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ABSTRACT: Chorion hardening is triggered by the contents of cortical alveoli that are released upon fertilization of medaka (Oryzias latipes) eggs. We purified the chorion hardening-inducing activity as a single protein from the exudate of cortical alveoli of medaka eggs. This activity was co-purified with proteolytic activity of the chorion protein ZI-1,2. Based on the amino acid sequence of purified protein, we cloned the cDNA of this protein from a medaka ovarian cDNA library. Sequence analyses revealed typical sequence features, a zinc-binding motif and a methionine turn motif, of the astacin metalloproteinase family. We termed this protein "alveolin." Alveolin has a molecular mass of 21.5 kDa deduced by the amino acid sequence and neutral optimal pH range. Alveolin hydrolyzes ZI-1,2. Alveolin activity was strongly inhibited by metal-chelating agents but not by various proteinase inhibitors. To our knowledge, this is the first description of the isolation and identification of the chorion hardening-inducing factor from cortical alveoli exudate of teleost eggs.
Journal of Biological Chemistry 04/2000; 275(12):8349-54. · 4.77 Impact Factor
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ABSTRACT: Our previous findings suggest the activity of cytochrome P-450 aromatase (P-450arom), the enzyme which converts testosterone to estradiol-17beta, in the ovarian follicle of medaka (Oryzias latipes) is regulated at the transcriptional level. In this study, we cloned a cDNA encoding a FTZ-F1-like protein (mdFtz-F1) from ovarian follicles of medaka. In vitro translated mdFTZ-F1, and nuclear extract from medaka ovarian follicles, formed complexes with oligonucleotide probes containing putative orphan nuclear receptor binding motifs, which are present in the promoter region of the medaka P-450arom gene. The expression pattern of mdFtz-F1 transcripts during oogenesis coincides with that of P-450arom transcripts. Transfection assays further suggest a potential transcriptional regulatory activity of mdFTZ-F1 upon the medaka P-450arom promoter. Taken together, these results suggest a potential role of mdFTZ-F1 in the transcriptional regulation of P-450arom in the ovarian follicle of medaka.
Molecular and Cellular Endocrinology 04/1999; 149(1-2):221-8. · 4.19 Impact Factor
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ABSTRACT: We have previously shown that pertussis-toxin-sensitive inhibitory guanine-nucleotide-binding-regulatory proteins (G proteins) are involved in the signal transduction of steroidal maturation-inducing hormone (MIH) of rainbow trout (Oncorhynchus mykiss) oocytes, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP) [Yoshikuni, M. & Nagahama, Y. (1994) Dev. Biol. 166, 615-622]. In this study, we obtained five different cDNA fragments of G protein alpha subunits from medaka (Oryzias latipes) intact ovarian follicles (three subtypes of G(i alpha), G(i alpha a), G(i alpha b) and G(i alpha c); two subtypes of G(s alpha), G(s alpha d), and G(s alpha e)). Using a newly developed extraction method for medaka oocyte RNA, we demonstrated that oocytes expressed both G(i alpha a) and G(i alpha c), but not G(i alpha b). Full-length cDNA clones for G(i alpha a) and G(i alpha c) were then isolated from a medaka ovarian follicle cDNA library. The predicted amino acid sequences of G(i alpha a) and G(i alpha c) exhibited significant similarity with G(i alpha1) and G(i alpha2) of other species, respectively. Both G(i alpha a) and G(i alpha c) possessed a specific Cys residue in the C-terminal region that was the site for ADP-ribosylation by pertussis toxin. G(o alpha), another G protein that is ADP-ribosylated by pertussis toxin, was not detected in oocytes, although it was expressed in brain tissue. Western blot analyses using a specific antibody against G(i alpha1) and G(i alpha2) subunit proteins revealed that in both medaka and rainbow trout G(i alpha) subunit protein (40 kDa) contents were abundant in plasma membranes of postvitellogenic immature oocytes, decreased in mature oocytes, and were absent in ovulated eggs. Furthermore, specific 17alpha,20beta-DP binding to plasma membranes was higher in postvitellogenic immature oocytes than in ovulated eggs. Taken together, these results suggest that G(i alpha a) and/or G(i alpha c) may be involved in the transduction of the signal from 17alpha,20beta-DP receptors during oocyte maturation of fish oocytes.
