Identification and characterization of germ cell genes vasa and dazl in a protogynous hermaphrodite fish, orange-spotted grouper (Epinephelus coioides)

Journal Pre-proof Identification and characterization of germ cell genes vasa and dazl in a protogynous hermaphrodite fish, orange-spotted grouper (Epinephelus coioides) Ling Qu, Xi Wu, Meifeng Liu, Chaoyue Zhong, Hongyan Xu, Shuisheng Li, Haoran Lin, Xiaochun Liu PII:

S1567-133X(19)30167-X

DOI:

https://doi.org/10.1016/j.gep.2020.119095

Reference:

MODGEP 119095

To appear in:

Gene Expression Patterns

Received Date: 13 October 2019 Revised Date:

18 January 2020

Accepted Date: 25 January 2020

Please cite this article as: Qu, L., Wu, X., Liu, M., Zhong, C., Xu, H., Li, S., Lin, H., Liu, X., Identification and characterization of germ cell genes vasa and dazl in a protogynous hermaphrodite fish, orangespotted grouper (Epinephelus coioides), Gene Expression Patterns (2020), doi: https://doi.org/10.1016/ j.gep.2020.119095. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

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Identification and characterization of germ cell genes vasa and dazl in

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a protogynous hermaphrodite fish, orange-spotted grouper

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(Epinephelus coioides)

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Ling Qu a, Xi Wu a, Meifeng Liu a, Chaoyue Zhong a, Hongyan Xu b, Shuisheng Li a,

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Haoran Lin a, Xiaochun Liu a, ⁎

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a

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Guangdong Provincial Key Laboratory of Improved Variety Reproduction in Aquatic

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Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou,

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China

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b

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Guangzhou, China

State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and

Pearl River Fisheries Research Institute, Chinese Academic of Fisheries Sciences,

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⁎

Corresponding authors.

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E-mail addresses: Xiaochun Liu, [email protected]

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ABSTRACT

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Vasa and dazl genes have been reported to play pivotal roles in germ cell

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development and differentiation both in vertebrates and invertebrates; however, little

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is known about their functions in germ cell differentiation during gametogenesis and

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sex reversal in hermaphroditic fish. In the present study, vasa (Ecvasa) and dazl

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(Ecdazl) cDNA were cloned from orange-spotted grouper (Epinephelus coioides). The

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full-length cDNA sequences of Ecvasa and Ecdazl were 2162 and 2101 bp, and

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encoded 646 and 214 amino acid residues, respectively. Real-time PCR showed that

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Ecvasa and Ecdazl mRNA were highly expressed in the gonads, with higher

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expression levels in the testis than in the ovary. Further, in situ hybridization revealed

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that Ecvasa and Ecdazl RNA were dynamically expressed in germ cells at different

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stages during oogenesis, sex reversal, and spermatogenesis in orange-spotted grouper.

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Intriguingly, the signals for Ecvasa and Ecdazl mRNA became weaker in oocytes of

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ovo-testes gonads, indicating that the expression of germ cell genes could be

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suppressed in oocytes during sex reversal in the orange-spotted grouper. Our study is

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the first time to describe the expression profiles of vasa and dazl mRNA in germ cells

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during gametogenesis and sex reversal in the orange-spotted grouper. These findings

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will provide new insights into understanding the mechanisms through which vasa and

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dazl regulate germ cell differentiation in hermaphrodite fish species.

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Keywords Epinephelus coioides, Ddx4, deleted in azoospermia-like, germ cells, sex

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reversal

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Highlights •

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expression in testis. •

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Ecvasa and Ecdazl mRNA were highly expressed in gonads with higher

Expression of germ cell genes could be suppressed in oocytes during sex reversal.

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Vasa and dazl can be used as marker genes to trace the germ cells development during sex reversal in orange-spotted grouper.

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1. Introduction

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Germ cell development is the basis of animal reproduction. The formation and

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development of germ cells, which originate from primordial germ cells and are

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specialized during embryogenesis, are extremely complex processes. Processes from

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the formation of primordial germ cells to their maturation are regulated by many

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factors including genes, hormones, and the environment (Barske and Capel, 2008).

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Germ cell markers could be effectively used to trace the development of germ cells,

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and this would facilitate studying the mechanisms behind the specification and

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development of germ cells. Moreover, there have been many studies on gene markers

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of germ cells and other gonadal cells including the PGC specific gene nanos3 (Sun et

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al., 2017), the germline stem cell gene nanos2 (Sun et al., 2017), the Sertoli cell gene

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sox9 (Xia et al., 2007), and the germ cell genes vasa, dazl, and boule (Xu et al., 2005;

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Li et al., 2011; Bhat and Hong, 2014).

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Vasa (Ddx4, DEAD box polypeptide 4) is a well-known germ cell marker gene.

