Production Of 11-ketotestosterone Research Articles

Loss of Function of Vasoactive-intestinal Peptide Alters Sex Ratio and Reduces Male Reproductive Fitness in Zebrafish.

Vasoactive-intestinal peptide (Vip) is a pleiotropic peptide with a wide range of distribution and functions. Zebrafish possess 2 isoforms of Vip (a and b), in which Vipa is most homologous to the mammalian form. In female zebrafish, Vipa can stimulate LH secretion from the pituitary but is not essential for female reproduction, as vipa-/- females display normal reproduction. In contrast, we have found that vipa-/- males are severely subfertile and sex ratio of offspring is female-biased. By analyzing all aspects of male reproduction with wild-type (WT) males, we show that the testes of vipa-/- are underdeveloped and contain ∼70% less spermatids compared to WT counterparts. The sperm of vipa-/- males displayed reduced potency in terms of fertilization (by ∼80%) and motility span and duration (by ∼50%). In addition, vipa-/- male attraction to WT females was largely nonexistent, indicating decreased sexual motivation. We show that vipa mRNA and protein is present in Leydig cells and in developing germ cells in the testis of WT, raising the possibility that endogenous Vipa contributes to testicular function. Absence of Vipa in vipa-/- males resulted in downregulation of 3 key genes in the androgen synthesis chain in the testis, 3β-hsd, 17β-hsd1, and cyp11c1 (11β-hydrogenase), associated with a pronounced decrease in 11-ketotestosterone production and, in turn, compromised reproductive fitness. Altogether, this study establishes a crucial role for Vipa in the regulation of male reproduction in zebrafish, like in mammals, with the exception that Vipa is also expressed in zebrafish testis.

Adrenal gland involvement in 11-ketotestosterone production analyzed using LC-MS/MS.

11-ketotestosterone (11KT), which is derived by the bioconversion of testosterone via 11β-hydroxytestosterone (11OHT), is a potent agonist of the human androgen receptor. The adrenal gland is considered an important organ in 11KT production because CYP11B1, which catalyzes testosterone to 11OHT, is expressed in the adrenal glands. The present study aimed to demonstrate adrenal gland involvement in 11KT production in prepubertal children, a topic which has not yet been addressed by any previous studies. Three, retrospective, observational studies were performed. Study 1 enrolled patients aged 8 months to 7 years with severe Kawasaki disease (KD) who were treated with mPSL pulse. Studies 2 and 3 included patients who had received a corticotropin-releasing hormone (CRH) stimulation test and adrenocorticotropic hormone (ACTH) stimulation test, respectively. Samples were collected before and after treatment or drug administration, and serum 11KT, 11OHT, and other 11-oxygenated androgens were measured by LC-MS/MS. Steroid hormone values before and after medication were analyzed using the Wilcoxon signed rank test. Studies 1, 2, and 3 included twenty patients with severe KD, eight patients with a CRH stimulation test, and eight patients with an ACTH stimulation test, respectively. Study 1 demonstrated that the median (IQR) 11KT level was significantly higher before, than after, mPSL pulse (0.39 (0.28-0.47) nmol/L versus 0.064 (0.012-0.075) nmol/L; P < 0.001). Studies 2 and 3 indicated no significant difference in the median 11KT value before and after the CRH or ACTH stimulation test while the 11OHT value was significantly higher after the test. In conclusion, the mediation of 11KT production by ACTH demonstrated the importance of the adrenal glands in the synthesis of this androgen in prepubertal children.

The A-ring reduction of 11-ketotestosterone is efficiently catalysed by AKR1D1 and SRD5A2 but not SRD5A1

Testosterone and its 5α-reduced form, 5α-dihydrotestosterone, were previously thought to represent the only active androgens in humans. However, recent studies have shown that the potent androgen, 11-ketotestosterone, derived from the adrenal androgen precursor, 11β-hydroxyandrostenedione, may in fact serve as the primary androgen in healthy women. Yet, despite recent renewed interest in these steroids, their downstream metabolism has remained undetermined. We therefore set out to investigate the metabolism of 11-ketotestosterone by characterising the 5α- or 5β-reduction commitment step. We show that inactivation of 11-ketotestosterone is predominantly driven by AKR1D1, which efficiently catalyses the 5β-reduction of 11-ketotestosterone, committing it to a metabolic pathway that terminates in 11-ketoetiocholanolone. We demonstrate that 5α-reduction of 11-ketotestosterone is catalysed by SRD5A2, but not SRD5A1, and terminates in 11-ketoandrosterone, but is only responsible for a minority of 11-ketotestosterone inactivation. However, as 11-ketoetiocholanolone is also generated by the metabolism of the glucocorticoid cortisone, 11-ketoandrosterone should be considered a more specific urinary marker of 11-ketotestosterone production.

