Comprehensive proteomic characterization and functional annotation of common carp seminal plasma

Aquaculture of common carp

The common carp (Cyprinus carpio L.) is one of the most commercially important global aquaculture species. Historically, carp has been among the oldest freshwater species cultivated for food, playing a pivotal role in both Asian and European aquaculture. For example, in Germany and Poland, carp ranks second in production (https://www.aquaculture-welfare-standards.net/en/species/carp/, Myszkowski 2022) and is one of the key species in Asian aquaculture (Miao and Wang 2020). In aquaculture conditions, carps are raised in freshwater ponds and are often artificially reproduced using reproductive biotechniques, such as hormonal stimulation and artificial fertilization. As such, the production of high-quality gametes, including spermatozoa, is a prerequisite for successful reproduction. Sufficient knowledge regarding semen physiology and biochemistry is essential for improving reproductive biotechniques and for gaining insight into the functioning of carp reproductive system.

Seminal plasma – definition and function

The semen of vertebrates, including fish, has two components: the cellular component mainly composed of male gametes (spermatozoa), involved in fertilization, and immune cells, and the liquid component, which is the seminal plasma. In most teleost fish, seminal plasma is a secretory product of the testes and spermatic duct; some compounds may also originate from blood plasma (Ciereszko 2008; Dietrich et al. 2010a). Seminal plasma is uniquely composed of mineral and organic substances supporting spermatozoa. The main role of seminal plasma is to create an optimal environment for the storage of spermatozoa, often for prolonged periods, with special emphasis on maintaining optimal osmolality, keeping spermatozoa in the immotile state, and supporting sperm metabolism, antimicrobial and stress protection, and protection against xenobiotics (Ciereszko 2008). The composition of seminal plasma reflects species specificity of fish reproduction; for example, significant differences are observed between cold-water salmonids and warm-water cyprinids (Ciereszko et al. 2017).

Seminal proteins, their role, and proteomic approach

Proteins are an important organic composition of teleost fish seminal plasma and are involved in the protection of sperm viability (Lahnsteiner et al. 2004; Lahnsteiner 2007). Several major proteins have been purified and characterized, which allowed deeper insights into their functions. For example, protease inhibitors (Mak et al. 2004; Wojtczak et al. 2007a; Nynca et al. 2010a, 2011a) are involved in the protection of spermatozoa against proteolytic enzymes present in seminal plasma (Kowalski et al. 2003). Transferrin is a multitask protein with antioxidative and antimicrobial protection function as well as protective role against cadmium toxicity (Dietrich et al. 2010a, 2011). Parvalbumin is a calcium-binding protein that appears to be characteristic for carp semen, with the possible role in sperm motility activation (Dietrich et al. 2010b). Apolipoproteins are associated with the stabilization of sperm membranes and are characterized by antibacterial properties (Nynca et al. 2010b; Dietrich et al. 2014a), lipocalins are involved in the transportation of hydrophobic molecules (Nynca et al. 2011b), and hemopexin plays a pivotal role in antioxidative protection (Dietrich et al. 2020). Aside from major proteins that are quite easy to detect, there are hundreds of minor proteins in fish seminal plasma that can now be detected using a proteomic approach, which enables comprehensive and large-scale analysis of proteins (Ciereszko et al. 2012; Shaliutina et al. 2012; Dietrich et al. 2019a). This allows, for the first time, the acquisition of a comprehensive picture of seminal proteins and the characterization of their role by using bioinformatics tools, which opens new horizons in unraveling the new biological roles of seminal proteins. Furthermore, proteomics facilitates the selection proteins of interest for further detailed studies focusing on their structure and function.

Current knowledge and justification of the study

Earlier proteomic studies identified a limited number (22–137) of proteins in carp seminal plasma by using one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Shaliutina-Kolešová et al. 2016; Dietrich et al. 2014b, 2019a, 2019b, 2021). These proteins were analyzed using bioinformatics tools, which significantly enhanced basic knowledge regarding seminal plasma proteins and their potential role in fish reproduction, including the regulation of sperm motility, spermatogenesis, maintenance of sperm membrane lipid stability and antioxidative protection, as well as immune and stress responses. However, because of the limited number of proteins, the results of these studies are likely to be incomplete.

