New insights into. spermatogenesis.

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Phebe Lawrence
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1 New insights into mammalian spermatogenesis Harvesting the postgenomics bounty

Mitosis Spermatocytes (4n) Germ cells Meiosis Peritubular

2 Spermatogenesis in mammals Interstitial tissue Sertoli Cells Peritubular cells Basal lamina Spermatogonia (2n) Mitosis Spermatocytes (4n) Germ cells Meiosis Peritubular cells Spermatids (n) Spermiogenesis Spermatozoa (n) Seminiferous tubule

3 The Testicular Pilot Proteome Project Establish the repertoire of testicular proteins Identify key proteins in spermatogenesis Explore testicular pathologies (e.g., CIS) Protein markers for clinical applications

4 Differential proteomic analysis of rat spermatogenesis Spermatogonia Diploid Mitotic phase Spermatocytes (Pachytene stage) Tetraploid Meiotic phase Spermatids Haploid Spermiogenesis Identify stage-specific proteins Cytosolic proteins: DIGE Cytosolic proteins: DIGE Membraneous proteins: ICPL Nuclear proteins: ICPL An example of application: towards 3D DIGE

5 Experimental design DIGE minimal labeling Duplex comparisons with Cy2 used as internal standard Standard (A+B) Cy2 Sample A Spermatogonia diploid Spermatocytes tetraploid SPG / SPC / Mix SPT / SPG / Mix SPC / SPT / Mix Cy3 Sample B Cy5 Spermatids haploid SPG / SPC / Mix SPT / SPG / Mix SPC / SPT / Mix Rolland et al., J. Proteome Res. 2007; 6(2):

6 Experiment 1 - Results 359 3(2),5-biphosphate nucleotidase 12,48 16, tropomyosin 3, Unknown 2,85 7,13 2,5 391 Similar to leucine zipper transcription factor 3,28 3, Isocitrate dehydrogenase 3 (NAD+) alpha 3,3 409 Tropomyocin 4 2,81 4, Dimethylarginine dimethylaminohydrolase 1 4,91 4, Aldehyde reductase 1 3,13 4, L-lactate dehydrogenase B 4,01 4, Calpactin I heavy chain 3,85 4, Recombinant Rat Annexin V, Triple mutant 6,73 3, Copper chaperone for superoxide dismustase 3,58 4, Similar to esterase D 2,76 3,3 457 Glyceraldehyde-3-phosphate dehydrogenase 5,18 5,87 Spot n 471 Carbonyl Nom reductase de la protéine Ratio SPC/SPG2,61 Ratio SPT/SPG 2,94Ratio SPT/SPC 4 Oxygen 475 regulated similar to protein apoa-i binding protein 4,98 2,64 4,69 2,64 21 Similar 484 to Similar nuclear to autoantigenic 60S acidic ribosomal sperm protein P2 3,25 7,12 3,36 10,41 43 Similar 492 to Lactate osmotic dehydrogenase stress protein 3, C chain 6,7 12,37 3,3 (1,85) 44 Osmotic 499 stress Triosephosphate protein isomerase 1 9,06 2,79 10,71 2,54 49 Similar 515 to Phosphatidylethanolamine osmotic stress protein binding protein 10,96 (1,85) 17,4 3,12 (1,59) 55 Similar 523 to Protein endoplasmin carboxyl precursor methyltransferase 3,84 8,03 4,39 3,79 91 Thimet 527 oligopeptidase Glutathion S-transferase Yb-2 5,05 10,72 10,92 6,49 (2,16) 94 Thimet 528 ologopeptidase/hypothetical Unnamed protein product protein 3,54 3,68 10,01 2,89 (1,98) 116 DnaK-type 530 Glutathion molecular S-transferase chaperone hst70 Yb-1 subunit (2,12) 979 9, ,78 10, heat 562 shock Fertility 70kD protein 5SP22 2,93 (2,05) 3,28 3, Testis-specific 570 Peroxiredoxin heat shock 1protein-related gene hst7 3,51 4,24 2,7 128 Heat 574 shock ADP-ribosylation protein 1-like factor-like 3 5,15 6,64 22,7 7,46 4, kdq heqt Ubiquitin-conjugating shock protein enzyme E2N (2,38) 2,55 2, Testis-specific 636 heat lipid shock binding protein-related gene hst7 13,91 12,48 11,29 5, DnaK-type 639 Testis molecular lipid binding chaperone protein hst70 2,91 4,63 8,63 6,73 2, metalloendopeptidase 651 Chain D, Tsg101 domain in complex with ubiquitin 3,56 (2,07) 5,68 4, Myo-inositol 698 Chain 1-phosphate D, Tsg101 synthase domain in A1complex with ubiquitin 5,14 6,63 4,34 7, Myo-inositol 1-phosphate synthase A2 5,38 5, HSP60 protein (2,35) Glucose regulated protein, 58 kda (2,05) 3,28 (1,6) 181 Protein disulfide isomerase A3 precursor (1,9) 2,67 (1,4) 225 Tubulin beta 2 / / / id Protein disulfide isomerqse A6 precursor / / / 252 Ribonuclease/angiogenin inhibitor 2, Proliferation related acidic leucine rich protein PA 3,62 3, Aldehyde dehydrogenase family 1, member A1 21,87 19, I20Rik protein 4, Put. Beta-actin (aa ) 7,1 309 Put. Beta-actin (aa ) (2,24) 3,05 35 proteins validated as differentially expressed between the 3 cell types Dilute abundant proteins Amplify detection of discrete proteins Rolland et al., J. Proteome Res. 2007; 6(2):

