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.     

In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA.

Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.     

Gonadal development and associated changes in liver size and sexual steroids during the reproductive cycle of captive male and female Atlantic cod (Gadus morhua L.)

Gametogenesis in female and male Atlantic cod (Gadus morhua L.) was investigated by sampling blood plasma and gonadal tissue from 19 to 33-month-old fish. The reproductive cycles of both female and male Atlantic cod are characterized by distinct annual variations in gonadal size and developmental stage and these are associated with changes in sex steroids and liver size. I(H) did not change during early gonadal development, but both spent females and males had lower I(H) than late maturing females and spermiating males, respectively. In females I(G) was correlated to plasma E2 levels and they were highest in spawning females. The lowest levels during the reproductive cycle were observed in spent females. Plasma T levels were low throughout ovarian development, and were at a minimum in spent females. 11-ketotestosterone in plasma of males increased rapidly during spermiation, while T increased at earlier testicular stages and reached maximum during spermiation. High plasma levels of steroids in male and female cod during spawning serve to promote further development and growth of less advanced stages of germ cells.     

Immunological identification of two female-specific proteins from the plasma of Indian freshwater murrel,Channa punctatus (Bloch)

Existence of a non-phosphorylated female-specific protein (FS II), in addition to phosphorylated vitellogenin (FS I), in the plasma of murrel by exogenous administration of estradiol-17beta is reported. Polyspecific rabbit antibodies were raised against estrogen-inducible murrel plasma proteins. This antiserum was absorbed with normal male serum in order to obtain female-specific antiserum (FSAS). Radial immunodiffusion studies suggested that both the proteins (FS I and FS II) were present in the plasma of E2-treated and normal vitellogenic females and in the ovarian homogenate from gravid females, but absent in normal male plasma. Autoradiographic experiments demonstrated that phosphorus moiety was attached with FS I only. Further, immunoelectrophoretic analysis and peptide maps supported the observation that FS I and FS II were discrete, unrelated female-specific proteins.     

Gonadal morphology, enzyme histochemistry and plasma steroid levels during the annual reproductive cycle of male and female brown bullhead catfish, Ictalurus nebulosus Lesueur

The annual reproductive cycle of the brown bullhead catfish, Ictalurus nebulosus Lesueur, was investigated over a two-year period. In females, GSI increased in the spring as follicles enlarged and the granulosa became hypertrophied, dropped during spawning in August, then rose in the autumn as follicles enlarged slightly. 3β-Hydroxysteroid dehydrogenase (3β-HSD) activity was limited to thecal nests of large, vitellogenic follicles. Plasma testosterone and estradiol-17β levels increased in parallel with GSI. Levels of both steroids dropped prior to the spawning period, although a peak in estradiol-17β was evident during the spawning period. No 11-ketotestosterone was detected in female plasma. In males, GSI increased in the spring as spermatogenesis proceeded, and dropped during spawning. 3β-HSD activity was confined to Leydig cells and was most intense prior to spawning. Plasma testosterone and 11-ketotestosterone peaked during the pre-spawning period, dropped prior to spawning, then rose slowly during the autumn. A peak in estradiol-17β occurred during the spawning period. Significant differences in GSI and plasma steroid levels during the pre-spawning and spawning periods were observed between the two yearly cycles; they may be related to differences in rainfall during these periods.     

Regulation of vitellogenesis in Nereis virens (Annelida: Polychaeta): effect of estradiol-17beta on eleocytes

In the marine polychaete Nereis virens, the yolk protein precursor vitellogenin (Vg) is synthesized in specialized coelomic cells (eleocytes) during oogenesis. This process was visualized by immunohistochemistry using antibodies raised against the yolk protein. Transversal sections from male and female worms confirmed that eleocytes from females but not from males produce Vg. In order to investigate the hormonal regulation of Vg synthesis, eleocytes were incubated in vitro with estradiol-17beta (E(2)) at a concentration of 1 microg/l for up to three days. A strong increase in Vg secretion was detected by ELISA in culture media of treated eleocytes from vitellogenic females. In contrast, no response to the hormonal treatment was detectable in immature worms. Our results showed that Vg synthesis is under a complex regulation, which involves endocrine factors like estrogens. The role of E(2) in vitellogenesis of N. virens rather resembles the situation found in vertebrate than the one in insects.     

