Oocytes express an endogenous red fluorescent protein in a stony coral, Euphyllia ancora: a potential involvement in coral oogenesis.

Stony corals are indispensable in building the framework of coral reefs, which possess the highest levels of marine biodiversity on earth1. Sexual reproduction of corals is among the most important means of establishing new coral colonies, populations, and hence coral reefs. In sexual reproduction, the gametes or larvae released into the ocean allow corals to colonize different habitats and create genetic links between one reef region and another2. Sexual reproduction also allows corals to foster genetic diversity, an advantage in adapting to environmental changes. Gametogenesis is the initiation of sexual reproduction and creates mature gametes that contain all the material to drive fertilization and embryonic development. To date, coral gametogenesis has been investigated in many species and locations worldwide mainly from ecological and histological perspectives3,4,5. The common characteristics of gametogenesis (e.g., processes, specific site of gametogenesis within the polyp, and the cycle of gametogenesis) among species have been revealed3,4,5. Generally, the annual spawning pattern of corals exhibits a single annual gametogenic cycle that is completed in less than 12 months3,4. Corals develop their gametes in a specific site of each endodermal mesentery3,4. Although cnidarians (e.g. corals, sea anemones, hydras, and jellyfish) are generally thought to possess no true organs, the specific site of gamete production is often referred to as a gonad (testis/ovary)4. Oogenesis begins with the proliferation of oogonia alongside the gonadal mesoglea, which is a gelatinous extracellular matrix. After the oogonia differentiate into early oocytes by entering meiosis, the oocytes enlarge in size and infiltrate into the gonadal mesoglea. Consequently, the oocytes are enveloped by a thin mesogleal layer in which they increase in size by accumulating yolk until they reach the maturation period3. On the other hand, spermatogenesis begins with spermatogonial proliferation alongside the gonadal mesoglea6,7. After spermatogonia form clusters, they infiltrate into the gonadal mesoglea and form spermaries that consist of dozens of spermatogonia surrounded by a thin layer of mesoglea. Spermatogonial proliferation, meiotic differentiation, and spermiogenesis take place within each spermary3,6,7. Recently, several studies on coral gametogenesis from the molecular and cellular perspectives have been performed, and molecules related to coral gametogenesis have been identified8,9,10,11,12. Furthermore, the research bases required for detailed studies, such as a genomic database13 and molecular markers for germ cells6,7, have gradually been developed. However, the current understanding of the mechanisms underlying coral gametogenesis is quite limited. Because of increasing human concerns about reef degradation, the significance of coral reef conservation and coral biology have become topics of general interest. Therefore, further investigation of coral reproduction at the molecular and cellular levels would provide valuable information not only for the field of comparative biology but also for applied biology, such as the artificial induction of gametogenesis and spawning under aquaculture conditions. A differential screen (suppression subtractive hybridization [SSH]) is one of the reliable methodologies to identify genes that are differentially expressed between two samples, such as between a non-reproductive sample and a reproductive sample14. Previously, we reported a study describing the SSH-based identification and characterization of a novel yolk protein cDNA, named euphy, in a gonochoric stony coral, Euphyllia ancora15. It was demonstrated that the euphy protein is synthesized by ovarian somatic cells, transported to, and abundantly accumulated in oocytes during oogenesis15. Through the course of our SSH screening to explore oogenesis-related genes, we identified several clones that were up-regulated during the reproductive period of the female E. ancora. Preliminary homology searches in GenBank using BLAST (NCBI) showed that one of the clones, #2-6, exhibited similarities to the green fluorescent proteins of other corals. Because to date, the biological relationship between fluorescent proteins (FPs) and oogenesis has been relatively unexplored in corals, this prompted us to perform the further characterization of the clone. To the best of our knowledge, there are only a few studies that describe the results of microscopic observation of FPs in the released eggs of two coral species, Pocillopora verrucosa16 and Montipora capitata17,18. Here, we present the molecular identification and functional characterization of a novel red fluorescent protein that is endogenously, and almost exclusively, expressed in the oocytes from an early stage of oogenesis in the adult E. ancora.