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Vasa protein is an ATP-dependent RNA helicase, highly conserved across phyla,

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which also presents a characteristic DEAD-box (Asp-Glu-Ala-Asp) (Mochizuki et al.,

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2001). The vasa gene was initially identified as a maternal-effect gene, and mutations

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in this gene were found to cause abdominal deformities and developmental failure in

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Drosophila germ cells (Schüpbach and Wieschaus, 1986). Subsequently, a vasa-like

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gene was also identified in fish (Kobayashi et al., 2000), amphibians (Marracci et al.,

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2007), birds (Tsunekawa et al., 2000), and mammals (Toyooka et al., 2000). In Hydra

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magnipapillata, vasa was found to be expressed both in oocytes and nurse cells

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(Mochizuki et al., 2001). In addition, in medaka (Shinomiya et al., 2000), mice

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(Toyooka et al., 2000), and most other species, the vasa gene is expressed only in the

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germ cell lineage from the embryo stage to the adult gonad.

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Dazl (deleted in azoospermia-like), a maternal gene, is another germ cell marker

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gene in vertebrates (Johnson et al., 2001; Li et al., 2011; Li et al., 2016). The dazl

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gene belongs to the DAZ (deleted in azoospermia) family, which contains boule, daz,

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and dazl (Xu et al., 2001). These genes encode proteins with an RNA-binding domain.

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The dazl gene has been found in different species, including fish (Li et al., 2011),

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axolotl (Johnson et al., 2001), chickens (Lee et al., 2016), and humans (Reijo et al.,

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2000).

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Although vasa and dazl are widely defined as germ cell marker genes in many

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animals, their expression patterns vary and present species-specificity to a variable

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extent in different species. (Bhat and Hong, 2014; Cardinali et al., 2004; Dwarakanath

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et al. 2014; Li et al., 2017; Kobayashi et al., 2000; Úbeda-Manzanaro et al., 2014). In

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addition to their important roles in germ cell marker, Vasa and dazl can also play

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important roles in germ cell development, proliferation, and differentiation. The

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number of PGCs (primordial germ cells) was significantly reduced, and

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differentiation failure was induced when Xenopus laevis were microinjected with an

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anti-vasa antibody (Ikenishi and Tanaka, 1997). In medaka, vasa knockout resulted in

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a PGC migration defect but did not alter PGC numbers (Li et al., 2009), while

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zebrafish with vasa mutations developed exclusively into sterile males (Hartung et al.,

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2014). Interestingly, dazl might possess similar functions to vasa in germ cells. For

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example, the microinjection of a Dazl antibody significantly reduced the number of

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PGCs and even completely abolished PGC formation in medaka (Li et al., 2016). In

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both male and female mice, Dazl mutations resulted in the failure of germ cells to

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complete meiotic prophase (Saunders et al., 2003). In addition, dazl inhibited the

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development of PGCs by limiting the translation of core pluripotency factors,

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differentiation, and apoptosis, implying that dazl has an active role in large mRNA/protein interactive networks (Chen et al., 2014).

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In the grouper, a typical protogynous hermaphroditic fish, the gonad first

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develops as an ovary and then reverses into a testis during the course of the

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organism’s life history, making it an ideal model for investigating ovarian

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development and sex reversal in fish (Chen et al., 2011). The regulatory mechanism

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of sex differentiation and sex reversal for these species are far from been clarified.

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The discovery of specific germ cell marker genes may be of great help in the study of

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this process. In the present study, we identified and cloned two germ cell genes, vasa

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and dazl, from orange spotted-grouper (Epinephelus coioides) and analyzed their

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expression patterns in gonads at different developmental stages during sexual reversal.

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These will ultimately provide the basis for further studies on the mechanisms

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underlying the differentiation and migration of PGCs, gonadal development, sex

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differentiation, and sex reversal in grouper.

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2. Materials and Methods

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2.1. Animals and ethics

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Orange-spotted grouper were obtained from the Marine Fisheries Development

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Center of Guangdong Province (Huizhou, Guangdong, China). Fish were reared under

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controlled water temperatures of 22.7–27.7 ℃. The use of animals in this study was

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endorsed by the respective Animal Research and Ethics Committees of Sun Yat-Sen

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University, and all experiments were conducted in accordance with the guidelines of

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the committee.

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2.2. Cloning and sequence analysis of Ecvasa and Ecdazl

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Total RNA from the orange-spotted grouper samples was extracted using Trizol

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reagent (Invitrogen, USA). The RNA quality was assessed by 1.2% agarose gel

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electrophoresis and verified using an ultraviolet spectrophotometer (NanoDrop

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2000/2000c, USA) with the A260/A280 ratio between 1.8 and 2.0. Full-length cDNA

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sequences of Ecvasa and Ecdazl were cloned from the ovary using 5′ RACE (rapid

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amplification of cDNA ends) and 3′ RACE by the SMARTer™ RACE cDNA

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Amplification Kit (Clontech, USA). Based on the cDNA fragments of the E. coioides

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genome (unpublished data), two pairs of vasa primers, vasa-5′ R1/vasa-5′ R2 and

