Supplementary Table 2 List of all putative Leydig cell-specific transcripts identified using criteria set in the text and comparison to previous studies

Affymetrix Genbank Symbol control (raw Expression (percent control) after EDS Ranking in Sanz Leydig cell ratio†

expression)* 1d 3d 5d 8d 12d et al (2013)** development*** LC/WT SC/WT RS/WT PS/WT SPES/WT

1387139_at NM_032082 Hao2 hydroxyacid oxidase 2 (long chain) 9678 5.33 0.45 0.31 0.30 0.31 105 0.512 4.771 0.156 0.202 0.300

1387034_at NM_012619 Pah phenylalanine hydroxylase 4450 8.02 0.61 0.54 0.58 0.56 x 0.556 2.452 0.122 0.149 0.142

1387123_at NM_012753 Cyp17a1 cytochrome P450, family 17a1 6904 8.19 0.63 0.54 1.73 6.65 1134 0.823 2.251 0.046 0.041 5.053

1370363_at L46791 Ces3/Ces1d†† carboxylesterase 3 (1d) 10226 15.24 0.74 0.47 0.49 0.71 4 0.474 2.546 0.147 0.174 0.096

1388241_at AF139918 Insl3 insulin-like 3 10447 17.39 0.98 0.65 0.67 0.82 x 0.444 2.001 0.077 0.066 0.958

1368578_at NM_017265 Hsd3b1 hydroxysteroid dehydrogenase-1, delta5-3beta 5826 8.52 1.28 1.09 2.95 11.78 87 0.696 4.157 0.080 0.134 9.408

1368468_at NM_017286 Cyp11a1 cytochrome P450, family 11a1 4896 9.70 1.36 1.26 2.85 7.01 104 0.890 3.748 0.271 0.377 2.940

1370964_at BF283456 Ass arginosuccinate synthetase 6673 19.45 1.57 1.17 1.42 2.53 x 0.721 1.756 0.086 0.087 0.892

1370148_at NM_012582 Hp haptoglobin 7192 12.24 1.88 1.75 1.65 1.65 x 0.716 4.884 0.067 0.196 0.232

1388176_at AI717047 Cml5 camello-like 5 1194 2.08 2.74 2.77 2.61 2.87 116 0.715 1.119 0.117 0.122 2.733

1370065_at NM_053318 Hpx hemopexin 2648 9.06 3.02 2.30 2.11 2.23 x 0.170 1.429 0.639 0.549 0.485

1368621_at NM_022960 Aqp9 aquaporin 9 2907 5.53 3.02 2.16 3.17 3.04 x 0.987 1.187 1.043 1.985 0.451

1378133_at AW532618 Slc7a11 Solute carrier family 7, member 11 1967 25.36 3.29 3.45 4.60 7.43 194 0.960 1.660 0.185 0.306 0.914

1387465_a_at NM_031038 Gnrhr gonadotropin releasing hormone receptor 1043 6.25 4.26 3.87 3.87 4.03 x 1.016 1.456 0.215 0.161 0.702

1387423_at NM_012978 Lhcgr luteinizing hormone/choriogonadotropin receptor 615 5.95 4.69 4.25 4.31 5.10 5 3.573 1.291 0.448 0.523 5.440

1387973_at U39206 Cyp4f4 Cytochrome P450, family 4f4 678 9.44 5.42 5.59 6.19 5.51 x 1.823 2.901 0.774 0.953 0.759

1385500_at BF400284 LOC500945 similar to 1700012B09 Rn.59584 758 9.77 5.82 3.86 6.96 10.79 1.707 0.299 1.894 1.064 0.130

1369136_at NM_012542 Cyp2a3 Cytochrome P450, subfamily IIA3 501 8.78 5.99 6.39 6.19 5.59 x 1.485 1.322 1.612 1.344 1.121

1392664_at BF551274 Gpr182 G -coupled receptor 182 (adrenomedullin receptor) 466 5.79 6.01 5.58 6.01 7.08 1980 1.492 1.613 0.276 0.293 0.425

1368990_at NM_012940 Cyp1b1 cytochrome P450, family 1b1 7824 5.92 6.02 3.59 5.52 7.39 x 0.465 2.256 0.226 0.193 2.279

1367857_at NM_053445 Fads1 desaturase 1 5010 12.75 6.85 2.99 6.39 14.85 168 0.641 3.146 0.187 0.182 1.987

1367689_a_at AF072411 Cd36 CD36 molecule 5232 7.15 7.39 10.25 7.44 5.31 38 0.544 2.528 0.103 0.064 0.503

