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

Abstract

To date,the molecular and cellular mechanisms underlying coral sexual reproduction remain largely unknown. We then performed a differential screen to identify genes related to oogenesis in the stony coral Euphyllia ancora. We identified a clone encoding a novel red fluorescent protein cDNA of E. ancora (named EaRFP). Microscopic observation and quantitative RT-PCR revealed that EaRFP is almost exclusively expressed in the ovary of the adult coral. The combination of the ovarian-cell separation method and the RT-PCR analysis revealed that the oocytes, but not the ovarian somatic cells, are the cells expressing EaRFP. Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period. Furthermore, recombinant EaRFP was shown to possess an H2O2 degradation activity. These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis. The present study provides not only the first evidence for the potential involvement of FPs in coral oogenesis but also an insight into a cellular strategy underlying coral sexual reproduction.

Testicular somatic cells in the stony coral Euphyllia ancora express an endogenous green fluorescent protein

“…This study revealed that GFP is endogenously expressed in the testis of adult E. ancora . Because we have previously found that the oocytes of E. ancora express an endogenous RFP (Shikina et al, ), we originally assumed that GFP would be expressed in the male germ cells. Unexpectedly, however, our expression analysis clearly demonstrated the expression of endogenous GFP in somatic cells, but not germ cells, in the testis.…”

“…The pcDNA3.1(+)‐EaGFP plasmid or empty pcDNA3.1(+) vector was transfected into human embryonic kidney 293 (HEK293) cells using Lipofectamine 3000 (Thermo Fisher Scientific). Transfection of the plasmid into the HEK293 cell line and maintenance of the HEK293 cells were carried out according to a previously described methodology (Shikina et al, ). GFP expression of the transfected cells was observed under a fluorescence microscope (IX71 equipped with U‐MWIB2, Olympus).…”

“…Previously, using suppression‐subtractive‐hybridization screening method, the authors of the present study identified a gene encoding red fluorescent protein (RFP) that is almost exclusively expressed in the ovary of reproductive female colonies (Shikina et al, ). Detailed expression analysis demonstrated that RFP is specifically expressed in oocytes but not in ovarian somatic cells.…”

“…For instance, the single gametogenic cycle requires approximately half a year in males and a year in females, and annual spawning occurs during a short period in April or May every year (Shikina et al, ). In addition, techniques for the isolation of different parts of polyp tissues (e.g., testis, ovary, tentacle, and mesenterial filament) from living corals have been established in this species of coral (Shikina et al, ), facilitating investigations of differences among different parts of polyp tissues at mRNA and protein levels (Shikina et al, ).…”

“…Detailed expression analysis demonstrated that RFP is specifically expressed in oocytes but not in ovarian somatic cells. In addition, a basic technique for the separation of oocytes and ovarian somatic cells has been established by using the endogenous expression of RFP as an indicator of oocytes (Shikina et al, ). In an effort to explore the possibility that testicular somatic cells and male germ cells can be separated using a similar methodology, we have recently performed fluorescence microscopic observation of E. ancora testis.…”