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1.
J Steroid Biochem Mol Biol ; 127(3-5): 176-88, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21514383

ABSTRACT

Classically, the estrogen signaling system has two core components: cytochrome P450 aromatase (CYP19), the enzyme complex that catalyzes the rate limiting step in estrogen biosynthesis; and estrogen receptors (ERs), ligand activated transcription factors that interact with the regulatory region of target genes to mediate the biological effects of estrogen. While the importance of estrogens for regulation of reproduction, development and physiology has been well-documented in gnathostome vertebrates, the evolutionary origins of estrogen as a hormone are still unclear. As invertebrates within the phylum Chordata, cephalochordates (e.g., the amphioxus of the genus Branchiostoma) are among the closest invertebrate relatives of the vertebrates and can provide critical insight into the evolution of vertebrate-specific molecules and pathways. To address this question, this paper briefly reviews relevant earlier studies that help to illuminate the history of the aromatase and ER genes, with a particular emphasis on insights from amphioxus and other invertebrates. We then present new analyses of amphioxus aromatase and ER sequence and function, including an in silico model of the amphioxus aromatase protein, and CYP19 gene analysis. CYP19 shares a conserved gene structure with vertebrates (9 coding exons) and moderate sequence conservation (40% amino acid identity with human CYP19). Modeling of the amphioxus aromatase substrate binding site and simulated docking of androstenedione in comparison to the human aromatase shows that the substrate binding site is conserved and predicts that androstenedione could be a substrate for amphioxus CYP19. The amphioxus ER is structurally similar to vertebrate ERs, but differs in sequence and key residues of the ligand binding domain. Consistent with results from other laboratories, amphioxus ER did not bind radiolabeled estradiol, nor did it modulate gene expression on an estrogen-responsive element (ERE) in the presence of estradiol, 4-hydroxytamoxifen, diethylstilbestrol, bisphenol A or genistein. Interestingly, it has been shown that a related gene, the amphioxus "steroid receptor" (SR), can be activated by estrogens and that amphioxus ER can repress this activation. CYP19, ER and SR are all primarily expressed in gonadal tissue, suggesting an ancient paracrine/autocrine signaling role, but it is not yet known how their expression is regulated and, if estrogen is actually synthesized in amphioxus, whether it has a role in mediating any biological effects. Functional studies are clearly needed to link emerging bioinformatics and in vitro molecular biology results with organismal physiology to develop an understanding of the evolution of estrogen signaling. This article is part of a Special Issue entitled 'Marine organisms'.


Subject(s)
Chordata/metabolism , Estrogens/metabolism , Evolution, Molecular , Signal Transduction , Amino Acid Sequence , Animals , Aromatase/chemistry , Aromatase/genetics , Aromatase/metabolism , Chordata/genetics , Cloning, Molecular , Crystallography, X-Ray , DNA, Complementary , Humans , Models, Molecular , Molecular Sequence Data , Polymerase Chain Reaction/methods , Protein Binding , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Sequence Homology, Amino Acid , Transcription, Genetic
2.
J Mol Endocrinol ; 37(1): 105-20, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16901928

ABSTRACT

The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to the estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the diversity, function, and regulation of ERRs in non-mammalian species are not well understood. In this study, we report the cloning of four ERR cDNAs from the Atlantic killifish, Fundulus heteroclitus, along with adult tissue expression and estrogen responsiveness. Phylogenetic analysis indicates that F. heteroclitus (Fh)ERRalpha is an ortholog of the single ERRalpha identified in mammals, pufferfish, and zebrafish. FhERRbetaa and FhERRbetab are co-orthologs of the mammalian ERRbeta. Phylogenetic placement of the fourth killifish ERR gene, tentatively identified as FhERRgammab, is less clear. The four ERRs showed distinct, partially overlapping mRNA expression patterns in adult tissues. FhERRalpha was broadly expressed. FhERRbetaa was expressed at apparently low levels in eye, brain, and ovary. FhERRbetab was expressed more broadly in liver, gonad, eye, brain, and kidney. FhERRgammab was expressed in multiple tissues including gill, heart, kidney, and eye. Distinct expression patterns of FhERRbetaa and FhERRbetab are consistent with subfunctionalization of the ERRbeta paralogs. Induction of ERRalpha mRNA by exogenous estrogen exposure has been reported in some mammalian tissues. In adult male killifish, ERR expression did not significantly change following estradiol injection, but showed a trend toward a slight induction (three- to five-fold) of ERRalpha expression in heart. In a second, more targeted experiment, expression of ERRalpha in adult female killifish was downregulated 2.5-fold in the heart following estradiol injection. In summary, our results indicate that killifish contain additional ERR genes relative to mammals, including ERRbeta paralogs. In addition, regulation of ERRalpha expression in killifish apparently differs from regulation in mammals. Together, these features may facilitate determination of both conserved and specialized ERR gene functions.


