Four types of calcitonin are produced in salmonid fish, although their functional diversity is almost unknown. To explore the significance of these isoforms, we have characterized salmon-type calcitonin (sCT) mRNAs in the rainbow trout (Oncorhynchus mykiss), and examined their tissue distribution. In addition to the previously isolated sCT-I cDNAs, two new forms of sCT cDNA were cloned from the ultimobranchial gland, and one of them (sCT-IV cDNA) was predicted to encode an N-terminal peptide of 80 amino acid residues, a putative cleavage site Lys-Arg, sCT-IV, a cleavage and amidation sequence Gly-Lys-Lys-Arg, and a C-terminal peptide of 18 amino acids. The sCT-IV precursor was 78% identical with the rainbow trout sCT-I precursors. The other cloned cDNA encoded a precursor for a novel CT, sCT-V. The sCT-V peptide was different from sCT-IV by only one amino acid residue: Val at position 8 in the latter was replaced by Met. The sCT-V precursor had 80 and 90% identity with the sCT-I and -IV precursors respectively. No cDNA clones were obtained for sCTs-II or -III.Tissue distribution of sCT-I, -IV and -V mRNAs was examined by RT-PCR and specific cleavage with restriction enzymes. An amplified fragment from sCT-I mRNA was detected not only in the ultimobranchial gland, but also in the gills, testis and ovary. RT-PCR analysis coupled to restriction digestion further revealed that sCT-IV mRNA was expressed in both the testis and the ultimobranchial gland. The expression sites of sCT-IV mRNA were localized to the Leydig cells of the testis and to the parenchymal cells of the ultimobranchial gland, by in situ hybridization histochemistry. Although the amino acid sequence of sCT-V peptide was nearly the same as that of sCT-IV, the sCT-V gene showed a much wider pattern of expression: the band amplified by RT-PCR was detected in all the tissues examined except the kidney, gills and blood cells. The sCT-V mRNA was shown to be localized in the parenchymal cells of the ultimobranchial gland, but not in other tissues at the cellular level, suggesting very low expression of sCT-V mRNA in those tissues. Our results show different patterns of tissue expression of three types of sCT genes in the rainbow trout, suggesting that sCTs-I, -IV and -V might differ in their local actions.
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M Suzuki, K Kubokawa, H Nagasawa, and A Urano
We determined the nucleotide sequences of cDNAs encoding precursors of vasotocin (VT) from two cyclostomes, the lamprey Lampetra japonica and the hagfish Eptatretus burgeri, for estimation of their phylogenetic relationships. Although only 47% similarity was found between the VT cDNAs, the predicted VT precursors of the lamprey and the hagfish were both composed of a signal peptide, VT, Gly-Lys-Arg and a neurophysin, as has been shown for precursors of vasopressin (VP) family hormones, including VP, VT and molluscan conopressin.
The central region of the lamprey neurophysin was very similar to those of previously characterized gnathostome neurophysins. Conspicuously, all the positions of 14 Cys residues were conserved in the lamprey neurophysin. The C-terminal region did not have a distinctive Leu-rich core segment, which is always found in the glycopeptide (copeptin) moiety of VP precursors. In contrast, the hagfish neurophysin showed at least two insertions and one deletion in the conserved central region including 14 Cys residues, but contained a potential N-linked glycosylation site and had a high proportion of Leu residues in the C-terminal region, like the neurophysin of another hagfish, Eptatretus stouti.
The evolutionary relationships of the precursors of VP family hormones among the lamprey, hagfish, gnathostomes and a mollusc were estimated by a maximum likelihood method. The phylogenetic tree with the highest bootstrap probability showed that the lamprey VT precursor is more closely related to the gnathostome VT and VP precursors than to the hagfish VT precursors.
M. Suzuki, S. Hyodo, M. Kobayashi, K. Aida, and A. Urano
Gonadotrophin-releasing hormone (GnRH) is considered to have an important role in the control of reproduction in salmonid fish, although we do not have any direct evidence. To clarify this problem by molecular techniques, we first determined the nucleotide sequence of the mRNA encoding the precursor of salmon-type GnRH (sGnRH) from the masu salmon, Oncorhynchus masou.
The masu salmon sGnRH precursor was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP) which was connected to sGnRH by a Gly-Lys-Arg sequence. The amino acid sequence of sGnRH and Gly-Lys-Arg were highly conserved when compared with the corresponding regions of African cichlid sGnRH and mammalian GnRH precursors. However, the GAP region was markedly divergent, with a 66% amino acid similarity to African cichlid GAP and an 8·3–15% similarity to mammalian GAPs. Northern blot analysis indicated the presence of a single mRNA species of about 600 bases in the olfactory bulb and telencephalon and in the diencephalon. The signal was more intense in the former regions.
An in-situ hybridization study further revealed that sGnRH neurones were distributed in the olfactory nerve, the ventral part of the olfactory bulb, the ventral part of the telencephalon, the lateral preoptic area and the preoptic nucleus. The sGnRH neurones were thus longitudinally scattered between the olfactory nerve and the lateral preoptic area in the rostroventral part of brain. The intensity of the hybridization signals and the size of hybridization-positive somata were much greater in the olfactory nerve and the rostral olfactory bulb than in the other regions. Preoptic sGnRH neurones were scarcely detected in immature masu salmon, whereas they were more frequently observed in maturing animals. It is possible that the olfactory and the preoptic sGnRH neurones have different physiological roles in salmonid fish.
