Characterization, expression, and regulatory effects of nr0b1a and nr0b1b in spotted scat (Scatophagus argus)

https://doi.org/10.1016/j.cbpb.2021.110644Get rights and content

Highlights

  • The duplicated genes nr0b1a and nr0b1b were identified in spotted scat.

  • nr0b1b was retained in several fishes, but was secondarily lost in other vertebrates.

  • nr0b1a and nr0b1b exhibited differential spatiotemporal patterns during gonadal development.

  • Nr0b1a suppressed, but Nr0b1b stimulated Nr5a1-mediated cyp19a1a expression.

Abstract

Nuclear receptor subfamily 0 group B member 1 (Nr0b1) belongs to the nuclear receptor (NR) superfamily. It plays critical roles in sex determination, sex differentiation, and gonadal development in mammals. In this study, the duplicated genes nr0b1a and nr0b1b were identified in spotted scat (Scatophagus argus). Phylogenetic and synteny analyses revealed that, unlike nr0b1a, nr0b1b was retained in several species of teleosts after an nr0b1 gene duplication event but was secondarily lost in other fish species, amphibians, reptiles, birds, and mammals. In a sequence analysis, only 1.5 LXXLL-related repeat motifs were identified in spotted scat Nr0b1a, Nr0b1b, and non-mammalian Nr0b1a/Nr0b1, different from the 3.5 repeat motifs in mammalian Nr0b1. By qPCR, nr0b1a and nr0b1b were highly expressed in testes from stages IV to V and in ovaries from stages II to IV, respectively. Male-to-female sex reversal was induced in XY spotted scat by the administration of exogenous E2. A qPCR analysis showed that nr0b1b mRNA expression was higher in sex-reversed XY fish than in control XY fish, with no difference in nr0b1a. A luciferase assay showed that spotted scat Nr0b1a and Nr0b1b did not individually activate cyp19a1a gene transcription. As in mammals, spotted scat Nr0b1a suppressed Nr5a1-mediated cyp19a1a expression, despite containing only 1.5 LXXLL-related repeat motifs in its N-terminal region, while Nr0b1b stimulated Nr5a1-mediated cyp19a1a transcription. These results demonstrated that nr0b1a and nr0b1b in spotted scat have distinct expression patterns and regulatory effects and further indicate that nr0b1b might be involved in ovarian development by regulating Nr5a1-mediated cyp19a1a expression.

