long term survival of high quality sperm: insights into ... · proteomics of honeybee sperm 5 101...

Long term survival of high quality sperm: Insights 1

into the sperm proteome of the honeybee Apis 2

mellifera 3 4

5 6

7 8

Reza Zareie1,2, Holger Eubel 1,3, A. Harvey Millar1,2 & 9

Boris Baer1,2,4* 10

11 12 13

1 Centre for Integrative Bee Research (CIBER), ARC CoE in Plant Energy Biology, MCS 14 Building M316, The University of Western Australia, 6009 Crawley, Australia 15 16 2 Centre for Comparative Analysis of Biomolecular Networks, MCS Building M316, The 17 University of Western Australia, 6009 Crawley, Australia 18 19 3 Institute for Plant Genetics, Department of Plant Proteomcis, Leibniz University 20 Hannover, 30419 Hannover, Germany 21 22 4 Centre for Evolutionary Biology, School of Animal Biology (MO92), The University of 23 Western Australia, 6009 Crawley, Australia 24 25 26

E-mail contacts: 27

Reza Zareie: [email protected] 28

Holger Eubel: [email protected] 29

Harvey Millar: [email protected] 30

*Boris Baer: [email protected] (Corresponding author) 31

32

33

34

mailto:[email protected]

Proteomics of honeybee sperm 2

Abstract 35

In the social bees, ants and wasps, females (queens) only mate during a brief period early 36

in their lives and afterwards store a lifetime supply of sperm in a specialized organ, the 37

spermatheca. In some species, stored sperm can remain viable for several decades and is 38

used by queens to fertilize millions of eggs. The physiological adaptations that allow this 39

prolonged survival are unknown. To unravel them, we conducted proteomic analyses on 40

the sperm of the honeybee Apis mellifera to define proteins that are bee-specific or 41

highly-divergent from sequences in the sperm proteomes of flies or mammals, and which 42

might therefore be associated with long-term sperm survival. We identified a honeybee 43

sperm proteome of 343 members and define the subset of proteins or protein networks 44

that cannot be discerned in the sperm proteomes of fruit flies and humans. This subset 45

contained a significant number of proteins that are predicted to act in enzyme regulation, 46

or in nucleic acid binding and processing. From our analysis we conclude that long-term 47

survival of sperm in social insects could be underpinned by substantial changes in only a 48

specific subset of sperm proteins that allow physiological adaptation to storage. The 49

unexpected preponderance of proteins predicted to be involved in transcriptional 50

processes and enzyme regulation suggest these are the primary targets of this adaptation. 51

52

Keywords: Sperm viability, Sperm senescence, Comparative Proteomics, Sperm Proteins 53

54


Introduction 55

Sperm undergoes dramatic modifications during and after spermatogenesis making it a 56

highly specialized cell type 1, which evolved in response to natural and sexual selection to 57

maximize paternity success. The most obvious characteristic of sperm physiology is their 58

motility, but recent research has revealed that these cells are more than simple DNA 59

delivering vehicles 2. For example, sperm can react to environmental stimuli such as pH 60

and temperature and have evolved traits to compete against rival sperm as part of 61

postcopulatory sexual selection 1. The selective forces of natural and sexual selection 62

generated a spectacular variation in sperm form and functioning 3, 4 making sperm the 63

most diverse cell type known to date. We have ample knowledge about sperm 64

morphology and ultrastructure. However, sperm physiology and its molecular interplay 65

with male and female derived glandular secretions underlying the above-mentioned traits, 66

remain substantially understudied. 67

Eusocial hymenopteran insects, being the social bees, ants and wasps, provide unique 68

model systems to study the biology of sperm, since their social lifestyle resulted in the 69

evolution of a number of highly specialized reproductive traits 5, 6. For example, pair 70

formation and copulations only occur during a very short period and prior to colony 71

initiation 7, 8. As females (queens) never re-mate later in life they store a lifetime supply 72

of sperm in a specialized organ, the spermatheca. As a consequence social insect sperm 73

has to survive for prolonged periods of time, which can be up to several decades in some 74

ant species 9, 10 and queens have to economize sperm use so very few sperm are required 75

to fertilize an individual egg 10, 11. These characteristics of the mating biology of social 76

insects have resulted in the evolution of large ejaculate sizes and sperm of high quality 12, 77


13. Leaf cutting ants are a good example to illustrate the extreme levels of sperm survival, 78

quality and economy. In these species queens initially store several hundred million 79

sperm 14, 15, which bears significant costs for the queens 16. The sperm survives alongside 80

the queen for 20 or more years and is used to fertilize tens of millions of eggs 10. 81

