Matured black rockfish used in this study were obtained from Penglai, China. Ten sexually mature individuals in November were anesthetized with a 0.05% solution of 3-aminobenzoic acid ethyl ester methanesulfonate-222 (MS-222) (Sigma–Aldrich, United States). The tissues of ovary and testis were rapidly removed. Some samples were immediately immersed in liquid nitrogen for cDNA synthesis, while others were fixed overnight with 4% paraformaldehyde in phosphate-buffered saline (PBS) and then preserved in 70% ethanol for section in situ hybridization (SISH). Twenty sexually mature females in April to May were also anesthetized with MS-222. Moreover, the embryos of different development stages in the ovary were removed and fixed by 4% paraformaldehyde in PBS overnight and stored at −20°C in PBS with 50% formamide for whole mount in situ hybridization (WISH).

One-year-old and hatching fry of black rockfish were reared at Shenghang Sci-Tech Co., Ltd. (Shandong Province, China). The testes and ovaries of six 1-year-old individuals were removed after anesthesia and fixed in 4% paraformaldehyde as above. Starting from hatching, about 20 hatched fry were fixed with 4% paraformaldehyde every day for 1 month. After 1 day of fixation, the gonadal tissue samples and fry were stored at 70% ethanol for SISH.

All experiments were performed in accordance with the relevant national and international guidelines and approved by the Institutional Animal Care and Use Committee, Institute of Oceanology, Chinese Academy of Sciences.

A homology search of the amino acid sequence of Ssvas was carried out using the National Center for Biotechnology Information website¹. The amino acid sequences were aligned using the AlignX program in Vector NTI, Suite 8 software package (Life Technologies). Phylogenetic analysis was conducted with Mega4 software using the neighbor-joining method, and the bootstrap replicates were set to 1,000 (Saitou and Nei, 1987). Some potential regulatory motifs in the 3′UTRs of teleost vasa were searched by MEME motif analysis (Bailey and Elkan, 1994).

Total RNA was extracted from the ovaries of Sebastes schlegelii using RNA fast 200 (Fastagen, China). In accordance with the manufacturer’s instructions, the first-strand cDNA was synthesized by the PrimeScript^TM RT reagent kit with gDNA Eraser (Takara, Japan). Then, the cDNA was stored at −20°C as a probe synthesis template.

vasa mRNA distribution was detected in embryo and tissue by in situ hybridization (ISH), and the protocols followed the methods described previously (Lin et al., 2012b), with a few modifications as detailed below. Briefly, the 857 bp Ssvas fragment (nucleotides 1,540–2,396 bp; accession no. JN634874) was inserted into pGEM-T Easy vector (Promega, Madison, WI). The primers are shown in Table 1. Sense and antisense RNA probes were synthesized by in vitro transcription from a vector under the drive of the T7 or SP6 promoter with the DIG RNA Labeling Kit (Roche, Mannheim, Germany). The RNA probes were treated with RNase-free water and purified with SigmaSpin^TM Sequencing Reaction Clean-Up (Sigma–Aldrich). ISH was conducted by chemical stain with BCIP/NBT substrates on whole mount and sections samples.

For WISH, embryos were dechorionated, and dehydrated and rehydrated through a graded series of methanol after being taken out from 50% formamide in PBS. The embryos were digested with proteinase K (10 μg/ml) (5–10 min for embryo < 24 h; 15–30 min for embryo > 24 h) and refixed before hybridization. For SISH, samples were dehydrated through a graded series of ethanol, embedded in paraffin wax, and cut into 5 μm-thick sections for testis and 7 μm for ovary. The sections were de-waxed using two washes of xylene and rehydrated through an ethanol gradient. After being washed with PBST, they were refixed using 4% paraformaldehyde in PBS and digested with proteinase K (10 μg/ml) for 10 min. The WISH and SISH were performed with the probes at 65°C for 14 h. Stained samples were observed and photographed through a Nikon ENi microscope, using a Nikon DS-Ri2 imaging system.

Chimeric mRNAs which fused EGFP or mCherry to the 3′UTR of Sebastes schlegelii or Danio rerio vasa gene were synthesized by in vitro transcription, and the protocols followed the methods described previously (Zhou et al., 2019). The main process is as follows: firstly, template plasmids were constructed using Gibson assembly, and the 3′UTRs of EGFP-Pmvasa 3′UTR and mCherry-Omvasa 3′UTR plasmids in our lab were replaced by vasa 3′UTRs of black rockfish and zebrafish, respectively. Then, according to the plasmids, the transcription templates were synthesized by PCR. After purification, capped chimeric mRNAs were synthesized using mMESSAGE mMACHINE SP6/T7 kit (Ambion, United States). The primers for preparing mRNAs are also shown in Table 1. In the previous study, the EGFP-Pmvasa 3′UTR and mCherry-Omvasa 3′UTR mRNAs that contained red seabream and marine medaka vasa 3′UTRs have been synthesized in our lab (Zhou et al., 2019).

