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Cloning and characterization of rec8 gene in orange-spotted grouper (Epinephelus coioides) and Dmrt1 regulation of rec8 promoter activity

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Abstract

Meiosis is a specialized type of cell division critical for gamete production during sexual reproduction in eukaryotes. The meiotic recombination protein Rec8 has been identified as an important factor in germ cell meiotic initiation in vertebrates; however, its equivalent role in teleosts is poorly characterized. In this study, we cloned and sequenced the rec8 gene from orange-spotted grouper (Epinephelus coioides). The cDNA sequence consisted of 2244 base pairs (bp), including a 5′ untranslated region (UTR) of 198 bp and a 3′UTR of 284 bp. The open reading frame of grouper rec8 was 1752 bp, encoding 584 amino acids. Expression levels of rec8 were higher in the ovary, intersex gonad, and testis. A neighbor-joining phylogenetic tree based on the deduced amino acid sequence indicated a common origin for grouper and other teleost rec8 molecules. Immunohistochemistry using a polyclonal anti-Rec8 antibody localized the protein in the oogonia and primary oocytes in the ovary and in spermatogonia and spermatocytes in the intersex gonad and testis, suggesting that Rec8 may play an important role in the meiotic division and the development of grouper germ cells. In addition, we found that the transcription factor Dmrt1 increased rec8 promoter activity through the second binding site, based on dual-luciferase assays. Together, these results suggest that Rec8 plays a crucial role in meiosis and may be regulated by Dmrt1 to affect meiosis in groupers.

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Data availability

All data generated or analyzed during this study are included in this article.

Abbreviations

Rec8:

meiotic recombination protein

UTR:

untranslated region

DE3:

Escherichia coli

SG:

spermatogonia

SC:

spermatocyte

ST:

spermatid

PO:

primary oocyte

References

  • Burkhardt S, Borsos M, Szydlowska A, Godwin J, Williams SA, Cohen PE, Hirota T, Saitou M, Tachibana-Konwalski K (2016) Chromosome cohesion established by Rec8-cohesin in fetal oocytes is maintained without detectable turnover in oocytes arrested for months in mice. Curr Biol 26:678–685

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chelysheva L, Diallo S, Vezon D, Gendrot G, Vrielynck N, Belcram K, Rocques N, Márquez-Lema A, Bhatt AM, Horlow C, Mercier R, Mézard C, Grelon M (2005) AtREC8 and AtSCC3 are essential to the monopolar orientation of the kinetochores during meiosis. J Cell Sci 118:4621–4432

    CAS  PubMed  Google Scholar 

  • Eijpe M, Offenberg H, Jessberger R, Revenkova E, Heyting C (2003) Meiotic cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins SMC1beta and SMC3. J Cell Biol 160:657–670

    CAS  PubMed  PubMed Central  Google Scholar 

  • Feng R, Fang L, Cheng Y, He X, Jiang WT, Dong RR, Shi HJ, Jiang DN, Sun LN, Wang DS (2015) Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus). Sci Rep-UK 5:10131

    CAS  Google Scholar 

  • Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Ishiguro K, Watanabe Y (2007) Chromosome cohesion in mitosis and meiosis. J Cell Sci 120:367–369

    CAS  PubMed  Google Scholar 

  • Johnston SE, Huisman J, Pemberton JM (2019) A genomic region containing REC8 and RNF212B is associated with individual recombination rate variation in a wild population of Red Deer (Cervus elaphus). G3 8:2265–2276

    Google Scholar 

  • Kim S, Bardwell VJ, Zarkower D (2007) Cell type-autonomous and non-autonomous requirements for Dmrt1 in postnatal testis differentiation. Dev Biol 307:314–327

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kitajima TS, Yokobayashi S, Yamamoto M, Watanabe Y (2003) Distinct cohesin complexes organize meiotic chromosome domains. Science 300:1152–1155

    CAS  PubMed  Google Scholar 

  • Kleckner N (1996) Meiosis: how could it work? Proc Natl Acad Sci U S A 93:8167–8174

    CAS  PubMed  PubMed Central  Google Scholar 

  • Klein F, Mahr P, Galova M, Buonomo SB, Michaelis C, Nairz K, Nasmyth K (1999) A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell 98:91–103

    CAS  PubMed  Google Scholar 

  • Krentz AD, Murphy MW, Kim S, Cook MS, Capel B, Zhu R, Matin A, Sarver AL, Parker KL, Griswold MD, Looijenga LH, Bardwell VJ, Zarkower D (2009) The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ cell proliferation and pluripotency. Proc Natl Acad Sci USA 106(52):22323–22328

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lee JY, Orr-Weaver TL (2001) The molecular basis of sister-chromatied contain. Annu Rev Cell Dev Biol 17:753–777