Subject(s)
Estrogens/metabolism , Fundulidae/metabolism , Protein Isoforms/metabolism , Receptors, Estrogen/metabolism , Amino Acid Sequence , Animals , Female , Fundulidae/anatomy & histology , Humans , Likelihood Functions , Male , Molecular Sequence Data , Phylogeny , Protein Isoforms/genetics , Receptors, Estrogen/classification , Receptors, Estrogen/genetics , Sequence Alignment , Tissue Distribution
3.
J Steroid Biochem Mol Biol ; 78(5): 427-39, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11738553

ABSTRACT

Teleost fish are characterized by exceptionally high levels of neural estrogen biosynthesis when compared with the brains of other vertebrates or to the ovaries of the same fish. Two P450arom mRNAs which derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (b>>a) and ovary (a>>b) and have a different developmental program (b>>a) and estrogen upregulation (b only). A polymerase chain reaction (PCR)-based genomic walking strategy was used to isolate the 5'-flanking regions of the goldfish (Carassius auratus) cyp19 genes. Sequence analysis of the cyp19b gene approximately 1.8 kb upstream of the transcription start site revealed a TATA box at nucleotide (nt) -30, two estrogen responsive elements (EREs; nt -351 and -211) and a consensus binding site (NBRE) for nerve growth factor inducible-B protein (NGFI-B/Nur77) at -286, which includes another ERE half-site. Also present were a sequence at nt -399 (CCCTCCT) required for neural specificity of the zebrafish GATA-2 gene, and 16 copies of an SRY/SOX binding motif. The 5'-flanking region ( approximately 1.0 kb) of the cyp19a gene had TATA (nt -48) and CAAT (nt -71) boxes, a steroidogenic factor-1 (SF-1) binding site (nt -265), eight copies of the SRY/SOX motif, and two copies of a recognition site for binding the arylhydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) heterodimer. Both genes had elements previously identified in the brain specific exon I promoter of the mouse aromatase gene. Cyp19a- and -b/luciferase constructs showed basal promoter activity in aromatase-expressing rodent pituitary (GH3) cells, but differences (a>>b) did not reflect expression in fish pituitary in vivo (b>>a), implying a lack of appropriate cell factors. Consistent with the onset of cyp19b expression in zebrafish embryos, microinjection of a green fluorescent protein (GFP) reporter plasmid into fertilized eggs revealed labeling in neural tissues at 30-48 h post-fertilization (hpf), most prominently in retinal ganglion cells (RGC) and axon-like projections to the optic tectum. Expression of a cyp19a/GFP reporter was not detectable up to 72 hpf. Tandem analysis of cyp19a and cyp19b promoters in living zebrafish embryos can be a useful approach for identifying cis-elements and cellular factors involved in the correct tissue-specific, spatial, temporal and estrogen regulated expression of aromatase genes during CNS and gonadal development.


Subject(s)
Aromatase/genetics , Brain/metabolism , Goldfish/genetics , Goldfish/metabolism , Ovary/metabolism , Promoter Regions, Genetic , Animals , Animals, Genetically Modified , Base Sequence , Brain/growth & development , Cell Line , Cloning, Molecular , DNA/genetics , Female , Gene Expression Regulation, Developmental , Genes, Reporter , Goldfish/growth & development , Green Fluorescent Proteins , Luminescent Proteins/genetics , Molecular Sequence Data , Ovary/growth & development , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sequence Homology, Nucleic Acid , Tissue Distribution , Transfection , Zebrafish/genetics , Zebrafish/metabolism
4.
Comp Biochem Physiol B Biochem Mol Biol ; 129(2-3): 261-8, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11399458

ABSTRACT

Estrogen synthesized in the brain itself by the action of cytochrome P450 aromatase (P450arom) is known to have permanent organizing effects on the developing CNS. In fish, estrogen upregulates the predominant brain isoform (P450aromB), implying that xenoestrogens (XE) could act as neurodevelopmental toxicants by altering P450aromB. To test this hypothesis, zebrafish embryos were exposed to 17beta-estradiol (E(2)), diethylstilbestrol (DES, a potent agonist), and bisphenol A (BPA, a weak agonist). RT-PCR/Southern transfer analysis showed that E(2) (0.01-10 microM) upregulated P450aromB in a dose-response manner. The effect of DES (0.01 microM) was similar to 1 microM E(2) (three- to four-fold higher than control), but BPA was less effective (

Subject(s)
Aromatase/metabolism , Brain/metabolism , Estrogens/pharmacology , Zebrafish/embryology , Animals , Aromatase/genetics , Biomarkers/analysis , Brain/drug effects , Brain/enzymology , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , Estradiol/agonists , Estradiol/pharmacology , Estrogens/agonists , Isoenzymes/genetics , Isoenzymes/metabolism , Microscopy, Phase-Contrast , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Zebrafish/metabolism
5.
Endocrinology ; 142(2): 740-50, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11159846