Establishment of a stem Leydig cell line capable of 11-ketotestosterone production.

The deficiency or insufficiency of androgen can trigger a range of reproductive diseases as well as other symptoms. Stem Leydig cells (SLCs) are critical for the formation and maintenance of a functional androgen-producing cell (Leydig cell, LC) population throughout adult male life. However, to date, our knowledge about SLCs is poor. Here we report the derivation and characterisation of a clonal stem LC line (designated as TSL) capable of 11- ketotestosterone (11-KT) production from a 3-month-old Nile tilapia (Oreochromis niloticus) testis. The cells retained stable proliferation after 77 generations with normal karyotype and growth factor dependency. They expressed platelet-derived growth factor receptor-α (pdgfrα), nestin and chicken ovalbumin upstream promoter transcription factor II (coup-tfIIa), which are characteristic of SLCs. Upon induction in defined medium, TSLs could undergo differentiation into steroidogenically active LCs and produce 11-KT. When implanted into recipient Nile tilapia testes from which endogenous LCs had been eliminated by ethane dimethanesulphonate (EDS) treatment, the PKH26-labelled TSLs could colonise the interstitium, subsequently express steroidogenic genes and restore 11-KT production. Taken together, our data suggest that TSLs possess the ability of continuous proliferation and potential of differentiation into functional LCs invitro and invivo. To the best of our knowledge TSL might represent the first stem LC line capable of 11-KT production to date. Our study may offer new opportunities for investigating the self-renewal of SLCs and steroidogenesis invitro, and provide an invaluable invitro model for investigating endocrine disruptors.

17β-Hydroxysteroid dehydrogenase type 12a responsible for testicular 11-ketotestosterone synthesis in the Japanese eel, Anguilla japonica

The production of 11-ketotestosterone (11KT), an important steroid hormone in piscine spermatogenesis, is regulated by the pituitary gonadotropins [Gths: follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh)] and it is synthesized by catalytic reactions involving several steroidogenic enzymes. Among these enzymes, the role of 17β-hydroxysteroid dehydrogenases (Hsd17bs) that exhibited 17-ketosteroid reducing activity (17KSR activity) responsible for 11KT synthesis is still poorly understood. In the present study, for the deeper understanding of testicular 11KT biosynthesis, we first investigated the steroidogenic pathway to produce 11KT in Japanese eel testis. In vitro incubation of the testis with androstenedione (A4) and the subsequent analysis of the metabolites by thin-layer chromatography indicated that 11KT was synthesized from A4 via 11β-hydroxyandrostenedione (11OHA4) and 11-ketoandrostenedione (11KA4), which indicated that the steroidogenic enzyme exhibiting the 17KSR activity responsible for converting 11KA4 to 11KT is crucial for 11KT production. Subsequently, cDNAs encoding three candidate enzymes, Hsd17b type3 (Hsd17b3), Hsd17b type12a (Hsd17b12a), and 20β-hydroxysteroid dehydrogenase type2 (Hsd20b2), potentially with the 17KSR activity were isolated and characterized in the Japanese eel. The isolated hsd17b3, hsd17b12a, and hsd20b2 cDNAs putatively encoded 308, 314, and 327 amino acid residues with high homology to those of other vertebrate counterparts, respectively. The Hsd17b3, Hsd17b12a, and Hsd20b2 expressed either in HEK293T or in Hepa-E1 converted 11KA4 to 11KT. Tissue-distribution analysis by quantitative real time PCR revealed that hsd17b12a and hsd20b2 mRNAs were detected in the testis, while hsd17b3 mRNA was not detectable. Furthermore, we examined the effects of Gths on the 17KSR activity and the expression of the candidate genes in the immature testis. The 17KSR activity was upregulated by administration of Gths. Furthermore, only expression of hsd17b12a among three candidates was upregulated by Gths as well as the 17KSR activity. These findings strongly suggested that Hsd17b12a is one of the enzymes with 17KSR activity responsible for 11KT synthesis in the testis of Japanese eel.

