Disposition of genistein in rainbow trout (Oncorhynchus mykiss) and siberian sturgeon (Acipenser baeri).

Genistein (G) is a xenoestrogen from soy present in fish diet. In vivo, a 50-fold difference in sensitivity to genistein on vitellogenin (VTG) synthesis was found when comparing trout and sturgeon. This difference was not linked to the estrogen receptor affinity nor to the sensitivity of induction of the VTG pathway. The study was performed to check if differences in the G disposition in the two species could explain their difference of sensitivity to G. A pharmacokinetic analysis of radiolabeled G was performed to determine its bioavailability and metabolism in both species. G was used at levels corresponding to fish farm exposure. G plasma levels after chronic ingestion were found to be 15.6 times higher in sturgeon than in trout. Sturgeon primarily produces sulfate conjugates after G ingestion whereas trout mainly produces glucuronides. Sturgeon was able to excrete orobol glucuronide in bile. An important first pass effect was suggested in both species. No accumulation of G or its metabolites was observed in the two species. Trout muscles accounted only for 0.14 of radioactivity 48 h post-ingestion similarly to sturgeon. Trout viscera accounted for 15% of the radioactivity 48 h post-ingestion. In sturgeon, 48 h post-ingestion, viscera accounted for 21.5% of the radioactivity. These rates decreased rapidly thereafter. The study partly explains the difference in sensitivity to G, previously recorded between the two species. In addition, it shows that human exposure to G through farmed fish consumption is negligible.

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.

Effects of dietary phytoestrogens in vivo and in vitro in rainbow trout and Siberian sturgeon: interests and limits of the in vitro studies of interspecies differences.

A study of the effects of dietary genistein on trout and sturgeon in vivo showed that sturgeon was sensitive to 20 ppm of genistein, whereas trout was not. To analyze the origin of this interspecies difference in sensitivity, a cell culture technique was developed with hepatocytes from sturgeon and compared to results obtained with hepatocytes from trout in the same system. The hepatocyte culture proved to be useful as bioassay for estrogenicity. Vitellogenin (VTG), assayed by a specific enzyme-linked immunosorbent assay, was used as a biomarker of the estrogenic activity. 17 beta-Estradiol, its glucuronide and sulfate derivatives, and estradiol analogues (ethynylestradiol and diethylstilbestrol) were tested. Nonestrogenic compounds such as androgens, progesterone, and cortisol were tested as negative controls. VTG production was monitored at doses ranging from 1 nM to 10 microM estradiol. Phytoestrogens, from the isoflavone family, were tested individually at increasing doses exhibiting dose response curves for concentrations from 500 nM to 10 microM. With tamoxifen, an antagonist of estrogen receptors, the estrogenic effect was partially reduced. The effect was the same with ICI182,780 in sturgeon, whereas the effect was the opposite in trout. The estrogenic potency of the isoflavones ranged differently between the two species in the following order: biochanin A < daidzein = formononetin < genistein < equol in trout and biochanin A < genistein < daidzein < formononetin < equol in sturgeon. Further, in sturgeon, formononetin was the most potent phytoestrogen in vitro, whereas its activity was weakest in vivo. These data suggest that one must reconsider the relevance of heterologous estrogenic tests and of homologous in vitro tests for estrogenic potency of chemicals.

Effect of genistein-enriched diets on the endocrine process of gametogenesis and on reproduction efficiency of the rainbow trout Oncorhynchus mykiss.