Zebrafish and marine medaka were maintained at 28 ± 0.5°C (16 h light/8 h dark) at the fish facility of the Institute of Oceanology, Chinese Academy of Sciences. The marine medaka is a truly euryhaline species closely related to the medaka. Embryos were collected within 30 min after fertilization. To verify the labeling ability of Ssvas 3′UTR in zebrafish and marine medaka, the EGFP-Ssvasa 3′UTR and mCherry-Drvasa 3′UTR mRNAs were co-injected into zebrafish embryos at the 1–2 cell stage, and the EGFP-Ssvasa 3′UTR and mCherry-Omvasa 3′UTR mRNAs were co-injected into marine medaka embryos in the same manner. Similarly, to compare the ability of vasa 3′UTRs among Sebastes schlegelii and other teleost in marking zebrafish PGCs, the EGFP-Pmvasa 3′UTR and mCherry-Omvasa 3′UTR mRNAs were also microinjected into zebrafish embryos as above. Microinjected eggs were cultured in a biochemical incubator with 28°C for EGFP or mCherry observation and photography under fluorescent microscope (Nikon ENi, Japan) at different stages. The microscope equipped with a FITC filter or a TRITC filter and a DS-Ri2 imaging system.

The full-length cDNA of Ssvas had been isolated in the previous research. In this study, multiple alignment of different fish Vasa proteins showed that there were eight consensus motifs (I, IA, IB, IC, II, III, IV, V) in the DEAD-box protein family (Figure 1A). This also revealed that the DEAD domain was composed of 180 amino acids and the superfamily Helicase C-terminal (Helicase C) of 77 amino acids, which had high similarity in teleost (Figure 1A). The Sebastes schlegelii presented Vasa typical conserved domains (Figure 1A). Phylogenetic analysis of Vasa protein exhibited that Sebastes schlegelii belonged to Euteleostei species (Figure 1B). In phylogeny, compared to Osteriophysans species like Danio rerio, Sebastes schlegelii was closer to some Euteleostei fish, such as Thunnus orientalis, Scophthalmus maximus, Pagrus major, Oryzias latipes, and Oncorhynchus mykiss (Figure 1B).

The expression pattern of vasa during embryogenesis was analyzed by WISH. At the blastula stage, all blastomeres were strongly stained, without the four cell clusters like zebrafish (Figure 2A). In Sebastes schlegelii, the Ssvas transcripts failed in early localization during the cleavage stage. As the embryo developed, the distribution of the signal in the cell became non-uniform, and the areas of embryonic shield seemed to be darker than the rest at the 30% epiboly stage (Figure 2B). Until the 70% epiboly stage, some Ssvas-positive cells firstly appeared at the germ ring of the epiboly bottom (Figure 2C). According to morphology, these positive cells were early PGCs. At the 90% epiboly stage to early neurula stage, the PGCs gathered on the dorsal side of the narrow embryonic body and randomly aligned along the anterior of the body axes (Figure 2D). Then, they moved posterior-ward and lined along the anterior–posterior axis on both sides of the embryonic body at somite stage (Figure 2E). These cells formed two clusters and aligned bilaterally on the ventral side at heart beating stage (Figures 2F,G). Finally, the PGCs localized at the dorsal sides of the gut, ventral side of notochord at the hatching stage (Figures 2H,I).

The PGCs underwent a post-birth migration in Sebastes schlegelii. PGCs are characterized by large cell size and obvious nuclei, which are easily distinguished with somatic cells on tissue sections. At 2 days after birth (dab), PGCs localized in the upper part of the body cavity under the notochord, around the dorsal peritoneum (Figures 3A1,A2). A part of PGCs were covered by a single layer somatic cell at 8 dab (Figures 3B1,B2). PGCs surrounded by single somatic cells were found to migrate to the genital ridge at 9 dab (Figures 3C1,C2). By 10 dab, three PGCs were completely surrounded by somatic cells and arranged in a row to form elongated gonadal primordia, which appeared under the ventral kidney and dorsal sides of the gut (Figures 3D1,D2). Until 15 dab, the distinct primordial gonads were discovered in the posterior part of the body cavity, and the PGCs in gonads were strongly stained with Ssvas antisense probe by SISH (Figures 3E1–E3). Through the identification of Ssvas, the germline formation process of Sebastes schlegelii was summarized, as shown in the schematic illustration (Figure 4).

