RESUMO
Members of the transforming growth factor-beta superfamily, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), have crucial roles in primary follicle growth in mammals. To initiate investigations into their significance in teleost oogenesis, we set out to clone and characterise the cDNAs of gdf9 and bmp15 and analysed their patterns of gene expression during the ovarian reproductive cycle in the European sea bass (Dicentrachus labrax). Sea bass gdf9 and bmp15 cDNAs were 2200 and 2049 bp long, coding for 438 and 459 amino acids (aas), respectively, and were most similar to zebrafish gdf9 and bmp15 (64.4 and 56.1%, respectively). By Northern analysis, sea bass gdf9 and bmp15 mRNA transcripts were detected in the ovary only of the tissues analysed and their sizes were 2.2 and 2.1 kb, respectively. Dot-blot analysis revealed high levels of gdf9 and bmp15 expression in the ovary during primary oocyte growth and previtellogenesis (July to October), with a significant decline at the onset of vitellogenesis (November) and remaining low until the beginning of new oocyte growth (April/May). There was a highly significant positive correlation (r=0.939) between gdf9 and bmp15 gene expression in individual samples. The high levels of gdf9 and bmp15 mRNA transcripts in the ovary, especially during the previtellogenic growth period suggest an important role for these factors in early primary oocyte growth in the European sea bass.
Assuntos
Bass/fisiologia , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ciclo Menstrual/fisiologia , Ovário/fisiologia , Sequência de Aminoácidos , Animais , Bass/anatomia & histologia , Proteína Morfogenética Óssea 15 , Feminino , Fator 9 de Diferenciação de Crescimento , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/classificação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Dados de Sequência Molecular , Filogenia , Reprodução/fisiologia , Alinhamento de SequênciaRESUMO
In mammals, a multitude of studies have shown that anti-Müllerian hormone (AMH/AMH), apart from inducing Müllerian duct regression during male sexual differentiation, exerts inhibitory effects on male and female gonadal steroidogenesis and differentiation. However, in lower vertebrates like teleost fish, the function of AMH/AMH has been far less explored. As a first step to unravel its potential role in reproduction in teleost fish, we isolated and characterised the AMH gene in the European sea bass (sb), Dicentrachus labrax, determined putative regulatory elements of its 5'-flanking region, and analysed its gene expression and those of alternatively-spliced transcripts. The characterisation of sb-AMH revealed distinct features that distinguishes it from mammalian and bird AMH, suggesting a high rate of diversification of AMH during vertebrate evolution. It contained 7 exons that were divided by 6 introns, of which the last intron (intron vi) was localised only a few nucleotides upstream of the putative peptide cleavage site. The guanine and cytosine content of the open reading frame (ORF) was 52.7% and thus notably lower than that of bird and mammalian AMH. Sb-AMH cDNA was 2045 base pairs (bp) long, containing an ORF of 1599 bp encoding 533 amino acids. Deduced amino acid similarities of the conserved, carboxyterminal domain were highest with AMH in Japanese flounder (84.2%) and lowest with chicken AMH (45.5%). In the proximal promoter sequence of sb-AMH, a steroidogenic factor-1 (SF-1) binding site was present; however other regulatory sequences essential for transcriptional activation of AMH in mammals were absent. Likewise, there was no sequence homology to an SF3A2 sequence within the first 3200 bp upstream of the sb-AMH translation start site. Gene expression of sb-AMH and of alternatively-spliced sb-AMH transcripts were analysed in male and female juvenile and adult gonads as well as in somatic tissues of juvenile males. sb-AMH expression was highest in juvenile testis, but still remarkably high in juvenile ovaries and adult testis, as well as in brain, pituitary, and heart of juvenile male sea bass. Apart from adult ovary, levels of alternatively-spliced sb-AMHexonII/-99 were marginal in comparison with sb-AMH. In contrast, the transcript variant sb-AMHexonVII/+5 was expressed to a similar extent as sb-AMH in all tissues examined. The results of this work have provided the basis for future studies concerning the regulation and function of AMH/AMH in this species.