Three practical diets were formulated to contain 0, 500, or 1000 ppm genistein. The three diets were distributed for 1 year to groups of rainbow trout undergoing their first gametogenesis and until spawning. Growth performance of rainbow trout was not affected by dietary treatments. Plasma cholesterol levels were equivalent between groups. In males, a slight but constant induction of vitellogenin (VTG) synthesis and a decrease in testosterone levels were observed. A slight decrease in plasma levels of betaFSH and betaLH was noticed at the end of spermatogenesis in the male fish fed a diet with 500 ppm (genistein) (from 2.16 +/- 0.39 to 1.47 +/- 0.23 for betaFSH and from 0.44 +/- 0.09 to 0.31 +/- 0.09 for betaLH). There was a significantly reduced 17alpha,20beta(OH)(2)-progesterone (from 10.93 +/- 0.88 in control to 5.46 +/- 0.92 in males and from 251.22 +/- 21.40 to 183.22 +/- 13.48 in females). Testicular development was accelerated in genistein-fed fish, and sperm motility and concentration were decreased in a dose-dependent manner at spawning. In females, a significant increase in plasma VTG occurred only at the beginning and at the end of oogenesis. Testosterone levels were decreased at the beginning of oogenesis. Both betaFSH and betaLH were decreased by genistein (from 6.38 +/- 1.55 to 3.44 +/- 0.82 for betaFSH and from 15.18 +/- 3.00 to 6.93 +/- 0.99 for betaLH in females), whereas spawning was delayed only in females fed the diet with 500 ppm of genistein. Gamete quality was impaired only in this group, as underlined by a lower percentage of ovulating females (from 100 to 79% at the end of the trial), a lower fertilization rate, and a lower viability of fry. These results may be explained by the agonistic/antagonistic effect of genistein on estrogen function related to the tissue ratio between endogenous estrogens/genistein.

Seasonal variations and maturity stages in relation to differences in serum levels of gonadal steroids, vitellogenin, and thyroid hormones in the common dentex (Dentex dentex).

Seasonal variations in serum concentrations of 17beta-estradiol (E(2)), vitellogenin (Vg), testosterone (T), 11 ketotestosterone (11-KT), and thyroid hormones (T(4), l-thyroxine; and T(3), 3,5, 3'-triiodo-l-thyronine) were investigated during the first, second, and third reproductive cycles in intensively reared populations of common dentex, Dentex dentex, and correlated with gonadal development and spawning. In females, there were baseline E(2) values (<0.10 ng/ml) and negligible Vg concentrations during the postspawning and pregametogenesis period (June to December), and these increased thereafter to peak during the spawning period. Maximum T(3) and T(4) serum concentrations were found around spawning. There was a positive correlation during vitellogenesis and final maturation between Vg and T(3) (r(2) = 0.366). In addition, Vg and T(3) concentrations were statistically higher in the stages of vitellogenesis and final maturation than at the other stages (P<0.001). Minimum T(3) and T(4) concentrations (October) coincided with the decrease in water temperature and the associated decrease in the daily feeding rate and the specific growth rate. In males, as in females, seasonal changes in serum levels of T and 11-KT were well correlated with gonadal development. The presence of males in the stage of completed spermiogenesis in December coincided with the surge in both androgens and this increase lasted until the end of the spawning period. There were no significant differences in serum T(3) and T(4) levels among the maturity stages. The observed seasonal changes in serum gonadal steroids and Vg reflected the pattern of oocyte development and the spawning behavior of common dentex and were typical of the patterns described in most multiple spawners studied to date. Thyroid hormones may enhance early ovarian development and stimulate vitellogenesis in female dentex.

Synthesis of haptens and conjugates for ELISAs of phytoestrogens. Development of the immunological tests.

Seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein, and at the C8 position for a second series, with only formononetin and daidzein. The different haptens were coupled to bovine serum albumin (BSA) and to swine thyroglobulin (Thyr). Polyclonal antibodies were generated against the BSA conjugates. Enzyme-linked immunosorbent assays (ELISAs) were developed based on competition between free phytoestrogens and the Thyr-hapten conjugates for specific antibodies. IC(50) values of the standard curves ranged between 0.8 and 20 ng/mL that is, 0.3 and 9.2 pmol/well. The antibodies obtained should be useful for assays in vegetable matter as well as in biological fluids after a separation step. These ELISAs should be valuable also in the food industry to control phytoestrogen concentrations prior to and after processing.

Expression and localization of messenger ribonucleic acid for the vitellogenin receptor in ovarian follicles throughout oogenesis in the rainbow trout, Oncorhynchus mykiss.

