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To test the hypothesis that alternative splicing could be an adaptive mechanism for populations subject to multi-generational estrogenic exposures, we compared estrogen receptor alpha (ERα) splicing variants in two populations of killifish (Fundulus heteroclitus): one resident in an estrogenic polluted environment (New Bedford Harbor, NBH, MA, USA) and one from a relatively uncontaminated reference site (Scorton Creek, SC, MA, USA). In total we identified nineteen ERα variants, each with deletions of one or more coding exons. Four of the variants with potential functional relevance were analyzed by qPCR to test for population differences in expression by tissue type, site, sex, seasonal reproductive status and estrogen treatment. Significantly, a 5′-truncated short form variant (ERαS) was highly expressed in liver and ovary, and was associated with seasonal reproductive activity in SC but not NBH fish. Both ERαS and the full-length long variant (ERαL) were estrogen-inducible (ERαS > ERαL) but the induction response was lower in NBH than in SC fish. In contrast, NBH killifish were hyper-responsive to estrogen as measured by expression of two other estrogen responsive genes: vitellogenin (Vtg) and aromatase B (AroB). Most strikingly, two ERα deletion variants (Δ6 and Δ6–8), lacking ligand binding and activation function domains, were identified in a subset of NBH fish, where they were associated with reduced responsiveness to estrogen treatment. Together, these results support the hypothesis that alternative splicing of the esr1 gene of killifish could be an autoregulatory mechanism by which estrogen modulates the differential expression of ERα, and suggests a novel and adaptive mechanistic response to xenoestrogenic exposure.

It is well established that estrogen-like environmental chemicals interact with the ligand-binding site of estrogen receptors (ER) to disrupt transcriptional control of estrogen responsive targets. Here we investigate the possibility that estrogens also impact splicing decisions on estrogen responsive genes, such as that encoding ERα itself. Targeted PCR cloning was applied to identify six ERα mRNA variants in zebrafish. Sequencing revealed alternate use of transcription and translation start sites, multiple exon deletions, intron retention and alternate polyadenylation. As determined by quantitative (q)PCR, N-terminal mRNA variants predicting long (ERαL) and short (ERαS) isoforms were differentially expressed by tissue-type, sex, stage of development and estrogen exposure. Whereas ERαL mRNA was diffusely distributed in liver, brain, heart, eye, and gonads, ERαS mRNA was preferentially expressed in liver (female > male) and ovary. Neither ERαL nor ERαS transcripts varied significantly during development, but 17β-estradiol selectively increased accumulation of ERαS mRNA (~170-fold by 120 hpf), an effect mimicked by bisphenol-A and diethylstilbestrol. Significantly, a C-truncated variant (ERαS-Cx) lacking most of the ligand binding and AF-2 domains was transcribed exclusively from the short isoform promoter and was similar to ERαS in its tissue-, stage- and estrogen inducible expression. These results support the idea that promoter choice and alternative splicing of the esr1 gene of zebrafish are part of the autoregulatory mechanism by which estrogen modulates subsequent ERα expression, and further suggest that environmental estrogens could exert some of their toxic effects by altering the relative abundance of structurally and functionally distinct ERα isoforms. 2.674 JCR (2013) Q3, 64/123 Endocrinology & metabolism

Genetically distinct estrogen receptor (ER) subtypes (ERα and ERβ) play a major role in mediating estrogen actions in vertebrates, but their unique and overlapping functions are not entirely clear. Although mammals have 1 gene of each subtype (ESR1 and ESR2), teleost fish have a single esr1 (ERα) and 2 esr2 (ERβa and ERβb) genes. To determine the in vivo role of different ER isoforms in regulating estrogen-inducible transcription targets, zebrafish (Danio rerio) embryos were microinjected with esr-specific morpholino (MO) oligonucleotides to disrupt splicing of the exon III/intron III junction in the DNA-binding domain. Each MO knocked down its respective normal transcript and increased production of variants with a retained intron III (esr1 MO) or a deleted or mis-spliced exon III (esr2a and esr2b MOs). Both esr1 and esr2b MOs blocked estradiol induction of vitellogenin and ERα mRNAs, predominant hepatic genes, but esr2b was the only MO that blocked induction of cytochrome P450 aromatase B mRNA, a predominant brain gene. Knockdown of ERβa with the esr2a MO had no effect on estrogen induction of the 3 mRNAs but, when coinjected with esr1 MO, attenuated the effect of ERα knockdown. Results indicate that ERα and ERβb, acting separately or cooperatively on specific gene targets, are positive transcriptional regulators of estrogen action, but the role of ERβa, if any, is unclear. We conclude that MO technology in zebrafish embryos is an advantageous approach for investigating the interplay of ER subtypes in a true physiological context.

