High-Throughput Discovery of Germ-Cell-Specific Genes

ABSTRACT

As a specialized cell population that plays the unique role of transmitting genetic information to subsequent generations, germ cells have been intensively studied to unravel their unique physiology from the specification of primordial germ cells to the fateful reunion of gametes during fertilization. For their differential expression, germ-cell-specific genes are the keys to understanding these unique features. In the last decade, the emerging methodologies designed for large-scale and high-throughput analysis have created an ever-increasing amount of data. Among these methodologies, expressed sequence tag libraries, serial analysis of gene expression, and microarrays provide valuable expression data that can be further analyzed. Using the mouse as a model system, we describe a strategy starting from the quick identification of germ-cell-specific genes using public domain expression data to the functional characterization of the identified genes using targeted gene disruption. This strategy should accelerate the process to fill in the missing pieces of the germ cell physiology puzzle and the construction of genetic networks to help us to understand the etiology of infertility. Furthermore, these identified germ-cell-specific genes may lead to the development of new contraceptives targeted specifically to germ cells.

Germ-cell-specific genes - EST - SAGE - microarray - knockout mice

REFERENCES

• 1 Zhao G Q, Garbers D L. Male germ cell specification and differentiation.  Dev Cell. 2002;  2 537-547
• 2 Matova N, Cooley L. Comparative aspects of animal oogenesis.  Dev Biol. 2001;  231 291-320
• 3 Xu E Y, Lee D F, Klebes A, Turek P J, Kornberg T B, Reijo Pera R A. Human BOULE gene rescues meiotic defects in infertile flies.  Hum Mol Genet. 2003;  12 169-175
• 4 van der Weyden L, Adams D J, Bradley A. Tools for targeted manipulation of the mouse genome.  Physiol Genomics. 2002;  11 133-164
• 5 Committee on New Frontiers in Contraceptive Research .Board on Health Sciences Policy . Target Discovery and Validation. In: Nass SJ, Strauss JF 3rd New Frontiers in Contraceptive Research: A Blueprint for Action. Washington, DC; The National Academies Press 2004: 27-77
• 6 Lessard C, Pendola J K, Hartford S A, Schimenti J C, Handel M A, Eppig J J. New mouse genetic models for human contraceptive development.  Cytogenet Genome Res. 2004;  105 222-227
• 7 Greenbaum D, Luscombe N M, Jansen R, Qian J, Gerstein M. Interrelating different types of genomic data, from proteome to secretome: ‘oming in on function.  Genome Res. 2001;  11 1463-1468
• 8 Galperin M Y. The Molecular Biology Database Collection: 2004 update.  Nucleic Acids Res. 2004;  32(database issue) D3-D22
• 9 Leo C P, Hsu S Y, Hsueh A J. Hormonal genomics.  Endocr Rev. 2002;  23 369-381
• 10 Liang M, Cowley Jr A W, Greene A S. High throughput gene expression profiling: a molecular approach to integrative physiology.  J Physiol. 2004;  554(pt 1) 22-30
• 11 Hughes T R, Marton M J, Jones A R et al.. Functional discovery via a compendium of expression profiles.  Cell. 2000;  102 109-126
• 12 Shima J E, McLean D J, McCarrey J R, Griswold M D. The murine testicular transcriptome: characterizing gene expression in the testis during the progression of spermatogenesis.  Biol Reprod. 2004;  71 319-330
• 13 Maratou K, Forster T, Costa Y et al.. Expression profiling of the developing testis in wild-type and Dazl knockout mice.  Mol Reprod Dev. 2004;  67 26-54
• 14 Burns K H, Owens G E, Ogbonna S C, Nilson J H, Matzuk M M. Expression profiling analyses of gonadotropin responses and tumor development in the absence of inhibins.  Endocrinology. 2003;  144 4492-4507
• 15 Ellis P J, Furlong R A, Wilson A et al.. Modulation of the mouse testis transcriptome during postnatal development and in selected models of male infertility.  Mol Hum Reprod. 2004;  10 271-281
• 16 Matzuk M M, Lamb D J. Genetic dissection of mammalian fertility pathways.  Nat Med. 2002;  8(suppl) S33-S40
• 17 Cooke H J, Saunders P T. Mouse models of male infertility.  Nat Rev Genet. 2002;  3 790-801
