Background

Sturgeon ancestors existed more than 200 million years ago, and the extant species are living fossils [1] with unique reproductive systems, including gametes, compared to modern teleost fish. For example, sturgeon eggs are encased by a large and thick (50 μm) egg envelope composed of three or four distinct layers compared to the typical one or two envelope layers found in teleost fish. The different structures and numerous micropyles, from 3 to 15, of sturgeon eggs are unusual among fish [2] because the eggs of teleost fish possess only a single funnel-shaped micropyle. Sturgeons are important from an evolutionary perspective and highly valued for their black caviars and high-quality meat. Unfortunately, overfishing for meat and caviar production has led to a severe decline in the sturgeon population, and 27 sturgeon species are currently listed as endangered on the Red List. Therefore, gaining a deeper understanding of the molecular processes underlying sturgeon egg formation and quality is of great scientific and practical importance.

Ovarian fluid (OF) is the maternally derived fluid that surrounds fish eggs, and it plays an important role in creating an optimal environment for egg maturation and fertilization success. OF modulates sperm velocity as a mechanism of cryptic female choice [3]. The biochemical composition of sturgeon OF, including ions, proteins, amino acids and sugars, supports and protects fish gametes against the harmful effects of low freshwater osmolality [4]. The pH of OF and the contents of potassium, sodium and calcium ions are similar to other fish species, but osmolarity varies between fish species. Sturgeon OF has an average osmolality of approximately 200 mOsm kg− 1, and salmonids exhibit an average osmolality of greater than 250 mOsm kg− 1 [5]. The concentration of proteins in sturgeon OF is greater (2.41 ± 0.30 mg mL− 1 for Siberian sturgeon) [6] than that cyprinids (1.58 mg mL− 1 for bleak) [7]. There is a gap in our knowledge for the identification of particular proteins of sturgeon OF, which is a prerequisite for a better understanding of their specific roles.

Despite the importance of sturgeon oocytes for aquaculture, little information is available on their protein composition. Proteomic techniques are powerful tools for studying proteins on a large scale and have been successfully used to identify egg proteins in various fish species, such as pikeperch (1296 proteins) [8] and zebrafish (2535 proteins) [9]. Similarly, OF proteins have been identified in chinook salmon (174 proteins) [10], pikeperch (796 proteins) [8] and rainbow trout (54 proteins) [11]. However, only a limited number of proteins have been identified in the OF and eggs of sturgeon species, such as sterlets and Persian sturgeon, and protein counts ranged from 30 to 80 [12, 13]. A comprehensive proteomic study of sturgeon eggs and their environment is lacking because most studies focused solely on the OF or eggs. A comprehensive approach would elucidate the relationship between the OF and eggs. Only one recent proteomic study on pikeperch (a teleost fish) investigated OF and eggs and addressed this issue [8]. In-depth proteomic studies of eggs successfully identified potential biomarkers for egg quality in pikeperch [8] and zebrafish [9]. Comparative proteomic analysis of sturgeon OF and eggs allows determination of the important roles of proteins in the female reproductive system, development and ovarian physiology of fish.

The ecological and economic importance of sturgeon support a clear need to examine the proteomic composition of the OF and eggs of sturgeon species. Therefore, the present study provides a comprehensive overview of the OF and egg proteomes of Siberian sturgeon and compared their proteomes using an efficient LC‒MS/MS approach to elucidate their composition and functional significance. Western blot analysis was used to validate proteins specific to the OF and eggs. This knowledge provides new information on the factors influencing egg quality and reproductive success and may have implications for biomarker discovery and aquaculture practices.

Results

Characteristics of sturgeon OF and egg quality

The protein concentration, osmolality and pH of the OF were 2.49 ± 0.28 mg mL− 1, 235 ± 11 mOsm kg− 1 and 7.74 ± 0.01, respectively. The eggs collected for analysis were characterized by high quality with a fertilization rate at the second cleavage (4 h postfertilization) of 97.5 ± 0.3% and a hatching rate of 78.4 ± 6.5%. The parameters for all five females are provided in Supplementary Table S1.