The possibility that chronic, multigenerational exposure to environmental estrogens selects for adaptive hormone-response phenotypes is a critical unanswered question. Embryos/larvae of killifish from an estrogenic-polluted environment (New Bedford Harbor, MA [NBH]) compared with those from a reference site overexpress estrogen receptor alpha (ERα) mRNA but are hyporesponsive to estradiol. Analysis of ERα mRNAs in the two populations revealed differences in splicing of the gene encoding ERα (esr1). Here we tested the transactivation functions of four differentially expressed ERα mRNAs and tracked their association with the hyporesponsive phenotype for three generations after transfer of NBH parents to a clean environment. Deletion variants ERαΔ6 and ERαΔ6–8 were specific to NBH killifish, had dominant negative functions in an in vitro reporter assay, and were heritable. Morpholino-mediated induction of ERαΔ6 mRNA in zebrafish embryos verified its role as a dominant negative ER on natural estrogen-responsive promoters. Alternate long (ERαL) and short (ERαS) 5′-variants were similar transcriptionally but differed in estrogen responsiveness (ERαS ≫ ERαL). ERαS accounted for high total ERα expression in first generation (F1) NBH embryos/larvae but this trait was abolished by transfer to clean water. By contrast, the hyporesponsive phenotype of F1 NBH embryos/larvae persisted after long-term laboratory holding but reverted to a normal or hyper-responsive phenotype after two or three generations, suggesting the acquisition of physiological or biochemical traits that compensate for ongoing expression of negative-acting ERαΔ6 and ERαΔ6–8 isoforms. We conclude that a heritable change in the pattern of alternative splicing of ERα pre-mRNA is part of a genetic adaptive response to estrogens in a polluted environment.

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'.

Epidemiological, ecological, and laboratory-based studies support the hypothesis that endocrine disrupting chemicals (EDCs) in the environment are responsible for developmental and reproductive abnormalities. We have previously described a killifish population resident in a highly polluted Superfund site (New Bedford Harbor, NBH) that shows evidence of exposure to an estrogenic environment and endocrine disruption. Here, we compare NBH with a local reference population (Scorton Creek, SC) for developmental patterns and direct effects of exogenous estradiol on the estrogenic markers, brain cytochrome P450 aromatase (CYP19A2 or AroB), hepatic vitellogenin (Vtg), and hepatic estrogen receptor alpha (ER alpha). In contrast to our previous observation of elevated ER alpha in NBH embryos, developmental levels of AroB and Vtg mRNAs did not differ between the two sites, demonstrating that not all estrogen-responsive genes are upregulated in NBH embryos. A dose-response experiment showed that NBH larvae are less responsive (lower maximum induction, as measured by ER alpha) and less sensitive (higher EC(50) for induction, as measured by AroB) to estradiol than SC larvae, changes that would be adaptive in an estrogenic environment. In contrast, induction of Vtg mRNA is similar in the two populations, indicating that the adaptive mechanism is target gene-specific. Based on the lower basal levels of ER alpha mRNA in several tissues from adult NBH fish vs SC fish (Greytak and Callard, 2007), we predicted estrogen hyporesponsiveness; however, induction of ER alpha by estradiol exposure in reproductively inactive males did not differ between the two sites. Moreover, AroB was more responsive and Vtg induction was greater (2d) or similar (5d) in NBH as compared to SC males. Worth noting is the high inter-individual variability in estrogen responses of gene targets, especially in NBH killifish, which may indicate evolving preadaptive or adaptive mechanisms. In conclusion, although multi-generational exposure to a highly polluted environment is associated with changes in basal levels of ER alpha mRNA, this is not a simple predictor of estrogen responsiveness. We hypothesize that adaptation of killifish to the estrogenic and polluted environment may be occurring through diverse mechanisms that are gene-, tissue type- and life-stage-specific.

Epidemiological, wildlife, and laboratory studies support the hypothesis that chemicals released into the environment through anthropogenic activities are responsible for abnormalities of reproduction and development. Although the New Bedford Harbor (NBH) killifish population has survived and reproduced successfully for >50 yr ( approximately 20 generations), fish have high body burdens of the major NBH contaminants (polychlorinated biphenyls); elevated levels of P450 aromatase B and vitellogenin mRNA (markers of estrogen effect); and evidence of endocrine disruption. To investigate possible adaptive changes in the estrogen response system of NBH killifish, we cloned the estrogen receptors (ER) from killifish populations resident in NBH and a relatively unpolluted reference site (Scorton Creek MA, SC). ERalpha, -betaa, and -betab cDNAs encoding full-length polypeptides of 620, 543, and 672 amino acids, respectively, were identified. Each ER subtype had multiple splice variants, single nucleotide polymorphisms (SNPs), and a characteristic tissue distribution and developmental profile. As measured by real-time quantitative RT-PCR analysis, the overall tissue distribution of each ER was similar in NBH and SC fish, implying that the regulatory pathways which maintain tissue-specific expression are largely unchanged by long term pollutant exposure. Nonetheless, a striking difference was seen in the quantity of mRNA of the estrogen-inducible gene ERalpha, which was significantly lower in brain, liver and ovaries of reproductively active NBH as compared to SC females. Paradoxically, despite the "estrogenic" NBH environment, ERalpha mRNA levels did not differ in reproductively inactive NBH and SC females, or in males at the two sites at any time of year. We interpret results in NBH fish as due in part to a deficit of circulating estrogen, and in part to pollutant-mediated hyporesponsiveness of the ERalpha gene. In marked contrast to adult fish, ERalpha was elevated approximately 5-fold in NBH as compared to SC embryos/larvae, perhaps indicative of estrogenic chemicals in yolk. We conclude that contaminants in the NBH environment impact molecular components of the estrogen signaling pathways in resident killifish populations. Whether these changes are transient or heritable requires further study.

