Estradiol-17 Stimulates the Renewal of Spermatogonial Stem Cells in Males Takeshi Miura,* , † ,1 Chiemi Miura,* Takashi Ohta,* Manal R. Nader,* Takashi Todo,‡ and Kohei Yamauchi* *Department of Biology, Faculty of Fisheries, Hokkaido University, Hakodate 041-8611, Japan; †PRESTO, Japan Science and Technology Corporation; and ‡Sado Marine Biological Station, Niigata University, Sado Island, Niigata 952-2135, Japan Received September 3, 1999 In this work, we examined the functions of the fe- male hormone “estrogen” on spermatogenesis of the Japanese eel (Anguilla japonica). Estradiol-17 (E 2 ), a natural estrogen in vertebrates, was present in the serum and its receptor was expressed in the testis during the whole process of spermatogenesis. Sper- matogonial stem cell renewal was promoted by E 2 im- plantation but was suppressed by tamoxifen (an an- tagonist of estrogen). In vitro, 10 pg/ml of E 2 was sufficient to induce spermatogonial stem cell division in cultured testicular tissue, therefore confirming the in vivo observations. These results clearly show that estrogen is an indispensable “male hormone” in the early spermatogenetic cycle. © 1999 Academic Press It is widely accepted that “estrogen” is a “female” hormone. However, it has been reported that estrogen is present in some male vertebrates (1–3) and that it’s receptors are expressed in the male reproductive or- gans (4, 5). In mammals, estrogen appears to regulate the reabsorption of luminal fluid in the epididymis (6) and to affect sexual behavior (7, 8). Even though there has been a noticeable increase in reports analyzing the actions of estrogen in the testis recently, the clear functions of estrogens in male reproduction have not been established. Recently, environmental pollution by chemicals— collectively known as endocrine disrupters— have been shown to stimulate or block biological processes (9), and have been recognized to interfere with sensitive hormone pathways that regulate reproductive func- tions. This diverse group of chemicals includes envi- ronmental estrogens or exestrogens, compounds that evoke estrogenic responses by mimicking the actions of endogenous estrogen including estradiol-17 (E 2 ). In male animals, exposure to estrogenic compounds can lead, among others, to reduced gonad size, feminization of genetic males, low sperm count and/or quality (10, 11). Due to the lack of understanding of the mecha- nisms by which these actions are performed, it is im- portant to analyze the role of estrogen in male repro- duction. To better clarify the various functions of estrogen on spermatogenesis, male Japanese eels (Anguilla ja- ponica) were used as experimental animals. Under cultivated conditions, male Japanese eel have imma- ture testes containing only non-proliferated spermato- gonia (12). It has been proven that complete spermat- ogenesis in the Japanese eel can be induced by a single injection of human chorionic gonadotropin (hCG) in vivo (12), and by supplementation with either hCG (13) or 11-ketotestosterone (11-KT) (14) in vitro. In this study, we investigated the role of estrogen in spermat- ogenesis using the male Japanese eel system described above. MATERIALS AND METHODS Animals. Male cultivated Japanese eel (180 –200 g in body weight) were purchased from a commercial eel supplier, and kept in circulating fresh-water tanks with a capacity of 500 liters at 23°C. To 50 male eels, a single injection of hCG dissolved in saline (150 mM NaCl) was given intramuscularly at a dose of 5 IU per gram of body weight. Thirty fish were injected only with saline. Fish were sacrificed at 0, 1, 3, 6, 9, 12, 15, and 18 days after hCG or saline injection, and blood and testes were collected. Serum was immedi- ately separated and E 2 levels were measured by radioimmunoassay according to Kagawa et al. (15). Poly (A) + RNA was isolated from the testes using the First Track kit (Invitrogen, San Diego, CA). Northern and in situ hybridization. Poly (A) + RNA (1 g) was electrophoresed on a 1% (w/v) agarose denatured gel and blotted onto nylon membranes. The uniformity of sample loading and transfer were monitored by staining the blotted RNA with methylene blue. The eel estrogen receptor -like cDNA (16) and -actin cDNA (17) 1 To whom correspondence should be addressed. Fax: + 81-138- 40-5545. E-mail: takeshi@pop.fish.hokudai.ac.jp. Biochemical and Biophysical Research Communications 264, 230 –234 (1999) Article ID bbrc.1999.1494, available online at http://www.idealibrary.com on 230 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.