Proteomic profiling of the signal crayfish Pacifastacus leniusculus egg and spermatophore

Animal Reproduction Science 149 (2014) 335–344

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Proteomic profiling of the signal crayfish Pacifastacus leniusculus egg and spermatophore Hamid Niksirat a,∗ , Liselotte Andersson b , Peter James b , Antonín Kouba a , Pavel Kozák a a Research Institute of Fish Culture and Hydrobiology, South Bohemian Research Centre of Aquaculture and Biodiversity of Hydrocenoses, ˇ Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceské Bud˘ejovice, Zátiˇsí 728/II, 389 25 Vodnany, ˇ Czech Republic b Department of Immunotechnology (Hus 406), Medicon Village, Lund University, 223 81 Lund, Sweden

a r t i c l e

i n f o

Article history: Received 9 May 2014 Received in revised form 28 July 2014 Accepted 29 July 2014 Available online 7 August 2014 Keywords: Arthropod Cell defence Cell signaling Protease Respiration

a b s t r a c t Proteins of the signal crayfish Pacifastacus leniusculus egg and spermatophore were identified using in-gel digestion, mass spectrometry, and Mascot search. Forty-one and onehundred-fifty proteins were identified in egg and spermatophore, respectively. The proteins were classified into nine categories including cell defence, cell signaling, cytoskeleton, DNA related activity, metabolism and energy production, protease and protease inhibitor, respiration, transportation, and others and unknown. Twenty-two proteins were found in both egg and spermatophore. The respiration and cytoskeleton groups are the most diverse categories in the protein profiles of the egg and spermatophore, respectively. No protein was assigned to DNA related activity and cell defence categories in the protein profile of the crayfish egg. Differences between protein profiles of the crayfish egg and spermatophore show different functional priorities for each of gametes. Several proteins having possible roles in gametogenesis, capacitation, acrosome reaction, and fertilization were identified. This proteomic profile of signal crayfish gametes provides a basis for further investigation of functional roles of the identified proteins in aspects of reproduction such as capacitation and fertilization. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction Freshwater crayfishes make up a large and diverse group of ecologically and commercially important animals comprising 3 families, 33 genera, and 640 species (Crandall and Buhay, 2008). However, many aspects of their biology, especially reproduction, are not well known. Proteins play important roles in the reproduction process (Govindaraju et al., 2012), and identification of protein profiles of eggs and spermatozoa can offer insight into their function and

∗ Corresponding author. Tel.: +420 389034745. E-mail address: [email protected] (H. Niksirat).

provide an initial step in understanding the molecular mechanisms of complex reproductive processes such as spermatozoon capacitation and fertilization. The gamete proteome of a species allows the identification of conserved proteins and domains, providing relevant information on the essential conserved functions and evolution of gamete proteins (Lewis et al., 2003; Oliva et al., 2009). Chevaillier (1967) studied extra-nuclear basic proteins in decapods and designated them decapodine. The actin in the acrosome of the spermatozoon of the crab Cancer pagurus was studied by Tudge et al. (1994). Cytoskeletal proteins such as actin and tubulin in the spermatozoa of four decapod crabs have been investigated (Tudge and Justine, 1994), and Kurtz et al. (2008) addressed histones and nucleosomes

http://dx.doi.org/10.1016/j.anireprosci.2014.07.024 0378-4320/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/3.0/).

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in the spermatozoon of the cancer crab. Shui et al. (2012) identified proteins relevant to the ovarian development in the red swamp crayfish Procambarus clarkii. However, our understanding of the molecular basis of most aspects of crayfish reproduction is limited. The crayfish spermatozoon lacks a true flagellum and is non-motile (Tudge, 2009), making it a potential model for the study of molecular similarities and differences with motile-type spermatozoa. The aim of the present study was to identify and compare gamete proteins in male and female signal crayfish, and to discuss their potential roles, which may be useful for future studies of fertilization, evolution, and taxonomy of this diverse group of crustaceans. 2. Materials and methods 2.1. Gamete collection Male and female signal crayfish Pacifastacus leniusculus Dana, 1852 were collected from the Babaˇcka Brook (Sklené nad Oslavou, Czech Republic; 49◦ 26 N, 16◦ 2 E). Electrical stimulation (Jerry, 2001) was used for extrusion of freshly ejaculated spermatophores from six specimens. Egg samples were collected from the base of third walking legs of three females, immediately after natural ovulation and egg deposition. Gametes were stored at −80 ◦ C until processing. 2.2. Protein extraction and measurement Spermatophore and egg samples were immersed in liquid nitrogen, pulverized (Cryopulverizer 5901-4H, Biospec Product Inc.) and mixed with 1% sodium dodecyl sulfate (SDS) on ice. The supernatant was separated by centrifugation at 16100 rcf , for 30 min, and protein concentrations were determined using the Micro-Lowry Assay kit (Sigma–Aldrich). 2.3. In-gel trypsin digestion and mass spectrometry One hundred micrograms of protein from each sample were mixed with sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, 5% mercaptoethanol) and heated at 95 ◦ C for 5 min. The samples were run approximately 2 cm into a 12% SDSPAGE (Criterion Precasted TGX Gels, BioRad). The gel was stained using Gel Code Blue Stain Reagent (Pierce). Each of the lanes was cut into 10 slices, destained in 50% acetonitrile and 25 mM NH4 HCO3 , and dried using Speed-vac (SAVANT, SPD131DDA, Thermo Scientific). The gel slices were reduced with 10 mM dl-dithiothreitol in 100 mM NH4 HCO3 at 56 ◦ C for 1 h. Alkylation was performed by adding 55 mM iodoacetamide in 100 mM NH4 HCO3 and incubating for 45 min at room temperature in the dark. Gels were washed with 100 mM NH4 HCO3 , then in acetonitrile. This was repeated and gels were dried with Speed-vac. Trypsin (12.5 ng ␮l−1 modified Trypsin V5111, Promega Biotech AB) in 50 mM NH4 HCO3 was added to the dehydrated gels. The samples were left for 30 min at 4 ◦ C prior to incubation at 37 ◦ C overnight. The peptides were extracted from the gels by adding 5% trifluoroacetic

