Proteome Analysis of Silkworm, Bombyx mori, Larval Gonads

Article pubs.acs.org/jpr

Proteome Analysis of Silkworm, Bombyx mori, Larval Gonads: Characterization of Proteins Involved in Sexual Dimorphism and Gametogenesis Jin-e Chen,†,‡ Jian-ying Li,†,§ Zheng-ying You,† Li-li Liu,⊥ Jian-she Liang,∥ Ying-ying Ma,∇ Ming Chen,⊥ Hua-rong Zhang,∇ Zhen-dong Jiang,† and Bo-xiong Zhong*,† †

College of Animal Sciences, Zhejiang University, Hangzhou 310058, PR China Institute of Sericultural Research, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, PR China § Institute of Developmental and Regenerative Biology, Hangzhou Normal University, Hangzhou 310036, PR China ⊥ College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China ∥ College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, PR China ∇ Zhejiang California International NanoSystems Institute (ZCNI), Zhejiang University, Hangzhou 310029, PR China ‡

S Supporting Information *

ABSTRACT: Sexual dimorphism is initialed by the components of the sex determination pathway and is most evident in gonads and germ cells. Although striking dimorphic expressions have been detected at the transcriptional level between the silkworm larval testis and the ovary, the sex-dimorphic expressions at the protein level have not yet been well characterized. The proteome of silkworm larval gonads was investigated using a shotgun-based identification. A total of 286 and 205 nonredundant proteins were identified from the silkworm testis and ovary, respectively, with a false discovery rate (FDR) lower than 1%. Only 40 and 16 proteins were previously identified, and 246 and 189 proteins were newly identified in the silkworm testis and the ovary, respectively. The gametogenesis mechanism of silkworm was demonstrated using the protein expression profile and bioinformatics analysis. Cellular retinoic acid binding protein (CRABP) showed to be highly abundant in testis, while tubulins were abundant in ovary. Several homologies of Drosophila essential proteins for gametogenesis were identified in silkworm, such as male meiotic arrest gene product ALY and VISMAY in testis, and maternal mRNA localization protein exuperantia and SQUID in ovary. The gene ontology (GO) annotation and pathway analysis provide system-level insights into the sexual dimorphism and gametogenesis. KEYWORDS: Bombyx mori, larvae, testis, ovary, shotgun proteomics, spermatogenesis, oogenesis

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INTRODUCTION

Proteomics technology has become a useful tool for profiling insect tissues and stages proteins revealing specific physiological states.4−8 Previous proteome research led to the identification of 56 and 33 proteins in silkworm larval testis9 and pupal ovary,10 respectively, using a 2-DE and mass spectrometry method. These identifications provided significant insight into silkworm spermatogenesis and oogenesis. However, gametogenesis is a complex process, and larger identification is required for fully understanding this process. Shotgun proteomics, which led to hundreds or even thousands of identifications, has proven to be an effective method for the analysis of more complex protein mixtures. This highthroughput method has been used in investigations on the human testis proteome,11 as well as on the zebrafish adult gonads proteome.12

Silkworm, raised for silk production, is a domesticated insect of economic and agricultural importance. It is also used in fundamental research and applied biotechnology as a Lepidopteran model for understanding other insects and organisms.1,2 Sexual dimorphism, which is initialed by the components of the sex determination pathway, is a fundamental aspect in sexual reproduction. It is a result of the sex-dimorphic differences in gene expression in various tissues. Gonads and germ cells have the most prominent dimorphism.3 Investigations on sexual dimorphism and gametogenesis in silkworm are attractive not only for providing knowledge on insect sexrelated events including sex determination and reproduction, but also for the economical aspects of silkworm, e.g., the male silkworm being easier to raise, having the high transformation efficiency of leaf to silk and the more superior silk grade compared to the female silkworm. © XXXX American Chemical Society

Received: September 29, 2012

A

dx.doi.org/10.1021/pr300920z | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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Silkworm gametogenesis occurs throughout the larval and the adult stage. In silkworm larva, each testis contains 4 follicles. The primary spermatogonia are located near the blind end of each follicle. After six cycles of mitotic division with incomplete cytokinesis, spermatogonia produce 64 primary spermatocytes enclosed by cyst cells. All the spermatocytes of a cyst are joined by ring canals. The spermatocyte undergoes meiotic division and consequently generates a cyst with 256 spermatids.13 Male silkworm produce dimorphic spermatozoan, as other Lepidopteran insects, namely, eupyrene and apyrene sperm. The eupyrene sperm serves for fertilization. The role of the apyrene sperm remains unclear, although it is suggested that apyrene sperm may serve as a nutrient and assist the eupyrene in fertilizing the egg.14,15 These two kinds of sperm differentiate at the following stages: the eupyrene sperm is obtained at the final (the fifth) larval instar, whereas the apyrene sperm is derived after pupation.16 Therefore, a testis of fifth instar larva contains all the stages of spermatogenesis from stem cell to the eupyrene sperm. Similarly in the female silkworm larva, each ovary contains 4 ovarioles. The developing oocyte develops as a cluster of 8 interconnected cells following three cycles of mitosis with incomplete cytokinesis. It is enclosed by a layer of follicle cells to form an egg chamber as a functional unit during oogenesis. The meiotic prophase starts in the third larval instar.17,18 The silkworm oogenesis is specified as 12 stages based on follicles morphologic characters from 1-day-old of fourth instar larvae to 10-days-old of pupae.19 During the larval stage, the period from the fourth to the fifth instar is called the previtellogenesis stage. Therefore, an ovary of fifth instar larva contains all the stages of oogenesis from stem cell to the previtellogenesis stage. The characteristics of previtellogenesis stage are mitotically active, and in this stage the yolk components appeared first as lipid droplets then as glycogen granules at the end of the fifth instar. The general process of spermatogenesis and oogenesis is similar, from stem cell division to germ cell proliferation, while the function and morphology of sperm and egg are so different. Sperm delivers a haploid genome to the egg. To fulfill this mission, it should have motility and other critical functions including egg activation and delivery of paternal factors. Egg prepares the embryo pattern formation and reserves the necessary substances for an independent development of the embryo. There should be numerous genes involved in sexspecific aspects during spermatogenesis and oogenesis. A striking difference was showed in the gene expression of silkworm gonads on the third day of the fifth instar. There were 2462 dimorphism genes detected using DNA microarray technology.20 However, sex-specific difference in the protein expression is still unclear, since mRNA is not always well correlated with proteins due to regulation at various levels. For example, translational regulation is a prevalent phenomenon and plays an essential role in spermatogenesis and oogenesis.21,22 The mRNAs of those proteins required at later transcriptional inactive stages will be synthesized in advance. In this study, we unfolded the proteome profiles of the testis and ovary of the silkworm larvae on the fifth day of the fifth instar using shotgun strategy. The function and involvement of the identified proteins in sexual dimorphism and gametogenesis was explored using bioinformatics analysis.

Article

MATERIALS AND METHODS

Silkworm and Tissues

Testes and ovaries were obtained from the polyvoltine silkworm strain P50. The silkworms were raised on fresh mulberry leaves under standard conditions (25 °C and 80% humidity). The testes and ovaries were dissected and collected from the silkworms on day 5 of the fifth instar under a stereoscopic microscope. The dissected samples (Figure 1A) were snap-frozen with liquid nitrogen after being rinsed in PBS three times and stored at −80 °C for subsequent use.

Figure 1. Protein identification in silkworm testis and ovary. (A) The silkworm testis and ovary on the fifth day of the fifth instar. (B) SDSPAGE results of protein extracts from silkworm testis and ovary. Lane 1−2 were for testes samples; lane 3−4 for ovaries. (C) The Venn diagram for the identified proteins. The blue circle represents identifications from testis, and the red one represents those from ovary.

