Supplementary Material for. Disruption of Ttll5/Stamp gene in male mice causes sperm malformation and infertility

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1 Supplementary Material for Supplementary Material for Lee et al., Page 1 Disruption of Ttll5/Stamp gene in male mice causes sperm malformation and infertility Geun-Shik Lee, Yuanzheng He, Edward J. Dougherty, Maria Jimenez-Movilla, Matteo Avella, Sean Grullon, David S. Sharlin, Chunhua Guo, John A. Blackford, Jr., Smita Awasthi, Zhenhuan Zhang, Stephen P. Armstrong, Edra C. London, Weiping Chen, Jurrien Dean, and S. Stoney Simons, Jr.* From the Steroid Hormones Section, NIDDK/LERB, Laboratory of Cellular and Developmental Biology, NIDDK, Adipocyte Biology and Gene Regulation Section, NIDDK/LERB, Nuclear Receptor Biology Section, NIDDK/LERB, Genomic Core, NIDDK, and National Institutes of Health, Bethesda, MD *Address correspondence to Dr. S. Stoney Simons, Jr., Bldg. 1, Room 8N-37B, NIDDK/LERB, NIH, Bethesda, MD ( stoneys@helix.nih.gov; Phone: ; FAX: )

2 Supplementary Material for Lee et al., Page 2 Tables Table S1: Oligonucleotide primers for RT-PCR quantitation of known genes involved in spermatogenesis. Oligonucleotide sequences PCR product Gene Gene bank ID Forward (5' to 3') Reverse (5' to 3') size (bp) Gopc NM_53187 TGTGCATGGTATTGGAGCAT AAAGCTTGGGAACAGCTCAA 17 Agfg1 (Hrb) NM_1472 CCTCGGTTCATGCGTCTATT TCTCCCAACAGGGATTTCAG 88 Csnk2a2 NM_9974 TGCTCATCCATACGCTCTTG AGCAGAGACCCTGCACATCT 83 Gba2 NM_ CAGGCGGTATACACGGTTCT GTTCTGCCAGGCTGAGATTC 112 Tekt2 NM_1192 CCCAAAGACTCCACCACACT TCTGAGCGTCCAGTTCATTG 148 Tekt4 NM_27951 ACAAGCCGTGGACCAGATAC TGGGTGCTCTCATTGGTGTA 126 Vdac3 NM_11696 ATAAACCTGGCATGGACAGC TCGGAGGGTCTGCGTATAAC 141 Sepp1 NM_ TGACAAAATCAGCCCATGAA TGAGTTGGGGAGAGGTATGC 125 Akap4 NM_ GTGAGAGCCCTTTCAGTTGC GGCATTGCTCTTCAGGTCTC 111 Spag6 NM_15773 TCGCAGCTGATTGTTAATGC CAGAATGAGCAGCCACGTAA 118 Gapdhs NM_885 CCAAGCCTGCTTCTTACTCG GACCACCTGGTCCTCTGTGT 98 Adcy1 (sac) NM_17329 AACGTTCGTGGAGTTGGTTC GACCTCGACAGAGGCTTGAC 119 Catsper1 NM_13931 CGGGTCCATGAGAAGTTGTT CGCTGCTTCACTGTCATGTT 134 Catsper2 NM_15375 TATCTTCATGCATCCCACCA AGGCTCGAGTCCTTCAACAA 134 Catsper3 NM_29772 GTTGATGGCTGGACTGACCT TGAAGGATGCAAGCAAGATG 97 Catsper4 NM_1133 CCTTATCTGCGAGGTTCTGC CCCATGTTCTGGAAATGCTT 1 Tnp1 NM_97 GCATGAGGAGAGGCAAGAAC AGGACGCTCTTCCGGTATTT 82 Tnp2 NM_13694 TCGACACTCACCTGCAAGAC CTTGTATCTTCGCCCTGAGC 148 H1fnt (H1t2) NM_2734 CAAGGAAAAGCATGCACAAA TTGGAGCCCATATGGAAAAG 115 Crem NM_11185 ACTAGCACGGGGCAATACAC TCTGCTAGTTGCTGGGGACT 134 Tbpl1 NM_113 GCACTGGAGCAACAAGTGAA GCCAAAACATTCACCACCTT 118 Papolb (Tpap) NM_19943 TGCACTGAGTGAAAGCATCC GGATTGGGTATGTTCGTTGC 15 Piwil1 (Miwi) NM_21311 AACCCTCTTCCAGGGACAGT CAGGCCACTGCTGTCATAGA 138 Spem1 NM_28855 TGTTACCCCTTGGTCTCTGG TTTTGTCCCCAGTCACCTTC 93

