[Show abstract][Hide abstract] ABSTRACT: Interactions between the thyroid hormone (TH) and corticosteroid (CS) hormone axes are suggested to regulate developmental processes in vertebrates with a larval phase. To investigate this hypothesis, we isolated three nuclear receptors from a larval acanthomorph teleost, the red drum (Sciaenops ocellatus), and established their orthologies as thraa, thrb-L and gra-L using phylogenomic and functional analyses. Functional characterization of the TH receptors in COS-1 cells revealed that Thraa and Thrb-L exhibit dose-dependent transactivation of a luciferase reporter in response to T3, while SoThraa is constitutively active at a low level in the absence of ligand. To test whether interactions between the TH and CS systems occur during development, we initially quantified the in vivo receptor transcript expression levels, and then examined their response to treatment with triiodothyronine (T3) or cortisol. We find that sothraa and sothrb-L are autoregulated in response to exogenous T3 only during early larval development. T3 did not affect sogra-L expression levels, nor did cortisol alter levels of sothraa or sothrb-L at any stage. While differential expression of the receptors in response to non-canonical ligand hormone was not observed under the conditions in this study, the correlation between sothraa and sogra-L transcript abundance during development suggests a coordinated function of the TH and CS systems. By comparing the findings in the present study to earlier investigations, we suggest that the up-regulation of thraa may be a specific feature of metamorphosis in acanthomorph teleosts.
General and Comparative Endocrinology 03/2012; 176(1):39-51. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Among the most conserved osmoregulatory hormone systems in vertebrates are the renin-angiotensin system (RAS) and the natriuretic peptides (NPs). We examined the RAS and NP system in the euryhaline Atlantic stingray, Dasyatis sabina (Lesueur). To determine the relative sensitivity of target organs to these hormonal systems, we isolated cDNA sequences encoding the D. sabina angiotensin receptor (AT) and natriuretic peptide type-B receptor (NPR-B). We then determined the tissue-specific expression of their mRNAs in saltwater D. sabina from local Texas waters and an isolated freshwater population in Lake Monroe, Florida. AT mRNA was most abundant in interrenal tissue from both populations. NPR-B mRNA was most abundant in rectal gland tissue from both populations, and also highly abundant in the kidney of saltwater D. sabina. This study is the first to report the sequence of an elasmobranch angiotensin receptor, and phylogenetic analysis indicates that the D. sabina receptor is more similar to AT(1) vs. AT(2) proteins. This classification is further supported by molecular analysis of AT(1) and AT(2) proteins demonstrating conservation of AT(1)-specific amino acid residues and motifs in D. sabina AT. Molecular classification of the elasmobranch angiotensin receptor as an AT(1)-like protein provides fundamental insight into the evolution of the vertebrate RAS.
Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 12/2010; 157(4):423-31. · 1.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rate-limiting and regulated step in steroidogenesis, the conversion of cholesterol to pregnenolone, is facilitated by the steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage (P450scc). We have isolated cDNAs encoding StAR and P450scc from the Atlantic stingray, Dasyatis sabina, and characterized the steroidogenic activity of the encoded proteins using a heterologous expression system. Green monkey kidney (COS-1) cells cotransfected with D. sabina StAR and human P450scc/adrenodoxin reductase/adrenodoxin fusion (F2) constructs produced significantly more pregnenolone than cells transfected with the F2 construct alone. COS-1 cells transfected with a modified F2 construct (F2DS) in which human P450scc is replaced by D. sabina P450scc had higher rates than cells transfected with D. sabina P450scc alone. In other vertebrates, the stress peptide adrenocorticotropic hormone (ACTH) elicits its effects on corticosteroidogenesis in part through regulation of StAR and P450scc mRNAs. In vitro incubation of D. sabina interrenal tissue with porcine ACTH significantly increased intracellular cAMP and corticosteroid production. As demonstrated by quantitative PCR, ACTH also induced significant increases in mRNA abundance of both StAR and P450scc. Our results suggest that, as in higher vertebrates, chronic ACTH-induced glucocorticoid synthesis in elasmobranchs is mediated by regulation of primary steroidogenic mRNAs. This study is the first to demonstrate steroidogenic activity of an elasmobranch P450scc protein and express a composite elasmobranch steroidogenic pathway in a heterologous cell line. Also, the regulation of StAR and P450scc mRNAs has not previously been demonstrated in elasmobranch fishes.