Introduction

The nuclear receptor (NR) superfamily plays important roles in many physiological and biochemical processes, including steroid hormone production and metabolism (Volle and Lobaccaro, 2007), lipid metabolism (Sinal et al., 2000), cellular differentiation and development (Jetten, 2009), sex differentiation, and gonadal development (Achermann et al., 1999; Fujii et al., 2014; Zanaria et al., 1994). The NR superfamily consists of seven subfamilies (NR0–NR6) (Germain et al., 2006; Laudet et al., 1992), and typical NR superfamily members consist of five conserved domains, A/B (N-terminal domain), C (DNA-binding domain; DBD), D (hinge domain), E (ligand-binding domain; LBD), and F (C-terminal domain) (Robinson-Rechavi et al., 2003). Moreover, the AB or N-terminal region and E or C-terminal domain of the nuclear receptors contain a ligand-independent (AF-1) and ligand-dependent transcriptional activation domain (AF-2), respectively (Dowhan and Muscat, 1996; Masuyama et al., 1997; Wang et al., 2002). An unusual NR0B1/Nr0b1 (Nuclear receptor subfamily 0 group b member 1), belonging to the NR0 subfamily, has been identified in humans (Zanaria et al., 1994), mice (Swain et al., 1996), chickens (Smith et al., 2000), alligators (Western et al., 2000), frogs (Sugita et al., 2001), and fish (Li et al., 2013; von Schalburg et al., 2014; Wang et al., 2002). Unlike most NRs, NR0B1/Nr0b1 lacks conserved functional DBD domains and has a unique N-terminal domain containing LXXLL-related repeat motifs (Bae et al., 1996; Guo et al., 1996; Wang et al., 2002; Zanaria et al., 1994). In vertebrates, Nr0b1 showed rapid evolutionary change after its relocation from an autosomal position in non-mammalian taxa to an X-linked location in mammals. In mammals, 3.5 LXXLL-related repeat motifs are found in the Nr0b1 N-terminal region (Guo et al., 1996; Wang et al., 2002; Zanaria et al., 1994). In all non-mammals, Nr0b1 contains only 1.5 LXXLL-related repeat motifs in the N-terminus (Nakamoto et al., 2007; Stickels et al., 2015; Wang et al., 2002; Xia et al., 2018). The 3.5 LXXLL repeat motifs are critical for protein–protein interactions in mammals (Iyer et al., 2007; Suzuki et al., 2003). Yeast two-hybrid and transient-transfection assays have confirmed that three LXXLL-related motifs within the 3.5 repeats in the Nr0b1 N-terminal region are essential for the interaction with and repression of Ad4BP/SF-1 (NR5A1, Nuclear receptor subfamily 0 group a member 1) target gene expression (Iyer and McCabe, 2004; Iyer et al., 2007; Suzuki et al., 2003). Nr0b1 functions as an inhibitor to suppress the expression of NR5A1 target genes, including Cyp19a (which encodes Aromatase, a key enzyme for estrogen synthesis), StAR, Cyp17, 3β-HSD, and Cyp11a1 (Gurates et al., 2002; Gurates et al., 2003; Jo and Stocco, 2004; Peng et al., 2003). In medaka (Oryzias latipes), Nr0b1a inhibits Nr5a1-mediated cyp19a1a expression in vitro, although only 1.5 LXXLL-related motifs have been identified in its N-terminal region (Nakamoto et al., 2007). Therefore, it is necessary to characterize Nr0b1 in other fish species.

Dax1 (dosage sensitive sex reversal (DSS), adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1) is encoded by Nr0b1. It plays crucial roles in sex determination, sex differentiation, gonad development, and male fertility (Iyer and McCabe, 2004; Smith et al., 2000; Swain et al., 1996; Ravel et al., 2014; Zanaria et al., 1994). In male mammals, Nr0b1 mutations and deletions cause hypogonadotropic hypogonadism with a primary gonadal defect characterized by cellular obstruction of the seminiferous tubules and efferent ductulus, leading to germ cell death and infertility (Bardoni et al., 1994; Bouma et al., 2005; Iyer and McCabe, 2004; Ludbrook et al., 2012; Meeks et al., 2003a, Meeks et al., 2003b; Stickels et al., 2015; Swain et al., 1996). However, in female mice, the loss of Nr0b1 results in follicles with irregular borders and multi-oocytes, with no corpus luteum production; however, it does not affect female fertility (Park et al., 2005; Yu et al., 1998). In teleosts, a previous study of zebrafish (Danio rerio) has shown that mutations in nr0b1a cause female-to-male sex reversal but do not affect testis development (Chen et al., 2016). The nr0b1a gene has been cloned and characterized by reverse transcription PCR (RT-PCR), quantitative real-time polymerase chain reaction (qPCR), and in situ hybridization (ISH) in various species of teleosts, including medaka, Pengze crucian carp (Carassius auratus), rice-field eel (Monopterus albus), Atlantic cod (Gadus morhua), and European seabass (Dicentrarchus labrax) (Chen et al., 2016; Haugen et al., 2012; Hu et al., 2015; Li et al., 2013; Martins et al., 2007; Nakamoto et al., 2007). Interestingly, in tilapia (Oreochromis niloticus) and fugu (Takifugu rubripes), two nr0b1 genes, nr0b1a and its duplicated gene nr0b1b, have been characterized (Cheng et al., 2015; Wang et al., 2002); however, the duplicated gene have not been detected in other species. Increasing genomic data provide a basis for determining whether the nr0b1b genes were maintained in the genomes of other species. The cloning of nr0b1b in teleosts as well as analyses of its expression and regulation are needed. Unlike the extensive tissue distribution of nr0b1a in teleosts (Hu et al., 2015; Li et al., 2013; Martins et al., 2007; Wang et al., 2002; Wang et al., 2015; Xia et al., 2018), tilapia nr0b1b is exclusively expressed in the gonads, with higher expression in the ovary than in the testis (Cheng et al., 2015; Wang et al., 2002). In spotted scat (Scatophagus argus), Chen et al. have cloned and studied the wide tissue distribution of nr0b1a (Chen et al., 2015); however, the sequence, expression characteristics, and gene regulation of the two nr0b1 genes have not been determined.