• 18 de Rooij D G, de Boer P. Specific arrests of spermatogenesis in genetically modified and mutant mice.  Cytogenet Genome Res. 2003;  103 267-276
• 19 Rothstein J L, Johnson D, DeLoia J A, Skowronski J, Solter D, Knowles B. Gene expression during preimplantation mouse development.  Genes Dev. 1992;  6 1190-1201
• 20 Rothstein J L, Johnson D, Jessee J et al.. Construction of primary and subtracted cDNA libraries from early embryos.  Methods Enzymol. 1993;  225 587-610
• 21 McCarrey J R, O'Brien D A, Skinner M K. Construction and preliminary characterization of a series of mouse and rat testis cDNA libraries.  J Androl. 1999;  20 635-639
• 22 Rajkovic A, Yan C, Klysik M, Matzuk M. Discovery of germ cell-specific transcripts by expressed sequence tag database analysis.  Fertil Steril. 2001;  76 550-554
• 23 Yan W, Rajkovic A, Viveiros M M, Burns K H, Eppig J J, Matzuk M M. Identification of Gasz, an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper.  Mol Endocrinol. 2002;  16 1168-1184
• 24 Yan W, Ma L, Burns K H, Matzuk M M. HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis.  Proc Natl Acad Sci USA. 2003;  100 10546-10551
• 25 Yan W, Ma L, Burns K H, Matzuk M M. Haploinsufficiency of kelch-like protein homolog 10 causes infertility in male mice.  Proc Natl Acad Sci USA. 2004;  101 7793-7798
• 26 Suzumori N, Yan C, Matzuk M M, Rajkovic A. Nobox is a homeobox-encoding gene preferentially expressed in primordial and growing oocytes.  Mech Dev. 2002;  111 137-141
• 27 Rajkovic A, Lee J H, Yan C, Matzuk M M. The ret finger protein-like 4 gene, Rfpl4, encodes a putative E3 ubiquitin-protein ligase expressed in adult germ cells.  Mech Dev. 2002;  112 173-177
• 28 Roy A, Yan W, Burns K H, Matzuk M M. Tektin3 encodes an evolutionarily conserved putative testicular microtubules-related protein expressed preferentially in male germ cells.  Mol Reprod Dev. 2004;  67 295-302
• 29 Yan W, Burns K H, Ma L, Matzuk M M. Identification of Zfp393, a germ cell-specific gene encoding a novel zinc finger protein.  Mech Dev. 2002;  118 233-239
• 30 Velculescu V E, Zhang L, Vogelstein B, Kinzler K W. Serial analysis of gene expression.  Science. 1995;  270 484-487
• 31 Ko M S. Embryogenomics: developmental biology meets genomics.  Trends Biotechnol. 2001;  19 511-518
• 32 Schlecht U, Primig M. Mining meiosis and gametogenesis with DNA microarrays.  Reproduction. 2003;  125 447-456
• 33 Boguski M S, Lowe T M, Tolstoshev C M. dbEST-database for “expressed sequence tags”.  Nat Genet. 1993;  4 332-333
• 34 Evans S J, Datson N A, Kabbaj M et al.. Evaluation of Affymetrix Gene Chip sensitivity in rat hippocampal tissue using SAGE analysis. Serial analysis of gene expression.  Eur J Neurosci. 2002;  16 409-413
• 35 Takahashi N, Ko M S. Toward a whole cDNA catalog: construction of an equalized cDNA library from mouse embryos.  Genomics. 1994;  23 202-210
• 36 Stanton J L, Green D P. Meta-analysis of gene expression in mouse preimplantation embryo development.  Mol Hum Reprod. 2001;  7 545-552
• 37 Sharov A A, Piao Y, Matoba R et al.. Transcriptome analysis of mouse stem cells and early embryos.  PLoS Biol. 2003;  1 E74
• 38 Virlon B, Cheval L, Buhler J M, Billon E, Doucet A, Elalouf J M. Serial microanalysis of renal transcriptomes.  Proc Natl Acad Sci USA. 1999;  96 15286-15291
• 39 St Croix B, Rago C, Velculescu V et al.. Genes expressed in human tumor endothelium.  Science. 2000;  289 1197-1202
• 40 Vilain C, Vassart G. Small amplified RNA-SAGE.  Methods Mol Biol. 2004;  258 135-152
• 41 Neilson L, Andalibi A, Kang D et al.. Molecular phenotype of the human oocyte by PCR-SAGE.  Genomics. 2000;  63 13-24
• 42 Yao J, Chiba T, Sakai J et al.. Mouse testis transcriptome revealed using serial analysis of gene expression.  Mamm Genome. 2004;  15 433-451
• 43 Akmaev V R, Wang C J. Correction of sequence-based artifacts in serial analysis of gene expression.  Bioinformatics. 2004;  20 1254-1263
• 44 Saha S, Sparks A B, Rago C et al.. Using the transcriptome to annotate the genome.  Nat Biotechnol. 2002;  20 508-512
• 45 Schena M, Shalon D, Davis R W, Brown P O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray.  Science. 1995;  270 467-470