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vasa-3′ F1/vasa-3′ F2 were designed (Supplementary Table 1). Two rounds of PCR

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were conducted for the 5′ and 3′ RACE amplification. The vasa-5′ R1 and UPM

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(universal primer) comprising long-UPM and short-UPM were mixed at a ratio of 1:5

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for the first round 5′ RACE primers, whereas vasa-3′ F1 and UPM were used as the

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first round primers for 3′ RACE. vasa-5′ R2 and NUP (nested universal primer), as

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well as vasa-3′ F2 and NUP, were used as the second-round primers for 5′ and 3′

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RACE, respectively. The same method was used to clone the full-length cDNA of

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dazl. PCR was performed using a KOD-Plus-Neo (Toyobo, Japan) according to the

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manufacturer’s instructions. All PCR products were separated on 1.2% agarose gels,

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purified using a Gel Extraction Kit (Omega), and then ligated into PGEM®-T

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(Promega, USA). Five different positive clones were selected for sequencing.

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Putative amino acid and multiple sequence alignments were performed using

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DNAMAN version 8 (USA). Phylogenetic trees were generated with MEGA version

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4.0 (USA) using the neighbor-joining method.

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2.3. Tissue distribution of Ecvasa and Ecdazl mRNA

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The tissue expression patterns of Ecvasa and Ecdazl mRNA in orange-spotted

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grouper were analyzed by real-time PCR. Eight tissues, including pituitary, stomach,

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spleen, kidney, liver, gonad, intestine and heart, were collected from adult females

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(n=5) and males (n=5), respectively. The fish was anesthetized with 0.05% MS222

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before being euthanized. Tissues were quickly dissected, frozen immediately in liquid

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nitrogen, and kept at -80℃ for RNA extraction.

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2.4. Expression patterns of Ecvasa and Ecdazl in different stages of gonad

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development

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The expression patterns of Ecvasa and Ecdazl during gonad development were

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analyzed using real-time PCR. Gonad samples were collected from orange-spotted

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grouper of three-month-old to three-year-old. The fish was anesthetized before being

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sacrificed. Gonad tissues were quickly dissected, frozen immediately in liquid

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nitrogen, and stored at -80℃ for RNA extraction. Part of the gonad was conserved in 4%

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PFA (paraformaldehyde) for in situ hybridization. A piece from the central part of the

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gonad of each fish was fixed in Bouins’ solution for histology analysis. The

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development stages of the gonad of each fish was determined according to the

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histological criteria described in our previous study (Chen et al., 2011). Seven stages

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of gonads were collected in the present study, including gonadal stage-I: the ovary

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with proliferative oogonia and an ovarian lumen; gonadal stage-II: the ovary with

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mostly primary growth-stage oocytes; gonadal stage-III: the ovary with cortical

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alveolus-stage oocytes; gonadal stage-IV: the ovary with vitellogenic-stage oocytes;

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gonadal stage-V: the early bisexual gonad; gonadal stage-VI: the late bisexual gonad;

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gonadal stage-VII: the testis. Gonad tissue at each developmental stage was collected

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from five to eight fish.

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2.5. In situ hybridization

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In situ hybridization was performed in accordance with a previously reported

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procedure (Xu et al., 2005). Briefly, gonadal tissues were dissected and fixed in 4%

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PFA at 4 °C overnight, washed with PBS (phosphate buffered saline) at room

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temperature, dehydrated with gradient methanol/PBS, and stored in 100% methanol at

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-20 °C overnight. Then, the tissues were rehydrated with gradient methanol/PBS,

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immersed in 30% (w/v) sucrose–PBS at 4 °C overnight, embedded in optimal cutting

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temperature compound (Sakura, USA), cryosectioned at 6–10 µm for ovaries and 4–5

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µm for testis using a Leica RM2135 Microtome (Leica, Germany), and pasted

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on polylysine-coated slides (Fishery, USA).

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Probes were prepared using the DIG RNA labeling kit (Roche). The lengths of

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the vasa and dazl probes were 1041 and 707 bp, respectively (primers shown in

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Supplementary Table 1). The probes were treated with DNaseI (Ambion, USA) for 15

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min at 37 °C, then purified with lithium chloride and ethanol. Frozen testis and ovary

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sections were pre-hybridized for a minimum of 1 hour, then hybridized with either the

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sense or antisense probes at 65 °C for 12–18 hours. The fluorescence and chemical in

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situ hybridization signals were developed by staining with anti-Digoxigenin-POD

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(Roche, Germany) and anti-Digoxigenin-AP (Roche, Germany), then incubated in

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TSATM PLUS Fluorescein (PerkinElmer, USA) and NBT/BCIP (Roche, Germany) as

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the chromogenic substrates, respectively.

