Etude du sécrétome germinal testiculaire in situ par Omique combinatoire.

International audienc

An integrative omics strategy to assess the germ cell secretome and to decipher Sertoli-germ cell crosstalk in the mammalian testis

International audienceMammalian spermatogenesis, which takes place in complex testicular structures called seminiferous tubules, is a highly specialized process controlled by the integration of juxtacrine, paracrine and endocrine information. Within the seminiferous tubules, the germ cells and Sertoli cells are surrounded by testicular fluid (TF), which probably contains most of the secreted proteins involved in crosstalk between these cells. It has already been established that germ cells can modulate somatic Sertoli cell function through the secretion of diffusible factors. We studied the germ cell secretome, which was previously considered inaccessible, by analyzing the TF collected by microsurgery in an "integrative omics" strategy combining proteomics, transcriptomics, genomics and interactomics data. This approach identified a set of proteins preferentially secreted by Sertoli cells or germ cells. An interaction network analysis revealed complex, interlaced cell-cell dialog between the secretome and membranome of seminiferous cells, mediated via the TF. We then focused on germ cell-secreted candidate proteins, and we identified several potential interacting partners located on the surface of Sertoli cells. Two interactions, APOH/CDC42 and APP/NGFR, were validated in situ, in a proximity ligation assay (PLA). Our results provide new insight into the crosstalk between germ cells and Sertoli cells occurring during spermatogenesis. Our findings also demonstrate that this "integrative omics" strategy is powerful enough for data mining and highlighting meaningful cell-cell communication events between different types of cells in a complex tissue, via a biological fluid. This integrative strategy could be applied more widely, to gain access to secretomes that have proved difficult to study whilst avoiding the limitations of in vitro culture

Details of datasets and the methods used to reconstruct dialog between Sertoli and germ cells.

<p>(A) Conversion of the rat and ram UniProt identifiers (UPIDs) into mouse Entrez Gene identifiers (EGIDs). (B) Definition of the germ cell and Sertoli cell transcriptomes (PSIDs: Probeset identifiers) (C) Selection of loci (mouse Entrez gene IDs) encoding secreted or potentially secreted proteins (RSIDs: RefSeq identifiers UPIDs: UniProt identifiers; ENSIDs: Ensembl identifiers) (D) Selection of genes encoding proteins associated with a “plasma membrane” and/or “cell surface” location. (E) Selection of proteins secreted by one type of cell (germ or Sertoli cell) and interacting with membrane proteins of the other type of cell from BioGRID, HPRD, IntAct, MINT and NCBI databases.</p

<i>In situ</i> detection of APOH/CDC42 and APP/NGFR interactions in the rat testis by Duolink PLA.

<p>(A, E) Abundant PLA signals (red dots) were detected in the seminiferous tubules of rat testis when specific primary antibodies were used, reflecting the close proximity of APOH/CDC42 or APP/NGFR close proximity. (B, C, F, G) Negative controls, with only one primary antibody for the targeted protein-protein interaction. (D, H) Quantification of PLA signals in testis sections for APOH/CDC42 (** Student's <i>t</i>-test <i>p</i> = 0.0016) or for APP/NGFR (Student's <i>t</i>-test <i>p</i> = 0.16). Scale bars = 50 µm. A nonspecific background nuclear signal was observed for a few tubule sections (see B).</p

Experimental design and integrative omics workflow.

<p>A schematic diagram of the strategy used to access germ cell and Sertoli cell secretomes and to highlight potential protein-protein interactions. BTB: blood testis barrier; GC: germ cells; LC: Leydig cells; PC: peritubular cells; SC: Sertoli cells; Spg: spermatogonia; pSpc: pachytene spermatocytes; rSpt: round spermatids; eSPT: elongated spermatids.</p

Integration of “omics” data to establish a network of molecular interactions between germ cells and Sertoli cells mediated by the TF.

<p>This integrated network focuses on proteins produced and secreted by germ cells (GCs) or Sertoli cells (SCs) and interacting with membrane proteins of the other type of cell. Nodes symbolizing GC-secreted and Sertoli cell-secreted factors are indicated in light green and purple, respectively, whereas GC-membrane and Sertoli cell-membrane proteins are represented by red and light blue nodes, respectively.</p