1388037_at J05087 Atp2b3 ATPase, Ca++ transporting, plasma membrane 3 759 8.72 7.48 12.37 10.50 8.19 2010 1.546 2.550 0.665 0.671 2.697

1381920_at AI502118 Transcribed locus 634 9.89 7.65 6.91 7.61 7.53 0.750 0.582 0.237 0.357 1.060

1371100_at AA859049 Ces1c Carboxylesterase 1C 712 12.64 7.72 6.46 7.02 8.71 x 0.885 0.774 0.520 0.323 0.372

1390790_a_at AA964219 hypothetical LOC361349 1731 18.88 8.35 7.08 7.66 9.57 0.592 1.397 0.383 0.215 4.223

1387197_at NM_031817 Omd osteomodulin 879 8.69 8.37 13.95 33.87 38.95 694 0.885 1.236 0.404 0.394 6.095

1368265_at NM_134369 Cyp2t1 Cytochrome P450, family 2, subfamily t, polypeptide 1 437 8.89 8.42 8.11 7.66 7.94 x 0.969 4.063 1.250 1.217 1.848

1379491_at AW524012 Mrap Melanocortin 2 receptor accessory protein 418 15.10 9.14 9.14 9.26 9.48 135 1.365 8.241 0.556 0.647 0.772

1382137_at BG662490 Abhd3 abhydrolase domain containing 3 1853 7.99 9.55 13.01 17.16 16.78 251 0.699 3.597 0.054 0.173 4.073

1368565_at NM_019225 Slc1a3 solute carrier family 1, member 3 3313 25.64 9.68 6.07 6.44 7.04 1188 0.543 1.481 0.339 0.519 1.651 1370255_at AI009659 Sftpc surfactant associated protein C 347 12.97 10.37 9.80 10.37 11.53 687 0.418 0.301 1.013 1.186 0.830

1370310_at M33648 Hmgcs2 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 10550 10.18 10.81 10.29 9.80 11.61 143 0.377 3.467 0.061 0.026 2.585

1390146_at BF414998 Tmem150c Transmembrane protein 150C 266 10.90 11.65 15.79 12.41 15.41 x 1.061 1.850 2.125 0.408 0.902

1368021_at NM_130780 Adh1 alcohol dehydrogenase 1 10324 15.96 11.88 9.14 11.83 18.77 30 0.499 3.336 0.167 0.179 2.404

1369553_at NM_054007 Hsd17b3 hydroxysteroid (17-beta) dehydrogenase 3 345 14.49 11.88 11.30 10.72 11.30 66 1.974 2.648 1.102 1.102 1.172

1391490_at BF394332 Transcribed locus 430 17.51 11.91 16.22 42.73 31.07 2.688 3.096 0.676 0.728 0.841

1369118_a_at 345 13.62 12.17 11.30 11.59 11.01 1.140 1.048 0.851 0.811 1.128

1387655_at AF189724 Cxcl12 chemokine (C-X-C motif) ligand 12 1062 8.79 12.23 8.94 11.41 20.20 713 0.310 3.359 0.332 0.278 6.406

1382953_at AI043711 Tbx3 T-box 3 360 11.27 12.52 12.47 13.12 12.77 1079 0.352 12.344 1.719 2.057 8.948

1368453_at NM_031344 Fads2 2 565 22.48 12.74 12.21 29.03 27.61 413 2.450 2.984 0.570 0.755 2.036

1383757_at AI044802 Transcribed locus 454 26.65 12.78 24.67 34.36 27.31 0.171 3.068 1.364 1.449 2.321

1367887_at NM_017024 Lcat lecithin cholesterol acyltransferase 492 19.31 12.80 15.24 15.04 16.46 183 1.560 2.062 0.836 0.709 3.275

1386998_at NM_012497 Aldoc aldolase C, fructose-biphosphate 1371 16.48 12.98 11.45 12.91 15.39 11 1.387 4.325 0.179 0.164 0.941

1389161_at AW144193 Transcribed locus 540 21.11 13.15 16.67 15.19 17.22 1.167 2.207 1.028 0.467 2.444

1378471_at BE111819 Raver2 Ribonucleoprotein, PTB-binding 2 327 11.96 13.22 11.31 11.22 13.48 1679 0.788 5.033 0.132 0.155 2.975

1389412_at AA800693 Transcribed locus 1666 20.23 13.41 12.64 14.02 14.99 0.644 1.361 0.632 0.680 2.316

1371817_at BI285489 RGD:1359423 similar to myo-inositol 1-phosphate synthase A1 4361 14.98 13.49 8.77 14.94 20.61 0.325 0.712 0.752 0.995 0.231