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2.6. Real-time PCR

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Real-time PCR was performed on an ABI 7900 real-time PCR System. Total

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RNA was digested with DNaseI (Ambion, USA) and reverse transcribed into cDNA

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using the ReverTra Ace qPCR RT Kit (Toyobo). The PCR reactions are performed

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using SYBR Green I (Toyobo, Japan) and the primers were presented in

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Supplementary Table 1. All experiments were performed according to the

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manufacturers’ instructions. The PCR cycling conditions were as follows: 120

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seconds at 50 °C and 120 seconds at 95 °C, followed by 40 cycles of 15 seconds at

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95 °C, 30 seconds at 60 °C, and 30 seconds at 72 °C, with final steps for 15 seconds at

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95 °C and 30 seconds at 60 °C. The β-actin gene was used as an internal control.

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2.7. Histological examination of gonadal developmental stages

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Gonadal tissues were fixed in Bouins’ solution overnight, dehydrated with serial

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grades of ethanol, treated with xylene, then embedded in paraffin. The gonadal tissues

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were serially-sectioned at 5–8 µm and stained with hematoxylin and eosin (Chen et al.,

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2011). The classification of gonadal stages was performed by light microscopy

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(Nikon IQ50, Japan).

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2.8. Statistical analysis

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All data are expressed as the mean values ± SEM. Statistical analysis was carried

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out by one-way analysis of variance (ANOVA) or a Student’s t-test, and P < 0.05 was

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considered statistically significant. All statistics were performed using GraphPad

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Prism 5.0 (GraphPad Software, USA).

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3. Results

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3.1. Identification of vasa and dazl cDNA in orange-spotted grouper

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The full-length cDNA sequences of vasa and dazl were cloned by 5′ and 3′

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RACE. The Ecvasa complete cDNA sequence (GenBank accession no. MK655479)

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was 2162 bp in length and consisted of 1961 bp of ORFs (open reading frames), 92 bp

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of 5′-UTR (untranslated region), 129 bp of 3′-UTR, and encoded a 646-amino acid

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protein (Supplementary Fig. 1). The full-length Ecdazl sequence (GenBank accession

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no. MK655477) was 2101 bp, consisted of a 645 bp ORF encoding 214 amino acid

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residues, a 96 bp 5′-UTR and a 1359 bp 3′-UTR (Supplementary Fig. 2).

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Multiple sequence alignment revealed that EcVasa shared high identity with fish

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Vasa sequences, ranging from 72–95% (Supplementary Table 2). EcVasa protein

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contained eight conserved motifs as follows: motifI (AQTGSGKT), motifIa

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(PTRELI), motifIb (TPGR), motifII (DEAD), motifIII (SAT), motifIV (RGLD),

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motifV (RGLD), and motifQ (GYVKPTPVQ) (Fig. 1). The EcDazl shared 54–63%

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identity with fish Dazl sequences and 38–51% with other species (Supplementary

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Table 2). EcDazl protein also had highly conserved RNP1 and RNP2 motifs in the

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RNA recognition motif (Fig. 2).

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Based on the phylogenetic tree, EcVasa and Vasa homologs in fish were

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clustered into a single clade (Fig. 3). In addition, EcVasa was clustered together with

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the Vasa homologs of brown-marbled grouper, tilapia, and medaka. The Dazl proteins

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were clustered into two separate clades, fish and other vertebrates (Fig. 4). Moreover,

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EcDazl was clustered together with tilapia and medaka Dazl proteins.

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3.2. Tissue-specific expression of Ecvasa and Ecdazl mRNA

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The expression of Ecvasa and Ecdazl was detected in tissues, the results showed

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that Ecvasa was specifically expressed in ovary and testis tissues, and not detected in

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the other somatic tissues examined (Fig. 5). However, the Ecdazl was highly

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expressed in gonads, and was also slightly expressed in non-gonad tissues such as the

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heart and intestines. Both Ecvasa and Ecdazl were expressed at higher levels in the

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testis than in the ovary.

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3.3. Expression patterns of Ecvasa and Ecdazl in different gonadal stages

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The mRNA expression levels of Ecvasa and Ecdazl in gonads at different

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development stages were detected (Fig. 6), and the corresponding histological details

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of gonads are shown in Supplementary Fig. 3. The Ecvasa mRNA was lowly

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expressed in ovary at gonadal stage-I, but significantly increased in ovary at gonadal

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stage-II and decreased in ovary at gonadal stage-IV. During sex reversal from ovary

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to testis, the expression of Ecvasa was significantly increased. The mRNA expression

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level of Ecdazl was very low in ovary at gonadal stage-I, significantly increased in

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ovary at gonadal stage-II. And the Ecdazl expression level was also significantly

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increased during sex reversal.

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3.4. Localization of Ecvasa and Ecdazl in germ cells during gametogenesis

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Ecvasa mRNA signals were restricted to germ cells and were not detected in the

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negative control (Fig. 7a). In the ovary, Ecvasa mRNA was predominantly detected in

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the oogonia (Og), primary oocytes (O1), and cortical–alveolus stage oocytes (O2) but

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was scarcely detected in vitellogenic-stage oocytes (O3) (Fig. 7–8). Signals of Ecvasa

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mRNA in the oogonia were moderate and uniformly distributed in the cytoplasm (Fig.

