Synthesis of vitellogenin in cultures of male and female frog liver regulated by estradiol treatment in vitro.

Using the frog Xenopus laevis, we show that the addition of physiological concentrations of estradiol to cultures of liver from untreated males rapidly induces the synthesis of large amounts of vitellogenin. Sustained synthesis of vitellogenin requires continuous exposure to estradiol. A nonestrogenic steroid, dexamethasone, does not induce vitellogenin synthesis but does induce increased synthesis of a different protein in liver cultures.     

Gonadotropin stimulation of estrogen and yolk precursor synthesis in juvenile rainbow trout

Radioimmunoassays for vitellogenin and estradiol were used to examine the response of immature rainbow trout (Salmo gairdneri) to pituitary gonadotropic preparations. Vitellogenin was induced into serum of both male and female fish by two injections of estradiol. Repeated injection of an extract of salmon, Oncorhynchus tshawytscha, pituitaries (PE) induced a time-dependent increase in both estradiol and vitellogenin. Low concentrations of serum vitellogenin were found in all control groups but a 16-day treatment with PE significantly elevated serum estradiol and vitellogenin concentrations which were positively correlated. A longer term experiment resulted in higher vitellogenin concentration in the serum of trout treated with PE or the glycoprotein fraction of pituitaries prepared with concanavalin A-Sepharose. Treatment for a short time with a high dose of the fraction of the pituitary extract which had no affinity to Con A-Sepharose resulted in a slight stimulation of vitellogenin synthesis but concurrent treatment with antibody to the glycoprotein gonadotropin eliminated this. These results show that the carbohydrate-rich gonadotropin stimulates vitellogenin synthesis but the carbohydrate-poor gonadotropin does not, and support a hypothesis of action by two gonadotropins in regulation of teleost vitellogenesis.     

Proteins associated with poly(A)+RNA of cockerel liver: effects of estradiol stimulation.

The protein population of poly(A)-containing messenger ribonucleoprotein (mRNP) from cockerel liver was analyzed before and after estradiol induction. Polysomal and free mRNP were isolated by thermal elution from oligo(dT)-cellulose. The proteins were separated by two-dimensional electrophoresis (of which the first dimension was a nonequilibrium pH gradient) and were visualized by silver staining. In order to determine similarities and differences between proteins of various mRNP fractions, trace amounts of 125I-labeled protein from one fraction were coelectrophoresed with stainable amounts of protein from another fraction on the same gel. Of 27 proteins analyzed, 15 were common to polysomal and free mRNP, 7 were specific to polysomal mRNP, and 5 were specific to free mRNP. Moreover, estradiol strongly influenced 11 proteins. Six proteins changed either in relative intensity or in distribution between polysomal and free mRNP fractions. Three major proteins appeared in both fractions, and two additional proteins disappeared from the free mRNP fraction after estradiol treatment. The results suggest that the protein population in polysomal mRNP is quite complex and that the profound influence of estradiol on protein synthesis in cockerel liver may be connected to changes in the protein composition of mRNP.     

Coordination of ribosome content and polysome formation during estradiol stimulation of vitellogenin synthesis in immature male chick livers.

To elucidate the mechanisms by which protein synthesis is affected by estradiol, we characterized cockerel liver polysomal profiles during hormone induction and withdrawal. We describe a method for isolating intact polysomes which results in preparations that are stable even after storage in solution at 10 degrees for 16 hr. In addition, our procedure eliminates the necessity for starving animals prior to experiments. Recovery of radioactive polysomes indicated that yield is about 90% and that our polysomal preparations appear to represent polysome distribution in vivo. Using this approach we show that estradiol injection stimulates ribosome content 6-fold and that formation of polysomes is coincident with the induction of vitellogenin synthesis. We also demonstrate that the size and number of polysomes increase and decrease in a coordinated fashion with the rate of vitellogenin synthesis. The kinetics of ribosome synthesis and the fact that at least 80% of the newly synthesized ribosomes are directly recruited into polysomes indicate that ribosomes might be limiting the rate of protein synthesis during the stimulatory phase of the hormone cycle.     

Induction of vitellogenin synthesis in goldfish by massive doses of androgens.