3 Supplementary Material for Lee et al., Page 3 Table S2: Microarray results from testes of wt and Stamp tm/tm mice of heterogeneous genetic background showing genes altered at the whole gene level. p-value Fold-Change Gene Symbol GeneBank ID (TM vs. Wt) (TM vs. Wt) Tshr NM_ E Prps1l1 NM_ E N1Rik AK Egr2 NM_ Tdgf1 NM_ Scn1a NM_ E Acp1 NM_ Snapc1 NM_ Gtf2a1 NM_ Ier3 NM_ Egr1 NM_ Ier2 NM_ Rhbg NM_ Serpina3c NM_ Hdac9 NM_ Fbxw15 NM_ Heg1 NM_ Nr4a1 NM_ A3Rik ENSMUST Usp48 NM_ Dio2 NM_ Eif4e NM_ Fam5b BC Pik3r4 NM_ Rrs1 NM_ Atp1a2 NM_ Cd84 NM_ Skiv2l2 NM_ Skint9 NM_ BC Arg2 NM_ Abhd14a NM_ Nme6 NM_ E-5-2.1

4 Supplementary Material for Lee et al., Page 4 References for the androgen-regulated genes (underlined with bold lettering) are as follow: Egr1 (5), Nr4a1 (2), Dio2 (7), Pik3r4 (4), Atp1a2 (1), and Arg2 (3, 6). References 1. Bolduc C, Yoshioka M, St-Amand J. 7. Transcriptomic characterization of the long-term dihydrotestosterone effects in adipose tissue. Obesity (Silver Spring) 15: DePrimo SE, Diehn M, Nelson JB, Reiter RE, Matese J, Fero M, Tibshirani R, Brown PO, Brooks JD. 2. Transcriptional programs activated by exposure of human prostate cancer cells to androgen. Genome Biol 3:RESEARCH Doane AS, Danso M, Lal P, Donaton M, Zhang L, Hudis C, Gerald WL. 6. An estrogen receptor-negative breast cancer subset characterized by a hormonally regulated transcriptional program and response to androgen. Oncogene 25: Nakamura Y, Suzuki T, Igarashi K, Kanno J, Furukawa T, Tazawa C, Fujishima F, Miura I, Ando T, Moriyama N, Moriya T, Saito H, Yamada S, Sasano H. 6. PTOV1: a novel testosterone-induced atherogenic gene in human aorta. J Pathol 9: Seenundun S, Robaire B. 7. Time-dependent rescue of gene expression by androgens in the mouse proximal caput epididymidis-1 cell line after androgen withdrawal. Endocrinology 148: Velasco AM, Gillis KA, Li Y, Brown EL, Sadler TM, Achilleos M, Greenberger LM, Frost P, Bai W, Zhang Y. 4. Identification and validation of novel androgenregulated genes in prostate cancer. Endocrinology 145: Xu Y, Chen SY, Ross KN, Balk SP. 6. Androgens induce prostate cancer cell proliferation through mammalian target of rapamycin activation and post-transcriptional increases in cyclin D proteins. Cancer Res 66:

5 Supplementary Material for Lee et al., Page 5 Table S3 Exon expression in Stamp tm/tm vs. Stamp +/+ mice (heterogeneous background) Probeset ID Gene Symbol RefSeq p-value (tm vs. wt) Fold-Change (tm vs. wt) Aass NM_ Aass NM_ Abcc9 NM_ Abcc9 NM_ Abcc9 NM_ Abhd14a NM_ Abhd14a NM_ Abhd14a NM_ Abhd14a NM_ Abhd14a NM_ Abhd14b NM_ Abhd14b NM_ Actr1 NM_ Actr1 NM_ Aebp1 NM_ Aebp1 NM_ Aim1 NM_ Aim1 NM_ Aim1 NM_ Aim1 NM_ Aim1 NM_ Aldoc NM_ Aldoc NM_ Ambp NM_ Ambp NM_ Aox3 NM_ Aox3 NM_ Arg2 NM_ Arg2 NM_ Arg2 NM_ Arg2 NM_ Arg2 NM_ Arg2 NM_ Arg2 NM_ Arhgap1 NM_ Arhgap1 NM_ Arhgap1 NM_ Arid4a NM_ Arid4a NM_ Atp1a2 NM_ Atp1a2 NM_ Atp1a2 NM_ Atp1a2 NM_ Atp1a2 NM_ Atp1a2 NM_ Atp1a2 NM_ Atp2c1 NM_ Atp2c1 NM_ Atp2c1 NM_ Atp2c1 NM_