LC‒MS/MS identification of OF and egg proteins

In the OF, a total of 732 proteins, containing 3519 peptides, were identified from 12,721 MS/MS matched spectra, and 692 proteins, comprised of 3150 peptides, were identified in eggs from 12,406 MS/MS matched spectra with high confidence (FDR < 1%). Among these identified proteins, 617 and 565 proteins with a minimum of two peptides were detected in at least three of five biological replicates of the OF and eggs, respectively (Supplementary Table S2). The mass spectrometry proteomics data were deposited into ProteomeXchange with the dataset identifier PXD044168. The list of the 10 most abundant proteins (exponentially modified protein abundance index (emPAI) ≥ 9) in the OF and eggs is presented in Table 1.

Table 1 The 10 most abundant proteins in the ovarian fluid (OF) and eggs of Siberian sturgeon (exponentially modified protein abundance index (emPAI) ≥ 9)
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Functional annotation of the OF and egg proteomes

To elucidate the biological functions of the sturgeon OF and egg proteins, we performed a search in the NCBI database and mapped 602 and 553 proteins from the OF and eggs, respectively, to 465 and 471 unique human gene homologs, respectively due to the presence of multiple homologs of the same human protein. Among the identified proteins, gene homologs were not found for 30 and 16 proteins of the OF and eggs, respectively (mostly vitellogenins (VTGs), fish-egg lectin-like, riboflavin-binding protein, protein rapunzel-like, type-4 ice-structuring protein, cell-surface glycoprotein 1, microtubule-associated protein futsch, high choriolytic enzyme 1, gonadal soma-derived growth factor, glycine-rich cell wall structural protein 1.8, stonustoxin and 15 uncharacterized proteins).

GO analysis of biological process terms revealed that most of the OF proteins were associated with exocytosis and secretion (162 proteins) and immune system processes (182 proteins), especially neutrophil-, leucocyte- and granulocyte-mediated immunity, and various cellular metabolic and catabolic process-related terms (182 proteins) and protein localization, targeting and translation (48 proteins) were enriched among the egg proteins (Fig. 1). Enrichment analysis using Ingenuity Pathway Analysis (IPA) identified 40 and 58 canonical pathways that were significantly enriched in the OF and eggs, respectively. The top 10 canonical pathways are presented in Table 2. KEGG pathway analysis highlighted similar pathways. The full functional annotations for the OF and eggs are available in Supplementary Tables S3 and S4, respectively.

Fig. 1
figure 1

GO biological process enrichment analysis of total identified proteins in the ovarian fluid (OF) and eggs of Siberian sturgeon. Most OF proteins were associated with exocytosis and secretion and immune system processes, but the top-ranked biological processes were cellular metabolic and catabolic processes in eggs

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Table 2 Top 10 canonical pathways significantly enriched in ovarian fluid (OF) and egg proteins
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Comparison of OF and egg proteins

Using the emPAI-based estimates of the quantitative relationships within each type of sample, a total of 772 differentially abundant proteins (DAPs) between the OF and eggs were identified, including 365 proteins that were more abundant in the OF and 407 proteins that were more abundant in eggs (Supplementary Table S5). Of these DAPs, 339 proteins were exclusively detected in OF samples, and 287 were unique to eggs (i.e., not detected in the OF samples) (Fig. 2A; Supplementary Tables S6). Among the DAPs shared by the OF and eggs, the top proteins (FC > 20) in OF were albumin (ALB) (140-fold), serotransferrin (TF) (72-fold), hemopexin (HPX) (51-fold), alpha-1-antitrypsin homolog (SERPINA1) (30-fold), and cytosolic nonspecific dipeptidase (CNDP2) (29-fold), where as zona pellucida sperm-binding proteins (ZPs) (40-fold), peroxiredoxin 4 (PRDX4) (31-fold), vitellogenin-like (VTG2) (23-fold), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) (23-fold), and endoplasmin (HSP90B1) (20-fold) were highly abundant in the eggs (Supplementary Table S7).