Our recent comparative study focusing on bacterial Aeromonas salmonicida infection of male carps and changes in seminal plasma protein abundance in relation to infection-provided insights into the immune response of male reproductive system, including acute-phase response, complement activation response, complement activation and coagulation, and inflammation (Dietrich et al. 2022). In this study, we focused only on 44 differentiated proteins vital for infection response. This is only a fraction of all identified proteins in this study because using high-throughput liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based proteomic analysis and updated Cyprinus carpio genome annotation, 1402 seminal plasma proteins were identified; this value is significantly higher than that previously reported for carp. This provides, for the first time, unique opportunity to conduct broad analysis on carp seminal plasma proteins as such a high number of proteins enhances the strength of bioinformatic analysis, potentially resulting in the accomplishment of comprehensive knowledge regarding male carp reproduction. Therefore, our study aimed to conduct comprehensive bioinformatics analysis of seminal plasma proteome of male controls from the previous experiment (Dietrich et al. 2022) by using advanced bioinformatics tools. To obtain better understanding of the protein function, we introduced a novel classification system by categorizing proteins into three groups on the basis of their cellular localization for (1) secretory proteins, (2) proteins with either secretory properties and localization to different cellular compartments, and (3) proteins without secretory properties. We assumed that secretory proteins are presumably specific seminal plasma proteins and that intracellular proteins have a multicellular origin from either the spermatozoa or diploid cells, particularly leucocytes, of the carp reproductive system. Furthermore, we provided more information on proteins involved in extracellular vesicles (EVs) owing to the emerging importance of EVs in seminal plasma function. In addition, we identified proteins specific for fish and discussed their possible role in male fish reproduction.

Characteristics of common carp semen

Sperm concentration was measured using the spectrophotometric technique (Ciereszko and Dabrowski 1993) and sperm motility via computer-assisted sperm analysis, as described by Wojtczak et al. (2007b).

LC–MS analysis

For the purpose of conducting LC–MS analyses, we used seminal plasma obtained from healthy carps (n = 5) from the control group of a previously conducted experiment by Dietrich et al. (2022). LC–MS analysis and sample preparation were performed at the Mass Spectrometry Laboratory at the Institute of Biochemistry and Biophysics of Polish Academy of Sciences (PAS). We used an LC–MS system consisting of ultraperformance liquid chromatograph (M-class, Waters) coupled with a Q Exactive mass spectrometer (Thermo Scientifi, Waltham, Massachusetts, USA) using a Flex nano electrospray ionization ion source (Thermo Scientific), as previously described (Dietrich et al. 2022). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2025) partner repository, with the dataset identifier PXD065911 and 10.6019/PXD065911.

Bioinformatics

We obtained information from UniProt annotations (The UniProt Consortium 2023) for each protein to divide the proteins into three groups by using subcellular location criteria. Secretory proteins were defined as proteins located outside the cell membranes. The second group of proteins were characterized as both secretory and intracellular, such as cell membrane, endoplasmic reticulum, lysosome, Golgi apparatus, cytoplasmic vesicles, and extracellular space. The third group of proteins were non-secretory and had various intracellular localizations. To evaluate the potential similarity of seminal plasma to blood protein, we searched The Human Protein Atlas (Human Protein Atlas, proteinatlas.org; Uhlén et al. 2015) to compare the results of our identified proteins with those listed in the database as secreted to blood.

We conducted separate analyses for protein groups divided according to subcellular location criteria. In addition, to gain a broader perspective, we analyzed all identified proteins and conducted a functional enrichment analysis of networks associated with EVs and reproduction.

We performed the functional profiling of the identified proteins from the selected groups into three gene ontology (GO) categories (biological processes, cellular components, and molecular functions) by using the Search Tool for the Retrieval of Interacting Genes (STRING) database (ver. 10.5, http://string-db.org/) with a medium confidence score cutoff of 0.4.

Characteristics of common carp semen

The sperm concentration was 19.2 ± 0.8 × 10⁹ mL⁻¹, and the sperm motility was 91 ± 6%, with the following sperm kinetic parameters: curvilinear velocity (VCL) 119.7 ± 9.6 μm s⁻¹, straight-line velocity (VSL) 66.0 ± 6.7 μm s⁻¹, and amplitude of lateral head displacement (ALH) 6.9 ± 1.1 μm. The seminal plasma osmolality was 279.0 ± 3.8 mOsm kg⁻¹, and the protein concentration was 1.34 ± 0.10 mg mL⁻¹.

