Pou5f1 and Nanog Are Reliable Germ Cell-Specific Genes in Gonad of a Protogynous Hermaphroditic Fish, Orange-Spotted Grouper (Epinephelus coioides)

Pluripotency markers Pou5f1 and Nanog are core transcription factors regulating early embryonic development and maintaining the pluripotency and self-renewal of stem cells. Pou5f1 and Nanog also play important roles in germ cell development and gametogenesis. In this study, Pou5f1 (EcPou5f1) and Nanog (EcNanog) were cloned from orange-spotted grouper, Epinephelus coioides. The full-length cDNAs of EcPou5f1 and EcNanog were 2790 and 1820 bp, and encoded 475 and 432 amino acids, respectively. EcPou5f1 exhibited a specific expression in gonads, whereas EcNanog was expressed highly in gonads and weakly in some somatic tissues. In situ hybridization analyses showed that the mRNA signals of EcNanog and EcPou5f1 were exclusively restricted to germ cells in gonads. Likewise, immunohistofluorescence staining revealed that EcNanog protein was limited to germ cells. Moreover, both EcPou5f1 and EcNanog mRNAs were discovered to be co-localized with Vasa mRNA, a well-known germ cell maker, in male and female germ cells. These results implied that EcPou5f1 and EcNanog could be also regarded as reliable germ cell marker genes. Therefore, the findings of this study would pave the way for elucidating the mechanism whereby EcPou5f1 and EcNanog regulate germ cell development and gametogenesis in grouper fish, and even in other protogynous hermaphroditic species.


Introduction
Germ cell development is indispensable for animal reproduction and fertility. The complex process from the formation of primordial germ cells (PGCs) to the differentiation into gametes is strictly regulated by many factors, such as hormones and reproduction-related genes [1][2][3]. Germ cell marker genes can be used for exploring PGCs formation and migration, germ cell development, and the mechanism behind gametogenesis. In teleost fish, some germ cell-specific genes have been characterized, including PGC-specific gene Nanos3 [4], mitotic germ cell-specific gene Ly75 [5], spermatogonium-specific gene Plzf [6,7], oocyte-specific gene Slbp2 [8], as well as the well-known germline-specific genes Vasa, Dazl and Piwi [9,10].
Pou5f1 (also known as Oct4) and Nanog are closely related to each other and characterized as core transcription factors essential for embryogenesis and the maintenance of Table 1. Primers for synthesizing RNA probes, cloning the full-length cDNAs, and analyzing gene expression levels.

Total RNA Extraction and PCR
Total RNA of tissue was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality was evaluated by agarose gel electrophoresis. The cDNA was synthesized with 1 µg total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Real-time quantitative PCR (RT-qPCR) was performed on a Roche LightCycler 480 System (Roche Diagnostics, San Francisco, CA, USA) with SYBR Green Realtime PCR Master Mix (Toyobo). Produce was as follows: 30 s at 95 • C, followed by 40 cycles of 5 s at 95 • C, 5 s at 58 • C, and 15 s at 72 • C, with final step for 15 s at 95 • C and 30 s at 60 • C. Semi-quantitative PCRs for about 240 bp DNA fragments were performed with Taq PCR StarMix (Genstar, Shanghai, China), and PCR procedure was as follows: initial denaturation at 94 • C for 2 min, followed by 35 cycles of 30 s at 94 • C, 30 s at 58 • C, and 15 s at 72 • C, finally 72 • C for 5 min. Semi-quantitative PCR for the 1299 bp open reading frame (ORF) of EcNanog was performed with KOD One PCR Master Mix-Blue (Toyobo), and PCR procedure was as follows: initial denaturation at 94 • C for 2 min, followed by 35 cycles of 5 s at 98 • C, 5 s at 60 • C, and 5 s at 68 • C, finally 68 • C for 5 min. After gel electrophoresis, bands were photographed with a Tanon 1600 image system (Tanon, Shanghai, China). The β-Actin was used as an internal control. Genetic expression quantifications were normalized to β-Actin. Primers were listed in Table 1.