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)ERRα is an ortholog of the single ERRα identified in mammals, pufferfish, and zebrafish. FhERRβa and FhERRβb are co-orthologs of the mammalian ERRβ. Phylogenetic placement of the fourth killifish ERR gene, tentatively identified as FhERRγb, is less clear. The four ERRs showed distinct, partially overlapping mRNA expression patterns in adult tissues. FhERRα was broadly expressed. FhERRβa was expressed at apparently low levels in eye, brain, and ovary. FhERRβb was expressed more broadly in liver, gonad, eye, brain, and kidney. FhERRγb was expressed in multiple tissues including gill, heart, kidney, and eye. Distinct expression patterns of FhERRβa and FhERRβb are consistent with subfunctionalization of the ERRβ paralogs. Induction of ERRα 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 ERRα expression in heart. In a second, more targeted experiment, expression of ERRα 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 ERRβ paralogs. In addition, regulation of ERRα expression in killifish apparently differs from regulation in mammals. Together, these features may facilitate determination of both conserved and specialized ERR gene functions.

In teleost fish, the predominant brain form of cytochrome P450 aromatase (P450aromB) is a neural marker of estrogen effect, and an entry point for studying the role of hormonal and environmental estrogens on neurodevelopment and neuroplasticity. As part of a project using zebrafish to investigate these issues, we developed and validated a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay for quantifying and comparing P450aromB and P450aromA expression in unfertilized eggs, embryos/larvae, and dissected tissues of adult fish. Results confirm that P450aromB and -A predominate in brain and ovary, respectively, and further show that the degree of overlapping expression (ratio, B:A) is 100:1 in brain, 1:50 in ovary, 1:1 in eye, and 2:1 in testis. Sex differences were observed in eye only (female > male). When compared to whole ovaries, unfertilized eggs had similar levels of P450aromA but enrichment of P450aromB, which suggests preferential synthesis or accumulation in mature oocytes. Both of the maternally derived aromatase isoforms were rapidly degraded post-fertilization, but the onset of embryonic P450aromB expression (5 hpf) was much earlier than P450aromA (48 hpf), and reached higher maximum levels (e.g., 10-fold at 72 hpf). Consistent with earlier reports, P450aromB but not -A was estrogen-inducible, but the estrogen response system in embryos was far more robust than in adults (>100- vs.

Populations of killifish (Fundulus heteroclitus) persist in many different highly polluted environment indicative of adaptation or tolerance. In this study, we sought to determine whether long term, multigenerational exposures to environmental contaminants has affected reproductively relevant genes and biological processes. A homology cloning strategy was used to isolate the killifish cytochrome P450 aromatase (P450arom, estrogen synthetase) cDNAs. Consistent with previous fish studies, killifish were found to have two P450arom forms, which segregated into A- and B-gene clades and were differentially expressed in brain (B >> A) and ovary (A >> B). Comparison of killifish from highly polluted (New Bedford Harbor, NBH) and unpolluted (Scorton Creek, SC) environments revealed no site-related differences in P450arom coding sequences or in overall tissue distribution patterns. As measured by real-time quantitative PCR (QPCR) analysis, however, P450arormB (a known marker of estrogen effect) was approximately two-fold higher in the brain of NBH than of SC fish, a difference seen in reproductively active and inactive males and females. Providing further evidence of exposure to estrogen-like pollutants or metabolites in NBH, vitellogenin (vtg) mRNA and protein were elevated in seasonally active and inactive males, and in reproductively inactive females, when compared to SC fish. By contrast, during the period of reproductive activity, NBH females had a lower gonadosomatic index, lower plasma estrogen, a decreased hepatosomatic index, and reduced vtg expression as compared to SC females, indicating that the female hypothalamic-pituitary-gonadal (HPG)-liver axis is impaired in the polluted environment. As measured by a decrease in plasma androgen (but not GSI), the male HPG axis was impaired in reproductively active NBH versus SC fish. In agreement with reports that NBH killifish are resistant to dioxin-like chemicals (DLC) that activate arylhydrocarbon receptor (AhR) signaling, ovarian P450aromA (a marker of dioxin-like effect in zebrafish embryos) did not differ in SC and NBH fish. In conclusion, the killifish population at the NBH Superfund site maintains a level of reproductive competence in the face of evidence of exposure to estrogen-like pollutants and endocrine disruption.

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.

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.