1367733_at NM_019291 Ca2 carbonic anhydrase 2 1142 18.83 13.66 27.67 21.54 14.71 x 1.042 2.681 0.273 0.308 2.702

1377084_at BE106598 Transcribed locus 1937 17.91 13.68 14.35 12.65 13.42 0.855 0.952 0.511 0.415 1.350

1367615_at NM_133516 Svs5 seminal vesicle secretion 5 491 27.28 13.71 12.78 14.11 19.55 1733 5.731 2.344 0.235 0.215 7.296

1377429_at BI292231 Lpo lactoperoxidase 313 17.25 14.38 14.06 14.70 14.06 x 0.716 0.769 1.181 1.254 1.419

1383414_at BG373072 RGD1308117 similar to 9930012K11Rik protein 1055 13.65 14.88 18.48 22.65 30.71 1.209 1.235 1.126 1.150 2.288

1369033_at 126 16.67 15.08 16.67 15.87 16.67 1.314 1.115 0.808 0.761 3.722

1386881_at NM_012588 Igfbp3 insulin-like growth factor binding protein 3 1288 50.70 15.30 23.06 29.11 35.02 x 0.280 1.538 0.563 0.305 7.374

1368061_at NM_031742 Kcnh1 potassium voltage-gated channel, subfamily H1 381 41.47 15.49 13.91 15.75 19.16 x 0.857 0.580 1.644 1.328 2.487

1373110_at AI407487 Transcribed locus 1206 7.05 15.67 14.26 13.02 15.01 0.648 1.366 0.748 0.658 7.101

1372372_at BG372437 Cmbl Carboxymethylenebutenolidase homolog 1343 7.81 15.74 21.20 16.07 15.49 75 1.012 4.173 0.132 0.333 1.165

1369640_at BG378227 Gja1 gap junction membrane channel protein alpha 1 291 21.99 15.81 21.31 27.84 20.96 735 1.004 8.471 0.326 0.578 0.904

1368060_at NM_031714 Hrsp12 heat-responsive protein 12 3643 18.69 16.03 12.90 32.94 53.03 573 0.533 2.238 0.371 0.525 9.436

1367994_at NM_031027 Dpyd dihydropyrimidine dehydrogenase 386 12.44 16.06 20.47 14.51 16.32 120 0.876 1.194 0.552 0.606 1.107

1380267_at AI029135 Tekt3 tektin 3 1171 9.65 16.25 6.38 20.89 20.73 x 0.792 0.073 1.462 0.662 0.196

1367648_at NM_013122 Igfbp2 insulin-like growth factor binding protein 2 9193 19.10 16.40 16.31 17.67 22.52 306 0.540 2.813 0.199 0.147 3.874

1387020_at BG664123 Cyp51 cytochrome P450, subfamily 51 956 58.45 16.68 15.38 19.29 28.47 966 0.511 1.507 0.987 0.412 2.454

1377660_at NM_053373 Pglyrp1 peptidoglycan recognition protein 1 3062 31.42 16.72 11.63 19.24 26.13 x 0.242 0.825 0.594 0.624 0.141

1378063_at AW524795 Transcribed locus 275 16.36 16.73 18.18 18.18 18.18 0.822 0.441 0.861 0.714 1.214 1390783_at BG671264 Abca8a ATP-binding cassette, sub-family A (ABC1), member 8a 596 6.87 16.88 25.74 23.61 30.03 x 0.501 2.292 1.979 1.220 12.048

1387183_at J02844 Crot carnitine O-octanoyltransferase 1403 13.47 17.03 16.39 17.11 15.61 669 1.521 3.471 0.626 0.885 1.602

1393756_at AI060002 Dmp1 dentin matrix protein 1 245 17.96 17.14 16.33 16.73 15.10 x 1.018 1.101 0.938 0.617 0.700

1387706_at NM_080586 Gabrg1 gamma-aminobutyric acid (GABA) A receptor, gamma 1 257 16.34 17.51 13.62 16.73 15.95 x 0.712 0.779 1.374 1.170 1.433

1389725_at BM390364 Tm7sf2 transmembrane 7 superfamily member 2 615 26.18 17.56 17.07 20.00 29.92 100 0.693 6.300 2.200 8.118 8.761

1376191_at AA848820 Hpgd 15-hydroxyprostaglandin dehydrogenase 195 12.82 17.95 16.41 13.33 13.85 240 4.152 1.736 0.405 0.539 2.229

1371976_at AI102758 Fam195a Family with sequence similarity 195, member A 993 29.10 18.03 21.55 33.74 29.20 1143 0.889 1.339 1.266 0.629 1.054

