Scleractinian gonadal transcriptome assembly
Since scleractinian gametogenesis occurs exclusively in gonads, isolated gonads (but not whole polyps) are useful to explore genes associated with gametogenesis. However, gonad isolation is technically difficult in many scleractinians due to small polyp sizes. Gonad isolation not only requires an understanding of polyp anatomy, but also technical skill. The present study applied previously established techniques for gonad isolation from E. ancora polyps  to the current transcriptomic study. Bioinformatics methods to eliminate contigs from symbiotic dinoflagellates or other contaminants were also employed . 60.2% of the contigs in the E. ancora gonadal transcriptome assembly showed similarities to entries in the SWISS-PROT database (Fig. 3). Specifically, 68% were similar to Stylophora pistilata gene models  and 59.7% to Pocillpora damicornis gene models  in SWISS-PROT. In addition, conserved Pfam protein domains were detected in 66.2% of contigs in the E. ancora gonadal transcriptome (Fig. 3). Conserved Pfam protein domains were detected in 54% of sequences in the Heliopora coerulea transcriptome assembly . Other transcriptome assemblies showed similar percentages: Dendrophyllia sp. (48.8%), Eguchipsammia fistula (45.4%), and Rhizotrochus typus (51.3%) . The E. ancora gonadal transcriptome is clearly comparable to other coral genomic or transcriptomic datasets. The present transcriptome assembly allowed us to identify sex-specific and gonadal phase-specific upregulated genes as well as evolutionarily conserved genes associated with germ cell development. The resulting dataset will provide a foundation for future research investigating molecular and cellular mechanisms of gametogenesis in scleractinians.
Characteristics of premature/mature ovaries as assessed by anatomical and histological analyses
The observed growth of oocytes and the loss of germinal vesicles in oocytes of premature/mature ovaries suggest that oocytes were still actively accumulating essential materials (e.g., yolk and other components) for survival and development of embryos until just before maturation. Also, the oocyte maturation process, including germinal vesicle breakdown (GVBD) and resumption of meiosis occurred in some oocytes.
Upregulated genes in premature/mature ovaries
Yolk formation and accumulation is one of the most important aspects of oogenesis for oviparous animals. In scleractinian eggs, several major yolk proteins, including vitellogenin (Vg), a female-specific phosphoglycolipoprotein, and large amounts of lipids (e.g., wax esters, fatty acids, phosphatidylethanolamines, and phosphatidylcholines) have been identified to date [12, 13, 19, 20, 38, 39]. The present study found that transcripts encoding 3 major yolk proteins were upregulated (Vg, Egg protein, and Euphy, Fig. 5, Table 4), in agreement with histological observations, indicating that oocytes were actively accumulating yolk materials. Those yolk proteins are produced by ovarian somatic cells adjacent to oocytes [12, 13]. However, little is known about the uptake mechanisms of yolk proteins by oocytes. Although receptor-mediated endocytosis has been hypothesized, related receptor molecules have not been identified yet . The present study also identified transcripts encoding two types of low-density lipoprotein receptor-related proteins (Lrps) as upregulated genes in premature/mature ovaries (Fig. 5, Table 4). In some teleosts, a member of Lrps, Lrp13, serves as one of the Vg receptors expressed on oocyte membranes [40, 41]. Thus, the Lrps identified here may be involved in uptake mechanisms of yolk materials in scleractinians, and are promising candidate receptors for Vg and/or other lipoproteins in future studies.
In addition to the major yolk materials, eggs of scleractinians are assumed to accumulate materials essential for larval development. Among the upregulated genes in premature/mature ovaries, we identified several sequences similar to components of skeletal organic matrix proteins found in A. digitifera , A. millepora , and S. pistillata . Since no skeleton formation occurs in ovaries, it is likely that these gene products (mRNA and proteins) are stored in oocytes during oogenesis to be used for skeleton formation during larval development. We cannot rule out the possibility that the identified genes may have other functions in oocyte development/maturation.