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 (

Cadmium (Cd) is an established spermatotoxicant. Using the shark (Squalus acanthias) testis model, we investigated stagerelated patterns of intratesticular Cd accumulation and effect. After a single injection of 109 CdCl2, tracer was rapidly eliminated from plasma but accumulated and was retained in testis for at least 7 days. Intratesticular 109 Cd 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 . 160fold 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 5 PoM . GZ 5 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.

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.

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.

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.

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.

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. Images Figure 2.

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 [ 3 H]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 ( 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. By contrast, EGIF was effective throughout the year. Results indicate that a spermatogonial growth regulatory factor emanating from a testis-associated immune tissue enters the testis proper via vascular pathways to form a gradient of inhibitory bioactivity countercurrent to the spermatogenic progression. This gradient may be part of a mechanism accounting for the orderly diametric arrangement of maturing spermatocysts and the seasonal onset and arrest of spermatogenesis characteristic of this model. The strict temporal and spatial arrangement of successive germ cell stages in the testis of all vertebrates suggests that a mechanism of this type may have general relevance.

The ability to convert androgens to estrogens is an ancient and highly conserved characteristic of the vertebrate brain but is lacking in amphioxus and all other invertebrates, indicating that a functional form of the enzyme first evolved in the brain of No data (2012) UEM

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.

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.

To investigate the relationship between steroidogenesis and spermatogenesis, two key enzymes of androgen biosynthesis, 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) and 5α-reductase, were compared at premeiotic (PrM), meiotic (M) and postmeiotic (PoM) stages. Staged tissues were obtained by dissection from the testis of the spiny dogfish Squalus acanthias, in which spermatogenesis is a simple diametric progression. Microsomal 3β-HSD activity was measured by conversion of [3H] dehydroepiandrosterone (DHEA) to androstenedione (AE). Reaction constants were: Km = 3.2 μM and Vmax = 243 pmol/min/mg protein. 3β-HSD increased progressively with maturation, resulting in three- to four-fold higher levels in PoM than in PrM stages. Absolute values and stage-related differences were the same, whether microsomes were derived from whole testis or from isolated spermatocysts (germ cell/Sertoli cell units), thus supporting microscopic studies showing that Sertoli cells are the primary steroidogenic elements of dogfish testis. In vitro conversion of [3H]testosterone to [3H]dihydrotesterone (DHT) was used to estimated 5α-reductase activity. Apparent substrate affinity was similar to that of 3β-HSD (Km = 2.9 μM), but maximal product yields were two to three orders of magnitude lower (Vmax = 208 fmol/min/mg protein). Also, the stage-related pattern of 5α-reductase activity (PrM > PoM >> M) differed from that of 3β-HSD (PoM >> M > PrM). Together with previously measured steroidogenic enzyme activities using the shark testis model, these results predict that de novo androgen biosynthesis is highest in testicular regions with mature spermatids and fully differentiated Sertoli cells, and also suggest that availability of T versus DHT for androgen receptor binding may be developmentally regulated during spermatogenesis.

It is well-established that neurons in the adult mammalian central nervous system (CNS) are terminally differentiated and, if injured, will be unable to regenerate their connections. In contrast to mammals, zebrafish and other teleosts display a robust neuroregenerative response. Following optic nerve crush (ONX), retinal ganglion cells (RGC) regrow their axons to synapse with topographically correct targets in the optic tectum, such that vision is restored in ~21 days. What accounts for these differences between teleostean and mammalian responses to neural injury is not fully understood. A time course analysis of global gene expression patterns in the zebrafish eye after ONX can help to elucidate cellular and molecular mechanisms that contribute to a successful neuroregeneration. To define different phases of regeneration after ONX, alpha tubulin 1 ( tuba1) and growth-associated protein 43 ( gap43), markers previously shown to correspond to morphophological events, were measured by real time quantitative PCR (qPCR). Microarray analysis was then performed at defined intervals (6 hours, 1, 4, 12, and 21 days) post-ONX and compared to SHAM. Results show that optic nerve damage induces multiple, phase-related transcriptional programs, with the maximum number of genes changed and highest fold-change occurring at 4 days. Several functional groups affected by optic nerve regeneration, including cell adhesion, apoptosis, cell cycle, energy metabolism, ion channel activity, and calcium signaling, were identified. Utilizing the whole eye allowed us to identify signaling contributions from the vitreous, immune and glial cells as well as the neural cells of the retina. Comparisons between our dataset and transcriptional profiles from other models of regeneration in zebrafish retina, heart and fin revealed a subset of commonly regulated transcripts, indicating shared mechanisms in different regenerating tissues. Knowledge of gene expression patterns in all components of the eye in a model of successful regeneration provides an entry point for functional analyses, and will help in devising hypotheses for testing normal and toxic regulatory factors.

Due to a cystic mode of spermatogenesis and zonal testes, the spiny dogfish shark (Squalus acanthias) and salamander (Necturus maculosus) are advantageous for studying the relationship between steroidogenesis and germ cell development. Direct analysis of steroidogenic enzymes and steroid receptors in staged tissue samples, together with light and electron microscopic observation, suggests that androgens and estrogens produced by Sertoli cells or Leydig cells immediately before or after spermiation serve as intratesticular regulators of events specific to premeiotic stages of spermatogenesis. These correlative data are consistent with limited observations in mammalian testis and provide a conceptual framework for direct testing of functional interactions between spermatogenesis and steroidogenesis in future studies.