Protein Sample Preparation and 1-D SDS-PAGE

The gonads tissues from 80 silkworms were pooled, and total protein content was solubilized in lysis buffer (2.5% SDS, 10% glycerin, 5% β-mercaptoethanol and 62.5 mM Tris-HCl pH 6.8). The extraction methods were conducted as previously reported.4 Protein concentrations were measured by 2-D Quant Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions. An amount of 200 μg of protein was used per lane for the SDS-PAGE (a 5% stacking gel overlying a 12.5% resolving gel) with two replicates for each sample. The resulting gel bands were visualized with Coomassie Brilliant Blue R250 staining (Sigma, St. Louis, MO, USA) (Figure 1B). B



Article

Table 1. Proteins Identified with at Least Two Unique Peptides in Silkworm Testis accession number gi|112982661 gi|112982669 gi|112982701 gi|112982800 gi|112982812 gi|112982828 gi|112982844 gi|112982880 gi|112983028 gi|112983032 gi|112983092 gi|112983318 gi|112983342 gi|112983416 gi|112983420 gi|112983462 gi|112983479 gi|112983487 gi|112983493 gi|112983495 gi|112983501 gi|112983546 gi|112983556 gi|112983562 gi|112983600 gi|112983696 gi|112983736 gi|112983782 gi|112983898 gi|112983926 gi|112983958 gi|112984000 gi|112984012 gi|112984078 gi|112984098 gi|112984112 gi|112984274 gi|112984292 gi|112984336 gi|112984370 gi|112984376 gi|112984390 gi|112984394 gi|112984422 gi|112984454 gi|112984502 gi|114050749 gi|114050993 gi|114051013 gi|114051117 gi|114051229 gi|114051245 gi|114051600 gi|114051634 gi|114051740 gi|114051966 gi|114052072 gi|114052088 gi|114052160 gi|114052242

protein description ribosomal protein S6a ribosomal protein S2 40S ribosomal protein S14 ribosomal protein L4a ribosomal protein L6a heat shock cognate protein ribosomal protein L7a translationally controlled tumor protein homologue glutathione S-transferase sigma 1 calreticulin precursor ALYa beta-tubulin beta-tubulina ras proteina heat shock protein hsp 19.9a ribosomal protein L7A alpha-tubulina ribosomal protein L8 protein kinase c inhibitora ribosomal protein L9a alpha-tubulin ribosomal protein L12 90-kDa heat shock protein ribosomal protein L13a cellular retinoic acid binding protein SQUID protein homologue eukaryotic translation initiation factor 4Aa cyclophilin A elongation factor 1 gamma arginine kinasea Annexin IX isoform A ribosomal protein S18 heat shock 70 kD protein cognate 40S ribosomal protein S4a 40S ribosomal protein S3aa ribosomal protein S3a ribosomal protein L23 ribosomal protein L22 ribosomal protein P2 ribosomal protein L13Aa ribosomal protein L14 elongation factor 1-alpha ribosomal protein S16a 60S ribosomal protein L17a protein disulfide isomerase low molecular 30 kDa lipoprotein PBMHP-6 precursor chaperonin subunit 6a zetaa proteasome zeta subunita histone H3a calcyphosphine isoform 2a microtubule-associated protein RP/EB family member 3a proteasome alpha 3 subunita LRP16 proteina mitochondrial inorganic phosphate carriera H+ transporting ATP synthase gamma subunita mitochondrial aldehyde dehydrogenase H+ transporting ATP synthase beta subunit isoform 1 vacuolar ATP synthase subunit Ea proteasome subunit alpha type 6-Aa glutathione S-transferase omega 1 C

protein probability

total independent spectra

number of unique peptides

NSAF (× 10−3)

1 1 1 1 1 1 1 0.9989 0.9946 1 1 0.9857 0.9908 0.9998 1 1 1 1 0.9464 1 0.9999 1 1 1 1 0.9999 0.9999 1 1 1 1 1 0.9997 1 1 1 1 1 1 0.9999 1 1 0.9967 0.9994 0.9999 1 1 1 1 1 1 1 1 1 1 1 1 0.9989 1 1

8 9 9 5 5 14 13 2 3 5 2 4 5 2 19 10 6 5 2 5 8 6 36 9 230 3 3 9 3 7 7 6 2 15 5 23 2 6 2 2 5 15 5 3 3 28 4 3 17 7 3 7 3 2 3 15 14 3 4 10

5 5 2 2 3 8 7 2 2 2 2 2 2 2 7 6 4 3 2 4 3 2 12 6 18 2 3 2 3 5 5 2 2 8 3 13 2 3 2 2 2 6 2 2 2 6 3 3 3 6 3 3 3 2 3 8 10 3 4 5

4.775 5.171 8.907 1.731 2.769 3.244 7.251 1.753 0.527 1.889 1.18 1.486 1.712 1.632 16.102 5.598 2.068 2.916 2.342 3.954 2.679 5.478 7.554 6.136 263.293 1.571 1.073 8.169 1.065 2.299 3.244 6.574 0.459 9.204 2.851 14.226 2.145 6.136 2.691 1.468 4.538 4.865 4.948 2.409 0.913 16.394 1.132 1.425 18.761 5.041 1.289 4.146 1.848 0.95 1.524 4.62 4.075 2.001 2.445 5.9



Article

Table 1. continued accession number gi|114052252 gi|114052278 gi|114052328 gi|114052408 gi|114052472 gi|114052504 gi|114052545 gi|114052751 gi|114052967 gi|114053127 gi|114053151 gi|114053173 gi|114053181 gi|114053215 gi|114053221 gi|114053253 gi|148298648 gi|148298685 gi|148298693 gi|148298717 gi|148298726 gi|148298736 gi|148298750 gi|148298752 gi|148298800 gi|148298875 gi|148298878 gi|151301010 gi|153791847 gi|153792203 gi|153792609 gi|153792659 gi|156119322 gi|158631166 gi|160333413 gi|160333678 gi|160333861 gi|163838694 gi|164420679 gi|164420683 gi|187281708 gi|187281814 gi|187281844 gi|219362829 gi|237648982 gi|237648984 gi|284813565 gi|350534642 BGIBMGA000103 BGIBMGA000398 BGIBMGA000672 BGIBMGA000710 BGIBMGA000803 BGIBMGA001119 BGIBMGA001983 BGIBMGA002005 BGIBMGA002337 BGIBMGA002569 BGIBMGA002570 BGIBMGA002640

protein description calmodulin ATP synthase hydroxypyruvate isomerasea mitochondrial aldehyde dehydrogenasea peptidylprolyl isomerase Ba proteasome 25 kDa subunit chaperonin subunit 4 delta GTP-binding nuclear protein Rana elongation factor Tua aspartate aminotransferasea electron-transfer-flavoprotein beta polypeptidea bmp-2 proteina histone H2A-like protein eukaryotic translation initiation factor 3 subunit Fa prohibitin protein WPH 6-phosphogluconate dehydrogenasea ribosomal protein S13a fructose 1,6-bisphosphate aldolasea heat shock protein hsp20.8 vacuolar ATP synthase subunit B ADP-ribosylation factor annexin isoform 1a 6-phosphogluconolactonasea 14-3-3 epsilon protein enolase ribosomal protein L31 vacuolar ATP synthase catalytic subunit A beta-tubulina abnormal wing disc-like protein vesicle amine transport protein tropomyosin-2 isoform 4a actin-depolymerizing factor 1 low molecular 30 kDa lipoprotein PBMHPC-20 precursora ADP/ATP translocase fumarylacetoacetasea glutathione S-transferase sigma 2 ribosomal protein L10a cytochrome c1a fibroinasea ribosomal protein S11 isoform 1 triosephosphate isomerase actin, muscle-type A1a actin, cytoplasmic A4 clathrin heavy chain histone H2B-like protein histone H4-like protein aliphatic nitrilasea DJ-1 beta hypothetical protein [Danaus plexippus]a CG1140 [Drosophila melanogaster] knockdown [Drosophila melanogaster]a Vap-33-1 [Drosophila melanogaster]a viral IAP-associated factor [Drosophila melanogaster]a GDP dissociation inhibitor [Drosophila melanogaster] serine protease inhibitor 3 [Drosophila melanogaster] stress-sensitive B [Drosophila melanogaster]a enolase [Drosophila melanogaster]a CaBP1 [Drosophila melanogaster] CG33303 [Drosophila melanogaster] ubiquitin carboxy-terminal hydrolase [Drosophila melanogaster]a D

protein probability



NSAF (× 10−3)

1 1 0.9999 1 1 1 1 0.9721 1 0.994 0.9857 1 1 1 1 1 0.9999 0.9981 0.999 1 0.9999 0.9416 0.9998 1 1 1 1 0.9968 1 1 1 1 1 1 1 1 0.9957 1 1 0.977 0.9998 0.9975 1 0.9922 0.9996 1 1 0.9999 1 0.9999 1 1 0.9999 1 1 1 1 1 1 0.9925

4 9 2 5 3 2 4 2 2 2 2 4 9 2 5 3 4 6 2 7 2 2 2 69 10 4 5 2 21 7 8 11 14 21 3 6 2 3 2 5 2 33 6 3 2 23 4 3 2 3 2 3 2 3 3 8 7 4 3 3

2 6 2 5 2 2 4 2 2 2 2 3 2 2 5 3 2 3 2 5 2 2 2 14 7 2 5 2 5 5 8 2 4 8 3 4 2 3 2 2 2 2 3 2 2 4 4 2 2 3 2 3 2 3 3 5 6 4 3 3

