Fish Androgen Receptor: cDNA Cloning, Steroid Activation of Transcription in Transfected Mammalian Cells, and Tissue mRNA Levels Takashi Todo, 1 Toshitaka Ikeuchi, Tohru Kobayashi, and Yoshitaka Nagahama 2 Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan Received November 30, 1998 We have previously identified 11-ketotestosterone (11KT, a portent androgen in teleosts) as the spermatogenesis-inducing hormone of the Japanese eel. In this study, a cDNA encoding the eel androgen receptor (AR) was isolated from the Japanese eel tes- tis. This cDNA contains a complete open reading frame encoding 848 amino residues. The amino acid se- quence of the eel AR shows high homology with other ARs. In transient transfection assays of mammalian cells, the eel AR showed androgen-dependent activa- tion of transcription from the androgen-responsive MMTV promoter. Of the endogenous androgens found in the Japanese eel, 11KT was the most effective acti- vator of transcription. These results indicate that the cloned cDNA encodes a functional eel AR, and its ma- jor native ligand is 11KT. This is the first isolation of an AR molecule from fish, and is the first evidence that 11KT acts via a nuclear receptor. © 1999 Academic Press Androgens play essential roles in vertebrate sper- matogenesis, but their specific regulatory actions have not been fully clarified. Recent studies in our labora- tory, using a teleost, the Japanese eel (Anguilla ja- ponica), have identified 11-ketotestosterone (11KT), a major androgen in some fish, as the spermatogenesis- inducing hormone (1,2,3). 11KT can induce the entire process of spermatogenesis from spermatogonial pro- liferation to sperm formation in both cultured testicu- lar fragments (2) and cocultured germ and somatic cells (3) from immature eels. 11KT activates Sertoli cells to stimulate production of activin B which has been identified as the spermatogonial proliferator in the Japanese eel (4). However, the mechanisms under- lying the regulation of eel spermatogenesis by 11KT are not fully understood. Androgens generally act on target cells through a nuclear androgen receptor (AR). The AR is a ligand- dependent transcription factor which belongs to a large family of nuclear receptors (5). Nuclear receptors pos- sess highly conserved DNA binding domains (DBDs) and moderately conserved ligand binding domains (LBDs). It is assumed that the regulation of eel sper- matogenesis by 11KT is mediated by an AR. However, classical radioligand binding experiments in several fish species have failed to identify a specific 11KT re- ceptor, although high affinity binding sites for testos- terone (T) have been described (6). Moreover, no AR molecule or its cDNA have been isolated from fish. In this context, it is of considerable interest, both from the standpoint of investigation of the molecular mecha- nisms underlying 11KT-regulated eel spermatogenesis and with respect to evolutionary and structural aspects of the AR, to isolate and characterize a cDNA clone encoding eel AR. Here, we describe the isolation and characterization of an eel cDNA containing an entire AR coding region. The transactivation function of the eel AR was deter- mined by expressing the cDNA in transiently trans- fected human kidney 293 cells. The AR mRNA levels in various tissues from gonadotropin-treated and un- treated male eels were measured by quantitative com- petitive reverse transcription-polymerase chain reac- tion (RT-PCR). MATERIALS AND METHODS Animals and treatment. Cultivated Japanese eel males (150-200 g in body weight) were purchased from a commercial eel supplier. They were kept in recirculating freshwater tanks with a capacity of 500 l at 20°C. Fish were not fed throughout the experimental period. A single injection of human chorionic gonadotropin (HCG) dissolved in saline (150 mM NaCl) was given intramuscularly at a dose of 5 IU per g body weight (1). 1 Present address: Sado Marine Biological Station, Faculty of Sci- ence, Niigata University, Tassha 87, Aikawa, Sado Island, Niigata 952-2135, Japan. 2 To whom correspondence should be addressed at Laboratory of Reproductive Biology, National Institute for Basic Biology, 38 Ni- shigonaka, Myodaiji, Okazaki 444-8585, Japan. Fax: (81) (564) 55- 7556. E-mail: nagahama@nibb.ac.jp. Biochemical and Biophysical Research Communications 254, 378 –383 (1999) Article ID bbrc.1998.9919, available online at http://www.idealibrary.com on 378 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.