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12 pages, 6 figures, 2 tables, supplementary data https://dx.doi.org/10.1016/j.marenvres.2016.03.010, Specimens of Solea solea and Solea senegalenesis at different developmental stages were obtained from seven fishing grounds along the NW Mediterranean. Gonad development in males was classified into five stages, from early spermatogenesis to recovery, while four stages were considered in females, from growth to maturation. Vitellogenin (VTG) and sex steroid levels including an estrogen (estradiol, E2), two androgens (testosterone, T and 11-ketotestosterone, 11KT) and a progestin (17,20β-dihydroxy pregn-4-en-3-one, 17,20β-P or maturation inducing steroid, MIS) were analysed in plasma. Their levels were more clearly related to the developmental stage of the gonads than to the sampling site characteristics. In addition, enzyme activities in gonads, such as acetylcholinesterase (AChE) and carboxylesterase (CbE) were gender-dependent and higher in males than in females. Gonadal glutathione S-transferase (GST) activity was enhanced in the most anthropogenic impacted sites. VTG was absent in males and very low or undetectable in immature females, while mature females exhibited high VTG levels, clearly related to the gonado-somatic index. Sex steroid levels (ng/ml) varied in males and females regardless of the species. E2 levels in females ranged from 0.22 to 6.98 while in males ranged from 0.11 to 0.27. T varied from 0.12 to 0.93 in females and from 0.56 to 1.36 in males, while 11KT in females fluctuated from 0.03 to 0.57 and from 0.26 to 6.42 in males. Similarly, MIS in females ranged from 0.75 to 3.71 and from 1.12 to 5.61 in males. The lack of endocrine disturbances was confirmed by histological examination of the gonads. This study informs on basal sex hormone levels and enzyme activities during gonadal maturation of wild Solea spp. that can be useful in the identification and further remediation of possible pollution events, This work was financed by the Ministry of Science and Innovation of Spain (ref CTM2010-16611)

8 pages, 4 figures, 3 tables, Drospirenone (DRO) is one of the most widely used progestins in contraceptive treatments and hormone replacement therapies. The pharmacokinetics and potential toxicological effects of DRO were investigated in juvenile sea bass (Dicentrarchus labrax) exposed through the diet (0.01–10 μg DRO/g) for up to 31 days. DRO was detected in the blood (4–27 ng/mL) of fish exposed to the highest concentration, with no significant bioaccumulation over time and no alteration of hepatic metabolizing enzymes, namely, CYP1A and CYP3A-catalysed activities and UDP-glucuronyltransferase (UGT). Pregnenolone (P5), progesterone (P4), 17α-hydroxyprogesterone (17P4), 17α-hydroxypregnenolone (17P5), androstenedione (AD) and testosterone (T) were determined in plasma and gene expression of cyp17a1, cyp19a1a and cyp11β analysed by qRT-PCR in gonads. The significant increase in plasmatic levels of 17P5, 17P4 and AD detected after 31 days exposure to 10 ng DRO/g together with the increased expression of cyp17a1 in females evidence the ability of DRO to alter steroid synthesis at low intake concentrations (7 ng DRO/day). However, the potential consequences of this steroid shift for female reproduction remain to be investigated, Maria Blanco acknowledges a pre-doctoral fellowship (FPI, BES-2012-054438) from the Spanish Government and Paula Medina the pre-doctoral fellowship MECE2, ANT0806. Denise Fernandes acknowledges a post-doctoral fellowship (SFRH/BPD/34289/2006) from the Portuguese Fundação para a Ciência e Tecnologia (FCT) of the Ministry of Science and Technology of Portugal. This study was supported by the Spanish National Plan of Research (Project Ref. CGL2011-24538)

