Interrelationship between signal transduction pathways and 1,25(OH)2D3 in UMR106 osteoblastic cells.

In this study, the interrelationship between signal transduction pathways and 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] action was examined in UMR106 osteoblastic cells. Treatment of these cells with 8-bromo-cAMP (1 mM) resulted in an upregulation of the vitamin D receptor (VDR) and an augmentation in the induction by 1,25(OH)2D3 of 25(OH)D3 24-hydroxylase [24(OH)ase] and osteopontin (OPN) mRNAs as well as gene transcription. Transfection with constructs containing the vitamin D response element devoid of other promoter regulatory elements did not alter the cAMP-mediated potentiation, suggesting that cAMP-enhanced transcription is due, at least in part, to upregulation of VDR. Treatment with phorbol ester [12-O-tetradecanoyl-phorbol-13-acetate (TPA) 100 nM], an activator of protein kinase C, significantly enhanced 1,25(OH)2D3-induced OPN mRNA and transcription but had no effect on VDR or on 24(OH)ase mRNA or transcription. Studies using OPN promoter constructs indicate that TPA-enhanced OPN transcription is mediated by an effect on the OPN promoter separate from an effect on VDR. Thus interactions with signal transduction pathways can enhance 1,25(OH)2D3 induction of 24(OH)ase and OPN gene expression, and, through different mechanisms, changes in cellular phosphorylation may play a significant role in determining the effectiveness of 1,25(OH)2D3 on transcriptional control in cells expressing skeletal phenotypic properties.

Expression of three vitelline envelope protein genes in arctic char.

Several studies have shown effects of estrogenic substances on endocrine and reproductive systems in wildlife. Measurement of plasma vitellogenin (VTG) is a commonly used method to determine exposure to estrogenic substances in fish. There is, however, a growing need for additional sensitive and accurate methods to detect estrogenic substances in vivo. The vitelline envelope proteins (VEPs) have been suggested, in other studies, as suitable biomarkers for estrogenic substances. The present study investigates the induction of VEPs in juvenile Arctic char (Salvelinus alpinus). The results demonstrate that VEP mRNA exhibits earlier induction than estrogen receptor mRNA or VTG mRNA following injection of juvenile Arctic char with a single dose of 17beta-estradiol (E2; 10 mg/kg bw). These results indicate that the VEPs have a higher sensitivity for E2 than VTG. However, an early and sex-independent expression of VEPbeta in estrogen-unchallenged juvenile Arctic char was observed. These findings suggests that the regulatory mechanisms of VEPs might be more complex than previously thought, which in turn may have implications for the usage of VEPs as biomarkers for xenoestrogen exposure.

Cloning of rainbow trout egg envelope proteins: members of a unique group of structural proteins.

All vertebrate eggs are surrounded by an extracellular envelope that protects the egg and is vital for a successful fertilization. The terminology and functions of the egg envelope vary in different vertebrate groups, but the envelope itself is consistently composed of a few major proteins that are deposited around the oocyte during oocyte growth. Here, we describe the deduced amino acid sequences and tissue expression patterns of the three major egg envelope proteins for rainbow trout (Oncorhynchus mykiss). All three vitelline envelope proteins (VEPs) are expressed in the livers of both male and female fish, with higher expression in females. In addition, VEPgamma mRNA is also detected in the female gonads. To our knowledge, this is the first time that expression of a VEP protein gene has been demonstrated to occur in more than one organ. Sequence comparison reveals that all three VEP proteins share distinct homology with their amphibian, avian, and mammalian counterparts. Whereas mammalian zona pellucida protein 3 isoforms contain two conserved serines needed for sperm binding, these are not conserved in teleost species, in which sperm entry is restricted to the micropyle. Besides the difference in VEPgamma sperm-binding function, the high sequence homology suggests that the egg envelope proteins from these distinct vertebrate groups share a common ancestry and form a unique group of structural proteins.

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.

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.

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.

Differences in metallothionein gene expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line.

Primary cultures of rainbow trout, Salmo gairdneri, hepatocytes were used to study the expression of metallothionein (MT) genes in response to steroid hormone treatment. The expression pattern was compared to that of an immortal cell line (RTH-149). MT mRNA accumulated in both cell cultures after exposure to zinc while 17 beta-oestradiol had no effect in either system. Treatment with cortisol and corticosterone resulted in a 2-fold increase of metallothionein mRNA levels in the primary cultures but had no effect in the RTH-149 cell culture. Primary cultures that were exposed to zinc or cortisol showed a high temporal correlation (r = 0.974) between MT mRNA and MT protein levels. The basal level expression was 3-4-fold higher in primary cultures than in RTH-149 cells. The present study demonstrates the inducibility of rainbow trout MT genes in response to glucocorticoids. It further indicates that primary cultures are to be preferred to immortal cell lines when investigating the inducibility of MT mRNA.

Cortisol induction of metallothionein in primary culture of rainbow trout hepatocytes.

In the present experiment, metabolically active primary cultures of rainbow trout hepatocytes were utilized to study the inducibility of metallothionein after cortisol treatment. Glucocorticoid induction of metallothionein has previously only been demonstrated in mammalian systems. We now demonstrate the inducibility of rainbow trout metallothionein by cortisol treatment of primary hepatocytes. A 90% elevation above control levels was achieved within 8 days of treatment. Zinc treatment was performed to evaluate the system, and 100 microns zinc in the culture medium resulted in a 350% increase of the metallothionein levels. We conclude that primary culture of rainbow trout hepatocytes constitutes an efficient system for studies of MT induction kinetics.