Marine Biotechnology

, Volume 20, Issue 2, pp 206–219 | Cite as

Gonad Transcriptome Analysis of the Pacific Oyster Crassostrea gigas Identifies Potential Genes Regulating the Sex Determination and Differentiation Process

  • Chenyang Yue
  • Qi Li
  • Hong Yu
Original Article


The Pacific oyster Crassostrea gigas is a commercially important bivalve in aquaculture worldwide. C. gigas has a fascinating sexual reproduction system consisting of dioecism, sex change, and occasional hermaphroditism, while knowledge of the molecular mechanisms of sex determination and differentiation is still limited. In this study, the transcriptomes of male and female gonads at different gametogenesis stages were characterized by RNA-seq. Hierarchical clustering based on genes differentially expressed revealed that 1269 genes were expressed specifically in female gonads and 817 genes were expressed increasingly over the course of spermatogenesis. Besides, we identified two and one gene modules related to female and male gonad development, respectively, using weighted gene correlation network analysis (WGCNA). Interestingly, GO and KEGG enrichment analysis showed that neurotransmitter-related terms were significantly enriched in genes related to ovary development, suggesting that the neurotransmitters were likely to regulate female sex differentiation. In addition, two hub genes related to testis development, lncRNA LOC105321313 and Cg-Sh3kbp1, and one hub gene related to ovary development, Cg-Malrd1-like, were firstly investigated. This study points out the role of neurotransmitter and non-coding RNA regulation during gonad development and produces lists of novel relevant candidate genes for further studies. All of these provided valuable information to understand the molecular mechanisms of C. gigas sex determination and differentiation.


Gonad transcriptome Sex determination and differentiation Reproductive regulation Weighted gene correlation network analysis Crassostrea gigas 



This work was supported by the grants from National Natural Science Foundation of China (31772843, 31672649), Shandong Province (2016ZDJS06A06), Fundamental Research Funds for the Central Universities (201762014), and Major Project for Tianjin Seed Technology (15ZXZYNC00050).

Author Contributions

CY carried out the molecular genetic studies, participated in the data analysis, and drafted the manuscript. QL conceived of the study, participated in experimental design and coordination, and contributed to the manuscript preparation. HY participated in the data analysis. All authors read and approved the final manuscript.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

