Two cytochrome P450 aromatase genes in the hermaphrodite ricefield eel Monopterus albus: mRNA expression during ovarian development and sex change

Previously, the ricefield eel (Monopterus albus) was speculated to have only one cytochrome p450 aromatase gene. In this study, however, the cDNAs encoding two distinct cytochrome p450 aromatases, cyp19a1a and cyp19a1b, were isolated. The genomic organizations of both cyp19 genes were conserved when compared with other teleosts. Northern blot detected an abundant expression of cyp19a1a in the ovary, and cyp19a1b in the hypothalamus. RT-PCR coupled with Southern blot showed that cyp19a1a was expressed predominantly in the gonads of both sexes, with higher levels in the ovary than testis, while cyp19a1b was expressed in all the tissues examined in the male, but only in the brain and pituitary in the female. The levels of cyp19a1a mRNA in the ovary were increased significantly during vitellogenesis, but decreased significantly at mature stage. The levels of cyp19a1b mRNA in the brain and pituitary did not vary significantly during vitellogenesis. As ovarian development shifted from vitellogenesis to maturation, the levels of cyp19a1b mRNA was decreased significantly in the brain, but increased significantly in the pituitary. During natural sex change from female to male, the levels of cyp19a1a mRNA in the gonad were significantly decreased. The levels of cyp19a1b mRNA in the hypothalamus were significantly increased at the early intersexual phase, whereas the expression levels in the pituitary were significantly decreased at the intersexual phases. Taken together, these results showed a novel sexual dimorphism of cyp19a1b mRNA tissue distribution, and both CYP19 genes were associated with the ovarian development and natural sex change of the ricefield eel.

High-affinity peptide transporter PEPT2 (SLC15A2) of the zebrafish Danio rerio: functional properties, genomic organization, and expression analysis

Solute carrier 15 (SLC15) membrane proteins PEPT1 (SLC15A1) and PEPT2 (SLC15A2) have been described in great detail in mammals. In contrast, information in lower vertebrates is limited. We characterized the functional properties of a novel zebrafish peptide transporter orthologous to mammalian and avian PEPT2, described its gene ( pept2) structure, and determined mRNA tissue distribution. An expressed sequence tag (EST) cDNA (Integrated Molecular Analysis of Gene Expression; IMAGE) corresponding to zebrafish pept2 was completed by inserting a stretch of 75 missing nucleotides in the coding sequence to obtain a 3,238-bp functional clone. The complete open reading frame (ORF) was 2,160 bp and encoded a 719-amino acid protein. Electrophysiological analysis after cRNA injection in Xenopus laevis oocytes suggested that zebrafish PEPT2 is a high-affinity/low-capacity transporter ( K0.5 for glycyl-l-glutamine ∼18 μM at −120 mV and pH 7.5). Zebrafish pept2 gene was 19,435 kb, thus being the shortest vertebrate pept2 fully characterized so far. Also, zebrafish pept2 exhibited 23 exons and 22 introns, whereas human and rodent pept2 genes contain 22 exons and 21 introns only. Zebrafish pept2 mRNA was mainly detected in brain, kidney, gut, and, interestingly, otic vesicle, the embryonic structure that develops into the auditory/vestibular organ, homolog to the higher vertebrate inner ear, of the adult fish. Characterization of zebrafish pept2 will contribute to the investigation of peptide transporters using a well-established genetic model and will allow the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful marker to screen mutations that affect choroid plexus and inner ear development.

Hypothyroidism induces type 2 iodothyronine deiodinase expression in mouse heart and testis

In the present study we show the expression profiles of both type 1 and type 2 iodothyronine deiodinases (D1 and D2) in a wide spectrum of mouse tIssues, and D2 regulation by thyroid status. A characteristic tIssue-specific expression for each isoform was observed. D2 transcripts were detected in most tIssues with variable levels of expression. The observed D2 mRNA tIssue distribution was similar to that described in rats and is in agreement with the view of different patterns of expression between rodents and humans. However, it is interesting to note that despite the low levels of D2 transcripts in mouse heart and testis in the euthyroid state, the induction of hypothyroidism caused a significant increase in D2 activity in these tIssues. Similar results were also obtained in adult rats. These results suggest a previously unrecognized role for type 2 deiodinase in controlling intracellular triiodothyronine levels in rodent heart and testis during states of thyroid hormone deficiency.