6 Supplementary Material for Lee et al., Page Brip1 NM_ Brip1 NM_ Bst2 NM_ Btg2 NM_ Btg2 NM_ Btg2 NM_ Btg2 NM_ C3 NM_ C3 NM_ C3 NM_ Camkv NM_ Camkv NM_ Car5a NM_ Car5a NM_ Ccl22 NM_ Ccl22 NM_ Cd84 NM_ Cd84 NM_ Cd84 NM_ Cd84 NM_ Cds2 NM_ Cds2 NM_ Cln3 NM_ Cln3 NM_ Cln3 NM_ Cntn6 NM_ Cntn6 NM_ Col1a1 NM_ Col1a1 NM_ Col4a1 NM_ Col4a1 NM_ Cyb561d2 NM_ Cyb561d2 NM_ Cyb561d2 NM_ Cybb NM_ Cybb NM_ Dctd NM_ Dctd NM_ Dhx3 NM_ Dhx3 NM_ Dio2 NM_ Dio2 NM_ Dio2 NM_ Dio2 NM_ Dio2 NM_ Dio2 NM_ Dmkn NM_ Dmkn NM_ Dock11 NM_ Dock11 NM_ Dpp1 NM_ Dpp1 NM_ Efnb1 NM_ Efnb1 NM_ Efnb1 NM_ Egr1 NM_

7 Supplementary Material for Lee et al., Page Egr1 NM_ Egr1 NM_ Egr1 NM_ Egr1 NM_ Egr2 NM_ Egr2 NM_ Egr2 NM_ Eif2ak4 NM_ Eif2ak4 NM_ Ell3 NM_ Ell3 NM_ Errfi1 NM_ Errfi1 NM_ Errfi1 NM_ Ets2 NM_ Ets2 NM_ Evc NM_ Evc ENSMUST Fads1 NM_ Fads1 NM_ Fads1 NM_ Fam179b BC Fam179b BC Fam55b NM_ Fam55b NM_ Fbxw15 NM_ Fbxw15 NM_ Fmn2 NM_ Fmn2 NM_ Fos NM_ Fos NM_ Fos NM_ Fosl2 NM_ Fosl2 NM_ Gad2 NM_ Gad2 NM_ Gldc NM_ Gldc NM_ Gpr98 NM_ Gpr98 NM_ Hdac9 NM_ Hdac9 NM_ Hdac9 NM_ Hdac9 NM_ Hdac9 NM_ Hemk1 ENSMUST Hemk1 NM_ Hlcs NM_ Hlcs NM_ Hsdl1 NM_ Hsdl1 NM_ Hspa12a NM_ Hspa12a NM_ Hspa12a NM_ Ier2 NM_ Ier2 NM_

8 Supplementary Material for Lee et al., Page Ier3 NM_ Ier3 NM_ Igf2bp1 NM_ Igf2bp1 NM_ Igh AY Igh AY Il1b NM_ Il1b NM_ Il31ra NM_ Il31ra NM_ Il31ra NM_ Il31ra NM_ Il31ra NM_ Itih2 NM_ Itih2 NM_ Itpr3 NM_ Itpr3 NM_ Kcnh5 NM_ Kcnh5 NM_ Kcnh5 NM_ Kcnh5 NM_ Kcnj9 NM_ Kcnj9 NM_ Klc1 NM_ Klc1 NM_ Kremen1 NM_ Kremen1 NM_ Kremen1 NM_ Kremen1 NM_ Kremen1 NM_ Lcat NM_ Lcat NM_ Ldlr NM_ Ldlr NM_ Ldlr NM_ Ldlr NM_ Lipi NM_ Lipi NM_ Lipi NM_ Lrrc9 NM_ Lrrc9 NM_ Ltbp3 NM_ Ltbp3 NM_ Mdga2 NM_ Mdga2 NM_ Mdga2 NM_ Mdga2 NM_ Mdga2 NM_ Mdga2 NM_ Mdga2 NM_ Mfap4 NM_ Mfap4 NM_ Mpped1 NM_ Mpped1 NM_ Mrpl3 NM_ Mrpl3 NM_