As part of an ongoing program of research using the testis of the dogfish shark (Squalus acanthias) to characterize morphologic and functional changes during spermatogenesis, we have developed procedures for culturing intact spermatocysts (germ cell/Sertoli cell clones) and isolated Sertoli cells from premeiotic, meiotic, and postmeiotic stages of development. Phase contrast and light microscopy confirmed the stage and cellular composition of spermatocysts and showed that they retained their closed, spherical configuration for at least 15 d in culture. Stage-related variations in [3H]thymidine incorporation (premeiotic much greater than meiotic = postmeiotic) were observed, a pattern that was the same quantitatively and qualitatively after one or seven days of culture. [3H]Leucine-labeled protein synthesis was twofold greater in cultures with premeiotic spermatocysts than in cultures with more mature stages, whether medium or cysts were analyzed. Sertoli cells isolated from spermatocysts of different stages differed in size, shape, cytological appearance, ability to form flattened monolayers, and rate of DNA synthesis. One day after seeding, [3H]thymidine labeling of Sertoli cells corresponded to the pattern obtained with intact spermatocysts (premeiotic much greater than meiotic = postmeiotic); however, 7 days in culture effected a 40- to 200-fold increase in this parameter and altered the stage-dependent pattern (premeiotic = meiotic greater than postmeiotic). Also, when [3H]leucine-labeled macromolecules secreted by Sertoli cells from premeiotic versus meiotic stages were analyzed by polyacrylamide gel electrophoresis (PAGE), banding patterns differed. Initial results demonstrate the feasibility and potential of this in vitro system for studying qualitative and quantitative changes during spermatogenesis.

Although testosterone (T) stimulates aggressive and reproductive behaviors in males of many vertebrate species, it is now known that the full expression of T action in the brain requires aromatization to estradiol (E2) and subsequent interaction of locally formed E2 with nuclear estrogen receptors. In experiments reported here, we used a behavioral test which quantifies the response of an individual male Japanese quail (Coturnix coturnix japonica) to the visual stimulus of a conspecific. We have called this behavior aggression because it shares many features in common with traditional measures of aggression, e.g., predicting dominance and subordinance. Nevertheless, the behavior probably also combines a complex steroid-sensitive masculine behavior. The advantage of this test is that it allows the discrimination of individual differences in masculine behavior but avoids fighting and sexual encounters per se, thereby reducing effects of learning, a problem with previous tests of avian aggression. In addition, this test has been applied usefully to identify neuroendocrine correlates to male behavior. Using this test, the arousal of reproductively inactive males (hereafter referred to as aggression) is activated by administration of T or estradiol benzoate (EB), but not by 5 alpha-dihydrotestosterone (DHT). T-induced aggression was blocked by the aromatase inhibitor 4-hydroxyandrostenedione (OHA), an effect partially reversed by treatment with EB. In addition, OHA or the estrogen receptor blocker CI-628 reduced aggressiveness of reproductively active males whereas the androgen receptor blocker flutamide had no effect. Results with the 5 alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 alpha-carboxyamide (4-MA) were equivocal. Additionally, treatment of reproductively inactive quail with T or E2 but not DHT increased aromatase activity in the hypothalamus-preoptic area (HPOA). We conclude, therefore, that T to E2 conversion is essential for the activation of aggressiveness in this species. Although locally formed estrogen exerts its effects on aggression in part by increasing activity of aromatase per se, analysis of the time course of behavioral induction or suppression by the various treatments suggests that the response has multiple components, including both short latency, receptor-independent and long latency, receptor-dependent events.

Estrogen formation in brain and pituitary mediates certain androgen actions in central targets. Goldfish (Carassius auratus) and quail (Coturnix coturnix japonica) have been advantageous for studying the role of locally formed estrogen in autoregulating aromatization and in controlling estrogen receptor occupancy, androgen receptor levels, and behavioral expression. Data from these two experimental models reveal a molecular basis for androgen-estrogen synergism in neuroendocrine tissues and for alterations in androgen sensitivity/responsiveness. These mechanisms are essential components of seasonal reproduction in the test species and may have wider relevance for cyclicity in other vertebrates, including mammals.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

The brain and ovarian aromatase isozymes of goldfish (Carassius auratus) are encoded by different CYP19 genes. This study measured aromatase activity in the goldfish brain tissues. For a direct comparison of the properties of the two aromatase isozymes, Chinese hamster ovary cells were stably transfected with brain- and ovary-derived cDNAs (respectively, p450 arom B and -A) and the properties of the expressed isozymes were compared. The kinetic parameters of the two isozymes were determined using androstenedione and testosterone as substrates and compared to those of human aromatase. Inhibition profile analyses on the two isozymes were performed using seven inhibitors [4-hydroxyandrostenedione, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione, bridge (2,19-methyleneoxy)androstene-3,17-dione, aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole]. Except for AG, the compounds tested were found to be much stronger inhibitors against the ovary enzyme than the brain enzyme. In addition, the ovary isoform was more sensitive to two phytoestrogens, chrysin and 7,8-dihydroxyflavone, than the brain form. These studies reveal that catalytic properties of the goldfish aromatase isoforms are significantly different from those of human aromatase. In addition, differences in the K(i) values of aromatase inhibitors for the two goldfish isoforms suggest structural variance in the active sites of these isozymes.