The expression and localization of vitellogenin (VTG) receptor (VTGR) mRNA were identified throughout ovarian development in the rainbow trout, Oncorhynchus mykiss. Northern blot confirmed the presence of a transcript (approximately 3.9 kilobases [kb]) that was specific to the ovary. The expression of VTGR mRNA varied throughout ovarian development and was highest in previtellogenic ovaries and in ovaries at the onset of vitellogenesis containing ovarian follicles (OF) from 35 to 600 microm in diameter. In situ hybridization using 35S riboprobes showed that the transcription of the VTGR gene was initiated in OF measuring 45-50 microm in diameter, with transcripts being exclusively localized in the ooplasm. A dramatic increase in mRNA synthesis occurred during previtellogenic growth (OF from 50 to 200 microm); this was followed by a gradual decrease during the vitellogenic growth phase. VTGR mRNA was not detected in OF greater than 1000 microm in diameter (oocytes actively sequestering VTG). Immunocytolocalization of yolk proteins derived from VTG demonstrated that oocytes started to sequester VTG when they were around 300 microm in diameter, shortly after the time of maximal density of VTGR mRNA in the ooplasm. The timing of transcription of the VTGR gene, predominantly during previtellogenesis, suggests that the VTGR is recycled to the oocyte surface during the vitellogenic growth phase.

Evolution of oogenesis: the receptor for vitellogenin from the rainbow trout.

Receptors that transport vitellogenin (VTG) into oocytes are of vital importance to egg-laying species because they mediate a key step in oocyte development. Here we describe the cloning of the first piscine oocyte-specific receptor cDNA, i.e., that encoding the VTG receptor from the rainbow trout (Oncorhynchus mykiss). The receptor, a 826-residue type-I membrane protein, is a member of the low density lipoprotein receptor (LDLR) superfamily. It closely resembles the mammalian so-called very low density lipoprotein receptors, in that its aminoterminal ligand binding domain consists of a cluster of 8 cysteine-rich repeats. The short intracellular portion contains the internalization signal typical for the LDLR superfamily, Phe-Glu-Asn-Pro-Val-Tyr. Notably, the receptor lacks a domain with a high density of potential O-glycosylation sites often found in somatic cell-specific members of the LDLR family. A specific transcript of 3.9 kb is abundant in ovary, but undetectable in muscle and heart, which are the major sites of expression of very low density lipoprotein receptors in mammals. In vitro translation of the full-length cDNA produced a 97-kDa protein, and transient expression in COS-1 cells showed that the cDNA encodes a protein of the same size that binds vitellogenin in ligand blots. As revealed by in situ hybridization, transcripts are present in previtellogenic oocytes, indicating that production of receptor protein precedes the phase of yolk deposition. Our results in fish, together with those in birds (Bujo, H., et al. 1994. EMBO J. 13: 5165-5175) suggest that vitelogenesis provides a prime model for the study of ligand/receptor systems designed to sustain reproduction.

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.

Identification of vitellogenin receptors in the ovary of a teleost fish, the Mediterranean sea bass (Dicentrarchus labrax).

The characterization of vitellogenin (VTG) receptors in ovarian membranes from vitellogenic female sea bass (Dicentrarchus labrax) is described. Incubation of membrane proteins with radiolabeled VTG (125I-VTG) after SDS-electrophoresis showed specific binding of 125I-VTG to a protein band of 100 kDa. Filter binding assays showed that binding of 125I-VTG to membrane receptors was saturable with increasing amounts of 125I-VTG. Scatchard analysis of the saturation data revealed a single class of binding sites with an apparent KD of 1.04 x 10(-8) M. The specificity of the VTG receptors was tested in competition assays; binding of 125I-VTG to ovarian membranes was completely abolished with an excess of purified sea bass VTG (cold VTG, VTG degree) or plasma from estradiol (E2)-treated fish, while the addition of control male plasma (without VTG) caused negligible effect.

Vitellogenin receptors during vitellogenesis in the rainbow trout Oncorhynchus mykiss.