European Journal of Biochemistry 12/1997; 249(3):846-53. · 3.58 Impact Factor
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ABSTRACT: We have cloned a full length cDNA coding for the activin type IIB receptor (GactRIIB) from the goldfish ovary. GactRIIB shares 73 and 70% amino acid identity in the extracellular domain, and 78 and 80% identity in the intracellular domain with the type IIB receptors of the mouse and Xenopus respectively. The intracellular domain of GactRIIB contains two serine kinase consensus sequences, DFKSRN and GTRRYMAPE, in agreement with the reports in other vertebrates that serine/threonine phosphorylation is involved in activin signal transduction. The identity of GactRIIB was confirmed by transient expression in the COS cells followed by activin binding. Iodinated human activin A bound to the GactRIIB-transfected cells and the binding could be completely inhibited by unlabeled activin. Affinity labeling revealed a band of about 85 kDa, which is in agreement with the reported type II receptors in other vertebrates. Together with the fact that activin is expressed in the goldfish ovary, the cloning of activin receptors from the ovary suggests paracrine and autocrine roles for activin in the goldfish ovarian functions.
Journal of Molecular Endocrinology 09/1997; 19(1):47-57. · 3.48 Impact Factor
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ABSTRACT: We isolated and characterized a cDNA encoding testicular 11beta-hydroxylase, cytochrome P450(11beta) from the Japanese eel (Anguilla japonica) testis. The cDNA contains an open reading frame that encodes a protein of 511 amino acids. The predicted amino acid sequence shares 38-48% homology with those of adrenal P450(11beta) from mammals and frog. Transient expression in COS 1 cells confirmed that the protein encoded by this cDNA had P450(11beta) activity. Northern blotting revealed a single 1.8 kb long transcript of P450(11beta). This transcript was not found in immature eel testes prior to an injection with human chorionic gonadotropin (hCG), but it was present in eel testes after hCG injection.
FEBS Letters 12/1996; 397(2-3):250-2. · 3.54 Impact Factor
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ABSTRACT: Two cDNA clones of cytochrome P-450 aromatase (P-450arom) were isolated from a medaka (Oryzias latipes, a daily spawner) ovarian follicle cDNA library using a medaka P-450arom genomic DNA fragment as a probe. The first, 1,809-bp insert (S81f) contains an 1,554-bp open reading frame encoding a 518-amino-acid polypeptide. The second, 1,852-bp (S52f) insert possesses an open reading frame identical to that of the S81f insert, except for the absence of the heme-binding region as the result of an additional A residue at the position of nucleotide 1,827. Expression of the S81f cDNA, but not of the S52f cDNA, in nonsteroidogenic COS-1 cells leads to production of a steroidogenic enzyme which is capable of converting exogenous testosterone to estrogen. The P-450arom genomic DNA fragment hybridizes to a single mRNA in medaka ovarian follicle RNA. Changes in level of P-450arom transcripts and P-450arom enzyme activity were determined in medaka ovarian follicles collected at 16 stages of development. A close correlation was found between these two profiles, both being high in midvitellogenic follicles and low in postvitellogenic follicles collected during oocyte maturation. These findings, together with those of our previous study showing that actinomycin D prevented gonadotropin-induced aromatase activation by medaka ovarian follicles, suggest that aromatase activity is regulated at the transcriptional level in medaka vitellogenic follicles.
Molecular Reproduction and Development 12/1996; 45(3):285-90. · 2.53 Impact Factor
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ABSTRACT: We isolated the structural gene encoding cytochrome P-450 aromatase (P-450arom), for the first time from a nonmammalian vertebrate, the medaka (a teleost fish, Oryzias latipes), using the rainbow trout P-450arom cDNA as a probe. The structure of the entire P-450arom gene, the nucleotide sequence of its 5' flanking region, and the transcriptional initiation sites were determined. The medaka P-450arom gene consists of nine exons, but spans only 2.6 kb, being much smaller than the human P-450arom gene (at least 70 kb), as a result of extremely small introns (medaka, 73-213 bp vs. human, 1.3-10 kbp). The splicing junctions are located at exactly the same positions as those found in the human P-450arom. The deduced amino acid sequence is 51-52% identical to those of mammals and chicken, and 75% identical to the rainbow trout amino acid sequence. Genomic Southern blots revealed the presence of a single medaka gene. Promoter analyses indicated two major transcription initiation sites 60 and 61 bp upstream from a putative initiation codon. The promoter region of medaka P-450arom gene also contains potential Ad4BP sites and estrogen responsive element (ERE) half-sites. These results suggest that the basic structural organization of P-450arom genes and the regulatory mechanisms of expression are well conserved throughout the vertebrates.