Assuntos
Processamento Alternativo , Bass/genética , Perfilação da Expressão Gênica , Glicoproteínas/genética , Hormônios Testiculares/genética , Região 5'-Flanqueadora , Sequência de Aminoácidos , Animais , Hormônio Antimülleriano , Sítios de Ligação , Clonagem Molecular , Éxons , Feminino , Íntrons , Masculino , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
AIM: To determine whether the ultrastructure of the capillary system in human skeletal muscle changes during advancing senescence, we evaluated the compartmental and subcompartmental organization of capillaries from vastus lateralis muscle (VL) biopsies of 41 non-diseased persons aged 23-75 years. METHODS: From each VL biopsy, 38-40 randomly selected capillaries were assessed by transmission electron microscopy and subsequent morphometry with a newly established tablet-based image analysis technique. RESULTS: Quantification of the compartmental organization revealed most indicators of the capillary ultrastructure to be only non-significantly altered (P > 0.05) over age. However, the peri-capillary basement membrane (BM) was thicker in the older participants than in the younger ones (P ≤ 0.05). Regression analysis revealed a bipartite relationship between the two parameters: a homogenous slight increase in BM thickness up to the age of approximately 50 years was followed by a second phase with more scattered BM thickness values. In 44.5% of the capillary profiles, projections/filopodia of the pericytes (PCs) traversed the BM and invaded endothelial cells (ECs) visible as PC pegs in pale cytoplasm holes (EC sockets). Strikingly, PC pegs were often in proximity to the EC nucleus. In PC profiles, sockets were likewise detected in 14.2% of the capillaries. Within these PC sockets, cellular profiles were frequently seen, which could be assigned to EC filopodia, internal PC curling or PC-PC interactions. Quantification of the occurrence of peg-socket junctions revealed the proportions of empty EC sockets and empty PC sockets to increase (P ≤ 0.05) during ageing. CONCLUSION: Our investigation demonstrates advancing senescence to be associated with increase in BM thickness and loss of EC and PC filopodia length in skeletal muscle capillaries.
Assuntos
Envelhecimento/fisiologia , Capilares/ultraestrutura , Músculo Esquelético/irrigação sanguínea , Adulto , Idoso , Membrana Basal/ultraestrutura , Citoplasma/ultraestrutura , Células Endoteliais/fisiologia , Células Endoteliais/ultraestrutura , Feminino , Humanos , Hipertensão/patologia , Processamento de Imagem Assistida por Computador , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Músculo Esquelético/fisiologia , Neovascularização Fisiológica/fisiologia , Pericitos/fisiologia , Doença Arterial Periférica/patologia , Adulto JovemRESUMO
Three oestrogen receptor [ER] subtypes have been described in teleost fish, namely ERalpha, and two ERbeta subtypes, called ERbeta1 and ERbeta2 (or ERbeta and ERgamma in Atlantic croaker). Their expression during embryonic development and gonadal growth has evoked interest in their potential role in sexual differentiation and gonadal development in fish. We cloned three oestrogen receptors from adult liver (sb-ERalpha cDNA) and ovary (partial sb-ERbeta1 and sb-ERbeta2 cDNAs) of the European sea bass, and according to their phylogenetic relatedness to other ERs in teleosts, named them sea bass [sb-] ERalpha, ERbeta1 and ERbeta2. Deduced amino acid numbers for sb-ERalpha, sb-ERbeta1 and sb-ERbeta2 were 639, 517 and 608, respectively, representing in the case of sb-ERbeta1 and sb-ERbeta2 about 90% of the open reading frame. Highest amino acid identities were found for sb-ERalpha with eelpout ERalpha (88.7%), for sb-ERbeta1 with Atlantic croaker ERgamma (85.8%), and for sb-ERbeta2 with Atlantic croaker ERbeta (90.1%). Southern analysis confirmed that all three sea bass oestrogen receptors (sb-ERs) are the products of three distinct genes. In adult sea bass, ERalpha was predominantly expressed in liver and pituitary, while sb-ERbeta1 and sb-ERbeta2 were more ubiquitously expressed, with highest expression levels in pituitary. In a mixed-sex population of juvenile sea bass, sb-ERalpha expression was significantly elevated in gonads at 200 days posthatch (dph), while for sb-ERbeta1 and sb-ERbeta2 highest expression levels were observed in gonads at 250 dph. For sb-ERbeta2, expression was also significantly higher in the brain at 250 dph. The cloning of these three ER subtypes in the European sea bass together with the results obtained on expression levels in adult and juvenile animals has given us the foundation to investigate their possible role in sexual differentiation and development in this species in future studies.