Functional analysis of recombinant single-chain Japanese eel Fsh and Lh produced in FreeStyle 293-F cell lines: Binding specificities to their receptors and differential efficacy on testicular steroidogenesis

Pituitary gonadotropins, follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), play central roles in the control of gonadal development of vertebrates. In mammals, Fsh and Lh exclusively activate their respective cognate receptors: Fsh receptor (Fshr) in the Sertoli cell and Lh/choriogonadotropin receptor (Lhcgr) in the Leydig cell. In teleosts, the distinct functions of Fsh and Lh and information on cellular localization of their receptors are still poorly understood. Recently we established FreeStyle 293-F cell lines producing recombinant Japanese eel Fsh and Lh (reFsh and reLh), which form a single chain consisting of a common α-subunit and β-subunits. In this study, we conducted functional analyses of reFsh and reLh, focusing on the binding specificities to their receptors and effects on testicular steroidogenesis in vitro. Assays with gonadotropin receptors-expressing COS-7 cells indicated reFsh stimulated its cognate receptor, meanwhile reLh activated both receptors. Although results of in vitro incubations showed that reFsh and reLh induced testicular 11-ketotestosterone production in a dose and time-dependent manner by upregulating expression of steroidogenic enzymes, the effective doses of reLh were apparently lower and the effects of reLh emerged faster in comparison with reFsh. Results of quantitative real-time PCR using testicular cell fractions showed that fshr and lhcgr1 mRNA were detected both in Sertoli and Leydig cells. These analyses revealed that reFsh and reLh were biologically active and hence will be useful for future studies. Moreover, our data showed that both eel Fsh and Lh acted as steroidogenic hormones through their receptors in testicular somatic cells; however, Lh was more potent on androgen production, implying differential functions on spermatogenesis.

The 11β-hydroxysteroid dehydrogenase isoforms: pivotal catalytic activities yield potent C11-oxy C19 steroids with 11βHSD2 favouring 11-ketotestosterone, 11-ketoandrostenedione and 11-ketoprogesterone biosynthesis

The 11β-hydroxysteroid dehydrogenase (11βHSD) types 1 and 2 are primarily associated with glucocorticoid inactivation and reactivation. Several adrenal C11-oxy C19 and C11-oxy C21 steroids, which have been identified in prostate cancer, 21-hydroxylase deficiency and polycystic ovary syndrome, are substrates for these isozymes. This study describes the kinetic parameters of 11βHSD1 and 11βHSD2 towards the C11-keto and C11-hydroxy derivatives of the C19 and C21 steroids. The apparent Km and Vmax values indicate the more prominent 11βHSD2 activity towards 11β-hydroxy androstenedione, 11β-hydroxytestosterone and 11β-hydroxyprogesterone in contrast to the 11βHSD1 reduction of the C11-keto steroids, as was demonstrated in the LNCaP cell model in the production of 11-ketotestosterone and 11-ketodihydrotestosterone. Data highlighted the role of 11βHSD2 and cytochrome P450 17A1 in the contribution of C11-oxy C21 steroids to the C11-oxy C19 steroid pool in the C11-oxy backdoor pathway. In addition, 11βHSD2 activity, catalysing 11-ketotestosterone biosynthesis, was shown to be key in the production of prostate specific antigen and in the progression of prostate cancer to castration resistant prostate cancer. The study at hand thus provides evidence that 11βHSD isozymes play key roles in pathophysiological states, more so than was previously put forward.