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7b, Fig. 8a1–a3), highest in the primary oocytes and cortical-alveolus stage oocytes

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(Fig. 7c–d, Fig. 8b1–b3), while Ecvasa mRNA exhibited non-uniform distribution in

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the cytoplasm that formed condensed patches (Fig. 7c–d), but were scarcely detected

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in vitellogenic-stage oocytes (Fig. 7d, Fig. 8b1–b3). Similar to Ecvasa, Ecdazl mRNA

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expression was moderate in the oogonia (Fig. 7h, Fig. 8a4–a6), peaked in primary

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oocytes and cortical-alveolus stage oocytes (Fig. 7i-j, Fig. 8b4–b6), and was weakly

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detected in the vitellogenic-stage oocytes (Fig. 7j, Fig. 8b4–b6). Ecdazl signals could

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not be detected in the negative control (Fig. 7g) and somatic cells of the ovary (Fig.

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7–8).

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In the testis, Ecvasa expression was detected from the spermatogonium to

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spermatid stages (Fig. 7–8). Ecvasa mRNA expression was strong in spermatogonia,

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but weak in spermatocytes and spermatids. Moreover, part of the Ecvasa signal was

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concentrated in one or more dots in the cytoplasm of spermatogonia, spermatocytes

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and spermatids, whereas the others were uniformly distributed in the cytoplasm. All

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Ecvasa signals in spermatids were concentrated in the patches (Fig. 8). Ecdazl signals

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in the testis were also observed throughout spermatogenesis, with being the highest in

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the spermatogonia, strong in spermatocytes, and moderate in spermatids (Fig. 7–8).

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In the bisexual gonad, Ecvasa and Ecdazl mRNA signals became degraded in

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oocytes, and were even undetectable in some of the oocytes; however, Ecvasa and

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Ecdazl mRNA could be easily detected in spermatocytes (Fig. 7e, k).

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4. Discussion

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4.1. Characterization of Ecvasa and Ecdazl cDNA

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In this study, Ecvasa and Ecdazl cDNA were cloned. Sequence analyses showed

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that EcVasa contains eight conserved motifs, showing the highest homology with

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previously described Vasa proteins (Xu et al., 2005). EcDazl had an RNA recognition

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motif, closely resembling to those reported in DAZ family members in other species

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(Johnson et al., 2001). Phylogenetic analysis showed that both EcVasa and EcDazl

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were clustered with Vasa and Dazl homologs, respectively. These analyses also

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indicated that EcVasa appears to be a Vasa homolog and that EcDazl is a Dazl

297

homolog in the orange-spotted grouper. In addition, Vasa or Dazl homologs of

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orange-spotted grouper, tilapia, and medaka clustered into a single clade, whereas

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those of zebrafish and rainbow trout formed another clade, indicating that these

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proteins in orange-spotted grouper are closer to those of tilapia and medaka than those

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of zebrafish and rainbow trout. The findings imply that the functions of these genes in

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grouper might be more similar to those of the tilapia and medaka homologs

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(Kobayashi et al., 2000; Li et al., 2009; Li et al., 2016).

304 305

4.2. The expression profiles of Ecvasa and Ecdazl are associated with germ cells

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differentiation

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In this study, we observed that Ecvasa was exclusively expressed in gonads and

308

not in somatic tissues. Therefore, vasa is gonad-specific gene in the orange-spotted

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grouper, this is similar to the previously reported in other species (Tsunekawa et al.,

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2000; Johnson et al., 2001; Li et al., 2017). However, the studies in the European sea

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bass revealed that vasa mRNA was also expressed in some of somatic tissues other

312

than gonads (Blázquez et al., 2011). Likewise, the Ecdazl mRNA was highly

313

expressed in gonads, also slightly detected in somatic tissues such as the heart and

314

intestines. In Atlantic Salmon, dazl-1 was also shown to be expressed in gonads and

315

non-gonadal tissues (Kleppe et al., 2017). On the contrary, adult tissues in various fish

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species including Asian seabass (Dwarakanath et al., 2014), medaka (Xu et al., 2007),

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tilapia (Bhat and Hong, 2014) had shown that dazl was restricted to gonads. These

318

results suggest that vasa and dazl might play a multifunctional role, not only in the

319

gonads but also in some nongonadal tissues.

320

The expression profile of both the Ecvasa and Ecdazl in the ovary exhibited

321

sexually dimorphic which is similar to those reported in tilapia (Kobayashi et al.,

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2000; Bhat and Hong, 2014) and medaka (Shinomiya et al., 2000; Xu et al., 2007).