LC‒MS/MS analysis of seminal plasma proteins

LC–MS/MS analysis and sample preparation were performed at the Mass Spectrometry Laboratory at the Institute of Biochemistry and Biophysics of PAS. From a total of 386,204 MS/MS spectra and 5182 peptides, 2009 proteins were identified in carp seminal plasma in at least three individuals, with a minimum of two unique peptides and false discovery rate (FDR) of ≤1% (Supplementary Table S1). After removing duplicates and triplicates, the total number of proteins was 1402, among which 1354 (Table S2) were annotated to unique human gene homologs and 49 (Table 1) were fish-specific.

Functional annotation of seminal plasma proteome

To gain insights into the biological roles of proteins in carp seminal plasma, we categorized the proteins by using the UniProt database into three groups on the basis of their cellular localization. Of the 1354 proteins, 233 were identified as secretory, which represents 17.13% of the total identified proteins. Among these secretory proteins, 141 were classified as exclusively secretory (Table 2), 92 as both secretory and intracellular (Table 3), and the remaining as intracellular (Fig. 1).

Fig. 1.

Percentage distribution of proteins unique to human gene homologs, categorized on the basis of their localization.

Exclusively secretory proteins

Among the 141 secretory proteins, all were found to contain signal peptides. Majority of the proteins (109) have also been indicated as blood proteins in human (Table 2).

GO analysis of biological processes showed that these proteins are involved in 222 biological processes. Major biological processes were related to immune response, response to stress, extracellular matrix organization, and lipid transport and metabolism (Fig. 2a). We also identified proteins involved in the regulation of vesicle-mediated transport and body fluid levels.

Fig. 2.

The gene ontology (GO) analysis of biological process (a), molecular function (b) and cellular component classification (c) of exclusively secretory proteins identified in carp seminal plasma.

Molecular function analysis showed mostly the activities of proteolytic enzymes and their regulators. Binding activities were also identified for protease, signaling receptor, glycosaminoglycan, calcium ions, and complement. Other molecular functions include extracellular matrix structural constituent and phosphatidylcholine-sterol O-acyltransferase activator activity (Fig. 2b).

According to the cellular component criteria, secretory proteins were mainly located in different extracellular regions, secretory granules, and lumens of several cell structure (Fig. 2c). Moreover, secretory proteins were associated with the endoplasmic reticulum, Golgi apparatus, vesicles, and membranes. They were also associated with various extracellular protein complexes, such as serine-type endopeptidase complex, high-density lipoprotein particle, very-low-density lipoprotein particle, and chylomicrons.

The complete results of the GO analysis of exclusively secretory proteins are presented in Table S3.

Secretory proteins with various intracellular localizations

A subset of 92 proteins with both secretory and intracellular localizations included 40 proteins also classified as blood proteins in humans (Table 3).

GO analysis showed 244 biological processes associated with these proteins. Major biological processes (Fig. 3a) were related to various immune responses and responses to stress. Several processes were also associated with response to stimulus, which is defined as any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.). Other processes were related to organismal process and development; cell differentiation, migration, and adhesion; and blood vessel morphogenesis.

Fig. 3.

The gene ontology (GO) analysis of (a) biological process, (b) molecular function and (c) cellular component classifications of proteins characterized by both secretory and intracellular localizations, and proteins exclusively localized intracellularly, identified in carp seminal plasma.

Molecular function analysis uncovered various binding activities, including those involving proteins and protein-containing complexes, signaling receptors, proteoglycans, and calcium ions (Fig. 3b). According to the cellular component criteria, proteins were mainly located in different extracellular regions, membranes, exosomes, and vesicles as well as in the endoplasmic reticulum, lysosome, and Golgi apparatus (Fig. 3c). The complete results of the GO analysis are presented in Table S4.

Proteins related to extracellular vesicles

According to the cellular component criteria, we analyzed the entire protein set and performed functional enrichment of the cellular component GO:1903561 (EV; PPI enrichment P-value: <1.0 × 10⁻¹⁶), which comprised 636 molecules (Supplementary Fig. S1).