Spotted scat is an important aquaculture fish in China with an XX-XY sex-determination system (Mustapha et al., 2018). The genotypic sexes have been successfully determined using two sex-linked DNA markers, spotted scat dmrt1 and dmrt3, developed by our group (Mustapha et al., 2018; Jiang et al., 2019). We have previously published its genome and gonadal transcriptome (He et al., 2019; Huang et al., 2021). Based on the sex-linked DNA markers combined with comparative genomics and gonadal transcriptome data, we obtained a normal dmrt1 gene (located on the Y chromosome in males) and a truncated mutant dmrt1b (located on the X chromosome in both males and females). Further analysis revealed that the Y-chromosome-specific dmrt1 has putative functions in sex determination (Huang et al., 2019; Mustapha et al., 2018). Moreover, it is possible to obtain substantial numbers of fertilized eggs by artificial breeding. Therefore, spotted scat is a good model fish for studies of gene expression and function during gonadal development in both males and females. In this study, we identified two nr0b1 genes, nr0b1a and nr0b1b, and analyzed the spatial expression profiles at different stages of gonadal development and after sex-reversal induced by 17β-estradiol (E2) by qPCR. Moreover, the transcriptional regulation of cyp19a1a by Nr5a1 and Nr0b1a/Nr0b1b were investigated by promoter analyses. Our findings provide a valuable dataset for studies of the roles of spotted scat nr0b1a and nr0b1b during gonadal development and improve our understanding of the regulatory effects of these genes on cyp19a1a in fish.

Section snippets

Animals

The spotted scat individuals were obtained from Zhanjiang Donghai Island Cultivation Base (Zhanjiang, Guangdong, China). All animal experiment protocols were authorized by the Institutional Animal Care and Use Committee of GuangDong Ocean University (Zhanjian, China). All fish were anesthetized with 100 mg/l tricaine methane sulfonate (MS 222; Sigma-Aldrich, St. Louis, MO, USA) and dissected.

Phylogenetic and synteny analyses of Nr0b1s

Sequences were obtained from the NCBI (http://blast.ncbi.nih.gov/) and Ensemble (//www.ensembl.org/index.html

Identification and sequence analysis

Nr0b1/nr0b1 was detected in the human, mouse, chicken, lizard, and Xenopus genomes, and nr0b1a was found in the genomes of all bony fishes. We detected a duplicate of nr0b1a, named nr0b1b, in the spotted scat (Perciformes: Scatophagidae) genome. Similarly, nr0b1b was identified in some species of the families Sparidae, Cichlidae, Latidae, Moronidae; Bovichtidae, Serranidae, Pomacentridae, Carangidae, Sciaenidae, Apogonidae, Echeneidae, and Ambassidae (order Perciformes), Holocentridae (order

Phylogenetic analyses of Nr0b1

Nr0b1 is an essential factor involved in sex determination, sex differentiation, gonad development, and male fertility in vertebrates. Previously, Nr0b1/nr0b1 has been isolated in humans, mice, chickens, lizards, and Xenopus, and nr0b1a has been found in all bony fishes. Interestingly, the duplication of nr0b1a yielded nr0b1b in tilapia and fugu (Cheng et al., 2015; Wang et al., 2002). With increasing genomic data, nr0b1b has been identified in the genomes of increasing teleosts, including

