Supplemental Figure 1. Partial purification of ovarian membrane androgen binding moiety used to generate an antibody for library screening.

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1 a b c Supplemental Figure 1. Partial purification of ovarian membrane arogen biing moiety used to generate an antibody for library screening. Croaker ovaries were homogenized a the plasma membrane fraction was isolated by centrifugation. Following centrifugation, treatment with 10 mm Triton X-100 for 30 min followed by removal of the detergent with polyabsorbents for 1 h resulted in extraction of a solubilized protein from the membrane fraction with significant amounts of high affinity a displaceable specific T biing (83% recovery of membrane [ 3 H]-T biing). a.) DEAE chromatography with a continuous NaCl gradient between 0 a 0,1M NaCl resolved the solubilized ovarian proteins into two major UV-absorbing peaks. The fractions were concentrated to 1 mg of protein per ml with a 10,000- Da molecular weight cutoff centrifuge concentrator a thereafter checked for [ 3 H]-T biing. Fractions that did not display [ 3 H]- T biing were discarded. Analyses of the partially purified proteins by SDS/PAGE showed major bas at 40 kda in both fractions (b.), but only protein in Fraction 2 displayed high specific [ 3 H]- T biing (c). Therefore, this partially purified receptor fraction was used to immunize mice.

2 Croaker MDDFSSISLLSVAMLVGCYVAGTIPLAVNFSEEKLKLITVLGAGLLCGTALAVIIPEGVHAIYEEILEGGHHSHG 75 Fugu MDDFSSISLLSLAMLVGCYVAGIIPLAVNFSEEKLKLVTVLGAGLLCGTALAVIIPEGVHALYEELLEGGHQNHG 75 Human MDDFISISLLSLAMLVGCYVAGIIPLAVNFSEERLKLVTVLGAGLLCGTALAVIVPEGVHALYEDILEGKH--HQ 73 Croaker QAGVVEVSEAKGDAESVLSGGGKHEHSHEQLHACIGVSLVLGFVFMLLVDQIGSSHVHSTEDPESARVTSSKITT 150 Fugu HSGAAEVSETKAEVEAVLSTGGKHEHAHEQLHACIGVSLVLGFVFMLLVDQIGSSHVHSTEDPESARAASSKITT 150 Human ASETHNVIASDKAAEKSVVHEHEHSHDHTQLHAYIGVSLVLGFVFMLLVDQIGNSHVHSTDDPEAARSSNSKITT 148 Croaker TLGLVVHAAADGVALGAAASTSQTSVQLIVFVAIMLHKAPAAFGLVSFLMHAGLERNRIRKHLLVFALAAPVLAM 225 Fugu TLGLVVHAAADGVALGAAASTSQTSVQLIVFVAIMLHKAPAAFGLVSFLMHAGLERNRIRKHLLVFALAAPVLAM 225 Human TLGLVVHAAADGVALGAAASTSQTSVQLIVFVAIMLHKAPAAFGLVSFLMHAGLERNRIRKHLLVFALAAPVMSM 223 Croaker LTFLGLSQSSKEALSDINATGVAMLFSAGTFLYVATVHVLPEVGGGGHSHAASGGNGGKGLSKVEVVALVLGCLI 300 Fugu LTFLGLSQSSKEALSDINATGVAMLFSAGTFLYVATVHVLPEVGAGGHSHAPAGGSGNKGLSKVEVLALVLGCLI 300 Human VTYLGLSKSSKEALSEVNATGVAMLFSAGTFLYVATVHVLPEVGGIGHSHKP-DATGGRGLSRLEVAALVLGCLI 297 Croaker PLVLSVGHHH 310 Fugu PLVLSVGHHH 310 Human PLILSVGHQH 307 * Supplemental Figure 2. Alignmentsof amino acid sequences of croaker, Fugu a human ZIP9s using the ClustalW multiple alignment program. Amino acid differences are shown in red. * amino acid in croaker ZIP9 that results in a prediction of 7TMs instead of 8TMs as in Fugu a human ZIP9s.

