Diethylstilbestrol induces fish oocyte maturation Toshinobu Tokumoto*†‡, Mika Tokumoto*†, Ryo Horiguchi§¶, Katsutoshi Ishikawa*, and Yoshitaka Nagahama†§ *Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan; †CREST Research Project, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan; §Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan; and ¶Department of Molecular Biomechanics, Graduate University for Advanced Studies, Okazaki 444-8585, Japan Communicated by Howard A. Bern, University of California, Berkeley, CA, January 6, 2004 (received for review August 6, 2003) An endocrine-disrupting chemical, diethylstilbestrol (DES), a non- Materials and Methods steroidal estrogen, triggers oocyte maturation in fish. The mor- Materials. Goldfish were purchased from a local supplier and phology (the time course of the change in germinal vesicle break- maintained at 15°C until used. Zebrafish were maintained at down) and an intracellular molecular event (the de novo synthesis 28.5°C on a 14-h light͞10-h dark cycle (16). 17,20␤-DHP, DES, of cyclin B) induced by DES were indistinguishable from those DES dimethyl ether (DM-DES), DES dipropionate (DP- induced by a natural maturation-inducing hormone, 17␣,20␤-di- DES), and 17␤-estradiol were purchased from Sigma. Di- hydroxy-4-pregnen-3-one (17,20␤-DHP). A synergistic action of methylstilbestrol (DMS) was a generous gift from J. Katzenel- DES on 17,20␤-DHP-induced oocyte maturation was observed. lenbogen (University of Illinois, Urbana). 17␣-Estradiol, Both 17,20␤-DHP- and DES-induced oocyte maturation was inhib- ethynylestradiol, butyl benzyl phthalate, di(2-ethylhexyl)- ited by an antibody against the maturation-inducing hormone phthalate, and pentachlorophenol were obtained from Wako receptor. The structural requirement for the action of DES is Pure Chemical (Osaka). Other chemicals were purchased as discussed based on results obtained with DES analogs. follows: hexestrol (HEX; ICN); trans,trans-dienestrol(␣- dienestrol) (DIES; U.S. Pharmacopeia, Rockville, MD); res- maturation-inducing hormone ͉ endocrine-disrupting chemical ͉ veratorol (Calbiochem); DDTs (AccuStandard, New Haven, goldfish ͉ zebrafish CT); bisphenol A (Nacalai Tesque, Kyoto); p-nonylphenol (Kanto Chemical, Tokyo); 4-octylphenol (Aldrich). ocyte maturation in lower vertebrates is triggered by mat- Oocyte Preparation and in Vitro Culture. Ovaries of goldfish were Ouration-inducing hormone (MIH), which acts on receptors isolated from killed females and placed in fresh goldfish located on the oocyte membrane and induces the activation of Ringer’s solution (125 mM NaCl͞2.4 mM KCl͞0.28 mM maturation-promoting factor in the oocyte cytoplasm (1–4). ͞ ͞ ͞ ͞ MgSO4 0.89 mM MgCl2 2.4 mM CaCl2 2 mM Hepes 5.6 mM During the course of maturation, oocytes undergo drastic mor- glucose͞100 units/ml penicillin͞0.2 mg/ml streptomycin, pH phological changes associated with progression of the meiotic 7.5) and washed three times with the same solution. Full-grown cell cycle, in which breakdown of the oocyte nuclear envelope immature oocytes were exposed in vitro by incubating ovarian [germinal vesicle breakdown (GVBD)] occurring at the fragments (each containing 5–20 oocytes) in 4 ml of goldfish prophase͞metaphase transition is usually regarded as a hallmark Ringer’s solution containing each agent (from a 1,000-fold of the progress of oocyte maturation. Two MIHs, 17␣,20␤- stock in ethanol) at room temperature with gentle agitation (40 dihydroxy-4-pregnen-3-one (17,20␤-DHP) and 17␣,20␤,21- rpm). To assess maturation processes, germinal vesicles in trihydroxy-4-pregnen-3-one (20␤-S), have been identified in full-grown oocytes were examined under a binocular micro- several fish species (5, 6). In goldfish, 17,20␤-DHP has been scope (SMZ645, Nikon) after placing the oocytes in clearing shown to induce oocyte maturation by stimulating the de novo solution (17). The nuclear state (%) at each time point was synthesis of cyclin B, a regulatory subunit of maturation- determined in 40 oocytes. The morphology of oocytes was promoting factor (7). Although progestins including 17,20␤- photographed with a digital microscope (VH8000, Keyence, DHP and 20␤-S are the most potent steroid inducers of oocyte Osaka). maturation in fish, other hormones such as deoxycorticosterone Ovaries of zebrafish were isolated from killed females and ͞ and testosterone, but not estradiol or its analogs, are also placed in fresh zebrafish Ringer’s solution (116 mM NaCl 2.9 ͞ ͞ effective (8). mM KCl 1.8 mM CaCl2 5 mM Hepes, pH 7.2) and washed three Several endocrine-disrupting chemicals, Kepon and dichlo- times with the same solution. Immature oocytes were exposed in rodiphenyldichloroethane, have been reported to antagonize vitro by incubating ovarian fragments (each containing 2–10 MIH-induced meiotic maturation of fish oocytes in vitro (9). oocytes) in 4 ml of zebrafish Ringer’s solution containing each