Comparative proteomic analysis of the ovarian fluid and eggs of Siberian sturgeon.

BACKGROUND: Sturgeon species are living fossils that exhibit unique reproductive characteristics, and elucidation of the molecular processes governing the formation and quality of sturgeon eggs is crucial. However, comprehensive data on the protein composition of sturgeon ovarian fluid (OF) and eggs and their functional significance are lacking. To address this knowledge gap, the aim of the present study was to conduct a comprehensive comparative proteomic analysis of Siberian sturgeon OF and eggs using liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: A total of 617 proteins were identified in OF, and 565 proteins were identified in eggs. A total of 772 proteins showed differential abundance. Among the differentially abundant proteins, 365 were more abundant in OFs, while 407 were more abundant in eggs. We identified 339 proteins unique to OFs and 287 proteins specific to eggs, and further investigated the top 10 most abundant proteins in each. The functional annotation of the OF proteins highlighted their predominant association with immune system processes, including the complement and coagulation cascade, neutrophil and leukocyte-mediated immunity, cholesterol metabolism, and regulation of the actin cytoskeleton. Analysis of egg proteins revealed enrichment in metabolic pathways, such as oxidative phosphorylation and fatty acid metabolism, and protein ubiquitination and translation. OF-specific proteins included extracellular matrix and secretory vesicles, and eggs were enriched in proteins localized to mitochondria and ribosome components. CONCLUSIONS: This study presents the first comprehensive characterization of the protein composition of sturgeon OF and eggs and elucidates their distinct functional roles. These findings advance our understanding of sturgeon reproduction, OF-egg signaling and the origin of OF proteins. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD044168 to ensure accessibility for further research.

Proteomic analysis of carp seminal plasma provides insights into the immune response to bacterial infection of the male reproductive system.

Aeromonas salmonicida is recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. However, the mechanism of immunity to these bacteria in common carp is still not well understood, especially the immune regulation in the gonad to bacterial infection. The aims of our study were to analyze changes in the seminal plasma proteome following A. salmonicida infection in carp males. The observed pathological changes in the tissue (liver, spleen, kidney and testis) morphology and upregulation of immune-related genes (tnfa2, il6a) confirmed the successful infection challenge. Using mass spectrometry-based label-free quantitative proteomics, we identified 1402 seminal plasma proteins, and 44 proteins (20 up- and 24 downregulated) were found to be differentially abundant between infected and control males. Most differentially abundant proteins were involved in the immune response mechanisms, such as acute phase response, complement activation and coagulation, inflammation, lipid metabolism, cell-cell and cell-matrix adhesion, creatine-phosphate biosynthesis and germ cell-Sertoli cell junction signaling. Bacterial infection also caused profound changes in expression of selected genes in the testis and hematopoietic organs, which contributed to changes in seminal proteins. The altered seminal proteins and bacterial proteins in seminal plasma may serve as valuable markers of infection in the testis.

2D-DIGE proteomic analysis of blood plasma reveals changes in immune- and stress-associated proteins following hormonal stimulation of carp males.

In carp aquaculture, hormonal manipulation with an analog of GnRH (Ovopel) and carp pituitary extract (CPE), which act at different levels of the hypothalamic-pituitary-gonadal axis, is a routine practice to enhance sperm production. Our recent studies revealed that hormonal stimulation of male carp was associated with changes in the seminal plasma proteome, including blood origin proteins. Here, we explored whether Ovopel and CPE could affect the blood proteome of male carp. Both preparations induced increases in semen volume, total number of sperm, and testosterone level. However, hormonal stimulation did not affect the plasma cortisol and glucose levels. A comparative proteomic analysis of carp blood plasma between the control (PBS) and the hormonally treated males revealed significant changes (>1.2 <-1.2-fold change, P < 0.05) in the abundance of 30 spots (14 up- and 16 downregulated) and 44 spots (28 up- and 16 downregulated) upon CPE and Ovopel treatment, respectively. The most significantly affected pathways were acute phase response signaling, the coagulation system, LXR/RXR and FXR/RXR activation; however, there were different sets of proteins in Ovopel- and CPE-treated males. The majority of differentially abundant proteins were involved in the regulation of the immune defense response, the response to stress, and complement activation. Moreover hormonal stimulation with CPE markedly increased the bactericidal activity of blood and both preparations caused profound changes in gene expression in hematopoietic organs. This work is important in understanding the biological processes behind the protein-based response to hormonal stimulation of sperm production in fish.

Cryoprotectant-specific alterations in the proteome of Siberian sturgeon spermatozoa induced by cryopreservation.

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods-liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability.

Application of two-dimensional difference gel electrophoresis to identify protein changes between center, margin, and adjacent non-tumor tissues obtained from non-small-cell lung cancer with adenocarcinoma or squamous cell carcinoma subtype.

Lung cancer is responsible for the most cancer-related mortality worldwide and the mechanism of its development is poorly understood. Proteomics has become a powerful tool offering vital knowledge related to cancer development. Using a two-dimensional difference gel electrophoresis (2D-DIGE) approach, we sought to compare tissue samples from non-small-cell lung cancer (NSCLC) patients taken from the tumor center and tumor margin. Two subtypes of NSCLC, adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were compared. Data are available via ProteomeXchange with identifier PXD032736 and PXD032962 for ADC and SCC, respectively. For ADC proteins, 26 significant canonical pathways were identified, including Rho signaling pathways, a semaphorin neuronal repulsive signaling pathway, and epithelial adherens junction signaling. For SCC proteins, nine significant canonical pathways were identified, including hypoxia-inducible factor-1α signaling, thyroid hormone biosynthesis, and phagosome maturation. Proteins differentiating the tumor center and tumor margin were linked to cancer invasion and progression, including cell migration, adhesion and invasion, cytoskeletal structure, protein folding, anaerobic metabolism, tumor angiogenesis, EMC transition, epithelial adherens junctions, and inflammatory responses. In conclusion, we identified several proteins that are important for the better characterization of tumor development and molecular specificity of both lung cancer subtypes. We also identified proteins that may be important as biomarkers and/or targets for anticancer therapy.