Rainbow trout vitellogenin receptors have been characterized by ligand blotting and Scatchard analysis. Their evolution has been studied over a reproductive cycle in a broodstock of 2-year-old females. The receptors were prepared from ovarian membrane homogenates and were solubilized using n-octyl-beta-D-glucopyranoside. The visualization of the receptor by ligand blotting using 125iodine-vitellogenin after sodium dodecyl sulfate electrophoresis revealed the existence of one major binding component corresponding to a protein of 113 kDa. The Scatchard transformation of the binding data revealed a single class of binding sites with an apparent Kd of 1.8 x 10(-8) M/L. The variations of the binding characteristics (Kd and maximum binding) were investigated during vitellogenesis. This study revealed that the Kd was not affected by oocyte growth during vitellogenesis, but was highly decreased in ovulated eggs. The receptor number increased during the same period from 35 to 860 fM per oocyte, while the receptor number per mm2 of oocyte membrane surface was doubled during the same period.

Occurrence and in vitro biosynthesis of 11-ketotestosterone in Siberian sturgeon, Acipenser baeri Brandt maturing females.

High levels of 11-ketotestosterone (11KT) were found (49 to 160 ng ml(-1)) in plasma of Siberian sturgeon females during the end of their reproductive cycle. These levels were measured either by specific radioimmunoassay, or both by specific radioimmunoassay and by UV absorption after HPLC (isocratic conditions, 33% methanol, 26% acetonitrile, 41% water). In order to find the origin of 11KT synthesis, ovaries were incubated (30 min and 2h at 20°C) with tritiated 17-hydroxyprogesterone (17OHP) or with tritiated androstenedione (A4). Testosterone (conversion rate from tritiated 17OHP: 4%) and 11-ketotestosterone (conversion rate from tritiated A4: 1.6%) were identified as metabolites of respectively 17OHP and A4 (TLC, HPLC and crystallization). 11ß-hydroxyandrostenedione (11ßOHA4) and 11ß-hydroxytestosterone (11ßOHT) were suggested to be intermediate metabolites. Besides interrenal and blood cells were incubated respectively with tritiated cortisol and tritiated A4. 11ßOHA4 was identified in interrenal incubation (yield from tritiated cortisol: 1.2%). 11KT in interrenal (yield from tritiated cortisol: 0.14%), and 11ßOHA4 and 11KT in blood cells (yield from tritiated A4: 1.6%), were suspected to be synthesized (TLC, HPLC, acetylation). No significant metabolization of tritiated cortisol could be found in liver. The possible contribution of each of these tissues to high 11KT levels found in plasma is discussed.

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.

In vivo estrogen induction of hepatic estrogen receptor mRNA and correlation with vitellogenin mRNA in rainbow trout.

We have previously described the cloning, sequencing and in vitro expression of a full-length rainbow trout estrogen receptor cDNA (rtER cDNA). This full cDNA randomly labelled was used to study the estrogen induction of hepatic rtER mRNA in correlation with vitellogenin (Vg) mRNA in different physiological situations. In this paper, we show that in the liver two mRNA species are under hormonal control and their level increases about 8-fold after estrogen stimulation. These two mRNAs are expressed and induced in the liver as early as the hatching stage in correlation with the expression of Vg mRNA. A long-term analysis of rtER mRNA after estradiol (E2) injection shows a transient induction of the nuclear ER and its mRNA which recover to the basal level after 2 weeks. Nevertheless, a memory effect was observed on the expression of the Vg gene which does not appear to be directly related to the estrogen receptor level.

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.

Plasma kinetics of ingested tritiated estradiol and the influence on estradiol plasma levels in the cultured Siberian sturgeonAcipenser baeri.

Plasma kinetics of tritiated estradiol (E2) were studied in the Siberian sturgeonAcipenser baeri in order to explain the large amount of E2 found in plasma of 4, 5 and 10-year-old cultured males. This work presents two approaches. The first is based on a single ingestion of(3)H-labelled E2, which allowed us to plot model curves of resorption and elimination processes. The second deals with chronic ingestion of(3)H-labelled E2 during a five day period, twice a day, based on a rhythm copied from the feeding practice on a fish farm. Three different doses were tested, based on the amount present in fish diets. Oral administration of E2 to sturgeon leads to E2 accumulation and a saturation of the metabolic processes generally involved in the elimination of aromatic xenobiotics. An explanation of this progressive accumulation of E2 in sturgeon plasma has to take into account the steroid binding proteins. Their synthesis could be induced by the orally administrated E2 and protect it from metabolism.

