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ABSTRACT: Serotonin (5-HT) is well known for modulating the release of GnRH and gonadotropin in teleosts. Reports on increased female:male ratio after the blockade of 5-HT biosynthesis proposed a role for 5-HT in brain sex differentiation. Two types of tryptophan hydroxylase (Tph), rate-limiting enzyme in the biosynthesis of 5-HT were cloned from vertebrates. In the present study, we cloned Tph from brain and evaluated its importance during early development of XX and XY Nile tilapia. Tph cloned from tilapia brain is 1888 bp in length and it encodes predicted protein of 462 amino acid residues. Tph activity of tilapia was confirmed by demonstrating the conversion of L-tryptophan to 5-hydroxy tryptophan by the recombinant protein after transient transfection of this cDNA clone in COS-7 cells. Northern blot identified single transcript around 2kb in male brain. Tissue distribution of Tph revealed high abundance in brain, kidney, liver and testis. Semi-quantitative RT-PCR revealed exclusive expression of Tph in the male brain from 5 to 20 days post hatch (dph) while in the female brain, it was from 25 dph. These results were authenticated by localization of Tph transcripts in olfactory bulb-telencephalon region of 11 dph male brain using in situ hybridization. Tph immunoreactivity (-ir) was also evident in the nucleus preopticus-periventricularis area of male brain as early as 12 dph. However, Tph-ir was observed in several regions of both male and female brain without any distinction from 30 dph. Dimorphic expression pattern of Tph during early brain development around the critical period (7-21 dph) of gonadal sex determination and differentiation may implicate a role for Tph in brain sex differentiation of tilapia.
General and Comparative Endocrinology 11/2009; 166(2):320-9. · 3.27 Impact Factor
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ABSTRACT: Multiple forms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) and their differential tissue expression pattern have not been shown in any lower vertebrates. In the present study, we report cloning of two novel 3beta-HSDs and two variants from gonads of the Nile tilapia. 3beta-HSD cDNAs encode two peptides of 375 (3beta-HSD type-I/variant 1) and 367 (3beta-HSD type-II/variant 1) amino acid residues that share 31.9% homology. 3beta-HSD type-I/variant 1 shared high homology with other piscine counterparts while 3beta-HSD type-II/variant 1 exhibited homology to mammalian DeltaC27-3beta-HSD and multifunctional viral 3beta-HSD. The latter seems to be ancient form among vertebrates. Transiently transfected 3beta-HSDs' open reading frames in COS-7 cells converted exogenous pregnenolone/androsta-5-ene-3beta-17beta-diol to progesterone/testosterone. Tissue distribution pattern of 3beta-HSDs by RT-PCR revealed varied expression pattern. Northern blot analysis of 3beta-HSDs demonstrated steady or gradual rise in transcripts level at different gonadal stages. These data revealed the importance of novel 3beta-HSDs in teleosts and also provided phylogenetic significance.
Molecular and Cellular Endocrinology 12/2008; 299(2):146-52. · 4.19 Impact Factor
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ABSTRACT: Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.
Journal of Experimental Zoology Part A Ecological Genetics and Physiology 08/2008; 309(7):419-26. · 1.64 Impact Factor
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ABSTRACT: In order to elucidate the roles of 17beta-HSDs in fish gonadal steroidogenesis, three types of 17beta-HSDs (17beta-HSD1, 17beta-HSD8 and putative 17beta-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17beta-HSD type 1, 8 and 12 were 1504, 1006 and 1930 bp long, with open reading frames encoding proteins of 289, 256 and 314 aminoacids, respectively. Tissue distribution pattern analyzed by RT-PCR and Northern blot showed that 17beta-HSD1 was dominantly expressed in the ovary, while the putative 17beta-HSD12, one of the two duplicates found in fish, is a male specific enzyme and expressed exclusively in testis (detected by RT-PCR only). On the other hand, 17beta-HSD8 was expressed in the brain, gill, heart, liver, intestine, gonad, kidney and muscle of both male and female. Enzymatic assays of the three types of 17beta-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17beta-HSD1 expressed in E. coli had the preference for NADP(H) as cofactor and could catalyze the inter-conversion between estrone and estradiol efficiently as well as the inter-conversion between androstenedione and testosterone, but less efficiently. Tilapia 17beta-HSD8 recombinant protein expressed in HEK 293 cells could catalyze the conversion of testosterone to androstenedione, as well as the inter-conversion between estrone and estradiol. However, the putative 17beta-HSD12 expressed in E. coli or in HEK 293 cells showed no conversion to any of the four substrates tested in this study. Based on enzyme characterization and tissue distribution, it is plausible to attribute crucial roles to 17beta-HSDs in the gonadal steroidogenesis of teleosts.