Oral administration of massive doses of methyltestosterone into goldfish caused an extensive proliferation of the rough endoplasmic reticulum, hypertrophy of the Golgi apparatus, and the production of numerous secretory granules in the liver, suggesting the induced synthesis of some secretory proteins. The electrophoretic pattern of serum proteins was prominently altered, showing three bands which were absent in normal, immature fish. All of these changes were similarly observed in estradiol-treated fish. In order to identify the hormone-induced serum proteins, they were purified from estradiol- and methyltestosterone-treated fish and were subjected to chemical and immunological analyses. The results indicated that the serum proteins induced by methyltestosterone and by estradiol are identical in the contents of lipids, phospholipids, calcium, and protein-bound phosphorus and the reactivity to antiserum elicited to the estradiol-induced serum proteins. Furthermore, it was found that the antibody forms a single, connecting precipitin line with the hormone-induced proteins, sera of vitellogenic fish, and ovary extracts of matured fish, but not with sera of normal males. In view of the above findings, it was concluded that the serum proteins induced by methyltestosterone are vitellogenin. Ethynyltestosterone and methylandrostenediol were also effective, but testosterone, dihydrotestosterone, and methyldihydrotestosterone were much less effective in inducing vitellogenin synthesis.     

Isolation and identification of female-specific serum lipophosphoprotein (vitellogenin) in the catfish, Heteropneustes fossilis (Bloch)

A female-specific serum lipophosphoprotein, vitellogenin, was identified in the female catfish during vitellogenesis, and its synthesis was induced in the female or male catfish following administration of estradiol. A crude preparation of vitellogenin was isolated from the serum of estradiol-treated male or female catfish following gel filtration on Ultrogel AcA 34. Estimation of alkali-labile protein phosphorus was found to be a reliable index of the presence of vitellogenin in the serum of the catfish. Polyacrylamide gel electrophoresis study showed that a new protein band was present in the serum of estradiol-treated female catfish.     

Vitellogenin synthesis by fat body and ovary in the terrestrial isopod, Armadillidium vulgare

Vitellogenin synthesis in Armadillidium vulgare was investigated in tissue cultures. The synthesis of vitellogenin was assayed by the incorporation of [35S]methionine into precipitin with anti-vitellin serum. The forms of synthesized vitellogenin were analyzed by polyacrylamide gel electrophoresis and fluorography. The fat body synthesized vitellogenin and its rate was correlated with the molting cycle: a maximum level at stage D and lower levels at A-C and E of the molting cycle. Four forms of vitellogenin (Vg 1-4) were synthesized; the larger forms (Vg 1-2) were prominent. The ovary also synthesized a slight amount of vitellogenin at the end of the molting cycle. The smaller forms (Vg 3-4) were synthesized under cultured condition. Through vitellogenesis in A. vulgare, the fat body must be the principal site of vitellogenin synthesis. Most vitellogenin may be transported from the fat body to the ovary through hemolymph at stage D of the molting cycle.     

Translation in vitro of rat brain messenger RNA coding for tubulin and actin.

A partially purified fraction of poly(a)-rich brain mRNA coding for tubulin and actin was obtained by stepwise elution from an oligo(deoxythymidylate)-cellulose column and was efficiently translated in a wheat-germ cell-free system. The newly synthesized tubulin and actin migrated along with the authentic proteins on sodium dodecyl sulfate polyacrylamide gels and on sodium dodecyl sulfate-urea polyacrylamide gels, where tubulin alpha- and beta-subunits are separated. The two proteins synthesized in vitro were found to be biologically active; they could be induced to polymerize and were both precipitated by vinblastine. In addition, specific binding of tubulin to an affinity column of colchicine-Sepharose and actin to myosin were demonstrated.     

Hormonal control of in vitro vitellogenin synthesis in Rana esculenta liver: effects of mammalian and amphibian growth hormone.

Estradiol 17-beta is known to induce hepatic synthesis and secretion of vitellogenin in all species studied and in Rana esculenta, previous experiments demonstrated the involvement of pituitary in these processes; indeed, in addition to estradiol 17-beta, homologous pituitary homogenate directly stimulated male and female liver to produce vitellogenin in tissue cultures. Therefore, the effect of ovine growth hormone (o-GH) and Rana catesbeiana growth hormone (f-GH) on hepatic vitellogenin synthesis was investigated. In the present in vitro experiments, both o-GH and f-GH positively stimulated vitellogenin synthesis, in female and male liver, in a dose-related fashion. No significant differences were found in VTG levels induced by o-GH and f-GH. The GH stimulatory effects, found during the different phases of the reproductive cycle, displayed different trends related to season and sex.     