Distribution of calcium,magnesium and inorganic phosphate in plasma of estradiol-17β treated rainbow trout

Summary Freshwater rainbow trout,Salmo gairdneri, were injected with different doses of estradiol-17 in order to induce the synthesis of a protein, regarded as identical to vitellogenin. The plasma levels of free and protein-bound calcium, magnesium and inorganic phosphate were studied in control and estradiol-17 treated fish, using an ultrafiltration method. Estradiol-17 caused a dose-dependent increase in plasma vitellogenin levels, which strongly correlated to protein-bound levels of calcium and magnesium in plasma. Calcium and magnesium were bound to vitellogenin in a ratio of 9:1, which was considerably higer than the protein-binding ratio of these ions in normal plasma (5.2:1). The dose-dependent increase in total plasma levels of calcium, magnesium and inorganic phosphate during estradiol-17 treatment was solely due to an increase in the protein-bound fraction of these ions. It is concluded that the physiologically important plasma levels of free calcium, magnesium and inorganic phosphate are effectively regulated at normal levels during vitellogenin synthesis.     

Effects of 2,2'4,4'5,5'-hexachlorobiphenyl (PCB 153) on plasma sex steroids and vitellogenin in rockfish (Sebastes schlegeli)

We examined the estrogenic effect of 2,2'4,4'5,5'-hexachlorobiphenyl (PCB 153) on the rockfish, Sebastes schlegeli. We measured levels of plasma estradiol-17beta (E(2)) and testosterone (T) by radioimmunoassay (RIA). Plasma concentrations of T and 17alpha-hydroxyprogesterone in female and male fish injected with PCB 153 using two dosages (0.16 mg/kg body weight. and 0.57 mg/kg) did not differ significantly between sexes or from sham-injected controls of the same sex. Plasma concentrations of E(2) in females injected with PCB 153 (both levels) increased at 12 and 24 h. Concentrations of plasma vitellogenin (VTG) in females increased 72 h after injection with PCB 153 and reached 0.38 and 1.25 mg/mL, respectively. No VTG was detected in males injected with the same dosage. These results suggest that PCB 153 may lead to the production VTG in female rockfish through a synergistic effect with E(2), resulting in indirect disruption of the aromatization process.     

Development and validation of chemiluminescent immunoassay for vitellogenin in five salmonid species.

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of vitellogenin (Vg) in five salmonids. The CLIA for salmon Vg was performed using the two-site method, with anti-masu salmon beta'-component as primary antibody and chemiluminescent acridinium-labeled anti-rainbow trout lipovitellin F(ab)'(2) as the second antibody. Using cutthroat trout Vg as the standard, the working range of the CLIA was from 60 pg to 500 ng Vg/ml. Intra- and inter-assay coefficients of variation ranged from 3.04 to 6.67% and 3.23 to 5.86%, respectively. For the various salmonid species, serially diluted samples of serum from vitellogenic fish ran parallel to their purified Vg standard curve in the CLIA. In male cutthroat trout maturing during the 4 months before spawning, serum Vg levels ranged from 1.56 to 8000 ng/ml. High levels of Vg in some individuals may have resulted from temporary elevation of estradiol-17beta levels in the same fish during December or January (1-2 months before spawning). This is the first report on changes in serum Vg levels in maturing male trout using CLIA, the most sensitive assay for Vg yet developed.     

Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta)

Vitellogenin synthesis is induced in juvenile rainbow trout (Salmo gairdneri) and juvenile sea trout (Salmo trutta) by estradiol-17 beta. A purification procedure for vitellogenin from trout plasma by precipitation with MgCl2-EDTA and subsequent anion exchange chromatography on DEAE-Sephacel is described. The total lipid contents of purified rainbow trout and sea trout vitellogenins are 18 and 19%, respectively. Approximately 2/3 of the lipids are phospholipids, while the remainder consists of triglycerides and cholesterol. Phosphorus determinations on delipidated vitellogenin yield a phosphorus content of 0.63% in rainbow trout and 0.58% in sea trout vitellogenin. Native (dimeric) vitellogenins from rainbow trout and sea trout both have an apparent molecular weight of 440,000, when estimated by gel filtration on Sepharose 6B.     