8.074 2.443 1.153 1.472 2.256 1.284 1.075 1.407 0.638 0.7 1.185 2.378 10.303 1.092 2.739 0.996 3.959 2.474 1.623 2.131 1.649 0.944 1.289 39.494 3.471 4.831 1.296 0.677 20.518 2.36 4.794 11.175 8.197 10.562 1.565 4.404 1.37 1.461 0.882 3.854 1.724 13.252 2.409 0.268 2.435 33.284 1.538 2.372 0.235 0.873 0.646 1.819 1.407 1.018 1.195 3.883 2.429 1.434 0.973 1.958



Article

Table 1. continued accession number

protein description

BGIBMGA002644 BGIBMGA003335 BGIBMGA003361 BGIBMGA003564 BGIBMGA003901 BGIBMGA004376 BGIBMGA004395 BGIBMGA004397 BGIBMGA004399 BGIBMGA004400 BGIBMGA004415 BGIBMGA004454 BGIBMGA004603 BGIBMGA004655 BGIBMGA004711 BGIBMGA004847 BGIBMGA004960 BGIBMGA005116 BGIBMGA005395 BGIBMGA005537 BGIBMGA005727 BGIBMGA005784 BGIBMGA006022 BGIBMGA006363 BGIBMGA006583 BGIBMGA006613 BGIBMGA006823 BGIBMGA006966 BGIBMGA007103 BGIBMGA007349 BGIBMGA007390

14-3-3zeta [Drosophila melanogaster] proteasome beta1 subunit [Drosophila melanogaster]a Karyopherin beta 3 [Drosophila melanogaster] ornithine aminotransferase precursor [Drosophila melanogaster] ATP synthase-beta [Drosophila melanogaster]a proteasome beta5 subunit [Drosophila melanogaster]a 30K protein 6 [Bombyx mori]a 30 kDa protein [Bombyx mori]a 30K protein 7 [Bombyx mori]a 30K protein 3 [Bombyx mori]a short stop [Drosophila melanogaster]a 30K protein 15 [Bombyx mori]a beta-tubulin at 56D [Drosophila melanogaster]a CG3523 [Drosophila melanogaster] pontin [Drosophila melanogaster]a sec13 [Drosophila melanogaster]a CG12173 [Drosophila melanogaster] glutamyl-prolyl-tRNA synthetase [Drosophila melanogaster]a loquacious [Drosophila melanogaster]a CG9391 [Drosophila melanogaster]a CG5840 [Drosophila melanogaster] lethal (2) essential for life [Drosophila melanogaster] La autoantigen-like [Drosophila melanogaster] CG6971 [Drosophila melanogaster]a CG1532 [Drosophila melanogaster]a hypothetical protein [Danaus plexippus]a CG7433 [Drosophila melanogaster]a nuclear transport factor-2 [Drosophila melanogaster]a CG4365 [Drosophila melanogaster]a heat shock protein 60 [Drosophila melanogaster] microsomal triacylglycerol transfer protein [Drosophila melanogaster] putative Pantothenate kinase 4 [Danaus plexippus]a CG42260 [Drosophila melanogaster]a CG4390 [Drosophila melanogaster] glutamate carboxypeptidase [Danaus plexippus]a alpha spectrin [Drosophila melanogaster] nucleosome remodeling factor-38kD [Drosophila melanogaster]a CG1516 [Drosophila melanogaster]a hypothetical protein [Danaus plexippus]a porin [Drosophila melanogaster]a 26−29kD-proteinase [Drosophila melanogaster] proteasome 35kD subunit [Drosophila melanogaster]a oscillin [Drosophila melanogaster]a putative plasma membrane associated protein, S3−12-like protein [Danaus plexippus]a small glutamine-rich tetratricopeptide containing protein [Drosophila melanogaster]a male fertility factor kl2 [Drosophila melanogaster] CG7470 [Drosophila melanogaster]a CG10672 [Drosophila melanogaster]a Tcp-1eta [Drosophila melanogaster] terribly reduced optic lobes [Drosophila melanogaster] CG6287 [Drosophila melanogaster] CG7261 [Drosophila melanogaster]a glycoprotein 93 [Drosophila melanogaster]a helicase at 25E [Drosophila melanogaster]a flotillin 2 [Drosophila melanogaster]a SF2 [Drosophila melanogaster]a immune-related Hdd13 [Hyphantria cunea]a phosphoglyceromutase [Drosophila melanogaster]a

BGIBMGA007521 BGIBMGA007588 BGIBMGA007723 BGIBMGA007728 BGIBMGA007948 BGIBMGA007988 BGIBMGA008442 BGIBMGA008554 BGIBMGA008955 BGIBMGA009139 BGIBMGA009234 BGIBMGA009467 BGIBMGA009933 BGIBMGA010000 BGIBMGA010475 BGIBMGA011064 BGIBMGA011785 BGIBMGA012182 BGIBMGA012240 BGIBMGA012309 BGIBMGA012514 BGIBMGA012753 BGIBMGA012772 BGIBMGA012838 BGIBMGA013096 BGIBMGA013098 BGIBMGA013244

E

protein probability



NSAF (× 10−3)

1 1 0.9998 1 1 1 1 1 1 1 1 1 0.9999 1 1 0.9408 0.9955 0.9998 1 1 1 1 1 1 1 0.9976 0.9919 0.9982 0.9813 0.999 1

29 3 2 14 9 2 11 16 7 7 2 5 7 5 4 2 2 2 2 4 2 7 2 4 4 2 3 2 2 4 2

5 3 2 9 7 2 4 7 4 4 2 5 4 5 4 2 2 2 2 3 2 3 2 3 4 2 3 2 2 2 2

13.902 2.001 0.298 5.162 3.048 1.062 6.273 9.368 4.098 2.739 0.24 3.156 2.355 0.353 1.317 0.959 0.181 0.426 0.843 2.199 1.073 5.9 0.777 2.474 2.116 0.198 0.949 2.307 1.175 1.051 0.374

1 0.9418 1 0.9991 1 1 1 1 1 1 1 1 0.9977

15 2 4 3 9 5 2 2 27 2 4 6 2

3 2 3 3 9 5 2 2 12 2 3 4 2

14.563 1.519 2.333 0.969 0.55 2.609 0.25 0.763 14.381 0.616 2.351 3.241 0.385

1

4

3

2.052

1 1 1 1 1 1 1 1 0.9999 1 1 0.9954 1

3 2 3 8 2 8 2 9 2 2 3 3 7

3 2 3 7 2 4 2 5 2 2 3 2 6

0.134 0.385 1.819 2.215 0.0774 2.783 0.285 1.63 0.707 1.247 2.092 1.763 4.114



Article

Table 1. continued accession number BGIBMGA013341 BGIBMGA013342 BGIBMGA013364 BGIBMGA013413 BGIBMGA014040 BGIBMGA014214 a

protein probability

protein description Retinoid- and fatty acid-binding glycoprotein [Drosophila melanogaster]a Retinoid- and fatty acid-binding glycoprotein [Drosophila melanogaster] hypothetical protein [Danaus plexippus]a alpha-tubulin at 84B [Drosophila melanogaster] viking [Drosophila melanogaster]a thiolase [Drosophila melanogaster]a



NSAF (× 10−3)

1

10

4

2.6

1

68

43

3.935

1 0.9999 0.9999 1

3 10 2 6

3 3 2 4

1.667 3.342 0.17 3.711

Indicates the testis-specific proteins.

In-Gel Digestion and LC−MS/MS Analysis

PeptideProphet and the ProteinProphet programs. The FDR was estimated using the following formula: 2 × hit number from decoy database/(hit number from the target−decoy database) × 100. For an estimation of relative protein abundance, the normalized spectral abundance factor (NSAF) of each protein were calculated on the basis of its spectra count and protein length and then normalized by total SAF.26,27

Each gel band was cut into 12 fractions and then was in-gel tryptically digested.23 The LC−MS/MS analysis followed as described by Li et al.24 Separation of digested peptides was performed by the Ettan MDLC nanoflow/capillary LC system (GE healthcare) consisting of a trapping column (300-μm inner diameter × 5 mm, 3 μm particles, 100 Å pore, PepMap C18, P/ N 160454, Sunnyvale, CA, USA) and a nanocolumn (75-μm inner diameter × 15 cm, 3 μm particles, 100 Å pore, PepMap C18, P/N 160321, Sunnyvale, CA, USA). The digest was desalted on the trapping column for 7 min at a 10 μL/min flow rate with buffer A (5% ACN, 0.1% formic acid in water), then transferred to the nanocolumn equilibrated with 95% buffer A (0.1% formic acid in water) and 5% buffer B (84% ACN, 0.1% formic acid in water), and eluted at a constant flow rate of 300 nL/min in solvent B gradient delivered from 5 to 60% for 50 min, then from 60 to 95% for 10 min, and held at 95% for 15 min. Peptides were detected using a hybrid LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Full scan MS spectra were acquired over the 300−1600 m/z range with the resolution of 60 000. The MS/MS were conducted in data dependent acquisition mode with the dynamic exclusion setting: repeat count 2, repeat duration 30 s, exclusion duration 180 s. The five most intense ions were sequentially selected for collision-induced dissociation MS/MS with normalized collision energy of 35% and 2 Da isolation width. The full scan mass spectra were collected in profile mode, and MS/MS data were collected in centroid mode.