Conclusions

We identified two nr0b1 genes, nr0b1a and nr0b1b, in spotted scats, and found that these genes show different expression profiles, with high nr0b1a mRNA expression in the testis and nr0b1b mRNA expression in the ovaries of spotted scats. The expression of nr0b1b was higher in sex-reversed XY fish than in control XY fish, but nr0b1a showed no differences. A promoter analysis showed that neither spotted scat Nr0b1a nor Nr0b1b alone influences cyp19a1a gene transcription. Moreover, Nr0b1a

Author contributions

Shi H.J. conceived, designed the experiments and analyzed the data; Ru X.Y. performed the experiments. Mustapha U.F., Huang Y. and Pan S.H. feed the fish in Zhanjiang Donghai Island Cultivation Base. Shi H.J. and Li G.L. wrote the manuscript, Jiang D.N. review and editing. All authors have read and agreed to the published version of the manuscript.

Animal ethics

All animal experiments throughout the study were conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals for the Science and Technology Bureau of China. Experiments involving spotted scat were approved by the Animal Research and Ethics Committees of the Institute of Aquatic Economic Animals of Guangdong Ocean University (201903004).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was funded by the Key Project of “Blue Granary Science and Technology Innovation” of the Ministry of Science and Technology, grant number 2018YFD0901203; the Nation Natural Science Foundation of China, grant number 32002367; the Natural Science Foundation of Guangdong Province, grant number 2018B030311050; the Department of Education of Guangdong Province, grant number 2018KTSCX090 and 2018KQNCX106, the Guangdong Provincial Special Fund For Modern Agriculture Industry Technology

References (75)

  • H.F. Liu et al.

    Characterization and gonadal expression of FOXL2 relative to Cyp19a genes in spotted scat Scatophagus argus

    Gene.

    (2015)
  • B. Mandal et al.

    Gonadal recrudescence and annual reproductive hormone pattern of captive female spotted scats (Scatophagus argus)

    Anim. Reprod. Sci.

    (2020)
  • U.F. Mustapha et al.

    Male-specific Dmrt1 is a candidate sex determination gene in spotted scat (Scatophagus argus)

    Aquaculture.

    (2018)
  • C. Ravel et al.

    Are human male patients with DAX1/NR0B1 mutations infertile?

    Ann. Endocrinol. (Paris).

    (2014)
  • C.J. Sinal et al.

    Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis

    Cell.

    (2000)
  • A.K. Singh

    Introduction of modern endocrine techniques for the production of monosex population of fishes

    Gen. Comp. Endocrinol.

    (2013)
  • J. Sugita et al.

    Expression of Dax-1 during gonadal development of the frog

    Gene.

    (2001)
  • D. Vizziano-Cantonnet et al.

    Sexual dimorphism in the brain aromatase expression and activity, and in the central expression of other steroidogenic enzymes during the period of sex differentiation in monosex rainbow trout populations

    Gen. Comp. Endocrinol.

    (2011)
  • D.H. Volle et al.

    Role of the nuclear receptors for oxysterols LXRs in steroidogenic tissues: beyond the "foie gras", the steroids and sex?

    Mol. Cell. Endocrinol.

    (2007)
  • K.R. Von Schalburg et al.

    Sex-specific expression and localization of aromatase and its regulators during embryonic and larval development of Atlantic salmon

    Comp. Biochem. Physiol. B Biochem. Mol. Biol.

    (2014)
  • D.S. Wang et al.

    Molecular cloning of DAX1 and SHP cDNAs and their expression patterns in the Nile tilapia, Oreochromis niloticus

    Biochem. Biophys. Res. Commun.