Western Blotting
The monoclonal anti-Nanog antibody from medaka was provided by Professor Hongyan Xu [36]. The total protein of tissues was extracted with RIPA Lysis Buffer (Beyotime, Shanghai, China) and mixed with SDS-PAGE Sample Loading Buffer (Beyotime). After being boiled for 5 min, 10 µL protein buffer was loaded into a lane, electrophoresed through 10% SDS-PAGE gels, and electroblotted onto polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA, USA) by an electroblotter (BioRad, Hercules, CA, USA). The membrane was blocked with 5% BSA (Solarbio) for 1 h. After being washed with TBS (Solarbio), the membrane was incubated with anti-β-Actin antibody (Bioss, Beijing, China) or anti-Nanog antibody (1:1000 dilution in TBS) at 4 • C overnight. Then, the membrane was washed with TBS and incubated with HRP-conjugated goat anti-rabbit IgG (Bioss) (1:2000 dilution in TBS) for 2 h. Finally, protein blots were colored with Chemiluminescent Substrate for Western blotting Kit (Cyanagen, Bologna, Italy) and imaged by an Alliance MINI HD9 system (Uvitec, Cambridge, UK). The β-Actin was used as an internal control.

In Situ Hybridization (ISH)
ISH protocol was described in our previous study [37]. Briefly, after being fixed in 4% paraformaldehyde (Sangon, Shanghai, China) at 4 • C overnight, gonads were dehydrated with gradient methanol/PBS from 20% to 100% methanol, and then stored at −20 • C until further use. Gonads were rehydrated with gradient methanol/PBS up to 100% PBS, immersed in 30% (w/v) sucrose at 4 • C overnight, and embedded in OCT compound (SAKURA Tissue-Tek, Atlanta, GA, USA). These gonad samples were cryosectioned at 4 µm using a Leica RM2135 Microtome (Leica, Wetzlar, Germany). Probes were synthesized using DIG RNA labeling kit (Roche, Mannheim, Germany). Lengths of EcPou5f1 and EcNanog probes were 1037 bp and 1299 bp, respectively. Ingredients of hybridization buffer were as follows: 50% deionized formamide, 5 × saline sodium citrate (SSC), 0.5 mg/mL salmon sperm RNA, 1 × Denhart's solution, and 5% dextran sulphate. Gonadal sections were pre-hybridized with hybridization buffer for 2 h and then hybridized with 1 µg/mL DIG probes at 65 • C for 15 h. Subsequently, sections were washed with SSC and blocked with Blocking Reagent (Roche) for at least 1 h. Sections were incubated with anti-Digoxigenin-AP (Roche), then colored with NBT/BCIP (Roche). Photographs were imaged by a Leica DMI8 microscope (Leica).

Dual-Label ISH
Dual-label ISH protocol referred to our previous study [38]. The Vasa probe was 1040 bp and synthesized using Fluorescein RNA labeling kit (Roche). Gonadal sections were pre-hybridized for 2 h, then hybridized by 1 µg/mL Vasa probe and one of EcPou5f1 and EcNanog probes at the same time at 65 • C for 15 h. Subsequently, sections were washed with SSC and blocked with Blocking Reagent for at least 1 h. The Vasa probe was incubated with anti-Fluorescein-POD (Roche), then stained red fluorescence for 10 min with TSA TM PLUS Fluorescein system (PerkinElmer, Shelton, USA). After being washed, the EcPou5f1 or EcNanog probes were incubated with anti-Digoxigenin-POD (Roche) and were stained green fluorescence for 10 min. Finally, gonadal sections were counterstained by DAPI (Solarbio) for cell nuclei staining. Sections were imaged with a Zeiss LSM 800 microscope (Zeiss, Jena, Germany) or a Leica TCS SP5 microscope (Leica).