Real-Time Quantitative PCR (qRT-PCR)

Total RNA of each sample was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA extracts were treated with Recombinant DNase I (RNase-free; TaKaRa Code: D2270A) and then reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA) with random primers followed by PCR amplification using specific gene primers. The gene-specific primers (Table S1, Supporting Information) were designed using the Primer Express software (version 2.0; Applied Biosystems, Foster City, Calif.) and Beacon designer (Palo Alto, CA, USA). The qRT-PCR was performed using an iQTM 5 Multicolor Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and the SYBR Premix Ex Taq (Perfect Real Time, Takara Bio) kit. Three replications of the cDNAs of each sample and Rpl27a gene (accession no. NM_001044057) were performed in the same plate. The relative expression level of each gene was calculated using 2(Ct internal control−Ct target gene) by normalizing with the Rpl27a gene according to the R = 2−ΔΔCt method.28 Mean ± SD was calculated on the basis of the three replicate samples. Bioinformatics Analysis

Database Construction and Protein Identification

The protein sequences were subjected to BLAST query against the AmiGO database (http://amigo.geneontology.org/cgi-bin/ amigo/blast.cgi) online. The corresponding gene ontology (GO) terms were extracted from the most homologous proteins using a Perl program. The GO annotation results were plotted using the Web Gene Ontology Annotation Plot (WEGO http://wego.genomics.org.cn/cgi-bin/wego/index.pl) tool by uploading compiled WEGO native format files containing the obtained GO terms.29 Chromosome mapping was performed in physical map in KAIKObase, an integrated silkworm genome database (http:// sgp.dna.affrc.go.jp/KAIKObase/) using corresponding cds with a Blastn search. The chromosome loci of the genes mapped onto the sex chromosome were showed using SilkMap tool (http://silkworm.swu.edu.cn/silkdb/doc). The regulatory network of proteins was built by using Pathway Studio software (version 7.0, Ariadne Genomics, Inc., Rockville, MD). The protein names of Drosophila melanogaster homologies were searched against the Drosophila database, which contains the relationship of the protein interaction and the regulatory network published in the literature.30 The D.

An in-house protein sequence database was constructed containing silkworm reference protein sequences recently released by the NCBI (November 1, 2011; 2114 entries) and the predicted protein sequences (14 623 entries) from silkworm genome database (http://www.silkdb.org/silkdb/ doc/download.html), supplemented with common contaminants (e.g., keratins) (247 entries) (http://maxquant.org/ contaminants.zip) maintained by Max Planck Institute of Biochemistry, Martinsried. A composite target−decoy database was built with the forward and reverse sequences for calculating the FDR.25 The MS/MS raw files were searched against the in-house database using a local installation of Mascot (Matrix Science; Version 2.3). The parent ion mass tolerances were set to 50 ppm with fragment ion set to 0.6 Da. Two missing cleavages were allowed. Carbamidomethyl on cysteine and oxidation on methionine were selected as fixed modification and variable modifications, respectively. The search results were exported as *.dat files and statistically analyzed using the software TransProteomic Pipeline (TPP, v4.0 JETSTREAM rev 2) with the F



Article

Table 2. Proteins Identified with at Least Two Unique Peptides in Silkworm Ovary accession number gi|112982669 gi|112982671 gi|112982701 gi|112982828 gi|112982880 gi|112982932 gi|112982996 gi|112982998 gi|112983028 gi|112983032 gi|112983080 gi|112983246 gi|112983276 gi|112983314 gi|112983318 gi|112983322 gi|112983366 gi|112983418 gi|112983448 gi|112983487 gi|112983501 gi|112983505 gi|112983546 gi|112983556 gi|112983600 gi|112983659 gi|112983782 gi|112983816 gi|112983898 gi|112983958 gi|112984000 gi|112984022 gi|112984182 gi|112984216 gi|112984230 gi|112984274 gi|112984292 gi|112984298 gi|112984310 gi|112984376 gi|112984390 gi|112984454 gi|112984502 gi|114050833 gi|114050901 gi|114051115 gi|114051191 gi|114051313 gi|114051770 gi|114051966 gi|114052072 gi|114052224 gi|114052252 gi|114052278 gi|114052462 gi|114052504 gi|114052545 gi|114052561 gi|114052571 gi|114052587

protein description

protein probability



NSAF (× 10−3)

ribosomal protein S2 ribosomal protein S12a 40S ribosomal protein S14 heat shock cognate protein translationally controlled tumor protein homologue ferritin mitochondrial aldehyde dehydrogenasea superoxide dismutase [Cu−Zn]a glutathione S-transferase sigma 1 calreticulin precursor repressor splicing factor 1a small GTP-binding proteina 60S ribosomal protein L5a ras-related GTP-binding protein Rab11a beta-tubulin transitional endoplasmic reticulum ATPase TER94 protein disulfide-isomerase like protein ERp57a glycerol-3-phosphate dehydrogenase-1a phenoloxidase subunit 2 precursor, prophenoloxidase-2sa ribosomal protein L8 alpha-tubulin ribosomal protein S10 ribosomal protein L12 90-kDa heat shock protein cellular retinoic acid binding protein DNA supercoiling factora cyclophilin A glyceraldehyde-3-phosphate dehydrogenasea elongation factor 1 gamma Annexin IX isoform A ribosomal protein S18 ribosomal protein S9a ribosomal protein L30a ribosomal protein L27a ribosomal protein L26a ribosomal protein L23 ribosomal protein L22 ribosomal protein L21 ribosomal protein L18Aa ribosomal protein L14 elongation factor 1-alpha protein disulfide isomerase low molecular 30 kDa lipoprotein PBMHP-6 precursor transketolasea 14-3-3 protein zeta ubiquitin conjugating enzyme E2a peroxiredoxina chaperonin containing t-complex polypeptide 1 beta subunita 26S proteasome non-ATPase regulatory subunit 13a mitochondrial aldehyde dehydrogenase H+ transporting ATP synthase beta subunit isoform 1 adenylate cyclasea calmodulin ATP synthase glutamate dehydrogenasea proteasome 25 kDa subunit chaperonin subunit 4 delta cytosolic malate dehydrogenasea lysophospholipasea mitochondrial matrix protein p33a

1 0.965 0.9991 1 1 1 0.9993 0.9988 0.9993 0.9999 1 0.9999 0.9999 0.9999 1 0.9798 1 0.9993 0.9609 1 0.9984 1 0.9982 0.9991 1 0.9998 1 1 0.9999 0.9968 0.95 1 1 0.9677 0.9969 1 1 0.906 0.9652 0.9998 1 1 0.9995 1 0.9947 0.952 0.9996 1 1 0.9994 1 0.9893 1 1 1 1 1 0.9996 0.9999 0.9898

11 5 6 9 7 8 5 4 6 9 4 7 3 4 74 6 19 3 6 9 35 18 4 10 35 7 15 9 8 4 3 9 8 3 8 8 11 2 4 4 22 17 8 7 6 5 4 9 5 5 10 3 8 21 15 4 9 3 4 3

5 2 2 5 3 4 2 2 2 4 2 3 2 2 10 3 6 3 2 4 4 6 2 4 11 4 3 3 4 3 2 5 2 2 5 4 2 2 2 2 7 8 2 4 3 4 2 4 3 3 6 2 4 7 6 2 3 2 2 2

8.535 7.295 8.019 2.816 8.53 9.363 5.205 5.245 1.424 4.592 5.278 7.006 2.051 3.801 33.619 1.521 8.865 1.689 1.758 7.089 15.831 22.877 4.933 2.834 54.11 4.408 18.387 5.5 3.836 2.504 4.439 9.417 14.287 4.536 10.976 11.59 15.192 2.542 4.578 4.903 9.636 6.988 6.326 3.318 3.884 6.683 5.115 3.409 2.649 2.08 3.931 3.492 21.807 7.7 5.49 3.467 3.265 1.85 3.453 2.121