    (2002)
  • P.S. Western et al.

    Temperature-dependent sex determination in the American alligator: expression of SF1, WT1 and DAX1 during gonadogenesis

    Gene.

    (2000)
  • J.C. Achermann et al.

    Mutational analysis of DAX1 in patients with hypogonadotropic hypogonadism or pubertal delay

    J. Clin. Endocrinol. Metab.

    (1999)
  • D.S. Bae et al.

    Characterization of the mouse DAX-1 gene reveals evolutionary conservation of a unique amino-terminal motif and widespread expression in mouse tissue

    Endocrinol.

    (1996)
  • B. Bardoni et al.

    A dosage sensitive locus at chromosome Xp21 is involved in male to female sex reversal

    Nat. Genet.

    (1994)
  • G.J. Bouma et al.

    Gonadal sex reversal in mutant Dax1 XY mice: a failure to upregulate Sox9 in pre-Sertoli cells

    Development.

    (2005)
  • J.H. Chen et al.

    Molecular characterization of dax1 and SF-1 and their expression analysis during sex reversal in spotted scat, Scatophagus argus

    J. World Aquacult. Soc.

    (2015)
  • D.H. Dowhan et al.

    Characterization of the AB (AF-1) region in the muscle-specific retinoid X receptor-gamma: evidence that the AF-1 region functions in a cell-specific manner

    Nucleic Acids Res.

    (1996)
  • J. Felsenstein

    Confidence limits on phylogenies: an approach using the bootstrap

    Evolution.

    (1985)
  • J. Fujii et al.

    Involvement of androgen receptor in sex determination in an amphibian species

    PLoS One

    (2014)
  • V. Gennotte et al.

    The sensitive period for male-to-female sex reversal begins at the embryonic stage in the Nile Tilapia and is associated with the sexual genotype

    Mol. Reprod. Dev.

    (2014)
  • P. Germain et al.

    Overview of nomenclature of nuclear receptors

    Pharmacol. Rev.

    (2006)
  • W. Guo et al.

    Genomic sequence of the DAX1 gene: an orphan nuclear receptor responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism

    J. Clin. Endocrinol. Metab.

    (1996)
  • B. Gurates et al.

    WT1 and DAX-1 inhibit aromatase P450 expression in human endometrial and endometriotic stromal cells

    J. Clin. Endocrinol. Metab.

    (2002)
  • T. Haugen et al.

    Sex differentiation in Atlantic cod (Gadus morhua L.): morphological and gene expression studies

    Reprod. Biol. Endocrinol.

    (2012)
  • F.X. He et al.

    Comparative transcriptome analysis of male and female gonads reveals sex-biased genes in spotted scat (Scatophagus argus)

    Fish Physiol. Biochem.

    (2019)
  • Q. Hu et al.

    Molecular cloning and characterization of amh and dax1 genes and their expression during sex inversion in rice-field eel Monopterus albus

    Sci. Rep.

    (2015)
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      As described below: in testes, stage I: spermatogonial proliferation stage; stage II: spermatocyte growth stage; stage III: spermatocyte maturation; stage IV: sperm cell metamorphosis stage and stage V: sperm maturation stage. In ovaries, stage I: filled with oogonium or/and phase I oocytes (atchromatin nucleolar); stage II: mainly filled with phase II oocytes at the perinuclear stage; stage III: mainly filled with phase III oocytes forming the vitelline vesicle; stage IV: mainly filled with phase IV oocytes at lipidic and proteic vitellogenesis stage; and stage V: mainly filled with phase V mature oocyte (Cui et al., 2013; Cui et al., 2017; Shi et al., 2021c). Gonadal H&E staining was performed in control and E2 injected spotted scat before RNA-seq, and the stage IV gonads of female and male fish were observed in control and E2 injected groups, with spermatogonia, spermatocytes and spermatids filled in the testes and phase II-IV oocytes in the ovaries (Fig. S2).

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