    CAS  PubMed  Google Scholar 

  • Lee J, Iwai T, Yokota T, Yamashita M (2003) Temporally and spatially selective loss of Rec8 protein from meiotic chromosomes during mammalian meiosis. J Cell Sci 116:2781–2790

    CAS  PubMed  Google Scholar 

  • Lin FM, Tong F, He Q, Xiao SQ, Liu XC, Yang HR, Guo Y, Wang Q, Zhao HH (2020) In vitro effects of androgen on testicular development by the AR-foxl3-rec8/fbxo47 axis in orange-spotted grouper (Epinephelus coioides). Gen Comp Endocrinol 292:11343

    Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Ma G, Zhang W, Liu L, Chao WS, Gu YQ, Qi L, Xu SS, Cai X (2018) Cloning and characterization of the homoeologous genes for the Rec8-like meiotic cohesin in polyploid wheat. BMC Plant Biol 18:224

    CAS  PubMed  PubMed Central  Google Scholar 

  • Matson CK, Murphy MW, Griswold MD, Yoshida S, Bardwell VJ, Zarkower D (2009) The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. Dev Cell 19(4):612–24

    Google Scholar 

  • Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, Morrey CE, Shibata N, Asakawa S, Shimizu N, Hori H, Hamaguchi S, Sakaizumi M (2002) DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature 417:559–563

    CAS  PubMed  Google Scholar 

  • Matta SL, Vilela DA, Godinho HP, França LR (2002) The goitrogen 6-n-propyl-2-thiouracil (PTU) given during testis development increases Sertoli and germ cell numbers per cyst in fish: the tilapia (Oreochromis niloticus) model. Endocrinology 143:970–978

    CAS  PubMed  Google Scholar 

  • Murphy MW, Zarkower D, Bardwell VJ (2007) Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA. BMC Mol Biol 8:58

    PubMed  PubMed Central  Google Scholar 

  • Nasmyth K (2001) Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu Rev Genet 35:673–745

    CAS  PubMed  Google Scholar 

  • Parisi S, McKay MJ, Molnar M, Thompson MA, van der Spek PJ, van Drunen-Schoenmaker E, Kanaar R, Lehmann E, Hoeijmakers JH, Kohli J (1999) Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21p family conserved from fission yeast to humans. Mol Cell Biol 19:3515–3528

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pasierbek P, Jantsch M, Melcher M, Schleiffer A, Schweizer D, Loidl J (2001) A Caenorhabditis elegans cohesion protein with functions in meiotic chromosome pairing and disjunction. Genes Dev 15:1349–1360

    CAS  PubMed  PubMed Central  Google Scholar 

  • Quagio-Grassiotto I, Grier H, Mazzoni TS, Nóbrega RH, de Arruda Amorim JP (2011) Activity of the ovarian germinal epithelium in the freshwater catfish, Pimelodus maculatus (Teleostei: Ostariophysi: Siluriformes): germline cysts, follicle formation and oocyte development. J Morphol 272:1290–1306

    PubMed  Google Scholar 

  • Raymond CS, Shamu CE, Shen MM, Seifert KJ, Hirsch B, Hodgkin J, Zarkower D (1998) Evidence for evolutionary conservation of sex-determining genes. Nature 391:691–695

    CAS  PubMed  Google Scholar 

  • Raymond CS, Kettlewell JR, Hirsch B, Bardwell VJ, Zarkower D (1999) Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development. Dev Biol 215:208–220

    CAS  PubMed  Google Scholar 

  • Raymond CS, Murphy MW, O’Sullivan MG, Bardwell VJ, Zarkower D (2000) Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Genes Dev 14:2587–2595

    CAS  PubMed  PubMed Central  Google Scholar 

  • Roeder GS (1997) Meiotic chromosomes: it takes two to tango. Genes Dev 11:2600–2621

    CAS  PubMed  Google Scholar 

  • Schulz RW, Menting S, Bogerd J, Franca LR, Vilela DAR, Godinho HP (2005) Sertoli cell proliferation in the adult testis: evidence from two fish species belonging to different orders. Biol Reprod 73:891–898

    CAS  PubMed  Google Scholar 

  • Schulz RW, de França LR, Lareyre JJ, Le Gac F, Chiarini-Garcia H, Nobrega RH, Miura T (2010) Spermatogenesis in fish. Gen Comp Endocrinol 165:390–411

    CAS  PubMed  Google Scholar 

  • Smith CA, Roeszler KN, Ohnesorg T, Cummins DM, Farlie PG, Doran TJ, Sinclair AH (2009) The avian Z-linked gene DMRT1 is required for male sex determination in the chicken. Nature 461:267–271

    CAS  PubMed  Google Scholar 

  • Tachibana-Konwalski K, Godwin J, van der Weyden L, Champion L, Kudo NR, Adams DJ, Nasmyth K (2010) Rec8-containing cohesin maintains bivalents without turnover during the growing phase of mouse oocytes. Genes Dev 24:2505–2516