G



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protein description

gi|114052621 gi|114052707 gi|114052783 gi|114053141 gi|114053277 gi|120444903 gi|124430725 gi|148298693 gi|148298697 gi|148298717 gi|148298726 gi|148298748 gi|148298752 gi|148298796 gi|148298800 gi|148298878 gi|151301000 gi|153791817 gi|153792023 gi|153792203 gi|153792270 gi|153792659 gi|158631166 gi|160333678 gi|164420683 gi|166706856 gi|169234669 gi|169234936 gi|182509200 gi|187281708 gi|187281844 gi|219362829 gi|237648982 gi|237648984 gi|304307739 gi|309384283 gi|350534642 BGIBMGA000398 BGIBMGA000715 BGIBMGA001119 BGIBMGA001406 BGIBMGA001983 BGIBMGA002462 BGIBMGA002569 BGIBMGA002570 BGIBMGA003212 BGIBMGA003361 BGIBMGA003564 BGIBMGA004172 BGIBMGA004655 BGIBMGA004950 BGIBMGA004960 BGIBMGA005559

myosin light polypeptide 9 isoform Aa receptor for activated protein kinase C RACK 1 isoform 2a serine hydroxymethyltransferasea exuperantia H+ transporting ATP synthase O subunit isoform 1a chaperonina sex-specific storage-protein 2a heat shock protein hsp20.8 eukaryotic initiation factor 5C vacuolar ATP synthase subunit B ADP-ribosylation factor H+ transporting ATP synthase subunit ea 14-3-3 epsilon protein thioredoxin-like proteina enolase vacuolar ATP synthase catalytic subunit A ribosomal protein S24a S-adenosyl-L-homocysteine hydrolasea MLE proteina vesicle amine transport protein malate dehydrogenasea actin-depolymerizing factor 1 ADP/ATP translocase glutathione S-transferase sigma 2 ribosomal protein S11 isoform 1 aubergine proteina cytochrome P450 9G3a sex-specific storage-protein 1 precursora muscle glycogen phosphorylasea triosephosphate isomerase actin, cytoplasmic A4 clathrin heavy chain histone H2B-like protein histone H4-like protein tudor staphylococcus/micrococcal nuclease kinesin heavy chaina DJ-1 beta CG1140 [Drosophila melanogaster] CG12262 [Drosophila melanogaster]a putative rab gdp-dissociation inhibitor [Danaus plexippus] cctgamma [Drosophila melanogaster]a serine protease inhibitor 3 [Drosophila melanogaster] CG11089 [Drosophila melanogaster]a CaBP1 [Drosophila melanogaster] CG33303 [Drosophila melanogaster] Rpn7 [Drosophila melanogaster]a predicted protein [Drosophila melanogaster] ornithine aminotransferase precursor [Drosophila melanogaster] CG7896 [Drosophila melanogaster]a CG3523 [Drosophila melanogaster] pugilist [Drosophila melanogaster]a CG12173 [Drosophila melanogaster] mesencephalic astrocyte-derived neurotrophic factor [Drosophila melanogaster]a CG5840 [Drosophila melanogaster] La autoantigen-like [Drosophila melanogaster] CG17838 [Drosophila melanogaster] predicted protein [Drosophila melanogaster]a heterogeneous nuclear ribonucleoprotein at 98DE [Drosophila melanogaster]a

BGIBMGA005727 BGIBMGA006022 BGIBMGA006260 BGIBMGA006263 BGIBMGA006405

H

protein probability



NSAF (× 10−3)

0.9964 1 0.9997 0.9913 0.9657 0.9999 0.9986 1 0.9998 1 1 0.9998 1 0.9807 1 1 0.9947 1 0.9999 1 0.999 1 1 1 0.993 0.9926 0.9804 1 1 0.9989 1 0.9974 1 1 0.9769 0.9988 0.9752 0.9998 0.9069 1 0.9922 0.9973 1 0.9999 0.9999 0.964 1 0.9991 0.9999 1 1 1 0.9999

4 4 3 3 3 3 6 5 5 15 8 6 17 7 9 7 8 3 9 38 4 12 10 9 4 4 9 19 5 5 14 17 12 24 4 5 2 7 4 6 4 3 5 3 6 3 8 4 4 12 6 6 6

2 2 2 2 2 2 3 3 3 4 4 3 6 2 5 4 2 2 3 6 2 4 3 5 2 2 3 7 4 3 6 5 4 9 2 2 2 3 2 3 2 2 3 3 3 2 4 3 2 5 3 3 3

4.408 2.542 1.308 1.577 3.062 2.932 1.731 5.599 2.437 6.166 8.91 14.287 13.141 13.427 4.218 2.45 12.276 1.546 1.43 17.302 1.493 16.463 6.792 8.921 4.164 0.904 3.498 5.166 1.207 4.11 7.592 2.054 19.73 46.905 0.92 0.989 2.136 2.752 1.914 2.75 1.504 1.614 1.724 1.452 2.628 1.594 1.612 1.992 1.143 1.145 1.307 0.735 7.023

0.9965 0.9855 0.9876 0.9621 0.9986

6 3 4 3 6

3 2 2 2 3

4.346 1.573 2.395 1.096 3.369



Article


protein description

BGIBMGA007208 BGIBMGA007349 BGIBMGA007390

futsch [Drosophila melanogaster]a heat shock protein 60 [Drosophila melanogaster] microsomal triacylglycerol transfer protein [Drosophila melanogaster] phosphoglycerate kinase [Drosophila melanogaster] alpha spectrin [Drosophila melanogaster] heat shock protein cognate 3 [Drosophila melanogaster] Rpn2 [Drosophila melanogaster]a CG2918 [Drosophila melanogaster]a 26−29kD-proteinase [Drosophila melanogaster] T-complex chaperonin 5 [Drosophila melanogaster]a ubiquitin activating enzyme 1 [Drosophila melanogaster]a male fertility factor kl2 [Drosophila melanogaster] bicoid stability factor [Drosophila melanogaster]a 30K protein 12 [Bombyx mori]a CG6359 [Drosophila melanogaster]a puromycin sensitive aminopeptidase [Drosophila melanogaster]a UK114 [Drosophila melanogaster]a karst [Drosophila melanogaster]a Tcp-1eta [Drosophila melanogaster] terribly reduced optic lobes [Drosophila melanogaster] CG6287 [Drosophila melanogaster] hypothetical protein [Daphnia pulex]a Retinoid- and fatty acid-binding glycoprotein [Drosophila melanogaster] alpha-tubulin at 84B [Drosophila melanogaster] CG6188 [Drosophila melanogaster]a ATP citrate lyase [Drosophila melanogaster]a histone H1 [Drosophila melanogaster]

BGIBMGA007681 BGIBMGA007948 BGIBMGA007950 BGIBMGA008426 BGIBMGA008551 BGIBMGA009139 BGIBMGA009699 BGIBMGA009911 BGIBMGA010475 BGIBMGA010538 BGIBMGA010876 BGIBMGA011317 BGIBMGA011674 BGIBMGA011698 BGIBMGA012171 BGIBMGA012182 BGIBMGA012240 BGIBMGA012309 BGIBMGA012651 BGIBMGA013342 BGIBMGA013413 BGIBMGA013705 BGIBMGA014446 BGIBMGA014578 a

protein probability



NSAF (× 10−3)

0.9995 0.9981 1

10 4 17

3 2 6

0.77 1.419 4.295

0.973 1 0.9986 0.9997 0.9992 0.9448 0.9999 0.9992 0.9914 0.9974 0.9998 0.994 0.99 0.9999 0.9966 1 0.9999 0.9999 1 1

4 16 3 5 4 3 5 8 5 3 4 4 3 4 4 11 12 24 12 67

3 9 2 2 3 2 2 3 3 2 2 2 2 3 2 3 5 3 4 19

1.918 1.322 0.929 2.577 0.894 1.248 1.873 1.558 0.302 0.327 1.862 4.874 0.704 4.818 0.196 4.113 0.627 11.274 12.881 5.236

1 0.9479 0.9982 1

60 4 5 5

6 2 2 2

27.08 2.771 2.885 3.622

Indicates the ovary-specific proteins.

melanogaster homologies were obtained following a Blastp search against the RefSeq protein database (18556 sequences) of D. melanogaster, available on NCBI Blast home (http://blast. ncbi.nlm.nih.gov/Blast.cgi). Three independent networks were constructed on the basis of the common and sex-specific proteins. The FASTA protein sequences of identified proteins were searched against KEGG GENES database using a BLASTP program (http://blast.genome.jp/). The corresponding KEGG pathways were extracted.

■

RESULTS AND DISCUSSION

Protein Identification

Protein expression profiles of silkworm testis and ovary were identified using SDS-PAGE (Figure 1B) coupled with LC− MS/MS. To control the FDR, the results were filtered with high peptide and protein probability at 0.8 and 0.9, respectively. Redundancy was prevented two ways: first, the nonredundant sequence collection of the in-house database was ensured by downloading only the reference sequences from the NCBI database. Second, for group proteins that showed shared peptides, the selection was made manually to decrease redundancy. Proteins in the same group showing distinct peptides were all listed, while there was no report for proteins that could only be identified by subsets of peptides matching to other identified proteins. Identification by a single peptide often represents the identification of low molecular weight or low abundance proteins and is often sufficient to conclude the presence of a product of a certain gene in the sample.31,32

Figure 2. Real-time quantitative PCR results of some genes. The relative expression level of each gene was calculated using 2(Ct internal control−Ct target gene) by normalizing with the Rpl27a gene. Data represent the mean ± standard deviation from three replicate samples.