acid and 75% acetonitrile (ACN) and incubating for 30 min at room temperature. This was repeated twice to obtain three extractions, and a fourth extraction was obtained using only ACN with 10 min incubation. The extracts were combined, the volume reduced using Speed-vac, and dissolved in 10 ␮l 0.1% formic acid (FA). The samples were separated and analyzed on an Eksigent 2D NanoLC system (Eksigent Technologies, Dublin, CA, USA) coupled to an LTQ Orbitrap XL (Thermo Electron, Bremen, Germany). Peptides were loaded with a constant flow of 10 ␮l min−1 onto a pre-column (PepMap 100, C18, 5 ␮m, 5 mm × 0.3 mm, LC Packings, Amsterdam, Netherlands) and subsequently separated on a RP-LC analytical column (10 ␮m fused silica emitter, 75 ␮m × 16 cm) (PicoTipTM Emitter, New Objective, Inc. Woburn, MA, USA), packed inhouse with Reprosil-Pur C18-AQ resin (3 ␮m Dr. Maisch Gmbh, Ammerbuch-Enteringen, Germany) with a flow rate of 300 nl min−1 . The peptides were eluted with a 60 min linear gradient of 3–35% ACN (0.1% FA) followed by a 10 min linear gradient from 35 to 90% ACN (0.1% FA). The four most intense ions were selected for fragmentation with the dynamic exclusion duration of 2 min. 2.4. Mascot search Searches were done against the decapod section of Uniprot database using Mascot version 2.3. The mass tolerance was set as 5 ppm for parent ions and 0.5 Da for fragment ions, and one missed protease cleavage was allowed. Cys carbamidomethylation was set as fixed modification, and Met oxidation as variable. Significance threshold was set as p < 0.01. 2.5. Classification of proteins The proteins were classified into nine groups: cell defence, cell signaling, cytoskeleton, DNA related activity, metabolism and energy production, protease and protease inhibitor, respiration, transportation, and others and unknown, based on their known functions in the Uniprot database and the scientific literature. Proteins with more than one function were categorized according to their most well-defined function. 3. Results and discussion Forty-one and one-hundred-fifty proteins were identified (p < 0.01) by Mascot search in the signal crayfish egg and spermatophore, respectively. Twenty-two proteins were found in both egg and spermatophore (Tables 1 and 2). The respiration and cytoskeleton groups are the most diverse categories in the protein profiles of the egg and spermatophore, respectively. No protein was assigned to DNA related activity and cell defence categories in the protein profile of crayfish egg (Table 3). 3.1. Cell defence Zero and 16.7% of identified proteins of the crayfish egg and spermatophore were ascribed to the cell defence category, respectively. Steps in the process of producing

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Table 1 The proteins identified in the egg of signal crayfish (1: cell defence, 2: cell signaling, 3: cytoskeleton, 4: nucleic acids related activity, 5: metabolism and energy production, 6: protease and protease inhibitors, 7: respiration, 8: transportation, 9: others and unknown). Proteins with star (*) were identified only in the egg. Accession number

Protein name

Source organism

Function

F5A6B8* G9BIX3 Q8WQT2 B1A186 C0STY3 B0FRF9*, E2JE30*

Actin 3 Alpha-2-macroglobulin Apolipocrustacein Arginine kinase (fragment) Arginine kinase 2 Arginine kinase

Cytoskeleton3 Endopeptidase inhibitor6,1 Lipid transporter activity8 Kinase activity5 Kinase activity5 Kinase activity5

Q0P0L6 Q9UAR3* A0PGI9* Q26180* Q6Y0Z1 Q9NFR6∗ , A1YL84*

Beta actin Clotting protein Farnesoic acid O-methyltransferase Hemocyanin Hemocyanin 2 Hemocyanin alpha subunit (fragment) Hemocyanin B chain

