Abstract
Bivalve mollusks exhibit hermaphroditism and sex reversal/differentiation. Studies generally focus on transcriptional profiling and specific genes related to sex determination and differentiation. Few studies on sex reversal/differentiation have been reported. A combination analysis of gonad proteomics and transcriptomics was conducted on Chlamys nobilis to provide a systematic understanding of sex reversal/differentiation in bivalves. We obtained 4258 unique peptides and 93,731 unigenes with good correlation between messenger RNA and protein levels. Candidate genes in sex reversal/differentiation were found: 15 genes differentially expressed between sexes were identified and 12 had obvious sexual functions. Three novel genes (foxl2, β-catenin, and sry) were expressed highly in intersex individuals and were likely involved in the control of gonadal sex in C. nobilis. High expression of foxl2 or β-catenin may inhibit sry and activate 5-HT receptor and vitellogenin to maintain female development. High expression of sry may inhibit foxl2 and β-catenin and activate dmrt2, fem-1, sfp2, sa6, Amy-1, APCP4, and PLK to maintain male function. High expression of sry, foxl2, and β-catenin in C. nobilis may be involved in promoting and maintaining sex reversal/differentiation. The downstream regulator may not be dimorphic expressed genes, but genes expressed in intersex individuals, males and females. Different expression patterns of sex-related genes and gonadal histological characteristics suggested that C. nobilis may change its sex from male to female. These findings suggest highly conserved sex reversal/differentiation with diverged regulatory pathways during C. nobilis evolution. This study provides valuable genetic resources for understanding sex reversal/differentiation (intersex) mechanisms and pathways underlying bivalve reproductive regulation.






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Adell T, Muller WE (2004) Isolation and characterization of five Fox (Forkhead) genes from the sponge Suberites domuncula. Gene 334:35–46
Akasaka M, Harada Y, Sawada H (2010) Vitellogenin C-terminal fragments participate in fertilization as egg-coat binding partners of sperm trypsin-like proteases in the ascidian Halocynthia roretzi. Biochem Biophys Res Commun 392:479–484
Akasaka M, Kato KH, Kitajima K, Sawada H (2013) Identification of novel isoforms of vitellogenin expressed in ascidian eggs. J Exp Zool B Mol Dev Evol 320:118–128
Anderson K, Burnell FJ, Roiko A, Andrew M, Connor WAO, Elizur A (2010) Development of a method for identifying elevated vitellogenin gene expression in the Sydney rock oyster (Saccostrea glomerata) as an indicator of endocrine disruption on the Sunshine Coast. Ecol Manag Restor 11:143–146
Arnaud-Haond S, Monteforte M, Blanc F, Bonhomme F (2003) Evidence for male-biased effective sex ratio and recent step-by-step colonization in the bivalve Pinctada mazatlanica. J Evol Biol 16:790–796
Beukeboom LW, Perrin N (2014) The evolution of sex determination. Oxford, London.