Therefore, protein identification from a single unique peptide was allowed in our identification for increasing sensitivity. The annotated tandem mass spectra for single peptide identifications in testis and ovary were provided in Figures S1 and S2 (Supporting Information), respectively. The obvious protein contaminants including trypsin and keratin were removed. Finally, 286 proteins and 205 proteins were identified for testis and ovary with FDR of 0.7 and 0.98%, respectively. Among them, 184 and 145 proteins were identified with more than 2 unique peptides for testis and ovary, respectively (Table I



Article

Figure 3. Gene ontology categories of the common and sex-specific expressed protein genes from silkworm testis and ovary. Nutrient reservoir showed to be the specific category for ovary.

Figure 4. Chromosome distribution of the common and sex-specific genes from silkworm testis and ovary.

and those only detected in testis or ovary as the testis- and ovary-specific ones. Results showed that 92 proteins were common proteins, 194 proteins were testis-specific proteins, and 113 were ovary-specific ones (Figure 1C). The protein lists were compared to published identification in silkworm larval testis9 and pupal ovary,10 where 56 proteins in testis and 33 proteins in ovary were identified using twodimensional electrophoresis (2-DE) and MALDI-TOF-MS, respectively. Among them, there were 40 proteins in testis and 16 proteins in ovary overlapped by our identification. Therefore, 246 and 189 proteins were newly identified in the silkworm testis and the ovary, respectively. Overview of Silkworm Gonads Proteome

The most enriched protein family was ribosomal proteins (RPs) both in testis and ovary, 35 RPs in testis and 29 RPs in ovary. Among those, 14 were commonly expressed between testis and ovary. The enrichment of ribosomes is an indication that the germ cells undergo cell growth and proliferation. Spectral counts (SCs) have been demonstrated as a good measure of protein relative abundance.33 However, the SCs should be divided by the protein mass or length, for the larger proteins will generate more peptides and therefore more SCs than smaller proteins.27 Cellular retinoic acid binding protein

Figure 5. The chromosome mapping of the identified proteins on the Z chromosome. Three red stars indicate the best hit (two red stars) with the score above 400 (one red star). (A) The common proteins mapped onto the Z chromosome. (B) The testis-specific proteins mapped onto the Z chromosome. (C) The ovary-specific proteins mapped onto the Z chromosome.

1 and Table 2); 102 and 60 were with single peptide (Table S2, Supporting Information). The proteins detected in both testis and ovary were considered as the common expressed proteins, J



Article

Table 3. Testis-Specific Protein Genes Harbored on the Sex Chromosome accession number BGIBMGA000528-PA BGIBMGA000672-PA BGIBMGA000704-PA BGIBMGA000710-PA BGIBMGA002005-PA BGIBMGA002103-PA BGIBMGA002114-PA BGIBMGA003865-PA gi|112983920|ref| NP_001036847.1| gi|114053253|ref| NP_001040525.1|

e-value

description

biological process

molecular function

eukaryotic translation initiation factor 4 gamma [Apis mellifera] citrate synthase [Aedes aegypti]

4× 10−120 0

RNA metabolic process

Binding

Carbohydrate metabolic process

Procollagen lysyl hydroxylase [Drosophila melanogaster] putative VAMP-associated protein [Danaus plexippus] stress-sensitive B [Drosophila melanogaster]

5× 10−129 2× 10−109 3× 10−178 0 0 8 × 10−63

Oxidation−reduction process

Citrate (Si)-synthase activity Iron ion binding

alpha-tubulin at 84B [Drosophila melanogaster] laminin B2 [Drosophila melanogaster] glycine cleavage system h protein [Danaus plexippus] Chitinase-like protein EN03 precursor [Bombyx mori] 6-phosphogluconate dehydrogenase [Bombyx mori]

ADP transport

0

Antimicrobial humoral response Embryonic morphogenesis Glycine decarboxylation via glycine cleavage system Biological process

0

Pentose biosynthetic process

Structural molecule activity ATP:ADP antiporter activity GTP binding

Molecular function NADP binding

Figure 6. The biological network for testis-specific proteins. This network was built by Pathway studio software (Ariadne Genomics) based on D. melanogaster biological network (BIN) database. All the molecules are located in a virtual cell. The protein name was abbreviated in the network. The full names of those proteins and correspondences to the silkworm homologies were listed in Table S6 (Supporting Information). The different shapes of the nodes in the network represent different molecules or classes. The arrow represents the regulatory interaction. Positive regulation is indicated as “+” and negative regulation is indicated as “−”.

(RA) in silkworm spermatogenesis and oogenesis. RA is a signaling molecule participating in several processes of gene expression by binding to its nuclear receptors.34 During

(CRABP) should be a highly abundant protein in testis and ovary, especially in the testis for high NSAF value as the 2-DE pattern showed,9 indicating the critical role of retinoic acid K



Article

Table 4. Proteins with Low Sequence Similarity to D. melanogaster’s common testisspecific

ovaryspecific

accession number

protein description

gi|112984502|ref| NP_001037486.1| BGIBMGA000302PA

low molecular 30 kDa lipoprotein PBMHP-6 precursor [Bombyx mori] PREDICTED: constitutive coactivator of PPAR-gamma-like protein 1 homologue [Bombus terrestris] 30k protein 6 [Bombyx mori]

BGIBMGA004395PA BGIBMGA004397PA BGIBMGA004399PA BGIBMGA004400PA BGIBMGA004454PA BGIBMGA008554PA BGIBMGA013098PA gi|156119320|ref| NP_001095196.1| gi|156119322|ref| NP_001095197.1| BGIBMGA010876PA BGIBMGA012651PA gi|151301117|ref| NP_001093085.1|

exuperantia (exu).39 Localization of osk and grk mRNA required RNA-binding protein SQUID.44,45 We identified exuperantia and SQUID protein homologue in silkworm ovary. Furthermore a homologue of bicoid stability factor (the predicted protein BGIBMGA010538-PA) was also identified in silkworm ovary. This may be an indication that the localization way of morphogen may be conservative between B. mori and D. melanogaster, although there was evidence that those three morphogens may not exist in silkworm.46,47 A homologue (the predicted protein BGIBMGA007948-PA) of Drosophila fusome-enriched protein alpha-spectrin was identified both in silkworm testis and ovary. Fusome is an important organelle and plays a coordination role in mitotic divisions both in males48 and females.49 It also regulates meiotic division in males.48 In female Drosophila, fusome also has a role in oocyte determination.50 Furthermore, several other fusomeassociated proteins, such as calreticulin precursor, protein disulfide isomerase (PDI), ferritin, and transitional endoplasmic reticulum ATPase TER94, were also identified. Those proteins were all commonly expressed proteins, indicating some common features of fusome between male and female silkworm. We also identified a small GTPase Rab11 in silkworm ovary, which is required for normal fusome structure, cyst formation and germ stem cell maintenance during Drosophila oogenesis.51 Thirty-three highly homologous proteins (Table S3, Supporting Information) were found by comparing with Drosophila sperm proteome.52 There include some proteins involved in Drosophila sperm development, such as dynein heavy chain at 36C, which has a role in sperm motility, αtubulin at 84B in spermatid development, actin 5C in sperm individualization and β-tubulin at 56D in cytoskeleton organization. However, some differences were found between B. mori and D. melanogaster. The leucyl aminopeptidase was showed to be the most abundant protein in Drosophila sperm proteome, while in our identification, it is not. The results at transcriptional level also reveal that it may not be the abundant protein in silkworm testis.20 Our results indicated that the most abundant protein in silkworm testis may be CRABP. It was also showed in the 2DE pattern.9