3 a b c d e ZIP9 ab 1:5000 ZIP9 ab 1:5000 ZIP9 ab 1:5000 blocking 1: Supplemental Figure 3. Specificity of antibody for croaker ovarian Fraction 2 a croaker ZIP9. a.) Western blot analysis of croaker ovarian fractions using the antibody. Lane 1) Fraction 2 from purification, Lane 2). Fraction 1 from purification, Lane 3) whole ovary extract. The results show a single immunoreactive ba of the predicted size for ZIP9 in the [ 3 H]-T biing membrane fraction b.) ICC of nontransfected a croaker ZIP9-transfected SKBR3 cells. The results show increased immunostaining of ZIP9-transfected cells compared to nontransfected controls which was blocked by pretreatment with the antigen (Fraction 2), further demonstrating the specificity of the antibody for ZIP9. c.) IP of ovarian fractions a subsequent [ 3 H]-T biing assay show that the antibody pulls down a protein that bi [ 3 H]-T from both membrane fraction as well as Fraction 2, thereby showing specificity for the purified protein with an arogen biing function. d) Western blot analysis of ZIP9-transfected SKBR3 cell membranes a Fraction 2 membranes using the antibody. The results show that NT cells do not express ZIP9 (Lane 1), whereas transfected (Tr) cells show a strong ba (Lane 2) at the same size as the purified ZIP9 protein (Lane 3). Collectively, these results iicate the the antibody is specific for croaker ZIP9 a the protein that bis [ 3 H]-T in Fraction 2. e.) Western blot analysis of ovarian membranes a Fraction 2 with a nar antibody (RabMab ab52615, Abcam) 3. Bas of the correct size for nar were detected in whole ovary extract (lanes 1 & 2), while no signal were seen in plasma membranes (lane 3 & 4) a Fraction 2 containing ZIP9 (lane 5 & 6), iicating the absence on nar in these fractions. Additional characterization of the specificity of the antibody is presented in the manuscript : (Fig. 2b Organ specificity, Western blots. Fig. 3b, 3c- ZIP9 protein expression in tranfected scells, Western blot, ICC. 3d- Liga blot shows T biing to same position as antibody biing on Western blot. 3f- Pull-down assay IP shows that the protein pulled down by the antibody bi[ 3 H]-T. 6a ZIP9 sirna decreases expression of the specific ba on Western blot).

4 a b Supplemental Figure 4. Regulation of Atlantic croaker ZIP9 expression a [ 3 H]-T biing by hormones a during ovarian reproductive cycle. a.) Ovarian expression of ZIP9 protein (light grey bars) as well as specific testosterone biing (dark grey bars) are upregulated by hormone treatments in vitro. Ovarian fragments were exposed to different concentration of the hormones (hcghuman chorionic gonadotropin, T-Testosterone, E2-17β-estradiol,) for 48 hours prior to isolation of membrane fraction/rna for biing analysin, QPCR a Western blot analysis b.) Expression of the arogen biing protein in ovaries varied during the reproductive cycle, consistent with a physiological role for the putative arogen receptor/zinc transporter (ZIP9) in regulation of reproduction in Atlantic croaker.

5 Supplemental Table 1-Structural analyses of vertebrate ZIP9s Accession number SOSUI TMpred DAS TMHMM HMMtop N-terminal C-terminal Human FJ * EC EC Sea trout KF * EC IC Atlantic Croaker KF * EC IC Zebrafish NM_ * EC EC Fugu ENSTRUT ^ EC EC Medaka ENSORLT ^ EC EC Stickleback ENSGACT ^ EC EC Cod ENSGMOT ^ EC EC Pufferfish ENSTNIT ^ EC EC Platyfish ENSXMAT ^ EC EC Tilapia ENSONIT ^ EC EC Multiple structural analyses of vertebrate ZIP9 amino acid sequences were coucted using DAS, HMMtop, TMHMM, TMpred a SOSUI computer programs. The number of transmembrane domains that each program predicts is shown. Localization of N- a C-terminal domains are iicated with EC; extracellular or IC; intracellular. Protein sequences obtained from * GenBank a ^ Ensembl.

6 Competitor RBA (%) T P M M DHT 0.96 Arostenedione 0.56 Flutamide 0.38 E2 F 11-KT Epitestosterone 20ß-S 11-KA Mibolerone Vinclozilin Supplemental Table 2. Relative biing affinity (RBA) of various steroids a xenobiotics for membrane fractions from SKBR-3 cells transfected with ZIP9. iicates substances that did not display 50 % displacement of [ 3 H]- T from its receptor in the range of concentrations examined. M2, M1: metabolites of vinclozilin