Histological and transcriptomic effects of 17α-methyltestosterone on zebrafish gonad development

Lee et al. BMC Genomics (2017) 18:557 DOI 10.1186/s12864-017-3915-z RESEARCH ARTICLE Open Access Histological and transcriptomic effects of 17α-methyltestosterone on zebrafish gonad development Stephanie Ling Jie Lee1*, Julia A. Horsfield2, Michael A. Black3, Kim Rutherford1, Amanda Fisher2 and Neil J. Gemmell1 Abstract Background: Sex hormones play important roles in teleost ovarian and testicular development. In zebrafish, ovarian differentiation appears to be dictated by an oocyte-derived signal via Cyp19a1a aromatase-mediated estrogen production. Androgens and aromatase inhibitors can induce female-to-male sex reversal, however, the mechanisms underlying gonadal masculinisation are poorly understood. We used histological analyses together with RNA sequencing to characterise zebrafish gonadal transcriptomes and investigate the effects of 17α-methyltestosterone on gonadal differentiation. Results: At a morphological level, 17α-methyltestosterone (MT) masculinised gonads and accelerated spermatogenesis, and these changes were paralleled in masculinisation and de-feminisation of gonadal transcriptomes. MT treatment upregulated expression of genes involved in male sex determination and differentiation (amh, dmrt1, gsdf and wt1a) and those involved in 11-oxygenated androgen production (cyp11c1 and hsd11b2). It also repressed expression of ovarian development and folliculogenesis genes (bmp15, gdf9, figla, zp2.1 and zp3b). Furthermore, MT treatment altered epigenetic modification of histones in zebrafish gonads. Contrary to expectations, higher levels of cyp19a1a or foxl2 expression in control ovaries compared to MT-treated testes and control testes were not statistically significant during early gonad development (40 dpf). Conclusion: Our study suggests that both androgen production and aromatase inhibition are important for androgen-induced gonadal masculinisation and natural testicular differentiation in zebrafish. Keywords: Sex differentiation, Gonad differentiation, Androgens, Zebrafish Background Natural and synthetic steroid hormones influence gonadal sex differentiation and sex ratios in teleost fish [1–5]. In particular, androgens regulate testicular development, spermatogenesis, male secondary sex characteristics, sexual behaviour and maintenance of male sexual phenotype in teleosts [4, 6–9]. The major natural androgen in teleost fish is 11-ketotestosterone [4], which is converted from androstenedione and testosterone via the steroidogenic enzymes 11β-hydroxylase (Cyp11c1) and 11β-hydroxysteroid dehydrogenase (Hsd11b2) [10–13]. The effects of these androgens are mediated by androgen receptors [10, 14]. * Correspondence: 1 Department of Anatomy, University of Otago, Dunedin, Otago, New Zealand Full list of author information is available at the end of the article Synthetic androgens are structural mimics of the native androgen receptor ligands testosterone and 11-ketotestosterone (11-KT). They include 17αmethyltestosterone (MT), 17α-methyldihydrotestosterone, 17β-trenbolone, mibolerone and mesterolone [15]. Treatment with exogenous androgens at the labile stage during early development triggers testicular development, stimulates precocious spermatogenesis along with phenotypic and behavioural masculinisation, skews sex ratios towards males in gonochoristic fish [16–24] and accelerates female-to-male sex change in protogynous hermaphroditic fish [25–29]. Studies suggest that androgen-induced female-to-male phenotypic sex reversal is functional and persistent [30–33]. Androgen induction of female-to-male sex reversal [15, 34] is routinely used for sex control in © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. BMC Genomics (2017) 18:557 aquaculture [35], with MT being the most frequently used androgen for production of all-male populations in commercial settings [15] because of its high masculinising potency [15]. Two hypotheses exist for the mode of action of androgens for induction of testicular differentiation: (1) indirect, via aromatase inhibition which reduces estrogen production [36–38] and (2) direct, via interactions with androgen receptor [36]. Prior studies of androgeninduced gonadal masculinisation in European seabass [39], Japanese flounder [19], groupers [26, 29], Nile tilapia [40] and rainbow trout [41–45] using candidate gene and microarray approaches [46] support the aromatase inhibition hypothesis. The gonadal masculinisation observed in zebrafish following treatment with aromatase inhibitor also strongly supports this hypothesis [38]. However, recent studies in tilapia and grouper implicate androgen receptor in androgen-induced phenotypic masculinisation [36, 47]. Overall, the molecular mechanisms underpinning androgen-induced gonadal masculinisation in fishes remain to be clarified. Zebrafish (Danio rerio) is a popular toxicological model and has been used extensively to study the effects of endocrine-disrupting chemicals on gonad development and sexual phenotype [24, 30–32, 48–57]. The primary sex determining region present in wild zebrafish strains appear to have largely been lost in laboratory strains through the domestication process [58]. Almost all zebrafish in the world are derived from domesticated strains, with most from the AB strain and its derivatives except for the WIK strain. Domesticated zebrafish use a polygenic sex determination system [59] vulnerable to the effects of high temperature [60, 61], hypoxia [62], rearing density [63, 64], inbreeding, out-crossing [65, 66] and hormones [30]. Zebrafish is a juvenile hermaphrodite, wherein all individuals initially develop a nonfunctional ‘juvenile ovary’ [67–70]. Oocytes undergo apoptosis in juvenile ovaries of presumptive males during juvenile ovary-to-testis gonadal transformation [71]. In contrast, oocytes continue oogenesis and oocyte maturation in presumptive females [68] via unknown ovarian aromatase (Cyp19a1a)-mediated mechanisms [70]. Depending on the dosage, treatment with exogenous androgens can induce female-to-male sex reversal [24, 30–33] and paradoxical feminisation in zebrafish [72]. The Tg(vas:egfp) zebrafish line exhibits sexually dimorphic expression of green fluorescent protein which facilitates in vivo sex identification during early gonad development [73]. It has been used to study estrogenic effects in zebrafish [74]. Little is known about the direct transcriptional effects of MT during gona (...truncated)