Journal of Molecular Endocrinology 09/2005; 35(1):103-16. · 3.48 Impact Factor
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ABSTRACT: Using semi-quantitative reverse transcriptase polymerase chain reaction we analyzed the ontogenic expression patterns of several nuclear receptors (estrogen receptors [ERalpha and beta], androgen receptors [ARalpha and beta], Ad4BP/SF-1 and Dax-1) and cytochrome P450 aromatases (brain and ovarian types) in whole brain and gonads of the Nile tilapia. ERalpha and beta transcripts were evident in both sexes with a high expression of ERalpha in females at 0 day after hatching (0 dah). ARalpha appeared early (0 dah) in males and while in females at 25 dah. Among the two types of cytochrome P450 aromatases, the expression of the brain type (bP450arom) but not the ovarian type (oP450arom) was evident from 0 to 90 dah in the whole brain of both males and females. Expression of Ad4BP/SF-1 in female brain began from 0 dah but in male brain at 5 dah. Expression of Dax-1 began at 0 dah and it was higher throughout in male brain than that of the female brain. In gonads, ERalpha and beta transcripts were evident in both sexes with slight variation. In females, both oP450arom and Ad4BP/SF-1 amplicons were evident at 15 dah. In males, although faint expressions of Ad4BP/SF-1 amplicons were evident at early duration of development, oP450arom did not appear until 90 dah. Conversely, expression of bP450arom was observed throughout in the developing testis with varied pattern while in developing ovary it was evident till 15 dah and reappeared only after 90 dah. Taken together, present results suggest that brain acts merely as a synchronizer in the sex differentiation process initiated by gonadal cues/factors in the Nile tilapia.
Fish Physiology and Biochemistry 04/2005; 31(2-3):129-35. · 1.53 Impact Factor
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ABSTRACT: Gonadal development and steroidogenesis in teleosts is regulated by two gonadotropic hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Earlier studies in tilapia have shown that FSH-beta and LH-beta appear by 14 days after hatching (dah), results from the current study corroborate with these previous reports in tilapia. Here we demonstrate the appearance of LH in pituitary between 14 and 20 dah. In addition to this the present study primarily focuses on any possible differences in appearance of LH-beta and FSH-beta immunoreactivity between XX and XY population of Nile tilapia. LH immunoreactivity was found to be lower in pituitary of XX fish when compared to XY fish. The development of FSH-beta immunoreactivity in pituitary of the Nile tilapia is also presented. Overall, it remains to be established what significance these findings on the appearance of gonadotropins hold for sex differentiation in tilapia.
Fish Physiology and Biochemistry 04/2005; 31(2-3):177-81. · 1.53 Impact Factor
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ABSTRACT: Estrogens are essential for many reproductive and non-reproductive functions. In teleosts, it is well-known that several subtypes of estrogen receptors are required for the precise action of estrogens. Present study describes the cloning of the third estrogen receptor, ER- beta2, from the Nile tilapia by EST sequencing coupled microarray. The cloned ER-beta2 showed 77.7% amino acid identity with the reported Atlantic croaker ER-beta. Three ERs, ER-alpha, ER-beta1 and ER-beta2, from the fugu genome were also isolated to analyze their gene structures. Comparison of the intron/exon boundaries and exon numbers of fugu, tilapia, rainbow trout and zebrafish, and phylogenetic analysis of 63 ER sequences revealed that ER-beta probably underwent two successive lineage-specific duplications in teleost. The former took place only in zebrafish lineage, and the latter took place in advanced teleosts without the zebrafish lineage, whereas no duplication of the ER-alpha gene has been detected. Tissue distribution analysis by RT-PCR revealed that tilapia ER-alpha and ER-beta1 were expressed ubiquitously, whereas ER-beta2 is expressed only in the pituitary, liver, intestine, kidney and gonads, with the highest expression in the testis and the lowest level in the ovary. Northern blot analysis detected a single transcript of about 3.4 kb in the testis but not in the ovary mRNAs. In transient transfection assays using human embryonic kidney 293 (HEK293) cells, tilapia ER-beta2 showed estrodiol-17beta dependent transactivation.