Comparative analysis of follistatin-, activin beta A- and activin beta B-mRNA steady-state levels in diverse porcine tissues by multiplex S1 nuclease analysis

OBJECTIVE: The relation of activins (dimers of the beta-subunits of inhibin) and follistatin (FS) (their binding protein) affect the growth and differentiation of many cell types. Activin- and FS-mRNAs show a widespread co-expression throughout the organism, indicating an essential role for the FS/activin system in diverse physiological processes. The present study was performed to investigate FS-, activin betaA-, and activin beta B-mRNA expression in porcine tissues and to compare the relative mRNA tissue distribution by a newly developed multiplex S1 nuclease protection assay. METHODS: Twenty micrograms total RNA from different porcine tissues were subjected to multiplex S1 analysis. Specific mRNA expression was determined by measurements of optical densities on autoradiographs. RESULTS: Activin beta A-mRNA expression was abundant in the ovary, adrenal gland, fat, vein, artery and uterus, activin beta B-mRNA was highly expressed in the ovary, pituitary, uterus, placenta, aorta and cerebellum. FS-mRNA showed a widespread expression with high levels in ovary, uterus, cerebellum, placenta and fat. The comparison of relative activin beta A-, activin beta B- and FS-mRNA expression within a certain tissue showed a predominance of activin beta A-mRNA in the adrenal gland, fat, artery, spinal cord, cerebrum and colon and of activin beta B-m RNA in pituitary, testis and placenta, while FS-mRNA levels exceeded those of activin subunits in epididymis, liver, lymphoid tissue, muscle, intestine, cerebellum, ovary and uterus. CONCLUSIONS: The presented data provide an overview of FS-, activin beta A-, and activin beta B-mRNA steady state levels in porcine tissues.

Cloning and molecular characterisation of the trout (Oncorhynchus mykiss) vacuolar H(+)-ATPase B subunit

The current model of transepithelial ion movements in the gill of freshwater fish incorporates an apically oriented vacuolar H(+)-ATPase (H(+)V-ATPase; proton pump) that is believed to facilitate both acid excretion and Na(+) uptake. To substantiate this model, we have cloned and sequenced a cDNA encoding the B subunit of the rainbow trout (Oncorhynchus mykiss) H(+)V-ATPase. The cloning of the B subunit enabled an examination by northern analysis of its tissue distribution and expression during external hypercapnia. Degenerate oligonucleotide primers to the B subunit of the H(+)V-ATPase were designed and used in a semi-nested polymerase chain reaction (PCR) to amplify an 810 base pair (bp) product from a trout gill/kidney cDNA library. This PCR product was cloned and sequenced and then used to screen the same cDNA library. The assembled 2262 bp cDNA included an open reading frame coding for a deduced protein of 502 amino acid residues. A BLAST search of the GenBank nucleotide database revealed numerous matches to other vertebrate and invertebrate H(+)V-ATPase B subunits. Protein alignment demonstrated that the trout H(+)V-ATPase B subunit is more than 85 % identical and more than 90 % similar to those in other vertebrate species. An initial analysis of H(+)V-ATPase mRNA tissue distribution revealed significant expression in blood. Although a comparison of perfused tissues (blood removed) with non-perfused tissues demonstrated no obvious contribution of the blood to total tissue H(+)-ATPase mRNA levels, all subsequent experiments were performed using perfused tissues. Levels of H(+)V-ATPase mRNA expression were high in the gill, kidney (anterior or posterior), intestine, heart and spleen, but lower in liver and white muscle. Exposure of the fish to 12 h of external hypercapnia (water P(CO2)=7. 5 mmHg; 1 kPa) was associated with a transient increase (at 2 h) in the levels of H(+)V-ATPase B subunit mRNA in gill and kidney; liver mRNA levels were unaffected. These results are consistent with the hypothesis of an apically localised plasma membrane H(+)V-ATPase in the freshwater trout gill and that the expression of this proton pump is increased during periods of acidosis, at least in part because of an increased steady-state level of H(+)V-ATPase mRNA.

Cloning and expression of rat pancreatic β-cell malonyl-CoA decarboxylase

To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic β-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3ʹ of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic β-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues.

The sodium iodide symporter gene and its regulation by cytokines found in autoimmunity

Iodide concentration by the thyroid gland, an essential step for thyroid hormone synthesis, is mediated by the Na+/I- symporter (NIS). To identify factors that may regulate this process, we have studied NIS gene expression in the Fisher rat thyroid cell line (FRTL-5) by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. Increasing concentrations of bovine TSH (0.1, 1, 10, 50 and 100 mU/l), with or without tumour necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) or interleukin-1 alpha (IL-1 alpha) were added to FRTL-5 cells previously deprived of TSH for a minimum of 5 days. RNA was extracted and samples were studied for NIS expression. TSH enhanced NIS mRNA expression in a dose-dependent manner, with induction evident at 0.1 mU/l, reaching a peak at 50 mU/l, an effect detected after 6 h of stimulation, but not in the first 2 h. Both TNF alpha and, to a lesser extent, IL-1 alpha inhibited basal and TSH-induced NIS expression. High concentrations of IFN gamma also downregulated TSH-stimulated NIS mRNA expression. Using the same technique, we also investigated NIS mRNA tissue distribution in two male and one female Wistar rats. High levels of NIS expression were detected in the thyroid, stomach, and mammary gland, lower levels were found in the intestine, adipose tissue and liver, borderline levels were expressed in the salivary gland, and no expression was detected in the kidneys. In summary, we have shown that TSH upregulates rat NIS gene expression in vitro, and this induction can be modulated by cytokines. Analysis of the distribution of rat NIS mRNA ex vivo demonstrated variable levels of NIS transcription in different tissue samples.