General and Comparative Endocrinology 08/2010; 168(1):121-32. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is thought that a single corticosteroid, 1alpha-hydroxycorticosterone (1alpha-B), is both a glucocorticoid and mineralocorticoid in the elasmobranch fishes. We investigated the putative mineralocorticoid role of 1alpha-B by examining regulation of interrenal 1alpha-B synthesis by osmoregulatory hormones in the euryhaline stingray Dasyatis sabina. Using synthesized steroid, a commercial enzyme-linked immunoassay was validated for the quantification of 1alpha-B. In interrenal cultures, the antinatriuretic peptide angiotensin II (ANG II) was potently steroidogenic, whereas C-type natriuretic peptide had no effect on 1alpha-B titers. However, both peptides significantly decreased abundance of rate-limiting steroidogenic mRNAs (steroidogenic acute regulatory protein, StAR; cholesterol side-chain cleavage, P450scc). We also isolated cDNAs encoding ANG II from three species of elasmobranch, verifying heterogeneity among elasmobranch peptides at the first amino acid position. Potential implications of this heterogeneity were investigated by examining the effects of homologous and heterologous ANG II on interrenal steroid production and steroidogenic mRNAs. Changes at amino acid position three, but not position one, of ANG II significantly affected steroidogenic potency. Conversely, changes at position one, but not position three, significantly affected the potency of ANG II to alter levels of steroidogenic mRNAs. This study is the first to demonstrate regulation of elasmobranch steroidogenic mRNAs by osmoregulatory peptides.
The Journal of steroid biochemistry and molecular biology 03/2010; 120(4-5):149-54. · 3.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The current study sought to clarify the role of cholecystokinin (CCK) in the digestion of larval red drum (Sciaenops ocellatus) in order to better characterize the processes limiting the utilization of microparticulate diets at first feeding. The red drum CCK cDNA, isolated from adult anterior intestine and pyloric caeca, contains a 414 base pair (bp) open reading frame encoding a deduced amino acid sequence of 138 residues which is highly similar to preprocholecystokinin from other vertebrates. The mature CCK octapeptide has the same amino acid sequence as that found in mammals and in Atlantic herring (Clupea harengus). Tissue distribution analysis of adult and juvenile red drum using primers specific for red drum CCK mRNA revealed bright bands in samples from the brain, pyloric caeca, anterior intestine, and gonad with fainter bands seen in all other tissues. Immunohistochemical analysis of larval red drum showed that CCK-immunoreactive (CCK-IR) cells were present as early as 3 days post hatch (DPH) in some fish and were present in all fish by 6 DPH. CCK-IR cells were found in the anterior midgut in early larvae and had spread to the first bend of the gut by day 6. In older larvae (18+ DPH), CCK-IR cells were found in large numbers in the anterior intestine and in the developing pyloric caeca. The sequence and distribution of CCK mRNA along with the presence of CCK-IR cells in early red drum larvae suggest that CCK is present and may be capable of regulating pancreatic secretion in early red drum larvae.
General and Comparative Endocrinology 11/2009; 166(1):152-9. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although cortisol plays an important role in teleost development, the onset of cortisol production and the cortisol stress response in teleosts remain poorly understood. Here we have reported basal cortisol levels and the development of the cortisol stress response in larval red drum (Sciaenops ocellatus). We isolated partial nucleic acid sequences encoding two key corticosteroidogenic enzymes, CYP11B and CYP21 and assessed ontogenetic patterns of their mRNA levels relative to basal and stress-induced cortisol production. Basal cortisol was first detected 3 days post-hatch (DPH) and reached a maximum at 9 DPH. Cortisol did not increase in response to an acute stressor prior to 6 DPH. From 6 DPH forward, stress caused significant increases in larval cortisol content. Stress-induced cortisol levels in 6-9 DPH larvae were highest 1h post-stress. In larvae 11 DPH and older, the highest cortisol measurements occurred 0.5h post-stress. Elevated cortisol was still evident after 3h in 6 DPH larvae. From 11 DPH onward, basal cortisol levels were reestablished in larvae by 1h post-stress. CYP11B and CYP21 transcripts were detected in red drum 12h prior to hatching and in all post-hatch larvae examined. Changes in CYP11B and CYP21 mRNA levels did not occur in association with the ontogenetic appearance of cortisol, or the onset of the stress response. As larvae developed, the dynamics of the cortisol stress response matured from a low magnitude, slow recovery response, to a response similar to that observed in juvenile and adult fish.