In mammals, a single Sertoli cell nurtures 3-4 successive generations of germ cells. Thus, it is not possible to study this cell type at a single spermatogenic stage. In the dogfish shark Squalus acanthias, a single cohort of Sertoli cells remains associated with a germ cell clone throughout its development. Moreover, different germ cell stages are topographically segregated within the testis and can be easily staged by transilluminationmicroscopy. Recently, we have developed methods for the isolation and culture of spermatocysts (Sertoli/germ cell units) and Sertoli cells only from pre-meiotic, meiotic, and post-meiotic stages of germ cell development. Here, we present data that illustrate the feasibility of using the Squalus testis model for characterizing stage-related biochemical changes in Sertoli cells.

Production and development of mature male gametes is essential for normal reproductive efficiency and survival of species. Epidemiological research and observations of wildlife have reported an increase in male reproductive abnormalities, including declining sperm counts in human males, thus opening a debate as to the role of environmental pollutants and other factors. The complex organization of the testis of man and common laboratory mammals, and the lack of a good in vitro spermatogenesis system for direct study, has hampered routine chemical testing and frustrated attempts to define the extent of the problem. The challenge is further exacerbated by our incomplete understanding of normal testicular control mechanisms, despite intense research activity over the last 25 years and the acquisition of powerful new tools of cellular and molecular biology for accumulating data. Future progress in understanding human health effects, and the impact of environmental factors on survival of species and ecosystems, has been linked to the development of new animal models and test systems.

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.

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.

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. © 1995 Wiley-Liss, Inc.

Primarily from studies in mammals, it is evident that testicular functions have complex control mechanisms that include several categories of endocrine, paracrine, and autocrine mediators and involve crosstalk among germinal elements and multiple somatic cell types (e.g., Sertoli cells, Leydig cells, peritubular myoid cells, and fixed and transient leucocytes). As the only somatic cell element intimately in contact with germ cells, Sertoli cells have a pivotal role. What has hampered efforts to define functions of Sertoli cells during spermatogenic development is the organizational complexity of the mammalian testis. By contrast, the testis of certain lower vertebrates is much simpler in organization and has advantageous anatomic or functional characteristics that facilitate observation and experimental manipulation stage by stage. As part of a program of research examining the functional interdependence of spermatogenesis and steroidogenesis, we have carried out many studies using the spiny dogfish shark (Squalus acanthias) and have developed methods for studying spermatogenesis and its regulation using intact germinal units in vitro. Reviewed in this chapter are published and unpublished results from this laboratory.

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.

Due to exceptionally high brain aromatase activity, teleost fish are advantageous for studying neural aromatase regulation, localization, and physiology. To determine the molecular mechanism of enhanced expression, we have isolated, cloned and sequenced a 3 kb full-length aromatase cDNA from a goldfish (Carassius auratus) brain library using a human placental aromatase cDNA as probe. The deduced sequence of goldfish aromatase is 510 amino acids (predicted Mw, 58 kDa) with 69% overall sequence similarity, when compared to human placental aromatase, and higher homologies in presumptive functional domains. A major 3 kb mRNA species was abundant in brain and low or non-detectable in non-neural tissues, reflecting the order of enzyme activities. To determine the cellular basis of high enzyme activity in goldfish brain, a human placental aromatase antibody was used to immunolocalize labeled cells. This antibody immunoprecipitated a single 56 kDa in vitro translation product of goldfish brain poly(A+)RNA and revealed discrete clusters of intensely stained neurons, processes, and terminals concentrated in, but not limited to, reproductive brain centers. Close proximity of aromatase- and androgen receptor-positive neurons in certain regions provides anatomic evidence of a functional relationship between direct and indirect pathways of neural androgen action. Aromatase-positive neurons and fibers formed interconnected networks in novel loci (e.g. retina-->optic tract-->optic tectum), and catalytic activity was confirmed biochemically in these tissues, indicating that neuroestrogen may have a role in visual input and integration. Availability of goldfish-specific nucleotide and antibody probes will facilitate further studies using this model.