Procambarus clarkii Pacifastacus leniusculus Penaeus semisulcatus P. clarkii Neocaridina denticulata Litopenaeus vannamei, Portunus trituberculatus Fenneropenaeus chinensis P. leniusculus P. monodon L. vannamei P. leniusculus Homarus americanus Pontastacus leptodactylus, Panulirus interruptus Palinurus vulgaris P. leniusculus, P. vulgaris, Cherax quadricarinatus, Portunus pelagicus, F. chinensis H. americanus, P. vulgaris Eriocheir sinensis C. sapidus Penaeus japonicus Metapenaeus ensis

Respiration7,1

P83180*, P10787 Q7M4G8* Q8MUH8, P80888*, 17FMY1*, A2I5Y9, B9VR33*

Hemocyanin chain b (fragments) Hemocyanin

Q95P17, P82298* K4EJG5 Q9NGL5* B0L612 A9Q7C4*

Hemocyanin subunit 3 (fragment) Hemocyanin subunit 6 Hemocyanin subunit Hemocyanin subunit Y Hemocyanin-like protein (fragment) Sarcoplasmic calcium-binding protein (fragment) Tubulin beta-1 chain Vitellogenin

P86909 Q25009 Q9GSG2, A3RKP7, Q95P34*, A1X8W2, A1XFT3, Q5XXZ5, Q7Z017, D2SSM3, Q1L7X1*, Q4W8F1, Q762M9*

and depositing gametes are vulnerable to exploitation by parasites, pathogens, and environmental stress. Copulation may lead to transmission of infections that impair health or reproduction. Therefore, parental investment in cell defence is necessary to ensure survival of gametes (Hathaway et al., 2010). Heat shock proteins are induced in cells exposed to elevations in temperature, chemical or physical stress, viral infection, drugs, and transforming agents, where they mediate cytoprotective effects through maintenance of protein homeostasis and blockade of caspase dependent apoptosis (Lindquist and Craig, 1988; Beere, 2004). It has been shown that heat shock proteins are essential to spermatogenesis, with a crucial function in sperm maturation in red claw crayfish Cherax quadricarinatus (Fang et al., 2012). Kamaruddin et al. (2004) reported presence of heat shock protein 70 and its redistribution in the spermatozoon during capacitation and acrosome reaction in mature bull spermatozoa. Oxygen consumption generates derivatives, active forms of oxygen metabolites and peroxidized molecules, also called reactive oxygen species (ROS) that are toxic to the cell (Chabory et al., 2010). Superoxide dismutase, which protects spermatozoon plasma membrane phospholipids

Cytoskeleton3 Lipid transporter activity8 Methyltransferase activity9 Respiration7,1 Respiration7,1 Respiration7,1

Respiration7,1 Respiration7,1

Respiration7,1 Respiration7,1 Respiration7,1 Respiration7,1 Respiration7,1

Chionoecetes opilio

Calcium cell signalling2

H. americanus C. quadricarinatus, H. americanus, Macrobrachium rosenbergii, C. sapidus, F. chinensis, Charybdis feriatus, L. vannamei, Fenneropenaeus merguiensis, P. trituberculatus, P. japonicas, Pandalus hypsinotus

Cytoskeleton3 Lipid transporter activity8,1

against the deleterious effects of oxidative stress (Cassani et al., 2005), was identified in the crayfish spermatophore. Chabory et al. (2010) reviewed the role of glutathione peroxidase in preventing lipid peroxidation for maintenance of mammalian spermatozoa integrity. Catalase, also present in the signal crayfish spermatophore, may play a protective role against oxidant-induced DNA damage as shown in the mammalian spermatozoon (Libman et al., 2010). 3.2. Cell signaling Cell signaling proteins made up 2.4% and 6% of the crayfish egg and spermatophore protein profiles, respectively. The 14-3-3 proteins present in the crayfish spermatophore are highly conserved acidic proteins that occur in a wide range of eukaryote cells and organisms and are thought to act as adaptor proteins in cell signaling. They are likely to be key molecules interacting with cellular phosphoproteins such as those involved in fertilization and acrosome reaction in bovine sperm (Puri et al., 2008). Protein phosphorylation is associated with sperm capacitation, which is necessary for spermatozoa to acquire the capability of spermatozoon-oocyte binding and acrosome reaction (Urner and Sakkas, 2003).

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Table 2 The proteins identified in the spermatophore of the signal crayfish (1: cell defence, 2: cell signaling, 3: cytoskeleton, 4: nucleic acids related activity, 5: metabolism and energy production, 6: protease and protease inhibitors, 7: respiration, 8: transportation, 9: others and unknown). Proteins with star (*) were identified only in the spermatophore. Accession number

Protein name

Source organism

Function

G8XR26* J7I3M4* F8TPA2* D9I8L2* Q8MWE3*

14-3-3 epsilon 14-3-3 14-3-3 protein (fragment) 14-3-3 zeta 14-3-3 zeta-like type I (fragment) 14-3-3-like protein 70 kDa heat shock protein form 1 (fragment) 70 kDa heat shock protein 70 kDa heat shock protein form 2 (fragment) 70 kDa heat shock-like protein (fragment) Actin Actin 1 Actin 2 Actin D (fragment) Actin T2 ALG-2 interacting protein x Alpha-2-macroglobulin

Penaeus monodon Portunus trituberculatus P. monodon Fenneropenaeus merguiensis P. monodon

