Comprehensive proteomic characterization and functional annotation of Siberian sturgeon seminal plasma proteins
Introduction
Sturgeon ancestors existed >200 million years ago, and the extant species are called living fossils (Birstein et al., 1997). Sturgeons belong to the order Acipenseridae, family Chondrostei, and occupy a key evolutionary position between teleost and cartilaginous fishes. The population of sturgeons has dramatically decreased because of overfishing and water pollution (Dettlaff et al., 1993). As a result, most sturgeons are considered endangered species (27 species of sturgeon are on the Red list). Siberian sturgeon (Acipenser baerii) is considered the most valuable species in aquaculture, as they are a source of caviar and high-quality meat with the current production in aquaculture of about 442 tons in 2020 (FAO, 2022). Compared to other sturgeon species, Siberian sturgeon shows a rapid growth rate, resistance to pathogens, and a relatively short reproductive cycle (7–8 years). Regarding its commercial importance and special evolutionary status, it is worth studying its reproduction to improve restocking programs, which are of great interest for this species (Babaei et al., 2017).
The biology and physiology of sturgeon semen differ substantially from those of teleost fish by a more complex structure of spermatozoa, longer sperm motility duration, lower sperm concentration, sperm maturation process, atypical testicular morphology and characteristics of seminal plasma. Sturgeon spermatozoa, which have an acrosome, may represent a transitional stage before the simplification of sperm structure in teleost fishes, which leads to the loss of the acrosome (Pšenička and Ciereszko, 2018). Moreover, in sturgeon, sperm maturation occurs outside the testis via dilution of sperm by urine during passage of semen through the kidneys to the Wolffian ducts, while in teleosts, sperm maturation occurs due to factors in the environment of the spermatic duct, such as pH and bicarbonate ions (Dzyuba et al., 2014a). Correspondingly, seminal plasma and spermatozoa proteins in teleostean and chondrostean fishes have evolved adaptations in response to the reproductive environment (Li et al., 2009). The uniqueness of sturgeon semen implicates the need for studying semen composition at the protein level. In addition, seminal plasma might be a rich source of biomarkers of reproductive physiology and/or disorders in male fishes. To identify potential protein biomarkers in seminal plasma, construction of a comprehensive protein catalogue in this fluid is a necessary first step.
Sturgeon seminal plasma has a specific chemical and biochemical composition and plays an important role in supporting and protecting viability and creating an optimal environment to maintain spermatozoa in the quiescent state. It is characterized by low concentration of substantial mineral compounds (Na+, K+, Ca2+, Mg2+), low protein concentrations (not higher than 1 mg ml−1) and low osmolality (50–80 mOsmol kg−1). Interestingly, the composition of seminal plasma is very similar to that of urine (Dzyuba et al., 2014b). To date, knowledge about sturgeon seminal plasma proteins is limited to the characterization of a few enzymes, such as lactic dehydrogenase (LDH), arylsulfatase (ARS), acid phosphatase (ACP), β-N-acetylglucosamisase (BetaGlcNAc), and antioxidative enzymes (Sarosiek et al., 2004; Shaliutina et al., 2013; Alavi et al., 2014). In addition, gelatinases (serine proteases and metalloproteases), acrosin and serine protease inhibitors were studied using zymography and western blot in sturgeon seminal plasma (Słowińska et al., 2015). However, in most cases, such proteins have not yet been specifically identified.
To study proteins on a large scale, proteomic methods are necessary. Proteomic techniques have been widely used to identify seminal plasma proteins of mammalian species, for example, in humans (Pilch and Mann, 2006) and dolphins (Fuentes-Albero et al., 2021), and recently in several fish species, such as carp (Dietrich et al., 2014), rainbow trout (Nynca et al., 2014), Chinook salmon (Gombar et al., 2017) and pikeperch (Dietrich et al., 2021). It should be emphasized that the composition of seminal plasma proteins varies among teleost fish species, likely reflecting species-specific characteristics of reproduction in fish. To our knowledge, there are no reports on the in-depth characterization of sturgeon seminal plasma proteins, and this knowledge is limited to the spermatozoa proteome (Li et al., 2010, Li et al., 2011; Li et al., 2017a, Li et al., 2017b; Horokhovatskyi et al., 2018). Therefore, the proteomic characterization of Siberian sturgeon seminal plasma is important for better understanding the male reproductive processes in sturgeon and creating possibilities for improving artificial reproductive techniques, including short- and long-term semen storage and fertilization.
The aim of this study was to identify, describe, and classify for the first time the seminal plasma proteins of Siberian sturgeon and analyze their possible functions. Two complementary proteomic approaches: gel-based (2DE and 2D blue native (BN)/SDS–PAGE) and gel-free (LC–MS/MS) were applied to maximize the identification of proteins present in sturgeon seminal plasma. The presented protein catalogue provides new information on the composition of seminal plasma and its potential utility in diagnosis of reproductive disorders.
Section snippets
Experimental design and workflow
For in-depth characterization of protein composition of sturgeon seminal plasma we applied three different proteomic strategies, including 1) two-dimensional gel electrophoresis (2D SDS–PAGE) combined with identification of proteins using matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (MALDI TOF/TOF), 2) liquid chromatography-based analysis coupled with MS/MS (LC-MS/MS) in wide ion mass range of 300–2000 m/z, and three narrow mass ranges (i.e.,
Characteristics of sturgeon semen
Semen parameters for all six males are provided in Supplementary Table S1. Briefly, the mean semen volume was 35 ± 4.6 ml, the sperm concentration was 1.83 ± 1.25 × 109 ml−1, the sperm viability was 98.8 ± 0.3% and the sperm motility 89 ± 3% with the following sperm kinetic parameters VCL 157.8 ± 18.7 μm s−1, VAP 141.6 ± 23.3 μm s−1, VSL 130.1 ± 24.2 μm s−1, LIN 78.4 ± 7.6%, and ALH 4.8 ± 0.6 μm. The seminal plasma characteristics were as follows: osmolality 83 ± 18 mOsm kg−1 and protein
Discussion
This work represents the first in-depth proteomic characterization of sturgeon seminal plasma protein and, to the best of our knowledge, the largest seminal plasma proteome (665 proteins) reported for sturgeons (Actinopterygii). A major drawback to the identification of Siberian sturgeon seminal plasma proteins is a lack of a complete genome of this species. The number of protein sequences in NCBInr Acipenser baerii database is still extremely limited (566 sequences) compared to Homo sapiens
CRediT authorship contribution statement
Natalia Kodzik: Formal analysis, Data curation, Visualization, Methodology, Writing – original draft. Andrzej Ciereszko: Conceptualization, Writing – original draft, Writing – review & editing. Mirosław Szczepkowski: Resources. Halina Karol: Investigation. Sylwia Judycka: Investigation. Agata Malinowska: Formal analysis, Data curation. Bianka Świderska: Formal analysis, Data curation. Mariola A. Dietrich: Conceptualization, Methodology, Writing – original draft, Writing – review & editing,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by funds from the National Science Centre granted on research project 2019/35/B/NZ9/03501. The equipment used for proteomic analysis was sponsored in part by the Centre for Preclinical Research and Technology (CePT), a project cosponsored by the European Regional Development Fund and Innovative Economy, The National Cohesion Strategy of Poland.
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