• 46 Lockhart D J, Dong H, Byrne M C et al.. Expression monitoring by hybridization to high-density oligonucleotide arrays.  Nat Biotechnol. 1996;  14 1675-1680
• 47 Brazma A, Parkinson H, Sarkans U et al.. ArrayExpress-a public repository for microarray gene expression data at the EBI.  Nucleic Acids Res. 2003;  31 68-71
• 48 Edgar R, Domrachev M, Lash A E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository.  Nucleic Acids Res. 2002;  30 207-210
• 49 Brazma A, Hingamp P, Quackenbush J et al.. Minimum information about a microarray experiment (MIAME)-toward standards for microarray data.  Nat Genet. 2001;  29 365-371
• 50 Schulze A, Downward J. Navigating gene expression using microarrays-a technology review.  Nat Cell Biol. 2001;  3 E190-E195
• 51 Benes V, Muckenthaler M. Standardization of protocols in cDNA microarray analysis.  Trends Biochem Sci. 2003;  28 244-249
• 52 Lipshutz R J, Fodor S P, Gingeras T R, Lockhart D J. High density synthetic oligonucleotide arrays.  Nat Genet. 1999;  21 20-24
• 53 Chen Z J, Gate L, Davis W, Ile K E, Tew K D. Sensitivity and fidelity of DNA microarray improved with integration of Amplified Differential Gene Expression (ADGE).  BMC Genomics. 2003;  4 28
• 54 Pang A L, Taylor H C, Johnson W et al.. Identification of differentially expressed genes in mouse spermatogenesis.  J Androl. 2003;  24 899-911
• 55 Yu Z, Guo R, Ge Y et al.. Gene expression profiles in different stages of mouse spermatogenic cells during spermatogenesis.  Biol Reprod. 2003;  69 37-47
• 56 Schlecht U, Demougin P, Koch R et al.. Expression profiling of mammalian male meiosis and gametogenesis identifies novel candidate genes for roles in the regulation of fertility.  Mol Biol Cell. 2004;  15 1031-1043
• 57 Rossi P, Dolci S, Sette C et al.. Analysis of the gene expression profile of mouse male meiotic germ cells.  Gene Expr Patterns. 2004;  4 267-281
• 58 Hamatani T, Carter M G, Sharov A A, Ko M S. Dynamics of global gene expression changes during mouse preimplantation development.  Dev Cell. 2004;  6 117-131
• 59 Wang Q T, Piotrowska K, Ciemerych M A et al.. A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo.  Dev Cell. 2004;  6 133-144
• 60 Schultz N, Hamra F K, Garbers D L. A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets.  Proc Natl Acad Sci USA. 2003;  100 12201-12206
• 61 Miller D, Tang P Z, Skinner C, Lilford R. Differential RNA fingerprinting as a tool in the analysis of spermatozoal gene expression.  Hum Reprod. 1994;  9 864-869
• 62 Ostermeier G C, Dix D J, Miller D, Khatri P, Krawetz S A. Spermatozoal RNA profiles of normal fertile men.  Lancet. 2002;  360 772-777
• 63 Wang H, Zhou Z, Xu M et al.. A spermatogenesis-related gene expression profile in human spermatozoa and its potential clinical applications.  J Mol Med. 2004;  82 317-324
• 64 Miki R, Kadota K, Bono H et al.. Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays.  Proc Natl Acad Sci USA. 2001;  98 2199-2204
• 65 Bono H, Kasukawa T, Hayashizaki Y, Okazaki Y. READ: RIKEN Expression Array Database.  Nucleic Acids Res. 2002;  30 211-213
• 66 Su A I, Cooke M P, Ching K A et al.. Large-scale analysis of the human and mouse transcriptomes.  Proc Natl Acad Sci USA. 2002;  99 4465-4470
• 67 Yoshimizu T, Sugiyama N, De Felice M et al.. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice.  Dev Growth Differ. 1999;  41 675-684
• 68 Ohbo K, Yoshida S, Ohmura M et al.. Identification and characterization of stem cells in prepubertal spermatogenesis in mice.  Dev Biol. 2003;  258 209-225
• 69 Diehn M, Sherlock G, Binkley G et al.. SOURCE: a unified genomic resource of functional annotations, ontologies, and gene expression data.  Nucleic Acids Res. 2003;  31 219-223
• 70 Kent W J, Sugnet C W, Furey T S et al.. The human genome browser at UCSC.  Genome Res. 2002;  12 996-1006
• 71 Karolchik D, Baertsch R, Diekhans M et al.. The UCSC Genome Browser Database.  Nucleic Acids Res. 2003;  31 51-54
• 72 Dube J L, Wang P, Elvin J, Lyons K M, Celeste A J, Matzuk M M. The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes.  Mol Endocrinol. 1998;  12 1809-1817
• 73 Yan W, Ma L, Zilinski C A, Matzuk M M. Identification and characterization of evolutionarily conserved pufferfish, zebrafish, and frog orthologs of GASZ.  Biol Reprod. 2004;  70 1619-1625