323

Moreover, during ovary development, the expression levels of vasa and dazl mRNA

324

showed dynamic patterns: vasa and dazl mRNA were detected low in the early stage

325

of gonad, such as the stage-I (the ovary with proliferative oogonia and an ovarian

326

lumen), increased in gonadal stage-II (the ovary with mostly primary growth-stage

327

oocytes) and gonadal stage-III (the ovary with cortical alveolus stage-oocytes). In

328

addition, the analysis of in situ hybridization showed that mRNA expression of

329

Ecvasa and Ecdazl were moderate in the oogonium, abundant in primary oocytes and

330

cortical-alveolus stage-oocytes, but scarce in vitellogenic-stage oocytes. Similar

331

expression patterns were also observed for vasa transcript in tilapia (Kobayashi et al.,

332

2000), medaka (Shinomiya et al., 2000), and gibel carp (Xu et al., 2005), as well as

333

dazl mRNA in medaka (Xu et al., 2007) and tilapia (Bhat and Hong, 2014). Together,

334

these results suggest that vasa and dazl genes might play an important role in the

335

developing oocytes during oogenesis. However, the expression level of vasa in the

336

oocytes of gilthead bream (Cardinali et al., 2004) and dazl in the oocytes of the Asian

337

seabass (Dwarakanath et al., 2014) was found to be increased as the oocytes

338

developed to the mature gametes.

339

Furtherly, the analysis of in situ hybridization showed that the Ecvasa and

340

Ecdazl signals became decreased in male germ cells through spermatogonia till

341

spermatid in the testes of the orange-spotted grouper, thus demonstrating the similar

342

expression patterns to those of medaka (Shinomiya et al., 2000) and tilapia

343

(Kobayashi et al., 2000). Moreover, the Ecdazl signal was highest in spermatogonia,

344

strong in spermatocytes, and moderate in spermatids, resulting in a similar expression

345

pattern to that documented in rainbow trout (Li et al., 2014). These data suggest that

346

both the vasa and dazl genes play important roles in spermatogenesis. Additionally,

347

Ecvasa and Ecdazl mRNA were restricted to germ cells in gonads, and thus, they

348

could be used as marker genes to trace the germ cells development in the

349

orange-spotted grouper. However, vasa mRNA signal in the Chinese soft-shell turtle

350

(Li et al., 2017) and DAZL transcript in human (Reijo et al., 2000) were found to be

351

weak in spermatogonia and strong in spermatids. The findings indicated that vasa and

352

dazl might play species-specific roles involved in germ cells’ differentiation across

353

phyla.

354

Importantly, the vasa and dazl RNA exhibited obvious subcellular distributions

355

in grouper germ cells: most of vasa and dazl mRNA signals were concentrated into

356

particles in the perinuclear cytoplasm of germ cells. This kind of particles might to be

357

the Balbiani body which has been widely studied in other fish species (Li et al., 2013;

358

Xu et al., 2014; Riccia et al., 2018). To verify this issue, it needs more extensive

359

investigations in the future.

360

4.3. Expression levels of Ecvasa and Ecdazl are associated with sex reversal

361

Less expression of DAZL and VASA could cause azoospermic in men (Guo et al.,

362

2007; Lin et al., 2001). The Dazl mutation would affect the amount, motility, and

363

morphology of sperm in mice (Hsu et al., 2010). Moreover, it was reported that the

364

overexpression of VASA and/or DAZL could induce stem cells (human embryonic

365

stem cells and induced pluripotent stem cells) to differentiate into primordial germ

366

cells and promote meiotic in germ cells (Medrano et al., 2012). DAZL can promote

367

the entry of germ cells into meiosis in human (Jung et al., 2017). The orange-spotted

368

grouper can undergo sex reversal, from female to male. During sex reversal, Ecvasa

369

and Ecdazl expression levels gradually increase and are higher in the testis than the

370

ovary. Previous studies have also reported that the expression levels of vasa and dazl

371

are higher in the testis (Úbeda-Manzanaro et al., 2014; Zhu et al., 2018). These results

372

indicate that the dazl and vasa may play conserved roles in vertebrates, which also

373

imply that dazl and vasa could promote mitosis and meiosis during gametogenesis in

374

the orange-spotted grouper, similar to the functions of these two genes in the

375

mammals. Additionally, the products of dazl and vasa genes would regulate the germ

376

cells’ development and directly or indirectly modulate the sexual reversal in the

377

orange-spotted grouper. Intriguingly, in the ovo-testis gonads, Ecvasa and Ecdazl

378

mRNA signals were found to become dramatically decreased or even undetectable in

379

oocytes, while strong in the spermatogonia and spermatocyte. This indicates that sex

380

reversal in the orange-spotted grouper might begin with the degeneration of female

381

germ cells owing to the inhibition of these germline genes’ expression, which

382

maintains oocyte development. This was similar to our previous report that in the

383

early phase of sex reversal gonads, the male and female germ cells coexist, and genes

384

related to the female pathway are suppressed while genes related to the male pathway

385

are activated (Wang et al., 2017).