7 Sample pre-fractionation Objectives: Decomplexify samples prior to labeling Use a technique allowing high standardization % 100 Reversed-phase chromatography 4 sub-proteomes OD (Ar rbitrary units s) % % 41 % 48 % 57 % 75 % I II III IV Fractionation of rat germ cell cytosolic extracts t by RP-HPLC. Cell cytosolic extracts t from spermatogonia, spermatocytes, t and spermatids were fractionated t by RP-HPLC on a Vd Vydac C4 column, as described in the Materials and Methods. Proteins were eluted using a sequential 5 min step-gradient, at final acetonitrile (ACN) concentrations of 41, 48, 57, and 75%, into four chromatographic fractions (fractions I, II, III, and IV) and optical density (OD) was monitored at 210 nm. The fractions, each of 12.5 ml and containing equivalent amounts of protein, were lyophilized and used as the starting material for DIGE in experiment 2. The figure shown here is representative run of cytosolic extract from spermatogonia. Rolland et al., J. Proteome Res. 2007; 6(2):

8 Experiment 2 - Results Spermatogonia diploid Spermatocytes tetraploid rbitrary unit) OD (ar % 30 % 41 % 57 % 48 % 75 % 100 % ACN (% %) Spermatids haploid Fractionation of protein extracts by reversed-phase HPLC 0.0 I II III IV Time (min) 3 cell types x 4 fractions X replicate experiments 0 DIGE analysis No fractionation : 35 differential proteins MW Da ph 3-10 RP-HPLC fractionation : 977 unique differential proteins I II Rolland et al., J. Proteome Res. 2007; 6(2): II I IV

9 Differential proteomic analysis of rat spermatogenesis 977 Differentially expressed proteins with ratio > proteins with differential expression with ratio > expression clades SPT (42) SPG (65) SPG SPC SPT SPC (17)

10 Validation of DIGE results DIGE Western blotting Immunohistochemistry A Annexin V B GAPDH C MCM7 D PTBP2 E Diablo F Grp58 Rolland et al., J. Proteome Res. 2007; 6(2):

11 In situ validation of DIGE results Verify the expression of proteins in situ! No quantitative input needed Exclusion of candidate proteins if expressed by contaminating cells rspt SPG Diablo SPG espt SPG SPC SC espt Grp58 SPG rspt SPG SC SPC

The core meiotic transcriptome in mammals

norvegicus 7071 differentially expressed

clusters 1113 conserved genes covered on

differentially expressed genes displaying

SE SG SC ST TU TT CC SM SC ST TU TT SE SG

12 The core meiotic transcriptome in mammals M. musculus H. sapiens R. norvegicus 7071 differentially expressed mouse genes MITOTIC S OMATIC 4 expression clusters 1113 conserved genes covered on three GeneChips EIOTIC 957 conserved and differentially expressed genes displaying very similar patterns in rodents POST-M M SE SG SC ST TU TT CC SM SC ST TU TT SE SG SC ST TU TT Chalmel et al., Proc Natl Acad Sci USA 2007

13 Proteome versus Transcriptome R. norvegicus gènes genes Annexin V 5269 Differentially expressed GAPDH SE SG SC ST TU TT MCM7 124 proteins (ratio >2.5) 108: proteins and mrna "matching" 16: genes not present yet on arrays 65: identical profile Protein/mRNA 43: different profile 19: known delay between transcription and translation in SPC and SPT PTBP2 Diablo Grp58 Western blot Micro array

14 Spermatogenesis: Transcriptome vs Proteome CLPH mrna Proteins mrna 61 % Correlated profiles SPG SPC SPT SPG SPC SPT Protein SPG SPC SPT SPG SPC SPT Lztfl1 30 % Uncorrelated profiles Delay between transcription and translation mrna SPG SPC SPT SPG SPC SPT SPG SPC SPT 9 % Uncorrelated profiles No explanation Protein SPG SPC SPT SPG SPC SPT

15 Clph, a new testicular-specific gene Major gene expression in post-meiotic germ cells Rat Mouse Human

16 CLPH, a new disordered protein involved in mammalian spermatogenesis Expression features : CLPH protein is specific to the elongating spermatids ax m MP A cy m ax B Calvel et al., J. Proteome Res. 2009; in Press

