Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

Abstract

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

Keywords: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 40S ribosomal protein S18; AcN; Amh; CE; CHAPS; COMM domain containing 7; Cdc20; Comm7; DTT; Development; Dmrt1; EGFP; Ef1α; FA; FGF9; Fog2; Foxl2; Gapdh; Gata4; IAA; IL; Ilf2; Ilf3; LDLR; MS; MS222; Mass spectrometry; MeOH; Mif; MudPIT; NI; PTP; RA; RDA; Raldh2; Raldh3; Retsatl; Rps18; SRM; SRY box-containing 9a; SRY box-containing 9b; Selected reaction monitoring (SRM); Sept5a; Sept7b; Sept8a; Sept9b; Sexual differentiation; Sox9a; Sox9b; Spata4; TCEP; Targeted proteomics; Tuba1b; WD repeat 1; Wdr1; Zebrafish; acetonitrile; anti-mullerian hormone; cell division cycle protein 20 homolog; collision energy; days post fertilization; dithiothreithol; doublesex and mab3-related transcription factor 1; dpf; elongation factor 1 alpha; enhanced green fluorescent protein; fibroblast growth factor 9; forkhead box L2; formic acid; friend-of-GATA isoform 2; glyceraldehyde phosphate dehydrogenase; interleukin; interleukin enhancer-binding factor 2; interleukin enhancer-binding factor 3; iodoacetamide; low density lipoprotein receptor; macrophage migration inhibitory factor; mass spectrometry; methanol; multidimensional protein identification technology; normalized intensity; proteotypic peptide; representational difference analysis; retinaldehyde dehydrogenase type 2; retinaldehyde dehydrogenase type 3; retinoic acid; retinol saturase like; selected reaction monitoring; septin 5a; septin 7b; septin 8a; septin 9b; spermatogenesis associated 4; tricaine methane sulfonate; tris(2-carboxyethyl)-phosphine hydrochloride; tubulin alpha 1b; zinc finger transcription factor GATA isoform 4.