Assuntos
Bass/genética , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Gônadas/metabolismo , Sequência de Aminoácidos , Animais , Bass/metabolismo , Clonagem Molecular , Receptor alfa de Estrogênio/classificação , Receptor alfa de Estrogênio/fisiologia , Receptor beta de Estrogênio/classificação , Receptor beta de Estrogênio/fisiologia , Feminino , Expressão Gênica , Masculino , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Diferenciação Sexual , Fatores Sexuais , Distribuição TecidualRESUMO
Oestrogens are key regulators in sexual differentiation and development in higher vertebrates. P450 aromatase (p450arom) is the steroidogenic enzyme responsible for the synthesis of oestrogens from aromatisable androgens. Effects of endocrine disrupting chemicals on steroidogenic enzyme gene expression have received little attention so far, yet it is potentially a major pathway for sexual disruption. In this 14-day study the effects of exogenous 17beta-oestradiol (E2) at environmentally relevant concentrations were assessed on gene expression of p450aromB in the gonad and brain of maturing male and female fathead minnows (FHM). Exposure to E2 resulted in an oestrogenic response as shown by a dose-dependent induction of plasma vitellogenin (VTG) in female and male fish and a dose-dependent inhibition of testis growth. There was an effect of exposure to E2 on p450aromB mRNA expression in the gonads; E2 up-regulated p450aromB mRNA expression in the testis and ovary in a dose-response manner after 14 days of exposure. In male brain, p450aromB mRNA concentrations were significantly reduced in fish exposed to 100 and 320 ng E2/l on day 4, but on day 14 were elevated in males exposed to both 32 and 100 ng E2/l. No effects of E2 on p450aromB mRNA expression occurred in the brain of females. The results of this study show that concentrations of E2 found in the environment can have disruptive effects on key steroidogenic enzyme pathways that control sexual development in fish.
Assuntos
Aromatase/genética , Cyprinidae/metabolismo , Estradiol/farmacologia , RNA Mensageiro/biossíntese , Vitelogeninas/biossíntese , Animais , Aromatase/biossíntese , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Cyprinidae/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Masculino , Ovário/efeitos dos fármacos , Ovário/enzimologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Maturidade Sexual , Testículo/efeitos dos fármacos , Testículo/enzimologia , Vitelogeninas/sangue , Vitelogeninas/genéticaRESUMO
N-vinylpyrrolidone dimer (VPD), a novel vehicle for preclinical toxicity studies, was evaluated in a standard 28-day oral toxicity study in rats including a 4 week recovery period. In addition, a subgroup of animals was dosed for 13 weeks. In the 28-day study arm, daily dosages of 0 (saline control, 3mL/kg), 300, 1000 or 3000mg/kg at respective dose volumes of 0.3, 1 and 3mL/kg were administered. In the 13 week study arm, animals received daily doses of 0 or 300mg/kg. No test item related mortality or changes in body weight, food consumption, ophthalmology and clinical pathology parameters were observed after 28-days or 13 weeks of administration. VPD induced transient salivation at all tested dose levels after each dose and increased water consumption at doses ≥1000mg/kg (28-day arm). After 28-days of administration, urinalysis revealed slightly higher mean specific gravity in all treated groups. Relevant organ weight changes consisted of increased mean liver weights. Histopathology revealed hepatocellular centrilobular hypertrophy at a dose-related incidence and severity, minimal to slight follicular cell hypertrophy in male thyroids, hypertrophy of basophil/chromophobe cells in pituitaries and an increase in kidney hyaline droplets consistent with α(2µ)-globuline immunohistochemistry. After a 4-week recovery, all changes were partially or completely reversible. Administration of VPD at 300mg/kg for 13 weeks caused similar histopathological findings observed after 28-days dosing. Overall, in rats, repeated high oral doses of VPD produced changes in the liver, thyroid and pituitary, most likely secondary to hepatic microsomal enzyme induction and stimulation of the thyroid via disruption of the hypothalamic-pituitary-thyroid axis. In conclusion, our data suggest that VPD is a promising vehicle for preclinical studies. However, in rats, findings secondary to hepatic microsomal enzyme induction and stimulation of the thyroid need to be taken into consideration, depending on dose and study duration. In order to develop VPD as a preclinical excipient, additional preclinical toxicity studies (non-rodents, different administration routes, chronic and reproductive toxicity) would be beneficial.