• 74 Burns K H, Viveiros M M, Ren Y et al.. Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos.  Science. 2003;  300 633-636
• 75 Wu M H, Rajkovic A, Burns K H, Yan W, Lin Y N, Matzuk M M. Sequence and expression of testis-expressed gene 14 (Tex14): a gene encoding a protein kinase preferentially expressed during spermatogenesis.  Gene Expr Patterns. 2003;  3 231-236
• 76 Wu X, Viveiros M M, Eppig J J, Bai Y, Fitzpatrick S L, Matzuk M M. Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for the oocyte-to-embryo transition.  Nat Genet. 2003;  33 187-191
• 77 Wu X, Wang P, Brown C A, Zilinski C A, Matzuk M M. Zygote arrest 1 (Zar1) is an evolutionarily conserved gene expressed in vertebrate ovaries.  Biol Reprod. 2003;  69 861-867
• 78 Nagy A. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press 2003
• 79 Woltjen K, Bain G, Rancourt D E. Retro-recombination screening of a mouse embryonic stem cell genomic library.  Nucleic Acids Res. 2000;  28 E41
• 80 Zhang P, Li M Z, Elledge S J. Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step.  Nat Genet. 2002;  30 31-39
• 81 Valenzuela D M, Murphy A J, Frendewey D et al.. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.  Nat Biotechnol. 2003;  21 652-659
• 82 Adams D J, Biggs P J, Cox T et al.. Mutagenic Insertion and Chromosome Engineering Resource (MICER).  Nat Genet. 2004;  36 867-871
• 83 Skarnes W C, von Melchner H, Wurst W et al.. A public gene trap resource for mouse functional genomics.  Nat Genet. 2004;  36 543-544
• 84 D'Haeseleer P, Liang S, Somogyi R. Genetic network inference: from co-expression clustering to reverse engineering.  Bioinformatics. 2000;  16 707-726
• 85 Pe'er D, Regev A, Elidan G, Friedman N. Inferring subnetworks from perturbed expression profiles.  Bioinformatics. 2001;  17(suppl 1) S215-S224
• 86 Slonim D K. From patterns to pathways: gene expression data analysis comes of age.  Nat Genet. 2002;  32(suppl) 502-508
• 87 Jansen R, Yu H, Greenbaum D et al.. A Bayesian networks approach for predicting protein-protein interactions from genomic data.  Science. 2003;  302 449-453
• 88 Marcotte E M, Pellegrini M, Thompson M J, Yeates T O, Eisenberg D. A combined algorithm for genome-wide prediction of protein function.  Nature. 1999;  402 83-86
• 89 Vidal M. A biological atlas of functional maps.  Cell. 2001;  104 333-339
• 90 Noordewier M O, Warren P V. Gene expression microarrays and the integration of biological knowledge.  Trends Biotechnol. 2001;  19 412-415
• 91 Brazhnik P, de la Fuente A, Mendes P. Gene networks: how to put the function in genomics.  Trends Biotechnol. 2002;  20 467-472
• 92 Fraser A G, Marcotte E M. A probabilistic view of gene function.  Nat Genet. 2004;  36 559-564
• 93 Hwang S Y, Oh B, Knowles B B, Solter D, Lee J S. Expression of genes involved in mammalian meiosis during the transition from egg to embryo.  Mol Reprod Dev. 2001;  59 144-158
• 94 Primig M, Wiederkehr C, Basavaraj R et al.. GermOnline, a new cross-species community annotation database on germ-line development and gametogenesis.  Nat Genet. 2003;  35 291-292
• 95 Wiederkehr C, Basavaraj R, Sarrauste de Menthiere C et al.. GermOnline, a cross-species community knowledgebase on germ cell differentiation.  Nucleic Acids Res. 2004;  32(database issue) D560-D567
• 96 Leo C P, Vitt U A, Hsueh A J. The Ovarian Kaleidoscope database: an online resource for the ovarian research community.  Endocrinology. 2000;  141 3052-3054
• 97 Ben-Shlomo I, Vitt U A, Hsueh A J. Perspective: the ovarian kaleidoscope database-II. Functional genomic analysis of an organ-specific database.  Endocrinology. 2002;  143 2041-2044
• 98 Ward J O, Reinholdt L G, Hartford S A et al.. Toward the genetics of mammalian reproduction: induction and mapping of gametogenesis mutants in mice.  Biol Reprod. 2003;  69 1615-1625
• 99 Kent Hamra F, Schultz N, Chapman K M et al.. Defining the spermatogonial stem cell.  Dev Biol. 2004;  269 393-410
• 100 Rinn J L, Rozowsky J S, Laurenzi I J et al.. Major molecular differences between mammalian sexes are involved in drug metabolism and renal function.  Dev Cell. 2004;  6 791-800