The key molecular mechanisms of how social insect sperm are able to achieve such long-82

term storage and delay their senescence remain largely unknown. It seems obvious that 83

sperm must have evolved specific adaptations that allow them to achieve such 84

exceptional life history traits. Furthermore, such sperm traits have coevolved with the 85

physiological environments the sperm operate in such as the male’s seminal fluid 17 and 86

the secretions provided to sperm by queens during storage 18. The recent availability of 87

sperm proteomes of other species such as the fruit flies or humans now allows us to 88

compare sperm components from different species to identify the specific proteins or 89

protein networks that distinguish social insect sperm and underpin their long term 90

survival. 91

Here we used the honeybee Apis mellifera as a model where queens mate with up to 90 92

males during one of few nuptial flights. Ejaculates compete against each other for storage 93

inside the female’s sexual tract and proteins within the seminal fluid target sperm of rival 94

males and kill them, a process known as sperm incapacitation 19. Only about 3-5% or 95

around 6 million of the sperm initially acquired during copulations will ultimately 96

become stored in the spermatheca 5. This allows queens to consequently fertilize more 97

than 1.5 million eggs over a time frame of up to seven years. Whilst in storage, queens 98

support sperm through glandular secretions from the spermathecal gland 18, and sperm 99

appear to undergo further developmental processes to accommodate storage 20. We have 100


previously identified a number of honeybee sperm proteins as part of other experiments 101

17, 20, but these earlier datasets were too small to gain a general understanding of the full 102

componentry of bee sperm or to identify honeybee-specific sets of proteins that might be 103

present. Here we performed an extended proteomic analysis of honeybee sperm using gel 104

and non-gel based proteomic approaches with the aim of identifying a large number of 105

honeybee sperm proteins. We then used comparative approaches to identify a subset of 106

proteins, and predicted their associated biochemical functions, which are found in 107

honeybees and have not been found in sperm proteomes of fruit fly or human. 108

109

Material & Methods 110

Male breeding and sperm sampling 111

All males used for experimental work originated from colonies of Apis mellifera that we 112

kept in an animal yard at the University of Western Australia. We collected sperm from 113

males at an age of 2-3 weeks after enclosure to ensure that males had reached sexual 114

maturity. Sperm was collected by using a technique previously developed to artificially 115

inseminate honeybees 20, 21. In brief, males were anaesthetized with chloroform to initiate 116

male ejaculation, which was proceeded by gently squeezing the male abdomen between 117

two fingers. As soon as the ejaculate appeared at the tip of the male’s endophallus it was 118

collected with a glass capillary connected to a syringe. To separate sperm from its 119

surrounding seminal fluid, we applied a previously developed method 20. In short, semen 120

was diluted in Hayes solution (9.0 g/l NaCl, 0.2 g/l CaCl2, 0.2 g/l KCl, 0.1 g/l, NaHCO3, 121

pH 8.7), briefly mixed and centrifuged for 25 minutes at 3000 rpm (850 xg) and 4°C. 122

This procedure was repeated three times. Final sperm pellets were re-suspended in 50µl 123


of Hayes solution and frozen at -80°C prior to further analyses. To identify proteins 124

present in honeybee sperm we used both gel based and non-gel based approaches as 125

follows: 126

127

Gel based analysis of the honeybee sperm proteome 128

To separate sperm proteins on 2D-PAGE gels we used a previously developed protocol 17, 129

20. In brief 100 μl of sperm (equivalent to ~1 mg protein from ~250 drones) collected as 130

described above was acetone-precipitated, and dissolved in IEF solubilisation buffer. The 131

sample was loaded onto a 24-cm non-linear pH 3-11 IPG strip (GE) and run using the 132

following settings: 12 h at 30 V (rehydration step), 1 h at 500 V, 1 h gradient from 500 to 133

1000 V, 1 h gradient from 1000 to 3000 V, 2 h gradient from 3000 to 8000 V and 5 h at 134

8000 V. The strip was reduced and alkylated following the manufacturer instruction and 135

resolved on a 12% SDS polyacrylamide gel. Protein spots were visualized with 136

Coomassie Blue (G 250) colloidal staining. To identify proteins using tandem mass 137

spectrometry, individual spots were cut out of the gel, digested with Sequencing Grade 138

Modified Trypsin (Promega, V5111) and peptides identified on an Agilent LC/MSD Trap 139

XCT Ultra 6330 mass spectrometer coupled to an Agilent 1100 Series capillary LC 140

system. Peptides were resolved on a 0.5 x 50 mm Microsorb (Varian) C18 column eluted 141

over 15 minutes with a 5 to 60% acetonitrile gradient and 0.1% formic acid at 10 μl/min. 142