9 Supplementary Material for Lee et al., Page Mtmr7 NM_ Mtmr7 NM_ Mtmr7 NM_ Mtus1 NM_ Mtus1 NM_ Nek11 NM_ Nek11 NM_ Nme6 NM_ Nme6 NM_ Nme6 NM_ Nme6 NM_ Nphp3 NM_ Nphp3 NM_ Nphp3 NM_ Nr4a1 NM_ Nr4a1 NM_ Nr4a1 NM_ Nr4a1 NM_ Nr4a1 NM_ Nrxn3 NM_ Nrxn3 AK Nrxn3 NM_ Oasl1 NM_ Oasl1 NM_ Oma1 NM_ Oma1 NM_ Osbpl1 NM_ Osbpl1 NM_ Osmr NM_ Osmr NM_ Oxsr1 NM_ Oxsr1 NM_ Oxsr1 NM_ Pcnt NM_ Pcnt NM_ Pcnt NM_ Pde2a NM_ Pde2a NM_ Pik3r4 NM_ Pik3r4 NM_ Pik3r4 NM_ Pik3r4 NM_ Pkhd1l1 NM_ Pkhd1l1 NM_ Plk3 NM_ Plk3 NM_ Plk3 NM_ Plk3 NM_ Prkch NM_ Prkch NM_ Prkch NM_ Prkch NM_ Prkch NM_ Prkch NM_ Prkch NM_ Prmt3 NM_

10 Supplementary Material for Lee et al., Page Prmt3 NM_ Prps1l1 NM_ Prps1l1 NM_ Prps1l1 NM_ Prps1l1 NM_ Ptprv NM_ Ptprv NM_ Ptprv NM_ Ptprv NM_ Ptprv NM_ Ptprv NM_ Ptprv NM_ Rbm34 BC Rbm34 BC Ren1 NM_ Ren1 NM_ Ren1 NM_ Ren1 NM_ Rhbg NM_ Rhbg NM_ Rhbg NM_ Rhbg NM_ Rhbg NM_ Rhoj NM_ Rhoj NM_ Ros1 NM_ Ros1 NM_ Ros1 NM_ Rrp9 NM_ Rrp9 NM_ Runx1t1 NM_ Runx1t1 NM_ Sc4mol NM_ Sc4mol NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Scn1a NM_ Serpina3b NM_ Serpina3b NM_ Serpina3b NM_ Serpina3c NM_ Serpina3c NM_

11 Supplementary Material for Lee et al., Page Serpina3c NM_ Serpina3c NM_ Serpina3g NM_ Serpina3g NM_ Serpina3g NM_ Serpina3g NM_ Shisa5 NM_ Shisa5 NM_ Skiv2l2 NM_ Skiv2l2 NM_ Skiv2l2 NM_ Skiv2l2 NM_ Skiv2l2 NM_ Skiv2l2 NM_ Slamf7 AF Slamf7 NM_ Slc25a41 NM_ Slc25a41 NM_ Slc38a1 NM_ Slc38a1 NM_ Slc38a1 NM_ Slc38a1 NM_ Slc38a1 NM_ Slc38a1 BC Slc39a14 NM_ Slc39a14 NM_ Slc47a1 NM_ Slc47a1 NM_ Slc7a2 NM_ Slc7a2 NM_ Slc7a2 NM_ Slc7a2 NM_ Slit2 NM_ Slit2 NM_ Snapc1 NM_ Snapc1 NM_ Snapc1 NM_ Snapc1 NM_ Snapc1 NM_ Snapc1 NM_ Speg NM_ Speg NM_ Spna1 NM_ Spna1 NM_ Spna1 NM_ Ston2 NM_ Ston2 NM_ Ston2 NM_ Ston2 NM_ Ston2 NM_ Ston2 NM_ Susd4 NM_ Susd4 NM_ Tbc1d9 NM_ Tbc1d9 NM_ Tbc1d9 NM_

12 Supplementary Material for Lee et al., Page Tdgf1 NM_ Tdgf1 NM_ Tep1 NM_ Tep1 NM_ Tep1 NM_ Tg NM_ Tg NM_ Tgfbi NM_ Tgfbi NM_ Thbs1 NM_ Thbs1 NM_ Thbs1 NM_ Thbs1 NM_ Thbs1 NM_ Tm7sf2 NM_ Tm7sf2 NM_ Tmem115 NM_ Tmem115 NM_ Tmem115 NM_ Tmem195 NM_ Tmem195 NM_ Trf NM_ Trf NM_ Tshr NM_ Tshr NM_ Tshr NM_ Tshr NM_ Tshr NM_ Tshr NM_ Tshr NM_ Tshr NM_ Tshr NM_ Tshr NM_ Tshr NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_