The localization of Ssvas RNA was investigated in fish gonads by SISH. In accordance with adjacent histology, the stages of stained cells were identified. For 1-year-old black rockfish, in the ovary, there were mainly oogonia, early previtellogenic phase (I), and late previtellogenic phase (II) (Figure 5A1). Ssvas-specific expression was observed in all germ cells but not somatic cells (Figure 5A2). In oogonia, Ssvas mRNA was distributed evenly in the cytoplasm (Figure 5A2). In I–II oocytes, Ssvas transcripts were concentrated into several patches primarily in the perinuclear region (Figure 5A2). For sexually mature females, Ssvas transcripts were researched throughout oogenesis and showed a dynamic localization pattern in the ovary. A large number of early vitellogenesis phase (III) oocytes were present in the November individuals, but there was no Ssvas expression (Figures 5B1,B2). The tissue section hybridized with the sense probe was not stained, proving that this signal represents the expression of Ssvas (Figure 5B3).

Histological sections of the testis demonstrated that Sebastes schlegelii was lobular-type (Figures 5C1, D1). In the testis of 1-year-old black rockfish, there were spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatids, without spermatozoon (Figure 5C4). For sexually mature males more than 3 years old, a lot of spermatozoon were found (Figure 5D4). The testis was composed of many testicular lobules, and the Ssvas signals were only present in spermatogonia and primary spermatocytes, which localized at the edge of the lobules and spread throughout the whole testis except the vas deferens (Figures 5C2,D2). The signals in matured testis were significantly weaker than 1-year-old testis because Ssvas transcripts were predominantly detected in early germ cells, such as spermatogonia and spermatocytes, and no signal was detected in spermatids and spermatozoon (Figures 5C3,D3). As control, the tissue section hybridized with the sense probe was not stained (Figure 5D5).

In order to confirm that the labeling ability of Sebastes schlegelii vasa 3′UTR, EGFP-Ssvasa 3′UTR mRNA and mCherry-Drvasa 3′UTR mRNA were co-microinjected into zebrafish 1–2 cell stage fertilized eggs. After microinjection, mCherry and EGFP firstly expressed in the blastoderm at the 30–50% epiboly stage, and bright fluorescence accumulated in the area where the embryonic shield formed (Figures 6A1–A3). As embryogenesis proceeded, fluorescence concentrated on the embryonic body was more obvious, but PGCs were not distinguished from somatic cells until the early segmentation period. At the somite stage, as the fluorescence of somatic cells reduced gradually, some brighter cells moved and gathered on both sides of embryonic body (Figures 6B1–B3). The rounder and larger fluorescent cells were PGCs. PGCs migrated axial-ward and aggregated under the trunk at the heart beating stage (Figures 6C1–C3). Eventually, PGCs settled on the dorsal part of the gut at one day after hatching (Figures 6D – D3).

In the same way, the EGFP-Ssvasa 3′UTR and mCherry-Omvasa 3′UTR mRNAs were co-microinjected into marine medaka embryos. EGFP and mCherry firstly appeared at the 30% epiboly stage (Figures 6E1–E3). At the somite stage, some cells with strong fluorescence aligned on both sides of the trunk from the first somite to tail bud region (Figures 6F1–F3). These fluorescent cells were PGCs.

To compare the labeling ability of vasa 3′UTRs among Sebastes schlegelii and other teleost in marking zebrafish PGCs, the chimeric mRNAs of EGFP-Pmvasa 3′UTR and mCherry-Omvasa 3′UTR were also microinjected into zebrafish fertilized eggs. The mCherry and EGFP fluorescence firstly appeared in blastoderm at the early gastrula stage after microinjection (Figures 7A1–A3). A high level of fluorescence expression was found in the zebrafish embryonic body at the early somite stage (Figures 7B1–B3), but PGCs were not distinguished from somatic cells during the whole embryonic development (Figures 7C1–D3). In order to avoid misjudgment of results, a large number of embryos at heart beating stage were observed (Figures 7C2′,C3′). Although some fluorescent aggregates were found in the extension of the yolk sac at heart beating stage and 1 day after hatching, PGCs could not be distinguished (Figures 7C1–C3, D1–D3).

The teleost of Sebastes schlegelii, Pagrus major, Oryzias latipes, Oryzias melastigma, and Danio rerio were comparative analysis in this study. Phylogenetic analysis showed that Sebastes schlegelii and Pagrus major were clustered in one clade, while Oryzias latipes and Oryzias melastigma were clustered in one clade, both of which belonged to Euteleostei species (Figure 8A). And Danio rerio was grouped into a single branch, belonging to Osteriophysans species (Figure 8A).

M1–M20 motifs of vasa 3′UTRs were compared among the five teleost species. Sebastes schlegelii and Pagrus major had more similar conservative elements, while Oryzias latipes and Oryzias melastigma had more similar motifs (Figures 8B,C). Moreover, Danio rerio showed obvious different motifs with above species (Figures 8B,C). The results of MEME analysis were totally consistent with those of the phylogenetic analysis. However, in comparison with other three species, the motifs of M10, M12, and M19 specifically existed in Sebastes schlegelii and Danio rerio (Figures 8B,C).