Eluents were sprayed into the mass spectrometer under positive ion mode via an ESI low-143

flow nebulizer and analyzed with the MS scan over 300 to 1400 m/z at the speed of 5500 144

m/z per second and ion charge control of 150000. Ions were selected for MS/MS after 145

reaching an intensity of 20000 cps. 146


147

Non-gel based analysis of the honeybee sperm proteome 148

Protein samples were also analyzed using peptide mixture LC-MS/MS analyses. To do 149

this we used 100 µl of sperm sample that we first centrifuged at 800 xg for 1 min. The 150

pellet was re-suspended in 500 µl of digestion buffer (10 mM NH4HCO3, 100 µg 151

Trypsin/ml) and incubated overnight at 37°C. This step was repeated in order to increase 152

the peptide yield. For the gel-free analysis disulfide-forming cysteine residues were 153

intentionally left unmodified to exclude highly abundant cysteine-rich protamine-like 154

proteins from subsequent experiments 23,25. After the second digestion, insoluble 155

components were removed by centrifugation at 20’000 xg for 5 min. The supernatant was 156

dried by vacuum centrifugation and stored at 4°C before peptide fractionation. For this, 157

the peptide pellet was suspended in SCX buffer A (10 mM KH2PO4 in 25% acetonitrile, 158

pH 3.0) and bound to the column according to the manufacturer’s instructions. Separation 159

of peptides into ten fractions was achieved by step-elution with 10%-100% of buffer B 160

(1M KCl in 10 mM KH2PO4 and 25% acetonitrile, pH 3.0) in buffer A on a SCX column 161

(4.6mm x 50mm, Optimize Technologies, Oregon, USA). The eluent fractions were 162

desalted using C18 cartridges (Nest Group, MA, USA) and dried by vacuum 163

centrifugation. We found measurable amounts of peptides in the fractions containing 164

10%, 20%, 30% and 40% of buffer B based on the Bradford assays and pooled all 165

remaining fractions into a single fraction. All fractions were analyzed using an Agilent 166

6510 Q-TOF mass spectrometer (Agilent Technologies) with an HPLC Chip Cube 167

source. The Chip consisted of a 40 nl enrichment column (Zorbax 300SB-C18 5 u) and a 168

150 mm separation column (Zorbax 300SB-C18 5 u) driven by an Agilent Technologies 169


1100 series nano/capillary liquid chromatography system. Both systems were controlled 170

by a MassHunter Workstation Data Acquisition for Q-TOF (ver B.01.02, Build 65.4, 171

Patches 1,2,3,4, Agilent Technologies). Peptides were eluted from the enrichment column 172

and run through the separation column during a 1h-gradient (15% (v/v) acetonitrile – 173

60% (v/v) acetonitrile containing 0.1% formic acid) directly into the mass spectrometer 174

running in positive ion mode and scanning over 275 to 1500 m/z at 4 spectra x sec-1. 175

Precursor ions were selected for auto MS/MS at an absolute threshold of 500 and a 176

relative threshold of 0.01, with maximum 3 precursors per cycle, and active exclusion set 177

at 2 spectra and released after 1 minute. Precursor charge-state selection and preference 178

was set to 2+ and then 3+ and precursors selected by charge then abundance. Resulting 179

MS/MS spectra were analyzed further using MassHunter Workstation Qualitative 180

Analysis software (ver B.01.02, Build 1.2.122.1, Patches 3 Agilent Technologies) and 181

MS/MS compounds detected by “Find Auto MS/MS” using default settings. The 182

resulting compounds were then exported as mzData.xml files for subsequent analyses as 183

outlined below. 184

Following an initial run in peptide mixture analysis, the resulting mzdata.xml files were 185

then searched against the honeybee proteome using Mascot 2.2. The resulting peptides 186

matching with ion scores ≥ 37 were exported along with their respective peptide charge 187

into a .csv file. This file was then used to construct an exclusion list, based on peptide 188

(m/z) and charge (z). Isolation width was set to ‘medium~4 m/z’ precursor type set to 189

‘Exclude’, retention time set to “0” and Δm/z set to ‘100 ppm’. This table was then 190

loaded into the MassHunter Workstation Data Acquisition for Q-TOF (ver B.01.02, Build 191

65.4, Patches 1, 2, 3, 4, Agilent Technologies) software and a next sample of peptides 192


was run on the mass spectrometer. After each MS-MS run the new list of excluded 193

peptides was added to the previous list and the new list loaded for the consequent runs. 194

195

Bioinformatics analyses 196

Spectra from the gel based experiment were analyzed using ProteinScape™ version 2.1.0 197

(Bruker Daltonics, Bremen, Germany) that in turn triggered Mascot 2.2 core algorithm 198

(Matrix Science) to match the data against the honeybee sequences (an in-house database 199

build from 10618 Apis mellifera protein sequences from NCBI RefSeq release 48 plus 200

common contaminants) with the following options: scoring: standard, enzyme: semi-201

trypsin with 1 missed cleavage, fixed modification: cysteine carbamidomethylation, 202

variable modifications: Met oxidation and Gln and Asn deamidation, precursor-ion 203

tolerance: 100 ppm, fragment-ion tolerance: 0.5 Da. To further improve the identification 204

confidence of proteins with significant Mascot scores below 70 and above 50, proteins 205

were only accepted if their observed molecular masses were within 30% of the calculated 206

values. Identifications with Mascot scores above 70 were accepted regardless of their 207

positions on the gel 57. 208

Spectra from the non-gel based analyses were converted using MSConvrt (ProteoWizard 209

release-2_1_2705 51) with the default options except for the vendor peak picking filtering. 210