Estradiol-induced accumulation of vitellogenin mRNA and secretion of vitellogenin in liver cultures of Xenopus

Expiants of male Xenopus liver maintained in a serum-free culture medium respond to stimulation by 2 × 10^?8 M 17β-estradiol with an increasing rate of accumulation of vitellogenin mRNA, as revealed by hybridization of cDNA to the total cytoplasmic RNA extracted from the cultures. A similar response is observed for secretion of ³²PO₄-labeled vitellogenin into the culture medium. The in vitro response is improved in liver tissue of prestimulated animals, and by adaptation of liver expiants to the culture medium prior to hormone treatment, but attains only about 10% of the in vivo response. Since essential features of the in vivo response are maintained in liver expiants, organ culture appears suitable for investigating initial events of estradiol action leading to enhanced synthesis of vitellogenin.     

Molecular weight forms of adrenocorticotropic hormone secreted by primary cultures of mouse anterior pituitary.

Extracts of mouse anterior pituitary cells in monolayer culture were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to separate the molecular weight forms of ACTH. The gels were sliced and each segment was eluted. The eluates were assayed for ACTH immunoactivity. Approximately 10% of the immunoactivity in the extracts was found to be present as 20,000--32,000 mol wt ACTH. The remainder of the immunoactivity was equally distributed between two forms of ACTH with apparent molecular weights of 11,800 and 4,500. This distribution is very similar to that found in extracts of mouse anterior pituitary. Mouse anterior pituitary cultures were incubated for 2 h in serum-free tissue culture medium. ACTH was concentrated from the medium and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The medium was found to contain predominantly the 4,500 and 11,800 forms of ACTH. When vasopressin was added to these cultures (100 ng/ml), the rate of secretion of ACTH was more than doubled in serum free medium. Analysis of the medium from vasopressin-stimulated cultures showed that the 4,500 and 11,800 mol wt forms of ACTH were again the predominant forms present.     

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.     

Estrone and estradiol participation during exogenous vitellogenesis in the female rainbow trout, Salmo gairdneri

Ovarian steroids were treated for their ability to induce vitellogenin synthesis in the liver of the rainbow trout, Salmo gairdneri. These steroids were chosen because they had been reported to induce vitellogenin in the plasma of teleosts, i.e., estrone, estradiol, and testosterone, or because they were synthesized during vitellogenesis, as was the case with the above steroids and androstenedione and 17α-hydroxyprogesterone. Immature trout were ovariectomized and injected daily for 7 days. The administered doses of steroid were 100, 250, and 500 ng/g body wt. Only estradiol and estrone induced vitellogenin in the plasma; estrone had about 5% of the potency of estradiol. A dose-effect curve was determined for estradiol in the range from 25 to 1000 ng/g body wt. A maximal amount of 7 mg vitellogenin/ml plasma was found following the administration of a dose of 250 ng/g estradiol. Vitellogenin was not present in the plasma of animals treated with saline, nor could it be detected in the liver, not even after the administration of estradiol. Estradiol administration increased the total plasma protein concentration from 35 to maximally 51 mg/ml and decreased the total piver protein concentration from 163 to minimally 118 mg/g. The relative weight of the liver increased from 12.9% to maximally 22.4%. Vitellogenin was not detected in the liver of any of the experimental animals, indicating a low storage and rapid secretion of vitellogenin. The other steroids influenced some of the variables, but never was the total pattern of effect comparable to that of estradiol. Estradiol is found to be the ovarian steroid that physiologically regulates the synthesis of vitellogenin in the liver of the rainbow trout; estrone is less active. Experiments undertaken to determine the effects of the combined presence of estrone and estradiol revealed that estrone was capable of boosting the vitellogenic effect of estradiol when compared to the induced vitellogenin levels following treatment with a combination of testosterone and estradiol. The vitellogenin concentrations induced by a certain dose of one of the combinations of estrone and estradiol approximated the concentrations induced by the same dose of estradiol alone. This effect was independent of the ratio in which estrone and estradiol were administered. These findings could not be explained by conversions of estrone into estradiol or by the vitellogenic activity of estrone alone. The estrone, estradiol, and vitellogenin concentrations in the plasma of the experimental animals were in the same range as determined previously in untreated mature vitellogenic females. Vitellogenin and estradiol levels were found to correlate in experimental animals treated with estradiol (r = 0.627; N = 20). This was not the case in animals treated with combinations of estrone and estradiol. In these animals, however, the sum of estrone and estradiol levels in the plasma correlated with vitellogenin levels (r = 0.724; N = 42). Vitellogenin was not detected in the liver of any of the experimental animals, which indicates a low storage and rapid secretion of vitellogenin. The importance of viewing the sum of estrone and estradiol plasma levels in connection to physiological studies of the regulation of exogenous vitellogenesis in the rainbow trout is discussed.     