Functional enrichment of the selected GO term identified 713 biological processes, including those involving more than 400 proteins, such as cellular process, biological process regulation, metabolic process and biological regulation. In addition, the biological functions involving over 200 proteins include, response to stimulus, cellular metabolic process, nitrogen compound metabolic process, organic substance metabolic process, primary metabolic process, protein metabolic process, developmental process, and response to stress. Other important activities comprised response to organic substance, cellular response to chemical stimulus, regulation of protein metabolic activity, negative and positive regulations of the biological process, regulation of metabolic function, anatomical structure development, cellular metabolic process, cell differentiation, multicellular organismal process, and multicellular organism development (Table S8).

We also identified 155 significant molecular functions, including those with participation of more than 200 proteins, such as binding, protein binding, as well as hydrolase and catalytic activities (Table S8).

Table S8 presents 185 cellular component categories with involvement of more than 100 proteins, such as extracellular exosome, EV secretory granule, secretory vesicle, cytoplasmic vesicle, cytosol, endomembrane system, cell junction, intracellular anatomical structure, intracellular organelle, intracellular membrane-bound organelle, cell periphery, and protein-containing complex.

Proteins related to reproduction

To conduct a detailed analysis of proteins related to reproduction, we analyzed the entire protein set and performed functional enrichment of the networks for reproductive processes (GO:0022414; PPI enrichment P-value: <1.0 × 10⁻¹⁶) and sexual reproduction (GO:0019953; PPI enrichment P-value: <1.0 × 10⁻¹⁶), which showed 340 and 106 significant biological processes respectively.

We found that seminal plasma proteins were associated with gamete generation, spermatogenesis, and spermatozoa motility apparatus. The biological processes of both networks are presented in Table S9.

Fish-specific proteins

We identified 49 fish-specific proteins (Table 1). Among the identified proteins, the majority were molecules associated with immune responses. In addition, we noted the presence of vitellogenin group proteins, proteins characterized by other functions, and seven uncharacterized proteins.

Most abundant carp seminal plasma proteins are secretory and also present in blood plasma

The most abundant proteins of carp seminal plasma were established in our previous studies (Dietrich et al. 2014b; Ciereszko et al. 2017), and the results of the present study clearly indicated that these proteins are of secretory origin, including transferrin, apolipoprotein (APO) E, 14-kDa APOA2, APOA1, complement C3, retinol-binding protein, serpin 1 (alpha-1 antitrypsin), fetuin, and hemopexin. Heat shock protein 90-alpha has been classified as secreted and at different cellular localizations. These proteins are likely to be mainly produced by spermatic duct and/or originate from blood because majority of carp seminal proteins are also present in blood plasma (Lahnsteiner et al. 1993, 1995; Lahnsteiner 2003; Dietrich et al. 2014b); see also Table 2. This suggestion is supported by our recent results that found the expression of major proteins, such as APOA1, APOA2, and CR, both in carp testis and liver, kidney, and spleen (Dietrich et al. 2022).

Immune response

The results of our study clearly suggest that participation in immune response, both innate and humoral, is a major role of fish seminal plasma. The introduction of proteomic approach to studies on fish reproduction clearly indicated that acute-phase proteins are major proteins of fish semen, which highlights the importance of immune response in male fish reproduction (Dietrich et al. 2022). The acute-phase response is the first rapid mechanism of protection against microbes and pathogens, tissue damage repair, and inactivation of proteases in fish (Bayne and Gerwick 2001). The immunological processes are precisely regulated, often by proteolysis, which is clearly highlighted by the presence of several proteases and their inhibitors, such as inhibitors of serine proteases (serpins) and metalloproteases (TIMP).

Notably, response to wounding and wound healing were also indicated as significant biological processes. As such, these results suggest that the male reproductive system is equipped with a protective mechanism against injuries that can occur during spawning runs and male fight during spawning.

Lipid transport and metabolism

We detected several processes related to lipid transport and metabolism, including remodeling of lipoprotein, phospholipid efflux, and cholesterol transport, with apolipoproteins being the major proteins involved in these processes (Dietrich et al. 2014a). These results agree with our previous finding indicating that the presence of lipid-/cholesterol-binding proteins is a specific feature of carp seminal plasma because the abundance of these proteins is higher in seminal plasma than in blood (Dietrich et al. 2014b). Interestingly, molecular function analysis highlighted phosphatidylcholine-sterol O-acyltransferase activator activity. This enzyme is a major source of cholesterol ester in human plasma and can be activated by apolipoprotein E (Steinmetz et al. 1985). The latter is a secretory protein in carp seminal plasma identified in our study. The main role of lipids is likely to stabilize sperm membranes and provide energy source to spermatozoa. Our recent data also suggest that lipoproteins play a role in the reproductive tract of carp during bacterial infection (Dietrich et al. 2022).

