Effects of 17 beta-estradiol on levels and distribution of metallothionein and zinc in squirrelfish.

Females of the squirrelfish family (Holocentridae) accumulate higher levels of zinc in the liver than any other known animal. This zinc accumulation is made possible by high expression of the zinc-binding protein, metallothionein (MT). In the present study, the squirrelfish (Holocentrus ascensionis) MT cDNA was cloned and sequenced. The deduced amino acid sequence was very similar to other teleost MT. The role of estrogens on zinc metabolism was investigated by injecting male and immature female squirrelfish with 17 beta-estradiol (E(2)). E(2) treatment triggered transient increases in plasma zinc and vitellogenin (VTG) levels, and both of these variables showed very similar time courses. These results suggest that E(2) is responsible for the large hepatoovarian translocation of zinc observed in female squirrelfish and that VTG might be a vehicle for zinc. E(2) did not directly alter the levels of zinc or MT mRNA in the liver. However, the hepatic MT protein concentration increased differentially in the nuclear fraction. Thus E(2) is probably responsible for the association of MT with the nuclear fraction previously observed in untreated mature female squirrelfish.

Improved high-performance liquid chromatographic method for the separation and quantification of lipid classes: application to fish lipids.

An improved straight-phase HPLC method for the separation and quantification of lipid classes is described. Two binary gradient solvent systems were used, one for polar and one for neutral lipids, and detection was performed with a light-scattering detector. The developed HPLC methods were highly reproducible and allowed base-line separation of all investigated polar lipid classes (phosphatidic acid, diphosphatidylglycerol. phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylinositol and lysophosphatidylcholine) and neutral lipid classes (triacylglycerol, free fatty acid, diacylglycerol, cholesterol and monoacylglycerol) except of cholesterol ester and wax ester. Application of the chromatographic systems demonstrated that the methods are suitable for quantitative analysis of the major lipid classes present in lipid extracts from livers and eggs of Atlantic salmon (Salmo salar).

Amino acid composition and endocrine control of vitelline envelope proteins in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata).

The vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol-17 beta. As males of both species synthesize these proteins after treatment with estradiol-17 beta, the origin is not restricted to the ovaries. Vitellogenin of both European sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa.

Induction of vitelline envelope proteins by estradiol-17 beta in 10 teleost species.

Induction of vitelline envelope proteins by estradiol-17 beta was investigated in 14 teleost species from five systematic groups to assess whether estradiol-17 beta controls the synthesis of vitelline envelope proteins in teleost fish. Vitelline envelope proteins were detected in plasma from fish treated with estradiol-17 beta using three different antisera directed against vitelline envelope proteins from three teleosts. Induction of vitelline envelope proteins was demonstrated in 10 species. In 6 of these species males were available and used to demonstrate that the synthesis of vitelline envelope proteins is not restricted to the ovaries. The immunoreactivity of the proteins varied considerably within and between species. It is suggested that the synthesis of vitelline envelope proteins is controlled by estradiol-17 beta in the majority of teleost species.

Formation of the vitelline envelope precedes the active uptake of vitellogenin during oocyte development in the rainbow trout, Oncorhynchus mykiss.

In order to study the initial formation of the vitelline envelope and the appearance of vitellogenin in oocytes of rainbow trout, females were sampled monthly from 19 to 5 mo before ovulation. Immunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 microns. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol-17 beta increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 microns) present just inside the oolemma, when the oocytes reached a diameter of 600 microns. This was interpreted as an active uptake of vitellogenin. Oocytes of this size were first found in females sampled 9 mo before ovulation at the time when estradiol-17 beta levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 microns had an immunoreactive vitelline envelope with a thickness of about 3 microns. It is apparent that the initial formation of the vitelline envelope starts before the active uptake of vitellogenin and that the low previtellogenic plasma levels of estradiol-17 beta observed in females are of physiological significance.

The interrelation between photoperiod, growth hormone, and sexual maturation of adult Atlantic salmon (Salmo salar).