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Q, Huang M, Peng C, Wang X, Xiao L, Wang D, Chen J, Zhao H, Zhang H, Li S, Yang H, Liu Y, Lin H, Zhang Y (2018) MT-feeding-induced impermanent sex reversal in the orange-spotted grouper during sex differentiation. Int J Mol Sci 19:2828

    PubMed Central  Google Scholar 

  • Watanabe Y, Nurse P (1999) Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Nature 400:461–464

    CAS  PubMed  Google Scholar 

  • Wockner LF, Morris CP, Noble EP, Lawford BR, Whitehall VLJ, Young RM, Voisey J (2015) Brain-specific epigenetic markers of schizophrenia. Transl Psychiatry 5:e680

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xia W, Zhou L, Yao B, Li CJ, Gui JF (2007) Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers. Mol Cell Endocrinol 263:156–172

    CAS  PubMed  Google Scholar 

  • Xu H, Beasley M, Verschoor S, Inselman A, Handel MA, McKay MJ (2004) A new role for the mitotic RAD21/SCC1 cohesin in meiotic chromosome cohesion and segregation in the mouse. EMBO Rep 5:378–384

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xu H, Beasley MD, Warren WD, van der Horst GT, McKay MJ (2005) Absence of mouse REC8 cohesin promotes synapsis of sister chromatids in meiosis. Dev Cell 8:949–961

    CAS  PubMed  Google Scholar 

  • Yokobayashi S, Yamamoto M, Watanabe Y (2003) Cohesins determine the attachment manner of kinetochores to spindle microtubules at meiosis I in fission yeast. Mol Biol Cell 23:3965–3973

    CAS  Google Scholar 

  • Yoon SW, Lee MS, Xaver M, Zhang L, Hong SG, Kong YJ, Cho HR, Kleckner N, Kim KP (2016) Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure. Nucleic Acids Res 44:9296–9314

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Ume-hara C, Matsuda Y, Takamatsu N, Shiba T, Ito M (2008) A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis. Proc Natl Acad Sci U S A 105:2469–2474

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yu HG, Dawe RK (2000) Functional redundancy in the maize meiotic kinetochore. J Cell Biol 151:131–141

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yu J, Liang Q, Wang J, Wang K, Gao J, Zhang J, Zeng Y, Chiu PW, Ng EK, Sung JJ (2017) REC8 functions as a tumor suppressor and is epigenetically downregulated in gastric cancer, especially in EBV-positive subtype. Oncogene 36:182–193

    CAS  PubMed  Google Scholar 

  • Zarkower D (2001) Establishing sexual dimorphism: conservation amidst diversity? Nat Rev Genet 2:175–185

    CAS  PubMed  Google Scholar 

Download references

Funding

This work was supported by the Youth Fund Project of the National Natural Science Foundation of China (No. 41806151), the National Natural Science Foundation of China (No. 31972768), the Guangdong Provincial Natural Science Foundation (2017A030313099), and the Provincial Projects with Special Funds for Promoting Economic Development of Marine and Fisheries Department of Guangdong (SDYY-2018-05).

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Authors and Affiliations

  1. College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, People’s Republic of China

    Qing Wang, Fangmei Lin, Qi He, Qifeng Huang, Xuzhuo Duan, Xiaochun Liu, Shiqiang Xiao, Huirong Yang & HuiHong Zhao

  2. Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, People’s Republic of China

    Qing Wang, Huirong Yang & HuiHong Zhao

  3. Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, Guangzhou, 510642, People’s Republic of China

    Qing Wang & HuiHong Zhao

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  1. Qing Wang
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  8. Huirong Yang
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  9. HuiHong Zhao
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Contributions

QW, HR-Y, and HH-Z designed the experiments. FM-L constructed the plasmid. QH carried out immunohistochemistry. QF-H conducted the cell experiments and dual-luciferase studies. XZ-D examined the gonadal histology. XC-L and SQ-X performed real-time PCR and gene cloning. QW and HH-Z wrote the manuscript, with feedback from all other authors. All authors participated in the interpretation of data, editing, and approval of the manuscript.

Corresponding authors

Correspondence to Huirong Yang or HuiHong Zhao.

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All animal experiments were conducted in accordance with the guidelines and with the approval of the Animal Research and Ethics Committees of South China Agriculture University.

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The authors declare that they have no conflict of interest.

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All international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

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Wang, Q., Lin, F., He, Q. et al. Cloning and characterization of rec8 gene in orange-spotted grouper (Epinephelus coioides) and Dmrt1 regulation of rec8 promoter activity. Fish Physiol Biochem 47, 393–407 (2021). https://doi.org/10.1007/s10695-020-00920-7

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