Spectra were searched against honeybee sequences, as previously explained except that 211

decoy (reversed) sequences were also included, using Mascot 2.2, X! Tandem (GPM) and 212

Omssa (NCBI) with the following options: enzyme: semi-trypsin with 1 missed cleavage, 213

fixed modification: none, variable modifications: Met oxidation and Gln and Asn 214

deamidation, precursor-ion tolerance: 100 ppm for Mascot and 1.1 Da for X! Tandem and 215


Omssa, fragment-ion tolerance: 0.5 Da. Results were subsequently combined and 216

validated by the Trans Proteomic Pipeline (TPP) v4.4 with default conditions 54, 55. The 217

use of multiple search engines was found to increase the number of confidently identified 218

proteins compared to when only one search engine was used, similar to what has been 219

reported in the literature 55, 56. Protein identities with P values of at least 95% (or a false 220

discovery rate of ~0.1%) were accepted. 221

RefSeq sequence databases (release 48) were obtained from the NCBI Web site 222

(http://www.ncbi.nlm.nih.gov). Databases of the honeybee tissue proteome survey 22 and 223

a predicted honeybee proteome database were obtained from the Peptide Atlas site 224

(http://www.peptideatlas.org/). Fruit fly and human sperm protein lists were obtained 225

from the literature 23-25 while their sequences were acquired from the Internet sites of the 226

International Protein Index (ftp://ftp.ebi.ac.uk/pub/databases/IPI/) and FlyBase 227

(http://flybase.org/static_pages/downloads/ID.html). 228

Homology search and comparisons were conducted using BLAST 2.2.24+ release, 229

obtained from the NCBI FTP site (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+). To 230

determine homologous proteins in databases with 10’000 – 100’000 sequences we used a 231

cut-off value of E ≤ 1e-11 (for functional homology) or E ≤ 1e-23 (for the more stringent 232

bioprocess homology). When using databases with 1’000 to 10’000 sequences, we used a 233

cut-off value of E ≤ 1e-12 for functional homology and E ≤ 1e-24 for bioprocess 234

homology49 (and the statistics of sequence similarity scores in BLAST, see 235

http://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html for further details). When 236

comparing two databases against each other, the smaller database was used as the query 237

and we only used the best match reported for each query. 238

http://www.ncbi.nlm.nih.gov/

http://www.peptideatlas.org/

ftp://ftp.ebi.ac.uk/pub/databases/IPI/

http://flybase.org/static_pages/downloads/ID.html

ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+


Functional prediction analyses were performed using PANTHER, Pfam and Prosite. 239

PANTHER version 1.03 was downloaded from the PANTHER FTP site 240

(ftp://ftp.pantherdb.org/). Pfam version 1.3 was downloaded from the Sanger Institute 241

FTP site (ftp://ftp.sanger.ac.uk/pub/databases/Pfam). Prosite Revision: 1.75 was 242

downloaded from the Expasy FTP site (ftp://ftp.expasy.org/databases/prosite/). 243

The PANTHER database already contained GO terms but both Pfam and Prosite required 244

conversion tables to convert their functional predictions into GO terms. These conversion 245

tables and the GO standard definition file (Revision: 1.2329 dated 16:10:2011) were 246

downloaded from the GO consortium FTP site (ftp://ftp.geneontology.org/pub/go/). 247

When a GO term was assigned to a protein all its parent entries were also included in the 248

assignment. Redundant assignments resulting from the use of multiple predictive 249

algorithms were then removed and the resulting list was used for all subsequent analyses. 250

Pathways were mapped based on the Reactome Pathway Analysis tool 251

(http://www.reactome.org/ReactomeGWT/entrypoint.html) and Kegg Pathway Database 252

(http://www.kegg.jp/kegg/pathway.html). Reactome pathways are searchable by gene 253

names whereas Kegg pathways may be mapped using protein GI numbers. When needed, 254

translation between protein and gene identifiers were carried out using BridgeDB 255