Effects of environmentally relevant metformin exposure on Japanese medaka (Oryzias latipes)

Metformin is one of the most prevalent pharmaceuticals in both surface and waste waters, yet little is known about the bioavailability and/or effects of developmental exposure on early life stage (ELS) fish. Here, we demonstrate that embryo-larval stages of medaka are capable of taking up metformin from the aquatic environment, provided exposure occurs prior to chorion hardening (∼6-hpf). Once transferred to clean water, ELS medaka are able to completely depurate metformin in <24-hours. Furthermore, ELS medaka exposed to a range of relevant concentrations of waterborne metformin (from 6 hpf through 28-days post hatch) had significantly reduced growth metrics, altered metabolomes, and changes in the expression of genes associated with cell growth. The range of concentrations investigated were 1.0, 3.2, 10, 32, and 100 μg·L−1. To examine effects of chronic, low level metformin exposure across the full medaka life-cycle, we exposed newly fertilized embryos to 3.2 μg L−1 waterborne metformin for 165-days. The weight and length of adult fish were examined, as were effects on the production of some steroid hormones, specifically a significant increase (control females: 0.161 ± 0.023 pg/mg; metformin treated females: 3.42 ± 0.543) in the production of 11-ketotestosterone was observed in adult female medaka. Collectively, these results suggest that current environmental exposure scenarios may be sufficient to cause effects on developing fish.

Assessing recovery of in vitro steroid production in male rainbow darter (Etheostoma caeruleum) in response to municipal wastewater treatment plant infrastructure changes.

The present study examined in vitro 11-ketotestosterone and testosterone production by the testes of rainbow darter (Etheostoma caeruleum) collected from selected reference sites and downstream of 2 municipal wastewater treatment plants (MWWTPs; Waterloo and Kitchener) on the central Grand River (Ontario, Canada), over a 6-yr period (2011-2016). The main objective was to investigate if infrastructure upgrades at the Kitchener MWWTP in 2012 resulted in a recovery of this response in the post-upgrade period (2013-2016). Two supporting studies showed that the fall season is appropriate for measuring in vitro sex steroid production because it provides stable detection of steroid patterns, and that the sample handling practiced in the present study did not introduce a bias. Infrastructure upgrades of the Kitchener MWWTP resulted in significant reductions in ammonia and estrogenicity. After the upgrades, 11-ketotestosterone production by MWWTP-exposed fish increased in 2013 and it continued to recover throughout the study period of 2014 through 2016, returning to levels measured in reference fish. Testosterone production was less sensitive and it lacked consistency. The Waterloo MWWTP underwent some minor upgrades but the level of ammonia and estrogenicity remained variable over time. The production of 11-ketotestosterone and testosterone in rainbow darter below the Waterloo MWWTP was variable and without a clear recovery pattern over the course of the present study. The results of the present study demonstrated that measuring production of sex steroids (especially 11-ketotestosterone) over multiple years can be relevant for assessing responses in fish to environmental changes such as those resulting from major infrastructure upgrades. Environ Toxicol Chem 2018;37:501-514. © 2017 SETAC.

Molecular initiating events of the intersex phenotype: Low-dose exposure to 17α-ethinylestradiol rapidly regulates molecular networks associated with gonad differentiation in the adult fathead minnow testis