Fluorescent Immunostaining
Gonadal sections were dried in a drying oven and washed with PBS. After being blocked with 5% goat serum (Sangon) for 1 h, sections were incubated with the anti-Nanog antibody for 2 h (1:200 dilution in PBS containing 2% goat serum). After being washed with PBS, sections were incubated with HRP-conjugated goat anti-rabbit IgG (1:3000 dilution in PBS) for 1 h. Signals were developed using the TSA TM Plus Fluorescence System. The nucleus was colored by PI (Solarbio). Photographs were imaged by a Leica DMI8 microscope.

Statistical Analysis
All data were shown as the mean values ± SEM. Statistical analysis was implemented by one-way ANOVA and Student's t-test. A probability level less than 0.05 (p < 0.05) was considered statistically significant. All statistics were carried out using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA).

Identifications of EcPou5f1 and EcNanog
Full-length cDNA of EcPou5f1 was 2790 bp with a 1428 bp ORF, a 269 bp 5 -untranslated region (UTR), and a 1093 bp 3 -UTR, as well as encoded a predicted protein of 475 amino acids containing POUs and POUhD ( Figure 1A). Full-length cDNA of EcNanog was 1820 bp with a 1299 bp ORF encoding a 432 amino acid peptide with an HD domain, a 143 bp 5 -UTR, and a 378 bp 3 -UTR ( Figure 1B). Sequences of EcPou5f1 and EcNanog were deposited in GenBank with accession numbers OL439940 and OK415852, respectively.
Multiple sequence alignments showed that EcPou5f1 protein had the conserved POUs and POUhD, as well as a nuclear localization motif of RKRKR ( Figure 2). Full-length polypeptide of EcPou5f1 showed very high homology to fish Pou5f1 homologs from 70% to 91%, while low similar to tetrapod Pou5f1 homologs from 44% to 58% ( Figure 2). In addition, POU domains showed a conserved feature in diverse vertebrates, with an identity ranging from 66% to 100% ( Figure 2).
EcNanog protein had a conserved HD domain with a nuclear localization motif of YKQVKTWFQN ( Figure 3). EcNanog shared a low identity with tetrapod Nanog homologs, ranging from 30% to 34%, while a high identity with fish Nanog homologs ranging from 49% to 76% ( Figure 3). Likewise, the greatest similarity appeared on the most important functional domains, i.e., HD domain, ranging from 49% to 90% (Figure 3). Multiple sequence alignments showed that EcPou5f1 protein had the conserved POUs and POUhD, as well as a nuclear localization motif of RKRKR ( Figure 2). Full-length polypeptide of EcPou5f1 showed very high homology to fish Pou5f1 homologs from 70% to 91%, while low similar to tetrapod Pou5f1 homologs from 44% to 58% ( Figure 2). In addition, POU domains showed a conserved feature in diverse vertebrates, with an identity ranging from 66% to 100% ( Figure 2).  EcNanog protein had a conserved HD domain with a nuclear localization motif of YKQVKTWFQN ( Figure 3). EcNanog shared a low identity with tetrapod Nanog homologs, ranging from 30% to 34%, while a high identity with fish Nanog homologs ranging from 49% to 76% ( Figure 3). Likewise, the greatest similarity appeared on the most important functional domains, i.e., HD domain, ranging from 49% to 90% ( Figure 3).  In phylogenetic tree analysis, EcPou5f1 and EcNanog were respectively clustered into a single clade with fish homologs and separated from other POU and HD proteins, including Pou1, Pou3, Nkx2.5, and Msx1 ( Figure 4A,B).
In phylogenetic tree analysis, EcPou5f1 and EcNanog were respectively clustered into a single clade with fish homologs and separated from other POU and HD proteins, including Pou1, Pou3, Nkx2.5, and Msx1 ( Figure 4A,B).