Biochemical features : CLPH shares common properties with the proteins of the SCPP cluster The mammalian secreted calcium-binding phosphoproteins (SCPPs): High affinity for calcium ions, high level

17 Biochemical features : CLPH shares common properties with the proteins of the SCPP cluster The mammalian secreted calcium-binding phosphoproteins (SCPPs): High affinity for calcium ions, high level of phosphorylation High structural plasticity (presence of disordered regions) CLPH recclph protein is phosphorylated by Casein Kinase 2 in vitro Circular dichroism analysis of CLPH protein shows a high propensity for disordered regions Presence of a conserved EFhand Ca 2+ binding site : CD analysis of Ca 2+ -incubated recclph shows no drastic structural changes Calvel et al., J. Proteome Res. 2009; in Press

18 CLPH is a phosphoprotein CID spectra of the unmodified tryptic peptide eluting at 37.3 min, 271 AKDNPEDPLTDEEPADGVNTWVEK 294 (upper panel) and of its T phosphorylated analog eluting at 38.5 min (lower panel). *, phospho-threonyl fragment ions; **, loss of H 3 PO 4 (-98 Da). Calvel et al., J. Proteome Res. 2009; in Press

Transcriptomics and proteomics differential

Dev. 2006; 73(7):866-877. Rolland et al.

19 Transcriptomics and proteomics differential expression profiles of Minichromosome Maintenance Protein 7 (MCM7) during rat spermatogenesis A SPG SPC SPT C T S P L 3.2 kb 2.4 kb B 1.6 kb D 82 KDa E VII plspc pspc BSPG pspc S ispg espt rspt VI Uncorrelated mrna/protein profile explained pspc III-IV Com et al., Mol. Reprod. Dev. 2006; 73(7): Rolland et al., 2008 In: Molecular Mechanisms in spermatogenesis. CY Cheng Ed. Landes Bioscience, Austin, TX, USA. 2008, Vol 636, pp

20 GO enrichment on 124 proteins with expression ratio > 2,5 Global SPG SPC SPT SPG (64) SPC (17) SPT (42) SPG SPC SPT AMEN software - Chalmelet al., 2006 BMC Bioinformatics

21 Assessing protein enrichment by sample prefractionation Interactions between proteins belonging to the two groups : With and Without fractionation AMEN sotfware: Annotation Mapping Expression and Networks

22 Exploring spermatogenesis: what to do next? Datamining to go beyond expression profile Data mining: Going beyond expression profiles. A) Filtering strategy. Cross-species comparison and tissue profiling identified 80 testisspecific orthologs with conserved expression patterns in mouse, rat and human spermatogenesis. Genes differentially expressed during spermatogenesis were identified for each species. Differentially expressed genes corresponding to orthologs represented on microarrays for all 3 species were identified, using the HomoloGene database. Finally, expression data for 17 mouse somatic tissues downloaded from the GEO web server were used to select testisspecificgenes.se,spg,sc,st,tuandttcorrespondtosertolicell, spermatogonia, pachytene spermatocyte, early spermatid, seminiferous tubule and total testis samples, respectively. SO, MI, MEI and PM correspond to the somatic, mitotic, meiotic and postmeiotic ti expression clusters, respectively. B) Interaction ti network analysis. Data from IntAct, MINT and BioGRID were used to monitor interactions between conserved and testis-specific gene products. Some proteins important for male reproduction were found within these networks and were found to associate with a large number of interacting factors. As not all the interacting factors were detected on microarray analyses, interaction network analysis might help to extend expression data. C) Promoter analysis. Automated multi-step promoter analysis showed mouse testis-specific postmeiotic gene promoter regions to be specifically enriched in the CRE motif. We show here the results for Capza3, a gene also identified as present within the interaction node of conserved and testis-specific genes. Chalmel et al., Ann N Y Acad Sci 2007

23 org

24 Florence Aubry Christine Kervarrec Myriam Guerrois Antoine Rolland Pierre Calvel Michael Pi Primigi Frédéric Chamel Aurélie Lardenois Bertrand Evrard James Moore Emmanuelle Com Régis Lavigne Blandine Charoy Mélanie Lagarrigue Djibril Ousmanou

25 Molecular image of sperm transit in the rat epididymis by MALDI imaging mass spectrometry. A80µmspatial resolution. Peptides specific to maturing spermatozoa or epididymal ducts morphology can be visualized simultaneously. Mass signal overlay of three specific peptides of 5470, 6177 and Da is visualized in different colors.

La protéomique de l éjaculat

12 èmes journées sur l Assistance Médicale à la Procréation de l Hôpital Américain de Paris - 24-25 novembre 2011 La protéomique de l éjaculat Projet Fertichip charles.pineau@rennes.inserm.fr Inserm U1085