ABSTRACT

As a first step toward understanding estrogen's role in neurodevelopment, a PCR cloning strategy was used to isolate complementary DNAs encoding two distinct cytochrome P450 aromatase isoforms in adult zebrafish (Danio rerio) brain and ovary (termed P450aromB and P450aromA, respectively). Sequence and phylogenetic analysis showed that the zebrafish P450arom forms are orthologs of previously identified cyp19b and cyp19a genes in goldfish. On Northern blots, a single 4.4-kb transcript of the P450aromB subtype was identified in brain, and a 2.1-kb transcript of the P450aromA subtype in ovary, but RT-PCR showed a degree of overlapping expression. Both messenger RNA (mRNA) forms were detected in unfertilized eggs and 1.5 hpf (cleavage stage) embryos but declined by 12 hpf, indicating maternal transfer. A secondary rise in mRNAs between 12-24 hpf indicated the onset of embryonic cyp19b and -a transcription. Both mRNA species accumulated progressively to 120 hpf (early larval stage), but the relative magnitude and pattern of change was isoform specific. Estradiol (E(2,) 1 microM) advanced and amplified the developmentally programmed accumulation of P450aromB mRNA, and ICI164.384 decreased expressed levels, implying blockade of an endogenous estrogen mediated regulatory component. Conversely, E(2) had no effect or decreased P450aromA mRNA. The early embryonic expression of P450aromB and P450aromA isoforms, and differences in developmental programming and estrogen regulation, imply independent regulatory mechanisms and unique functions during major morphogenetic and differentiative events.


Subject(s)
Aging/metabolism , Aromatase/metabolism , Brain/enzymology , Embryo, Nonmammalian/metabolism , Estrogens/physiology , Isoenzymes/metabolism , Ovary/enzymology , Zebrafish/metabolism , Amino Acid Sequence/genetics , Animals , Aromatase/genetics , Brain/embryology , DNA, Complementary/genetics , Estradiol/pharmacology , Female , Isoenzymes/genetics , Larva/metabolism , Molecular Sequence Data , Ovary/embryology , Ovum/metabolism , Phylogeny , RNA, Messenger/metabolism , Zebrafish/embryology , Zebrafish/growth & development
6.
J Steroid Biochem Mol Biol ; 79(1-5): 305-14, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11850237

ABSTRACT

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromB>>A) and ovary (P450aromA>>B) and have a different developmental program (B>>A) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24-48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (alpha, beta, and gamma). The 5'-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (a>>b) are opposite to fish pituitary (b>>a). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30-48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.


Subject(s)
Aromatase/genetics , Estrogens/metabolism , Goldfish/genetics , Goldfish/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Animals , Brain/enzymology , Brain/growth & development , Cell Line , DNA-Binding Proteins , Female , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Goldfish/growth & development , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Biological , Ovary/enzymology , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear , Receptors, Estrogen/metabolism , Tissue Distribution , Transcription Factors , Transfection , Zebrafish/growth & development , Zebrafish Proteins
7.
Gen Comp Endocrinol ; 113(3): 388-400, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10068500

ABSTRACT

The brain of many teleost fish species, including the goldfish Carassius auratus, expresses exceptionally high levels of cytochrome P450 aromatase (estrogen synthetase). To begin investigating the molecular and cellular targets of estrogen action in goldfish brain, a polymerase chain reaction (PCR) cloning strategy was used to isolate an estrogen receptor (ER) complementary DNA (cDNA). The 2283-bp cDNA isolated from goldfish liver encoded a protein of 568 amino acids (aa) with an estimated molecular weight of 63,539. The goldfish ER had high overall sequence identity when compared to other vertebrate ER sequences: eel (64%), human beta subtype (59%), human alpha subtype (46%), medaka (46%), and rainbow trout (47%). The highest degree of conservation was seen in the DNA-binding (94-100%) and ligand-binding (67-79%) domains. Phylogenetic analysis of the ER gene family indicated that the goldfish and eel ER are most closely related to mammalian ERbeta subtypes, whereas previously identified fish, amphibian, and avian ER forms cluster separately with mammalian ERalpha subtypes. Using the goldfish ER cDNA (here designated gfERbeta), multiple mRNA species (3.1- 8.6 kb) were detected by Northern blot analysis in goldfish liver and ovary but expression was below detection in brain. Using reverse transcription-PCR analysis, gfERbeta mRNA was detected in forebrain, mid/hindbrain, pituitary, retina, liver, ovary, and testis. Further studies are required to determine whether an additional ERalpha subtype is present in the goldfish and whether ERalpha or ERbeta forms have evolutionary precedence in vertebrates.


Subject(s)
Aromatase/biosynthesis , Goldfish/metabolism , Receptors, Estrogen/biosynthesis , Amino Acid Sequence , Animals , Aromatase/genetics , Base Sequence , Blotting, Northern , Cloning, Molecular , DNA/biosynthesis , DNA/metabolism , DNA Primers , Estrogen Receptor beta , Gene Expression Regulation, Enzymologic/physiology , Humans , Molecular Sequence Data , Phylogeny , RNA, Messenger/biosynthesis , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , Receptors, Estrogen/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Tissue Distribution
8.
Biol Reprod ; 60(1): 14-22, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9858481