Fish Physiology and Biochemistry 04/2005; 31(2-3):255-66. · 1.53 Impact Factor
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ABSTRACT: In the present study, thiourea-induced thyroid hormone depletion and thyroxine (T(4)) 'overdose' were used as a strategy to understand the influence of thyroid hormones on ovarian recrudescence of juvenile (3-months-old), immature (8-months-old) and adult (1-year-old) air-breathing catfish, Clarias gariepinus. Thiourea-induced thyroid hormone depletion in juvenile catfish impaired ovarian development, but no significant effect was observed in immature catfish and during late stage of ovarian recrudescence of mature catfish. T(4) treatment in females undergoing late stages of ovarian recrudescence induced rapid oocyte growth by promoting its early entry into maturational phase as evident from the presence of more number of vitellogenic and post-vitellogenic follicles, decrease in aromatse immunoreactivity and reduced estradiol-17beta levels. Hence, thyroid hormones have an important role to play during early stages of ovarian development and vitellogenesis of catfish and also indicating that thyroid has a stage dependent effect on ovary.
Fish Physiology and Biochemistry 04/2005; 31(2-3):267-70. · 1.53 Impact Factor
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ABSTRACT: The Japanese eel (Anguilla japonica) and Nile tilapia (Oreochromis niloticus) 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) cDNAs were isolated from their respective testes cDNA libraries. The cDNAs predict two peptides of 436 and 406 amino acid residues that share about 42% homology with mammalian 11beta-HSD type 2 proteins. Analysis of the tissue distribution pattern by RT-PCR reveals that 11beta-HSD2 is expressed in a wide variety of tissues in tilapia, with higher expression in kidney and gill of both sexes, and with the highest expression in testis. 11beta-Dehydrogenase activity of the eel 11beta-HSD2 was confirmed by demonstrating the conversion of cortisol to cortisone by the recombinant protein after transient expression of this cDNA clone in COS-1 cells. Bands of approximately 2.7 and approximately 3.8 Kb were detected in Northern blot of eel and tilapia testes respectively, which is consistent with the cloned cDNA sizes of the two species. Northern blot analysis also revealed that the expression of the eel testis 11beta-HSD2 gene could be induced by human chorionic gonadotropin (hCG) injection, implying a role of 11beta-HSD2 in hCG-induced 11-ketotestosterone production and spermatogenesis in the Japanese eel.
Journal of Molecular Endocrinology 11/2003; 31(2):305-15. · 3.48 Impact Factor
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ABSTRACT: To clarify the importance of endogenous estrogens during sex differentiation in a teleost fish, the Nile tilapia, we examined the target events for endogenous estrogens and their role during gonadal sex differentiation. The expression of CYP19a (P450arom) precedes any morphological gonadal sex differentiation. Further to these findings, the treatment of XX fry with non-steroidal aromatase inhibitor (AI), Fadrozole, from seven to 14 days after hatching caused complete sex reversal to functional males. The XX sex reversal induced by AI was rescued completely with simultaneous estrogen treatment. We also found that XY fry treated with estrogen, before the appearance of morphological sex differences, caused complete sex reversal from males to females. Taken together, these results suggest that endogenous estrogens are required for ovarian differentiation. To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. Two out of ten gene products were expressed in germ cells, whereas the others were expressed in somatic cells.
Cytogenetic and Genome Research 02/2003; 101(3-4):289-94. · 1.53 Impact Factor
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ABSTRACT: A 548bp partial cDNA fragment of 17-hydroxysteroid dehydrogenase (17-HSD1) was obtained by RT-PCR from the ovary of Nile Tilapia. The expression of 17-HSD1 was high from 0 to 11 days after spawning, but there was a sharp decline at the day of spawning (day 14) indicating its involvement in ovarian cycle.
Fish Physiology and Biochemistry 01/2003; 28(1):381-382. · 1.53 Impact Factor
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ABSTRACT: Two subtypes (alpha and beta) of androgen (AR) and progestogen receptors (PR) are present in the testis of Japanese eel (Anguilla japonica). Amino acid homology of the open reading frames between alpha and beta in AR or PR is approximately 40%, but the DNA- and ligand-binding domains show high homology between subtypes. Judging from these structures, alpha and beta are not isoforms derived from translational initiation at two in-phase ATG codons, alternative splicing, or tetraploidy. In transient transfection assays using a reporter construct containing a steroid-responsive promoter, each subtype showed its corresponding hormone-dependent transactivation. The ligand affinity for transactivation between AR and PR subtypes was similar for physiological ligands. Tissue distribution of both subtype mRNAs was different. Protein interaction between subtypes was demonstrated in vitro by GST pull-down assays. These results clearly indicate that two functional subtypes of AR and PR exist in eel. These findings will advance our understanding of the mechanisms underlying sex steroid signaling.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 07/2001; 129(2-3):449-55. · 1.92 Impact Factor
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ABSTRACT: Vas (a Drosophila vasa homologue) gene expression pattern in germ cells during oogenesis and spermatogenesis was examined using all genetic females and males of a teleost fish, tilapia. Primordial germ cells (PGC) reach the gonadal anlagen 3 days after hatching (7 days after fertilization), the time when the gonadal anlagen was first formed. Prior to meiosis, no differences in vas RNA are observed in male and female germ cells. In the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in auxocytes and then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, followed by a decrease from vitellogenic to postvitellogenic oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes. No vas RNA expression is evident in either diplotene primary spermatocytes, secondary spermatocytes, spermatids or spermatozoa. The observed differences in vas RNA expression suggest a differential function of vas in the regulation of meiotic progression of female and male germ cells.