Intersex, or the presence of oocytes in the testes, has been documented in fish following exposure to wastewater effluent and estrogenic compounds. However, the molecular networks underlying the intersex condition are not completely known. To address this, we exposed male fathead minnows to a low, environmentally-relevant concentration of 17alpha-ethinylestradiol (EE2) (15ng/L) and measured the transcriptome response in the testis after 96h to identify early molecular initiating events that may proceed the intersex condition. The short-term exposure to EE2 did not affect gonadosomatic index and proportion of gametes within the testes. However, the production of 11-ketotestosterone and testosterone from the testis in vitro was decreased relative to controls. Expression profiling using a 8×60K fathead minnow microarray identified 10 transcripts that were differentially expressed in the testes, the most dramatic change being that of coagulation factor XIII A chain (20-fold increase). Transcripts that included guanine nucleotide binding protein (Beta Polypeptide 2), peroxisome proliferator-activated receptor delta, and WNK lysine deficient protein kinase 1a, were down-regulated by EE2. Subnetwork enrichment analysis revealed that EE2 suppressed transcriptional networks associated with steroid metabolism, hormone biosynthesis, and sperm mobility. Most interesting was that gene networks associated with doublesex and mab-3 related transcription factor 1 (dmrt1) were suppressed in the adult testis, despite the fact that dmrt1 itself was not different in expression from control males. Transcriptional networks involving forkhead box L2 (foxl2) (transcript involved in ovarian follicle development) were increased in expression in the testis. Noteworthy was that a gene network associated to granulosa cell development was increased over 100%, suggesting that this transcriptome network may be important for monitoring estrogenic exposures. Other cell processes rapidly downregulated by EE2 at the transcript level included glucose homeostasis, response to heavy metal, amino acid catabolism, and the cyclooxygenase pathway. Conversely, lymphocyte chemotaxis, intermediate filament polymerization, glucocorticoid metabolism, carbohydrate utilization, and anterior/posterior axis specification were increased. These data provide new insight into the transcriptional responses that are perturbed prior to gonadal remodeling and intersex following exposure to estrogens. These data demonstrate that low concentrations of EE2 (1) rapidly suppresses male hormone production, (2) down-regulate molecular networks related to male sex differentiation, and (3) induce transcriptional networks related to granulosa cell development in the adult testis. These responses are hypothesized to be key molecular initiating events that occur prior to the development of the intersex phenotype following estrogenic exposures.

Nodavirus Colonizes and Replicates in the Testis of Gilthead Seabream and European Sea Bass Modulating Its Immune and Reproductive Functions

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases.

Effects of model aromatizable (17α-methyltestosterone) and non-aromatizable (5α-dihydrotestosterone) androgens on the adult mummichog (Fundulus heteroclitus) in a short-term reproductive endocrine bioassay

Androgens originating from pulp mill processing, sewage treatment facilities and agricultural activities have the potential for discharge into aquatic receiving environments. To assess androgen effects on reproductive endocrine status in fish in estuarine environments, male and female adult northern mummichog (Fundulus heteroclitus macrolepidotus) were exposed to an aromatizable androgen (17α-methyltestosterone; MT) and a non-aromatizable androgen (5α-dihydrotestosterone; DHT) in a short-term reproductive endocrine bioassay. Fish were nominally exposed to 10μg/L or 100μg/L DHT, or 0.1μg/L or 1μg/L MT for 14days during gonadal recrudescence. Actual concentrations of androgens, as measured by enzyme immunoassay (EIA), were 10–49% of nominal MT 0.1, 3–6% of nominal MT 1, 5–10% of nominal DHT 10 and 3–25% of nominal DHT 100. Female mummichog were impacted to a greater degree by androgen exposure, with increased plasma testosterone (T) at 100μg/L DHT, depressed plasma 17β-estradiol (E2) at both DHT concentrations and at 1μg/L MT, as well as depressed in vitro E2 at both MT concentrations and 100μg/L DHT. Males had depressed plasma T in the 10μg/L DHT treatment and depressed in vitro 11-ketotestosterone production for both MT concentrations and 10μg/L DHT. Ovarian aromatase gene expression was induced in females exposed to 1μg/L MT. DHT at 100μg/L increased hepatic vitellogenin-1 (VTG1) expression in males and depressed VTG1 expression in females. The range of responses between sexes and among species provides evidence for modes of actions and potential impacts of androgens in aquatic receiving environments.

Characterization of tilapia (Oreochromis niloticus) gonadotropins by modeling and immunoneutralization

In fish, both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in reproduction. Here we explored the structure and differential specificity of tilapia (t) gonadotropins (GTHs) to delineate their physiological relevance and the nature of their regulation. We generated structural models of tGTHs and GTH receptors (R) that enabled us to better understand the hormone–receptor interacting region. In tilapia, FSH release is under the control of the hypothalamic decapeptide GnRH, an effect that was abolished by specific bioneutralizing antisera [anti-recombinant (r) tFSHβ]. These antisera also reduced the basal secretion and delayed GnRH-stimulated production of 11-ketotestosterone (11KT), and dramatically reduced LH levels. Immunoneutralization of tLH using anti-rtLHβ significantly reduced its GnRH-stimulated levels. Basal 11KT and FSH levels were also reduced. Taken together, these results suggest a feedback mechanism between FSH and LH release in tilapia.

