Proteomics of ionomycin‐induced ascidian sperm reaction: Released and exposed sperm proteins in the ascidian Ciona intestinalis

Abstract

Sperm proteins mediating sperm-egg interaction should be exhibited on the sperm surface, or exposed or released when sperm approach an egg. In ascidians (protochordates), sperm undergo a sperm reaction, characterized by enhanced sperm motility and mitochondrial swelling and shedding on contact with the vitelline coat (VC) or by treatment with Ca(2+) ionophore. Here, proteomic analysis was conducted on sperm exudates and sperm surface proteins using ionomycin-induced sperm reaction and cell-impermeable labeling in Ciona intestinalis type A (C. robusta). In the exudate from sperm treated with ionomycin, membrane proteins including a possible VC receptor CiUrabin were abundant, indicating the release of membranous compartments during sperm reaction. Among the surface proteins XP_009859314.1 (uncharacterized protein exhibiting homology to HrTTSP-1) was most abundant before the sperm reaction, but XP_004227079.1 (unknown Ig superfamily protein) appears to be most abundantly exposed by the sperm reaction. Moreover, proteins containing a notable set of domains, astacin-like metalloprotease domain and thrombospondin type 1 repeat(s), were found in this fraction. Possible roles in fertilization as well as localizations and behaviors of these proteins are discussed.

Peanut agglutinin specifically binds to a sperm region between the nucleus and mitochondria in tunicates and sea urchins

“…We previously reported (Nakazawa, Shirae‐Kurabayashi, Otsuka, & Sawada, ) that a PNA‐Alexa Fluor conjugate labels an intracellular region alongside the nucleus rather than the acrosome in sperm of the ascidian Ciona robusta (Hoshino & Tokioka, ) (formerly referred to as C. intestinalis type A; see [Brunetti et al, ; Pennati et al, ] for details), in contrast to our expectation that it would detect the acrosome. The existence of a novel PNA‐binding region in C. robusta suggests that glycans similar to those present in the mammalian acrosome are also accumulated in this sperm region.…”

“…Exposure of the PNA‐binding region of C. robusta sperm following ionomycin treatment (Nakazawa et al, ) was not observed under our experimental conditions. This was puzzling given that the phenomenon was observed at least twice in our previous experiments (e.g., Figure d).…”

“…We previously reported that the PNA‐binding region is exposed by 10 μM ionomycin in C. robusta sperm (Nakazawa et al, ). However, the PNA‐binding region seems unlikely to be exposed by ionomycin because no morphological change around the mitochondrial region during the acrosome reaction has been reported by electron microscopic observation (Fukumoto, ).…”

“…For comparative permeabilization (as shown in Figure b), sperm were treated with 0.1% Tween 20 (typical for cytochemistry), 0.5% Triton X–100 (following (Nakazawa et al, ), 0.1% SDS (some sperm structures are visualized well after treatment with SDS; (Haraguchi et al, ), and 100% methanol (a typical condition for staining mammalian sperm with PNA; (Lybaert et al, ).…”

A Brief Description of Surface Structure and Composition of the Pseudo-Snail Shell Formed by a Sea Anemone Stylobates sp. Symbiotic with Hermit Crabs from the Deep-Sea Floor

“…CBB-stained protein bands were excised from the polyacrylamide gel and subjected to in-gel digestion, LC/MS, and a database search, as described previously (Nakazawa et al, 2015). Briefly, samples were reduced with dithiothreitol and carbamidometh-ylated and carbamidomethylated, digested with modified trypsin (V511A; Promega, Madison, WI, USA), separated in an octadecyl silica gel column over a gradient of acetonitrile containing 0.1% formic acid, and analyzed with a mass spectrometer (LTQ-XL; Thermo Fisher Scientific) equipped with an electrospray ionization unit.…”