Tissue Distributions of EcPou5f1 and EcNanog
Semi-quantitative PCR results showed that a 245 bp fragment of EcPou5f1 was limited to gonads, whereas a 240 bp fragment of EcNanog was highly detected in gonads and weakly in other tissues, including brain, pituitary, head kidney, kidney, stomach, liver, and muscle ( Figure 5A). However, the 1299 bp ORF of EcNanog was only amplified in gonads ( Figure 5A). In Western blotting, EcNanog signals were detected as three strong bands between 40 and 55 kDa in intestine, a distinct band between 40 and 55 kDa in gonads and stomach, a single band about 35 kDa in muscle, a weak band between 40 and 55 kDa in kidney, as wells as no bands in other somatic tissues ( Figure 5B). In RT-qPCR

Tissue Distributions of EcPou5f1 and EcNanog
Semi-quantitative PCR results showed that a 245 bp fragment of EcPou5f1 was limited to gonads, whereas a 240 bp fragment of EcNanog was highly detected in gonads and weakly in other tissues, including brain, pituitary, head kidney, kidney, stomach, liver, and muscle ( Figure 5A). However, the 1299 bp ORF of EcNanog was only amplified in gonads ( Figure 5A). In Western blotting, EcNanog signals were detected as three strong bands between 40 and 55 kDa in intestine, a distinct band between 40 and 55 kDa in gonads and stomach, a single band about 35 kDa in muscle, a weak band between 40 and 55 kDa in kidney, as wells as no bands in other somatic tissues ( Figure 5B). In RT-qPCR analyses, EcPou5f1 and EcNanog were extremely expressed in gonads with a higher level in ovary than in testis ( Figure 5C,D). analyses, EcPou5f1 and EcNanog were extremely expressed in gonads with a higher level in ovary than in testis ( Figure 5C,D).

Chemical ISH of EcPou5f1 and EcNanog in Gonads
The sense riboprobes of EcPou5f1 and EcNanog showed no specific signal in ovary and testis ( Figure 6A,B,F,G). In testis, the antisense probe signal of EcPou5f1 was intense in spermatogonia, moderate in spermatocytes, and no signal could be detected in spermatids ( Figure 6C). In ovary, the antisense probe signal of EcPou5f1 was obviously observed in oogonia, primary growth stage oocytes, and cortical-alveolus stage oocytes, but scarcely detected in vitellogenic stage oocytes ( Figure 6D,E). In some primary growth stage oocytes, EcPou5f1 mRNA signals were unevenly distributed in perinuclear speckles and nuclei ( Figure 6D). The distribution of EcNanog mRNA in gonads had some differences from EcPou5f1 mRNA. In testis, EcNanog mRNA signal was observed in the spermatogenic cells from spermatogonium to spermatid ( Figure 6H). In ovary, EcNanog mRNA signal was detected in oogonia, primary growth stage oocytes, cortical-alveolus stage oocytes, and early vitellogenic stage oocytes, but scarcely in late vitellogenic stage oocytes ( Figure 6I,J). In addition, it was observed that a few small oval cells, possibly oogonia or oogonial stem cells, were EcPou5f1 or EcNanog-positive ( Figure 6D,I).

Chemical ISH of EcPou5f1 and EcNanog in Gonads
The sense riboprobes of EcPou5f1 and EcNanog showed no specific signal in ovary and testis ( Figure 6A,B,F,G). In testis, the antisense probe signal of EcPou5f1 was intense in spermatogonia, moderate in spermatocytes, and no signal could be detected in spermatids ( Figure 6C). In ovary, the antisense probe signal of EcPou5f1 was obviously observed in oogonia, primary growth stage oocytes, and cortical-alveolus stage oocytes, but scarcely detected in vitellogenic stage oocytes ( Figure 6D,E). In some primary growth stage oocytes, EcPou5f1 mRNA signals were unevenly distributed in perinuclear speckles and nuclei ( Figure 6D). The distribution of EcNanog mRNA in gonads had some differences from EcPou5f1 mRNA. In testis, EcNanog mRNA signal was observed in the spermatogenic cells from spermatogonium to spermatid ( Figure 6H). In ovary, EcNanog mRNA signal was detected in oogonia, primary growth stage oocytes, cortical-alveolus stage oocytes, and early vitellogenic stage oocytes, but scarcely in late vitellogenic stage oocytes ( Figure 6I,J). In addition, it was observed that a few small oval cells, possibly oogonia or oogonial stem cells, were EcPou5f1 or EcNanog-positive ( Figure 6D,I).

