Comprehensive proteomic characterization and functional annotation of Siberian sturgeon seminal plasma proteins

Siberian sturgeon (Acipenser baerii) is considered the most valuable species in aquaculture. The uniqueness of sturgeon reproduction implicates the need for studying semen composition at the protein level. Seminal plasma supports and protects the viability, motility and fertilizing capacity of spermatozoa by creating the optimal environment for storage and might be a rich source of biomarkers of reproductive physiology and/or disorders in male fishes. To our knowledge, there are no reports on the in-depth characterization of sturgeon seminal plasma proteins. The present study is the first to describe the in-depth proteomic characterization of Siberian sturgeon seminal plasma using two complementary proteomic approaches: gel-based (2DE and 2D blue native (BN)/SDS–PAGE) and gel-free (LC–MS/MS). LC–MS/MS allowed the identification of 657 proteins and 339 protein spots were identified after 2DE (almost 60% were present in various proteoforms). Combining the two proteomic strategies, we identified 665 proteins with high confidence (false positive rate below 1%), producing the largest catalogue of proteins from sturgeon seminal plasma to date. For the first time, we demonstrated interactions between seminal plasma proteins; four multiprotein complexes (MC1-MC4) were identified after 2D BN/SDS–PAGE, including (MC1) serotransferrin-2 (TF) and fish-egg lectin (FEL); (MC2) serum albumin 2 (ALB) and retinol-binding protein 4 (RBP4); (MC3) ALB and TF; and (MC4) apolipoprotein A-I (APOA1), riboflavin-binding protein (RfBP) and myoglobin (MB). BN/SDS–PAGE also revealed that immunoglobulin and TF formed homomultimeric (dimer, trimer, tetramer and pentamer) complexes in seminal plasma. ALB, TF, beta-Ala-His dipeptidase (CNDP1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), IGH, APOA1, hemopexin (HPX), type-4 ice-structuring protein (AFP4), FEL, RfBP, ovochymase 2 (OVCH2) and glycogen phosphorylase, liver form-like (PYGL) were selected as major seminal plasma proteins. The functional analysis of the identified proteins indicated their involvement mainly in immune system processes and response to stimulus, metabolism, vesicle-mediated transport, proteolysis, and cadherin binding involved in cell–cell adhesion. Moreover, 69 proteins were associated with reproductive processes, including spermatogenesis, fertilization, acrosomal reaction and sperm motility. Comparative proteomic analysis of sturgeon seminal plasma showed common proteins between fish and humans and 172 proteins specific for sturgeons, which may reflect the specificity of sturgeon reproduction. To conclude, this integrative proteomic view offers a deeper insight into the physiological function of seminal plasma and processes occurring in the sturgeon reproductive tract. The availability of a catalogue of sturgeon seminal plasma proteins opens the way to future research into development of new biomarkers and possible application in the improvement of methods of controlled reproduction. The mass spectrometry proteomics data have been deposited into ProteomeXchange with the identifier PXD035351.