24 p., y bibliografía, As fish are ectothermic animals, water temperature can affect their basic biological processes such as larval development, growth and reproduction. Similar to reptiles, the incubation temperature during early phases of development is capable to modify sex ratios in a large number of fish species. This phenomenon, known as thermolabile sex determination (TSD) was first reported in Menidia menidia, a species belonging to the family Atherinopsidae. Since then, an increasing number of fish have also been found to exhibit TSD. Traditionally, likewise in reptiles, several TSD patterns have been described in fish, however it has been recently postulated that only one, females at low temperatures and males at high temperatures, may represent the “real” or “true” TSD. Many studies regarding the influence of temperature on the final sex ratios have been focused on the expression and activity of gonadal aromatase, the enzyme involved in the conversion of androgens into estrogens and encoded by the cyp19a1a gene. In this regard, teleost fish, may be due to a whole genome duplication event, produce another aromatase enzyme, commonly named brain aromatase, encoded by the cyp19a1b gene. Contrary to what has been described in other vertebrates, fish exhibit very high levels of aromatase activity in the brain and therefore they synthesize high amounts of neuroestrogens. However, its biological significance is still not understood. In addition, the mechanism whereby temperature can induce the development of a testis or an ovary still remains elusive. In this context the present review is aimed to discuss several theories about the possible role of brain aromatase using fish as models. The relevance of brain aromatase and therefore of neuroestrogens as the possible cue for gonadal differentiation is raised. In addition, the possible role of brain aromatase as the way to keep the high levels of neurogenesis in fish is also considered. Several key examples of how teleosts and aromatase regulation can offer more insight into basic mechanisms of TSD are also reviewed., The work was supported by a project from the Spanish Government “TERMOSEX” PET-2007-0390 to MB and from the Argentine Scientific Agency PICT 16-38206 to GMS.

9 pages, 5 figures, 1 table.-- PMID: 15607525 [PubMed].-- Available online Nov 25, 2004., Sex hormone binding globulin (SHBG) binds and transports androgens and estrogens in the blood of vertebrate species including fish. We have used oligonucleotide primers corresponding to highly conserved regions of the SHBG coding sequences within the zebrafish and fugufish genomes to obtain a 1528 bp cDNA encoding SHBG from tissue RNA extracts from the European sea bass. Amino-terminal sequence analysis of recombinant sea bass SHBG indicated that its deduced precursor polypeptide includes a 35-residue secretion signal polypeptide, and the 361-residue mature sea bass SHBG sequence exhibits 45–67% sequence identity with SHBGs from other fish species that have been determined directly (for zebrafish) or deduced (for rainbow trout, medaka and fugufish) from sequences within public databases. The sea bass SHBG (39,894 Da) comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites, and exists as a 118,300 ± 11,500 Da homodimer. Sea bass SHBG exhibits a high affinity (Kd = 8.8 nM for 17β-estradiol) and specificity for gonadal steroids and their precursors (e.g., 17β-estradiol > testosterone > dehydroepiandrosterone > 5α-dihydrotestosterone > androstenedione > 11-ketotesterone). Interestingly, the affinity of sea bass SHBG for the synthetic estrogen, 17α-ethynylestradiol was found to be essentially identical to that for 17β-estradiol. The availability of SHBG sequences in sea bass and other fish set the stage for detailed studies of SHBG function in fish reproductive physiology, as well as its potential role as a target of endocrine disruptors., Supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.

17 pages, 5 figures, 2 tables, Twenty-day exposures to 17α-methyltestosterone (MT) (10 mg kg-©1 food) starting at 86 or 106 days post fertilization (DPF) resulted in a complete masculinization of sea bass Dicentrarchus labrax, as opposed to 46% females present in the controls. Earlier exposures failed to suppress ovarian development, resulting in a variable number of females (range 10·5–49·5%). All treatments assayed between 110 and 210 DPF induced a complete masculinization, regardless of the androgen or the dose tested. However, a dose dependent increase in the number of fish with intratesticular oocytes was observed after MT but not after 17α-methyldihydrotestosterone (MDHT) administration. This might be a reflection of the capability of MT and not of MDHT to be converted into oestradiol by the action of aromatase. One hundred-day exposures to either of the androgens (10 mg kg©1 food) starting at 60 or 160 DPF resulted in a total suppression of ovarian development and a partial induction of sterility. Complete sterility was accomplished after 200 days of exposure starting at early ontogenesis (60–260 DPF). These deleterious effects on gonadal development were later confirmed by a dramatic reduction of the gonadosomatic index. In addition, around the first year of age, growth was significantly depressed in all groups except those for which treatments started at the latest (160–260 DPF; experiment 1) or the earliest and shortest (46–66 DPF; experiment 2). Hence, the critical period of androgen-inducible masculinization is located between 96 and 126 DPF and coincides with a rapid proliferation of primordial germ cells in the sexually undifferentiated gonad, We thank S. Massuti, C. Marín, R. Simó and J. Monfort for technical help. M.B. and A.F. were supported by scholarships from the Generalitat Valenciana. This work was supported by an EU grant AIR2-CT93-1543, a CICYT grant AGF94-1321-CE and a contract from GEDISA to SZ