386 387

5. Conclusions

388

In summary, the vasa and dazl cDNAs were cloned and characterized in a typical

389

protogynous hermaphroditic fish, the orange-spotted grouper. Ecvasa and Ecdazl

390

mRNA were highly expressed in the gonads and specifically expressed in the germ

391

cells. Moreover, Ecvasa and Ecdazl mRNA exhibited dynamic expression patterns in

392

germ cells at different stages during gonadal development. The findings of this study

393

would provide the basis for further investigations on the factors regulating gonadal

394

development and sex reversal in the orange-spotted grouper.

395

Acknowledgments

396

This work was supported by National Key Research and Development Program

397

(2018YFD0900200), National Natural Science Foundation of China (u1401213),

398

Agriculture Research System of China (ARS-47), Special Fund for Agro-scientific

399

Research in the Public Interest (201403011), YangFan Innovative & Entrepreneurial

400

Research Team Project (No.201312H10), Program of the China-ASEAN Maritime

401

Cooperation Fund of Chinese government, and the National Science Foundation of

402

China (31802266).

403

Figure legends.

404

405

Fig. 1. Amino acid alignments of Ecvasa with the homologues from other vertebrates.

406

The GenBank for Vasa proteins used for this study are as follows: Homo sapiens

407

(BC047455.1), Mus musculus (AAI37602.1), Gallus gallus (NP_990039.2), Xenopus

408

tropicalis (NP_001016823.1), Pelodiscus sinensis (KT934805.1), Danio rerio

409

(NM_131057.1),

410

(KU695222.1),

411

(ACT35657.1), Epinephelus coioides (MK655479).

Oryzias Drosophila

latipes

(AB063484.1),

melanogaster

Epinephelus

(CAA31405.1),

fuscoguttatus

Haliotis

asinina

412

413 414

Fig. 2. Amino acid alignments of Ecdazl with the homologues from other vertebrates.

415

The GenBank for Dazl proteins used for this study are as follows: Homo sapiens

416

(NP_001177740.1), Capra hircus (AFR36910.1), Mus musculus (NP_034151.3),

417

Rattus norvegicus (NP_001102884.1), Gallus gallus (NP_989549.1), Xenopus laevis

418

(NP_001081772.1), Pelodiscus sinensis (XP_006133320.1), Acipenser sinensis

419

(AIU39884.1), Oryzias latipes (NP_001098269.1), Danio rerio (AAH76423.), Salmo

420

salar (XP_014033836.1), Oncorhynchus mykiss (ADW41782.1), Carassius gibelio

421

(ACN94469.1), Oreochromis niloticus (XP_005453868.1), Epinephelus coioides

422

(MK655477).

423 424

Fig. 3. Phylogenetic tree generated with the neighbor-joining method of EcVasa.

425

426 427 428

Fig. 4. Phylogenetic tree generated with the neighbor-joining method of EcDazl.

429

430 431

Fig. 5. Analysis of Ecvasa and Ecdazl mRNA expression pattern in tissues by

432

RT-PCR. The data were presented as the mean±SEM (n=5).

433

434 435

Fig. 6. Analysis of Ecvasa and Ecdazl mRNA expression pattern in gonads at

436

different development stages by RT-PCR. (I) gonadal stage-I, the ovary with

437

proliferative oogonia and ovarian lumen; (II) gonadal stage-II, the ovary with mostly

438

primary growth-stage oocytes; (III) gonadal stage-III, the ovary with cortical alveolus

439

stage oocytes; (IV) gonadal stage-IV, the ovary with vitellogenic stage oocytes; (V)

440

gonadal stage-V, the early bisexual gonad; (VI) gonadal stage-VI, the late bisexual

441

gonad; (VII) gonadal stage-VII, the testis. The results were presented as the mean±

442

SEM (n=5-8) and the values with different letters (a-f) were significant different in

443

pairwise comparisons (P < 0.05).

444

445 446

Fig. 7. Ecvasa and Ecdazl RNA expression in the gonadal by Chemical in situ

447

hybridization. (a-f) vasa mRNA expression in the gonad; (g-l) dazl mRNA expression

448

in the gonad; (a and g) the signal with sense probe; (b-f and h-l) the signal with

449

antisense probe; (b and h) the ovary with proliferative oogonia and an ovarian lumen;

450

(c and i) the ovary with cortical alveolus stage oocytes; (d and j) the ovary with

451

vitellogenic stage oocytes; (e and k) the bisexual gonad; (f and l) the testis. Og,

452

Oogonia; O1, previtellogenic stage oocyte; O2, cortical-alveolus stage oocyte; O3,

453

vitellogenic stage oocyte; Sg, Spermatogonia; Sc, Spermatocyte; St, spermatid. The

454

purplish brown is the vasa and dazl mRNA signal and the red is PI. Scale bar, 50µm.