The plasma profiles of growth hormone (GH) in adult male and female Atlantic salmon were determined in relation to manipulation of the photoperiod and to the development and timing of sexual maturation. Fish were exposed to natural light (NL) or NL + 24L:0D additional light over the netpens from January (ALJ) or March (ALM) to July. Thereafter, these groups were brought indoors, subdivided, and subjected to simulated natural photoperiod (SNP), continuous light (24L), or short day (8L). Assay of salmon GH by RIA in monthly plasma samples revealed that GH levels were generally < 1 ng ml-1 during January to June and were only slightly affected by additional light in January or March. ALJ-24L treatment, and to a lesser extent, ALM-24L treatment, was effective in preventing sexual maturation, and GH levels of immature fish continued to be < or = 1.5 ng ml-1. On the other hand, in sexually maturing fish, GH levels increased to 2-5 ng ml-1 months prior to ovulation. Short-day photoperiod (8L) from July advanced ovulation and spermiation, whereas continuous light from July delayed these processes. The timing of the increase of GH levels was shifted in a parallel manner, indicating a functional relationship between plasma GH levels and the process of sexual maturation.

Properties of cod metallothionein, its presence in different tissues and effects of Cd and Zn treatment.

One isoform of the low-molecular-weight metal-binding protein metallothionein (MT) has been isolated from the liver of Atlantic cod by size-exclusion and ion-exchange chromatography. Cod MT contained 33% cysteine, no aromatic amino acids or arginine. As is the case for other piscine MTs, the N-terminus of cod MT lacked the asparagine in position 4 which is present in mammalian MTs. In addition, cod MT differed from all other vertebrate MTs described in that the N-terminal methionine was not acetylated. Antibodies were raised in rabbits against hepatic MT from cod by repeated injections of native protein mixed with adjuvant. Anti-cod MT antisera cross reacted with similarly-sized proteins in liver, brain, anterior kidney, posterior kidney, spleen, intestine, gills and ovaries. The putative MT in cod brain migrated differently to that of the other tissues in native gel electrophoresis. Intraperitoneally injected Cd (1 mg/kg) was nearly entirely associated with the MT-peak in hepatic and renal cytosols, whereas a single injection of Zn (10 mg/kg) resulted in increases in all cytosolic Zn pools of the liver and no apparent change in cytosolic Zn, Cu, Ni or Cd in kidney.

Absence of direct regulation of prolactin cells by estradiol-17 beta in rainbow trout (Oncorhynchus mykiss).

The effects of estradiol-17 beta (E2) implants on plasma prolactin (PRL) concentrations, pituitary PRL content and pituitary PRL mRNA levels were examined in rainbow trout (Oncorhynchus mykiss). Intact immature fish treated with 1 mg estradiol-17 beta did not show significant changes in both PRL mRNA levels and pituitary PRL content after 3 days of treatment. In a similar experiment, no changes were observed in plasma PRL levels followed during 7 days. Similarly, lack of estradiol-17 beta effect on plasma PRL levels and on final PRL pituitary content was observed in ovariectomized female rainbow trout treated during 48 days with 25 mg estradiol-17 beta and in mature male fish over a 3-week treatment period. Localization of estradiol receptor (ER) mRNAs in the pituitary was carried out by Northern blot analysis using a full-length rainbow trout estrogen receptor (rtER) cDNA as a probe. The rostral pars distalis of the pituitary which contained mostly PRL cells showed the lower amount of rtER mRNA when compared to other parts of the pituitary. Moreover, two mRNAs of different size (3.5 and 1.4 kb) were detected in different parts of the pituitary. Further hybridization experiments using probes containing part of the rtER cDNA (E domain or C and D domains) indicated that the small-sized mRNA (1.4 kb) probably encodes a truncated ER protein lacking hormone binding domain or an ER-related protein. Thus, only the 3.56 kb mRNA appeared to be involved in the regulation of pituitary function by estradiol. In situ hybridization analysis allowed a more precise localization of this rtER mRNA in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical detection of the major vitelline envelope proteins in the plasma and oocytes of the maturing female rainbow trout, Oncorhynchus mykiss.