Changes in reproductive parameters during the spawning migration of pink salmon, Oncorhynchus gorbuscha (Walbaum)

The hormones 17β-estradiol, 17α-hydroxy-20β-dihydroprogesterone(17α, 20β-P), 11-ketotestosterone, testosterone, gonadotropin and also vitellogenin, were determined during the spawning migration of wild pink salmon in the Fraser and Thompson Rivers in British Columbia. This stock of pink salmon takes approximately 2 weeks to migrate the 333 km upstream to the spawning grounds. Both sexes were at an advanced stage of sexual development when they entered fresh water. In females both the 17β-estradiol and vitellogenin levels fell precipitously during the migration, to be very low at spawning, whereas the 17α,20β-P level rose rapidly, to be highest at arrival on the spawning grounds. The gonadotropin level also rose rapidly during the migration, and was highest in spent fish. Testosterone was at a high level throughout, although this level decreased steadily during migration. In many respects similar endocrine changes were observed in the male. For example, in the case of androgen levels, both testosterone and 11-ketotestosterone fell steadily during migration but were still relatively high at spawning, whereas both gonadotropin and 17α, 20β-P levels rose markedly as migration progress. However, although the qualitative changes were often similar between the sexes, the levels of 17α, 20β-P, testosterone, and gonadotropin were considerably higher throughout in females than in males. It is concluded that this stock of pink salmon is at an advanced stage of sexual development when it enters fresh water. The endocrine changes observed during this study represent those controlling the final stages of reproduction, specifically final oocyte maturation and ovulation in females, and the final stages of spermatogenesis and spermiation in males.     

Characterization of estrogen binding sites in the liver of the newt Pleurodeles waltl (Urodela:Amphibia): demonstration of a sex-linked heterogeneity

Saturation analysis over a wide range of [3H]estradiol-17 beta concentrations (1-40 nM) in cytosols prepared from liver of the newt Pleurodeles waltl of both sexes revealed a sex-linked heterogeneity of the estradiol-17 beta binding sites. In females, one type of binding site has been identified as a classical receptor. It exhibited a high affinity for estradiol-17 beta (Kd = 9 X 10(-9) M), had a high specificity for estrogenic compounds and was stabilized by monothioglycerol. In males, in addition to the receptor found in females, a second estrogen binding component was detected, not observed in female cytosols. It exhibited a Kd of 4.8 X 10(-8) M for estradiol 17 beta, higher capacity and displayed the same highly specific estrogen binding as does the estrogen receptor. It was affected by monothioglycerol and its binding was found to be significantly increased on cytosol dilution, as well as by estrogen-treatment.     

A biochemical method for distinguishing between the sexes of fishes by the presence of yolk protein in the blood

A method is described by which the adult females of marine and freshwater teleosts can be distinguished by a biochemical test performed on a blood sample. The test depends on the measurement in the blood plasma of alkali-labile protein-linked phosphorus, a specific measure of yolk protein (vitellogenin). In vitellogenic females, values of 20–100 μg protein phosphorus ml plasma⁻¹ are usually found, while in males, non-vitellogenic females and immature fish of both sexes the value is <7.5 and usually <5 μg ml⁻¹. At the appropriate season, most females can be positively identified. Good results can be obtained 2–3 months before spawning, and in some species for an undefined period after spawning. The advantages of this method over immunological techniques for the determination of vitellogenin, such as radioimmunoassay and immunoelectrophoresis, are the wide range of vertebrate species to which it can be applied, and its low cost. Its disadvantages are its lower sensitivity and the larger volume of plasma (0.5 ml) which must be used.     