ABSTRACT

Cadmium (Cd) is an established spermatotoxicant. Using the shark (Squalus acanthias) testis model, we investigated stage-related patterns of intratesticular Cd accumulation and effect. After a single injection of 109CdCl2, tracer was rapidly eliminated from plasma but accumulated and was retained in testis for at least 7 days. Intratesticular 109Cd was stage dependent, resulting in a 3- to 5-fold gradient: germinal zone (GZ) > premeiotic (PrM) > meiotic (M) > postmeiotic (PoM) stages. When measured as tissue:plasma ratios, the Cd-binding mechanism in GZ (71:1) was similar to that in liver (87:1) but lower than in kidney (381:1). The same intratesticular gradient was seen in untreated controls when tissue Cd levels were measured by atomic absorption spectroscopy, implying environmental exposure. A single CdCl2 injection (5 mg/kg i.v.) elevated testicular Cd > 160-fold in all stages but did not alter the direction or magnitude of the gradient. Intratesticular distribution of metallothionein-like Cd-binding protein was stage dependent (PrM = PoM > GZ = M), but the pattern differed from the Cd gradient. This binding component was Cd inducible in all but M stages, but induction did not alter the stage-dependent pattern of binding activity or Cd accumulation. Analysis of tissue subfractions after in vivo tracer injection indicated that the binding mechanism responsible for the intratesticular gradient is mainly cytosolic, but that a second less abundant component is associated with the nucleus. The functional significance of preferential Cd accumulation in GZ and PrM stages of spermatogenesis remains to be determined.


Subject(s)
Cadmium/metabolism , Dogfish/metabolism , Metallothionein/metabolism , Testis/metabolism , Animals , Cadmium/analysis , Cadmium Chloride/pharmacology , Cadmium Radioisotopes , Cell Membrane/chemistry , Cell Nucleus/chemistry , Cytosol/chemistry , Male , Organ Specificity , Testis/drug effects , Testis/ultrastructure , Tissue Distribution
9.
Mol Cell Endocrinol ; 138(1-2): 81-93, 1998 Mar 16.
Article in English | MEDLINE | ID: mdl-9685217

ABSTRACT

To investigate the molecular basis and physiological regulation of exceptionally high levels of aromatase (P450arom) activity in the brain of teleost fish, a 2927 bp P450arom cDNA encoding a 510 amino acid protein was isolated from a goldfish brain cDNA library. The brain-derived cDNA had 53% and 61-62% sequence identity when compared with human placental and fish ovarian P450arom forms, respectively, and higher homologies in conserved functional domains. Goldfish brain poly(A) RNA was translatable in vitro to a 56 kDa P450arom immunoprecipitation product. Northern blot analysis using the brain cDNA revealed a major 3.0 kb transcript of high abundance in brain (FB, forebrain > M/HB, mid/hindbrain), but no signal in ovary, testis or liver. P450arom mRNA varied seasonally in brain, with a peak at the onset of gonadal regrowth (February) that preceded the annual rise in enzyme levels and was 4-fold (FB) or 50-fold (M/HB) higher than during reproductive inactivity (July-December). Known markers of neurogenesis and estrogen action in brain (28S rRNA, beta-actin and beta-tubulin transcripts) each had unique seasonal patterns which differed from P450arom mRNA. In vivo steroid treatment showed that estrogen and aromatizable androgen increase FB and M/HB levels of P450arom mRNA 8- and 4-fold, respectively. P450arom mRNA in pituitary and retina had a different regulation. Southern analysis provided no evidence for multiple genes encoding the brain derived cDNA or for brain-specific gene amplification. Results imply that high accumulated levels of P450arom mRNA are the major determinant of high measured enzyme activity in goldfish brain, and that physiological regulation of mRNA expression in the natural environment is mediated by aromatization of androgen to estrogen.


Subject(s)
Aromatase/genetics , Brain/enzymology , DNA, Complementary , Gene Expression Regulation, Enzymologic , Amino Acid Sequence , Animals , Aromatase/biosynthesis , Aromatase/chemistry , Base Sequence , Female , Goldfish , Humans , Liver/enzymology , Male , Molecular Sequence Data , Organ Specificity , Ovary/enzymology , Placenta/enzymology , Pregnancy , Seasons , Sequence Alignment , Sequence Homology, Nucleic Acid , Testis/enzymology
10.
Endocrinology ; 139(4): 2179-89, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9529008

ABSTRACT

Evidence to date indicates that the gene encoding cytochrome P450 aromatase (P450arom) in humans is a single member of the CYPl9 family, but multiple CYPl9 loci and isoforms have been identified in pigs. Here we report the cloning and characterization of a second member of the CYP19 family in goldfish. A search for P450arom variants was prompted by studies showing that a full-length P450arom complementary DNA (cDNA) isolated from a goldfish brain cDNA library hybridizes with a high abundance 3 kb transcript in brain RNA but fails to detect a message in ovarian RNA. A stepwise PCR cloning strategy led to isolation of a 1.9-kb cDNA, which encodes a protein of 518 amino acids and has a predicted mol wt of 58.7K. The ovary-derived P450arom (-A) shares 68-72% sequence identity with ovarian aromatases of other fish species, but only 62% identity with the homologous brain-derived P450arom (-B). Amino acid differences are distributed throughout the two goldfish P450arom forms, but presumptive functional domains are highly conserved. Both P450aromA and -B are able to aromatize [3H]androgen to [3H]estrogen when expressed in nonsteroidogenic COS cells. Southern analysis and PCR-restriction analysis of genomic DNA using discriminating probes and primers indicates that a single locus encodes the brain-derived P450aromB (CYPl9B), whereas one or two different loci encode the ovarian form (CYPl9A). Northern blot analysis revealed two P450aromA messenger RNAs (1.9 >> 3.0 kb) in ovary. Simultaneous PCR amplification with A- and B-specific primer pairs confirms that P450aromA is the only form expressed in ovaries, but shows overlapping expression of the two genes in neural tissues. Whereas P450aromB messenger RNA predominates in brain (B/A, approximately 14:1), the ratios are reversed in retina (B/A, approximately 1:25). Further studies are required to resolve the evolutionary and functional implications of multiple CYPl9 genes and P450arom isozymes in goldfish, their differential expression in brain and ovary, and whether observations can be generalized to other vertebrates.