General and Comparative Endocrinology 08/2009; 165(2):269-76. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The steroidogenic acute regulatory protein (StAR), a member of the StAR-related lipid transfer domain (START) family, is critical to regulated steroidogenesis in vertebrates. We have isolated a cDNA encoding StAR from a well-studied model of teleost physiology, the Atlantic croaker Micropogonias undulatus. This cDNA (1204 nucleotides total length) contains an open reading frame of 858 nucleotides encoding a protein of 286 amino acids. Molecular phylogenetic analysis indicates the putative Atlantic croaker StAR protein is more closely related to StAR proteins (62-85% identity) than to the related START protein MLN-64 (28-31% identity). Green monkey kidney cells (COS-1) cotransfected with Atlantic croaker StAR and human cholesterol side chain cleavage (SCC) expression constructs are able to produce significantly more pregnenolone than cells transfected with SCC alone. StAR mRNA is detected in the Atlantic croaker head kidney by reverse transcriptase-polymerase chain reaction (RT-PCR) and in the kidney and hypothalamus in some individuals. Gonadal StAR gene expression is below the level of detection by RT-PCR in most individuals, but can be detected using fluorescent probes in quantitative RT-PCR. StAR mRNA is not detected in the Atlantic croaker brain. Six hour in vitro treatment of Atlantic croaker ovarian follicles with human chorionic gonadotropin (hCG) is insufficient to significantly alter StAR mRNA levels; however, 24 h hCG treatment induces StAR mRNA levels 17-fold over untreated controls. Neither 6 nor 24 h treatment with hCG significantly alters StAR mRNA levels in Atlantic croaker testicular minces. Likewise, 6h in vitro treatment with estradiol, testosterone or the maturation-inducing steroid 17,20beta,21-trihydroxy-4-pregnen-3-one is without effect on gonadal StAR mRNA levels.
General and Comparative Endocrinology 03/2007; 150(3):495-504. · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To better define the tissue- and sex-specific roles of aromatase in fishes, we have isolated a CYP19A1 cDNA sequence from a well-developed model of teleost reproduction, the Atlantic croaker (Micropogonias undulatus). This cDNA encodes a protein which has high identity (57-90%) to known CYP19A1 proteins and segregates with teleost CYP19A1 proteins in molecular phylogenetic analysis. In both sexes, the gene encoding Atlantic croaker CYP19A1 is expressed primarily in gonadal tissue, but also in the brain and other tissues at much lower levels, as determined relative to ribosomal 18S RNA expression by real-time quantitative RT-PCR. In females, the highest levels of CYP19A1 mRNA are found in the developing ovary compared to spawning, regressing and resting ovaries. In contrast, testicular CYP19A1 expression is lowest in developing testes and increases in spawning and regressing testes, although there were no statistically significant differences between stages. Brain CYP19A1 mRNA levels are lower in animals with developing gonads compared to spawning fish. In vitro treatment with human chorionic gonadotropin (10 IU/ml) for 6 or 24h increases CYP19A1 mRNA approximately 16- and 43-fold, respectively, in isolated Atlantic croaker ovarian follicles, but has no effect on CYP19A1 mRNA in testicular or brain minces. Six hour in vitro treatment with sex steroids (estradiol, testosterone or 17,20 beta,21-trihydroxy-4-pregnen-3-one; 290 nM) does not alter CYP19A1 mRNA in ovary, testis or brain. The regulation of CYP19A1 in the Atlantic croaker therefore differs in a tissue- and sex-specific manner.
General and Comparative Endocrinology 12/2006; 149(2):205-16. · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interrenal gland (adrenocortical homolog) of elasmobranchs produces a unique steroid, 1alpha-hydroxycorticosterone (1alpha-B). The synthesis of this and most other steroids requires both cholesterol side chain cleavage (CYP11A) and 3beta-hydroxysteroid dehydrogenase (HSD3). To facilitate the study of elasmobranch steroidogenesis, we isolated complementary DNAs encoding CYP11A and HSD3 from the freshwater stingray Potamotrygon motoro. The P. motoro CYP11A (2182 bp total length) and HSD3 (2248 bp total length) cDNAs harbor open reading frames that encode proteins of 542 and 376 amino acids (respectively) that are similar (CYP11A: 39-61% identical; HSD3: 36-53% identical) to their homologs from other vertebrates. In molecular phylogenetic analysis, P. motoro CYP11A segregates with CYP11A proteins (and not with related