Cell Type-Specific Expression of Testis Elevated Genes Based On
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Cell Type-Specific Expression of Testis Elevated Genes Based on Transcriptomics and Antibody-Based Proteomics Charles Pineau, Feria Hikmet, Cheng Zhang, Per Oksvold, Shuqi Chen, Linn Fagerberg, Mathias Uhlén, Cecilia Lindskog To cite this version: Charles Pineau, Feria Hikmet, Cheng Zhang, Per Oksvold, Shuqi Chen, et al.. Cell Type- Specific Expression of Testis Elevated Genes Based on Transcriptomics and Antibody-Based Pro- teomics. Journal of Proteome Research, American Chemical Society, 2019, 18 (12), pp.4215-4230. 10.1021/acs.jproteome.9b00351. hal-02280776 HAL Id: hal-02280776 https://hal-univ-rennes1.archives-ouvertes.fr/hal-02280776 Submitted on 28 Nov 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Page 1 of 53 1 2 3 1 Cell type-specific expression of testis elevated genes based on 4 5 2 transcriptomics and antibody-based proteomics 6 7 3 8 4 9 5 Charles Pineau1,2, Feria Hikmet3, Cheng Zhang4, Per Oksvold4, Shuqi Chen4, Linn Fagerberg4, 10 6 Mathias Uhlén4, Cecilia Lindskog3* 11 7 12 13 8 1 Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) 14 9 - UMR_S 1085, 35042 Rennes cedex, France 15 10 16 11 2 Protim, Univ Rennes, 35042 Rennes cedex, France 17 18 12 19 13 3 Uppsala University, Department of Immunology, Genetics and Pathology, Rudbeck 20 14 Laboratory, 75185 Uppsala, Sweden 21 15 22 16 4 Science for Life Laboratory, School of engineering Sciences in Chemistry, Biotechnology and 23 24 17 health, KTH - Royal Institute of Technology, 17121 Stockholm, Sweden 25 18 26 19 27 20 28 21 *Correspondence to: 29 30 22 Cecilia Lindskog 31 23 Department of Immunology, Genetics and Pathology 32 24 Rudbeck Laboratory manuscript 33 25 Uppsala University 34 26 751 85 Uppsala, Sweden. 35 36 27 E-mail: [email protected] 37 28 38 29 39 40 41 30 42 43 44 31 45 46 47 32 48 33 49 50 34 51 35 Accepted 52 53 54 55 56 57 58 1 59 60 Page 2 of 53 1 2 3 1 ABSTRACT 4 5 2 One of the most complex organs in the human body is the testis, where spermatogenesis takes 6 7 3 place. This physiological process involves thousands of genes and proteins that are activated and 8 9 4 repressed, making testis the organ with the highest number of tissue-specific genes. However, 10 11 5 the function of a large proportion of the corresponding proteins remains unknown and testis 12 13 14 6 harbors many missing proteins (MPs), defined as products of protein-coding genes that lack 15 16 7 experimental mass spectrometry evidence. Here, an integrated omics approach was used for 17 18 8 exploring the cell type-specific protein expression of genes with an elevated expression in testis. 19 20 9 By combining genome-wide transcriptomics analysis with immunohistochemistry, more than 500 21 22 10 proteins with distinct testicular protein expression patterns were identified, and these were 23 24 11 selected for in-depth characterization of their in situ expression in eight different testicular cell 25 26 12 types. The cell type-specific protein expression patterns allowed us to identify six distinct clusters 27 28 13 of expression at different stages of spermatogenesis. The analysis highlighted numerous poorly 29 30 14 characterized proteins in each of these clusters whose expression overlapped with that of known 31 32 15 proteins involved in spermatogenesis, including manuscript88 proteins with an unknown function and 60 33 34 35 16 proteins that previously have been classified as MPs. Furthermore, we were able to characterize 36 37 17 the in situ distribution of several proteins that previously lacked spatial information and cell type- 38 39 18 specific expression within the testis. The testis elevated expression levels both at the RNA and 40 41 19 protein level suggest that these proteins are related to testis-specific functions. In summary, the 42 43 20 study demonstrates the power of combining genome-wide transcriptomics analysis with antibody- 44 45 21 based protein profiling to explore the cell type-specific expression of both well-known proteins 46 47 22 and MPs. The analyzed proteins constitute important targets for further testis-specific research in 48 49 23 male reproductive disorders. 