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  1. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106CrossRefPubMedPubMedCentralGoogle Scholar
  2. Anders S, Pyl PT, Huber W (2015) HTSeq—a python framework to work with high-throughput sequencing data. Bioinformatics 31:166–169Google Scholar
  3. Aricescu AR, Hon WC, Siebold C, Lu W, Pa VDM, Jones EY (2014) Molecular analysis of receptor protein tyrosine phosphatase mu-mediated cell adhesion. EMBO J 25:701–712CrossRefGoogle Scholar
  4. Bixby JL (2003) Receptor protein tyrosine phosphatases as mediators of cellular adhesion. Front Biosci 8:d87–d99CrossRefPubMedGoogle Scholar
  5. Bork P, Beckmann G (1993) The CUB domain. A widespread module in developmentally regulated proteins. J Mol Biol 231(2):539–545CrossRefPubMedGoogle Scholar
  6. Chuang JC, Jones PA (2007) Epigenetics and microRNAs. Pediatr Res 61:24R–29RCrossRefPubMedGoogle Scholar
  7. Coe WR (1943) Sexual differentiation in mollusks. I. Pelecypods. Q Rev Biol 18:154–164CrossRefGoogle Scholar
  8. Costa FF (2008) Non-coding RNAs, epigenetics and complexity. Gene 410:9–17CrossRefPubMedGoogle Scholar
  9. 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:e36353CrossRefPubMedPubMedCentralGoogle Scholar
  10. Ebersbach G, Galli E, Møllerjensen J, Löwe J, Gerdes K (2010) Novel coiled-coil cell division factor ZapB stimulates Z ring assembly and cell division. Mol Microbiol 68:720–735CrossRefGoogle Scholar
  11. Enríquez-Díaz M, Pouvreau S, Chávez-Villalba J, Le Pennec M (2008) Gametogenesis, reproductive investment, and spawning behavior of the Pacific giant oyster Crassostrea gigas: evidence of an environment-dependent strategy. Aquac Int 17:491–506CrossRefGoogle Scholar
  12. Fang Q, Fang Y, Weng Y, Dai Y (2003) Distribution of neurotransmitters and regulatory peptide immunoreactive substances in ovary of Crassostrea gigas. J Oceanogr Taiwan Strait 22:299–302Google Scholar
  13. Guo X, Hedgecock D, Hershberger WK, Cooper K, Allen SK (1998) Genetic determinants of protandric sex in the Pacific oyster, Crassostrea gigas Thunberg. Evolution 52:394–402CrossRefPubMedGoogle Scholar
  14. Havrylov S, Redowicz MJ, Buchman VL (2010) Emerging roles of Ruk/CIN85 in vesicle-mediated transport, adhesion, migration and malignancy. Traffic 11:721–731CrossRefPubMedGoogle Scholar
  15. Hedrick PW, Hedgecock D (2010) Sex determination: genetic models for oysters. J Hered 101:602–611CrossRefPubMedGoogle Scholar
  16. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y (2008) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36:D480–D484CrossRefPubMedGoogle Scholar
  17. Langfelder P, Horvath S (2008) WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9:559CrossRefPubMedPubMedCentralGoogle Scholar
  18. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25CrossRefPubMedPubMedCentralGoogle Scholar
  19. Li Y, Zhang L, Sun Y, Ma X, Wang J, Li R, Zhang M, Wang S, Hu X, Bao Z (2016) Transcriptome sequencing and comparative analysis of ovary and testis identifies potential key sex-related genes and pathways in scallop Patinopecten yessoensis. Mar Biotechnol 18:453–465CrossRefPubMedGoogle Scholar
  20. Li Q, Liu W, Shirasu K, Chen W, Jiang S (2006) Reproductive cycle and biochemical composition of the Zhe oyster Crassostrea plicatula Gmelin in an eastern coastal bay of China. Aquaculture 261:752–759CrossRefGoogle Scholar
  21. 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 222:279–286CrossRefPubMedGoogle Scholar
  22. Mao X, Cai T, Olyarchuk JG, Wei L (2005) Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 21:3787–3793CrossRefPubMedGoogle Scholar
  23. Morishita F, Furukawa Y, Matsushima O, Minakata H (2010) Regulatory actions of neuropeptides and peptide hormones on the reproduction of molluscs. Can J Zool 88:825–845CrossRefGoogle Scholar
  24. Munger SC, Capel B (2012) Sex and the circuitry: progress toward a systems-level understanding of vertebrate sex determination. Wiley Interdiscip Rev Syst Biol Med 4:401–412CrossRefPubMedPubMedCentralGoogle Scholar
  25. Naimi A, Martinez AS, Specq ML, Diss B, Mathieu M, Sourdaine P (2009a) 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 154:134–142CrossRefPubMedGoogle Scholar
  26. Naimi A, Martinez AS, Specq ML, Mrac A, Diss B, Mathieu M, Sourdaine P (2009b) Identification and expression of a factor of the DM family in the oyster Crassostrea gigas. Comp Biochem Physiol A Mol Integr Physiol 152:189–196CrossRefPubMedGoogle Scholar
  27. Philippe D, Ababou A, Yang X, Ghosh R, Daviter T, Ladbury JE, Pfuhl M (2011) Making ends meet: the importance of the N- and C-termini for the structure, stability, and function of the third SH3 domain of CIN85. Biochemistry 50:3649–3659CrossRefPubMedGoogle Scholar
  28. Piferrer F (2013) Epigenetics of sex determination and gonadogenesis. Dev Dyn 242:360–370CrossRefPubMedGoogle Scholar
  29. Renault T, Faury N, Barbosa-Solomieu V, Moreau K (2011) Suppression substractive hybridisation (SSH) and real time PCR reveal differential gene expression in the Pacific cupped oyster, Crassostrea gigas, challenged with Ostreid herpesvirus 1. Dev Comp Immunol 35:725–735CrossRefPubMedGoogle Scholar
  30. Santerre C, Sourdaine P, Martinez AS (2012) Expression of a natural antisense transcript of Cg-Foxl2 during the gonadic differentiation of the oyster Crassostrea gigas: first demonstration in the gonads of a lophotrochozoa species. Sex Dev 6:210–221CrossRefPubMedGoogle Scholar
  31. Santerre C, Sourdaine P, Adeline B, Martinez AS (2014) Cg-SoxE and Cg-β-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 167:68–76CrossRefPubMedGoogle Scholar
  32. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108CrossRefPubMedGoogle Scholar
  33. 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 15:491CrossRefPubMedPubMedCentralGoogle Scholar
  34. Töpfer-Petersen E, Romero A, Varela PF, Ekhlasi-Hundrieser M, Dostàlovà Z, Sanz L, Calvete JJ (2009) Spermadhesins: a new protein family. Facts, hypoteses, and perpectives. Andrologia 30:217–224CrossRefGoogle Scholar
  35. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111CrossRefPubMedPubMedCentralGoogle Scholar
  36. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515CrossRefPubMedPubMedCentralGoogle Scholar
  37. Vandesompele J, Preter KD, Pattyn F, Poppe B, Roy NV, Paepe AD, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:research0034Google Scholar
  38. Xue J, Schmidt SV, Sander J, Draffehn A, Krebs W, Quester I, De Nardo D, Gohel TD, Emde M, Schmidleithner L, Ganesan H (2014) Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 40(2):274–288CrossRefPubMedPubMedCentralGoogle Scholar
  39. Yao B, Zhang J, Dai H, Sun J, Jiao Y, Tang Y, Wu J, Shi Y (2007) Solution structure of the second SH3 domain of human CMS and a newly identified binding site at the C-terminus of c-Cbl. Biochim Biophys Acta 1774:35–43CrossRefPubMedGoogle Scholar
  40. Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11:R14CrossRefPubMedPubMedCentralGoogle Scholar
  41. Zhang G, Fang X, Guo X, Li L, Luo R, Xu F, Yang P, Zhang L, Wang X, Qi H, Xiong Z (2012) The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490:49–54CrossRefPubMedGoogle Scholar
  42. Zhang N, Xu F, Guo X (2014) Genomic analysis of the Pacific oyster (Crassostrea gigas) reveals possible conservation of vertebrate sex determination in a mollusc. G3 (Bethesda) 4:2207–2217CrossRefGoogle Scholar
  43. Zhou H, Hu H, Lai M (2010) Non-coding RNAs and their epigenetic regulatory mechanisms. Biol Cell 102:645–655CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Mariculture, Ministry of EducationOcean University of ChinaQingdaoChina
  2. 2.Laboratory for Marine Fisheries Science and Food Production ProcessesQingdao National Laboratory for Marine Science and TechnologyQingdaoChina

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