1388133_at AA800228 Pippin RNA-binding protein pippin 484 16.32 18.18 20.87 41.12 36.16 x 0.959 2.032 0.536 0.461 0.774

1390518_at AW915157 Emid1 EMI domain containing 1 325 20.92 18.46 19.08 19.38 20.92 x 2.057 1.496 0.934 0.910 1.851

1369777_a_at NM_133440 Shank2 SH3/ankyrin domain gene 2 175 19.43 18.86 23.43 28.00 26.86 x 1.844 0.831 0.990 1.084 3.895

1392092_at AI101329 Rab33a RAB33A, member RAS oncogene family 683 18.45 18.89 19.18 15.81 17.86 x 0.584 0.855 1.486 1.288 2.287

1386953_at NM_017080 Hsd11b1 hydroxysteroid 11-beta dehydrogenase 1 11203 19.91 19.16 11.43 11.77 12.45 x 0.355 3.446 0.081 0.091 0.610

1383585_s_at AI043753 TL0ADA47YD01 TL0ADA47YD01 628 23.25 19.27 17.83 17.68 15.13 0.510 2.906 0.203 0.298 5.168

1387759_s_at J02612 Ugt1a UDP glycosyltransferase 1 family 2086 12.56 19.27 11.74 10.16 10.07 319 1.111 2.346 0.484 0.302 1.310

1388632_at BG665929 Rab6b RAB6B, member RAS oncogene family 469 15.35 19.40 22.39 22.81 25.80 x 0.810 1.159 0.681 0.463 2.018

1368180_s_at NM_017013 Gsta2 glutathione-S-, alpha type2 1521 35.83 19.72 23.08 20.25 17.69 330 1.775 4.397 0.313 0.312 0.163

1372536_at AI105042 Adck3 AarF domain containing kinase 3 1312 15.32 20.35 26.30 20.58 19.44 x 0.615 2.114 0.142 0.163 1.647

1369633_at 3304 19.01 20.58 16.71 18.95 29.96 0.529 1.399 0.335 0.328 4.527

1373748_at AW532566 Pdzrn3 PDZ domain containing RING finger 3 752 9.04 20.63 25.72 19.53 20.04 x 0.871 4.199 0.837 0.669 2.223

1373947_at BI278545 Dpt dermatopontin 3179 12.17 20.89 12.36 9.56 11.80 x 1.084 3.491 0.362 0.336 9.381

1376944_at AI407163 Prlr Prolactin receptor 851 3.17 21.04 19.74 13.74 17.98 37 0.988 2.180 0.253 0.250 1.896

1368304_at NM_053433 Fmo3 Flavin containing monooxygenase 3 979 7.92 21.15 18.47 24.66 33.16 x 0.756 1.250 0.081 0.115 2.347

1388949_at BI297693 Transcribed locus 353 8.34 23.31 18.99 16.52 14.05 1.093 1.189 0.488 0.503 1.605

1387669_a_at NM_012844 Ephx1 epoxide 1 4023 9.39 24.14 24.36 19.59 18.91 237 0.886 0.641 1.013 0.240 0.448

1370613_s_at AF461738 Ugt1a8 UDP-glucuronosyltransferase 1-8 precursor 3908 18.75 25.09 14.08 13.97 13.43 x 1.669 3.525 0.240 0.300 1.386

1370789_a_at 449 9.90 25.58 21.01 17.72 21.17 1.575 1.457 0.969 0.845 0.695

1377635_at BM389350 Fmo2 flavin containing monooxygenase 2 774 8.77 27.83 26.77 24.13 29.42 x 1.054 1.418 0.686 0.751 2.240

* Untransformed expression from Affymetrix arrays

** Transcripts from Table 1 were compared against Dataset 2 (Sanz et al 2013) which ranks putative Leydig cell transcripts in the order that they are enriched in the Leydig cell translatome.

The smaller the number the higher the enrichment in Sanz et al (2013). Transcripts marked with an x were not in Dataset 2 Sanz et al (2013).

*** Transcripts identified as upregulated during Leydig cell development by Stanley et al (2011).

† Ratio of transcript expression on Leydig cell (LC) arrays (Stanley et al, 2011) vs whole testis (WT) arrays (control data from this study) or Sertoli cell (SC), round spermatid (RS), pachytene spermatocyte (PS) or spermatogonia plus early spermatocyte (SPES) (Johnston et al 2008) vs Whole testis (WT) (Johnston et al 2008). ††Rat Ces1d was previously called Ces3 and is orthologous to mouse Ces3 (Holmes et al 2010)