30 kDa protein [Bombyx mori] 30k protein 7 [Bombyx mori] 30K protein 3 [Bombyx mori] 30K protein 15 [Bombyx mori] hypothetical protein [Bombyx mori] immune-related Hdd13 [Hyphantria cunea] low molecular 30 kDa lipoprotein PBMHP-12 precursor [Bombyx mori] low molecular 30 kDa lipoprotein PBMHPC-19 precursor [Bombyx mori] 30K protein 12 [Bombyx mori] hypothetical protein [Bombyx mori] Prpk-binding protein [Bombyx mori]

mammal testis development, RA may have a direct role in spermatogonia differentiation35 and be involved in meiotic initiation.36 The role of retinoic acid in silkworm reproduction is elusive, although there is evidence indicating that retinoic acid has the juvenile hormone-like role in stimulating vitellogenin synthesis.37 The other relative high abundance protein in silkworm testis was 14-3-3 epsilon protein. The 14-33 epsilon encoded by the Ywhae gene is the family member of 14-3-3 proteins, and involved in many crucial cellular processes via its widespread interactions with hundreds of partner proteins. The 14-3-3 epsilon has a regulation role in cell cycle38 and therefore is thought to participate in spermatocyte proliferation. β-Tubulin and BGIBMGA013413-PA (the homologue of αtubulin) may be the abundant proteins in ovary according to the relative abundance (NSAF values). Microtubules (MTs) are dynamically assembled from α- and β-tubulin heterodimers. MT dynamics are crucial to mitosis. In addition, MTs have a fundamentally important role in oogenesis, being involved in essential cellular processes such as oocyte specification and axis establishment through transport of maternal RNAs and proteins along MTs.39 Those additional roles of MTs in oogenesis may explain why these two tubulins were more abundant in ovary than in testis. Four types of tubulins were also identified specifically expressed in testis. Among those, two have been confirmed as testis-specific in other experiments.40 Those male-specific tubulins may be involved in aspects of specific structure and physiology related to sperm, such as components of the flagellar axonemes. In Drosophila, the body axes for embryo morphology pattern were established during midoogenesis by localization of three mRNA determinants: bicoid (bcd), oskar (osk), and gurken (grk).41−43 Expression and localization of bcd require

Correlation of mRNA Expression and Protein Abundances

Post-transcriptional regulation is important in gametogenesis. To explore the correlation of expression between mRNA and protein level, 11 genes representing common and sex-specific expression were selected for qRT-PCR analysis (Figure 2). The qRT-PCR results showed discrepancy of some genes between mRNA and protein level. The BmHSP90 may have a somatic role for growth and development of silkworm larvae,53 which is found to be expressed abundantly at transcriptional level. However, it was not the same at protein level comparing to other genes like Ywhae. Fibroinase (Bcp) and ALY showed testis-specific expression at protein level, while their mRNA abundance was higher in ovary than testis. MLE protein (MLE) and cytochrome P450 9G3 (Cyp9g3) showed ovary-specific at protein level, while expressed higher abundantly in testis than in ovary at transcriptional level. Those results suggest posttranscriptional regulation of those genes may occur. Our identification was compared with the microarray-based studies performed by Xia et al.20 The microarray-based results revealed that histone H2A-like protein, α-tubulin (BMTUA3), heat shock protein hsp 19.9, and stathmin were expressed highly both in testis and ovary at the transcriptional level. L



Article

Figure 7. Top 15 KEGG pathways involved by common and sex-specific protein genes. (A) Top 15 KEGG pathways involved by common proteins genes. (B) Top 15 KEGG pathways involved by testis-specific proteins. (C) Top 15 KEGG pathways involved by ovary-specific proteins.

Information). “Mitotic spindle elongation” and “translation” are the two most enriched BP terms both in testis and ovary, which is consistent with the active mitosis and the protein synthesis processes. The most enriched MF terms is binding (44.4% in testis, 45.4% in ovary). Of those ATP/GTP binding (15% in testis, 20% in ovary), DNA/RNA binding (7.3% in testis, 7.3% in ovary), and actin binding (4.5% in testis, 2.4% in ovary) are the top three categories. The enrichment of “ATP/GTP binding” indicates many processes are ATP dependent during spermatogenesis and oogenesis. Most of “RNA binding” proteins participated in mitotic and mRNA processes, for example, mitotic spindle elongation, mitotic cell cycle G2/M transition DNA damage checkpoint, mRNA cleavage and nuclear mRNA splicing. The enrichment of “actin binding” is consistent with the essential roles of actin during spermatogenesis and oogenesis. There is also a large category comprised of proteins without functional prediction (13.6% in testis, 10.7% in ovary). The most enriched CC terms are cytoplasm, ribosomal is the second, and lipid particle is the third enriched category. From the GO annotation, the functions of many predicted proteins were assigned on the basis of homology with other

However, despite histone H2A-like protein, the other three were all testis-specific at protein level. Pyruvate kinase, heat shock protein hsp20.8, transitional endoplasmic reticulum ATPase TER94, ATP synthase, beta-tubulin, ADP/ATP translocase, 14-3-3 protein epsilon, enolase, heat shock protein hsp20.1 were expressed specifically in testis. However, in the above-mentioned testis-specific transcripts, only the pyruvate kinase, ATP synthase and β-tubulin were also specifically expressed at protein level, while the others were all commonly expressed in testis and ovary at protein level. The discrepancy between mRNA and protein level indicated the occurrence of post-transtriptional regulations on those genes. There was also good correlation between protein and mRNA levels in several genes. For instance, two forms of silkworm storage proteins, termed sex-specific storage-protein 1 (SP1) and sex-specific storage-protein 2 (SP2), were detected to be ovary-specific both at the transcriptional level and the protein level. Gene Ontology Annotation

GO is a well-established structured vocabulary and used as a good tool in protein function annotation. Biological process (BP), molecular function (MF) and cellular component (CC) GO terms of all proteins were listed in Table S4 (Supporting M



Article

catalyzing the generation of ribulose-5-phosphate for synthesizing the nucleic acid and NADPH, which is indispensable for gamete fusion.63

proteins. Actin-based cell−cell adherens junctions are crucial for cell morphogenesis and differentiation,54 being involved in the moving events along with the spermatocyte differentiation, maturation and release into the tubule lumen at spermiation.55 Four proteins termed BP with “actin cytoskeleton organization” corresponded to the CC term “adherens junction” in testis. Those four proteins were predicted from the silkworm genome, having a similar sequence to Drosophila short stop, a Plakin family member containing both actin binding and microtubule binding domains.56 Three predicted proteins were identified related to the CP term as axonemal dynein complex in testis, indicating their roles in sperm motility. An integral comparison of GO categories between testis and ovary was performed and plotted by the WEGO tool (Figure 3) on the basis of the annotation results of the common and sexspecific proteins. The results show small differences between testis and ovary. However, some specific physiologies related to testis and ovary were revealed, such as nutrient reservoir being the specific functional category in ovary. This result is consistent with the fact that eggs should contain ample nutrients to support embryogenesis.

Biological Network Analysis

The study of individual genes and proteins has revealed many aspects of gametogenesis, while system-level network analysis will provide integrated description on gametogenesis processes. To assess the common targets involved by the common and sex-specific proteins, a regulatory network was delineated using the D. melanogaster biological network (BIN) database (Ariadne Genomics). In order to obtain more reliable results, only the cell processes involved in more than three proteins were retained (Figure 6; Figures S3 and S4, Supporting Information). The networks showed that apoptosis, cell cycle (cycle), differentiation (differentiate), embryogenesis, maturation, morphogenesis and proliferation were the enriched processes both in testis and ovary (Figure 6; Figures S3 and S4, Supporting Information). Those targets were all the essential processes during gametogenesis. Functional male and female gametes are the results of a precisely regulated progression of mitotic proliferation, differentiation, morphogenesis and maturation. Apoptosis has important functions in the regulation of normal development and of germ cell fate by eliminating unwanted or unneeded cells.64,65 Paternal and maternal factors are important in embryo development. Especially, the early embryo development is controlled by the maternal synthesized mRNA and proteins in many animals.66,67 Sex-specific processes, which have physiological bearings for the specific stage and the most unique processes between testis and ovary, were revealed too. For instance, there were more prevalent meiotic processes in testis than in ovary; it may be consistent with the status that there are larger numbers of cell divisions in spermatogenesis than in oogenesis. Localization of axis determinants is the unique processes during oogenesis. From the networks, it can be concluded that some essential processes in spermatogenesis and oogenesis should be conservative between B. mori and D. melanogaster, since we identified many D. melanogaster homologies, which have essential roles in D. melanogaster gametogenesis, such as ALY and VISMAY (the predicted protein BGIBMGA006116-PA) in spermatogenesis (Figure 6). The aly and vismay are the meiotic arrest genes. They are essential for Drosophila male meiosis.68,69 Several homologies of the essential regulators in Drosophila oogenesis were also identified, such as karst (kst), spaghetti squash (sqh), aubergine (aub), chickadee (chic) and moesin (moe) (Figure S4, Supporting Information). Karst70 and spaghetti squash71 are involved in cytoskeletal organization during follicle cell morphogenesis. Aubergine72 and chickadee73 (homology of silkworm profilin) play a role in localization of axis determinants. Moesin is involved in regulating actin-based morphogenesis and required for the antero-posterior polarity of the oocyte and cellular asymmetry of nurse cells during oogenesis.74 In comparison with the cell processes involved by the sexspecific proteins, larval development and endocytosis are the processes only participated by the common proteins (Figure S3, Supporting Information). The proteins lethal (2) 37Cc, SQUID, abnormal wing discs, and la autoantigen-like were involved in the larval development. In addition to having specific roles in gametogenesis, these proteins also have an essential role in growth and development. For example, in

