The Aromatase Inhibitor Fadrozole and the 5-Reductase Inhibitor Finasteride Affect Gonadal Differentiation and Gene Expression in the Frog <i>Silurana tropicalis</i>

Abstract

Aromatase (cyp19) and the 5α- and 5β-reductases (srd5alpha and srd5beta) are important enzymes for vertebrate sexual development. We investigated the effects of inhibition of cyp19 by fadrozole (FAD), and srd5alpha and srd5beta by finasteride (FIN) during anuran larval development. Chronic exposures of Silurana (Xenopus) tropicalis from Nieuwkoop-Faber stage 12 until stage 60 were performed using either 2 µM FAD or 25 µM FIN. Histological analysis of exposed metamorphic frogs revealed that both treatments induced intersex individuals (presence of testicular oocytes). FAD treatment resulted in 55% male, 30% female and 15% intersex, while FIN treatment produced 27% male, 53% female and 20% intersex. Real-time RT-PCR analysis of hepatic sex steroid- and thyroid hormone-related gene expression demonstrated that FAD-induced intersex animals had higher srd5alpha1, srd5alpha2 and eralpha mRNA levels than control and FAD males. In contrast, FIN-induced intersex had low srd5alpha1, srd5alpha2, srd5beta and dio3 and high dio2 mRNA levels. FIN-treated males exhibited high trbeta, dio2 and a lower dio3 mRNA levels. We conclude that chemically induced intersex animals display different gene expression profiles than non-exposed animals and that, although morphologically similar, intersex animals produced by different chemicals have different endocrine pathophysiologies.

Expression Profiles of Reproduction- and Thyroid Hormone-Related Transcripts in the Brains of Chemically-Induced Intersex Frogs

“…Finasteride significantly decreased hepatic srd5alpha2 and srd5beta mRNA levels in metamorphic S. tropicalis tadpoles (NF 60) and in whole larvae (NF 46) [Langlois et al, 2010]. Interestingly, the transcriptional regulation of srd5alpha3 mRNA was not affected by finasteride treatment in S. tropicalis in any tissue or developmental stage studied [NF 60 brain, present study; NF 60 liver Duarte-Guterman et al, 2009; NF 46 whole larvae Langlois et al, 2010]. Srd5alpha3 is a newly discovered enzyme and its regulation and function have not yet been fully explored [Tamura et al, 2007;Uemura et al, 2008], especially not in frogs.…”

“…Tadpoles of Silurana tropicalis were exposed to finasteride (25 M ) dissolved in ethanol (EtOH; 0.05% final concentration) and fadrozole (2 M ) dissolved in water from Nieuwkoop-Faber (NF [Nieuwkoop and Faber, 1994]) stage 12 until stage 60, along with their respective controls (EtOH and water controls) as previously described in Duarte-Guterman et al [2009]. At the end of the exposure, brains were dissected and stored at -80 ° C. Homogenization and disruption of individual brain samples was achieved using an MM301 Mixer Mill (Retsch, Newton, Pa., USA) at 20 Hz for 3 min.…”

“…Total cDNA was prepared from 1 g of total RNA and 0.2 g random hexamer primers using Superscript II reverse transcriptase (Invitrogen). Sex of the animals (male, female or intersex) was based on the gonadal analysis previously described in Duarte-Guterman et al [2009]. Intersex was defined as the presence of at least one oocyte in the testes.…”

Sex Reversal in Amphibians

“…Another inhibitor, fadrozole, induced masculinization of gonad tissues in X. laevis [Miyata and Kubo, 2000]. Fadrozole was also used in S. tropicalis [Duarte-Guterman et al, 2009;Olmstead et al, 2009]. In this species, when applied at 2 μ M from stage 12 to stage 60, 55% of the individuals differentiated as males, 15% were intersex having testicular oocytes, and 30% were females.…”

Transcriptomic profiling in Silurana tropicalis testes exposed to finasteride

“…Recently, adverse effects of using finasteride have become well established and data suggests that, in humans, it not only impairs sexual function, but also that it is associated with male breast cancer, various skin conditions, and depression, while in rodents, finasteride is linked to erectile dysfunction and change in prostate tissue histoarchitecture (reviewed in Traish, 2012). Aquatic amphibian species exposed to this 5a-reductase inhibitor also showed disrupted spermatogenesis, intersex animals, and a female biased sex ratio (Duarte-Guterman et al, 2009;Urbatzka et al, 2009). At the molecular level, gene expression endpoints have shown that finasteride alters the mRNA levels of gonadotropins-, androgen-, and thyroid hormone-related genes in the Western clawed metamorphosing frogs (Silurana tropicalis; Duarte-Guterman et al, 2009;Urbatzka et al, 2009;Langlois et al, 2011).…”