The zonal testis of the dogfish (Squalus acanthias) has proven advantageous to study biochemical changes in relation to stage of spermatogenesis, including information on steroidogenic enzymes and steroid receptors. To investigate whether sulfotransferase is part of a mechanism regulating the availability of biologically active hormone in close proximity to receptors, we measured in vitro conversion of [3H]estrone (E1) to sulfoconjugated metabolites in cytosolic subfractions of testes grossly dissected according to germ cell composition (premeiotic-PrM, meiotic-M, and postmeiotic-PoM stages). Assays were carried out in the presence of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) at 22 degrees C and optimized for time (60 min) and protein (500 micrograms/ml). Michaelis-Menten kinetics and saturation analysis gave the following reaction constants for [3H]E1: Km = 0.33 microM, Vmax = 2.5 pmol/min/mg; and for PAPS: Km = 33 microM, Vmax = 1.1 pmol/min/mg; competition studies carried out in the absence or presence of 1- or 5-fold excess radioinert steroids indicated that estrogen (E2 > E1) as well as androgens (T = DHEA > 5 alpha dihydrotestosterone, DHT) were effective inhibitors. Sulfotransferase activity was found to be stage-related, being highest in PoM regions (2.31 +/- 0.24 pmol/min/mg protein) when compared to M and PrM regions (1.22 +/- 0.22 and 1.28 +/- 0.21 pmol/min/mg protein, respectively). Sulfoconjugation and the intratesticular distribution of steroid sulfates were also measured in vivo by perfusion of the intact testis with [3H]androgen or -estrogen. The pathway of blood flow via the genital artery was epigonal organ-->PoM-->M-->PrM (mature-->immature). Perfused [3H]E2, T, and DHT were all extensively metabolized in a one-pass, 1 hr perfusion, less than 10% of perfused [3H] steroid being recovered from testicular tissues as unchanged steroid. In general, recovery of polar metabolites was greater than non-polar metabolites from all three substrates. Sequential hydrolysis with glucuronidase and glusulase indicated that sulfoconjugation is a minor component (< 20%) of several "inactivating" pathways, which include glucuronide conjugation, 17-ketosteroid synthesis, and pathways leading to unidentified polar metabolites. No consistent stage-related distribution patterns were observed for any of the metabolite subfractions; however, total recovered radioactive steroid (polar plus non-polar) formed a decreasing concentration gradient from point of entry of perfusate (PoM region) to point of exit (PrM region). These data support the conclusion that access to receptors by steroid ligands may be controlled by a balance between activating and inactivating pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

Using the dogfish shark (Squalus acanthias) testis model in which different germ cell stages are topographically separated, we previously observed that steroidogenic enzyme activities vary qualitatively and quantitatively during spermatogenesis. To determine whether these data, obtained by radiolabeled tracer analysis of testicular microsomes, accurately predict steroid secretion by intact cultured spermatocysts (germ cell/Sertoli cell units), steroids were radioimmunoassayed directly in spent medium. Steroid output in basal medium was low, but after addition of 25-hydroxycholesterol (25-OH chol, 60 microM), large amounts of progestins (P) and testosterone (T) accumulated. Dehydroepiandrosterone (DHEA), androstenedione (A), and estradiol (E2) were low or nondetectable in the presence or absence of 25-OH chol; however, addition of T (1 microM) as substrate elevated E2 above assay limits. T and P contents of media increased progressively over a 24-hr culture period (Day 1). Replacement and analysis of spent media at 24-hr intervals indicated that secretion was continuous up to 6 days after seeding, although secretory rates (T plus P) and T/P ratios changed with time in culture. Spermatocysts in premeiotic (PrM), meiotic (M), and post-meiotic (PoM) stages all secreted T and P; however, absolute values, T/P ratios, and stage-dependent patterns varied from one experiment to another with no obvious seasonal pattern. Also steroid secretion by staged cysts did not consistently agree with predictions based on steroidogenic enzyme activities measured in earlier cell-free assays, whether or not 25-OH chol was present. This discrepancy was not explained by stage-related patterns of steroid secretion vs retention or by addition of putative in vivo regulators. Agents that elevated P and/or T were dibutyryl cyclic AMP, 1-isobutyl-3-methylxanthine (IBMX), and forskolin, whereas ventral lobe extract and Ca2+ ionophore (A23817) were inhibitory. An autoregulatory feedback loop was indicated by a negative relationship between cyst concentration per well and steroid secretion rate. In contrast to T and P, E2 secretion in the presence of substrate (T, 1 microM) was as predicted from previously determined patterns of aromatase activity (M > PrM > PoM) and increased in response to IBMX. Added [3H]P, [3H]T and [3H]E2 all were extensively metabolized to polar products and P metabolism was stage related (PoM > M > PrM). These data indicate that analysis of free P, T, and E2 in static cyst cultures may underestimate true steroidogenic potential. Assuming further characterization of the system, we conclude that cultured spermatocysts from dogfish testis have potential for studying the multifactorial regulation of steroid synthesis and secretion stage-by-stage during spermatogenesis.

