Variability in the protein profiles in spermatozoa of two sturgeon species

Abstract

Conventional sperm analysis (i.e., motility and fertility) has been used to evaluate sperm quality. Understanding the quality of sperm on the molecular level in the sturgeons, Acipenser baerii and A. schrenckii, is essential for the improvement of the conservation of genetic resources and farming performance. In this study, we used the iTRAQ proteomics approach to perform proteomic profiling of spermatozoa associated with sperm quality in sturgeons (Data are available via ProteomeXchange with identifier PXD006108). The results showed 291 and 359 differentially expressed proteins in A. baerii and A. schrenckii, respectively, of which 72 were common to both species and all were upregulated in high quality compared with low quality samples. The differentially expressed proteins were mainly categorized into the generation of precursor metabolites and energy and oxidation, and they were localized to the mitochondria. Three distinguishing pathways, Arginine and proline metabolism, Pyruvate metabolism and the Citrate cycle (TCA cycle) were found to play an important role in energy metabolism, and some substrates could be used in the sperm medium for storage and cryopreservation. The quantity levels of two proteins, CKMT1 and LDHB, were verified by western blot analysis. Moreover, other potential biomarkers involved in oxidation reduction, ubiquitin-proteasome-dependent proteolysis, chaperones and binding activity were also discussed. Our study is the first to use the iTRAQ-based proteomics approach to analyse the sturgeon spermatozoa proteome, and the results that we obtained are valuable for the prediction of sperm quality and reproduction management in these threatened species.

Fig 1. Proteins differentially detected in spermatozoa protein extracts from high quality (H) samples of A. baerii (AB) and A. schrenckii (ASK) compared with low quality groups (L).

(A) Venn diagram showing the number of different proteins in AB and ASK groups. (B) Number of proteins with significant differences in AB-H/AB-L and ASK-H/ASK-L groups.

Fig 2. Gene ontology (GO) of the differentially expressed proteins identified in spermatozoa protein extracts from high quality samples compared with low quality groups.

(A) A. baerii. (B) A. schrenckii. (C) overlapped results from both species. Red columns represent upregulated proteins and green column represents downregulated proteins.

Fig 3. Cluster of orthologous group (COG) classification of the differentially expressed proteins identified in spermatozoa protein extracts from high quality samples compared with low quality groups.

(A) A. baerii. (B) A. schrenckii. (C) overlapped results from both species. Red columns represent upregulated proteins and green columns represent downregulated proteins. Classification: Information storage and processing [J] Translation, ribosomal structure and biogenesis [A] RNA processing and modification [K] Transcription [L] Replication, recombination and repair [B] Chromatin structure and dynamics Cellular processes and signalling [D] Cell cycle control, cell division, chromosome partitioning [Y] Nuclear structure [V] Defence mechanisms [T] Signal transduction mechanisms [M] Cell wall/membrane/envelope biogenesis [N] Cell motility [Z] Cytoskeleton [W] Extracellular structures [U] Intracellular trafficking, secretion, and vesicular transport [O] Posttranslational modification, protein turnover, chaperones Metabolism [C] Energy production and conversion [G] Carbohydrate transport and metabolism [E] Amino acid transport and metabolism [F] Nucleotide transport and metabolism [H] Coenzyme transport and metabolism [I] Lipid transport and metabolism [P] Inorganic ion transport and metabolism [Q] Secondary metabolites biosynthesis, transport and catabolism Poorly characterized [R] General function prediction only [S] Function unknown.

Fig 4. KEGG pathway enrichment of the differentially expressed proteins identified in spermatozoa protein extracts from high quality samples compared with low quality groups.

(A) A. baerii. (B) A. schrenckii. (C) overlapped results from both species.

Fig 5. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database.

Proteins from the pathways of Arginine and proline metabolism, the Citrate cycle (TCA cycle) and Pyruvate metabolism are shown. The red number represents proteins common to A. baerii and A. schrenckii. The blue number represents proteins unique to A. schrenckii. The black number represents proteins unique to A. baerii.The protein names are listed in Table 3.

Fig 6. Western blot validation of CKMT1 and LDHB proteins.

(A) CKMT1 and LDHB proteins in high quality (AB3, AB4, AB7, and AB10) and low quality (AB2, AB6, AB8, and AB9) samples of A. baerii spermatozoa, as well as high quality (ASK1, ASK2, and ASK6) and low quality (ASK3, ASK4, and ASK7) samples of A. schrenckii spermatozoa. Beta-actin was used as the loading control. (B) Quantification of protein levels in high and low quality of spermatozoa from A. baerii and A. schrenckii based on western blot analysis. *p<0.05, **p<0.01.