Assuntos
Excipientes/toxicidade , Pirrolidinonas/toxicidade , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Dimerização , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/patologiaRESUMO
Short-circuit current (I(sc)) and transepithelial conductance (Gt) were measured in guinea pig distal colonic mucosa isolated from submucosa and underlying muscle layers. Indomethacin (2 microM) and NS-398 (2 microM) were added to suppress endogenous production of prostanoids. Serosal addition of PGE2 (10 nM) stimulated negative I(sc) consistent with K secretion, and concentrations >30 nM stimulated positive I(sc) consistent with Cl secretion. PGE2 also stimulated Gt at low and high concentrations. Dose responses to prostanoids specific for EP prostanoid receptors were consistent with stimulating K secretion through EP2 receptors, based on a rank order potency (from EC50 values) of PGE2 (1.9 nM) > 11-deoxy-PGE1 (8.3 nM) > 19(R)-hydroxy-PGE2 (13.9 nM) > butaprost (67 nM) > 17-phenyl-trinor-PGE2 (307 nM) >> sulprostone (>10 microM). An isoprostane, 8-iso-PGE2, stimulated K secretion with an EC50 of 33 nM. Cl secretory response was stimulated by PGD2 and BW-245C, a DP prostanoid receptor-specific agonist: BW-245C (15 nM) > PGD2 (30 nM) > PGE2 (203 nM). Agonists specific for FP, IP, and TP prostanoid receptors were ineffective in stimulating I(sc) and Gt at concentrations <1 microM. These results indicate that PGE2 stimulated electrogenic K secretion through activation of EP2 receptors and electrogenic KCl secretion through activation of DP receptors. Thus stimulation of Cl secretion in vivo would occur either via physiological concentrations of PGD2 (<100 nM) or pathophysiological concentrations of PGE2 (>100 nM) that could occur during inflammatory conditions.
Assuntos
Cloretos/metabolismo , Colo/fisiologia , Dinoprostona/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Potássio/metabolismo , Xantonas , Animais , Bumetanida/farmacologia , Colo/anatomia & histologia , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/química , Diuréticos/farmacologia , Relação Dose-Resposta a Droga , Cobaias , Humanos , Técnicas In Vitro , Indometacina/farmacologia , Mucosa Intestinal/metabolismo , Masculino , Antagonistas de Prostaglandina/farmacologia , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/metabolismo , Xantenos/farmacologiaRESUMO
Distal colon from guinea pig was stimulated in vitro by aldosterone in Ussing chambers that allowed measurement of short-circuit current (Isc) and tissue conductance (Gt). The response to aldosterone was delayed by approximately 20 min and resulted in a negative Isc, consistent with K secretion. Approximately 1 h later the Isc began to increase and eventually became positive, consistent with subsequent stimulation of Na absorption. The Na-absorptive response could be inhibited by mucosal amiloride without altering the rate of K secretion. Similarly, K secretion could be inhibited by serosal bumetanide without altering Na absorption. In the presence of spironolactone, actinomycin D, or cycloheximide, aldosterone failed to stimulate both K secretion and Na absorption. A dose response to aldosterone provided an apparent Kd of 2.6 +/- 0.5 nM, consistent with a high-affinity receptor coupled to this secretory response. Stimulation by the K secretagogue epinephrine did not produce an additive increase in K secretion, suggesting that the same cell type responds to both aldosterone and epinephrine and that the protein induced by aldosterone was not one of the membrane proteins responsible for K secretion.