Fig. 2
figure 2

Functional analysis of proteins unique to ovarian fluid (OF) and eggs. Venn diagram representing the overlap of all identified proteins in the OF and eggs of Siberian sturgeon (A). GO biological processes and cellular components; KEGG pathway analysis of the OF (B) and egg (C) proteins. Pathways with many shared genes are clustered together. Larger dots indicate more significant P values

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GO and KEGG enrichment analyses of proteins specific to the OF and eggs

GO term enrichment analysis of the proteins unique to the OF and eggs revealed significant enrichment of 500 and 338 biological processes, respectively (Supplementary Table S8 and Table S9). We present the top 20 significantly enriched GO biological processes, which shows that most proteins unique to the OF were associated with exocytosis (61 proteins) and immune and stress response (107 proteins), especially leukocyte- and neutrophil-mediated immunity, complement activation and coagulation (Fig. 2B, C). In contrast, metabolic processes (100 proteins; including catabolic processes, such as RNA catabolic process and nitrogen compound catabolic process and energy derivation), protein localization and targeting (31 proteins), and translation (33 proteins) were enriched in eggs. KEGG pathway analysis revealed that proteins involved in complement and coagulation cascades (34 proteins), regulation of the actin cytoskeleton (17 proteins), cholesterol metabolism (9 proteins) and immune-related pathways were specifically enriched in the OF, and metabolic pathways (83 proteins), including oxidative phosphorylation (21 proteins), carbon metabolism (17 proteins), fatty acid degradation (10 proteins) and ribosomes (30 proteins), were most enriched in eggs (Fig. 2B, C). Among the proteins involved in the complement cascade, we identified proteins related to the classical, alternative and lectin pathways (Supplementary Figure S1). IPA highlighted similar pathways and emphasized the representation of proteins involved in eukaryotic initiation factor (EIF) signaling and protein ubiquitination pathways for egg-specific proteins. IPA revealed functional enrichment of OF proteins involved in organismal survival, inflammatory response, immune cell trafficking, hematological system development and organismal development, and egg-specific proteins were mostly involved in tissue morphology, embryonic development, organismal survival, connective tissue development and nervous system development (Table 3).

Table 3 Functional analysis of proteins specific to ovarian fluid (OF) and eggs using Ingenuity pathway analysis (IPA)
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Among the OF-specific proteins related to immune and stress responses, there was a high degree of connectivity between each other (Fig. 3). Among the egg-specific proteins linked to metabolism, six distinct interaction networks were found, among which 28 ribosomal proteins and proteins involved in oxidative phosphorylation (19 proteins) exhibited the highest degree of connectivity (Fig. 4).

Fig. 3
figure 3

Results of the Search Tool for the Retrieval of Interacting Genes/Proteins (v 11.0) analysis showing the protein association network of proteins specific to ovarian fluid assigned to immune system processes with a high confidence score. The nodes correspond to the proteins, and the edges represent the interactions (thick lines indicate a high score > 0.9; thin lines indicate a medium score > 0.7). Model statistics are presented on the left. An explanation of the edge colors is provided above the figure

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Fig. 4
figure 4

Results of the Search Tool for the Retrieval of Interacting Genes/Proteins (v 11.0) analysis showing the protein association network of proteins specific to eggs assigned to metabolic processes with a high confidence score. The nodes correspond to the proteins, and the edges represent the interactions (thick lines indicate a high score > 0.9; thin lines indicate a medium score > 0.7). Model statistics are presented on the left. An explanation of the edge colors is provided above the figure

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OF and egg proteins associated with reproduction

ShinyGo and GO analyses revealed that 102 OF proteins and 117 egg proteins were involved in reproductive processes. An additional search of OF and egg proteins using the Panther database (http://www.pantherdb.org/) revealed their involvement in the ovulation cycle, oocyte maturation, prevention of polyspermy, ovarian aging, fertilization, sperm-egg interaction and embryonic development. All identified proteins and their female reproductive functions are available in Supplementary Table S10.