The occurrence of GVBD in some oocytes of ovaries collected in April 2017 was an unexpected finding, because mature gametes were not observed in testes collected at the same time. It is possible that timing of oocyte maturation was split among oocytes and/or ovaries over April and May (or June) for unknown reasons. We cannot completely rule out the possibility that the GVBD was partially induced by isolation of ovaries from polyps, i.e., mechanical stress. Nevertheless, this study successfully identified two sequences similar to the serine/threonine-protein kinase mos (Mos) gene and the mitogen-activated protein kinase 1 (Mapk1) gene, which contribute to signaling pathways of oocyte maturation in a variety of animals, including cnidarians  (Fig. 5). Upregulation of these two genes in premature/mature ovaries implies that they may also function in oocyte maturation in scleractinians. Previous studies regarding oocyte maturation in scleractinians were limited to histological observations and focused on the presence and timing of GVBD [46, 47]. To the best of our knowledge, this is the first study to identify these candidate molecules in oocyte maturation of scleractinians.
In a variety of animals, hormones (i.e., steroids, growth factors, peptides, and other substances) are involved in these processes [48-55]. In Acropora species, transcriptomic studies suggest that melanopsin-like homolog and /or neuropeptides  and Rhodopsin-like receptors  are involved in the signaling pathway for spawning in scleractinians. Enriched BP terms in E. ancora premature/mature ovaries imply that neuronal activity is significantly higher than during other phases. Upregulation of transcripts similar to genes encoding monoamine receptors (e.g., octopamine receptors, dopamine receptors, adrenergic receptors, and serotonin receptors, Fig. 5, Table 4) also support this assumption. Recent studies show that some neurotransmitters (dopamine and serotonin) are also involved in regulation of scleractinian spawning. Treatment of Acropora tenuis with dopamine during the final phase of gametogenesis inhibited spawning . By contrast, treatments with serotonin and its precursor, L-5-hydroxytryptophan (5-HTP) induced spawning of Acropora cervicornis . Taking all these lines of evidence into account, the identified monoamine receptors may also be essential during the premature/mature phase of E. ancora oogenesis. It will be of interest to investigate whether treatment of female E. ancora with these neurotransmitters induces or inhibits oocyte maturation and spawning.
Of particular interest is the upregulation of three genes encoding neurogenic locus notch homolog proteins in premature/mature ovaries (Additional File 9). The Notch signaling pathway is conserved across animal taxa, and regulates cell-cell interactions and cell fate determination . One of the identified genes, neurogenic locus notch homolog protein 1, encodes Euphy, a novel major yolk protein in E. ancora oocytes identified in our previous study . The remaining 2 genes have not been previously reported. Although both sequences possess EGF-like domain repeats typifying notch homolog proteins, they are structurally distinct from Notch1 identified in vertebrates (e.g., human Notch1). These may be novel genes that emerged after gene duplication, domain shuffling, and rapid molecular evolution in cnidarian/scleractinian lineages [42, 43]. Interestingly, one of them, neurogenic locus notch homolog protein 3, was highly and significantly upregulated, and contains a zona pellucida (ZP) protein and transmembrane domains (Fig. 5, Additional File 9). The ZP is the extracellular matrix (ECM) surrounding mammalian oocytes, composed of four glycoproteins (ZP1-ZP4). ZP functions during oogenesis, fertilization, and preimplantation development in mammals . In jellyfish, a ZP domain-containing protein called mesoglein, which resembles mammalian ZP, was identified in the contact plate of oocytes . Although scleractinian oocytes have neither a protective coat nor a membrane surrounding them, this finding implies that the identified ZP domain-containing protein probably participates in oogenesis and subsequent fertilization processes.
GFP is one of the natural pigments of corals [63-66]. Although the natural functions of GFP remain obscure, proposed functions include photoprotection from high UVA/blue irradiation, photosynthetic enhancement, phototaxis of zooxanthellae [67-70], and antioxidant activity [71, 72]. We previously showed that E. ancora oocytes express an endogenous RFP with H2O2 degradation activity from early to mature stages of oocytes, and suggested a possible role of RFP in protecting oocytes from oxidative stress during oogenesis . Our finding implies that not only RFP, but also GFP may serve in oogenesis, particularly during the premature/mature phase (Fig. 5).