Cell signaling2 Cell signaling2 Cell signaling2 Cell signaling2 Cell signaling2

P. monodon Rimicaris exoculata

Cell signaling2 Response to stress1

Mirocaris fortunata R. exoculata

Response to stress1 Response to stress1

Procambarus clarkii

Response to stress1

Portunus pelagicus P. monodon P. monodon Litopenaeus vannamei L. vannamei P. monodon L. vannamei, Macrobrachium rosenbergii, Fenneropenaeus chinensis, Fenneropenaeus indicus, Pacifastacus leniusculus Homarus americanus H. americanus Gecarcinus lateralis F. chinensis P. leniusculus

Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cell signalling2 Endopeptidase inhibitor6,1

Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Protease activity6 Lipid transporter8 Kinase activity5

A0PJ13* A1XQQ6* A1XQQ5* B8XGP8* Q6UZ76* A2I5Y2* O96657* O96658* Q6RXK4* Q6DTY3* A5XB12* A0T1M0*, A0T1M1*, E6Y2A5*, E7D084*, G9BIX3 Q962V5* Q95VU1* O01941* D6BMV2* G9BIX4* O01942* O01943* O01944* I6LWU6* Q8MLY5* Q8MVU2* Q8MVU1* F5ANI9* C0MP63* Q8WQT2 B1A186, B3TNG0*, G3C6L4*

Alpha actin Alpha tubulin IV Alpha-1-tubulin Alpha2 macroglobulin isoform 2 Alpha-2-macroglobulin 2 isoform Alpha-2-tubulin Alpha-3-tubulin (fragment) Alpha-4-tubulin (fragment) Alpha-I tubulin Alpha-tubulin (fragment) Alpha-tubulin 1 (fragment) Alpha-tubulin 2 (fragment) Alpha-tubulin Angiotensin converting enzyme Apolipocrustacein Arginine kinase (fragment)

C0STY3 E0W6V9*, Q0GBZ3*

Arginine kinase 2 ATP synthase subunit beta

G. lateralis G. lateralis G. lateralis Cherax quadricarinatus P. monodon Homarus gammarus H. gammarus Eriocheir sinensis Pontastacus leptodactylus Penaeus semisulcatus P. clarkii, Oedignathus inermis, Pagurus bernhardus Neocaridina denticulata P. japonicus, P. leniusculus

Q0GBZ2*

ATP/ADP translocase

P. leniusculus

A5Y447* Q0P0L6, Q6JKG0* A7UMF7*, D9ZNA8* P81182*, E9K8G3*

Beta actin (fragment) Beta actin Beta tubulin (fragment) Beta-1,3-glucan-binding protein Beta-actin (fragment) Beta-I tubulin (fragment) Beta-tubulin (fragment) Bip Catalase Chitin deacetylase 1 Chitinase 3 (fragment) Cyclophilin (fragment) DEAD (Asp-Glu-Ala-Asp) box polypeptide 39 Double WAP domain-containing protein Enolase (fragment) Glucan pattern-recognition lipoprotein

Scylla serrata F. chinensis, C. quadricarinatus P. monodon, Cancer borealis L. vannamei, F. chinensis

C0KUK7* Q8I6V4* D9ZNB1* J7K1E9* Q6R2J1* D7NXS1* D6N9T3* Q5U8X7* K9J9V3* A9Y5J3* Q6QWP6* I6YF21*

Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Endopeptidase inhibitor6,1 Endopeptidase inhibitor6,1

Kinase activity5 Proton-transporting ATP synthase5 Transmembrane ATP/ADP transport5,8 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Immunity1,8

Penaeus japonicus

Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 ATP binding9 Antioxidant1 Carbohydrate metabolism5 Carbohydrate metabolism5,1 Protein folding9 ATP-dependent helicase activity4 Peptidase inhibitor6,1

C. sapidus F. chinensis

Glycolysis activity5 Immunity1

P. trituberculatus Callinectes sapidus C. borealis L. vannamei L. vannamei C. quadricarinatus P. monodon Farfantepenaeus paulensis Macrobrachium nipponense

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Table 2 (Continued) Accession number

Protein name

Source organism

Function

G0ZJ36*

Glutathione peroxidase (fragment) Glyceraldehyde 3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (fragment)

C. quadricarinatus

Response to oxidative stress1

E. sinensis, P. clarkii, H. americanus Cherax destructor, R. exoculata, P. bernhardus, P. japonicus, C. sapidus M. nipponense Cancer irroratus

Glucose metabolic process5

Response to stress1 Response to stress1

F. chinensis Cancer pagurus

Response to stress1 Response to stress1

Pachygrapsus marmoratus P. trituberculatus, F. chinensis, C. quadricarinatus, M. rosenbergii, P. monodon P. leniusculus Panulirus interruptus Panulirus interruptus P. pelagicus, M. nipponense, P. leniusculus Palinurus vulgaris