Dual-Label ISH for EcPou5f1 or EcNanog with Vasa in Gonads
In order to further verify the reliability of EcPou5f1 and EcNanog as germ cell-specific genes, we adopted Vasa, a well-known germ cell-specific gene [9], as a positive control in dual-label ISH. In testis and ovary, the mRNA signal of EcPou5f1 had an identical localization with Vasa mRNA signal in male and female germ cells (Figure 7). It was worth mentioning that the very faInt. fluorescence of EcPou5f1 mRNA could be observed in spermatids by Tyramide Signal Amplification system ( Figure 7B). Similar to the chemical ISH result of EcPou5f1 mRNA in ovary, the fluorescent signal of EcPou5f1 mRNA was obviously observed in the cytoplasm of oogonia, and the perinuclear speckles and nuclei of some primary growth stage oocytes ( Figure 7F). In ovary, the small oval cells, possibly oogonia or oogonial stem cells, with EcPou5f1 signal would be marked by Vasa signal (Figure 7E-G).

Dual-Label ISH for EcPou5f1 or EcNanog with Vasa in Gonads
In order to further verify the reliability of EcPou5f1 and EcNanog as germ cell-specific genes, we adopted Vasa, a well-known germ cell-specific gene [9], as a positive control in dual-label ISH. In testis and ovary, the mRNA signal of EcPou5f1 had an identical localization with Vasa mRNA signal in male and female germ cells (Figure 7). It was worth mentioning that the very faInt. fluorescence of EcPou5f1 mRNA could be observed in spermatids by Tyramide Signal Amplification system ( Figure 7B). Similar to the chemical ISH result of EcPou5f1 mRNA in ovary, the fluorescent signal of EcPou5f1 mRNA was obviously observed in the cytoplasm of oogonia, and the perinuclear speckles and nuclei of some primary growth stage oocytes ( Figure 7F). In ovary, the small oval cells, possibly oogonia or oogonial stem cells, with EcPou5f1 signal would be marked by Vasa signal (Figure 7E-G).

Immunohistofluorescence of Anti-Nanog Antibody in Gonads
Immunohistofluorescence was carried out for examining the localization of EcNanog protein in gonads of orange-spotted grouper. In testis, the signal of anti-Nanog antibody was mainly detected in the nucleus and cytoplasm of spermatogonia and the cytoplasm of spermatocytes, while slight in spermatids ( Figure 9A-C). In ovary, the signal of anti-Nanog antibody was predominantly detected in the nuclei of oogonia and primary growth stage oocytes, and then gradually spread in the cytoplasm of cortical alveolus stage oocytes, up to a homogeneous distribution in the whole vitellogenic stage oocytes ( Figure 9D-I).

Immunohistofluorescence of Anti-Nanog Antibody in Gonads
Immunohistofluorescence was carried out for examining the localization of EcNanog protein in gonads of orange-spotted grouper. In testis, the signal of anti-Nanog antibody was mainly detected in the nucleus and cytoplasm of spermatogonia and the cytoplasm of spermatocytes, while slight in spermatids ( Figure 9A-C). In ovary, the signal of anti-Nanog antibody was predominantly detected in the nuclei of oogonia and primary growth stage oocytes, and then gradually spread in the cytoplasm of cortical alveolus stage oocytes, up to a homogeneous distribution in the whole vitellogenic stage oocytes ( Figure 9D-I).