Bordukalo-Niksic T, Mokrovic G, Stefulj J, Zivin M, Jernej B, Cicin-Sain L (2010) 5HT-1A receptors and anxiety-like behaviours: studies in rats with constitutionally upregulated/downregulated serotonin transporter. Behav Brain Res 213:238–245
Boutet I, Moraga D, Marinovic L, Obreque J, Chavez-Crooker P (2008) Characterization of reproduction-specific genes in a marine bivalve mollusc: influence of maturation stage and sex on mRNA expression. Gene 407:130–138
Cheng W, Ip YT, Xu ZS (2013) Gudu, an armadillo repeat-containing protein, is required for spermatogenesis in Drosophila. Gene 531:294–300
Dheilly NM, Lelong C, Huvet A, Kellner K, Dubos MP, Riviere G, Boudry P, Favrel P (2012) Gametogenesis in the Pacific oyster Crassostrea gigas: a microarrays-based analysis identifies sex and stage specific genes. PLoS One 7:e36353
Doniach T, Hodgkin J (1984) A sex-determining gene, fem-1, required for both male and hermaphrodite development in Caenorhabditis elegans. Dev Biol 106:223–235
Finn RN, Kristoffersen BA (2007) Vertebrate vitellogenin gene duplication in relation to the “3R hypothesis”: correlation to the pelagic egg and the oceanic radiation of teleosts. PLoS One 2:e169
Furusawa M, Ohnishi T, Taira T, Iguchiariga SM, Ariga H (2001) AMY-1, a c-Myc-binding protein, is localized in the mitochondria of sperm by association with S-AKAP84, an anchor protein of cAMP-dependent protein kinase. J Biol Chem 276:36647–36651
Gagnidze K, Weil ZM, Pfaff DW (2010) Histone modifications proposed to regulate sexual differentiation of brain and behavior. BioEssays 32:932–939
Gamble T, Zarkower D (2012) Sex determination. Curr Biol 22:R257–R262
Garcia JLA, Munro ES, Monte MM, Fourrier M, Whitelaw J, Smail DA, Ellis AE (2010) Atlantic salmon (Salmo salar L.) serum vitellogenin neutralises infectivity of infectious pancreatic necrosis virus (IPNV). Fish Shellfish Immunol 29:293–297
Garcia-Ortiz JE, Pelosi E, Omari S, Nedorezov T, Piao Y, Karmazin J, Uda M, Cao A, Cole SW, Forabosco A, Schlessinger D, Ottolenghi C (2009) Foxl2 functions in sex determination and histogenesis throughout mouse ovary development. BMC Dev Biol 9:36
Gineitis A, Zalenskaya IA, Yau PM, Bradbury EM, Zalensky AO (2000) Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment. J Cell Biol 151:1591–1598
Gladfelter AS, Pringle JR, Lew DJ (2001) The septin cortex at the yeast mother-bud neck. Curr Opin Microbiol 4:681–689
Green AR, Backus LI (1990) Animal models of serotonin behavior. Ann N Y Acad Sci 600:237–248
Guerrier P, Leclercdavid C, Moreau M (1993) Evidence for the involvement of internal calcium stores during serotonin-induced meiosis reinitiation in oocytes of the bivalve mollusc Ruditapes philippinarum. Dev Biol 159:474–484
Hamida L, Medhioub M, Cochard JC, Pennec ML (2004) Evaluation of the effects of serotonin (5-HT) on oocyte competence in Ruditapes decussatus (Bivalvia, Veneridae). Aquaculture 239:413–420
Hanover JA, Love DC, Prinz WA (2009) Calmodulin-driven nuclear entry: trigger for sex determination and terminal differentiation. J Biol Chem 284:12593–12597
Hodgkin J (1986) Sex determination in the nematode C. elegans: analysis of tra-3 suppressors and characterization of fem genes. Genetics 114:15–52
Hong CS, Park BY, Saintjeannet JP (2007) The function of Dmrt genes in vertebrate development: it is not just about sex. Dev Biol 310:1–9
Iseli C, Jongeneel CV, Bucher P (1999) ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences. Proc Int Conf Intell Syst Mol Biol 1999:138–148
Kocer A, Pinheiro I, Pannetier M, Renault L, Parma P, Radi O, Kim K, Camerino G, Pailhoux E (2008) R-spondin1 and FOXL2 act into two distinct cellular types during goat ovarian differentiation. BMC Dev Biol 8:36–36