Response to stress1 Response to stress1

P. vulgaris

Respiration7,1

P. vulgaris

Respiration7,1

E. sinensis P. japonicus Macrobrachium malcolmsonii L. vannamei L. vannamei Stygiocaris lancifera, M. nipponense, Xiphocaris elongate, C. quadricarinatus, Menippe mercenaria L. vannamei P. clarkii, Metapenaeus ensis P. monodon

Respiration7,1 Respiration7,1 Nucleosome assembly4 Nucleosome assembly4 Nucleosome assembly4 Nucleosome assembly4

F. chinensis P. leniusculus

Unknown9 Protease inhibitor6,1

H. americanus Callinectes sapidus C. quadricarinatus E. sinensis P. leniusculus P. leniusculus Carcinus maenas, H. americanus

Proteolysis6 Cation: chloride symporter activity8 Cytoskeleton3 Unknown9 Peptidase inhibitor6,1 Unknown9 Proteolysis6

Alpheus vanderbilti

GTP binding9

Chionoecetes opilio

Calcium cell signalling2

P. leniusculus

Protease inhibitor6,1

P. leniusculus H. americanus H. americanus H. americanus P. leniusculus E. sinensis, C. quadricarinatus Alpheus utriensis

Serine-type endopeptidase6 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Oxidoreductase1 Oxidoreductase1 Compound biosynthetesis9

P. clarkii C. quadricarinatus

Cytoskeleton3 Cytoskeleton3

D7RF64*, Q7YT60*, F5A6F1*, P00357* B9URB4*, D6BP36*, G3C6W2*, Q4ZGB5*, Q6QWN6* Q0Z8X0* B8Y8L0* Q3L2Q2* B7ZEC1* B7ZEC0* B8YEL0*, D2DWR3*, G8XRI7*, H2B5Z8*, Q6S4R6*, Q71KW5* Q6Y0Z1 P04254* P10787 A2I5Y9, F5CEX2*, Q8MUH8 Q95P19* Q95P18* Q95P17 K4EJG5 B0L612 B8ZXE9* Q6PV61* P83863* B0YQY6*, B8ZXF3*, D4N4Z7*, G0ZJ48*, I1SM78* P83865* Q2PUK3*, Q1HGN3* Q1KS35* E9K8G2* C7T5D3*, C7T5D5*, C7T5D7* Q8MQV0* Q9U6A3* G0ZJ86* Q5QKQ7* P91776* Q26057* C8UZT5*, C8UZT6* B5TUY4* P86909 B5L664*

B2CW97* B6EAU2* B6EAU4* B6EAU5* Q9XYS0* K9J9J9*, H9BNW6* B5TV45* F1AQ57* G0ZJC9*

Heat shock cognate 70 Heat shock protein 70 (fragment) Heat shock protein 70 cognate Heat shock protein 70 kDa (fragment) Heat shock protein 70 kDa Heat shock protein 70

Hemocyanin 2 Hemocyanin A chain Hemocyanin B chain Hemocyanin Hemocyanin subunit 1 (fragment) Hemocyanin subunit 2 (fragment) Hemocyanin subunit 3 (fragment) Hemocyanin subunit 6 Hemocyanin subunit Y Histone 3 (fragment) Histone H2A Histone H2B (fragments) Histone H3 (fragment)

Histone H4 HSP70 Intracellular fatty acid binding protein K9 Kazal-type proteinase inhibitor (fragment) Muscle-specific calpain Na+ /K+ /2Cl− cotransporter Neural alpha2 tubulin Ovary protein Pacifastin light chain PAPI I protein Putative angiotensin converting enzyme (fragment) Putative GDP binding protein (fragment) Sarcoplasmic calcium-binding protein (fragment) Semigranular hemocyte specific kazal-type proteinase inhibitor Serine proteinase-like 2a Skeletal muscle actin 2 Skeletal muscle actin 4 Skeletal muscle actin 5 Superoxide dismutase [Cu–Zn] Superoxide dismutase Tetrahydrofolate synthase (fragment) Thymosin Thymosin-repeated protein 1

Glucose metabolic process5

Respiration7,1 Respiration7,1 Respiration7,1 Respiration7,1 Respiration7,1

Nucleosome assembly4 Response to stress1 Transporter activity8

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Table 2 (Continued) Accession number

Protein name

Source organism

Function

G8H4B8* Q25008* Q94570* Q94572* Q25009 Q94571* A1YL86*

Troponin C2 Tubulin alpha-1 chain Tubulin alpha-2 chain Tubulin alpha-3 chain Tubulin beta-1 chain Tubulin beta-2 chain Ubiquitin carboxyl-terminal hydrolase L5-like protein (fragment) Vitellogenin

L. vannamei H. americanus H. americanus H. americanus H. americanus H. americanus H. americanus

Calcium cell signaling2 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Cytoskeleton3 Ubiquitin-dependent protein catabolic process9

C. sapidus, Fenneropenaeus chinensis, H. americanus, F. merguiensis, C. feriatus, M. ensis, L. vannamei, C. quadricarinatus, P. japonicus

