F. Le Menn

The estrogenic activity of certain phytoestrogens in the siberian sturgeon Acipenser baeri

Various phytoestrogens such as formononetin, daidzein, genistein and equol were synthesized. Their purity was assessed by various analytical techniques including melting point determination, thin-layer chromatography (TLC), infra-red spectra (i.r. spectra), nuclear magnetic resonance (1H- and 13C-NMR) and gas chromatography coupled with mass spectrometry (GC-MS). The estrogenic activity of these compounds, as well as biochanin A and coumestrol, was biologically tested by the induction of vitellogenin secretion in yearling sturgeon and compared to the activity of estradiol-17 beta. Pure daidzein, biochanin A, genistein, equol and coumestrol all had estrogenic activity as assessed by their induction of hepatic synthesis of vitellogenin when administrated intraperitoneally to yearling Siberian sturgeon. Coumestrol seemed to be the most potent compound, inducing the most vitellogenin secretion with the lowest dose administered. Formononetin was inactive when administered by the intraperitoneal route. All the phytoestrogens tested were considerably less potent than estradiol-17 beta.

Enzyme immunoassay for 11-ketotestosterone using acetylcholinesterase as laberl: application to the measurement of 11-ketotestosterone in plasma of Siberian sturgeon

A competitive enzyme immunoassay using 11-ketotestosterone (11-KT) covalently coupled to acetylcholinesterase (AChE) from electric eel is developed. Ninety-six well microtiter plates are coated with pure mouse-anti-rabbit-IgG monoclonal antibody and competition is performed in one-step reaction by adding successively 11-KT standard or samples, enzymatic tracer and 11-KT-specific antiserum. This assay is more sensitive (detection limit is 7 pg/ml) than radioimmunoassays previously described, and specificity was not modified by the use of enzymatic tracer. This assay can be performed in less than 5 hr. Validation of the EIA was achieved in a fish model, Siberian sturgeon (Acipenser baeri), by comparative determinations of 11-KT levels in plasma extracts using EIA and RIA. Application to the measurement of 11-KT in plasma of farmed Siberian sturgeon during the first reproductive cycle is reported.

Stimulation of gonadotropin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol

In freshwater or seawater female silver eel, the release of gonadotropin (GTH) accumulated in the pituitary under estradiol (E2) influence could be stimulated by a conjugated treatment with a mammalian gonadoliberin agonist (GnRH-A = des-Gly10, (D-Ala6)-LH-RH ethylamide) and a blocker of dopamine receptor (pimozide). Furthermore, despite the GTH release, no reduction or even a significant increase in pituitary GTH levels were noted, indicating a stimulation of GTH synthesis. In consequence of the endogenous GTH release, a stimulation of ovarian development was induced, as demonstrated by the gonadosomatic index and histological study. Similar results were obtained with a combined treatment with GnRH-A and an inhibitor of catecholamine synthesis (L-alpha-methyl-3,4-dihydroxyphenylalanine). In contrast, no effect was produced by GnRH-A, pimozide, or L-alpha-methyl-DOPA, given alone. The results suggest that a double neuroendocrine mechanism (a lack of GnRH production and a dopaminergic inhibition of GnRH action) is involved in the prepubertal blockage of eel gonadotropic function before the reproductive migration.

Plasma Vitellogenin Levels during the Annual Reproductive Cycle of the Female Rainbow Trout (Oncorhynchus mykiss): Establishment and Validation of an ELISA

Rainbow trout, Oncorhynchus mykiss, vitellogenin (Vtg) was purified from plasma of E2-treated male by direct anion exchange chromatography and some of its biochemical characteristics were studied. Our results demonstrated that, under SDS-PAGE conditions, rainbow trout Vtg was composed of two molecular forms of 390 and 176 kDa representing, respectively, the dimeric form and the monomeric from of the molecule. The purified Vtg was used to raise a polyclonal antibody for Vtg (anti-Vtg). Using this anti-Vtg, a competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of rainbow trout Vtg. The practical sensitivity range of this ELISA was 20-320 ng/ml (80-20% of binding) and the detection limit was 9 ng/ml. The intra- and the inter-assay coefficients of variation (at 50% of binding) were estimated at 1.8% (n = 10) and 3.9% (n = 13), respectively. This ELISA was validated by detecting changes in Vtg levels in rainbow trout at different physiological stages, as well as in 2-year-old female rainbow trout throughout the reproductive cycle.

Estrogenic effect of dietary soya bean meal on vitellogenesis in cultured Siberian sturgeon Acipenser baeri

The unusual presence of vitellogenin in the plasma of male and nonvitellogenic female Siberian sturgeon has been demonstrated previously (Pelissero and Le Menn, 1988; Pelissero et al., 1989a) and was attributed to dietary effects. The present study examines estrogenic effects of dietary soya bean meal and of commercial trout diet on vitellogenesis in sturgeon. The 4-month study compared three diets, one commercial (T) and two experimental synthetic diets, one containing casein alone (SC for synthetic diet made on casein), and the other casein and soya bean (SS for synthetic diet made with soya bean) as protein sources. The dietary soya bean meal contained plant isoflavonic compounds which are well known to mimic the effects of estrogens in mammals. The SC diet, free of estrogenic compounds, served as the reference diet. When fed with the SC diet, sturgeons showed significantly lower plasma vitellogenin levels (0.0045 +/- 0.0012 mg/ml) compared with those fed the commercial diet (1.24 +/- 0.37 mg/ml). The SS diet had a very pronounced effect on the plasma vitellogenin level, which at the end of the experiment had reached 6 mg vitellogenin/ml. In no case was estradiol detectable in the plasma. Plasma androgen levels were high in all the three groups throughout the study period, and not significantly different from one another. Sturgeon fed the T diet had larger livers, with enlarged hepatocytes, compared with those fed the SS and SC diets.