An RNA-arbitrarily primed PCR differential display strategy was used to identify candidate genes in the pituitary that are up-regulated by endogenously activated gamma-aminobutyric acid (GABA) systems that may also be involved in the control of reproduction. Goldfish were injected with the GABA metabolism inhibitor gamma-vinyl-GABA (GVG), known for its high efficiency to specifically increase endogenous brain and pituitary GABA levels in this species, resulting in higher levels of circulating gonadotropin-II (GTH-II). Several transcripts related to hormone secretion, signal transduction pathways, and messenger RNA (mRNA) editing were shown to be up-regulated after GVG injection. Among these transcripts we characterized an mRNA coding for the secretory vesicle protein secretogranin-II (SgII), a member of the chromogranin family, which is the precursor of a novel 34 amino acid neuropeptide, goldfish secretoneurin (SN). A semiquantitative PCR developed to measure pituitary SgII mRNA levels showed a 5-fold increase in GVG treated fish vs. control fish. Moreover, GVG treatment specifically increased SgII mRNA levels in gonadotrophs, concomitant with a decrease in GTH-II cell content. In addition, i.p. injection of synthetic goldfish SN increased GTH-II release in goldfish pretreated with the dopamine antagonist domperidone. Activation of GABAergic neurons has two effects, enhancing in vivo GTH-II release and up-regulating SgII mRNA specifically in goldfish gonadotrophs. Together with our SN bioactivity data, this suggests the existence in the pituitary of an autocrine or paracrine mechanism linked to the regulated secretory pathway in the gonadotrophs.

This study investigated the effects of estrogens on sexual differentiation in sea bass (Dicentrarchus labrax L.), a gonochoristic marine teleost that under culture conditions has a histologically sexual undifferentiated period that covers most of the first year of life, after which most individuals develop as males. Sea bass that had no noticeable histological sign of sex differentiation were fed estrogens at two doses (5 or 10 mg kg-1 food) and for different periods ranging from 48 to 426 days post fertilization (DPF). Exposure to the synthetic estrogen 17α-ethynylestradiol (EE2) at 10 mg kg-1 food from 60 to 260 DPF, including the sensitive period to equivalent doses of synthetic androgens previously determined for this species (126-226 DPF), significantly (p < 0.05) more than doubled the number of juvenile females to 80%, compared to the control value of 33%, and completely suppressed gonadal development in the remaining 20% of the population. This suggests that the period during which sea bass gonads exhibit high sensitivity to androgens is also very sensitive to estrogens. A comparable exposure to the natural estrogen estradiol-17β (E2) resulted in 13% of the fish having suppressed gonadal development, but induced 57% of the fish to develop gonads with germinal tissue of both sexes, suggesting a pivotal role for E2 during this sensitive period. Earlier exposure to EE2 at 10 mg kg-1 food from 48-88 DPF, significantly (p < 0.05) increased the number of females to 62% from 36% in the control group, allowing for the normal testicular development in the remaining fish. In contrast, a later chronic exposure (226-426 DPF) to E2, at either 5 or 10 mg kg-1 food, starting when the gonads showed no sign of sexual differentiation but past the critical sensitive period, had no effect on the resulting overall sex ratios, indicating that after this period responsiveness of the gonads to estrogens decreases as gonadal sexual differentiation progresses. However, the consequences of this apparently innocuous exposure were later manifested in adults, exemplified by a significant dose-dependent reduction in the number of mature males at 626 DPF, coinciding with the second reproductive season, the time when males normally reach sexual maturation in cultured sea bass. This suggests that chronic exposure to E2 past the critical sensitive period may not affect the sex ratio, but could result in alterations in the male reproductive organs. This was later verified by histological analysis which revealed a significant (p < 0.05) dose-dependent reduction of the surface of the testicular lobules in the remaining males that did not mature. Together, these experiments illustrate both readily observable and subtle effects of estrogens on sex proportions, gonadal morphology and maturation rates, providing evidence that estrogen exposure can have delayed action in a teleost in a manner similar to the effects described for mammalian species. The possible existence of effects of this latter type in adult fish could be considered when evaluating the consequences of deliberate or accidental exposure to estrogens or putative estrogenic chemicals, particularly if such exposure had taken place during sex differentiation.