(http://www.bridgedb.org/, 26). Alternatively PICR (http://www.ebi.ac.uk/Tools/picr/, 27) 256

was used. Given the small number of honeybee proteins present in Kegg and in 257

Reactome, Drosophila-homologues for honeybee proteins were used to conduct this 258

analysis. To do this, honeybee sperm sequences were searched against Drosophila total 259

proteins (RefSeq, 22316 sequences). Resulting unique sequences with E ≤ 1e-23 were 260

filtered by removing sequences that had less than 30% relative homology length (as 261

ftp://ftp.pantherdb.org/

ftp://ftp.sanger.ac.uk/pub/databases/Pfam

ftp://ftp.expasy.org/databases/prosite/

ftp://ftp.geneontology.org/pub/go/

http://www.reactome.org/ReactomeGWT/entrypoint.html

http://www.kegg.jp/kegg/pathway.html

http://www.bridgedb.org/

http://www.ebi.ac.uk/Tools/picr/


defined by BLAST) of either query or hit protein lengths or their size ratios were >200% 262

or <50% (amino acid number). A similar approach was also used to obtain Drosophila-263

homologues of all honeybee proteins (RefSeq) that were then used to predict the 264

maximum number of honeybee proteins in all pathways. 265

266

267

Results 268

1. The honeybee sperm proteome (AmSp) 269

We identified a total of 121 honeybee sperm proteins within the gel spots cut from SDS-270

PAGE gels and 277 proteins from the gel free LC – MS/MS analysis (Supplemental 271

Table S1 and Figure S1). Combining the two data sets resulted in a final list of 343 272

individual sperm proteins that we refer to as the Apis mellifera sperm proteome (AmSp) 273

below. The protein sequences for 92 of the AmSp are annotated as hypothetical proteins 274

with no known function (Supplemental Table S1). The remaining 251 AmSp proteins 275

include 53 mitochondrial proteins and 30 cytoskeletal proteins and contain a number of 276

sperm specific proteins such as testis-specific tubulin alpha chain (RefSeq: XP|396338.2), 277

outer dense fiber protein 3 and 3B (RefSeq: XP|001123232.2 and XP_001121292.2), 278

sperm-associated antigen 6 (RefSeq: XP|394968.3), dnaJ homolog subfamily B member 279

13 (RefSeq: XP|001123348.1), and organic solute carrier partner 1 (RefSeq: 280

XP|001121796.2). We found that only 47% of the AmSp had previously been reported 281

through proteomics analysis in other honeybee tissues and only 31% had been found in 282

proteomic analysis of honeybee testes 22. Consequently our analyses of purified honeybee 283

sperm samples provides a substantially enlarged set of proteins linked to the reproductive 284


biology of honeybees. 285

286

2. Interspecific comparisons of the AmSp 287

When we compared the AmSp with the sperm proteome of Drosophila melanogaster 288

(DmSp) and Homo sapiens (HsSp) we found functional homologues (as defined in 289

Materials and Methods) for only 49% (168/343) of these proteins within DmSp and 45% 290

(155/343) of these proteins within HsSp (Figure 1.A). The number of AmSp without 291

homologs in the other two species is substantially larger than expected by random from 292

the total protein complement of each species. When we compared these total protein sets 293

of the three species, D. melanogaster homologs were found for 78% (8267/10618) of all 294

A. mellifera predicted proteins, and a similar number of homologs were even found for H. 295

sapiens (72% homologs (7644/10618)) (Figure 1.B). We then divided the AmSp into two 296

groups. The AmSp group for which we found functional homologues in DmSp and HsSp 297

we termed the common protein set and the 136 AmSp without homologs in either DmSp 298

or HsSp we termed the specific protein set (Supplemental Table S2). Parallel with this the 299

common proteins are frequently found in other honeybee tissues (averaged 19.1 300

tissues/protein) whereas the specific ones are detectable in markedly fewer other tissues 301

(averaged 2.4 tissues/protein) (Table S2). 302

When we conducted a biochemical pathway analysis on the entire set of honeybee sperm 303

proteins (based on the Kegg and Reactome databases as outlined in Materials and 304

Methods) we identified at least 13 pathways that were highly represented as shown in the 305

Supplemental Table S3.A (based on a confidence level ≥ 99% and at least 4 identified 306

proteins per pathway, see Supplemental Table S3.B for complete listing of all 3 307


proteomes). We found that honeybee sperm contain proteins involved in energy and 308

amino acid metabolism, maintaining the cytoskeleton, protein folding and defenses 309

against oxidative stress. We also identified the number of human and fruit fly sperm 310

proteins that could also be mapped to these pathways (see Supplemental Table S3.A). 311

Compared to these well-studied proteomes, fewer honeybee proteins mapped to these 312

pathways, likely because of the overall smaller number of proteins we identified in 313

honeybee sperm. The only exception to this was the glycine, serine and threonine 314

metabolism pathway that appears to be over-represented in honeybee sperm, relative to 315

these other species. 316

Only 10 out of 57 honeybee sperm proteins mapped to these pathways belonged to the 317

specific protein set. In order to gain more information about the nature and possible 318

function of the latter proteins we conducted a further set of analyses based on gene 319

ontology that did not rely on pre-processed mapped sequences that were required for 320

attribution of proteins to known pathways. 321

322

3. Comparative gene ontology 323

Gene ontology (GO) analysis of three sperm proteomes revealed that 76% (259/343) of 324