CYP11B proteins) and P. motoro HSD3 segregates with steroidogenic HSD3 proteins from other fishes. CYP11A and HSD3 mRNA is found only in interrenal and gonadal tissues, indicating de novo steroidogenesis is restricted to these tissues. Because 1alpha-B is thought to act in the elasmobranch response to hydromineral disturbances, we examined the effect of adapting P. motoro to 10 ppt seawater on mRNAs encoding steroidogenic genes. The P. motoro response to this salinity challenge does not include interrenal hypertrophy or an increase in the levels of interrenal CYP11A, HSD3 or steroidogenic acute regulatory protein (StAR) mRNA. This study is the first to isolate full length cDNAs encoding elasmobranch CYP11A and HSD3 and the first to examine the regulation of steroidogenic genes in elasmobranch interrenal cells.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 11/2006; 145(3-4):306-17. · 1.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The steroidogenic acute regulatory protein (StAR) is critical to the regulated synthesis of steroids in vertebrates. We have isolated cDNA sequences encoding StAR in the freshwater stingrays Potamotrygon hystrix and P. motoro. A single P. hystrix StAR transcript (3376 bp) and two overlapping P. motoro StAR transcripts (1272 and 3365 bp) were isolated. The P. hystrix and P. motoro StAR transcripts contain open reading frames encoding proteins of 284 amino acids that are 99% identical to each other and 56-64% identical to other StAR proteins. Pregnenolone synthesis by green monkey kidney (COS-1) cells transfected with an expression construct encoding a human cholesterol side chain cleavage/adrenodoxin reductase/adrenodoxin fusion protein was increased 16-fold by coexpression with a pCMV5/P. motoro StAR expression construct. Northern blot analysis revealed a single 4000 bp StAR transcript in the P. motoro interrenal gland, but RT-PCR indicates StAR mRNA is also expressed in the brain, gonads, atria, ventricle, gill (female only) and muscle (female only). Expression in extragonadal and extraadrenocortical tissues is an indication that StAR may be critical to processes other than steroidogenesis. The longest P. motoro StAR transcript contains a sequence with great similarity to short interspersed repetitive elements found in other elasmobranchs. This study is the first to isolate and characterize elasmobranch StAR cDNA sequences and to demonstrate the activity of a nonmammalian StAR protein in a heterologous expression system.
Journal of Molecular Endocrinology 01/2006; 35(3):557-69. · 3.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alternative splicing of exon 9 in human glucocorticoid receptor (hGR) transcripts yields two native hGR transcripts and proteins, hGRalpha and hGRbeta. We have now identified four novel hGRalpha transcripts that have various deletions of exon 2 sequences. Among these hGRalpha splice variants, three of them, 1A1/E2dist hGRalpha, 1A2/E2prox hGRalpha, and 1A3/E3 hGRalpha, arise from the hGR 1A promoter, while 1B/E3 hGRalpha comes from the hGR 1B promoter. When fused to Flag and enhanced green fluorescent protein (EGFP) tags at the carboxy terminus, all transcript variants can be correctly translated in vitro and in vivo. The Flag-tagged hGRalpha protein variants can functionally bind to a glucocorticoid response element (GRE) and can mediate hormonal stimulation of a pGRE-luciferase reporter gene. Compared to the "classical", native hGRalpha, these four variants exhibit a cell type-specific activation of a reporter gene, and this is influenced by the hGRalpha 3' untranslated region in the hGR transcript. When equal amounts of the cDNAs for these GRalpha variant proteins are transfected into cells, they can exhibit lower or higher transcriptional activation compared to the classical GR. Furthermore, the EGFP-tagged proteins are nuclear localized, even in the absence of hormone. Using quantitative reverse transcription PCR, we found that these transcripts exist at a low level in CEM-C7 cells and IM-9 cells, although the concentrations of the 1A3/E3 hGRalpha and 1B/E3 hGRalpha transcripts are higher than for hGRbeta transcripts, while 1A1/E2dist hGRalphaand 1A2/E2prox hGRalpha transcript levels are comparable to the 1A1 hGRalpha and 1A2 hGRalpha (without the exon 2 deletions) transcript levels, respectively. Because these novel hGR, N-terminal deleted, protein variants have altered biological activity, their expression could potentially affect the hormone sensitivity or resistance in leukemia and be useful in diagnosing hormone-sensitive or -resistant disease.