Blocking of progestin action disrupts spermatogenesis in Nile tilapia (Oreochromis niloticus).

In vitro studies have indicated that the maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP, DHP), probably through nuclear progestin receptor (Pgr), might be involved in the proliferation of spermatogonial cells and the initiation of meiosis in several fish species. However, further in vivo evidence is required to elucidate the role of DHP in spermatogenesis during sexual differentiation in teleosts. In this study, we cloned pgr and analyzed its expression in Nile tilapia (Oreochromis niloticus) and treated XY fish with RU486 (a synthetic Pgr antagonist) from 5 days after hatching (dah) to determine the role of DHP in spermatogenesis. Sequence and phylogenetic analyses revealed that the Pgr identified in tilapia is a genuine Pgr. Pgr was found to be expressed in the Sertoli cells surrounding spermatogonia and spermatids in the testis of tilapia. Real-time PCR analysis revealed that the expression of pgr in the testis was significantly upregulated from 10 dah, further increased at 50 dah, and persisted until adulthood in fish. In the testis of RU486-treated fish, the transcript levels of germ cell markers and a meiotic marker were substantially reduced. However, the expression of markers in Sertoli cells remained unchanged. Moreover, the production of 11-ketotestosterone and the expression of genes encoding various steroidogenic enzymes were also not altered. In contrast, the expression of cyp17a2, encoding one of the critical steroidogenic enzymes involved in DHP biosynthesis, declined significantly, possibly indicating the inhibition of DHP production by RU486. RU486 treatment given for 2 months did not affect spermatogenesis; however, treatment given for more than 3 months resulted in a decrease in spermatogonial cell numbers and depletion of later-phase spermatogenic cells. Simultaneous excessive DHP supplementation restored spermatogenesis in RU486-treated XY fish. Taken together, our data further indicated that DHP, possibly through Pgr, might be essential for spermatogonial cell proliferation and spermatogenesis in fish.

In Vitro Culture Conditions for Reproductive Endocrine Tissues of Fathead Minnow (Pimephales promelas)

Studies examined the effects of different culture conditions for the brain, gonads (testis and ovary), and liver of adult male and Female Fathead Minnow (Pimephales promelas) (FHM) during a 28 day assessment of viability and functionality. Five parameters were tested including media pH, incubation temperature, media composition, media exchange rate, and substrate. Within the ranges tested, incubation temperature was the most influential on tissue viability with dramatic improvements using cooler incubation temperatures (<18°C) over the entire 28 day test period. Tissues remained equally viable in five different common culture media, although highest viability with each medium was observed with a weekly media exchange over more frequent exchanges, alginate over polystyrene, and a media pH near physiological values. Use of these combined conditions resulted in continual function of testis and ovarian tissue over the entire 28 days, as indicated by continued production of 11-ketotestosterone (11-KT) by testes and estradiol (E2) by ovaries. Liver function was maintained through seven days, although vitellogenin production in response to added E2 eventually decreased over time. Consistent tissue viability over time periods commonly used for in vivo studies will enhance the link of in vitro tissue toxicology studies to whole-fish and population level impacts.

Undifferentiated Gonads of Pejerrey ( Odontesthes bonariensis ) Larvae Exposed to Masculinizing Temperatures Produce 11-ketotestosterone when Incubated with a Tritiated Precursor