Identification of androgen-binding protein from testis cytosol and sertoli cell culture medium of the cynomolgus monkey, Macaca fascicularis

The biochemical and immunological properties of monkey androgen-binding protein (mABP) from the cynomolgus monkey, Macaca fascicularis, have been examined in testis cytosol and medium of primary Sertoli cell-enriched cultures. mABP in testis tissue was separated from the serum protein testosterone-estradiol-binding globulin (mTeBG) by affinity chromatography on Concanavalin A-Sepharose (Con A). mTeBG from serum extracts was completely retained by the lectin and could be displaced with buffer containing alpha-methyl-D-glucoside. In contrast, mABP from testis extracts either did not interact with the Con A and appeared in the void volume or was partially retained by the column and could be eluted with buffer alone. A third component retained by the Con A may represent mTeBG contamination, a form of mABP which binds to Con A, or both. The specific 5 alpha-dihydrotestosterone (DHT)-binding activity in the void volume of the Con A column was designated mABP and was further studied. [3H]DHT binding to mABP was saturable and of limited capacity (0.163 +/- 19 pmol/mg protein). Scatchard analysis of the data was consistent with a single class of binding sites with an apparent dissociation constant (Kd) at 4 C of 2.6 +/- 0.2 X 10(-9) M. DHT was the most effective competitor of [3H]DHT binding to mABP, followed by 2-methoxyestradiol, testosterone, estradiol, and cyproterone acetate. Concentrated Sertoli cell culture medium subjected to steady state polyacrylamide gel electrophoresis produced a single peak of specifically bound [3H]DHT with a mobility similar to that of other androgen-binding proteins. [35S]Methionine-labeled medium proteins were immunoprecipitated with a rabbit anti-human TeBG antiserum. Two bands, corresponding to mol wt of approximately 46,000 and 48,000, were observed by fluorography, with the lighter component being more intense. After androgen affinity chromatography of radiolabeled medium proteins, these two bands were again observed on sodium dodecyl sulfate-urea-containing polyacrylamide gels. These results demonstrate that 1) mABP may be separated from mTeBG by lectin affinity chromatography, as in humans; 2) hTeBG and mABP are antigenically related; and 3) mABP consists of subunits of different mol wt in unequal ratios.     

Plasma vitellogenin and 17 beta-estradiol levels during the annual reproductive cycle of Podarcis s. sicula Raf.

Plasma vitellogenin and 17 beta-estradiol concentration were determined during the annual reproductive cycle of the female lizard Podarcis s. sicula Raf. living around Naples. Plasma vitellogenin was purified from estrogenized males for characterization and to raise specific immune serum. Using ELISA, plasma vitellogenin titers were determined in relation to ovary weight; plasma 17 beta-estradiol was measured by RIA method. Native vitellogenin was present as two polypeptide bands: alpha and beta. The electrophoretic patterns, studied in normal male and estrogenized male and female, showed vitellogenin to be a protein present in female and in estrogenized male plasma but not in normal males. Lizard monomeric VTG, determined by SDS-PAGE, was about 200 kDa. Correlations between seasonal ovarian weight variations and plasma vitellogenin and 17 beta-estradiol suggest that ovarian development in Podarcis depends on plasma vitellogenin synthesis, which in turn relies on plasma estradiol levels. The two ovulatory waves observed in this study coincided with the two peak values of plasma vitellogenin and 17 beta-estradiol.     