Assuntos
Aldosterona/farmacologia , Colo/metabolismo , Absorção Intestinal/efeitos dos fármacos , Potássio/metabolismo , Sódio/farmacocinética , Amilorida/farmacologia , Animais , Bumetanida/farmacologia , Cobaias , Masculino , Biossíntese de Proteínas , Receptores de Mineralocorticoides/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 microM) or histamine (10 microM) released contents from goblet granules. Stimulation with prostaglandin E(2) (PGE(2), 5 microM) or adenosine (10 microM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine ( approximately 160 fl/cell), but not with PGE(2) or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE(2) stimulated a prolonged fluid secretion that attained a rate of approximately 350 pl/min. Columnar cells lost approximately 50% of apical volume during maximal PGE(2) stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores.
Assuntos
Colecistocinina/farmacologia , Colo/citologia , Células Caliciformes/classificação , Células Caliciformes/metabolismo , Adenosina/farmacologia , Transporte Biológico/efeitos dos fármacos , Líquidos Corporais/metabolismo , Carbacol/farmacologia , Tamanho Celular , Agonistas Colinérgicos/farmacologia , Grânulos Citoplasmáticos/metabolismo , Dinoprostona/metabolismo , Células Caliciformes/efeitos dos fármacos , Histamina/farmacologia , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Matemática , Microscopia de Interferência , Microtomia/métodos , Muco/metabolismoRESUMO
Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 microM) or histamine (10 microM) released contents from goblet granules. Stimulation with prostaglandin E2 (PGE2, 5 microM) or adenosine (10 microM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine (approximately 160 fl/cell), but not with PGE2 or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE2 stimulated a prolonged fluid secretion that attained a rate of approximately 350 pl/min. Columnar cells lost approximately 50% of apical volume during maximal PGE2 stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores.
Assuntos
Colo/citologia , Colo/metabolismo , Células Caliciformes/metabolismo , Adenosina/farmacologia , Adulto , Idoso , Líquidos Corporais/metabolismo , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Colo/anatomia & histologia , Colo/efeitos dos fármacos , Dinoprostona/farmacologia , Feminino , Células Caliciformes/efeitos dos fármacos , Histamina/farmacologia , Humanos , Mucosa Intestinal/anatomia & histologia , Masculino , Pessoa de Meia-Idade , Muco/metabolismoRESUMO
Photoluminescence (PL) spectroscopy with subwavelength lateral resolution has been employed to probe individual localization centers in a thin InGaN/GaN quantum well. Spectrally narrow emission lines with a linewidth as small as 0.8 meV can be resolved, originating from the recombination of an electron-hole pair occupying a single localized state. Surprisingly, the individual emission lines show a pronounced blueshift when raising the temperature, while virtually no energy shift occurs for increasing excitation density. These findings are in remarkable contrast to the behavior usually found in macro-PL measurements and give a fundamental new insight into the recombination process in semiconductor nanostructures in the presence of localization and strong internal electric fields. We find clear indications for a biexciton state with a negative binding energy of about -5+/-0.7 meV.