Introduction: The involvement of androgens as early mediators of testis differentiation in fish is still a matter of debate [1]. Larval gonadal size limits the possibility to establish whether an early production of 11-oxigenated androgens precedes testicular differentiation. In a previous study we reported the expression of genes involved in androgen synthesis (i.e. cyp11a y la cyp11b) in larval trunks [2] and the production of 11-ketotestosterone was reported in larval trunks by EIA [3]. In this context the aim of the present work was to study the androgen synthesis capacity of gonads and peritoneum of Odontesthes bonariensis in larvae exposed to masculinizing temperatures (29oC) before morphological differentiation of the testis. First we validate the nature of androgens produced by adult testis, liver and spleen and then we studied the androgen production in larval gonads at 29oC. Methods: Pejerrey larvae were raise for 5 weeks at 29oC in order to obtain a whole male population. At 5 that moment, 100 fish were sampled and individually immerse in ice-cold water until movement ceased. Due to technological constrains in taking then apart, the gonads and peritoneum were dissected together under binocular microscope and pooled in ice cold L-15 medium. Adult testis at the onset of spermatogenesis, spleen and liver were also obtained and placed on L-15 medium. Tissue samples were incubated for 18 hs in 500 μL of L-15 medium in the presence of tritium labeled precursors. 17P was used for gonad and peritoneum, and A4 for spleen and liver. The reaction was arrested by ethanol addition to 80% followed by tissue disaggregation using a glass-glass grinder. The supernatant was evaporated under nitrogen until 20% its original volume qws reached and then extracted three times with dichloromethane (DM): methanol (M) (9DM:1M). This fraction was re-suspended and applied to a silica TLC plate, cleaned by a non-polar mobile phase run and then resolved by using benzene acetone (2:1). The resolved radioactive steroids were revealed by autoradiography, the characteristic Rf computed and radioactive zones isolated by scratching the silica followed by extraction using three rounds of 9DM: 1M. The bands extracts corresponding to 11-ketotestosterone (11-KT) and 11β-hydroxyandrostenedione (11β-OHA4) were separately analyzed by HPLC using a RP-C18 column and 0.5 mL fractions were obtained and their radioactivity content determined by scintillation counting. The 11β-OHA4 obtained from kidney extracts was analyzed by co-crystallization to constant specific activity.

Dimorphic Expression of Various Transcription Factor and Steroidogenic Enzyme Genes during Gonadal Ontogeny in the Air-Breathing Catfish, Clarias gariepinus

In the present study the expression of 13 genes known to be involved in sex differentiation and steroidogenesis in catfish was analyzed during gonadal ontogeny by quantitative real-time RT-PCR. Dmrt1 and sox9a showed exclusive expression in male gonads while ovarian aromatase (cyp19a1) and foxl2 were abundant in differentiating female gonads. Most of the genes related to steroidogenesis were expressed only after gonadal differentiation. However, genes coding for 3β-hydroxysteroid dehydrogenase (3β-hsd), 17α-hydroxylase/C17–20 lyase type 1 (cyp17) and steroidogenic acute regulatory protein (star) were barely detectable during gonadal differentiation. Ovarian aromatase, cyp19a1, which is responsible for estradiol-17β biosynthesis in females, was expressed very early in the undifferentiated gonads of catfish, around 30–40 days post hatch (dph). The steroidogenic enzyme, 11β-hydroxylase (cyp11b1) required for the production of 11-ketotestosterone (11-KT) was expressed only after differentiation of testis. These results suggest that estradiol-17β has a critical role in ovarian differentiation, while the role of 11-KT in testicular differentiation is doubtful. In conclusion, dimorphic expression of dmrt1 and sox9a in gonads during early development is required for testicular differentiation, and sex-specific expression of cyp19a1 and foxl2 in females plays a critical role in ovarian development. Our study reveals that the critical period of gonadal differentiation in catfish starts around 30–40 dph when sex-specific genes showed differential expression.

UNDIFFERENTIATED GONADS OF PEJERREY (ODONTESTHES BONARIENSIS) LARVAE EXPOSED TO MASCULINIZING TEMPERATURES PRODUCE 11-KETOTESTOSTERONE WHEN INCUBATED WITH A TRITIATED PRECURSOR