Subject(s)
Aromatase/genetics , Brain/enzymology , Goldfish/genetics , Isoenzymes/genetics , Ovary/enzymology , Amino Acid Sequence , Animals , Aromatase/chemistry , Base Sequence , COS Cells , DNA, Complementary/chemistry , DNA, Complementary/isolation & purification , Female , Glycosylation , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , RNA, Messenger/analysis , Sequence Alignment , Tissue Distribution , Transfection
11.
APMIS ; 106(1): 252-7; discussion 257-8, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9524587

ABSTRACT

The organisation of the testis of the dogfish shark is technically advantageous for stage-by-stage analysis of spermatogenesis in vivo and in vitro. Prior studies using this model show that total oestrogen receptors (ER) are concentrated in regions where spermatocysts ("follicle-like" germ cell-Sertoli cell units) are in stem cell and spermatogonial stages: respectively, germinal zone (GZ) and premeiotic (PrM) regions. By contrast, key enzymes regulating oestrogen (E) concentrations (aromatase, 17 alpha-hydroxylase) are maximal in meiotic (M) and postmeiotic (PoM) regions, respectively, which are upstream in the intratesticular vascular pathway. To investigate the hypothesis that E is part of a signalling mechanism between stages of development, studies were undertaken to test direct effects of oestradiol-17 beta (E2) on processes in ER-rich regions. As measured by [3H]thymidine (-Tdr) incorporation. DNA synthesis in GZ and PrM regions was inhibited by E2 (0-1000 nM) in a dose-response fashion. The maximal response (30-40%) was significant, reproducible and observed within 72 hr of treatment. Insulin differentially affected DNA synthesis and the response to E2 in GZ in GZ and PrM regions. As measured by [3H]Tdr release after prelabelling spermatocysts of GZ regions, apoptosis progressively decreased with increasing concentrations of E2. At the maximal dose of E2 used, there was no effect on total protein synthesis or secretion in combined GZ/PrM cysts, indicating that effects on DNA synthesis and cell death were authentically physiological, not pharmacological, and consistent with a state of developmental arrest. These results support the hypothesis that E synthesised within the testis is part of a negative feedback regulatory mechanism whereby more mature stages regulate the developmental advance of less mature stages. A growth control mechanism of this type could explain the strict temporal, spatial and quantitative order of succeeding stages characteristic of normal spermatogenesis in all vertebrates. Further study is required to determine whether E signalling in this model is restricted to Sertoli cells or has a germ cell component.


Subject(s)
Estrogens/biosynthesis , Spermatogenesis/physiology , Testis/cytology , Testis/physiology , Animals , Apoptosis/drug effects , Cells, Cultured , DNA/biosynthesis , Dose-Response Relationship, Drug , Estrogens/pharmacology , Feedback , Male , Receptors, Estrogen/physiology , Sharks , Spermatogenesis/drug effects
12.
Comp Biochem Physiol A Physiol ; 117(4): 471-6, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9219352

ABSTRACT

A 2061 bp cDNA encoding a goldfish (Carassius auratus) aldolase was isolated from a goldfish brain library. The deduced 362 amino acid sequence is more similar to vertebrate brain (aldolase C) and muscle aldolases (aldolase A) than to the liver isozymes (aldolase B). Northern blot analysis indicates strong expression of the mRNA in brain but not in liver or muscle, which indicates that this is aldolase C rather than aldolase A. Analysis of all known vertebrate aldolase amino acid sequences reveals five residues; Leu-57, Arg-314, Thr-324, Glu-332, and Gly-350 that are present exclusively in aldolase Cs. The goldfish clone possesses all five residues. The residues are primarily located in the carboxyl-terminal region of the enzyme and may play a role in determining the neuronal isozyme-specific properties of the enzyme. Furthermore, the existence of an aldolase C in a teleost fish has implications with respect to the timing of genome duplication events that are thought to have been critical in vertebrate evolution.