The major vitelline envelope proteins were detected in the plasma of female rainbow trout maturing under natural conditions by using the Western blot technique. Females were sampled every month from July until ovulation in January. The amount of vitelline envelope proteins in plasma increased markedly as the gonads increased in size from 0.4 to about 15% of the total body weight. The plasma level of oestradiol-17 beta largely followed the alterations in the amount of vitelline envelope proteins, indicating the endocrine control of vitelline envelope protein synthesis. In addition, plasma vitellogenin changed in a manner that resembled the changes in the amount of plasma vitelline envelope proteins. The appearance and growth of the vitelline envelope during oocyte development was demonstrated using immunohistochemical methods. The vitelline envelopes from oocytes at different stages of development were immunoreactive with the antibodies directed against the major vitelline envelope proteins. No immunoreactivity could be observed in the ooplasm or in the surrounding follicular cells, which indicated that the major vitelline envelope proteins were of extraovarian origin. The present study further supports the hypothesis that the major protein constituents of the vitelline envelope in teleosts are under the endocrine control of oestradiol-17 beta and that the site of synthesis is outside the ovary.

Eggshell zona radiata-proteins from cod (Gadus morhua): extra-ovarian origin and induction by estradiol-17 beta.

Using Atlantic cod (Gadus morhua) as a model organism, the aim of this report was to delineate whether teleostean eggshell zona radiata proteins have their origin, i.e., site of synthesis, in gonadal or somatic tissues. Estradiol-17 beta was administered intraperitoneally to one-year-old cod (Gadus morhua) with either undeveloped gonads or with differentiated gonads. By immunoblotting procedures estradiol-dependent protein induction was investigated using specific rabbit antisera directed against cod eggshell proteins and brown trout vitellogenin. No immunological cross-reactions were observed between the two antisera, and eggshell proteins and vitellogenin were detected in blood plasma and somatic tissues only in estradiol-treated cod. Three plasma-components were immunoreactive to antiserum directed against eggshell proteins, and these proteins possessed molecular weights of 78, 54 and 47 kDa, identical to the molecular weights of the cod eggshell alpha, beta and gamma zona radiata-proteins. These three immunoreactive plasma-components were observed after administration of estradiol-17 beta to both sexes, also in males having reached spermiation, and in juveniles of either sex without developed gonads. The data are interpreted to signify that cod eggshell zona radiata-proteins originate in an extra-ovarian tissue and are transported in the blood for deposition in the ovaries. We propose that oogenesis involves estradiol-17 beta regulation of both eggshell zona radiata-proteins and vitellogenin synthesis.

Evaluation of differential pulse polarography for the quantification of metallothionein--a comparison with RIA.

Two methods to quantify metallothionein (MT), differential pulse polarography (DPP) and radioimmunoassay (RIA), were compared for MT analysis of liver from Zn- and Cd-injected perch (Perca fluviatilis). Nine perch were intraperitoneally injected, twice a week during 2 weeks with ZnSO4 and CdCl2 to yield a total dose of 30 mg Zn and 3 mg Cd per kilogram body weight. Two samples, 100 and 200 mg from each liver, were homogenized separately and further prepared for DPP, RIA, and atomic absorption spectroscopy. MT values obtained by DPP were in good agreement with the MT values determined by RIA (r = 0.92). The relationship between the MT values analyzed with the two methods is described by the formula MTRIA = MTDPP x 0.99-0.048. Analysis of MT was not affected by sample size. MT values from individual liver samples plotted against the Cd and Zn content of the corresponding samples provided a high correlation. The correlation coefficient was 0.86 for MT values obtained by DPP and 0.92 for MT measured by RIA. It is concluded that DPP is a reliable method for analyzing MT in liver.

Oestradiol-17 beta induces the major vitelline envelope proteins in both sexes in teleosts.