Lipid transporter8,1

A1X8W2, A1XFT3, A3RKP7, D2SSM3, Q5XXZ5, Q6QQB2*, Q6RG02*, Q7Z017, Q7Z1G4*, Q9GSG2, Q9NDS1*, Q4W8F1

Sarcoplasmic calcium-binding protein, an important calcium cell signaling agent, was detected in both crayfish egg and spermatophore. The roles of calcium in spermatozoon capacitation, acrosome reaction, and egg activation have been described. During spermatozoon capacitation, intracellular calcium is increased in the acrosome region, which in turn activates related pathways. Egg-spermatozoon contact induces changes in spermatozoon calcium concentration that trigger pathways of the acrosome reaction (Breitbart, 2002). Several calciumbinding proteins have been reported as integral membrane proteins in the acrosome membrane (Sukardi et al., 2001; Nagdas et al., 2013). Upon fertilization, an increase in calcium at the point of spermatozoa entry propagates across the egg in a global wave (Stricker, 1999; Santella et al., 2004). 3.3. Cytoskeleton The proportion of cytoskeleton proteins is higher in the protein profile of the crayfish spermatophore (23.3%) compared to the egg (7.3%). Actin is one of the most abundant and conserved eukaryotic proteins (Egelman, 2004) and has been reported in spermatozoa of many vertebrates and invertebrates (Tudge and Justine, 1994). Actin filaments are present in the acrosome of the horseshoe crab, Limulus polyphemus, and undergo extension during the acrosome reaction (Sherman et al., 1999). Breitbart et al. (2005) reviewed roles of actin cytoskeleton in mammalian sperm capacitation and acrosome reaction. Thymosin has been reported to be localized at the acrosomal region of human spermatozoa and to increase its

penetration rate and fertilizing capacity by enhancing the acrosome reaction (Naz et al., 1992). Alpha-tubulin and beta-tubulin are components of microtubules. Most swimming or flagellate spermatozoa possess an axoneme, a tail with a structured 9 + 2 arrangement of microtubules. Among crustacean, true flagellate spermatozoa have been recorded only in the remipedia and in the maxillopodans. Although microtubules are present in many decapod spermatozoa, particularly in the long, and often numerous, microtubular arms, no true flagellum has been recorded (Tudge and Justine, 1994; Tudge, 2009).

3.4. DNA-related activity The protein profile of the crayfish spermatophore shows 6.7% of the proteins to be related to DNA activity. DNA in the nucleus of all spermatozoa is closely associated with histone proteins that are responsible for DNA compaction (Bloch, 1969). The decapod spermatozoa nuclear protein is unique (Tudge, 2009). Poljaroen et al. (2010) reported that nuclei of spermatozoa of the giant freshwater prawn, Macrobrachium rosenbergii, continuously lose histone proteins during spermatogenesis. It is possible that loss of these basic nuclear proteins results in unfolding and decondensation of chromatin, making the nucleus more mobile and flexible for the dynamic and explosive acrosome reaction in which the nucleus passes through the narrow perforatorial chamber in the acrosome vesicle. A condensed nucleus in which chromatin is highly compact would lack the flexibility necessary to allow it to pass through the narrow acrosomal canal (Kurtz et al., 2008).

Table 3 The proportion (%) of different groups of protein in the proteome profiles of the signal crayfish egg and spermatophore.

Cell defence Cell signaling Cytoskeleton DNA-related activity Metabolism and energy production Protease and protease inhibitors Respiration Transportation Others and unknown

Spermatophore (n = 150)

Egg (n = 41)

Identified in both (n = 22)

16.7 6 23.3 6.7 12.7 12 7.3 10 5.3

0 2.4 7.3 0 9.8 2.4 44 31.7 2.4

0 4.5 9.1 0 9.1 4.5 31.9 40.9 0

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3.5. Metabolism and energy production Twelve and seven tenths percent and 9.8% of the identified proteins of the crayfish spermatophore and egg protein profiles, respectively, fell into the metabolism and energy production category. Arginine kinase is a key enzyme for energy metabolism found throughout invertebrate species. It functions in the maintenance of ATP levels by regenerating depleted ATP supplies during periods of high energy demand (Kotlyar et al., 2000). Cell motility is a major source of energy consumption in the spermatozoon. In motile spermatozoa of horseshoe crab, L. polyphemus (Chelicerata: Xiphosura) arginine kinase is localized in the midpiece in the vicinity of the mitochondria and also along the length of the flagellum (Strong and Ellington, 1993). Spermatozoa of decapods are immotile (Tudge, 2009), and the energy source provided by arginine kinase may be consumed by other activities such as capacitation and the acrosome reaction. Gitlits et al. (2000) localized enolase mostly to the tail of mature spermatozoa of the rat, associated with microtubules. Since the decapod spermatozoon does not possess a tail, radial arms that are rich in microtubules (Tudge, 2009) can be speculated to be the site of enolase in the crayfish spermatozoon. The ADP/ATP translocase is an integral protein of the inner mitochondrial membrane that carries out the exchange between the extramitochondrial and intramitochondrial ADP and ATP, linking the processes of ATP production to those of ATP utilization (Houldsworth and Attardi, 1988). Anderson and Ellis (1967) reported that, in Cambarus sp. during the early spermatid stage, some mitochondria lose their matrix and are transformed into membranous lamellae. They proposed that these highly altered mitochondria are still capable of supplying energy to the spermatozoon. Therefore, membranous lamellae can be considered to be a possible site of ADP/ATP translocase in the crayfish spermatozoon. Chitin is present as a component of the exoskeleton in parasitic forms of organisms such as fungi, worms, and arthropods. It has been demonstrated that chitinase is a defence agent against those parasites in animals (Reese et al., 2007) and plants (Grover, 2012). This may also be a role of chitinase in the crayfish spermatophore, as the spermatophore is deposited on the body surface of the female, potentially exposing it to environmental parasitic organisms. 3.6. Protease and protease inhibitors Protease and protease inhibitors represent 12% and 2.4% of proteins identified in the crayfish spermatophore and egg, respectively. These proteins play key roles in most physiological processes, including cell migration, cell signaling, and cell surface and tissue remodeling (Barrett et al., 1998; Métayer et al., 2002). The equilibrium between proteases and their inhibitors appears to be critical for conserving the integrity of tissue and cell. Spermatozoa are rich in proteases, primarily localized within the acrosomal vesicle (Kohno et al., 1998; Tulsiani et al., 1998). The best known of the proteases is acrosin, a typical serine