Regulation of oogenesis: the piscine receptor for vitellogenin.

The receptor-mediated uptake of vitellogenin (VTG), a plasmatic lipophosphoglycoprotein, is crucial for oocyte growth in egg-laying animals. The plasma membrane receptor for VTG was characterized from oocytes of coho salmon, Oncorhynchus kisutch. In direct binding studies, the receptor exhibited high affinity (Kd, 180 nM) for salmonid VTG, and by ligand blotting with radiolabelled VTG it was visualized as a protein with an apparent Mr of 100,000, under non-reducing conditions. The fish VTG receptor was shown to share key structural elements with VTG receptors from chicken and Xenopus laevis. Namely, cross-reactivity at the level of ligand recognition was observed among VTG receptors from these species and immunological relatedness was demonstrated by immunoblotting with anti-chicken VTG receptor antibodies. In addition, as in chicken and Xenopus, binding of VTG to fish oocyte receptors was shown to be mediated by the lipovitellin domain of VTG. These results clearly indicate that regulation of oocyte growth at the level of yolk formation has been accomplished by the conservation of structural features of receptors required for internalization of VTG.

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.

Immunohistochemical investigation of the pituitary of the sturgeon (Acipenser baeri, Chondrostei).

An immunohistochemical study of the sturgeon (Acipenser baeri) pituitary was undertaken using antisera directed against hormones from various classes of vertebrates, including the only pituitary hormone available from sturgeon, gonadotrophin. A positive reaction was obtained after application of antisera towards the following hormones 1-24 synthetic ACTH (1-24 ACTH), melanophore stimulating hormone (MSH), ovine prolactin (oPRL), ovine growth hormone (oGH), salmon growth hormone (sGH), carp gonadotrophin (cGTH) and its beta subunit (ßcGTH), sturgeon gonadotrophin (aciGTH), carp thyrotrophin (cTSH) and ß subunit of the human thyrotrophin (ßhTSH). The results demonstrate that, in general, the sturgeon pituitary resembles that of teleosts as regards the distribution of the different cell types: ACTH and PRL cells in the rostral pars distalis, GTH, TSH and GH cells in the proximal pars distalis and MSH and PAS-cells in pars intermedia. In addition to the topographical organization of the sturgeon pituitary, this study provides data on the immunological relationships between sturgeon pituitary hormones and those of other vertebrates.

Effect of carp gonadotropin (cGTH) and a fraction unabsorbed on concanavalin A-Sepharose obtained from cGTH on vitellogenesis in the hypophysectomized marine teleost Gobius niger.

To investigate whether a differential potency on vitellogenesis exists between the carp gonadotropin (cGTH) and fraction I-cGTH (proteins from cGTH unbound on concanavalin A-Sepharose, which represent 5% of cGTH in weight), hypophysectomized Gobius niger were treated with the two hormonal preparations at the same level. Vitellogenesis was checked for synthesis of vitellogenin and yolk incorporation in the ovary by means of immunological studies and histological techniques (light and electron microscopy). In addition, increased synthesis of vitellogenin was induced by injection of estradiol 17 beta together with each gonadotropin to assess the action of the two hormonal preparations on vitellogenin incorporation. Oogenesis was enhanced by cGTH and fraction I-cGTH, and at the same dose levels both treatments produced a similar pattern of stimulation of vitellogenesis. Vitellogenin was found in all the blood samples of animals treated by the hormones (cGTH and fraction I-cGTH) alone. Vesicles of pinocytosis were detected by electron microscopy up to Stage IIIa of oogenesis. When a high synthesis of vitellogenin was induced by exogeneous estradiol 17 beta injections, the two gonadotropic preparations had similar effects in yolk incorporation. cGTH was not less potent than fraction I-cGTH in these processes even though the cGTH preparation contains only 5% of fraction I-cGTH. The contamination of cGTH by a small amount of material unbound on concanavalin cannot be solely responsible for the vitellogenic activity of cGTH which consists of 95% glycoproteins.