Discussion
Pluripotency markers Pou5f1 and Nanog are generally regarded as core transcription factors sustaining stem cell pluripotency and embryogenesis [11][12][13]. However, their roles are unclear in germ cell development and gametogenesis in teleost fish. In this study, the cDNA sequences and expression patterns of EcPou5f1 and EcNanog were characterized and analyzed in a protogynous hermaphroditic fish, orange-spotted grouper. Sequence analysis showed that EcPou5f1 protein possessed a conserved POU domain, and EcNanog protein had a conserved HD domain. The similarity of POU domain and HD domain is much higher than the full-length sequences in various species. This situation reflects the conserved nature of Pou5f1 and Nanog in the process of evolution to a certain extent. For instance, the Nanog required for inducing mouse IPSCs can be replaced with chicken or zebrafish Nanog [39], and other tetrapod Pou5f1 can maintain the pluripotency and selfrenewal of mouse ESCs [40,41]. Additionally, sequence alignments revealed that EcPou5f1 and EcNanog shared the highest homology with the orthologs in fish, in agreement with phylogenetic tree analysis. The greatest homology among fish implies that the functions of EcPou5f1 and EcNanog in orange-spotted grouper would be very similar to other fish homologs.
EcPou5f1 and EcNanog show two discrepant tissue distribution patterns. The protein and short DNA fragment of EcNanog were detected in gonads and some nongonadal tissues, e.g., stomach and muscle, whereas the ORF region of EcNanog was only detected in gonads. This is consistent with the PCR result of Nanog in blunt-snout bream, the short fragment is detected in gonads and nongonadal tissues, but the long fragment just exists in gonads [33]. In zebrafish, the transcript and protein of Nanog are distinctly detected in gonads, liver, and heart [42]. On the contrary, in adult Japanese flounder, Nanog is restrictively expressed in ovary and testis [25]. We deduce that the alternative splicing of EcNanog lead to the inconsistent PCR amplification between the short and long fragments, and the appearance of protein bands in some somatic tissues that may possess Nanog-positive stem cells [33,43,44]. Similar to the tissue distribution pattern of Japanese flounder Pou5f1 [30], EcPou5f1 was restrictively expressed in gonads. Nonetheless, some somatic tissues of medaka and Chinese sturgeon (Acipenser sinensis), for example, brain, can also express Pou5f1 [22,45]. Pou5f1 protein is a direct regulator of Nanog transcript [18]. Intriguingly, EcPou5f1 was not detected in those nongonadal tissues expressing the short DNA fragment or protein of EcNanog. We guess that other regulators, such as Sox2 and Klf4, and the auto-regulatory of Nanog might account for the absence of EcPou5f1 in nongonadal tissues [46,47]. In short, the tissue distributions of Pou5f1 and Nanog show species differences to a variable extent in diverse fish, and suggest that they play multifunctional roles in gonads and somatic tissues.
EcPou5f1 and EcNanog are dependable germ cell-specific genes in orange-spotted grouper. EcPou5f1 was restricted to germ cells, but scarce in vitellogenic stage oocytes. Using Tyramide Signal Amplification system, EcPou5f1 signal was faintly observed in spermatids, suggesting that a few EcPou5f1 transcripts existed in spermatids. These ISH results indicate that EcPou5f1 exists exclusively in germ cells of gonads and would downregulate dramatically from spermatocyte to spermatid. In medaka and Japanese flounder, Pou5f1 is limited to spermatogonia, oogonia, and most oocytes, but absent in spermatocytes and spermatids [22,30]. In testis of Nile tilapia, Pou5f1 protein shows a specific localization in undifferentiated spermatogonia [48,49]. However, in large yellow croaker, Pou5f1 is detected in spermatogonia and primary spermatocytes [32]. EcNanog was detected in all male germ cells and the female germ cells from oogonia to early vitellogenic stage oocytes. A similar expression pattern is also observed for Nanog in gonads of blunt-snout