During growth of the ovarian follicle, the teleost oocyte becomes surrounded by an acellular coat, the vitelline envelope. The nature, origin and number of the vitelline envelope proteins in fish appear to vary with species. In this work, polyclonal antibodies directed against vitelline envelope proteins from rainbow trout, brown trout and turbot were used to show that oestradiol-17 beta induces the major vitelline envelope proteins in juveniles, both males and females, from different species. The fact that males can synthesize vitelline envelope constituents shows that the origin of these proteins is not confined to the ovary. The vitelline envelope of rainbow trout eggs consists of three major proteins, designated alpha (60 kDa), beta (55 kDa) and gamma (50 kDa). The amino acid composition of each of the three proteins indicated that the three proteins are alike and the suggestion that these proteins represent a separate class of structural proteins is sustained.

The importance of metallothionein for the accumulation of copper, zinc and cadmium in environmentally exposed perch, Perca fluviatilis.

A field study of the role of metallothionein (MT) in the binding of heavy metals in perch (Perca fluviatilis), exposed to moderate levels of copper, zinc and cadmium, was performed. Perch were sampled at four sites in Sweden in September during two consecutive years. Two sites were located in the vicinity of a brassworks and two outside the emission range. The first year, fish from the two brassworks sites and from one of the uncontaminated sites were collected. The second year, fish from the most contaminated site and from the two uncontaminated sites were caught. The levels of hepatic copper, zinc and cadmium reflected the concentrations of these metals in water and were increased in fish from the two contaminated sites. The level of cadmium in liver was relatively low. MT was induced in liver of perch caught at the most contaminated site. The hepatic MT content in individual livers correlated well to the accumulation of copper (r = 0.85, P less than 0.001) and zinc (r = 0.75, P less than 0.001). There was a low but significant correlation between the levels of MT and cadmium in the liver (r = 0.48, P less than 0.001). The relationship between MT and metals was very similar both years. Subcellular fractionation of the metals in the liver revealed that an induction of MT was followed by an increased amount of copper, zinc and cadmium bound to the protein. The relative fraction of the total hepatic copper and cadmium bound to MT was increased at the most contaminated site, whereas there was no difference in subcellular distribution of zinc between the sites. In perch from the most contaminated site, the portions of hepatic copper, zinc and cadmium found in the cytosolic fraction were 70, 57 and 81%, respectively. Seventy-one % of the copper, 29% of the zinc and 84% of the cadmium found in hepatic cytosol of fish from the same site, eluted together with MT after gel filtration chromatography. The analysis of the subcellular distribution of copper, zinc and cadmium demonstrates that MT is responsible for the binding of a large amount of the total hepatic cellular content of copper and cadmium in perch.

Binding and detoxification of heavy metals in lower vertebrates with reference to metallothionein.

1. The metabolism of Cu, Zn, Cd and Hg in lower vertebrates is described, using fish as a model. 2. The main part of this review deals with metallothionein and the role of this protein for the storage and detoxification of these metals. 3. Factors influencing the bioavailability and probable uptake routes are identified. 4. The distribution of the metals within the organism is outlined. The distribution between tissues is described and the subcellular distribution discussed with reference to metallothionein.

Plasma growth hormone levels increase during seawater exposure of sexually mature Atlantic salmon parr (Salmo salar L.).

At the time of smoltification in May, smolts and sexually mature male parr were transferred to seawater (25% salinity) and sampled after 6 and 24 hr. Plasma levels of growth hormone (GH) were measured by radioimmunoassay. There was no difference in GH levels between smolts and mature parr in fresh water. GH levels did not change during exposure of smolts to seawater. In the mature male parr, plasma GH levels increased after 24 hr, when the levels were almost five times those of the freshwater controls. In the mature male parr, there was an increase in plasma osmolality, sodium, and magnesium after 24 hr in seawater; magnesium also increased after 6 hr. The levels of potassium and calcium did not change in either immature parr or mature male parr. The increase in plasma GH levels in the mature parr in seawater may be part of a mechanism to increase hypoosmoregulatory ability in fish not ready for seawater entry.

