胞最终成熟相关基因表达内参基 因的筛选

本研究检测了18S rRNA(18S)、28S rRNA(28S)、组织蛋白酶Z(cathepsin Z,CTSZ)、延伸因子1-α(elongationfactor 1-α,EF-1α)、3-磷酸甘油脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,gapdh)、β肌动蛋白(β-actin)6个候选内参基因在17α,2β双羟孕酮(DHP)诱导鲤(Cyprinus carpio)卵母细胞最终成熟过程的表达情况,并应用不同的内参分析软件对数据进行了分析.Bestkeeper软件分析表明EF-1α的变异系数和标准差是6个候选内参基因中最低的,且Bestkeeper指数在6个候选内参基因中最高,说明其表达稳定性最高,适合做为内参基因;geNorm软件分析认为需要同时使用EF-1α和CTSZ两个内参基因来校正目标基因的表达;NormFinder软件分析同样表明EF-1α是6个候选内参基因中表达最稳定的;最后通过RefFinder软件综合比较分析,确定EF-1α相对于其他5个候选内参基因,在17α,20β双羟孕酮诱导卵母细胞最终成熟阶段表达最为稳定,可作为内参校正17α,20β双羟孕酮诱导鲤卵母细胞最终成熟阶段各基因的表达情况.     

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.     

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.