AmSp, 81% of DmSp and 88% of HsSp had at least one GO annotation. Overall, the 325

major molecular functions and biological activities were similar between the proteomes, 326

indicating a substantial similarity in the machinery of the three sperm and thus their likely 327

physiological processes (Supplemental Tables S4.A and S5.A, see also Figures 2.A and 328

2.B). When we analyzed the molecular function terms of common and specific sets 329

separately we found that proteins with nucleotide (especially GTP) binding, nucleoside-330


triphosphatase, and peptidase activities were significantly more frequent in the common 331

AmSp while those with nucleic acid binding and enzyme regulator functions were 332

significantly over-represented in the specific AmSp set (Supplemental Table S4.B, see 333

Supplemental Table S6 for statistical analyses). Similarly, analyses of the biological 334

process terms for these protein sets indicated that two classes of processes related to 335

energy usage (glycolysis, alcohol, glucose and nucleotide metabolism), and ultra-336

structure (microtubule-based process, organelle morphogenesis and organization) are 337

found more often within the common set of honeybee sperm proteins. The specific sperm 338

protein set was enriched for proteins with biological process terms for cellular 339

macromolecule biosynthetic process, and nucleic acid (especially RNA) related processes 340

(Supplemental Table S5.B, also see Supplemental Tables S7.A and S7.B). Further 341

examination of the individual proteins in the latter group showed that these proteins can 342

be classified into two major sub-groups based on the predicted functions of their 343

domains. The first sub-group contained 14 proteins involved in nucleic acid protection 344

and expression, and a second sub-group of 6 proteins were involved in enzyme regulation 345

(Tables 1 and S7.B). 346

347

Discussion 348

Our identification of more than three hundred honeybee sperm proteins has allowed us to 349

gain new insight into the molecular machinery present in sperm and to provide a set of 350

analyses that can direct studies to define the mechanism of successful physiological 351

adaptation to long-term storage. Our comparative analysis found that a remarkably large 352

set of the proteins are highly specific to honeybee sperm. In most cases, these proteins 353


have not been identified previously through in-depth proteomic analysis in other tissues 354

of honeybees, including their reproductive organs, and could not readily be found to be 355

homologs of proteins in sperm of other species. We therefore provide the first empirical 356

evidence of the components in honeybee sperm that are likely to underpin its specific 357

adaptations in response to the demand to store large numbers of high quality sperm over 358

prolonged periods of time. The experimental data on the AmSp and its comparative 359

dissection provides the primary information required for future opportunities to 360

specifically study individual proteins of interest, and to unravel their effects on sperm 361

physiology and male reproductive success. 362

We found that the specific AmSp set contained honeybee sperm proteins that are 363

involved in nucleic acid interactions and enzyme regulation. Although these processes are 364

also important during mating or the early development of the zygote in all species, the 365

absence of clear homologs of the honeybee proteins in sperm of fruit flies and humans 366

suggests that these proteins are linked to the specific life history of honeybee sperm. 367

In general, sperm cells are characterised by a highly condensed nucleus and an extremely 368

reduced cytoplasm, which has led to the widespread idea that sperm is transcriptionally 369

and translationally silent. More recent studies, however, showed that mammalian 370

spermatozoa contain significant amounts of nuclear and mitochondrial mRNA 28-31 as 371

well as microRNA 32. Both mitochondrial and nuclear genes of mature sperm cells are 372

capable of generating transcripts 33, 34 suggesting that mRNA in sperm is not a leftover of 373

cytoplasmic condensation during spermatogenesis. Furthermore, such transcripts can be 374

translated into proteins, rather unexpectedly, by mitochondrial-type ribosomes 35, 36. 375

Finally, Liu et al. reported that sperm also contain the machinery that allows microRNA 376


to maturate and then form the RNA-induced silencing complex, RISC, which is important 377

for gene regulation and as an antiviral response 32. Consequently, while all sperm cells 378

seem to have some transcriptional or translational activity, its extent, biological relevance 379

and factors required for its maintenance during storage are unknown. Our identification 380

of a specific set of proteins in honeybee predicted to be involved in transcription and 381

RNA processing provide evidence of an adapted machinery for these processes that could 382

operate during storage. Further experimental work will be needed to confirm the activities 383

of these proteins and their role in honeybee sperm prior to, during and immediately after 384

storage. 385

In the specific AmSP group, a number of the proteins appear to be directly involved in 386

gene expression with four of them classified as transcription factors (TFs). These proteins 387

are expected to activate genes downstream and thereby act as initial triggers for more 388

complex physiological processes. Interestingly the presence of these transcription factors 389

and their involvement in gene regulation could explain why honeybee sperm is able to 390

survive in vitro with minimal intervention for up to nine months 37. In this context the 391

role of the sperm transcriptome may be also relevant. A survey of mammalian sperm 392

mRNA shows that a significant portion of the mRNAs code for nuclear and membrane 393

proteins involved in signal transduction as well as apoptosis and survival processes 29. 394