This study investigates the metabolism and mode of action of galaxolide (HHCB) in the European sea bass -Dicentrarchus labrax- following a single intraperitoneal injection of 50 mg HHCB/kg body weight. In addition, a group of fish was injected with 50 mg/kg of ketoconazole (KCZ), a fungicide that is known to interfere with different Cyp isoenzymes. HHCB was actively metabolised by sea bass and acted as a weak inhibitor of the synthesis of oxyandrogens in gonads of male fish. Both, HHCB and a hydroxylated metabolite were detected in bile. The fungicide ketoconazole was a strong inhibitor of Cyp11β and Cyp3a-catalyzed activities. The work contributes to the better understanding of the impact of synthetic musks on fish and proposes the determination of HHCB and/or its hydroxylated metabolite in bile as a tool to assess environmental exposure in wild fish. © 2012 Elsevier Ltd. All rights reserved., This study was supported by the Spanish Ministry of Science and Innovation under Project CGL2008-01888/BOS and CSIC project PIE 200930I037. Denise Fernandes acknowledges a postdoctoral fellowship (SFRH/BPD/34289/2006) from the Portuguese Fundaçao para a Ciencia e Tecnologia (FCT), and Giorgio Dimastrogiovanni a predoctoral fellowship (BES-2009-025271) from the Spanish Ministry of Science and Education.

14 pages, 5 figures, The purpose of this study was to develop a method for the hormonal masculinization of sea bass (Dicentrarchus labrax L.) reared under natural conditions of photoperiod and temperature. Sexually undifferentiated sea bass were fed 17α-methyltestosterone (MT) at 10 mg kg−1 food during three distinct periods of 100 days each: Period I, 126–226 days post fertilization (DPF) (July–November); Period II, 226–326 DPF (November–February); Period III, 326–426 DPF (February–May), and their combinations. Phenotypic sex ratios after hormone treatment were determined by histological examination of the gonads. MT significantly increased the proportion of juvenile males from controls (79%), to 93–100% in groups for which treatment included Period I. In contrast, no significant alterations in the phenotypic sex ratio were observed when MT treatment started after this period. In groups treated with MT for more than 100 days, including Period I, growth was significantly depressed. Furthermore, when MT was given for 300 days (126–426 DPF), 7% of the fish were intersex. The effects of MT on the developing gonads were confirmed by a significant reduction of the gonadosomatic index (GSI) when treatment included Period I regardless of treatment duration. The visceral fat index (VFI) decreased with increasing durations of MT administration, denoting a lipolytic effect. However, 200 or more days after MT withdrawal, the mean VFI of MT-treated groups showed no differences with respect to that of the controls, indicating that the effects of MT on visceral fat mobilization were transitory. Similarly, reduction of the hepatosomatic index (HSI) and increase of the carcass index were found to be related to the length of MT administration, but values similar to those of controls were obtained after suppression of hormonal treatments. Survival of the treated groups was not compromised (average 76% vs. 78% in controls), and no teratological phenomena were observed. No treatments starting before Period I or of less than 100 days of duration were tested in this study; however, the acquisition of complete and functional sex reversal without any deleterious effects after MT administration during this period and the lower effectiveness of MT treatment in progressively more advanced stages of development, shows that Period I is effective for the complete and permanent masculinization of sea bass, M. Blázquez was supported by a scholarship from the Generalitat Valenciana. This work was supported by EC Grant (Bridge no. 900188-C), CICYT Project (B1092-001 I-CE) to M.C., and by NATO Collaborative Research Grant (CRG-890958) to M.C. and E.M.D.