Journal of Biochemistry 05/1995; 117(4):719-25. · 2.37 Impact Factor
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ABSTRACT: A cDNA clone encoding 3 beta-hydroxysteroid dehydrogenase delta 5-4-isomerase (3 beta-HSD) was isolated from a cDNA library of rainbow trout ovarian thecal cells. Southern hybridization analysis of trout genomic DNA with the cDNA suggested the presence of a single gene encoding 3 beta-HSD in the rainbow trout showing a total genomic size of less than 4 kilobases (kb). The cDNA hybridized to a species of mRNA isolated from rainbow trout ovaries; the 1.4 kb transcripts were most abundant in trout ovaries during the later stages of oogenesis. The trout 3 beta-HSD expressed in non-steroidogenic monkey kidney tumor (COS-1) cells showed a unique enzymatic 3 beta-HSD activity. Dehydroepiandrosterone was a more favoral substrate of the trout 3 beta-HSD than 1- alpha-hydroxypregnenolone. Interestingly, the trout 3 beta-HSD expressed in COS-1 cells exhibited minimal ability to convert pregnenolone to progesterone.
FEBS Letters 09/1994; 350(2-3):309-13. · 3.54 Impact Factor
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ABSTRACT: A cDNA clone encoding cholesterol side-chain cleavage cytochrome P450 (P450scc) was isolated from a rainbow trout ovarian follicle cDNA library. The cDNA contains an open reading frame of 1,542 nucleotides encoding a protein of 514 amino acids. The predicted amino acid sequence of trout P450scc shows 48% homology with that of human, and 46% homology with that of rat, bovine and pig. P450scc activity was confirmed by transfected COS-1 monkey kidney tumour cells with an expression vector for trout P450scc cDNA and subsequent detection of conversion from 25-hydroxycholesterol to pregnenolone by radioimmunoassay. The cDNA only hybridized to a single 1.8 kb RNA transcript. The transcript was not found in early vitellogenic follicles, barely detected in postvitellogenic follicles, and abundant in postovulatory follicles.
FEBS Letters 04/1993; 319(1-2):45-8. · 3.54 Impact Factor
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ABSTRACT: cDNA inserts encoding 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) were, for the first time, isolated and cloned from a pig testis cDNA library using synthetic oligonucleotides deduced from the partially determined amino acid sequences. The cDNA contains an open reading frame predicted to encode 289 amino acid residues. Surprisingly, it has 85% amino acid homology to human carbonyl reductase. The purified enzyme exhibited carbonyl reductase activity. Adenine-rich sequence was located in the 3'-untranslated nine-rich sequence was located in the 3'-untranslated region, which may mean that the gene originates by retroposition. RNA transcripts of 1.3, 3, and 6 kilobases were detected in poly(A)+ RNA extracted from pig testis by Northern blot hybridization. The steady-state level of the RNA species increased to a maximum in testes from 10-day-old pigs, but rapidly declined thereafter to the same levels found in testes of mature animals.
Journal of Biological Chemistry 08/1992; 267(19):13451-5. · 4.77 Impact Factor
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ABSTRACT: A cDNA clone encoding cytochrome P-450c17 (17 alpha-hydroxylase/17,20-lyase) was isolated from a rainbow trout ovarian follicle cDNA library. The cDNA contained an open reading frame of 1,542 nucleotides encoding a protein of 514 amino acid residues. The amino acid sequence of trout P-450c17 shows a much greater homology with chicken P-450c17 than with that of human, bovine and rat. The trout P-450c17 expressed in non-steroidogenic mammalian COS-1 cells showed both 17 alpha-hydroxylase and 17,20-lyase activities. The cDNA only hybridized to a single species of mRNA (2.4 kb) isolated from rainbow trout ovaries; the 2.4 kb transcripts were abundant in trout ovaries during the later stages of oogenesis.
FEBS Letters 05/1992; 301(1):60-4. · 3.54 Impact Factor
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ABSTRACT: The enzyme aromatase P-450 (P450arom) catalyses the conversion of androgen to oestrogen. A cDNA insert encoding P450arom was isolated from a rainbow trout (Oncorhynchus mykiss) ovary cDNA library. The insert was sequenced and found to contain an open-reading frame predicted to encode a protein of 522 amino acid residues. The deduced polypeptide is 52% homologous with human, mouse and rat P450arom and 53% homologous with that of chicken. The insert was confirmed to encode P450arom by introducing it into COS-1 monkey kidney tumour cells (COS-1 cells) and detecting the conversion of testosterone to oestradiol-17 beta by radioimmunoassay. The N-terminal region of the deduced polypeptide was 19 amino acids longer than that of the other four species, and was found by hydropathy plotting to be very hydrophobic. Northern blot analysis revealed 2.6 kb RNA transcripts which were present in the trout ovary during vitellogenesis and hybridized to the cDNA insert. In preparations from subsequent stages of ovarian development, no RNA transcripts hybridized to the probe. Since the RNA transcripts are present only during the stage of oestradiol-beta production by the ovarian follicles, oestradiol-17 beta production may be regulated, in part, by the amount of P450arom mRNA present.
Journal of Molecular Endocrinology 03/1992; 8(1):53-61. · 3.48 Impact Factor