Although it was proposed that many of these mRNA are directly translated in the sperm 395

cell, it is impossible to discount their role early after fertilization. An example of such an 396

indirect role of sperm proteins was recently shown for the processing enzymes of a 397

microRNA that silences specific ovule genes post-fertilization 32. Alternatively, sperm 398

may contain a reservoir of (pioneer) transcription factors 48 required shortly after 399


fertilization to unlock either the sperm or ovule genetic archives. Long-lived transcription 400

factors, that maintain their function during storage, may thus be important for future 401

fertilization success. 402

The group of specific sperm proteins also contained a number of enzyme regulators that 403

have domains for signalling and signal transduction. Specifically, the enzyme regulator 404

GO grouping was dominated by GTPase regulators (RefSeq: Xp|00325039.1 405

XP|392541.4 and XP|397025.3) and kinase activity regulators (RefSeq: XP|001120113.1, 406

XP|396081.2 and XP|624139.3). All of these regulator proteins could be involved in 407

signalling pathways implicated in processes such as G-protein reception and vesicle 408

transport in sperm cells. Specifically, proteins with calcium binding and protein kinase A 409

(PKA) regulator activities may play roles in specific signalling steps in capacitation 410

and/or motility 38-40. While acrosome reaction and vesicular transport may be triggered by 411

G-protein coupled receptors and regulated by small GTPases 41-43. Alternatively, a 412

physiological role for these proteins during storage, or as long-lived agents for action 413

immediately after storage, is possible. Interestingly two of these proteins (RefSeq: 414

XP|003250394.1 and XP|001120113.1) are found to contain domains that would allow 415

the proteins to interact with each other, supporting the idea that this subset of proteins are 416

involved in related physiological processes. 417

A requirement in reducing sperm senescence in long term storage is to protect the 418

spermatozoa against oxidative damage. Both antioxidative enzyme activity and reduced 419

production of reactive oxygen species (ROS) associated with reduced metabolic rates are 420

implicated in achieving this 52,53. We found many antioxidative enzymes and one related 421

pathway in the honeybee sperm (Tables S1 and S3) but failed to identify any significant 422


differences between the antioxidative components of the 3 sperm proteomes. However, it 423

is conceivable that both translational/ transcriptional control and enzyme regulation 424

mechanisms are upstream elements for the control of the metabolic rate, ROS production, 425

antioxidative enzyme content, and also the DNA protective proteins reported here. A 426

protective system comprising of these elements should be able to (1.) maintain cell 427

viability while reducing its metabolic rate and thus limit the production of ROS during 428

the storage period; (2.) further protect the cellular components and its genetic load by 429

maintaining the levels of antioxidant and DNA protective elements in storage; and (3.) 430

when required (by appropriate signals) rapidly lift the cellular metabolic rate for 431

activation of the cellular functions, including the locomotion apparatus, while adjusting 432

the content of the cellular antioxidant and DNA protective proteins to a levels consistent 433

with the new ROS production. It could be also predicted that sperm with no need for long 434

term storage in their lifetime should possess a less sophisticated DNA-protecting 435

mechanism and less ability to significantly and safely alter their metabolic rates. 436

We consider that the use of multiple search engines and downstream statistical treatment 437

of the MS data to limit false-positive rate is a strong indicator of the presence of the 438

functional groups and pathways reported here. Future research on individual proteins of 439

interest could consequently benefit from the targeted proteomics approach used here, 440

such as multiple reaction monitoring (MRM) after the presence of individual proteins are 441

confirmed by appropriate techniques. 442

443

Conclusions: 444


We find that honeybee sperm consist of two distinct groups of proteins. Firstly there is 445

one group of well-known sperm proteins, which are clear homologs of proteins from 446

other species, and which are mainly involved in physiological processes that are 447

important for sperm metabolism and locomotion. However, we also find that more than 448

half of the sperm proteins we identified do not have homologs reported in sperm of other 449

species. A number of these honeybee specific sperm proteins are involved in transcription 450

and translation, enzyme regulation, and DNA protection. Consequently, a key adaptation 451

of social insect sperm to facilitate long term protection against cellular senescence could 452

be that these sperm possess a regulatory/signalling mechanism linked to a 453

translation/transcription control system with the ability to safely and quickly switch the 454

cellular metabolic rate from active to much less active with special attention to 455

adjustment of anti-senescence mechanisms, including antioxidative and DNA-protecting 456

components. The sperm of social insects, like other spermatozoa, is likely to have 457

maintained some level of de novo protein synthesis, during storage and/or immediately 458

afterwards but prior to fertilization but this is likely limited to absolutely crucial proteins 459

needed during storage. This represents a consolidating hypothesis of the relevant 460

observations to date and defines a path and a molecular component list for future research 461

aimed at better understanding the physiology of sperm storage in insects. 462

463

464

Additional data files 465

Further supplementary information is provided in an Excel file “Supplementary 466