28 p., 3 figures and references., The pharmaceutical fluoxetine, a selective serotonin reuptake inhibitor (SSRI), is often detected in municipal wastewater treatment plant effluents and surface waters within the ng/l range. There is, however, insufficient research evaluating potential hazards of fluoxetine in aquatic organisms at environmentally relevant concentrations. Taking into account that several SSRIs (fluoxetine, fluvoxamine) act as spawning inducers in bivalves, this study aimed at investigating the effects of fluoxetine exposure in the zebra mussel (Dreissena polymorpha) by assessing its potential to induce spawning at environmentally relevant concentrations (20 and 200. ng/l), as well as alterations of endogenous levels of testosterone and estradiol. Histological analyses of female and male gonads showed a concentration dependent decrease of oocyte and spermatozoan density, with a reduction in the number of oocytes per follicle of 40-70%, and spermatozoan density of 21-25%, relative to controls, following exposure to 20 and 200. ng/l of fluoxetine for 6 days, respectively. There was also a significant increase (1.5-fold) in the endogenous level of esterified estradiol in organisms exposed to 200. ng/l fluoxetine. Overall, the study shows that exposure to low levels of fluoxetine may effectively induce gamete liberation in the zebra mussel as well as alter endogenous levels of estradiol, and evidences the need of further investigating the potential of fluoxetine to alter the endocrine system of molluscs at environmentally relevant concentrations., This study was funded by the Ministerio de Medio Ambiente, Medio Rural y Marinom projects 041/SGTB/2007/1.1 and 042/RN08/03.4, the Spanish Ministry of Science and Education under Project Ref. CGL2008-01888/BOS, and a CSIC project (200930I037).

9 pages, 6 figures.-- PMID: 18983844 [PubMed].-- Available online Oct 18, 2008., The objective of this study was to contribute to our understanding of the role of sex steroids in fish sex differentiation and male maturation. Sexually undifferentiated sea bass were administered 17α-methyldihydrotestosterone (MDHT), estradiol-17β (E2), fadrozole (Fz), cyproterone acetate (CPA) or tamoxifen (Tx). MDHT produced 100% males whereas E2 and Tx resulted in 100% females. Fz produced 95% males while CPA did not alter sex ratios. E2 treatment did not affect gonadal aromatase (cyp19a) expression levels, supporting the possibility that the feminizing effect of exogenous E2 are not directly related to cyp19a regulation. Both MDHT and Fz decreased cyp19a expression. Moreover, androgen receptor (ar) expression levels increased during development in all but the MDHT group, suggesting that early exposure to an androgen down-regulates subsequent ar expression in males and that Fz does not interact with the androgen receptor. Together, these observations indicate that although MDHT and Fz result in a similar phenotype, the molecular pathways involved are likely different, and show that Fz masculinization is the consequence of inhibited ovarian differentiation rather than of a direct androgenic effect. Further, since CPA did not alter sex ratios when administered during the period of highest androgen sensitivity, we suggest that androgens are not required for initial testicular differentiation in the sea bass. MDHT and Fz did not alter the number of precocious males but reduced and increased, respectively, their gonadosomatic index (GSI). In addition, Fz had lasting effects on the GSI of precocious and non-precocious males, probably due to alterations of estrogen function in the testis, Supported by MEC Grants "SEXRATIO AGL-2002-02636" and "SEXGENE AGL-2006-01359", and a Grant from the Government of Catalonia to FP. L.N. was supported by a fellowship from MEC, BES-2003-0006 and M.B. was supported by a "Ramón y Cajal" contract from the Spanish Ministry of Science and Technology

