Molecular Cloning and Sexually Dimorphic Expression Analysis of nanos2 in the Sea Urchin, Mesocentrotus nudus

Abstract

Sea urchin (Mesocentrotus nudus) is an economically important mariculture species in China and the gonads are the solely edible parts to human. The molecular mechanisms of gonad development have attracted increasing attention in recent years. Although the nanos2 gene has been identified as a germ cell marker in several invertebrates, little is known about nanos2 in adult sea urchins. Hereinto, we report the characterization of Mnnano2, an M. nudus nanos2 homology gene. Mnnanos2 is a maternal factor and can be detected continuously during embryogenesis and early ontogeny. Real-time quantitative PCR (RT-qPCR) and section in situ hybridization (ISH) analysis revealed a dynamic and sexually dimorphic expression pattern of Mnnano2 in the gonads. Its expression reached the maximal level at Stage 2 along with the gonad development in both ovary and testis. In the ovary, Mnnanos2 is specifically expressed in germ cells. In contrast, Mnnanos2 is expressed in both nutritive phagocytes (NP) cells and male germ cells in testis. Moreover, knocking down of Mnnanos2 by means of RNA interference (RNAi) reduced nanos2 and boule expression but conversely increased the expression of foxl2. Therefore, our data suggest that Mnnanos2 may serve as a female germ cell marker during gametogenesis and provide chances to uncover its function in adult sea urchin.

Keywords: RNAi; germ cell; nanos2; sea urchins.

Figure 1

Amino acid alignments of MnNanos2 with the homologs from other species. The red box indicate the conserved posotion of zf-domain. The 8 invariant cysteine and histidine residues in zf-domin were marked with red color.

Figure 2

Sequence analysis of Nanos2 in M. nudus. (A) Multiple amino acid sequence alignment of Nanos2 ZF-domain, The 8 invariant cysteine and histidine residues in zf-domin were marked with red color. (B) phylogenetic tree of Nanos2.Nanos2 Amino acid of Mesocentrotus nudus was marked with red color. The phylogenetic tree was constructed with MEGA version 5.0 by bootstrap analysis using the maximum-likelihood method (1000 replicates) and human Nanos2 was used as the outgroup sequences.

Figure 3

Mnnanos2 expression in adult tissues and gonadal development stages. (A) RT-qPCR analysis of nanos2 expression in different adult tissues. Ubiquitin was used as the control. The expression of Mnnanos2 in Intestines was set to 1. Each bar represents mean ± standard deviation (SD) from three to five individuals. Tukey’s test was used to determine statistical analysis. Asterisks (*) indicate significant differences (p ≤ 0.05) between other tissues and intestines. (B) RT-qPCR analysis of nanos2 expression at different gonadal development stages. Ubiquitin was used as the control. The expression of Mnnanos2 at Stage 1 was set to 1. Each bar represents mean ± SD from three individuals. Data are from three independent experiments. Asterisks (*) indicate significant differences (p ≤ 0.05) between Stage 2–4 and Stage 1 determined by Tukey’s test. (C,G,K,O) Histological detection of the ovary at Stage 1–4. (E,I,M,Q) Histological detection of testis at Stage 1–4. The red boxed areas on the left are shown on the right at higher magnification (D,H,L,P,F,J,N,R). NP: nutritive phagocytes. Bar: (C,G,K,O,E,I,M,Q) 200 µm, and (D,H,L,P,F,J,N,R) 25 µm.

Figure 4

Mnnanos2 expression analysis by in situ hybridization in gonads at different gonadal development stages. (A–E) ovary, (F–J) testis. NP: nutritive phagocytes. Bar = 100 µm.

Figure 5

Expression analysis of Mnnanos2 in embryogenesis and early ontogeny. (A) RT-qPCR analysis of nanos2 expression in M. nudus embryos. Ubiquitin was used as the control. The expression of Mnnanos2 in Egg was set to 1. Each bar represents mean ± standard deviation (SD) from more than 50 individuals. Asterisks (*) indicate significant differences (p ≤ 0.05) between 4 cell—8 carpal and egg (B) RT-qPCR analysis of nanos2 expression in early ontogeny of M. nudus. Ubiquitin was used as the control. The expression of Mnnanos2 in 3 mpf was set to 1. Each bar represents mean ± SD from three individuals. Data are from three independent experiments. Asterisks (*) indicate significant differences (p ≤ 0.05) between 6–12 mpf and 3 mpf. Mpf: months post-fertilization. Tukey’s test was used to determine statistical analysis.

Figure 6

Detection of anti-MnNanos2 antibody and immunofluorescence localization of MnNaons2 in the ovary. (A) Western blot with the anti-MnNanos2 polyclonal antibody in ovary and testis extracts, (B) Western blot with the pre-immune rabbit serum in ovary and testis extracts, (C) Western blot with the anti-MnNanos2 antibody that had been pre-absorbed with the purified recombinant MnNanos2 protein in ovary and testis extracts, (D) GAPDH was used as the control, (E–G) immunofluorescence localization of MnNanos2 in the ovary, (H) immunofluorescence localization of pre-immuned serum in the ovary. Bar = 100 µm.

Figure 7

RNA interference (RNAi) of Mnnanos2. RT-qPCR measured the mRNA levels of Mnnanos2 and other sex-related genes after Mnnanos2 RNAi. (A) ovary and (B) testis. Ubiquitin was used as the control. The expression of Mnnanos2 in control group was set to 1. Each bar represents mean ± standard deviation (SD) from three individuals. Asterisks (*) indicate significant differences (p ≤ 0.05) between knockdown and control. The datasets were normal distribution by Shapiro–Wilk test. Student’s test was used for statistical analysis.