One of the environmental endocrine-disrupting chemicals agent (from a 1,000-fold stock in ethanol) at room temperature (EEDCs), diethylstilbestrol (DES) is a nonsteroidal substance with gentle agitation (40 rpm). To assess maturation processes, that was prescribed from the late 1940s to the early 1970s to germinal vesicles in full-grown oocytes were examined under a binocular microscope (SMZ645, Nikon) after placing the oo- pregnant women to prevent abortion, preeclampsia, and other cytes in clearing solution (17). The nuclear state (%) at each time complications of pregnancy. Male and female offspring ex- point was determined in Ͼ20 oocytes. posed in utero to DES may develop multiple and neoplastic lesions of the reproductive tract, along with other changes, Preparation of Oocyte and Egg Extracts. Groups of 20 oocytes were during development (10). Here we show that exposing fish washed in extraction buffer (0.1 M sodium ␤-glycerophos- oocytes to DES at a dose within a range similar to that used in experimental exposure to 17,20␤-DHP induces oocyte maturation. Estradiol-17␤ has been reported to be ineffective Abbreviations: 17,20␤-DHP, 17␣,20␤-dihydroxy-4-pregnen-3-one; MIH, maturation- in inducing fish oocyte maturation (11, 12) and even inhibitory inducing hormone; EEDC, environmental endocrine-disrupting chemical; DES, diethyl- stilbestrol; HEX, hexestrol; DIES, trans,trans-dienestrol(␣-dienestrol); DMS, dimethyl- in several teleost species (13–15). Thus, the stimulatory effect stilbestrol;, DM-DES, DES dimethyl ether; DP-DES, DES dipropionate; GVBD, germinal of DES to induce fish oocyte maturation observed in this study vesicle breakdown; DDT, dichlorodiphenyltrichloroethane; mPR␣, membrane progestin has not been published previously. This report shows that receptor ␣. EEDC can potentially induce oocyte maturation like an en- ‡To whom correspondence should be addressed. E-mail: [email protected]. dogenous MIH, 17,20␤-DHP. © 2004 by The National Academy of Sciences of the USA 3686–3690 ͉ PNAS ͉ March 9, 2004 ͉ vol. 101 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400072101 Downloaded by guest on September 25, 2021 Table 1. In vitro induction of GVBD in goldfish oocytes GVBD, % at concentration of Treatment 0.1 ␮M1␮M2␮M EtOH 0 17,20␤-DHP 83.3 Ϯ 6.3 98.3 Ϯ 2.9 98.3 Ϯ 2.9 DES 0 18.3 Ϯ 6.3 85.0 Ϯ 8.7 ␤-Estradiol 0 0 0 17␣-Estradiol 0 0 0 Ethynylestradiol 0 0 0 Resveratorol 0 0 0.8 Ϯ 1.4 o,pЈ-DDT 0 0 0 p,pЈ-DDT 0 0 0 Butyl benzyl phthalate 0 0 0 Di(2-ethylhexyl)phthalate 0 0 0 Bisphenol A 0 0 0 p-Nonylphenol 0 0 0 4-Octylphenol 0 0 0 Pentachlorophenol 0 0 0 The percentage of GVBD was calculated by determining the percentage of oocytes that had undergone GVBD in a group of 40 oocytes cultured in parallel for 6 h. Each value is the mean (Ϯ SD) of three separate experiments with ovaries of three separate females. ͞ ͞ ͞ ͞ phate 15 mM MgCl2 5 mM EGTA 20 mM Hepes 1mMDTT, pH 7.5) and transferred to a 1.5-ml Eppendorf microcentrifuge tube. After the excess buffer was removed, 200 ␮l of buffer was added. The samples were crushed with five strokes of a plastic pestle and centrifuged at 13,500 rpm for 10 min at 4°Cina fixed-angle rotor (MX-300 microcentrifuge, Tomy, Tokyo). The Fig. 1. DES induces genuine oocyte maturation. (A) The morphology of clear supernatant (100 ␮l) was collected for electrophoresis and oocytes after6hofeach treatment was photographed. Germinal vesicles were immunoblotting. seen near the center of oocytes after EtOH treatment, whereas they disap- peared after 17,20␤-DHP and DES treatments. (B) Extracts were prepared from SDS͞PAGE and Immunoblotting. Proteins were separated by 20 oocytes after incubation with EtOH, 17,20␤-DHP, or DES. Extracts of each PAGE under denaturing conditions (SDS͞PAGE with 10% treatment were electrophoresed under denaturing conditions (10.0% gel) gel) by the method of Laemmli (18) and transferred to and stained with Coomassie brilliant blue (CBBR) or immunostained with Immobilon membrane (Millipore). Membranes were blocked anti-goldfish cyclin B polyclonal antibody after electroblotting (␣-cycB). The in 5% nonfat powdered milk and incubated with primary 48-kDa band of cyclin B is indicated by an arrow. Molecular masses of standard antibodies for1hatroom temperature. Immunocomplexes proteins are indicated on the left. were visualized by using the ECL detection kit (Amersham Biosciences). inducing GVBD was investigated by using goldfish oocytes PHYSIOLOGY cDNA Cloning and Production of Recombinant Proteins. Recently, a (Table 1). Of the 13 agents tested, only DES was effective in inducing GVBD. As in the previous report (8), two other strong candidate for an MIH membrane receptor has been ␤ ␣ identified and characterized in spotted seatrout (19). We cloned estrogens ( -estradiol and ethynylestradiol) and 17 -estradiol cDNA for this membrane progestin receptor ␣ (mPR␣) from did not induce GVBD at the concentration tested.