Thyroid hormones are corequisites for estradiol-17β in vitro induction of Xenopus vitellogenin synthesis and secretion

Estradiol-17β administered to male frogs induces liver synthesis and secretion of vitellogenin, the precursor protein of the major egg yolk proteins. Estradiol-17β alone failed to induce this protein in cultures of liver tissue maintained for 1–2 weeks prior to addition of the hormone. If a “complex” defined culture medium, such as Coon's modified Ham's F12 medium, is used, efficient primary and secondary induction of vitellogenin synthesis and secretion occurs in the presence of estradiol-17β, triiodothyronine, and dexamethasone. Using Coon's medium we investigated the role of both triiodothyronine and dexamethasone as corequisites of estradiol-17β induction of secreted vitellogenin. Control cultures given no hormones showed a gradual decrease in the level of secreted albumin and fibrinogen. Addition of dexamethasone, alone, induced increased synthesis of secreted albumin and fibrinogen as well as other proteins. Cultures given thyroid hormones, alone, showed an increased level of secreted albumin and fibrinogen at early time points in the culture period. Thus, at early times thyroid hormones appear to enhance the activity of endogenous glucocorticoids. Independent of their interaction with glucocorticoids, thyroid hormones also enhance the activity of estrogens. Long-term cultures given estradiol-17β, alone, failed to synthesize and secrete vitellogenin. In contrast, cultures given the estrogen together with thyroid hormones showed vitellogenin synthesis. These results imply that similar interactions of several hormones occur in vivo in adult animals treated with estrogens. In the accompanying paper the interaction of dexamethasone with estradiol-17β and triiodothyronine is described (L. J. Wangh, 1982, Develop. Biol.89, 294–298).     

Development and Application of an Effective Detection Method for Fish Plasma Vitellogenin Induced by Environmental Estrogens

Vitellogenin is a protein induced by estrogens, including environmental chemicals with estrogenic activity. To measure the effects of environmental estogens, we developed an effective and rapid one-step method of detecting and purifying fish plasma vitellogenin using a high-performance anion-exchange chromatography column, POROS-HQ. Vitellogenin in a plasma of estradiol-treated male fish (mummichog and red sea bream) was eluted as a single peak with a retention time of 10 minutes from the column, which gives an almost pure preparation as assessed by SDS-PAGE. The lowest detectable amount of vitellogenin was 2 μg per assay. The method was used to analyze the plasma vitellogenin level of aquacultured red sea breams caught in August, when the spawning season is over, and usually no vitellogenin is detected in either females or males, physiologically. However, the data showed that in addition to a few females, some male fish synthesized vitellogenin, suggesting that some chemicals or unknown factors with estrogenic activity have induced fish in the ocean to produce vitellogenin.     

Validation of radioimmunoassay systems for the measurement of 11-keto- and 11 beta-hydroxytestosterone in teleost blood

Highly specific antisera for 11-keto- and 11 beta-hydroxytestosterone have been raised in sheep. Assay systems for the simultaneous measurement of 11-ketotestosterone, 11 beta-hydroxytestosterone, testosterone, progesterone and estradiol-17 beta were validated for Ictalurus nebulosus plasma and Carassius auratus serum. In males of both species 11-ketotestosterone and testosterone were the major steroids detected. In females, testosterone and estradiol-17 beta were the predominant steroids measured. Data from samples taken at different stages of the annual cycle suggest that seasonal fluctuations in gonadal steroid secretion occur in I. nebulosus and C. auratus.     

Ultrastructure and lipid content of the liver of the zebrafish,Brachydanio rerio,related to vitellogenin synthesis

The female zebrafish is capable of producing mature eggs on the fifth day of each reproductive cycle. During this five-day period the ultrastructure of hepatocytes undergoes several changes. The number of nuclear pores increases rapidly during spawning, followed by a proliferation of RER within 24 h. Two days after spawning, glycogen has disappeared and the liver contains large amounts of lipids. The lipid droplets are closely surrounded by elongated mitochondria. Golgi complexes are abundant, secreting dense bodies. Four days after spawning the hepatocytes tend to regain their pre-spawning appearance. It is suggested that the changes in the hepatocytes, which coincide with special phases of ovarian activity, are related to vitellogenin synthesis. Steroids, especially estradiol-17beta, may trigger this process in the liver.     

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.     

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. lmmunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 μm. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol-17β increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 μm) present just inside the oolemma, when the oocytes reached a diameter of 600 μm. 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 17β levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 μm had an immunoreactive vitelline envelope with a thickness of about 3 μm. 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-17β observed in females are of physiological significance. © 1994 Wiley-Liss, Inc.