Characteristics of mature testes as assessed by histological and cytological analyses
Spermiogenesis is a process by which haploid spermatids undergo a complex series of morphological changes, and eventually become elongated functional sperm. The presence of spermaries having both round spermatids and mature sperm in testes collected in June 2017 suggested that spermiogenesis was occurring in the testes at the time of collection, and that genes involved in regulation of spermiogenesis were being expressed in testes.
Upregulated genes in mature testes
Morphological changes of male germ cells during spermiogenesis include flagellum formation, nuclear DNA condensation, and elimination of organelles and cytoplasm. Scleractinian spermiogenesis is generally morphologically similar to that of vertebrates, except that male germ cells possess long flagella from early to late stages of development . Nevertheless, scleractinian male germ cells possess typical flagellar axonemes, characterized by a“9 + 2” arrangement of microtubules [21, 73]. In this study, further queries of genes associated with spermatid development (GO term:0007286), together with literature-based gene identification, allowed us to identify various candidate genes encoding proteins of flagellar components. The presence of a conserved molecular toolkit for spermiogenesis suggests that scleractinians and vertebrates share similar characteristics at both morphological and molecular levels.
Sperm motility is important for most scleractinians, which fertilize externally in seawater. Sperm of acroporid corals remain completely immotile in seawater until they come close to eggs, whereupon they acquire motility . The presence of chemoattractants and involvement of intracellular pH elevation and Ca2+-dependent signal transduction in sperm motility have been experimentally demonstrated [74, 75]. Molecules regulating flagellar motility still remain largely unexplored in scleractinians. This study identified a number of important genes encoding proteins involved in sperm motility and/or capacitation in mammals and sea urchins, such as cation channel sperm-associated protein 3 (CatSper3), sodium/hydrogen exchanger (sNHE), and adenylate cyclase type 10 (sAC) (Fig. 5, Table 5). These findings support the hypothesis of Romero and Nishigaki that CatSper3, sNHE, and sAC form prototypical machinery for sperm flagellar beating in metazoans . This study further identified the gene encoding creatine kinase, flagellar, which was first identified from flagella of sea urchin sperm, participating in energy transport from sperm heads to the flagella during sperm motility . Genes associated with sperm motility and/or capacitation in scleractinians suggest that these features were most likely present in the common ancestor prior to divergence of the cnidarian and bilaterian lineages.
Sex steroids are critical for sex differentiation, gametogenesis, and gamete maturation in vertebrates [78-82]. Sex steroids (e.g., estrogen, testosterone, and progesterone) have been demonstrated in several scleractinians, including E. ancora [83-86]. Additionally, the correlation between sex steroid levels and gametogenic cycles has led to the hypothesis that sex steroids may be involved in regulation of scleractinian reproduction [84, 86]. Steroid biosynthesis is catalyzed by various steroidogenic enzymes. Although steroid biosynthetic activities are known from extracts of some scleractinian tissues [84-89], only one gene encoding a steroidogenic enzyme, 17β-hydroxysteroid dehydrogenase type 14 (17β-hsd 14), has been identified and characterized so far . In the present study, a gene encoding steroid 17α-hydroxylase/17,20-lyase (Cyp17a) (Fig. 5, Table 5), a key enzyme in production of sex steroids and cortisol , was upregulated in mature testes. Although further analysis is required to clarify its activity, the presence of this enzyme implies that steroid biosynthesis occurs in mature testes, and the produced sex steroids/cortisol could be associated with maturation of male germ cells in scleractinians.