Mechanisms of Development 01/2001; 99(1-2):139-42. · 2.83 Impact Factor
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ABSTRACT: A cDNA encoding a nuclear 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP, spermiation-inducing hormone in fish) receptor (DPR) was, for the first time, isolated from an eel testis cDNA library. The amino acid sequence of DPR shows high homology with those of human and chicken progesterone receptors. The affinity of the bacterial recombinant DPR ligand binding domain protein for 17alpha,20beta-DP is higher than that of progesterone. In transfection experiments using COS7 cells, the DPR showed progestin-dependent activation of transcription. 17alpha,20beta-DP was the most effective activator of transcription. These results indicate that the cDNA encodes a functional eel DPR, and show that 17alpha,20beta-DP has a nuclear receptor-mediated action in eel testes.
FEBS Letters 02/2000; 465(1):12-7. · 3.54 Impact Factor
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ABSTRACT: There has been general acceptance that only one type of androgen receptor (AR) exists in an individual. This contrasts with other members of the nuclear receptor superfamily where multiple forms have been reported (e.g. estrogen receptor alpha/beta, thyroid hormone receptor alpha/beta, etc.). We have previously identified 11-ketotestosterone (a potent androgen in teleosts) as the spermatogenesis-inducing hormone of the Japanese eel and have cloned its receptor (eAR1) cDNA from eel testis. Here we report on the cloning of a cDNA encoding a second type of AR (eAR2) from the eel testis and the functional characterization of the encoded protein. This cDNA contains a complete open reading frame encoding 797 amino acid residues. The amino acid sequence of eAR2 shows high homology with other ARs, including eAR1, in the DNA-binding (98-88%) and ligand-binding (59-85%) domains, whereas the other domains show low homology (<35%). In transient transfection assays of mammalian cells, the eAR2 protein displayed androgen-dependent activation of transcription from the androgen-responsive murine mammary tumor virus promoter. Tissue distribution of its mRNA was different from that of eAR1. We conclude that eAR2 is a novel AR in the eel, which we suggest should be named eel ARbeta to distinguish it from eAR1 (eARalpha).
Journal of Biological Chemistry 09/1999; 274(36):25205-9. · 4.77 Impact Factor
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ABSTRACT: We have previously identified 11-ketotestosterone (11KT, a portent androgen in teleosts) as the spermatogenesis-inducing hormone of the Japanese eel. In this study, a cDNA encoding the eel androgen receptor (AR) was isolated from the Japanese eel testis. This cDNA contains a complete open reading frame encoding 848 amino residues. The amino acid sequence of the eel AR shows high homology with other ARs. In transient transfection assays of mammalian cells, the eel AR showed androgen-dependent activation of transcription from the androgen-responsive MMTV promoter. Of the endogenous androgens found in the Japanese eel, 11KT was the most effective activator of transcription. These results indicate that the cloned cDNA encodes a functional eel AR, and its major native ligand is 11KT. This is the first isolation of an AR molecule from fish, and is the first evidence that 11KT acts via a nuclear receptor.