Validation of mass spectrometry data using Western blotting

To validate the LC‒MS/MS results, we selected four proteins for further analysis using 1D Western blotting: ALB, fibrinogen beta chain (FGB), fibronectin 1 (FN1) and vitellogenin-like (XP_033858533.2), which shows 97.89% identity to vitellogenin AB2a from Acipenser schrenckii, corresponding to the fish VTG2 [14] (Supplementary Figure S2). As presented in Fig. 5, the changes in the abundance of the selected proteins were consistent with the changes in the LC‒MS/MS analysis. Western blot analysis confirmed the absence of FGB and FN1 proteins in the eggs and demonstrated their exclusive presence in the OF (Fig. 5A). A strong signal of ALB was also detected in OF. However, due to its low concentration in eggs (140 times lower than in the OF), it was not detected using the current conditions (i.e., below the limit of detection). Western blot analysis revealed a six-fold greater abundance of VTG2 in eggs compared to the OF (Fig. 5B). The full-length blots are presented in Supplementary Figure S2. The specificity of the antibodies used was confirmed using mass spectrometry analysis (Supplementary Table S11).

Fig. 5
figure 5

Validation of proteins specific to ovarian fluid (OF) and eggs, such as albumin (ALB), fibrinogen (FGB), fibronectin-1 (FN1), and vitellogenin 2 (VTG2), using Western blot analysis (A). The full-length blots are presented in Supplementary Fig. S2. A quantitative comparison of VTG2 in the OF and eggs (B). The data are presented as the means ± S.D. (n = 5 in OF and eggs). Statistical analysis was performed using Student’s t test; ****p ≤ 0.0001. The intensity of the protein bands on the TGX Stain-Free gels was analyzed using Image Lab 6 software (Bio-Rad)

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Discussion

The present study presents the first comprehensive characterization of the OF and mature egg proteomes of sturgeon and their comparison. Our findings contribute to the largest proteomic catalog of the OF and eggs in sturgeons (Actinopterygii), with a total of 602 and 553 proteins identified in the OF and eggs, respectively. Pathways related to mRNA translation (EIF signaling), protein degradation (ubiquitin‒proteasome pathway) and metabolic pathways (oxidative phosphorylation, glycolysis, fatty acid β-oxidation, and the sirtuin signaling pathway) were the most significant canonical pathways in eggs. OF was enriched in various pathways, such as LHR/RXR activation, acute phase response signaling, complement and coagulation system, actin cytoskeleton signaling, clathrin-mediated endocytosis signaling, and integrin and epithelial adherens junction signaling. We also identified proteins that were unique to the OF and eggs, which elucidated their distinct molecular profiles.

Most abundant proteins in the OF and eggs and their functions

The most abundant proteins in the sturgeon OF were predominantly involved in the humoral innate immune response, including acute phase proteins (APPs), antimicrobial peptides (AMPs) and pattern recognition receptors (PRRs). Acute phase response proteins (ALB, TF, HPX, SERPINA1 and apolipoprotein A-I (APOA1)) are multifunctional proteins that participate in various processes, such as iron ion homeostasis (TF, ALB, HPX), oxidative protection (TF, HPX, ALB, APOA1), lipid metabolism (APOA1) and protease activity regulation (SERPINA1). Histone H3 (H3) and hemoglobin subunit beta (HBB) are precursors of AMPs, which play a fundamental role in innate immunity by killing pathogens and modulating the immune response in fish [15]. Fish egg lectin (FEL) was previously identified in the eggs of several teleost fish species, and it acts as a PRR that specifically binds to bacteria and as an opsonin to protect develo** embryos/larvae from pathogenic invasion [16]. Among the abundant proteins in sturgeon OF, actin cytoplasmic 2 (ACTG1) and nucleoside diphosphate kinase A-like (NME1), which play important roles in oogenesis and embryo development in fish, were identified [17, 18]. The functions of ACTG1 and NME1 may also be linked to the immune response because ACTG1 is involved in the motility, phagocytosis and antigen presentation of immune cells [19], and NME1 is involved in T-cell activation [20]. Most of the dominant proteins in sturgeon OF (ALB, TF, HPX, HBB, SERPINA1, APOA1) were also found among the highly abundant proteins in the OF of teleost fish [8, 10, 11], which suggests that the general protective mechanisms of oocytes against pathogens and oxidative stress are evolutionarily conserved in the OF and similar to teleost fish. However, unlike teleost fish, the levels of histones H1 (H1), ACTG1, NME1 and FEL were more abundant in sturgeon OF, which indicates the importance of these proteins in the development and protection of sturgeon oocytes and embryos.

