The Role of the Heat Shock Cognate Protein 70 Genes in Sex Determination and Differentiation of Chinese Tongue Sole (Cynoglossus semilaevis)

Abstract

Fish sex determination can be affected by environmental temperature. This process relies on temperature-sensitive proteins such as heat shock proteins (HSPs). Our previous work found that heat shock cognate proteins (HSCs) may participate in high-temperature associated sex reversal of Chinese tongue sole (Cynoglossus semilaevis). However, the role of hsc genes in responding to high temperature and affecting sex determination/differentiation remains unclear. Here, by using C. semilaevis as model, we identified hsc70 and hsc70-like. hsc70 was abundant in the gonads with a testicular-higher expression at all gonadal development stages except for 6 months post fertilization (mpf). Intriguingly, hsc70-like showed higher expression in testes from 6 mpf on. Both long-term heat treatment during the temperature-sensitive sex-determining period and short-term heat stress at the end of this period caused different expression of hsc70/hsc70-like between sexes. The dual-luciferase assay results also suggested that these genes can respond to high temperature rapidly in vitro. Heat treatment of C. semilaevis testis cells overexpressed with hsc70/hsc70-like could affect the expression of sex-related genes sox9a and cyp19a1a. Our results indicated that hsc70 and hsc70-like were key regulators linking external high-temperature signals with sex differentiation in vivo and provide a new idea for understanding the mechanism by which high temperature affects sex determination/differentiation in teleosts.

Keywords: Cynoglossus semilaevis; high temperature; hsc70 genes; sex determination and differentiation.

Figure 1

Full-length cDNA and predicted protein sequences of C. semilaevis hsc70 and hsc70-like genes. (a) The mRNA and predicted protein sequence of hsc70; (b) The mRNA and predicted protein sequence of hsc70-like. The yellow shadows represented the conserved sequences of the HSP70 family, the green shaded EEVD is the cytoplasmic motif in the C-terminal region, the grey shadows represented the initiation codon and termination codon, and the asterisk (*) represented the termination codon. The poly (A) tail was underlined.

Figure 2

Multiple sequence alignment of HSC and HSC70-LIKE protein sequences from C. semilaevis and other vertebrates. GenBank accession numbers for protein sequences were as follows: XP_043872802.1 (Solea senegalensis), XP_020363711.1 (Oncorhynchus kisutch), XP_042153383.1 (Oncorhynchus tshawytscha), XP_019941072.1 (Paralichthys olivaceus), XP_043897279.1 (Solea senegalensis), XP_027129165.1 (Larimichthys crocea), XP_003448938.1 (Oreochromis niloticus), XP_024298732.1 (Oncorhynchus tshawytscha), XP_020358882.1 (Oncorhynchus kisutch), XP_002937574.2 (Xenopus tropicalis), NP_990334.2 (Gallus gallus), NP_112442.2 (Mus musculus), and XP_011541100.1 (Homo sapiens). The pink boxes indicated high similarity, while the blue boxes indicated completely identical residues.

Figure 3

Phylogenetic tree of target genes. The tree was constructed using nucleic acid sequences of hsc70 and hsc70-like from different species using the neighbor joining method with 1000 bootstrap replicates. Numbers at nodes indicated bootstrap support. C. semilaevis hsc70 and hsc70-like genes were red and labelled with asterisk (*).

Figure 4

Expression of hsc70 and hsc70-like genes in adult C. semilaevis tissues. (a,b) Detection of hsc70 (a) and hsc70-like (b) expression in C. semilaevis tissues by RT—qPCR. Means ± SEM from three independent individuals (n = 3) were shown. β-actin was used as the reference gene. Asterisks indicated statistically significant differences (* p <0.05; ** p < 0.01; *** p < 0.001).

Figure 5

Expression of hsc70 and hsc70-like genes in the gonads of male and female C. semilaevis at different stages. (a) Expression of hsc70 during male and female gonadal development; (b) Relative gene expression of hsc70-like during male and female gonadal development. Relative mRNA levels were shown as the means ± SEM (n = 3), and values were normalized using β-actin as the internal control. * p <0.05; ** p < 0.01; *** p < 0.001. mpf, months post fertilization.

Figure 6

Gonadal expression of hsc70 and hsc70-like genes in C. semilaevis under different high-temperature treatments. (a,b) Expression pattern of hsc70 in the gonads of 3 mpf C. semilaevis under short-term (a) and long-term (b) high-temperature treatment; (c,d) Expression pattern of hsc70-like in the gonads of 3 mpf C. semilaevis under short-term (c) and long-term (d) high-temperature treatment. The expression of target genes were shown as means ± SEM (n = 3). β-actin as the internal control. CT, control treatment; ST, short-term heat stress treatment; LT, long-term heat treatment. Different letters indicated statistically significant differences (p < 0.05).

Figure 7

High temperature activates promoter activity of hsc70 and hsc70-like genes in vitro. (a,b). Luciferase activity analysis of hsc70-promoter (a) /hsc70-like-promoter (b) under 42 °C heat shock in HEK293T cell and the pGL3-basic plasmid was used as a negative control; (c,d). gfp fluorescence analysis of hsc70-promoter (c) /hsc70-like-promoter (d) under 42 °C heat shock in HEK293T cell and the pEGFP-N3 plasmid was used as a negative control. The 42 °C heat shock treatment was conducted at 48 h post transfection. Luciferase activities and gfp relative expression were shown as means ± SEM (n = 3) and HEK293T β-actin was used as the internal reference (a–d). * p < 0.05.

Figure 8

Analysis of sex-related genes after the overexpression of target genes under heat shock treatment. (a,b) Relative expression of sox9a in C. semilaevis testis cell line overexpression with hsc70 (a) /hsc70-like (b); (c,d) Relative expression of cyp19a1a in C. semilaevis testis cell line overexpression with hsc70 (c) /hsc70-like (d). After transfection with pcDNA3.1-hsc70/pcDNA3.1-hsc70-like for 24 h, 28 °C heat shock treatment was conducted and lasted for 2 h. pcDNA 3.1 plasmid was set as negative control (NC). Relative expression results were shown as the means ± SEM (n = 3), and β-actin was normalized as an internal standard. Different lowercase letters indicated statistically significant differences (p < 0.05).