[Show abstract][Hide abstract] ABSTRACT: The newly described 1A promoter of the human glucocorticoid receptor (hGR) gene contains an interferon (IFN) regulatory factor element (IRF-E), a binding motif for the family of proteins termed IFN regulatory factors (IRFs) that are regulated by IFNs. To examine the in vivo role of IFNs in hGR gene regulation, human T cell lines (CEM-C7 and Jurkat) were treated with IFN gamma. IFN gamma rapidly induces the expression of IRF-1 proteins in a dose- and time-dependent manner. Luciferase expression is induced by IFN treatment in Jurkat cells transfected with an hGR 1A promoter IRF-E/luciferase reporter gene, but induction is lost with deletion of the IRF-E. Electrophoretic mobility shift and supershift analyses indicate an increase in the binding of IRF-1 to oligonucleotides containing the hGR 1A promoter IRF-E after IFN gamma treatment, whereas IRF-2 binding to this oligonucleotide is unchanged. Human IRF-1 and IRF-2 proteins expressed in Chinese hamster ovary cells bind to the hGR 1A promoter IRF-E; however, only IRF-1 activates transcription. Although IFNs clearly activate a transfected reporter gene containing the hGR 1A promoter in T cells, they do not alter the sensitivity of CEM-C7 cells to glucocorticoid-induced apoptosis. Additional studies revealed that the glucocorticoid steroid hormone, dexamethasone (DEX), completely blocks IFN induction of IRF-1 mRNA levels. This could explain the lack of any greater apoptotic response to a combination of DEX plus IFN compared with the response to DEX alone. In addition, treatment with IFN gamma alone does not alter endogenous GR mRNA levels (including exon 1A-containing transcripts derived from the hGR 1A promoter) in T lymphoblast cells, even though IRF-1 levels are induced. The difference in IRF-1-driven transcription between the hGR 1A reporter construct and the endogenous hGR 1A promoter could potentially be due to epigenetic effects, such as methylation.
[Show abstract][Hide abstract] ABSTRACT: At least three different promoter regions (1A, 1B, and 1C) are involved in the expression of the human GR gene. Promoters 1B and 1C are found in a 2800 bp region of DNA immediately upstream of the exon 1C transcriptional initiation site. Transcripts containing either exon 1B or 1C are expressed in a wide variety of human tissues and cultured cells. Luciferase reporter constructs were created containing promoter 1B plus 1C (-2738 to +19), promoter 1B (-2738 to -1046) alone, or promoter 1C (-1045 to +19) alone. All three constructs were equally effective in driving luciferase expression in HeLa (human cervical carcinoma) cells. In Jurkat (human T-cell acute lymphoblastic leukemia) cells, constructs containing promoters 1B plus 1C or promoter 1B were equally active, but the promoters 1B plus 1C construct was 35% more active than the promoter 1C construct. However, in HepG2 (human hepatoma) cells, the promoter 1C construct was as effective as promoters 1B plus 1C and more than twice as effective as promoter 1B. Sequences that reside proximal to the exon 1B transcriptional start site included three Sp1 (FP2-FP4) sites. Another site (FP1) contains the sequence TGATAG, which strongly resembles the consensus binding sequence for the GATA family of transcription factors. However, oligonucleotide competition and supershift analysis of FP1 indicates that this site is not a binding site for GATA proteins. These four sites are in addition to three YY1 and one Sp1 sites previously reported in promoter 1B. In HeLa cells, deletion of the three YY1 sites results in only a 30% loss of activity and substantial loss of activity occurs only after deletion of all four Sp1 sites, indicating the critical importance of Sp1 in GR expression in these cells. In contrast, the elimination of the three YY1 sites results in a dramatic decrease in promoter strength in both HepG2 and Jurkat cells (64 and 77%, respectively), while subsequent deletions of promoter elements do not result in substantial changes in promoter activity in these cell lines. This study shows that both promoters 1B and 1C are important for the ubiquitous expression of the human GR gene. Differences in the utilization of these promoters in various cell types are likely a reflection of different promoter availability and/or the levels of specific transcription factors in the cell. This could contribute to tissue-specific expression of GR levels in different cell types.
Molecular and Cellular Endocrinology 04/2002; 189(1-2):191-9. · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Traditionally, sciaenid eggs have been identified based on morphological characteristics such as size, number of oil globules
and/or pigmentation patterns. Identification of sciaenid eggs by these procedures is time consuming and often inaccurate due
to considerable egg size overlap among species. The utilization of molecular techniques for the identification of economically
important species has become a fundamental component in ecological studies involving fish eggs and larvae. This study reports
the development of a series of both multiplex and individual polymerase chain reactions to identify the eggs of 11 sciaenid
species commonly found in the Gulf of Mexico and estuaries near Port Aransas and Corpus Christi, TX, USA. Following method
development, the discriminatory power of the assay was first determined with samples from adult fish collected from Aransas
and Corpus Christi Bays, Galveston Bay and the lower Laguna Madre in northern Mexico. Most (97%) of these fishes were correctly
identified to the level of species. To demonstrate the applicability of the assay, wild fish eggs were collected and analyzed
from the Aransas Pass tidal inlet from September through December 2005. During this period, the eggs of four target species
were positively identified which was in keeping with current knowledge regarding the spawning areas and seasons of these sciaenids
based on the presence of mature females, eggs and/or larvae. Future use of this method can provide valuable information to
better discriminate spawning sites and seasons of these species.