Subject(s)
Brain/enzymology , Fructose-Bisphosphate Aldolase/chemistry , Neurons/enzymology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , Goldfish , Isoenzymes , Molecular Sequence Data
13.
Gen Comp Endocrinol ; 106(2): 155-68, 1997 May.
Article in English | MEDLINE | ID: mdl-9169111

ABSTRACT

Full expression of testosterone (T) actions in the brain requires both direct binding to androgen receptors (AR) and in situ aromatization to estradiol (E2). To determine the cellular basis of constitutively high aromatase and AR binding activities in teleost fish brain, and the neuroanatomic location and spatial relations of cells of each type, an immunocytochemical mapping study of goldfish (Carassius auratus) brain was carried out using antibodies to human placental aromatase and human/rat AR peptide and the avidin-biotin-peroxidase technique. Both antibodies specifically labeled cells that were neuronal in appearance and were most numerous in reproductive control centers: medial and ventral telencephalon (TEL) and preoptic and hypothalamic periventricular nuclei. Additional populations of aromatase- and AR-labeled cells were present in the olfactory bulbs, central telencephalon, and stratum periventriculare of the optic tectum. Anti-aromatase, but not anti-AR, labeled fiber tracts and fibrous layers in visual and auditory pathways, and perikarya and processes of premotor neurons known to integrate sensory input (reticulospinal neurons, Mauthner cells). Anti-AR selectively labeled lateral TEL regions, the nucleus ventromedialis thalami, and discrete cell clusters in the medial tegmental nucleus. Aromatase-immunoreactivity (-ir) was primarily cytoplasmic, whereas AR-ir was primarily nuclear, but relative intensity of nuclear vs cytoplasmic labeling with each antibody differed by brain region. Aromatase- and AR-ir cells were not obviously more numerous in goldfish brain than previously seen in birds and mammals, suggesting that enhanced expression occurs on a per cell basis. We conclude that T exerts its actions coordinately via direct and indirect pathways in most brain regions but independently via AR- or aromatase-mediated mechanisms in selected areas. These studies point to a wide role for androgen in modulating primary sensory signals as well as in classical reproductive processes.


Subject(s)
Aromatase/metabolism , Brain Chemistry/physiology , Goldfish/metabolism , Neurons/metabolism , Receptors, Androgen/metabolism , Animals , Brain/anatomy & histology , Female , Immunohistochemistry , Male , Neurons/enzymology
14.
J Steroid Biochem Mol Biol ; 61(3-6): 387-92, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9365215

ABSTRACT

Remarkably high levels of cytochrome P450 aromatase (P450arom) enzyme are expressed in the brains of teleost fish when compared to the ovaries of the same fish, or to the brain or ovaries of other vertebrates. Northern analysis using a full-length P450arom cDNA from a goldfish brain library indicates high accumulated levels of CYP19 mRNA in the brain but fails to detect P450arom mRNA in the ovary. The possibility of different brain and ovarian mRNA variants was investigated. Reverse transcription-polymerase chain reaction (RT-PCR) amplification of ovarian RNA using degenerate primers led to the isolation of a 243 bp P450arom cDNA fragment with approximately 20% of nucleotide and amino acid replacements when compared to the brain cDNA. Southern analysis with sequence-specific probes indicated two distinct CYP19 loci, and this was confirmed by PCR-restriction enzyme analysis of genomic DNA. Corresponding brain- and ovary-type genomic sequences were identified in a second, diploid fish species (zebrafish), evidence that two genes are not caused per se by tetraploidy in goldfish. RT-PCR analysis of different tissues with sequence-specific primers showed high levels of the brain mRNA variant and much lower levels of the ovarian variant in neural tissues with high enzyme activity. In contrast, the ovary expressed low levels of the ovarian mRNA variant exclusively. The data imply that the expression of two CYP19 genes in goldfish is controlled by distinct regulatory mechanisms. Further studies are required to determine whether the two genes lead to functionally distinct isozymes.


Subject(s)
Aromatase/genetics , Brain/enzymology , Gene Expression Regulation, Enzymologic , Ovary/enzymology , Amino Acid Sequence , Animals , Aromatase/biosynthesis , Biological Evolution , Female , Goldfish , Molecular Sequence Data , Sequence Alignment
15.
Environ Health Perspect ; 103 Suppl 7: 51-7, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8593875

ABSTRACT

Organizational and activational effects of estrogen (E) in the central nervous system (CNS) are exerted directly by circulating E and indirectly after aromatization of circulating androgen to E in the brain itself. Understanding an environmental chemical's ability to disrupt E-dependent neural processes, therefore, requires attention to both pathways. Because aromatase (Aro) is highly expressed in teleost brain, when compared to mammals and other vertebrates, fish are technically advantageous for localization and regulation studies and may also provide a model in which the functional consequences of brain-derived (neuro-)E synthesis are exaggerated. Recently, Aro was immunolocalized in cell bodies and fiber projections of second- and third-order neurons of the goldfish retina and in central visual processing areas. Authentic Aro enzyme activity was verified biochemically, suggesting a heretofore unrecognized role of sex steroids in the visual system. Initial studies show that in vivo treatment with aromatizable androgen or E increases calmodulin synthesis and calmodulin protein in retina and also affects retinal protein and DNA. Whether there are related changes in the processing of visual information that is essential for seasonal reproduction or in the generative and regenerative capacity of the goldfish visual system requires further investigation.


