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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

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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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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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