7 pages, 2 figures, 1 table.-- PMID: 18573255 [PubMed].-- Printed version published Aug 2008., The present study addresses the role of aromatase and estrogen receptors in sex differentiation and development. With this purpose, a sea bass female- and a male-dominant group were obtained by successive size gradings since in this species females are already larger than males at the time of sex differentiation. Changes in cyp19a and cyp19b gene expression and enzymatic activity were monitored by a validated real-time PCR and a tritiated water assay, respectively, during early development and sex differentiation. Changes in mRNA expression of estrogen receptors, both erb1 and erb2, were also assessed during this period. Results show clear sex-related differences in cyp19a gene expression and enzymatic activity in gonads, with females exhibiting significantly higher levels than males at 150 days post hatching (DPH), when histological signs of sex differentiation were evident. cyp19b gene expression and activity in brain were detectable during early ontogenesis at 50 DPH but no clear sex-related differences were observed. Both erb1 and erb2 showed higher gene expression levels in testis than in ovaries around 200–250 DPH, corresponding with the time of testicular differentiation and precocious male maturation, but no sex-related differences were found in the brain. Together these results indicate that in the European sea bass high expression levels of cyp19a are associated with ovarian differentiation and thus cyp19a can be considered as a suitable molecular marker of ovarian differentiation. However, the involvement of cyp19b in sex differentiation cannot be concluded. In addition, the higher levels of erb1 and erb2 in males versus females during sex differentiation, coinciding with precocious male maturation in the sea bass, suggest an important role for these receptors in testicular development and maturation., Supported by an EU Grant Q5RS-2000-31365 "PROBASS" to F.P. and C.M. M.B. was supported by a EU Postdoctoral contract and a "Ramón y Cajal" contract from the Spanish Ministry of Science and Technology. A.G. was supported by a fellowship from the Spanish Ministry of Science and Technology.

18 pages, 5 figures, 1 table.-- PMID: 17719171 [PubMed].-- Printed version published Sep 30, 2007., Sex hormone-binding globulin (SHBG) transports androgens and estrogens in the blood of vertebrate species, including fish, and regulates the bioavailability and metabolic clearance of these steroids. Liver is the major site of plasma SHBG synthesis, while an SHBG homologue, known as the androgen-binding protein, is produced in testes. When shbg gene expression was examined throughout European sea bass (Dicentrarchus labrax L.) development, SHBG mRNA was clearly detectable at 7 days post-fertilization and persisted throughout embryonic development. In male and female sea bass, the liver is the principal site of shbg gene expression, as determined by SHBG mRNA analyses. Immunoreactive SHBG is present in the liver and villous stroma of the intestine in both sexes. It is also present in the interstitial space between testicular lobules, and the connective tissue surrounding the ovary in the non-reproductive season and around post-vitellogenic oocytes. Plasma SHBG levels were measured over a 10-month period as male sea bass undergo sexual maturation. Immature females of the same age were also studied over the same time interval. The mean ± S.E.M. plasma SHBG levels in 2-year-old males and females are lower (80 ± 15 nM and 82 ± 16 nM, respectively) during the winter reproductive season (December–March) than the spring (April–June) months (144 ± 32 nM and 193 ± 18 nM, respectively). In both sexes, plasma SHBG levels start to decline 1–2 months before the reproductive season, coincident with a period of rapid weight gain, while increases after the reproductive season are not accompanied by significant changes in body weight. In addition, plasma SHBG in triploid (sterile) and diploid (fertile) male sea bass do not differ during the first spawning season. These data suggest that the decrease in plasma SHBG levels during sexual maturation in sea bass is related to nutritional or metabolic effects in relation to water temperatures and food intake, rather than changes in gonadal sex steroid production, Supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to GLH and a grant from the Aquaculture Research Network of Catalonia to FP. GLH holds a Tier 1 Canada Research Chair in Reproductive Health