50 51 Accepted 24 52 53 54 25 Key words: testis, reproduction, spermatogenesis, antibody-based proteomics, missing 55 26 proteins, protein evidence, immunohistochemistry, transcriptomics 56 57 58 2 59 60 Page 3 of 53 1 2 3 1 4 5 2 6 7 3 8 9 10 4 11 12 13 5 14 15 16 17 6 18 19 20 21 7 22 23 24 25 26 27 28 29 30 31 32 manuscript 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Accepted 52 53 54 55 56 57 58 3 59 60 Page 4 of 53 1 2 3 1 INTRODUCTION 4 5 6 2 Testis is one of the most complex organs in the mammalian body. It is divided into two 7 8 3 compartments: the seminiferous tubules and the interstitium. Seminiferous tubules are the site of 9 10 4 spermatogenesis and account for 60 to 80% of total testis volume in mammals. The seminiferous 11 12 13 5 tubules consist of germ cells at different stages of their development, interspersed with Sertoli 14 15 6 cells that constitute the seminiferous epithelium. Sertoli cells are known to have nursing properties 16 17 7 and to transduce hormonal signals, thus playing a central role in the control of spermatogenesis. 18 19 8 The seminiferous tubules are bounded by a basal membrane containing peritubular myoid cells 20 21 9 that provide structural integrity and take part in regulation of spermatogenesis and testicular 22 23 10 function. The interstitium, interspersed between the seminiferous tubules, is a connective tissue 24 25 11 matrix that contains blood and lymph vessels, Leydig cells, macrophages and occasionally other 26 27 12 cells (i.e., fibroblasts, lymphocytes and mast cells). The primary function of Leydig cells is the 28 29 13 production of androgens, e.g. testosterone which is crucial in development of male reproductive 30 31 14 tissues, in promotion of secondary sexual characteristics and for supporting spermatogenesis. 32 manuscript 33 34 15 Spermatogenesis is classically divided into three phases: 1) a proliferative mitotic phase in which 35 36 16 primitive germ cells, or spermatogonia, replenish their stock and undergo a series of mitotic 37 38 17 divisions; 2) a meiotic phase during which genetic recombination occurs in primary diploid 39 40 18 spermatocytes (preleptotene spermatocytes), which divide twice to produce haploid spermatids; 41 42 19 and 3) the final phase called spermiogenesis, in which spermatids morphologically transform into 43 44 20 spermatozoa. Spermatogenesis lasts 74 days in humans, controlled by juxtacrine, paracrine and 45 46 21 endocrine factors and conditioned by the successive activation and/or repression of thousands of 47 48 22 genes and proteins 1 49 50 23 51 Accepted 52 53 24 Previous studies based on deep RNA sequencing (RNA-Seq) have shown that testis has by far 54 2-4 55 25 the highest number of tissue-specific genes , with more than 2,000 genes showing some degree 56 57 58 4 59 60 Page 5 of 53 1 2 3 1 of elevated expression compared to other organs. One could speculate that this considerably high 4 5 2 number of testis elevated genes is due to the specialized functions and events occurring during 6 7 3 spermatogenesis and sperm maturation. The function and cell type-specific expression of the 8 9 10 4 corresponding proteins elevated in testis are however largely unknown, and although the different 11 12 5 steps of sperm maturation are well defined, all genes involved in the process are yet to be 13 14 6 characterized. “Missing proteins” (MPs) are defined as proteins that lack sufficient experimental 15 16 7 evidence of existence at the protein level. The Human Proteome Project (HPP) 5-6 is an initiative 17 18 8 aiming to define the human proteome. Together with its primary knowledgebase neXtProt 7, HPP 19 20 9 has set up criteria ranking proteins in a 1-5 tier system according to evidence of protein existence 21 22 10 (PE), suggesting that approximately 11% of the human proteins are considered to be MPs 8. In 23 24 11 the quest for identifying MPs, testis has been suggested as one of the organs harboring a 25 26 12 significant number of such proteins 9-11 and most specifically the post-meiotic germ cell lineage 27 28 13 including mature spermatozoa 10-12. 29 30 31 14 32 15 Antibody-based proteomics provides an advantageousmanuscript strategy for studying the spatial 33 34 35 16 distribution of proteins. The Human Protein Atlas project 4, 13 is a large-scale project with the aim 36 37 17 to map the entire human proteome using an integration of various omics technologies, including 38 39 18 immunohistochemistry and RNA-Seq. The publicly available database (www.proteinatlas.org) 40 41 19 contains more than 10 million high-resolution images showing the in situ localization of proteins 42 43 20 in a large set of human organs, tissues, cells and organelles, including samples of normal adult 44 45 21 testis.