455

456 457

Fig. 8. Ecvasa and Ecdazl RNA expression in the gonadal by fluorescence in situ

458

hybridization. (a1-a6) the early ovary (the ovary with proliferative oogonia and an

459

ovarian lumen); (b1-b6) the late ovary (the ovary with vitellogenic stage oocytes);

460

(c1-c6) the testis. Og, Oogonia; O1, previtellogenic stage oocyte; O2, cortical-alveolus

461

stage oocyte; O3, vitellogenic stage oocyte; Sg, Spermatogonia; Sc, Spermatocyte; St,

462

spermatid. The green is the vasa and dazl mRNA signal and the red is PI. Scale bar,

463

20µm.

464 465

Supplementary Figure legends.

466

467 468

Supplementary Fig. 1. Nnucleotide and deduced amino acid sequences of Ecvasa.

469 470 471

Supplementary Fig. 2. Nnucleotide and deduced amino acid sequences of Ecdazl.

472 473

Supplementary Fig. 3. Gonadal histology sections of orange-spotted grouper by

474

hematoxylin and eosin. (a) gonadal stage-I, the ovary with proliferative oogonia and

475

ovarian lumen; (b) gonadal stage-II, the ovary with with mostly primary growth stage

476

oocytes; (c) gonadal stage-III, the ovary with cortical alveolus stage oocytes; (d)

477

gonadal stage-IV, the ovary with vitellogenic stage oocytes; (e) gonadal stage-V, the

478

early bisexual gonad; (f) gonadal stage-VI, the late bisexual gonad; (g) gonadal

479

stage-VII, the testis. Og, Oogonia; O1, previtellogenic stage oocyte; O2,

480

cortical-alveolus stage oocyte; O3, vitellogenic stage oocyte; Sg, Spermatogonia; Sc,

481

Spermatocyte; St, spermatid. Scale bar, 50µm.

482

Supplementary Tables

483 484

Supplementary Table 1 Sequences of the primers used for PCR. Primer name

Purpose

Sequence(5,-3,)

vasa-3’ F1

RACE

ACCGTATTGGGAGAACTGGC

vasa-3’ F2

RACE

CAGGAAGGGAGGATCTTTCC

vasa-5’ R1

RACE

AATGGGTTTAGAGGAGGAAG

vasa-5’ R2

RACE

CTTCCTCCTCTAAACCCATT

dazl-3’ F1

RACE

AGGGGTATGGCACAACCTTC

dazl-3’ F2

RACE

AGCCACTACAGTCAGCCGTA

dazl-5’ R1

RACE

CAGCACGGTCTGTGATCAGT

dazl-5’ R2

RACE

CTCCCAAAGATCTCCCGCAT

UPM Long

RACE

CTAATACGACTCACTATAGGGCAAGCAGTGGT ATCAACGCAGAGT

UPM Short

RACE

CTAATACGACTCACTATAGGGC

NUP

RACE

AGCAGTGGTAACAACGCAGAGT

vasa F

RT-PCR

CTGGAATGCCACCCAAAGAG

vasa R

RT-PCR

TGCACAAATGACTGCTCCAC

dazl F

RT-PCR

CCATGATCCCACCTATGCCA

dazl R

RT-PCR

AGTCCACACAGTCCTGATCG

vasa F

ISH

GAGCCTGAGACCATCATC

vasa R

ISH

AGGACTCTTCACACTGTTG

dazl F

ISH

GAGCAGCAACCAGACTTCACCTT

dazl R

ISH

CATCAAGATGTCATCCTCTCCGAGTT

β-actin F

reference genes

TTCACCACCACAGCCGAGA

β-actin R

reference genes

TGGTCTCGTGGATTCCGCAG

vasa F

full PCR

GCAGCAAGCCTGACTGA

vasa R

full PCR

ACTCCAACAAGCTGAACACT

dazl F

full PCR

AAAGAAGCGCAACTGG

dazl R

full PCR

AACATCCAAACCCTGC

485 486

Supplementary Table 2 Comparison of identify between E. coioides and some

487

known species of Vasa protein and Dazl

488

Genbank species

Vasa

identify Dazl

Vasa

Dazl

Homo sapiens

BC047455.1

NP_001177740.1

67%

40%

Mus musculus

AAI37602.1

NP_034151.3

64%

39%

Gallus gallus

NP_990039.2

NP_989549.1

61%

38%

Xenopus

NP_001016823.1

NP_001081772.1

64%

51%

Pelodiscus sinensis

KT934805.1

XP_006133320.1

60%

39%

Drosophila melanogaster

CAA31405.1

Oryzias latipes

AB063484.1

NP_001098269.1

72%

56%

Oreochromis niloticus

AB032467.1

XP_005453868.1

74%

63%

Carassius gibelio

AAV70960.1

ACN94469.1

78%

54%

Oncorhynchus mykiss

AB032566.1

ADW41782.1

80%

60%

Danio rerio

NM_131057.1

AAH76423.1

76%

57%

Epinephelus fuscoguttatus

KU695222.1

95%

Haliotis asinina

ACT35657.1

59%

52%

489 490

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Declaration of Interest Statement

There is no financial/personal interest or belief that could affect their objectivity.