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protease with trypsin-like specificity, located in the acrosome and playing an important role in fertilization (Klemm et al., 1991). Spermatozoa of crayfish possess an acrosome appearing as a dense inverted cup-shape in the anterior of the cell (Tudge, 2009; Niksirat et al., 2013a, 2013b, 2014). Maintaining these sperm proteases in an inactive state until initiation of fertilization is also critical to maintaining cell integrity to ensure reproductive function (Uhrin et al., 2000). In Astacidae and Parastacidae, spermatophores are deposited on the ventral surface of the female and are exposed to the attack of other organisms and environmental chemicals. The spermatophores remain for several days with the female before initiation of ovulation and subsequent fertilization (Vogt, 2002), and protease inhibitors may play a protective role against external threats. Detection of Alpha-2-macroglobulin as a protease inhibitor in the sex products of mammals (Métayer et al., 2002) and in the crayfish spermatophore in the present study suggests a conserved function of these proteins throughout evolution. The present study revealed the presence of angiotensin converting enzyme (ACE) in the spermatophore of crayfish. Köhn et al. (1998) demonstrated that this enzyme is chiefly located at the plasma membrane of the acrosomal region, equatorial segment, post-acrosomal region, and midpiece of the human spermatozoon. Studies showed that ACE seems to be essentially involved in reproductive processes such as capacitation (Köhn et al., 1995) and acrosome reaction (Foresta et al., 1991). Calpains belong to a family of non-lysosomal Ca2+ dependent cysteine proteases widely expressed in tissues and cells (Molinari and Carafoli, 1997; Goll et al., 2003). In mammalian spermatozoa it is located within the acrosomal region between the plasma membrane and the outer acrosomal membrane (Schollmeyer, 1986; Yudin et al., 2000; Ben-Aharon et al., 2005). Bastián et al. (2010) showed that calpain modulates capacitation and acrosome reaction in guinea pig spermatozoa. 3.7. Respiration The proportion of respiration-related proteins in the proteome of the crayfish egg (44%) is higher than in the spermatophore (7.3%). Hemocyanins are respiratory proteins found in the hemolymph of molluscs and arthropods (van Holde and Miller, 1995). Identification of these proteins in crayfish gametes indicates the importance of respiration during spermatophore storage on the body of female and fertilization. In arthropods, the involvement of hemocyanin in defence against bacteria, fungi, and viruses has been shown (Jaenicke et al., 2009). The presence of dual-function proteins such as hemocyanins in the crayfish egg implies both respiratory function and immune protection, at least during the early stages of embryological development, especially before immune and respiratory cells are developed in the embryo. 3.8. Transportation Transportation proteins make up 31.7% and 10% of the protein of the crayfish egg and spermatophore, respectively. Apolipocrustacein, a major egg yolk precursor