Cytochrome P450 aromatase is the enzyme complex responsible for the synthesis of estrogens by the aromatization of androgens. In the vast majority of tetrapods examined so far, aromatase is the product of the Cyp19 gene, which exists as a single copy per haploid genome. In contrast, in teleosts there are two isoforms of the aromatase gene, Cyp19a and Cyp19b, which encode two structurally different proteins, P450aromA and P450aromB, respectively, with similar catalytic activities. The promoter region of both genes has been characterized in several teleost species and more than 20 different regulatory sites have been identified to date. These include response elements for members of the nuclear receptor superfamily, notably sex steroid receptors, and at least five transcription factors related to neurogenesis. This supports the idea that, besides other functions such as the control of reproduction, aromatase and therefore estrogens are actively involved in neurogenesis. Aromatase mRNA expression studies revealed that P450aromA and P450aromB are preferentially, but not exclusively, expressed in the gonads and brain, respectively. Other organs and tissues where aromatase is expressed, albeit at much lower levels include the pituitary, retina, anterior kidney, testis, liver and visceral fat, suggesting local actions of estrogens in several peripheral targets. Gene expression levels are usually matched by actual catalytic activity, with Km usually in the range 5¿ 50 nM and Vmax in the order of a few pmol/mg protein/h. The current challenge is to understand the regulation of both aromatase genes, especially in the context of sex differentiation and as a response to environmental factors, including temperature, social interactions, and endocrine disruptors, which is briefly reviewed. It is also important to gain a better understanding of the specific functions of estrogen in different tissues and key developmental and reproductive events throughout the fish life, Research at the authors’ laboratory supported by Spanish Ministry of Education and Science (MEC) Grants MAR96-1860 and AGL2002-02636 (‘‘SEXRATIO’’), and EU grant Q5RS-2000-31365 (‘‘PROBASS’’) to FP. M.B. was supported by a postdoctoral contract from the EU and from the MEC’s ‘‘Ramo´n y Cajal’’ program

12 pages, 5 figures, The teleost brain is characterized by exceptionally high levels of aromatase, the enzyme that converts androgens into estrogens, and by its continuous growth throughout life. Gonadal estrogens have been implicated in sex differentiation and the control of reproduction in adult fish, but the role of neural estrogens during early development is far from clear. The present study describes the isolation and characterization of the cDNA sequence from brain aromatase (P450aromB) in the European sea bass (Dicentrarchus labrax L.), a well established model for neuroendocrine research in fish. P450aromB was cloned from a brain cDNA library and encoded a predicted protein of 505 residues, with a calculated molecular weight of 57.2 kDa. Comparisons of the deduced amino acid sequence to that of the ovarian aromatase (P450aromA) in the same species revealed 62% identity, lower than the 84% identity shared between sea bass and tilapia brain aromatases. Phylogenetic analysis showed the occurrence of a gene duplication for P450arom in the teleost lineage after its divergence from the tetrapods. Moreover, the low percentage of identity between brain and ovarian forms within the same species suggests that both genes evolved separately right after the appearance of the teleosts. Tissue-specific expression of P450aromA and P450aromB mRNA was studied in adult sea bass. P450aromB was preferentially expressed in brain of both males and females but also present at much lower levels in testis, ovary and head kidney, an organ known for its steroidogenic capabilities in fish. However, P450aromA expression was restricted to testis and ovary. A semiquantitative PCR was developed to measure P450aromB mRNA levels. Analysis of the expression of P450aromB in the brain of juvenile sea bass showed that females exhibited higher mRNA levels than males at 200 days post fertilization (dpf), by the time of gonadal sex differentiation. A switch in expression occurred thereafter, between 200 and 250 dpf, with males exhibiting higher levels than females. This situation was maintained by 300 dpf and is in agreement with measured levels of enzymatic activity in adults. These changes and sex-related differences in expression profiles may imply differences in the functionality of the enzyme between males and females, suggesting an important role for P450aromB in sea bass sex differentiation. However, due to the continuous growth of the teleost brain throughout life, a role in neurogenesis for brain aromatase should also be considered, This work was supported by E.U. grant Q5RS-2000-31365 to F.P. M.B. was supported by a postdoctoral contract from the E.U.

Groups of sexually undifferentiated sea bass Dicentrarchus labrax were fed with the androgen 17α-methyltestosterone (MT) during sex differentiation. MT treatment increased males from 79 ± 3% in the controls (the usual 3:1 male:female sex ratio of cultured sea bass) to 100 ± 0%, implying that in the treated groups one out of each five resulting males was a masculinized female (neomale). Thirteen males from the MT treated groups were taken as the parental generation and their sperm used to individually fertilize a pool of eggs from unrelated females. The probability of having at least one neomale was 95% and most probably two or three of the males used were neomales. The offspring from each family were reared separately under the same environmental conditions. Samples were taken at 11 and 15 months of age, during and after sex differentiation, respectively. Results showed that females predominated among the larger fish whereas males and undifferentiated fish predominated among the smaller ones. Intersexes exhibited an intermediate size. All fish with a body length smaller than 12 cm were undifferentiated. These results suggest that sex differentiation is more dependent on length than on age. At 15 months, sex ratios were male-biased in all families, except one (females ranged from 5 to 50%) and only two families had sex ratios not significantly different from 1:1, suggesting that the mechanism of sex determination in the sea bass is not of a XX/XY or ZW/ZZ type since no family exhibited a female-biased progeny, as would be expected from both types. Results support the hypothesis that factors other than genetic, i.e., environmental, may act epigenetically on the sex determination mechanisms of sea bass, as has been demonstrated in other fishes, M.B. was supported by a scholarship from the Generalitat Valenciana. This work was supported by an EU grant AIR2-CT93-1543 and CICYT grants AGF93-0127 and AGF94-1321-CE to S.Z.