M Ashihara, M Suzuki, K Kubokawa, Y Yoshiura, M Kobayashi, A Urano, and K Aida
Salmon gonadotropin-releasing hormone (sGnRH) is considered to have an important role in the control of reproduction in salmonid fish. As a basis for understanding the physiological functioning of sGnRH at the molecular level, we characterized the nucleotide sequences of two types of cDNAs encoding the precursors of sGnRH in sockeye salmon (ss), Oncorhynchus nerka, by a cloning strategy based on reverse transcription-PCR. The two types of cDNAs are referred to as ss-pro-sGnRH-I and -II, and consisted of 435 and 481 bases respectively. Both precursors are predicted to contain a signal peptide, the hormone and a GnRH-associated peptide that is attached to the hormone via a Gly-Lys-Arg sequence. The presence of two types of mRNAs hybridizing with either cDNA was confirmed by Northern blot analysis of brain RNA from sockeye salmon, masu salmon, O. masou, and rainbow trout, O. mykiss. The ss-pro-sGnRH-I cDNA had 97·2% and 82·8% overall identity with sGnRH cDNA from masu salmon and putative sGnRH cDNA deduced from the gene of the Atlantic salmon, Salmo salar respectively, whereas the ss-pro-sGnRH-II cDNA had 80·0% and 91·2% overall identity with the former and the latter respectively. The nucleotide sequences of ss-sGnRH-I and -II cDNAs showed less similarity (79·3%). These results indicated that each salmonid species possesses two differing sGnRH genes. The results of Southern blot analysis using genomic DNA extracted from individuals support this evidence in sockeye salmon, masu salmon and rainbow trout.
M Tanaka, M Suzuki, T Kawana, M Segawa, M Yoshikawa, M Mori, M Kobayashi, N Nakai, and T R Saito
In addition to the known four alternative first exons E11, E12, E13 and E14 of the rat prolactin receptor (PRL-R) gene, a novel first exon, E15, was identified by cDNA cloning of the 5′-end region of PRL-R mRNA in the rat liver. Genomic fragments containing E15 and its 5′- or 3′-flanking regions were also cloned from rat kidney genomic DNA. A sequence search for E15 revealed that E15 is located 49 kb upstream of exon 2 of the PRL-R gene in rat chromosome 2q16. RT-PCR analysis revealed that E15 was preferentially expressed in the liver, brain and kidney. Expression profiles of E12-, E13- and E15-PRL-R mRNAs in the liver of male and female rats at 5 days of age and those at 8 weeks of age were examined by RT-PCR. The levels of E12-PRL-R mRNA in the female rat increased remarkably in rats at 8 weeks of age compared with those at 5 days of age, and the levels of E15-PRL-R mRNA in the male rat decreased markedly at 8 weeks of age compared with those at 5 days of age. In the female rat, the levels of E12-PRL-R mRNA at 8 weeks of age decreased with ovariectomy performed at 4 weeks of age and recovered with the administration of β-oestradiol. On the contrary, the levels of E15-PRL-R mRNA increased with ovariectomy and decreased with the oestrogen treatment. In the male rat liver, the levels of E12-PRL-R mRNA at 8 weeks of age increased strikingly with castration performed at 4 weeks of age and became undetectable with the administration of testosterone. The levels of E15-PRL-R mRNA increased slightly with castration and were restored by testosterone treatment. Removal of gonadal tissues and sex steroid hormone treatment had no effect on the expression levels of E13-PRL-R mRNA in both female and male rat livers. These results indicated that the expression of the PRL-R gene in the liver is regulated by the differential effects of sex steroid hormones on the transcription of the multiple first exons including the novel one.
S Miyagawa, A Suzuki, Y Katsu, M Kobayashi, M Goto, H Handa, H Watanabe, and T Iguchi
Developmental exposure to a synthetic estrogen, diethylstilbestrol (DES), induces carcinogenesis in human and laboratory animals. In mice, neonatal DES treatment induces persistent proliferation and keratinization of the vaginal epithelium, even in the absence of the ovaries, resulting in cancerous lesions later in life. To understand the mechanisms underlying this persistent cell proliferation and differentiation, we characterized the gene expression patterns in the neonatally DES-exposed mouse vagina using DNA microarray and real-time quantitative RT-PCR. We found that genes related to cellular signaling, which are candidates for mediating the persistent proliferation and differentiation, were altered, and genes related to the immune system were decreased in the neonatally DES-exposed mouse vagina. We also noted high expression of interleukin-1 (IL-1)-related genes accompanied by phosphorylation of JNK1. In addition, expression IGF-I and its binding proteins was modulated and led to phosphorylation of IGF-I receptor and Akt, which is one of the downstream factors of IGF-I signaling. This led us to characterize the expression as well as the phosphorylation status of IL-1 and IGF-I signaling pathway components which may activate the phosphorylation cascade in the vagina of mice exposed neonatally to DES. These findings give insight into persistent activation in the vagina of mice exposed neonatally to DES.