Protein−Gene Distribution on the Chromosome

Sex chromosomes are thought to have correlation with sex related events, such as sex determination and gametogenesis. Silkworm is female-heterogametic organism (ZZ in male, ZW in female), which have 28 pairs of chromosomes. In silkworms, the presence of the W chromosome has a dominant sex determination role.57 The W chromosome was thought to have no functional genes except the feminizing gene (fem), which is the dominant factor for female.58 The Z chromosome (Chromosome No. 1) showed a significantly higher number of testis-specific genes than the autosomes.59 To investigate the chromosome distribution of our identified proteins, all the common, testis- and ovary-specific proteins were mapped onto the silkworm chromosomes using the corresponding cds. Among 92 common protein genes, 87 were mapped onto the silkworm chromosome. In total, 184 from 194 testis-specific protein genes and 100 from 113 ovary-specific protein genes were mapped. The common and sex-biased genes distributed almost in all 28 chromosomes, and the number of genes on the Z chromosome for common, testis-, and ovary-specific was 4, 10, and 2, respectively (Figure 4; Figure 5; Table S5, Supporting Information). The testis-specific genes (10/194 × 100% = 5.15%) were higher than the ovary-specific genes (2/ 113 × 100% = 1.77%) on the sex chromosome. The Z chromosome is supposed to accumulate male advantageous genes in silkworm.59,60 Among the 10 testisspecific genes mapped on sex chromosome, most of them show possible role in silkworm spermatogenesis (Table 3). The predicted protein BGIBMGA000528-PA is the homology of the eukaryotic initiation factor 4G, which has a role in coordinating the initiation of meiotic division and differentiation in spermatocyte.61 BGIBMGA002005-PA (the homology of Drosophila stress-sensitive B) and BGIBMGA002103-PA (the homology of Drosophila α-tubulin at 84B) have been identified in Drosophila sperm proteome. The predicted protein BGIBMGA002114-PA is described as the homology of D. melanogaster laminin B2, which expressed in the epithelial wall of the testis tube, in the nuclei of primary spermatocytes and young spermatids, and in the heads and axonemes of elongated spermatids of D. melanogaster.62 6-Phosphogluconate dehydrogenase is the key enzyme for the pentose phosphate pathway, N



Article

abundance of CRABP in silkworm testis indicated the fundamental role of RA in silkworm gametogenesis. Many 30KP family members were detected in silkworm testis. Those lepidopteran-specific proteins may bear the order-specific meanings in spermatogenesis of lepidopteran insects.

addition to having a specific role in oogenesis by gurken mRNA localization and regulation, SQUID showed to be essential for other development processes.45 30K Proteins in Silkworm Spermatogenesis

Most of our identified proteins were significantly homologous to D. melanogaster; however, some proteins had no significant sequence similarity (e-value > 0.01, score < 40) with D. melanogaster’s (Table 4), which may be responsible for the difference between these two orders. Among those, most of them are expressed specifically in testis, and are 30K protein (30KP) family, indicating 30KPs may have an important role in silkworm spermatogenesis. The 30KPs were the major plasma proteins and classified into the lepidopteran-specific Lipoprotein_11 family.75,76 The roles of 30KPs in silkworm oogenesis and embryo development were identified as being the second major components of silkworm yolk protein (in addition to vitellin and egg-specific proteins)77 and being the amino acid source during embryo development.78 It is also suggested that 30KP has a role in inhibition of apoptosis.79,80 However, their roles in male germ lines remain unclear. We hypothesized that 30KPs play a role in the control of apoptosis during spermatogenesis, especially in spermatogonia proliferation. Lepidopteran males generate dimorphic sperm, eupyrene and apyrene (anucleate) sperm. Further investigation is required to confirm the possible relation between the 30KPs and the control of eupyrene and apyrene.

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KEGG Pathway Analysis

(1) Goldsmith, M. R.; Shimada, T.; Abe, H. The genetics and genomics of the silkworm Bombyx mori. Annu. Rev. Entomol. 2005, 50, 71−100. (2) Nagaraju, J.; Goldsmith, M. R. Silkworm genomicsprogress and prospects. Curr. Sci. 2002, 83, 415−425. (3) Parisi, M.; Nuttall, R.; Edwards, P.; Minor, J.; Naiman, D.; Lü, J.; Doctolero, M.; Vainer, M.; Chan, C.; Malley, J.; Eastman, S.; Oliver, B. A survey of ovary-, testis-, and soma-biased gene expression in Drosophila melanogaster adults. Genome Biol. 2004, 5, R40. (4) Li, J. Y.; Chen, X.; Fan, W.; Hosseini Moghaddam, S. H.; Chen, M.; Zhou, Z. H.; Yang, H. J.; Chen, J. E.; Zhong, B. X. Proteomic and bioinformatic analysis on endocrine organs of domesticated silkworm, Bombyx mori L. for a comprehensive understanding of their roles and relations. J. Proteome Res. 2009, 8, 2620−2632. (5) Zhou, Z. H.; Yang, H. J.; Chen, M.; Lou, C. F.; Zhang, Y. Z.; Chen, K. P.; Wang, Y.; Yu, M. L.; Yu, F.; Li, J. Y.; Zhong, B. X. Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet. J. Proteome Res. 2008, 7, 5103−5111. (6) Paskewitz, S. M.; Shi, L. The hemolymph proteome of Anopheles gambiae. Insect Biochem. Mol. Biol. 2005, 35, 815−824. (7) Zheng, A.; Li, J.; Begna, D.; Fang, Y.; Feng, M.; Song, F. Proteomic analysis of honeybee (Apis mellifera L.) pupae head development. PLoS One 2011, 6, e20428. (8) Takemori, N.; Yamamoto, M. T. Proteome mapping of the Drosophila melanogaster male reproductive system. Proteomics 2009, 9, 2484−2493. (9) Nie, H. Y.; Zhong, X. W.; Zou, Y.; Yi, Q. Y.; Zhao, P.; Xia, Q. Y. Identification of testis proteins of silkworm Bombyx mori using twodimensional electrophoresis and mass spectrometry. Acta Entomol. Sin. 2010, 53, 369−378. (10) Xu, Y. M.; Fu, Q.; Zhang, S.; Jia, L.; He, N. J. Construction of standard profiles of protein in ovarian tissue from Day 7 pupae of silkworm, Bombyx mori. Acta sericologica sinica 2009, 35, 768−755. (11) Guo, X.; Zhang, P.; Huo, R.; Zhou, Z.; Sha, J. Analysis of the human testis proteome by mass spectrometry and bioinformatics. Proteomics: Clin. Appl. 2008, 2, 1651−1657. (12) Groh, K. J.; Nesatyy, V. J.; Segner, H.; Eggen, R. I.; Suter, M. J. Global proteomics analysis of testis and ovary in adult zebrafish (Danio rerio). Fish Physiol. Biochem. 2011, 37, 619−647.

ASSOCIATED CONTENT

S Supporting Information *

Supporting tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-571-86971302. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the grants from National Basic Research Program of China (Grant No. 2012CB114601) and the National Natural Science Foundation of China (30972142). We thank Mr. Liang Chen for his kind help in data processing during bioinformatics analysis.

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KEGG pathway database has been used as a reference knowledge base for understanding higher-level functions of cellular processes and organism behaviors from large-scale molecular data set.81 Using the KEGG database, the top 15 pathways for common- and sex-specific expressed proteins between testis and ovary were shown (Figure 7). Ribosome is the top 1 pathway involved in both common and sex-specific proteins. The subsequent enriched pathway involved by common proteins is protein processing in endoplasmic reticulum, which is consistent with active protein synthesis in testis and ovary. The subsequent testis-specific enriched pathways are involved in obtaining energy, such as oxidative phosphorylation, pyruvate metabolism, and glycolysis/gluconeogenesis. Energy metabolism is a key factor for functional sperm, including sperm motility and active protein modification. The second enriched pathway in ovary is the citrate cycle, which is the final metabolism pathway of three nutrients, carboxyl, lipid, and amino acids, for intercross of their metabolism. There exists more active citrate cycle in ovary, which may be attributed to the nutrient synthesis and reservation in oocyte. The third enriched pathway in ovary is ubiquitin-mediated proteolysis, which may be consistent with the dynamics of cytoskeletal organization during oogenesis.

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CONCLUSIONS Shotgun-based identification enabled 286 and 205 identifications in silkworm larval testis and ovary, and among those, 246 and 189 proteins were newly identified in silkworm testis and ovary. Those identifications enriched the protein repertoire of silkworm gonads. The bioinformatics analysis provides the system-level view on the silkworm gametogenesis, and the common and specific features between Bombyx mori and Drosophila melanogaster were revealed on the basis of the protein homology. This study also put forward a number of questions for future research. For example, the most high O

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Proteome Analysis of Silkworm, Bombyx mori, Larval Gonads

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