Sea bass (Dicentrarchus labrax L.) vitellogenin. II Validation of an enzyme-linked immunosorbent assay (ELISA)

Abstract In this study, a competitive ELISA was developed for the quantification of vitellogenin (VTG) in sea bass. The purity of the antigen (VTG) and the specificity of the antibody (AbVTG) used in the assay, were assessed by ELISA and radial immunodiffusion, and no crossreactivity was observed with the male plasma. The sensitivity range of the ELISA was 1–60 ng/ml (85-20% of binding). The intra- and inter-assay variations (at 50% of binding) were 5.3% ( N = 5) and 9.8% ( N = 9), respectively. Annual VTG levels were quantified in animals reared under fish farming conditions and the VTG levels were correlated with the oocyte development.

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 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). 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 μl 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.

Effect of androgen mediated by the estrogen receptor of fish liver: Vitellogenin accumulation

We have investigated the action of high doses of androgens in Gobius niger L., a marine teleostean fish, by characterizing specific steroid receptors in liver and by assaying the plasma vitellogenin concentration under different hormonal treatments. Estrogen and androgen receptors were characterized in the liver nuclear extracts according to their binding specificity. The maximum binding capacity was 25 fmoles/mg protein for the estrogen and androgen receptors. In vivo, high doses of DHT()increased the concentration of plasmatic vitellogenin as assayed by immunodiffusion while low doses were inefficient. In spite of a similar number of estrogen and androgen nuclear receptor sites (25 fmoles/mg protein), DHT was at least 70 fold less active than E2 on yolk protein and vitellogenin induction both in male and female Gobius niger. In addition, the antiestrogen tamoxifen, which was inactive by itself, inhibited the E2 and the DHT induced accumulation of vitellogenin. Progesterone (2 mg/fish) was also totally inactive in inducing vitellogenin. We conclude that the induction of vitellogenin by DHT is mediated by the estrogen receptor rather than by the androgen receptor. In addition to the estradiol induced protein in rat uterus and to other estrogenic responses obtained by androgens in mammary cancer, fish vitellogenin is another estrogen regulated protein which can be induced by high doses of androgens.

Binding affinities of hepatic nuclear estrogen receptors for phytoestrogens in rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baeri)

Phytoestrogens are dietary estrogenic contaminants capable of inducing vitellogenin synthesis in rainbow trout and Siberian sturgeon. A competitive-binding assay on their hepatic estrogen receptors (ER) was performed to determine the relative affinity of phytoestrogens compared to estradiol (E(2)). Phytoestrogen concentrations used were 1000 times higher than for E(2), except for genistein and formononetin. For each compound, the competition with 50%-bound labelled E(2) (DC(50)) was considered in order to classify phytoestrogens according to their affinity for ER. The affinities are compared for each species. In rainbow trout, estradiol (DC(50): 7 nM)>formononetin (DC(50): 260 nM)>genistein (DC(50): 570 nM)>equol (DC(50): 5.3 microM)>daidzein (DC(50): 9 microM)>biochanin A (DC(50): 100 microM). In sturgeon, estradiol (DC(50): 5 nM)>genistein (DC(50): 220)>formononetin (DC(50): 1 microM)>equol>(DC(50): 8.3 microM)>daidzein>(DC(50): 80 microM)>biochanin A (DC(50): 100 microM). These results demonstrate that phytoestrogens, mimicking estradiol, can disturb the endocrine system by competing for ER. Also, the higher sensitivity to genistein observed in vivo in Siberian sturgeon (vitellogenin synthesis), compared to rainbow trout, is not due to a higher affinity of genistein for the hepatic ER. Thus, the metabolism of phytoestrogen could be species dependent and affect sensitivity.

Enzyme-linked immunosorbent assay (ELISA) for sole (Solea vulgaris) vitellogenin

1. 1. A specific and simple immunoassay for the sole (Solea vulgaris) vitellogenin (Vg) is described. This assay was developed using an antiserum to plasmatic Vg. 2. 2. The assay is based upon the competition between soluble Vg and Vg adsorbed on microtiter plates, for the rabbit anti-Vg antibody binding sites. 3. 3. The adsorbed Vg-antibody complexes are then revealed using the peroxidase-antiperoxidase technique. 4. 4. Following the revelation of the peroxidase activity by an appropriate chromogen, the resulting absorbance values are measured. The intensity of the reaction is proportional to the antibody linked to the adsorbed Vg. This assay can be performed in 6 hr. 5. 5. In our conditions a sensitivity range of 2.5–320 ng gave 90–10% of binding. Near 50% binding (20 ng) the intra-assay variation coefficient (CV) was 4.5% (n = 20) and the inter-assay CV was 7.5% (n = 12). 6. 6. This assay has been used for the quantitation of Vg in various biological samples. 7. 7. This enzyme immunoassay methodology could be a useful alternative to the use of radioactive substances.