Biochemical and Biophysical Research Communications 02/1999; 254(2):378-83. · 2.48 Impact Factor
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ABSTRACT: We produced an antibody that recognized only early stages of spermatogonia in Japanese eel testis. This antibody (anti-spermatogonia-specific antigen-1, anti-SGSA-1) recognized a band of about 38 kDa in Western blot analysis of extracts from eel testis. This antigen was observed by immunohistochemistry only in type-A and early type-B spermatogonia and could not be seen in the late type-B spermatogonia, which appeared after the initiation of spermatogenesis by a single injection of human chorionic gonadotropin. Immunoreactive SGSA-1 was absent in spermatocytes, spermatids, spermatozoa, Sertoli cells, and interstitial Leydig cells. Similarly, this antigen was also detected only in type-A/primary spermatogonia in the testes of two species of teleosts, medaka (Oryzias latipes) and tilapia (Oreochromis niloticus), as well as a toad (Xenopus laevis). These results imply that the disappearance of SGSA-1 in late type-B/secondary spermatogonia is a critical step in the progression of spermatogenesis, and indicate that anti-SGSA-1 is a useful marker for analysis of the molecular mechanism controlling the differentiation of spermatogonia in lower vertebrates.
Molecular Reproduction and Development 01/1999; 51(4):355-61. · 2.53 Impact Factor
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ABSTRACT: A recombinant expression vector containing Japanese eel (Anguilla japonica) testis cytochrome P450(11 beta) (11beta-hydroxylase) cDNA was introduced into COS-1 cells. Enzymatic activity of the expressed P450(11 beta) for corticosteroid synthesis was analysed by incubating transfected cells with 14C-labelled 11-deoxycorticosterone or 3H-labelled deoxycortisol as substrates. Thin layer chromatography of incubation medium revealed that a high percentage of 11-deoxycorticosterone was converted into corticosterone and 11-dehydrocorticosterone but no aldosterone was detected. Similarly, deoxycortisol was converted into cortisol and cortisone. These results show that eel P450(11beta) does not possess significant aldosterone synthesizing activity. Northern blot analysis detected a 1.8 kb transcript of P450(11beta) using RNA extracted from interrenals of untreated Japanese eel but no hybridization signal was apparent using RNA extracted from brain, spleen, heart, muscle or testis. Immunohistochemistry using an antiserum against P450(11beta) also revealed strong immunostaining in interrenal cells.
Molecular and Cellular Endocrinology 12/1998; 146(1-2):207-11. · 4.19 Impact Factor
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ABSTRACT: We examined the localization of steroidogenic cells in rainbow trout (Oncorhynchus mykiss) testis during spermatogenesis by using polyclonal antibodies generated against rainbow trout cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17alpha-hydroxylase/C17,21 lyase (P450c17), and aromatase cytochrome P450 (P450arom) as markers of steroid production. Since we had previously produced specific antibodies against 3beta-HSD and P450arom, antibodies against oligopeptides corresponding to C-terminal sequences of P450scc and P450c17, predicted from rainbow trout P450scc and P450c17 cDNAs, were produced in this study. These two antibodies recognized 54-kDa (P450scc) and 59-kDa (P450c17) bands specifically in several steroidogenic organs, i.e., testis, ovary, and interrenal tissue (head kidney) in Western blots. Immunohistochemically, immunoreactive P450scc, P450c17, and 3beta-HSD, but not P450arom, were found only in interstitial Leydig cells of immature and mature testes. Immunoreactive P450arom was not detected in either testis. This study suggests that Sertoli cells and germ cells of rainbow trout testis do not contain P450scc, P450c17, P450arom, or 3beta-HSD.
Cell and Tissue Research 07/1998; 292(3):573-7. · 3.11 Impact Factor
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ABSTRACT: A cDNA clone encoding the complete tilapia (a teleost fish, Oreochromis niloticus) cytochrome P450 aromatase (P450arom) was isolated from an ovarian follicle cDNA library. The deduced amino acid sequence (522 amino acid residues) had 72.2% and 59.5% homology with rainbow trout and catfish P450arom respectively, and about 50% homology with mammalian and avian P450arom. Expression of this cDNA in COS-7 cells produced a protein that converted exogenous testosterone to estrogens. Northern blots using a tilapia P450arom cDNA fragment and Western blots using an antiserum against a tilapia P450arom polypeptide fragment revealed a single P450arom mRNA (2.6 kb) and a single protein (59 kDa) in tilapia ovarian tissue respectively. These analyses also revealed that the levels of both P450arom mRNA and protein were low in early vitellogenic follicles, increased in midvitellogenic follicles, and declined to non-detectable levels in post-vitellogenic follicles. Changes in the ability of follicles to convert exogenous testosterone to estrogens (aromatase activity) were similar to those of P450arom mRNA and protein. These observations indicated that the capacity of tilapia ovarian follicles to synthesize estradiol-17 beta is closely related to the contents of P450arom mRNA and protein within them.
Journal of Molecular Endocrinology 03/1997; 18(1):57-66. · 3.48 Impact Factor