bream [33]. However, in testes of zebrafish, medaka, and Japanese flounder, Nanog is only expressed in spermatogonia [25,31,42]. The differential expression in differentiated male germ cells implies that EcPou5f1 and EcNanog may participate in spermatogenesis, whereas the Pou5f1 and/or Nanog of medaka, zebrafish, Japanese flounder, and Nile tilapia is mainly responsible for the pluripotency and self-renewal of spermatogonia. In accordance with the EcNanog ISH, EcNanog protein specifically existed in all male and female germ cells, includ-ing vitellogenic stage oocyte in which EcNanog mRNA was difficultly detected. EcNanog contained a motif of YKQVKTWFQN that had been identified as a nuclear localization motif in human Nanog [50]. As expected, fluorescent immunostaining revealed the nuclear localization of EcNanog in spermatogonia, oogonia, and primary growth stage oocytes. This is also supported by the nuclear localization of Nanog in zebrafish and blunt-snout bream [33,42]. Interestingly, we observed that EcNanog also existed in the cytoplasm of spermatocytes and spermatids. A similar localization of Nanog is observed in the spermatocytes and spermatids of pig testis [28]. The translocation of Nanog from nucleus to cytoplasm may imply the loss of function sustaining the pluripotency characteristics of spermatogonia. Although the subcellular localization of EcPou5f1 protein was not tested in this study, we speculated that EcPou5f1 might also locate in the nuclei of germ stem cells, on account of the nuclear localization signal RKRKR [50]. Vasa is a widely accepted germline-specific marker [51,52], and shows a specific expression in the germ cell lineage in many animals, e.g., medaka [53] and mouse [54]. Our previous study has demonstrated that Vasa is exclusively expressed in germ cells in gonads of orange-spotted grouper [9]. In adult testis of rhesus monkey (Macaca mulatta), Nanog is restrictively expressed in all spermatogenic cells, most of which are Vasa-positive [55]. In human fetal testis and ovary, Pou5f1 is limited to some fetal germ cells, whereas only a part of the cell shows the low intensity immunofluorescences of Pou5f1 and Vasa [20]. In this study, dual-label ISH analysis revealed that Vasa was co-localized with EcPou5f1 and EcNanog in gonads. Furthermore, in ovary, the small oval cells expressing EcPou5f1 or EcNanog would be marked by Vasa signal, suggesting that these cells exactly were oogonia or oogonial stem cells. On the basis of these results, we conclude that EcPou5f1 and EcNanog are reliable germ cell-specific markers in orange-spotted grouper.
Although there have been a few studies about the functions of Pou5f1 and Nanog in differentiated germ cells in mammals [26][27][28], it is widely accepted that Pou5f1 and Nanog are mainly responsible for the pluripotency of germline stem cells [20,21,56,57]. Conversely, the teleost Nanog and Pou5f1 are expressed in spermatogonia, oogonia, and a large proportion of oocytes, as well as have different expression patterns in the differentiated male germ cells in diverse fish [22,25,30,31,33,42,49]. Likewise, EcPou5f1 and EcNanog can be detected in all spermatogonia and oogonia, most oocytes, spermatocytes, and spermatid. The high expressions of teleost Nanog and Pou5f1 during oocyte development may be related to maternal inheritance, i.e., mRNA or protein delivery to the next generation [24,25,33]. The comparisons in differentiated male germ cells indicate that Pou5f1 and Nanog may have some differences in promoting spermatogenesis in different fish. Moreover, the differentials among diverse fish would provide a new perspective for understanding the evolution and conserved nature of teleost Pou5f1 and Nanog.

Conclusions
In summary, EcPou5f1 and EcNanog are germ cell-specific marker genes and play important roles in gametogenesis in a protogynous hermaphroditic fish, orange-spotted grouper. Our findings would contribute to further studies on the molecular mechanism underlying germ cell development, gametogenesis, and sex reversal in hermaphroditic fish.