The dominant egg proteins were associated with various functions, including development, immune response, antioxidative protection, metabolism and fertilization. Similar to teleost fish, numerous VTG family members were among the most abundant proteins in sturgeon eggs [3, 8, 10,11,12,13]. VTGs play multiple roles in providing an energy reserve for develo** embryos and protecting them against microbial attacks and oxidative stress [21]. Cystatin-B (CSTB) is a reversible inhibitor of cysteine proteases, and it is involved in cellular protection against proteolysis and immune defense mechanisms in fish eggs, which indicates internal and external protection of eggs against pathogens. The abundance of PRDX1, which is an enzyme that reduces hydrogen peroxide, highlights the importance of the peroxiredoxin system in protecting sturgeon eggs against oxidative stress. ZP3 is an important component of the white sturgeon egg envelope, and it plays a role in fertilization [12, 22]. ZP3 functions in teleosts are also related to oogenesis and embryonic development [23,24,25]. Cofilin-2 (CFL2) is an actin-depolymerizing factor that is essential for actin cytoskeleton organization in fish oocytes and eggs. CFL2 may function with ZPs in envelope hardening and egg adhesion after fertilization in sturgeon [69]. The expression of four proteins of interest, ALB, VTG2, FGB and FN1, was evaluated in the OF and eggs of sturgeon. The method of protein extraction from eggs was described above, and the OF was centrifuged at 3000 × g for 10 min at 4 °C. Equal amounts of protein (15 µg for FN1, 20 µg for VTG2 and ALB, 30 µg for FGB) were applied to Mini-Protean TGX Stain-Free 4–20% Gels (Bio-Rad). The quality of protein separation was checked after gel activation on a ChemiDoc instrument (Bio-Rad), and proteins were transferred to nitrocellulose membranes (0.22 μm) using a Mini Trans Biol Cell (Bio-Rad) in 20 mM Tris-HCl (pH 8.2), 150 mM glycine, and 10% methanol at 60 V for 90 min (4 °C). Nitrocellulose membranes were briefly rinsed in distilled water and blocked with 5% bovine serum albumin (Sigma‒Aldrich). The membranes were incubated overnight at 4 °C with primary polyclonal antibodies against ALB (1:1000), FGB (1:30000), FN1 (1:5000) and VTG2 (1:5000) (Supplementary Table S12). The membrane was rinsed to remove unbound primary antibodies and exposed to goat anti-rabbit antibodies (1:10000; Sigma‒Aldrich) linked to alkaline phosphatase. The products were visualized via incubation in a solution of alkaline phosphate buffer containing nitro blue tetrazolium (Sigma‒Aldrich) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma‒Aldrich) in the dark. The staining was stopped with 0.2 M EDTA. Antibody-bound proteins were detected via enhanced chemiluminescence using a ChemiDoc imaging system (Bio-Rad). The optical density of the protein bands detected on the membranes and the intensity of the protein bands on the TGX Stain-Free gels were analyzed using Image Lab 6 software (Bio-Rad). The image of the gel acquired before its transfer was used as a control for equal protein loading between samples. The volume density of each target protein band was normalized to its respective total protein content, and the total protein band density was normalized to the total protein loaded into each lane using stain-free technology. The data are expressed in arbitrary units according to the manufacturer’s instructions (Bio-Rad) and Posch et al. [69]. To confirm the specificity of the antibodies used, protein bands corresponding to the detected bands were manually removed from the gels and prepared for digestion and protein identification using MALDI-TOF/TOF [70].

Experimental design and statistical rationale

To evaluate the protein composition and differences between the proteomes of the OF and eggs, the present study performed five biological replicates using LC‒MS/MS analysis. The resulting MS/MS data were processed using Mscan software. The FDR thresholds for proteins were specified at 1%. To estimate the protein abundance, emPAI was calculated using unique precursors and normalized by total abundance. The differentially abundant proteins were calculated (IBB, Warsaw) (p < 0.05, fold change > 3). Gene Ontology (GO) enrichment, KEGG pathways, and IPA (Ingenuity Pathway Analysis) were used to examine the functional significance of the identified proteins. Protein–protein interaction network analysis was performed using the STRING database with a medium confidence score cutoff of 0.4. The expression of four identified proteins was confirmed by Western blotting using specific antibodies. All of the details are described above.