H Watanabe, A Suzuki, M Kobayashi, E Takahashi, M Itamoto, DB Lubahn, H Handa, and T Iguchi
In order to understand early events caused by estrogen in vivo, temporal uterine gene expression profiles at early stages were examined using DNA microarray analysis. Ovariectomized mice were exposed to 17beta-estradiol and the temporal mRNA expression changes of ten thousand various genes were analyzed. Clustering analysis revealed that there are at least two phases of gene activation during the period up to six hours. One involved immediate-early genes, which included certain transcription factors and growth factors as well as oncogenes. The other involved early-late genes, which included genes related to RNA and protein synthesis. In clusters of down-regulated genes, transcription factors, proteases, apoptosis and cell cycle genes were found. These hormone-inducible genes were not induced in estrogen receptor (ER) alpha knockout mice. Although expression of ERbeta is known in the uterus, these findings indicate the importance of ERalpha in the changes in gene expression in the uterus.
K. Ichikawa, K. Hashizume, Y. Nishii, T. Takeda, M. Kobayashi, S. Suzuki, and T. Yamada
Human thyroid hormone receptor (c-erb A protein) produced by Escherichia coli expression vector plasmid was purified sequentially using polyethylenimine precipitation of DNA, hydroxylapatite column chromatography, ammonium sulphate precipitation, Sephacryl S-300 gel filtration and mono Q-Sepharose column chromatography. These column procedures resulted in 41.3-fold purification of 3,5,3′-tri-iodo-l-thyronine (T3) binding activity over the initial E. coli extract. Purified protein as well as crude preparation showed high-affinity binding to T3. The c-erb A protein enriched by column purification was further purified by electroelution after electrophoresis. Rabbit antibody against the c-erb A protein was prepared and used for the Western blotting analysis. The antibody recognized c-erb A protein but not the bacterial proteins in crude E. coli extract. When partially purified rat hepatic nuclear thyroid hormone receptor was analysed, a 56kDa receptor was specifically recognized by the antibody.
H Watanabe, A Suzuki, M Goto, DB Lubahn, H Handa, and T Iguchi
Alkylphenols perturb the endocrine system and are considered to have weak estrogenic activities. Although it is known that nonylphenol can bind weakly to the estrogen receptor, it is unclear whether all reported effects of nonylphenol are attributable to its estrogen receptor-binding activity. In order to examine whether alkylphenols have similar effects to the natural hormone, estradiol, we used a mouse model to examine the effects of nonylphenol on gene expression and compared it with estradiol. DNA microarray analysis revealed that, in the uterus, most of the genes activated by this alkylphenol at a high dose (50 mg/kg) were also activated by estradiol. At lower doses, nonylphenol (0.5 mg/kg and 5 mg/kg) had little effect on the genes that were activated by estradiol. Thus, we concluded that the effects of nonylphenol at a high dose (50 mg/kg) were very similar to estradiol in uterine tissue. Moreover, since evaluation of estrogenic activity by gene expression levels was comparable with the uterotrophic assay, it indicated that analysis of gene expression profiles can predict the estrogenic activities of chemicals. In contrast to the similar effects of nonylphenol and estradiol observed in the uterus, in the liver, gene expression was more markedly affected by nonylphenol than by estradiol. This indicated that, in the liver, nonylphenol could activate another set of genes that are distinct from estrogen-responsive genes. These results indicated that nonylphenol has very similar effects to estradiol on gene expression in uterine but not in liver tissue, indicating that tissue-specific effects should be considered in order to elucidate the distinct effects of alkylphenols.
H Watanabe, A Suzuki, M Kobayashi, DB Lubahn, H Handa, and T Iguchi
Administration of physiological and non-physiological estrogens during pregnancy or after birth is known to have adverse effects on the development of the reproductive tract and other organs. Although it is believed that both estrogens have similar effects on gene expression, this view has not been tested systematically. To compare the effects of physiological (estradiol; E2) and non-physiological (diethylstilbestrol; DES) estrogens, we used DNA microarray analysis to examine the uterine gene expression patterns induced by the two estrogens. Although E2 and DES induced many genes to respond in the same way, different groups of genes showed varying levels of maximal activities to each estrogen, resulting in different dose-response patterns. Thus, each estrogen has a distinct effect on uterine gene expression. The genes were classified into clusters according to their dose-responses to the two estrogens. Of the eight clusters, only two correlated well with the uterotropic effect of different doses of E2. One of these clusters contained genes that were upregulated by E2, which included genes encoding several stress proteins and transcription factors. The other cluster contained genes that were downregulated by E2, including genes related to metabolism, transcription and detoxification processes. The expression of these genes in estrogen receptor-deficient mice was not affected by E2 treatment, indicating that these genes are affected by the E2-bound estrogen receptor. Thus, of the many genes that are affected by estrogen, it was suggested that only a small number are directly involved in the uterotropic effects of estrogen treatment.