RESUMO
The ability of the antagonists tolazoline, yohimbine and the combination of yohimbine with 4-aminopyridine to reverse the effects of the xylazine-component of the "Hellabrunn mixture" (125 mg/ml xylazine and 100 mg/ml ketamine) on nondomestic zoo ruminants is discussed. Arousal time, recovery time and changes in the parameter of circulatory and respiratory functions after antagonization are shown. Tolazoline is able to antagonize the xylazine effect completely within a short time. Using a dosage of 3-5 mg/kg there is a marked negative effect on the cardio-vascular system. Yohimbine in the used dosage of 0.25-0.3 mg/kg in non-domestic ruminants did not approve in its effects. Combining yohimbine (0.25-0.3 mg) with 4-aminopyridine (0.5 mg/kg) recovery time is about 30 minutes. The negative effect on the cardio-vascular system is less pronounced compared with tolazoline.
Assuntos
Anestesia/veterinária , Animais de Zoológico/fisiologia , Ketamina/antagonistas & inibidores , Ruminantes/fisiologia , Tiazinas/antagonistas & inibidores , Xilazina/antagonistas & inibidores , 4-Aminopiridina , Aminopiridinas/farmacologia , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Tolazolina/farmacologia , Ioimbina/farmacologiaRESUMO
Stimulation of Cl secretion by prostaglandin E2 (PGE2) was measured as the short-circuit current (Isc) across isolated epithelium of the rabbit distal colon. Cellular morphology of columnar and goblet cells during secretion was monitored using light and electron microscopy. Stimulation by PGE2 altered epithelial cell morphology only by a reduction of vacuolar space in the apical pole of crypt columnar cells, consistent with release of vacuole contents. Imaging of isolated crypts using differential interference microscopy confirmed the release of material from columnar cells during the onset of secretion. Inhibition of Cl secretion with the loop diuretic bumetanide did not block vacuole release. The actin filament-disrupting agent, cytochalasin, reduced the PGE2-stimulated Isc by 40% and blocked emptying of the vacuolar space. These electrical and morphological results indicate that the process of active ion secretion is associated with release of the macromolecular contents from apical vacuoles through a mechanism involving the cytoskeleton. In addition, this relationship supports the concept that vacuolated columnar cells of the crypts of Lieberkühn are the cell type that secretes Cl in response to PGE2.
Assuntos
Cloretos/metabolismo , Colo/metabolismo , Mucosa Intestinal/metabolismo , Animais , Cloretos/antagonistas & inibidores , Colo/citologia , Citocalasina B/farmacologia , Dinoprostona/farmacologia , Eletroquímica , Feminino , Mucosa Intestinal/citologia , Microscopia Eletrônica , Microvilosidades/metabolismo , CoelhosRESUMO
P450 17alpha-hydroxylase,17,20-lyase (P450c17) is a key steroidogenic enzyme in the production of androgens and, therefore, is also indispensable for the production of oestrogens (that are produced from the aromatisation of androgens). In this study, P450c17 cDNA was cloned from the ovary of the fathead minnow (FHM) and its gene expression was examined in the gonads and brains of male and female FHM at different stages of gonadal development with a view to developing an understanding of its involvement in the reproductive physiology in this species. The FHM-P450c17 cDNA sequence cloned was 1812 bp in length, with an open reading frame of 1554 nucleotides encoding a protein of 518 amino acids. Amino acid identity of FHM-P450c17 with P450c17s in other animals was up to 81.8% in other teleosts (channel catfish), 62% in elasmobranches (spiny dogfish), 64% in birds (chicken), and up to 48.8% in mammals (human). FHM-P450c17 gene expression occurred in the ovary, testis, and also in the brain (both male and female) at all stages of sexual development studied. Expression in the brain was at least 30-fold lower than in the gonads, but consistent in all fish life stages studied. In the testis, FHM-P450c17 gene expression was negatively correlated with gonadal development, but there was no obvious association between P450c17 gene expression and sexual development in the ovary, or brain (in both males and females). The results from this study demonstrate the expression of P450c17 in the brain for the first time in fish. Enzymatic studies are now needed to investigate the possible role of P450c17 in neurosteroid production in teleosts.