In Vitro Synthesis of Mouse Neuroblastoma Tubulin

Polyribosomes were isolated from a clonal line of mouse neuroblastoma grown in culture. In a heterologous in vitro system containing rat brain components, these polyribosomes were shown to direct the synthesis of neuroblastoma tubulin. Identification of the tubulin synthesized in vitro was achieved by coelectrophoresis with native neuroblastoma tubulin on sodium dodecyl sulfate polyacrylamide gels, immunoprecipitation, and demonstration of specific aggregation. Tubulin accounted for 2% of the total proteins synthesized. This in vitro protein synthesizing system offers a model for studying possible translational control mechanisms regulating the synthesis of proteins involved in nerve cell function.     

Control of fibronectin synthesis by rat granulosa cells in culture

The secreted and cellular [35S]methionine-radiolabeled proteins of cultured rat granulosa cells were separated by electrophoresis on sodium dodecylsulfate (SDS) polyacrylamide gradient slab gels. From 24 to 72 h of culture FSH increased the intensity of labeling of most of the secreted proteins. A 220,000-dalton protein, however, increased in intensity only in control cultures and became the major secreted protein after 72 h, comprising 20% of the total radiolabeled proteins. This protein was identified as fibronectin by immunoprecipitation. There was no increase in the secreted or cellular fibronectin in FSH- or testosterone- and insulin-treated cultures. These studies indicate that a component of extracellular matrix is a major secretory product of unstimulated immature granulosa cells. As hormones induce the differentiated functions of granulosa cells in culture, the secretion of fibronectin is inhibited.     

Vitellogenesis in the lizard Lacerta vivipara jacquin. I. Purification and partial characterization of plasma vitellogenin

The polypeptide moiety of lizard plasma vitellogenin was reproducibly dissociated and separated by SDS-PAGE into two subunits: Vg alpha 2-2.2 X 10(5) Da and Vg beta 1-1.1 X 10(5) Da. In estrogenized females, active incorporation of inorganic 32P into both vitellogenin chains present in the plasma, and in the culture medium of the liver, was identified following autoradiography of SDS-polyacrylamide gels. Lacerta vivipara vitellogenin was isolated from pooled plasma collected from heavily estrogenized males by the two-step precipitation procedure of H. S. Wiley, L. Opresko, and R. A. Wallace, (1980, Anal. Biochem. 97, 145-152), followed by chromatography on DEAE-cellulose. This preparation of L. vivipara vitellogenin was of sufficient purity to generate in rabbits an immune serum which cross-reacted very slightly with plasma free of vitellogenin (rocket immunoelectrophoresis). Using the double immunodiffusion procedure it was shown that the anti-vitellogenin serum recognized identical antigenic determinants in plasma from a vitellogenic female or from estrogenized lizards, and in crude vitellus. The immunodetection of L. vivipara native vitellogenin consistently allowed two circulating forms to be distinguished. After autoradiography of a rocket immunoelectrophoresis plate it was demonstrated that both native forms had incorporated inorganic 32P into the polypeptide moiety and/or the phospholipid moiety.     

Rapid estrogen metabolism and vitellogenin gene expression in xenopus hepatocyte cultures

Male hepatocytes metabolized estradiol-17β, 17-ethinylestradiol and mestranol extremely rapidly (t_1/2 = 40, 60 and 300 min, respectively), whereas these were more stable in cultures of female hepatocytes (t_1/2 = 120, 150 and 640 min, respectively). Vitellogenin mRNA accumulated for only 12 h after a single addition of 10^p-6 M estradiol to male hepatocyte cultures; mestranol, but not 17-ethinylestradiol or diethylstilbestrol, was more potent than the natural hormone. The level and rate of accumulation of vitellogenin mRNA were 5–15 times higher in female than in male hepatocytes, mestranol and estradiol being more potent than 17-ethinylestradiol and diethylstilbestrol. Ovariectomy, 60 days prior to cell culture, did not alter the metabolism of estradiol or the vitellogenic response of female hepatocytes. On the other hand, a single administration of estradiol in vivo to male Xenopus caused a long-lasting shift (at least 16 weeks) to the female pattern of its metabolism, although the enhanced inducibility of vitellogenin genes was partially reversed between 4 and 16 weeks after hormonal treatment. The addition of fresh estradiol every 4 h to male hepatocyte cultures to compensate for its rapid metabolism resulted in a continuous and sustained accumulation of vitellogenin mRNA at rates comparable to those attained in vivo. Our findings explain the requirement for high levels of estrogen to activate vitellogenin genes and establish Xenopus hepatocyte cultures as a reproducible system for analysing the expression of this multigene family.