Kotaka M, Gover S, Vandeputte-Rutten L, Au SW, Lam VM, Adams MJ (2005) Structural studies of glucose-6-phosphate and NADP+ binding to human glucose-6-phosphate dehydrogenase. Acta Crystallogr D Biol Crystallogr 61:495–504
Krantic S, Guerrier P, Dube F (1993) Meiosis reinitiation in surf clam oocytes is mediated via a 5-hydroxytryptamine5 serotonin membrane receptor and a vitelline envelope-associated high affinity binding site. J Biol Chem 268:7983–7989
Landoni M, De Francesco A, Galbiati M, Tonelli C (2010) A loss-of-function mutation in Calmodulin2 gene affects pollen germination in Arabidopsis thaliana. Plant Mol Biol 74:235–247
Li S, Ou XH, Wei L, Wang ZB, Zhang QH, Ouyang YC, Hou Y, Schatten H, Sun QY (2012) Septin 7 is required for orderly meiosis in mouse oocytes. Cell Cycle 11:3211–3218
Li HL, Zhang ZF, Bi Y, Yang DD, Zhang LT, Liu JG (2014) Expression characteristics of beta-catenin in scallop Chlamys farreri gonads and its role as a potential upstream gene of Dax1 through canonical Wnt signalling pathway regulating the spermatogenesis. PLoS One 9:e115917
Li Y, Zhang LL, Sun Y, Ma XL, Wang J, Li RJ, Zhang MW, Wang S, Hu XL, Bao ZM (2016) Transcriptome sequencing and comparative analysis of ovary and testis identifies potential key sex-related genes and pathways in scallop Patinopecten yessoensis. Mar Biotechnol 18:453–465
Liu XL, Zhang ZF, Shao MY, Liu JG, Muhammad F (2012) Sexually dimorphic expression of foxl2 during gametogenesis in scallop Chlamys farreri, conserved with vertebrates. Dev Genes Evol 222:279–286
Liu Y, Hui M, Cui Z, Luo D, Song C, Li Y, Liu L (2015) Comparative transcriptome analysis reveals sex-biased gene expression in juvenile Chinese mitten crab Eriocheir sinensis. PLoS One 10:e0133068
Longtine MS, Bi E (2003) Regulation of septin organization and function in yeast. Trends Cell Biol 13:403–409
Luber CA, Cox J, Lauterbach H, Fancke B, Selbach M, Tschopp J, Akira S, Wiegand M, Hochrein H, O’Keeffe M, Mann M (2010) Quantitative proteomics reveals subset-specific viral recognition in dendritic cells. Immunity 32:279–289
Markov GV, Tavares R, Dauphin-Villemant C, Demeneix BA, Baker ME, Laudet V (2009) Independent elaboration of steroid hormone signaling pathways in metazoans. Proc Natl Acad Sci U S A 106:11913–11918.
Martinez G, Mettifogo L, Perez MA, Callejas CR (2007) A method to eliminate self-fertilization in a simultaneous hermaphrodite scallop. 1. Effects on growth and survival of larvae and juveniles. Aquaculture 273:459–469
Matson CK, Murphy MW, Sarver AL, Griswold MD, Bardwell VJ, Zarkower D (2011) DMRT1 prevents female reprogramming in the postnatal mammalian testis. Nature 476:101–104.
Matson CK, Zarkower D (2012) Sex and the singular DM domain: insights into sexual regulation, evolution and plasticity. Nat Rev Genet 13:163–174
Matsumoto T, Yamano K, Kitamura M, Hara A (2008) Ovarian follicle cells are the site of vitellogenin synthesis in the Pacific abalone Haliotis discus hannai. Comp Biochem Physiol A Mol Integr Physiol 149:293–298
Matsumoto T, Nakamura AM, Mori K, Kayano T (2009) Molecular characterization of a cDNA encoding putative vitellogenin from the Pacific oyster Crassostrea gigas. Zool Sci 20:37–42
Matsumoto T, Masaoka T, Fujiwara A, Nakamura Y, Satoh N, Awaji M (2013) Reproduction-related genes in the pearl oyster genome. Zool Sci 30:826–850
Moon RT, Kohn AD, De Ferrari GV, Kaykas A (2004) WNT and beta-catenin signalling: diseases and therapies. Nat Rev Genet 5:689–699
Naimi A, Martinez AS, Specq ML, Diss B, Mathieu M, Sourdaine P (2009) Molecular cloning and gene expression of Cg-Foxl2 during the development and the adult gametogenetic cycle in the oyster Crassostrea gigas. Comp Biochem Physiol B Biochem Mol Biol 154:134–142