Data.xls”. The file includes 11 Tables with the following content: 467


• Table S1: A complete list of all sperm proteins identified in honeybee sperm. 468

• Table S2: AmSp homologues in DmSp and HsSp; and AmSp proteins detected in 469

other honeybee tissues and as reported in Peptide Atlas). 470

• Table S3.A: Proteins mapped to over-represented pathways of honeybee sperm. 471

This table also include numbers of proteins mapped to corresponding pathways in 472

DmSp and HsSp. 473

• Table S3.B: Pathways predicted in AmSp, DmSp and HsSp. 474

• Table S4.A: Molecular functions predicted in AmSp, DmSp and HsSp. 475

• Table S4.B: Major molecular functions predicted in honeybee sperm proteins with 476

ratios of common and specific proteins. 477

• Table S5.A: Biological processes predicted in AmSp, DmSp and HsSp 478

• Table S5.B: Major biological processes predicted in honeybee sperm proteins 479

with ratios of common and specific proteins. 480

• Table S6: Fisher's exact tests for distribution of AmSp proteins mapped to 481

molecular functions and biological processes. 482

• Table S7.A: Proteins mapped to GO terms overrepresented in the common Sp 483

group. 484

• Table S7.B: Proteins mapped to GO terms overrepresented in the specific AmSp 485

group. 486

• Figure S1: The 2D PAGE used to identify honeybee sperm proteins. 487

The mass spectrometry proteomics data have been deposited to the ProteomeXchange 488

Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner 489

repository with the dataset identifier PXD000169. 490


491

Acknowledgements 492

We were supported by the Australian Research Council (ARC) through a Queen 493

Elizabeth II and Future Fellowship to BB and an Australian Professorial and Future 494

Fellowship to AHM, an ARC Linkage Project to BB and AHM and the ARC Centre of 495

Excellence in Plant Energy Biology. We thank the honeybee keepers of Western 496

Australia, especially BetterBees of Western Australia for providing the necessary 497

honeybee material for this study. 498

499


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657


658

Figure legends 659

660

661

Figure 1: Number of homologue proteins in sperm (A) and total (B) proteomes of the 662

honeybee, fruit fly and human 663

664

665

666

Figure 2: Major sub-groups of molecular functions (A) and biological processes (B) 667

identified in honeybee sperm proteome 668

669

Table 1: Major classes identified in the specific proteins of honeybee sperm proteome. Classifications derived on protein’s predicted functions are shown in the two left columns. GO terms abundantly found in the AmSp specific group are in the next five columns (see Supplemental Table S7b for further details). Protein accessions are provided in the next column. Predicted protein functions and references used to predict these functions are shown in the last two columns.

Classification GO

:000

3676

GO

:003

0234

GO

:003

4645

GO

:009

0304

GO

:001

6070

RefSeq accession Predicted function Reference

Nucleic acid

binding and/or

metabolism

DNA protection

X X X XP|003249954.1 DNA repair CDD

X X XP|001121495.2 DNA replication and repair 22, 44, 45

X XP|003251300.1 Binds DNA for protection or repair Prosite, Pfam

Transcription and splicing

X X X X XP|392622.4 Transcription regulation Prosite, Pfam, PANTHER

X X X X XP|001120219.2 Transcription regulation Prosite

X X X X XP|003250668.1 Transcription regulation Prosite, PANTHER

X X X X XP|392215.4 RNA polymerase PANTHER,

X X X XP|394637.2 Histone acetylation (transcription regulation) Prosite, Pfam, PANTHER

X X X XP|397019.4 mRNA processing (perhaps splicing) Prosite, Pfam, PANTHER

X X XP|003251829.1 mRNA splicing PANTHER

Translation and post-

translational modification

X X X X XP|001123169.2 Seryl-tRNA synthetase (translation) Prosite, Pfam, PANTHER

X X X XP|394641.3 Cysteinyl-tRNA synthetase (translation) Pfam, PANTHER

X XP|003249197.1 mRNA binding and regulation Pfam, PANTHER, 50

X XP|624738.2 PTM and expression regulation Pfam, PANTHER, 46, 47

Enzyme regulation

Small GTPase

regulation

X XP|003250394.1 Small GTPase regulator (EF-hand calcium-binding) Prosite, PANTHER

X XP|397025.3 Activation of small GTPases Prosite, Pfam, PANTHER

X XP|392541.4 Small GTPase regulation, vesicle transport Prosite, Pfam, PANTHER

Kinase activity

regulation

X XP|624129.3 E3 ligase, promoting Cdk activity Prosite, Pfam, PANTHER

X XP|001120113.1 Regulation of PKA (binds EF-hand proteins) Prosite, Pfam, PANTHER

X XP|396081.2 kinase activator Pfam, PANTHER

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