Subject(s)
Brain/metabolism , Estrogens/biosynthesis , Visual Pathways/metabolism , Animals , Aromatase/biosynthesis , Brain/enzymology , DNA/analysis , Estrogens/analysis , Female , Goldfish , Male , Models, Biological , Nerve Tissue Proteins/metabolism , Retina/metabolism , Visual Pathways/enzymology
16.
Biol Reprod ; 53(2): 390-8, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7492692

ABSTRACT

To investigate factors controlling spermatogonial proliferation, we used premeiotic (PrM) spermatocysts (stage-synchronized germ cell/Sertoli cell clones) derived from the testis of the dogfish shark (Squalus acanthias) as an in vitro test system to estimate DNA synthesis by [3H]thymidine incorporation. Coculture of PrM spermatocysts with spermatocysts of the same (PrM) or more advanced stages (M, meiotic; PoM, postmeiotic) revealed the presence of an increasing gradient of inhibitory bioactivity from immature to mature stages (PoM > M > PrM). An even more potent and effective inhibition was detected when PrM spermatocysts were cocultured with equivalent amounts of epigonal organ, a lymphomyeloid tissue encapsulating the testis adjacent to the mature (PoM) region and immediately upstream in the vascular pathway. Inhibitory bioactivity also was present in spent media from cultured epigonal fragments and in cytosolic subfractions of epigonal homogenates but was undetectable in epididymis, muscle, serum, and red blood cells. Lower but significant inhibition was obtained with the white blood cell fraction of peripheral blood in one of two experiments. Effects of the epigonal growth-inhibitory factor (EGIF) were dose- and time-dependent, had a short response latency (3 h), were completely reversible (< 24 h after washout), and counteracted but did not block the stimulatory effects of insulin on [3H]thymidine incorporation by PrM spermatocysts. EGIF was equally inhibitory when tested on each of five PrM substages (stem cells-->preleptotene). Analysis of the entire series of experiments showed that testis-derived inhibitory activity varied seasonally, with maximum effectiveness correlated with periods of spermatogenic inactivity.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
DNA/biosynthesis , Dogfish , Growth Inhibitors/pharmacology , Meiosis , Spermatogenesis , Testis/chemistry , Animals , Cells, Cultured , DNA/antagonists & inhibitors , Dose-Response Relationship, Drug , Growth Inhibitors/administration & dosage , Kinetics , Male , Seasons , Spermatocytes/metabolism
17.
Dev Genet ; 16(2): 140-7, 1995.
Article in English | MEDLINE | ID: mdl-7736664

ABSTRACT

Control points of regulator action during spermatogenesis are not completely known. Using the shark testis model, which facilitates analysis of spermatogenesis stage-by-stage in vivo and in vitro, an early biochemical marker of programmed cell death (PCD) was detected. Nucleosomal oligomers were seen in DNA extracts of testis and isolated spermatocysts (clonal germ cell/Sertoli cell units) at premeiotic (PrM), but not meiotic (M) or postmeiotic (PoM), stages. Cell nuclei isolated from M stages of development were susceptible to cleavage by micrococcal nuclease, suggesting that developmental control of factors other than a nuclease-insensitive chromatin structure may account for stage specificity. Cytological features of apoptosis were seen in germ cells, but not Sertoli cells, of a subset of isolated PrM spermatocysts and appeared to be all-or-none in affected clones. In culture, DNA fragmentation occurred on schedule with or without various additives, but the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased accumulation of DNA breakdown products. Identification of the apoptotic form of PCD as a major, variable component of normal spermatogenesis and the use of PrM spermatocysts as an in vitro test system will allow further definition of mechanisms and developmental and physiological controls.


Subject(s)
Apoptosis , Spermatogenesis , Spermatozoa/cytology , Animals , DNA/metabolism , Dogfish , Male , Meiosis , Spermatozoa/metabolism
18.
J Neuroendocrinol ; 5(6): 635-41, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8680435

ABSTRACT

Using an animal model in which neural aromatase is apparently overexpressed (the goldfish, Carassius auratus) and an anti-human placental antibody which specifically crossreacts with goldfish brain aromatase, aromatase-immunoreactive neuronal cell bodies and fibers have been localized within the retina. These include a subset of horizontal cells, bipolar cells, and amacrine cells of the inner nuclear layer, some fibers of the outer and inner synaptic layers and certain cells of the ganglion cell layer; photoreceptors were never labeled. Some ganglion cell projections to the brain via the optic nerve and optic tract were aromatase-positive, as were small neurons of the stratum periventriculare (SPV) and fibers of two other strata of the optic tectum. Aromatase activity, as measured by [3H]androgen by tissue homogenates and cell cultures, confirmed the presence of aromatase in retina and in brain regions containing the optic tectum. This localization of the rate-limiting enzyme in estrogen biosynthesis suggests that neuroestrogen derived from circulating androgen m ay modulate transmission and integration of visual information important for reproduction in this species.