Ni JB, Zeng Z, Kong DZ, Hou L, Huang HQ, Ke CH (2014) Vitellogenin of Fujian oyster, Crassostrea angulata: synthesized in the ovary and controlled by estradiol-17 beta. Gen Comp Endocrinol 202:35–43
Osada M, Mori K, Nomura T (1992) In vitro effects of estrogen and serotonin on release of eggs from the ovary of the scallop. Nippon Suisan Gakkaishi 58:223–227
Osada M, Harata M, Kishida M, Kijima A (2004) Molecular cloning and expression analysis of vitellogenin in scallop, Patinopecten yessoensis (Bivalvia, Mollusca). Mol Reprod Dev 67:273–281
Paz M, Torrado M, Korochkin LI, Mikhailov AT (2005) Esterase-like and fibronectin-like polypeptides share similar sex-cell-biased patterns in the gonad of hermaphroditic and gonochoric species of bivalve mollusks. Cell Tissue Res 322:475–489
Penman DJ, Piferrer F (2008) Fish gonadogenesis. Part I: genetic and environmental mechanisms of sex determination. Rev Fish Sci 16:16–34
Pertea G, Huang X, Liang F, Antonescu V, Sultana R, Karamycheva S, Lee Y, White J, Cheung F, Parvizi B, Tsai J, Quackenbush J (2003) TIGR gene indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics 19:651–652
Piferrer F, Guiguen Y (2008) Fish Gonadogenesis. Part II: molecular biology and genomics of sex differentiation. Rev Fish Sci 16:35–55
Puinean AM, Rotchell JM (2006) Vitellogenin gene expression as a biomarker of endocrine disruption in the invertebrate, Mytilus edulis. Mar Environ Res 62:S211–S214
Qin Z, Li Y, Sun D, Shao M, Zhang Z (2012) Cloning and expression analysis of the vitellogenin gene in the scallop Chlamys farreri and the effects of estradiol-17β on its synthesis. Invertebr Biol 131:312–321
Reddiah K (1962) The sexuality and spawning of Manx pectinids. J Mar Biol Assoc UK 42:683–703
Santerre C, Sourdaine P, Adeline B, Martinez AS (2014) Cg-SoxE and Cg-beta-catenin, two new potential actors of the sex-determining pathway in a hermaphrodite lophotrochozoan, the Pacific oyster Crassostrea gigas. Comp Biochem Physiol A Mol Integr Physiol. 167:68–76
Shi Y, Wang Q, He MX (2014) Molecular identification of dmrt2 and dmrt5 and effect of sex steroids on their expressions in Chlamys nobilis. Aquaculture 426:21–30
Shimeld SM, Boyle MJ, Brunet T, Luke GN, Seaver EC (2010) Clustered fox genes in lophotrochozoans and the evolution of the bilaterian fox gene cluster. Dev Biol 340:234–248
Sironen A, Hansen J, Thomsen B, Andersson M, Vilkki J, Toppari J, Kotaja N (2010) Expression of SPEF2 during mouse spermatogenesis and identification of IFT20 as an interacting protein. Biol Reprod 82:580–590
Teaniniuraitemoana V, Huvet A, Levy P, Klopp C, Lhuillier E, Gaertner-Mazouni N, Gueguen Y, Le Moullac G (2014) Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes. BMC Genomics 15:491
Tong Y, Zhang Y, Huang JM, Xiao S, Zhang YH, Li J, Chen JH, Yu ZN (2015) Transcriptomics analysis of Crassostrea hongkongensis for the discovery of reproduction-related genes. PLoS One 10:e0134280
Tranter DJ (1958) Reproduction in Australian pearl oysters (Lamellibranchia). III. Pinctada albina (Lamarck): breeding season and sexuality. Mar Freshw Res 9:135–143
Tsai HW, Grant PA, Rissman EF (2009) Sex differences in histone modifications in the neonatal mouse brain. Epigenetics 4:47–53
Tu Q, Brown CT, Davidson EH, Oliveri P (2006) Sea urchin Forkhead gene family: phylogeny and embryonic expression. Dev Biol 300:49–62
Uhlenhaut NH, Jakob S, Anlag K, Eisenberger T, Sekido R, Kress J, Treier AC, Klugmann C, Klasen C, Holter NI, Riethmacher D, Schutz G, Cooney AJ, Lovell-Badge R, Treier M (2009) Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell 139:1130–1142
