Testis-specific expression pattern of dmrt1 and its putative regulatory region in the sea urchin (Mesocentrotus nudus)

Highlights

• Mndmrt1 exhibits testis-specific expression.

• Mndmrt1 is a male germ cells marker in sea urchin.

• Two regulatory regions have been identified in the promoter of Mndmrt1.

Abstract

Sea urchin (Mseocentrotus nudus) is an economically important mariculture species in several Asian countries. The growth rate and immunocompetence differ by sex in this species. However, the mechanisms of sex determination in M. nudus have remain unclear. In the present study, we focus on the dmrt1 gene of M. nudus (Mndmrt1) to investigate its dynamic expression pattern during different developmental stages. Real-time quantitative PCR (RT-qPCR) revealed that Mndmrt1 exhibits testis-specific expression and undetectable during the whole embryogenesis. With the development of ontogenetic, Mndmrt1 transcripts are first detected at 9 months post-fertilization (mpf). In addition, both the transcripts and protein of Mndmrt1 gene were specifically expressed in spermatogonia and spermatocytes, indicating that it might be a male germ cells marker in sea urchin. Significantly, the 1441 bp promoter sequence of Mndmrt1 gene was obtained by DNA walking, and one positive regulatory region at −1197/ −968 in the promoter, as well as one negative regulatory region at −1441/ −1198 have been identified by promoter activity analysis. Moreover, two regulatory regions contain multiple putative binding sites for transcription factors, including Sp1, Egr1, Sox5, CEBP, GATA and SRY. These findings suggest that Mndmrt1 may be related to testis differentiation and spermatogenesis in sea urchin and will provide an insight into understanding the regulatory mechanism of the dmrt1 gene.

Introduction

Mesocentrotus nudus is a commercially important mariculture species of sea urchin, and mainly distributed in the coastal regions of northern China, the Korean peninsula, the Russian far east coast, and northern Japan. Based on the increasing market demand, the scale aquacultural production of sea urchins are expanding. However, it is impossible to distinguish sex based on the phenotypic appearance in larval, juvenile, and adult individual, which complicates the genetic breeding of sea urchins (Sun et al., 2019; Zhang et al., 2019). In addition, the gonads are the only edible part of sea urchins for people, thus, the quality of gonads directly determines the economic value of sea urchins, such as gonad colour, gonadosomatic index, and taste (Takagi et al., 2017; Inomata et al., 2016). According to previously reports, the ovaries of sea urchins taste bitter and sour, while testes taste sweet and creamy (Phillips et al., 2010). Moreover, the gonadal growth and increasing body size of male sea urchins have advantage over that of female individuals(Gil et al., 2020). In light of the above, it seems that the commercial value of male sea urchins might be superior to that of female. Hence, it is valuable to understand the molecular mechanism of sex determination and sex differentiation in order to develop biotechnologies of sex control in sea urchin aquaculture.

Identifying sex-specific or sex-dominant markers is an effective method to reveal the genetic basis of sex determination and successfully develop monosexual populations in aquaculture (Liu et al., 2013; Chen et al., 2018; Mei and Gui, 2015). Doublesex and mab-3 related transcription factor 1 (Dmrt1) contains a conserved DNA binding motif and proved to be a conserved male-specific gene which lies downstream of the sex-determination regulatory pathway in various species (Kopp, 2012). For instance, in mammal (e.g., mouse), Dmrt1 is expressed in spermatogonia (Raymond et al., 2000), and knockout of Dmrt1 led to sex reversal in adult males (Inui et al., 2017; Zhang et al., 2016). In Aves (e.g., chicken), Dmrt1 is mapped to the Z chromosome and the sex of a chicken is determined by the dosage of Dmrt1(Smith et al., 2009). In Reptiles (e.g., turtle), Dmrt1 is expressed specifically in the sertoli cells in male gonad, and knockdown of Dmrt1 in ZZ embryos led to male to female sex reversal (Sun et al., 2017).In teleost (e.g., Japanese medaka), Dmy is a duplicated copy of dmrt1 which mapped on the Y chromosome (Ohmuro-Matsuyama et al., 2003), knock out or knock down Dmy can induce male to female sex reversal (Paul-Prasanth et al., 2006; Luo et al., 2015; Chakraborty et al., 2016), and over repression Dmy result in female-to-male gonadal reprogramming(Matsuda et al., 2007).In addition, the gonad-specific expression pattern of the dmrt1 gene and its role in sex determination has also been characterized in several invertebrate species. For example, in Malacostraca (e.g., swimming crab), Dmrt1 transcripts were highly expressed in testis and the expression levels of insulin-like androgenic gland hormone (IAG) was dynamic change after knockdown of Dmrt1 (Wang et al., 2020). Considering the above, Dmrt play conserved role in sex determination and sex differentiation in diverse species and may exhibit similar expression patterns and play similar functions in sea urchins.

In our previous studies, the dmrt1 gene has been identified from transcriptome database of sea urchin (Mesocentrotus nudus) and expressed specifically in testis (Sun et al., 2019). However, the dynamic expression pattern and function implication of dmrt1 in sea urchin remains unclear. In the present study, we analysed the Mesocentrotus nudus dmrt1 gene (Mndmrt1) expression patterns from the zygote to sexual maturity in order to reveal its dynamic expression change during gonad development. The cellular localization of Mndmrt1 mRNA and protein were examined in testis by in situ hybridization and immunohistochemistry. Furthermore, the 5′-flanking regions of the Mndmrt1 was isolated by DNA walking, and the putative upstream transcriptional regulatory factors were identified by searching online data bases and luciferase activity assay.