Elsevier

Aquaculture

Volume 472, 1 April 2017, Pages 81-92
Aquaculture

Fish semen proteomics — New opportunities in fish reproductive research

https://doi.org/10.1016/j.aquaculture.2016.03.005Get rights and content

Highlights

  • A proteomic approach has been introduced to study fish semen.

  • Major proteins in carp and rainbow trout semen have been identified and characterized.

  • The potential physiological role of seminal proteins has been addressed.

  • Specific seminal plasma proteins have been distinguished from blood proteins.

  • Cryodamage to the fish sperm proteome has been described.

Abstract

Proteomic approaches have recently been introduced to studies of male fish reproduction, providing new insights into the functioning of the reproductive system. Numerous previously unknown proteins have been identified, allowing for the comprehensive characterization of the mechanisms involved in the protection of spermatozoa in seminal plasma, especially against oxidative stress and microbial attack. The mechanisms responsible for supporting sperm survival during storage, especially the maintenance of sperm membranes, were also identified. Metabolic pathways, especially those related to sperm energy and metabolism, have been better characterized. A comprehensive comparative analysis of blood and seminal plasma proteins allows for a better understanding of the mechanisms of blood–testis barrier and the specificity of protein profiles of fish semen. Knowledge concerning the cryogenic damage done to sperm proteins has been greatly improved.

Statement of relevance

This is the first review to cover current knowledge of fish semen proteomics, based primarily on results published during the last two years. This new information has been integrated into the existing knowledge base of basic and applied research concerning fish reproduction. The identification of several new proteins sheds new light on the biochemistry and physiology of seminal plasma and spermatozoa, and allows for a better understanding of the relationship between blood and seminal plasma. The discovery of a plethora of newly identified proteins that are released from spermatozoa in response to cryogenic damage contributes to a better understanding of the mechanisms involved in cryopreservation. It also provides researchers with a list of potential biomarkers that are important for the evaluation of cryopreservation success. This study is relevant both for many types of scientists, including fish biologists and cryobiologists, and for practitioners, including fish management and hatchery personnel.

Introduction

Proteomics is a methodology that has recently revolutionized the biological sciences enabling the complex study of hundreds and thousands of proteins. The term “proteome” originates from PROTEin complement of the genOME. Proteomics is concerned with large comprehensive studies of protein products that are expressed by the genome, with a focus on the identification and quantification of proteins and their localization, post-translational modifications and interactions (Fields, 2001). The ultimate goal of proteomic research is to unravel protein functions.

Proteomics emerged as a leading technology driven field in the postgenomic era due to the central role played by proteins and protein–protein interactions in cell physiology. Because the results of transcriptomics studies often do not correlate well with protein expression, due to the actions of numerous post-transcriptional control mechanisms, proteomics is a desirable tool for the qualitative and quantitative evaluation of protein expression. The ultimate long-term goal of proteomics is to identify and quantify proteins and to evaluate the state of posttranslational modifications and interaction networks for every protein in the cell, tissue or body fluids (Wright et al., 2012). In this way, for the first time, breakthrough advances are possible to further our understanding of the way that information is coded by a low number of genes (20–30,000 in vertebrates), which can transform into hundreds of thousands of different protein species.

Previous reviews concerning fish seminal proteins have focused primarily on the identification and characterization of particular proteins (Ciereszko, 2008, Li et al., 2009, Ciereszko et al., 2011). Recently, we have reviewed information concerning the identification of seminal proteins in fish using a traditional approach, and we have introduced the subject of proteomic studies of fish semen (Ciereszko et al., 2012). This review is aimed at providing an overview of basic proteomic methodologies and recent advances in proteomic studies of fish semen. Special emphasis will be placed on the relationship between blood and seminal proteins, sperm and seminal plasma proteins and changes in the semen proteome following cryopreservation. Most of the information will focus on the proteome of rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio) — two important aquaculture species, worldwide, and also an important research model organisms within Teleostei.

Section snippets

The principles of proteomics

Basic proteomics follows five main steps (Fig. 1):

  • 1.

    The separation of proteins.

  • 2.

    The digestion of proteins into peptides by sequence-specific endopeptidases (usually trypsin).

  • 3.

    The measurement of the exact molecular weight of the digested peptides using sophisticated mass spectrometry.

  • 4.