Introduction: The involvement of androgens as early mediators of testis differentiation in fish is still a matter of debate [1]. Larval gonadal size limits the possibility to establish whether an early production of 11-oxigenated androgens precedes testicular differentiation. In a previous study we reported the expression of genes involved in androgen synthesis (i.e. cyp11a y la cyp11b) in larval trunks [2] and the production of 11-ketotestosterone was reported in larval trunks by EIA [3]. In this context the aim of the present work was to study the androgen synthesis capacity of gonads and peritoneum of Odontesthes bonariensis in larvae exposed to masculinizing temperatures (29oC) before morphological differentiation of the testis. First we validate the nature of androgens produced by adult testis, liver and spleen and then we studied the androgen production in larval gonads at 29oC. Methods: Pejerrey larvae were raise for 5 weeks at 29oC in order to obtain a whole male population. At 5 that moment, 100 fish were sampled and individually immerse in ice-cold water until movement ceased. Due to technological constrains in taking then apart, the gonads and peritoneum were dissected together under binocular microscope and pooled in ice cold L-15 medium. Adult testis at the onset of spermatogenesis, spleen and liver were also obtained and placed on L-15 medium. Tissue samples were incubated for 18 hs in 500 μL of L-15 medium in the presence of tritium labeled precursors. 17P was used for gonad and peritoneum, and A4 for spleen and liver. The reaction was arrested by ethanol addition to 80% followed by tissue disaggregation using a glass-glass grinder. The supernatant was evaporated under nitrogen until 20% its original volume qws reached and then extracted three times with dichloromethane (DM): methanol (M) (9DM:1M). This fraction was re-suspended and applied to a silica TLC plate, cleaned by a non-polar mobile phase run and then resolved by using benzene acetone (2:1). The resolved radioactive steroids were revealed by autoradiography, the characteristic Rf computed and radioactive zones isolated by scratching the silica followed by extraction using three rounds of 9DM: 1M. The bands extracts corresponding to 11-ketotestosterone (11-KT) and 11β-hydroxyandrostenedione (11β-OHA4) were separately analyzed by HPLC using a RP-C18 column and 0.5 mL fractions were obtained and their radioactivity content determined by scintillation counting. The 11β-OHA4 obtained from kidney extracts was analyzed by co-crystallization to constant specific activity.

Health status of native fish (Percilia gillissi and Trichomycterus areolatus) downstream of the discharge of effluent from a tertiary‐treated elemental chlorine‐free pulp mill in Chile

Few data exist on the possible effects of pulp and paper effluent discharge on native fish populations in the Southern Hemisphere, relative to the research done in the Northern Hemisphere. The present research examined two native fish species (Trichomycterus areolatus and Percilia gillissi) for effects at both the molecular and individual level due to the discharge of effluent from a tertiary treated elemental chlorine-free pulp mill into a fluvial system in Central Chile over three seasons (February 2007, October 2007, January 2008). Different responses were observed between species and between sexes. There was an increase in the production of gonadal 17β-estradiol in the females of both species but a drop in 11-ketotestosterone production in P. gillissi males. Female gonadal size was increased, especially during the summer period, with corresponding increases the frequency of advanced oocyte development, and in the oocyte diameter in both species. Hepatic ethoxyresorufin-O-deethylase (EROD) activity was elevated for both species downstream of the discharge point, although overall it was higher in P. gillissi than T. areolatus. Decreases in the frequency of smaller-sized fish for both species, as well as a drop in the size of the adults downstream of the discharge point, were observed. The present study is the first evidence of endocrine disruption in native freshwater fish associated with modern pulp mills in South America. This study establishes possible links in the reproductive alterations observed at the subindividual and individual levels that could explain the changes observed at the population level.

A potential role of male and female androgen in species recognition in a unisexual–bisexual mating complex

Hormones play a critical role in the regulation of vertebrate mating behavior, including receptivity, and several components of mate choice. However, less is known about the role of these chemical messengers in mediating behavior associated with premating reproductive isolation. The bisexual–unisexual mating complex of sailfin mollies, Poecilia latipinna, and Amazon mollies, Poecilia formosa (sexual parasites of sailfins) has been a model system for studying ultimate mechanisms of species recognition. However proximate mechanisms, such as variation in hormone levels, have not been examined. We paired male sailfin mollies with either female conspecifics or Amazon mollies and obtained water-borne hormone samples before and after mating for all fish. We measured 11-ketotestosterone, testosterone, and estradiol from the water samples. As expected from previous studies, males mated with conspecifics more frequently than with Amazon mollies. 11-Ketotestosterone production by males increased when they mated with female sailfin mollies who themselves also showed elevated production of 11-ketotestosterone. This increase in male and female 11-ketotestosterone levels was not seen when males mated with Amazon mollies. This unique endocrine interaction represents a potential proximate mechanism for species recognition by male sailfin mollies. We found no significant change in testosterone or estradiol under these conditions suggesting that a single hormone mediates bidirectional interactions between males and females during courtship.