Although testosterone (T) is essential for the normal completion of spermatogenesis, the exact T-sensitive control points are still unknown. Using staged tissues (premeiotic, PrM; meiotic, M; and postmeiotic, PoM) from zonal testes of the spiny dogfish Squalus acanthias, and standard [3H] steroid binding analysis, we characterized a T-binding component with physiochemical characteristics resembling classical androgen receptors (AR). [3H]T binding was of high affinity (dissociation constant = 4.4 x 10(-9) M), limited capacity (maximum binding, 94 fmol/g tissue) and relatively stable (t1/2 = 4 h at 4 C). The T-binding component was present in both cytosolic and nuclear extracts, adhered to DNA-cellulose, and displayed predicted sedimentation properties of an activated receptor in vivo or in vitro (5.06S). T, 5 alpha-dihydrotestosterone (DHT), and mibolerone (Mib), but not methyltrienelone (R1881), competed well for [3H]T binding; however, progesterone (P) was equivalent to T in its ability to displace tracer. Subsequent analysis of [3H] P binding revealed a P-binding component that was present in nuclear and cytosolic extracts, adhered to DNA, but differed from AR in its inability to bind Mib. Competition studies in which excess radioinert Mib was used to block AR revealed ligand specificity characteristics of progesterone receptors (PR): promegestone (R5020) greater than P greater than deoxycorticosterone, but T, DHT, dexamethasone, and corticosterone were ineffective competitors. Also, a nonlinear Scatchard plot was obtained, suggesting two P-binding activities, which differed in their binding affinities (dissociation constant = 0.88 vs. 5.9 x 10(-9) M) and capacities (66 vs. 210 fmol/g tissue). Conversely, using [3H]Mib to avoid interference from PR, we confirmed that T, DHT, and P were equivalent in their ability to displace ligand from AR. Comparison of tissues by stage of spermatogenesis revealed different distribution patterns for AR (PrM greater than M much greater than PoM) vs. PR (PoM much greater than M = PrM). These data provide definitive evidence for separate testicular T- and P-binding mechanisms and indicate the presence of temporally distinct sets of steroid-regulated genes.

A. A. Berthold (1849, cited in 1) is credited with pioneering the field of experimental endocrinology. In his classic experiments with roosters, Berthold observed that crowing, sexual behavior and aggressiveness could be eliminated by castration and restored by reimplanting testes, and he came to the important conclusion that the active principle was a blood-borne substance. Since the time of Berthold, the secreted products of the gonads have been identified as steroids, and our understanding of the molecular mechanisms and behavioral manifestations of steroid action in the central nervous system (CNS) has expanded considerably. Nonetheless, gaps in our knowledge remain, and steroid control of vertebrate behavior continues to be a focus of intense investigation. One important area of research has been focused on the hormonal basis of aggressive behavior. In this paper, we will review results of studies in this laboratory in which we have examined the hormonal basis of individual and sex-related differences in the intensity of aggressive displays in the Japanese quail (Coturnix coturnix japonica). As a preface to this detailed consideration of aggressive behavior and brain-steroid interactions in the control of behavior, it is appropriate to consider landmark technical and conceptual advances which are the foundation of present-day research.

Background Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. To identify housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish. Results QPCR analysis was used to quantify mRNA levels of bactin1, tubulin alpha 1(tuba1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), TATA-box binding protein (tbp), beta-2-microglobulin (b2m), elongation factor 1 alpha (elfa), and 18s ribosomal RNA (18s) during development (2 – 120 hr postfertilization, hpf); in different tissue types (brain, eye, liver, heart, muscle, gonads) of adult males and females; and after treatment of embryos/larvae (24 – 96 hpf) with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified 18s, b2m, and elfa as most stable during development and across tissue types, while gapdh, tuba1, and tpb were the most variable. Following chemical treatment, tuba1, bactin1, and elfa were the most stably expressed whereas tbp, 18s, and b2m were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using different reference genes to measure suppression of cyp19a1b by an estrogen receptor antagonist and induction of cyp1a by an arylhydrocarbon receptor agonist, the direction and magnitude of effects with stable and unstable genes differed. Conclusion This study provides data that can be expected to aid zebrafish researchers in their initial choice of housekeeping genes for future studies, but underlines the importance of further validating housekeeping genes for each new experimental paradigm and fish species.

1. 1. Although testosterone (T) stimulates aggressiveness in males of many vertebrate species, it is now known that the full expression of T actions in the central nervous system requires aromatization to estradiol (E2) and subsequent binding of formed E2 to its receptor. 2. 2. We have recently confirmed these as rate-limiting steps in the control of sex-related and individual differences in aggressiveness of the Japanese quail (Coturnix coturnix japonica). 3. 3. In this review, we describe some of the neuroendocrine factors which control aggression with a focus on our recent studies in quail.

Desert iguanas (Dipsosaurus dorsalis) were maintained at 10, 20, 30 or 40°C for one week. Animals were autopsied at zero time, and 30 and 90 minutes after a single injection of ACTH. Basal plasma corticosterone (B) level was similar in all lizards regardless of environmental temperature. Both blood glucose and liver glycogen levels showed highly significant correlations with environmental temperature. Injection of ACTH increased plasma B level in all temperature groups but the rate and magnitude of the response were temperature dependent. Liver glycogen was increased after ACTH injection at 20°C and plasma glucose response to ACTH was significantly correlated with temperature.