A radioimmunoassay for perch (Perca fluviatilis) metallothionein.

A sensitive radioimmunoassay (RIA) for the measurement of metallothionein (MT) from perch (Perca fluviatilis) has been developed. The method is a double-antibody RIA with rabbit anti-perch MT serum as first antibody, goat anti-rabbit immunoglobulin G as second antibody, and perch MT conjugated to 125I-labeled Bolton-Hunter reagent as tracer. The rabbit antiserum raised against perch MT recognizes rainbow trout (Oncorhynchus mykiss) MT, but shows little cross-reactivity with horse MT. At a dilution of 1:2000, the MT antibodies bind 36% of the tracer when no cold ligand is present. The sensitivity of the assay is 15 pg perch MT per tube and the practical working range is 0.15-250 ng perch MT per tube. The RIA allows determination of MT in plasma or lysed blood cells at concentrations as low as 3.0 ng/ml and in tissues at levels above 9.0 ng/g (wet weight). Intra- and interassay coefficients of variation were 6 and 10%, respectively.

Sex differences in hepatic cytochrome P-450 monooxygenase activities in rainbow trout during an annual reproductive cycle.

Hepatic microsomal cytochrome P-450 monooxygenase activities were investigated in rainbow trout during an annual reproductive cycle. The fish were kept in tanks supplied with fresh water at a constant temperature of 10 degrees C. The daily light and darkness cycle was adjusted to follow the natural photoperiod. Sampling was performed once every month for 1 year. Higher benzo(a)pyrene-hydroxylase (or aryl hydrocarbon hydroxylase; AHH), ethoxycoumarin-O-deethylase (ECOD) and ethylmorphine-N-demethylase (END) activities and cytochrome P-450 content were found during the late stage of sexual development in rainbow trout. When monooxygenase activities were expressed on a per cytochrome P-450 basis, sex-dependent differences were observed only for AHH and ECOD activities. It was thus found that sex-dependent variations of END were closely correlated with the total amount of cytochrome P-450. The results indicate that differences exist in hepatic cytochrome P-450 isoenzyme patterns between the sexes in rainbow trout. The similarity of the annual pattern of plasma levels of oestradiol and testosterone to that of sex-dependent differences in the cytochrome P-450 monooxygenases support the contention that sex steroids play a role in regulating the cytochrome P-450 system.

17 beta-estradiol increases plasma calcitonin levels in salmonid fish.

The functional role of calcitonin in teleost fish is in question. Data on the role of calcitonin in calcium regulation are inconsistent, and while a participation in some aspects of sexual maturation has been strongly indicated, the exact function is not known. To establish if there exists a functional relationship between 17 beta-estradiol (E2) and calcitonin in salmonid species, rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), and coho salmon (Oncorhynchus kisutch) were injected ip with a single or repeated doses of E2. It is concluded that E2 treatment increases plasma calcitonin levels directly or indirectly, and that it is possible that E2 is responsible for the rise in calcitonin levels during late sexual maturation of female salmonids. In accord with earlier studies, no correlation was found between changes in calcitonin levels and free plasma calcium levels. It seems clear that changes in free plasma calcium levels are not the primary cause of the plasma calcitonin changes in teleost fish. It is possible that calcitonin is involved in mobilizing calcium or directing its mobilization by protection of certain calcium pools during vitellogenesis. However, the increase in calcitonin occurs so close to ovulation that a reproductive role other than a calcium regulatory one is likely. The possibility of transfer of calcitonin itself to the developing oocytes and a subsequent role in embryonic development must also be considered.

Isolation of turbot (Scophthalmus maximus) vitellogenin by high-performance anion-exchange chromatography.

The use of high-performance anion-exchange chromatography on a Mono Q column for isolation of a glycolipophosphoprotein, vitellogenin, from turbot plasma has been evaluated. The method is an effective, rapid one-step procedure, which gives a pure preparation of vitellogenin as assessed by electrophoresis, [32P]orthophosphate incorporation and amino acid composition.