13 Supplementary Material for Lee et al., Page Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Ttll5 NM_ Tusc4 NM_ Tusc4 NM_ Tusc4 NM_ Vash1 NM_ Vash1 NM_ Vav1 NM_ Vav1 NM_ Vcam1 NM_ Vcam1 NM_ Vcam1 NM_ Vsig8 NM_ Vsig8 NM_ Vsig8 NM_ Xdh NM_ Xdh NM_ Zan NM_ Zan NM_ Zfp276 NM_ Zfp276 NM_ Zfp36 NM_ Zfp36 NM_ Zfp69 NM_ Zfp69 NM_ Zfp7 NM_ Zfp7 NM_ Zmynd1 NM_ Zmynd1 NM_

14 Supplementary Material for Lee et al., Page 14 Table S4 Abundance of transcribed STAMP exons (from chromosome 12) in TM vs WT mice Probe Start (bp) Probe Stop (bp) Probeset ID Alternative splicing Fold change TM vs. WT. Exon Exon id p value E E E E E E E E E E E E E E E E E Ave. fold reduction other than deleted exons 23 and 24: SD:.338 n: 31 SEM:.61

15 Supplementary Material for Lee et al., Page 15 Supplementary Figures Fig. S1: Movie of sperm from wt/wt and tm/tm STAMP in IVF solution and in proximity of oocytes. (See separate file labeled IVF movie.wmv )

16 Supplementary Material for Lee et al., Page 16 Fig. S2: Assignment of missing doublet in axonema of Stamptm/tm mice. Normal 9+2 microtubule axonemal structure of wt/wt sperm is shown in bottom right panel. Missing doublet 4 (in red) in tm/tm sperm was identified as described in Experimental Procedures of main text.

17 Supplementary Material for Lee et al., Page 17 Fig. S3: Comparison of spermatogenesis-relating gene expressions in Stamp tm/tm mice. WT Ht TM Gopc Agfg1 Csnk2a2 Gba2 Tekt2 Tekt Vdac3 Sepp1 Akap4 Spag6 Gapdhs Adcy Catsper1 Casper2 Catsper3 Catsper4 Tnp1 Tnp H1fnt Crem Tbpl1 Papolb Piwil1 Spem The expression levels of 24 spermatogenesis-related genes were measured in the testis of Stamp +/+ (black filled bar), Stamp +/tm (gray filled bar) and Stamp tm/tm (white filled bar) mice (n=3 each genotype) by RT-PCR (see Table 1 for list of primers) after total RNA was reverse-transcribed to first-stand complementary DNA (cdna) using SuperScript III, First- Strand Synthesis SuperMix for qrt-pcr (Invitrogen) according to the manufacturer s directions. Relative transcript abundance was normalized with glyceraldehyde-3-phosphate dehydrogenase and graphed (Average ± SEMs of duplicate values for all samples).

18 Supplementary Material for Lee et al., Page 18 Fig. S4: Histology of photoreceptors and cochlea of wt and Stamptm/tm mice of different ages.

19 Supplementary Material for Lee et al., Page 19 Methods: Retina and cochlea at specified ages were dissected and fixed in freshly prepared 2% paraformaldehyde/3% glutaraldehyde in phosphate buffered saline (PBS) for 24 hr at 4 C with mild rocking. Samples were then washed 3x in PBS at room temperature for 1 min each, followed by dehydration through a graded series of alcohol. Prior to dehydration, cochleas, but not retinas, were decalcified in 1 mm EDTA in PBS for 14 days refreshing the decalcification solution every 2-3 days. Retinal and cochlear tissues were then embedded in methacrylate plastic according to the manufactureʼs directions (Polysciences, Warrington, PA), sectioned at 4 μm using a rotary microtome, stained in aqueous Gillʼs #1 hematoxylin, and coverslipped in Permount. Groups of 2 cochlea from a single mouse, per age, and genotype, were analyzed. Images were captured using Nikon DS-Ri1 camera mounted on a Nikon Eclipse 8i microscope. Description: Histological examination comparing retina and cochlea from Stamp WT and MT mice at 3 and 7 months of age. Retinal and cochlear structures in Stamp MT mice were normal at both ages. No degeneration of photoreceptors or cochlear inner (open arrowheads) or outer (closed arrowheads) hair cells was evident. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inter plaxiform layer; GCL, ganglion cell layer; ohc, outer hair cell; ihc, inner hair cell; tm, tectorial membrane; is; inner sulcus; tc, tunnel of corti.