    The identification of proteins through peptide mass fingerprinting, using information obtained from protein and DNA sequence databases, often by the in silico digestion of sequences in genomic databases. Recently, de

Methodology of proteomic research

The two main methodological approaches are gel-based proteomics (2-DE) and gel-free mass spectrometry-based high throughput proteomics (Roe and Griffin, 2006). Gel-based proteomics is based on the separation of proteins in two-dimensional SDS-PAGE gels. Usually, the first dimension separates proteins according to isoelectric point (isoelectric focusing; proteins are reduced and alkylated after this step) and the second dimension separates proteins according to molecular mass (SDS-PAGE). Protein

Proteomics as a new tool for reproduction studies

Reproduction is a fundamental biological feature of living organisms, enabling the survival and evolution of species. Sexual reproduction is generally based on the fusion of haploid gametes and the production of embryos. Because transcription and translation are silenced (with some exceptions) in mature spermatozoa, it is implied that almost all macromolecular events in sperm cells take place at the level of proteins (Arnold and Frohlich, 2011). This also implies a potentially important role

Fish semen as an object for proteomic studies

Ray-finned fish (Actinopterygii) include more than 30,000 species, which represents half of all of the species richness of vertebrates (Near et al., 2012). Nearly all ray-finned fishes are teleosts. The semen of most teleost fish seems to be well suited for proteomic studies compared to higher vertebrates due to its simplicity. Seminal plasma is a secretory product of the testes and spermatic duct (Lahnsteiner et al., 1993, Lahnsteiner et al., 1995, Lahnsteiner, 2003, Ciereszko, 2008). Unlike

The fish seminal plasma proteome

The seminal plasma of fish is designed to provide an optimal environment for the storage of spermatozoa before spawning. This includes providing an energy source and protecting sperm motility and viability against proteolytic or oxidative attack (Ciereszko, 2008). Furthermore, it can be potentially important during external fertilization to create a microenvironment. Seminal plasma proteins have been shown to play an important role in sperm protection, and several of these proteins have been

Potential physiological role of seminal plasma proteins

Seminal plasma proteins have been shown to play an important role in sperm protection. However, more detailed information regarding the mechanisms involved in protection is lacking. An important finding from proteomic studies was the identification of major seminal proteins, including acute phase proteins (APPs), which may accompany inflammation (Jensen et al., 1997, Gabay and Kushner, 1999) and include transferrin, apolipoproteins, complement C3, alpha-1-antiproteinase, and hemopexin.

Fish sperm proteome

Proteomic studies of fish spermatozoa are a prerequisite to understanding the biochemical basis of sperm specific physiology. Such studies are also necessary to obtain complete information concerning fish semen, which is composed of seminal plasma and spermatozoa. Male reproduction varies between cyprinids and salmonids in sperm structure, complement of nuclear proteins, metabolism, mechanism of sperm activation, and parameters of sperm movement (Billard, 1992, Billlard et al., 1995, Jamieson,

Potential physiological role of sperm proteins

The majority of sperm proteins analysed by gene ontology (GO) analysis for biological process are classified as being involved in metabolic processes (62% and 54% for carp and rainbow trout, respectively). Within this group, enzymes of major metabolic pathways are recognized, including the tricarboxylic acid cycle, fatty acid oxidation, respiration, glycolysis, gluconeogenesis and amino acid metabolism. This domination of proteins involved in metabolism is consistent with the high energy

Relationship between seminal plasma and the sperm proteome

There is significant overlap between seminal plasma proteins and sperm proteins. In rainbow trout, approximately 25% of identified proteins were common to both (Nynca et al., 2014b), including multitask proteins (apolipoproteins, transferrin, and serum albumin), proteins related to the immune system (complements), antioxidant proteins (thioredoxin, superoxide dismutase, and GST), cytoskeletal proteins (tubulin and actin), family 14-3-3 proteins and enzymes (lactate dehydrogenase, creatine

Proteome changes in semen in response to cryopreservation

Sperm cryopreservation is a useful biotechnique that enables the long-term ex situ conservation of germplasm (Martínez-Páramo et al., 2009). The creation and maintenance of cryopreserved fish sperm banks may be an effective strategy to protect biodiversity in natural fish populations and commercial lines. Cryopreservation technology includes several steps, such as pre-freezing, dilution of semen with extender, freezing, cryogenic storage, thawing, and post-thaw storage (for review see Tiersch

Acknowledgements

This work was funded by EU COST Action AQUAGAMETE FA1205. This work was also supported by the Projects 2011/01/D/NZ9/00619 and 2011/01/D/NZ9/00628 from National Science Centre in Poland, the Projects IP2011 039071 and IP2011 009971 from the Polish Ministry of Sciences and Higher Education and funds appropriated to Institute of Animal Reproduction and Food Research.

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