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protein of decapod crustaceans and a member of the large lipid transfer protein superfamily, is homologous to insect apolipophorin II/I and vertebrate apolipoprotein B. The presence of apolipocrustacein and a clotting protein structurally related to vitellogenin in several shrimp species (e.g. Penaeus monodon) and apolipophorin II/I and vitellogenin in insect species (e.g. Apis mellifera), suggests that, in addition to their involvement in lipid metabolism, extracellular large lipid transfer proteins may have acquired other functions during metazoan evolution, such as involvement in clotting cascade and immune response (Avarre et al., 2007). Vitellogenin is the precursor of major yolk proteins in all oviparous organisms. It is cleaved to form yolk proteins that are used as nutrients by developing embryos and larvae (Li et al., 2008). The presence of vitellogenin in both crayfish egg and spermatophore suggests that it may be involved in other functions. Shyu et al. (1986) revealed that, in sea urchin, vitellogenin is synthesized in both females and males and suggested that its presence in males raises the possibility that it performs functions in addition to its role as a precursor of yolk protein, perhaps serving as an analog to the serum albumin of vertebrates, as a carrier protein, or as a store for amino acids. It has been shown that vitellogenin exhibits antioxidant activity in the honeybee (Seehuus et al., 2006) and nematode Caenorhabditis elegans (Nakamura et al., 1999). Vitellogenin is linked with immune defence through antibacterial or antifungal activity in the protochordate amphioxus and in fish (Zhang et al., 2005; Shi et al., 2006; Li et al., 2008). Immune and antioxidant functions of vitellogenin may be important in the crayfish spermatophore, especially when deposited on the body of the female, exposing it to environmental bacteria, fungi, and free radicals. Lack of some cell defence proteins in the signal crayfish egg (e.g. heat shock proteins, superoxide dismutase, etc.) that are present in the spermatophore can be compensated for by activity of proteins with dual function such as vitellogenin and apolipocrustacein, which are present in large amounts in egg. Male germ cell differentiation entails the synthesis and remodeling of membrane polar lipids and the formation of triacylglycerols, requiring fatty acid-binding proteins for intracellular fatty acid traffic (Oresti et al., 2013). One of those fatty acid-binding proteins is found between the inner acrosomal and the outer face of the nuclear envelope of the mammalian spermatozoon head, suggesting that it is required for attachment of the acrosome to the spermatozoon nucleus during spermatogenesis and fertilization (Oko and Morales, 1994). 3.9. Others and unknown Farnesoic acid O-methyltransferase was identified in the proteome of the crayfish egg but not in the spermatophore. This enzyme is involved in physiological processes such as gametogenesis, oocyte maturation, development, and metamorphosis of the shrimp (Gunawardene et al., 2002). The enzyme catalyses the final step in the methyl farnesoate (MF) biosynthetic pathway in crustaceans. A close relationship was observed between MF secretion and gametogenesis in female Libinia emarginata (Crustacean: Decapoda) (Borst et al., 1987).

The penetration of the egg by more than one spermatozoon, which is called polyspermy, causes death of the early embryo. It has been shown that activity of ubiquitin c-terminal hydrolase is involved in spermatozoon-egg interaction and anti-polyspermy defence (Yi et al., 2007). 4. Conclusion The higher diversity of respiratory proteins in the protein profile of the crayfish egg implies the importance of the respiration in the early stages of ovulation and fertilization. Lack of representatives of DNA related activity (e.g. histone) and cell defence (e.g. heat shock proteins, superoxide dismutase, etc.) groups in the protein profile of crayfish egg compared to the spermatophore suggests different ways for the management of DNA and cell defence in the crayfish gametes. Several proteins with potential roles in the gametogenesis, capacitation, acrosome reaction, and fertilization were identified in signal crayfish gametes. This proteomic profile provides a basis for further investigation of functional roles of the identified proteins in aspects of reproduction such as capacitation and fertilization. Conflict of interest statement None declared. Acknowledgments The Czech Science Foundation supported this work through project P502/12/P177. Partial funding was provided by the Ministry of Education, Youth and Sports of the Czech Republic (projects CENAKVA CZ.1.05/2.1.00/01.0024 and CENAKVA II – the results of the project LO1205 were obtained with a financial support from the MEYS of the CR under the NPU I program) and project 087/2013/Z by the Grant Agency of the University of South Bohemia. Open access was covered by the project CZ.1.07/2.3.00/20.0024 Strengthening of excellence scientific teams in USB FFPW. References Anderson, W.A., Ellis, R.A., 1967. Cytodifferentiation of the crayfish spermatozoon: acrosome formation, transformation of mitochondria and development of microtubules. Z. Zellforsch. Mikrosk. Anat. 77, 80–94. Avarre, J.-C., Lubzens, E., Babin, P.J., 2007. Apolipocrustacein, formerly vitellogenin, is the major egg yolk precursor protein in decapod crustaceans and is homologous to insect apolipophorin II/I and vertebrate apolipoprotein B. BMC Evol. Biol. 7, 3. Barrett, A.J., Rowlings, N.D., Woessner, J.F., 1998. Handbook of Proteolytic Enzymes. Academic Press, London, pp. 1666 pp. Bastián, Y., Roa-Espitia, A.L., Mújica, A., Hernández-González, E.O., 2010. Calpain modulates capacitation and acrosome reaction through cleavage of the spectrin cytoskeleton. Reproduction 140, 673–684. Beere, H.M., 2004. The stress of dying: the role of heat shock proteins in the regulation of apoptosis. J. Cell Sci. 117, 2641–2651. Ben-Aharon, I., Brown, P.R., Etkovitz, N., Eddy, E.M., Shalgi, R., 2005. The expression of calpain 1 and calpain 2 in spermatogenic cells and spermatozoa of the mouse. Reproduction 129, 435–442. Bloch, D.P., 1969. A catalog of sperm histones. Genetics 61 (Suppl.), 93–111. Breitbart, H., 2002. Intracellular calcium regulation in sperm capacitation and acrosomal reaction. Mol. Cell. Endocrinol. 187, 139–144. Breitbart, H., Cohen, G., Rubinstein, S., 2005. Role of actin cytoskeleton in mammalian sperm capacitation and the acrosome reaction. Reproduction 129, 263–268.

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