APPENDIX A

Compile germ-cell-specific gene candidate list using McCarrey Eddy round spermatid EST library as example.

1. Go to CGAP cDNA DGED (http://cgap.nci.nih.gov/Tissues/GXS).

2. Select [Organism-Mus musculus], [Library group-All EST libraries], [Mininum number-200], [Pool A (Tissue type-include testis; Tissue histology-normal)], [Pool B (Tissue type-exclude testis; Tissue histology-normal)], and submit query.

3. In pool A, unselect all libraries except [McCarrey Eddy round spermatid]; in pool B, use the browser command, Find, to locate and unselect [NIH_MGC_169], [NIA Mouse 15k cDNA clone set], and all libraries containing pooled tissues. Increase p value in statistical parameter to 0.7 to allow identification of singleton during in silico subtraction. Submit query to start subtraction.

4. When the result returns, select the [Full Text] link to access the whole list

5. Copy the result and convert it to a table using a text editor. By setting a cutoff at [1 sequence in pool A versus 2 sequences in pool B], a list of GCSGs from round spermatids is ready for further inspection.

6. Go to GNF SymAtlas (http://symatlas.gnf.org/SymAtlas/) and paste either UniGene cluster IDs or representative transcript accessions for batch query.

7. Available GNF multitissue microarray expression profiles can be browsed to validate the germ-cell-specific or gonad-specific expression patterns.

8. More expression profiles can be searched in NCBI GEO database (Table [3]). Using Hils1 as example, search term can be limited with Boolean qualifiers as [(Hils1 AND GSE640) OR (Hils1 AND GDS565)]. Genes that satisfy most criteria serve as good GCSG candidates for further analysis.

   Notes: (A) Human brain medulla library (Lib.9725) is contaminated with testis cDNAs and should be avoided for in silico subtraction of human data. (B) The human ovary tissue samples used by GNF were from two postmenopausal females (http://symatlas.gnf.org/GeneAtlasv2_sample_info.html); thus they are not applicable to the analysis of female germ-cell-specific genes since few germ cells remain. (C) The human tissue sample preparations, Testis Leydig Cell and Testis Interstitial, are contaminated with germ cells, judging by the high expression level of germ-cell-specific genes, such as protamines.