Molecules involved in fertilization remain largely unknown in scleractinians. We found that a gene similar to Hapless 2/Generative Cell Specific 1 (Hap2/Gcs1) was upregulated in mature testes (Fig. 5, Table 5). Hap2/Gcs1 was first identified as a male gamete-specific transmembrane protein in lilies . The coding gene is found in genomes of most major eukaryotic taxa (e.g., protozoa, plants, and animals) except fungi [93, 94]. Functional analysis with the mutant/gene targeting system showed that Hap2/Gcs1 are essential for gamete fusion in Arabidopsis , the protozoan parasite, Plasmodium , and the green alga, Chlamydomonas . Expression of Hap2/Gcs1 was also confirmed in male germ cells of some cnidarians, such as Hydra  and the starlet sea anemone, Nematostella vectensis , and its involvement in fertilization has been demonstrated in sea anemones . Upregulation of Hap2/Gcs1 in E. ancora mature testes suggests that Hap2/Gcs1 participates in scleractinian sperm-egg fusion. Most recently, we reported that receptor guanylate cyclase A (rGC-a) (also known as atrial natriuretic peptide receptor 1 in mammals) is expressed in E. ancora sperm flagella  (Fig. 5, Table 5). rGCs are expressed on sperm and serve as receptors for egg-derived sperm-activating and sperm-attracting factors in some echinoderms and mammals [98-101]. Taken together, evolutionarily conserved proteins underlie fertilization mechanisms of scleractinians.
Other major findings and potential applications
Genes encoding Histone H2B and Histone H2A were upregulated in premature/mature ovaries and mature testes, respectively (Fig. 5, Table 4, 5). Histones are the major protein components of chromatins in eukaryote cell nuclei. Five histone protein families exist: the core histone families (H2A, H2B, H3, and H4) and the linker histone family (H1) . Core histones are components of the nucleosome core, whereas linker histones are present in adjacent nucleosomes, where they bind to nucleosomal core particles, and stabilize both nucleosome structure and higher-order chromatin architecture [102, 103]. Various isoforms of each family have been identified as histone variants, and their importance in diverse cellular processes (e.g., transcriptional control, chromosome segregation, DNA repair and recombination, and germline specific translational regulation) have been revealed [104, 105]. In scleractinians, although sequences of the histone gene cluster have been identified in Acropora formosa (H3, H4, H2A, and H2B)  and Acropora gemmifera (H3, and H2B) , differences in gene expression levels between ovaries and testes have not been reported so far. This study revealed the existence of histone variants showing sexually dimorphic expression in scleractinians. In the cnidarian model organism, Hydractinia echinata, 19 genes encoding histones were identified, and some of them, such as histone H2A.X and five H2B variants, are specifically expressed in female and male germ cells, respectively . Our findings imply that the identified histone may control gene expression in female and male germ cells during scleractinian gametogenesis.
Studies of a variety of animals have revealed that a set of specialized and highly conserved genes govern germline specification, development, meiosis, and/or maintenance in metazoans [109, 110] (Additional File 5). In the gonadal transcriptome, we could identify many genes associated with germline specification and meiotic processes (Additional File 5). Although further spatiotemporal expression analyses and functional assays are required to clarify their functions, their expression in gonads implies that these genes participate in scleractinian germline development and meiosis.
The E. ancora gonadal transcriptome assembly includes a large number of genes without homology to sequences in the SWISS-PROT database. These findings suggest that although scleractinian gametogenesis shares many common molecular characteristics with gametogenesis in other metazoans, it also possesses characteristics that developed in evolutionarily unique ways. Further characterization and functional studies of these unannotated genes will clarify unique features in scleractinian gametogenesis, and this will eventually lead to comprehensive understanding of scleractinian gametogenesis.
The knowledge obtained in the present study will be useful for ecological studies and coral aquaculture. For instance, since scleractinian corals have no secondary sexual characteristics, histological analysis has traditionally been used to investigate polyp or colony sex, as well as to determine the status of germ cell development. However, histological analysis of scleractinians is time consuming. It generally requires decalcification steps, and the whole histological process sometimes takes 1-2 weeks. Identification of molecular markers for determining colony sex and germ cell development status offers a useful alternative process. Colony sex and germ-cell type could be determined faster using PCR with markers, than by histological means. Sex- and gonad phase-specific genes identified in this study would be candidates.