Regulation of body fluid levels

For the first time, we identified a biological process involved in the regulation of body fluid levels. This is particularly important for better understanding of the mechanism of hormonal stimulation of male carps raised under aquaculture conditions to enhance sperm production and quality (Dietrich et al. 2019a). Hydration of the testis and spermatic duct is a pre-requisite to enhancing spermiation by increasing seminal fluid production and sperm maturation (Mylonas et al. 2017). The mechanism of semen hydration is not well understood. Our results indicated the presence of 23 proteins that can regulate seminal fluid level (Table 4). Interestingly, among these proteins, we have identified plasma kallikrein (KLKB1), which can release bradykinin from kininogen (KNG1). Furthermore, we detected an angiotensin-I-converting enzyme (detected in proteins that were both secretory and intracellular), which is involved in bradykinin hydrolysis. Bradykinin is reportedly affected by sodium and water movements in tissues (Crocker and Willavoys 1975; Zaika et al. 2011). We also identified an angiotensin-like protein. These results suggest the involvement of the kallikrein–kinin system in the regulation of carp seminal plasma volume, presumably in semen hydration. This suggestion needs to be tested in further studies.

Secretory proteins with intracellular localizations

Analyses of proteins that were both secretory and intracellular showed that they had overlapping and extended biological functions, particularly secretory proteins, as observed in immune response and EV-related processes. This group is further characterized by several processes associated with development, such as organismal processes, blood vessel morphogenesis, as well as cell differentiation, migration, and adhesion. These findings suggest the involvement of these processes in spermatogenesis and sperm maturation. The latter may be supported by processes related to EVs, which play a pivotal role in sperm maturation (see below).

Proteins without secretory properties

Proteins related to extracellular vesicles

Our results clearly indicated the presence of EVs in carp seminal plasma, because we identified 636 proteins associated with these structures. Owing to the tight DNA packaging in male haploid gametes, support for sperm function cannot be exerted through transcription and translation (with the possible exception of translation of pre-existing nuclear transcripts by using mitochondrial translational machinery) (Gur and Breitbart 2008; Boguenet et al. 2021). Instead, post-translational modifications (PTMs), secretory proteins, and EVs are used (Samanta et al. 2018; Belleannée et al. 2022; Castillo et al. 2023; Parra et al. 2023).

In addition, we found molecules involved in their formation, secretion, as well as cargo uptake and sorting processes. Furthermore, we identified 19 GPI-anchored proteins associated with EVs, such as CD48, CD59, LYPD6B, and NT5E (Özhan et al. 2013; Klein-Scory et al. 2014; Karasu et al. 2018; Zhao et al. 2023). Proteins involved in EV formation mechanisms, driven by the endosomal sorting complexes required for transport (ESCRT), were also detected. These include ALG-2-interacting protein X, implicated in miRNA packaging, and vacuolar protein sorting-associated protein 4, essential for membrane remodeling during vesicle biogenesis (Alonso Y Adell et al. 2016; Iavello et al. 2016). Moreover, we demonstrated the presence of tetraspanin CD63, a widely recognized EV marker. CD63 regulates the ESCRT-independent pathway of endosomal sorting, is required for exosome formation, and acts as a crucial regulator of endosomal compartment fate as well as an inducer of the secretory pathway (Toribio and Yáñez-Mó 2022).

Interestingly, we found many members of Ras-associated binding (Rab) proteins regulating the intracellular signaling of EVs (e.g. Rab2A, Rab8a, Rab35, Rab 15, CDC42) and soluble N-ethylmaleimide-sensitive factor attachment protein receptors, membrane proteins that function in a complex and are essential for regulating the docking of granules and vesicles to target membranes, although they participate in the final step of exosome secretion (e.g. Vamp3, Vamp7, STX2, STXBP2). Moreover, we found that co-operation between members of these families is necessary for the proper regulation of membrane fusion and EV trafficking (Margiotta 2022).

Fish-specific proteins