Veitia RA (2010) FOXL2 versus SOX9: a lifelong “battle of the sexes”. BioEssays 32:375–380
Vizcaino JA, Cote RG, Csordas A, Dianes JA, Fabregat A, Foster JM, Griss J, Alpi E, Birim M, Contell J, O’Kelly G, Schoenegger A, Ovelleiro D, Perez-Riverol Y, Reisinger F, Rios D, Wang R, Hermjakob H (2013) The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res 41:D1063–D1069
Wang Q, He MX (2014) Molecular characterization and analysis of a putative 5-HT receptor involved in reproduction process of the pearl oyster Pinctada fucata. Gen Comp Endocrinol 204:71–79
Wang G, Hu CQ, Jiang T, Luo J, Hu J, Ling SH, Liu M, Xing GQ (2010) Overexpression of serotonin receptor and transporter mRNA in blood leukocytes of antipsychotic-free and antipsychotic-naïve schizophrenic patients: gender differences. Schizophr Res 121:160–171
Wang S, Wang Y, Ma J, Ding Y, Zhang S (2011) Phosvitin plays a critical role in the immunity of zebrafish embryos via acting as a pattern recognition receptor and an antimicrobial effector. J Biol Chem 286:22653–22664
Wang YP, Zhou LS, Zhao YZ, Wang SW, Chen LL, Liu LX, Ling ZQ, Hu FJ, Sun YP, Zhang JY, Yang C, Yang Y, Xiong Y, Guan KL, Ye D (2014) Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress. EMBO J 33:1304–1320
Williams VN, Reading BJ, Hiramatsu N, Amano H, Glassbrook N, Hara A, Sullivan CV (2014) Multiple vitellogenins and product yolk proteins in striped bass, Morone saxatilis: molecular characterization and processing during oocyte growth and maturation. Fish Physiol Biochem 40:395–415
Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6:359–362
Ye J, Fang L, Zheng HK, Zhang Y, Chen J, Zhang ZJ, Wang J, Li ST, Li RQ, Bolund L, Wang J (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:W293–W297
Zhang SC, Sun YN, Pang QX, Shi XD (2005) Hemagglutinating and antibacterial activities of vitellogenin. Fish Shellfish Immunol 19:93–95
Zhang Z, Tang WX, Zhou R, Shen XN, Wei ZY, Patel AM, Povlishock JT, Bennett J, Strauss JF (2007) Accelerated mortality from hydrocephalus and pneumonia in mice with a combined deficiency of SPAG6 and SPAG16L reveals a functional interrelationship between the two central apparatus proteins. Cell Motil Cytoskeleton 64:360–376
Zhang N, Xu F, Guo XM (2014) Genomic analysis of the Pacific oyster (Crassostrea gigas) reveals possible conservation of vertebrate sex determination in a mollusc. G3-Genes Genom Genet 4:2207–2217
Zheng H, Zhang Q, Liu H, Liu W, Sun Z, Li S, Zhang T (2012) Cloning and expression of vitellogenin (Vg) gene and its correlations with total carotenoids content and total antioxidant capacity in noble scallop Chlamys nobilis (bivalve: Pectinidae). Aquaculture 366:46–53
Zhou L, Charkraborty T, Yu X, Wu L, Liu G, Mohapatra S, Wang D, Nagahama Y (2012) R-spondins are involved in the ovarian differentiation in a teleost, medaka (Oryzias latipes). BMC Dev Biol 12:36–36
Acknowledgments
We would like to thank the Shanghai Applied Protein Technology Co., Ltd. for providing the technical support.
Funding
This project was supported by the Science and Technology Program of Guangzhou, China (201707010180), and the Science and Technology Planning Project of Guangdong Province, China (2014B030301064).
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Shi, Y., Liu, W. & He, M. Proteome and Transcriptome Analysis of Ovary, Intersex Gonads, and Testis Reveals Potential Key Sex Reversal/Differentiation Genes and Mechanism in Scallop Chlamys nobilis. Mar Biotechnol 20, 220–245 (2018). https://doi.org/10.1007/s10126-018-9800-1
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DOI: https://doi.org/10.1007/s10126-018-9800-1