Extract: The secretion of insulin in vivo and its synthesis and release by pancreatic islets in vitro were studied in rats at various stages of development. An oral glucose load caused a rapid and significant increase in the serum insulin level of pregnant rats and a slower response in their 21-day-old fetuses. Circulating free fatty acids (FFA) decreased in the dams, but did not change in the fetuses. A relative glucose intolerance was found in pups 1 hr, 5 days, and 10 days after birth. This was accompanied by delayed insulin response and by little or no decrease of circulating FFA. After weaning, the glucose tolerance and the insulin and FFA responses gradually improved. The oral administration of a 10-amino acid mixture caused a greater insulin release in newborn and nursing rats than in 21-day-old fetuses or in young adult rats. Total serum lipids were higher in 1-, 5-, 10-, and 20-day-old rats than in fetuses and adult animals, whether fed or fasted, but 18 hr of fasting decreased total lipids and phospholipids in all age groups. Thus, the age of relative hyperlipidemia corresponded with that of relative glucose intolerance. The secretion of insulin in vitro by pancreatic islets of 5-, 10-, 20-, 30-, and 45-day-old rats increased significantly when the concentration of glucose in the incubation medium was increased from 30 to 300 mg/ml. The islets of 30- and 45-day-old rats secreted the largest amount of insulin. The insulin content of pancreatic islets was higher and the percentage of insulin secreted in relation to the hormonal content was smaller in the islets of nursing rats. Islets of fetal, nursing, and adult rats incorporated L-[4,5-3H] leucine into proinsulin and insulin, but the conversion of proinsulin to insulin was minimal in the 1-day-old rat. This incorporation was greater when the glucose concentration of the medium or the incubation time were increased. Thus, birth appears to be associated with a temporary decrease in the ability to release insulin and to dispose of an exogenous glucose load. Speculation: Birth deprives the newborn pup of transplacental nutrition and exposes it to the danger of neonatal hypoglycemia, especially in the interval between birth and the first act of nursing. This danger is moderated by a previously described increase in the serum level of glucagon and by a decrease in the serum level of insulin. This change in the glucagon to insulin ratio favors hepatic glycogenolysis and gluconeogenesis and, possibly aided by the relative hyperlipidemia characteristic of this age, decreases the utilization of glucose by the relatively large masses of insulin-responsive tissues. Gluconeogenesis continues to be a major source of glucose throughout the nursing age, when the animal consumes a diet rich in protein and fat and low in carbohydrate. Although the fetal pancreas can synthesize insulin, the mechanism for its release and hence the ability to dispose of a glucose load do not reach full maturity until weaning, when the dietary intake of carbohydrate increases and the level of serum lipids decreases toward the values characteristic of the adult animal.

SummaryThe concentration of immunoreactive growth hormone (GH) in the serum of rats increased significantly from the 19th to the 21st day of fetal life, began to decrease on the last day of gestation, reached minimum values on the 20th day of extrauterine life and increased again after the pups were weaned. The relative rate of weight gain was greatest between the 19th and the 20th day of fetal life. After this time, it declined progressively, except for a brief period between the 5th and 10th day after birth. Thus, this parameter of growth followed a course that was more closely parallel to previously published changes in the level of serum insulin than in the changes in serum GH observed in these experiments. It is possible that, in fetal and young rats, growth may depend upon a combined action of insulin and GH in which the latter, rather than the former, plays the role of permissive factor.Addendum. Since this manuscript was submitted for publication, we found that the oral administration of glucose (...