Subject(s)
Aromatase/metabolism , Goldfish/physiology , Neurons/enzymology , Retina/enzymology , Superior Colliculi/enzymology , Visual Pathways/enzymology , Animals , Blotting, Western , Cells, Cultured , Estradiol/biosynthesis , Female , Immunohistochemistry , Male , Optic Nerve/cytology , Optic Nerve/enzymology , Retina/cytology , Retinal Ganglion Cells/enzymology , Superior Colliculi/cytology , Testosterone/metabolism , Visual Pathways/cytology
19.
J Exp Zool ; 267(2): 233-44, 1993 Oct 01.
Article in English | MEDLINE | ID: mdl-8409903

ABSTRACT

To investigate growth control mechanisms during spermatogenesis in vitro, [3H]thymidine incorporation into acid-insoluble macromolecules was used to quantify DNA synthesis in cultured spermatocysts (intact Sertoli cell/germ cell clones) derived from premeiotic (PrM), meiotic (M), and postmeiotic (PoM) regions of dogfish (Squalus acanthias) testis. Forty-eight hours after seeding in basal medium, DNA synthesis was > 7-fold higher in PrM cysts than in other stages, thus verifying the staging procedure. In autoradiograms, germ cells of PrM cysts (e.g., spermatogonial and preleptotene stages) were labeled all-or-none, but not all cysts were labeled, and later developmental stages (e.g., cysts with round or elongating spermatids) were never labeled. Fetal bovine serum (FBS, 10%) and insulin-transferrin-selenite (ITS, 10 micrograms-10 ng/ml) doubled DNA synthesis in PrM cyst cultures but had no effect at other stages. Bovine insulin (10 micrograms/ml) and human recombinant insulin-like growth factor-I (IGF-I, 15 ng/ml) also doubled [3H]thymidine uptake in PrM cultures, but lower doses were less effective and estradiol-17 beta, transferrin, adult shark serum, purified shark relaxin, and a variety of other known growth factors were neither stimulatory nor inhibitory at the doses and conditions tested. Sertoli cell monolayers derived from PrM- or M-stage spermatocysts displayed a dose-response increase in DNA synthesis after addition of IGF-I (15-75 ng/ml), with a maximal increment significantly greater than with 10% FBS. Using [3H]thymidine incorporation by PrM cysts as an end-point, stimulatory bioactivity was detected in the < 30,000 kDa fraction of spent media from PrM Sertoli cells, whereas the low molecular weight fraction of M-stage Sertoli cells was inhibitory. Gel electrophoretic analysis of the two fractions revealed qualitative and quantitative differences in protein banding patterns, reinforcing the view that secretory activity of Sertoli cells is stage related. Results of these studies implicate insulin/IGF-I in mechanisms governing proliferation of male germ cells and support the view that Sertoli cells have an autocrine or paracrine role as both targets and sources of growth regulatory factors.


Subject(s)
Insulin-Like Growth Factor I/physiology , Insulin/physiology , Sertoli Cells/cytology , Spermatocytes/cytology , Spermatogenesis/physiology , Analysis of Variance , Animals , Autoradiography , Cells, Cultured , DNA/biosynthesis , Dogfish , Male , Meiosis , Testis/cytology
20.
J Exp Zool ; 265(5): 522-32, 1993 Apr 01.
Article in English | MEDLINE | ID: mdl-8468541

ABSTRACT

Using shark (Squalus acanthias) testicular microsomes and [3H]progesterone ([3H]P) and [3H]17 alpha-hydroxyprogesterone ([3H]17 alpha-P) as substrates, two major products of 21-hydroxylase action were identified; respectively, 21-hydroxy-4-pregnen-3-one (11-deoxycorticosterone, DOC) and 17 alpha,21-dihydroxy-4-pregnen-3-one (11-deoxycortisol,S). Additional products of 17 alpha-hydroxylase/C-17,20-lyase action were detected: 17 alpha-hydroxyprogesterone (17 alpha P), testosterone (T), and androstenedione (AE). When microsomes derived from tissues in premeiotic (PrM), meiotic (M), and postmeiotic (PoM) stages of spermatogenesis were compared, maturation-related increases were observed with both 21-hydroxylase (3- to 20-fold) and 17 alpha-hydroxylase (2- to 6-fold). With [3H]P as tracer, the half-maximal substrate concentrations (Km = 0.2-1.0 microM) and maximal reaction velocities (Vmax = 9-25 pmol/mg protein/min) were similar for both enzymes when assayed in the same preparation, suggesting they compete for available substrate. Also, the presence of 1- or 10-fold molar excess radioinert DOC reduced conversion of [3H]P to 17 alpha-hydroxylated products. [3H]DOC itself was a substrate of 17 alpha-hydroxylation but not C-17,20-lyase action. Expression of 21-hydroxylase and 17 alpha-hydroxylase activities in cultured spermatocysts (intact germ cell/Sertoli cell units) was confirmed by detection of immunoreactive 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S),S,17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P), DOC, P and T in spent media. 20 beta-S and S secretion increased and 17 alpha,20 beta-P decreased progressively with stage of maturation, but DOC was similar in all stages. P secretion was maximal and T lowest in M-stage spermatocysts, but when DOC (0.1 microM) was added to PrM or PoM spermatocysts, T output decreased. Taken together, the data suggest that 21-hydroxylating pathways, via direct and indirect mechanisms, affect accumulation of bioactive steroids (P and T) differentially by stage of spermatogenic development. Whether 21-hydroxylated progestins produced in shark testis have paracrine or endocrine actions in their own right remains to be investigated.


Subject(s)
Hydroxyprogesterones/metabolism , Spermatogenesis , Testis/metabolism , Animals , Cells, Cultured , Crystallization , Desoxycorticosterone/pharmacology , Dogfish , Hydroxyprogesterones/chemistry , Kinetics , Male , Steroid 21-Hydroxylase/metabolism , Testis/cytology
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