Immunodetection of acetylated alpha‐tubulin in stony corals: Evidence for the existence of flagella in coral male germ cells

Abstract

The molecular and cellular characteristics of male germ cell development remain largely unknown in corals. This study focused on the expression pattern of acetylated α-tubulin (Ac-α-Tu), which is involved in male germ cell development in various animals across taxa, to gain a better understanding of male germ cell development in the stony coral Euphyllia ancora. Immunohistochemical analysis of the different stages of male germ cells showed the presence of filamentous Ac-α-Tu in the early to late stages of male germ cells-such as spermatogonia, spermatocytes, and spermatids-as well as in the flagella of mature sperm. Immunocytochemical and transmission electron microscope analyses demonstrated that early-stage male germ cells possess long flagella containing Ac-α-Tu. The presence of filamentous Ac-α-Tu was also immunohistochemically demonstrated in the male germ cells from 14 other coral species, implying that possession of flagella with Ac-α-Tu is a common characteristic of male germ cells in stony corals. Therefore, as a distinctive cellular characteristic of male germ cells, Ac-α-Tu could be used as a male germ cell marker in stony corals; indeed, immunolabeling for Ac-α-Tu may be a useful method to aid in the identification and morphological observation of male germ cells in various corals in basic and applied biology (e.g., aquaculture) as well as in ecological studies.

Discovery of a receptor guanylate cyclase expressed in the sperm flagella of stony corals

“…Tissue fixation and immunohistochemistry were performed according to the methodologies described elsewhere 59 . Anti- E. ancora GC-A antibody, anti- E. ancora Piwi antibody (EaPiwi) 40 and anti-acetylated alpha-tubulin antibody (Ac-α-Tu; Santa Cruz Biotechnology, Santa Cruz, CA) 41 were diluted 1:10,000, 1:4,000 and 1:4,000, respectively, in phosphate buffered saline with 0.1% Tween-20 (PBT) containing 2% skim milk and used for primary antibody reactions. A biotinylated goat anti-guinea pig IgG (Vector Laboratories, Burlingame, CA) or anti-mouse IgG antibody (Anaspec, San Jose, CA) was diluted 1:4,000 in PBT with 2% skim milk and used for secondary antibody reactions.…”

“…E. ancora possesses relatively large polyps (3–5 cm in diameter) that allow us to isolate specific types of tissues (e.g., testis, ovary, or tentacle) from living polyps. Moreover, reliable molecular markers for germline cells have been identified, and their expression patterns in gametogenesis have been thoroughly characterized 39–41 . These attributes allow us to identify the tissue and cell types that have the expression of target gene products.…”

De novo transcriptome assembly from the gonads of a scleractinian coral, Euphyllia ancora: Molecular mechanisms underlying scleractinian gametogenesis

“…Morphological changes of male germ cells during spermiogenesis include agellum 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 agella from early to late stages of development [21]. Nevertheless, scleractinian male germ cells possess typical agellar axonemes, characterized by a"9 + 2" arrangement of microtubules [21,73].…”

“…Scleractinian spermiogenesis is generally morphologically similar to that of vertebrates, except that male germ cells possess long agella from early to late stages of development [21]. Nevertheless, scleractinian male germ cells possess typical agellar 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 literaturebased gene identi cation, allowed us to identify various candidate genes encoding proteins of agellar components.…”

“…For instance, hormonal induction of gametogenesis and spawning under arti cial rearing systems would allow more e cient propagation of target species [14]. Sex-and stage-speci c molecular markers for germ cells would also enable us to monitor and to evaluate the developmental status of germ cells in corals cultured in captivity [21]. Moreover, because scleractinians belong to the phylum Cnidaria (e.g., corals, sea anemones, hydras, and jelly sh), which are regarded as evolutionarily basal in the animal kingdom, studies highlighting common mechanisms of sexual reproduction between scleractinians and advanced animals (e.g., vertebrates) should provide insights into the evolution of sexual reproduction in metazoans [14].…”