Comprehensive Egg Coat Proteome of the Ascidian Ciona intestinalis Reveals Gamete Recognition Molecules Involved in Self-sterility

Abstract

Despite central roles of egg coat proteins in gamete recognition, their functions and composition are poorly understood. Here, we report that the proteome of the egg coat in the solitary ascidian Ciona intestinalis, called vitelline coat (VC) fraction, contains more than 800 proteins identified by mass spectrometry-based analyses. Over 100 proteins were enriched in the VC fraction compared with the VC-free egg proteome. The most abundant component in the VC was an apolipoprotein-like protein. The VC contained multiple homologs of mammalian zona pellucida (ZP) proteins, the number of which was unexpectedly large and most of which possessed epidermal growth factor-like repeats. Furthermore, the present study revealed that two fibrinogen-like proteins, v-Themis-A and -B, both of which are expressed in the VC, are the molecules responsible for the two self-sterility loci that were identified by our previous genetic study in this species.Successful fertilization is a crucial process for sexual reproduction in living organisms. One of the key events in fertilization is a sperm-egg recognition step that takes place on the egg coat, an extracellular matrix of the egg. The egg coat provides both an adhesive scaffold and a formidable barrier to sperm so that only a single, congeneric and compatible sperm can penetrate through it. This recognition process mostly depends on a set of finely prearranged molecules that reside on the egg coat and sperm plasma membrane. Therefore comprehensive identification and characterization of such biomolecules are very important to understand the molecular mechanism of gamete recognition. Although several proteomic analyses have been performed on the sperm proteins in various organisms (1-3), proteomic studies on the egg coat proteins have been scarcely conducted because of technical difficulties in the sample preparation.A cosmopolitan ascidian, Ciona intestinalis, provides an ideal animal system for fertilization studies. The ascidian is a closest relative of vertebrates. Its egg coat is called vitelline coat (VC).4 C. intestinalis undergoes external fertilization. Compared with mammalian eggs, a large quantity of readily fertilizable eggs can be easily obtained. Moreover, draft genome sequences and a large set of transcriptomic data are available (4, 5).Another interesting feature of the C. intestinalis VC is its allorecognizable ability. Nearly all ascdians are hermaphroditic, releasing both sperm and eggs nearly simultaneously, but many species, including C. intestinalis, exhibit self-sterility. It is shown that sperm fertility is closely related to its capability to bind to the VC: self-sperm do not bind as firmly to the VC as nonself-sperm (6). This self/nonself-discriminating activity is obvious even between isolated (dead) VC and intact sperm, indicating that the VC contains female-side self-sterility factors.In this report, we identified more than 800 proteins in the VC fraction of C. intestinalis by mass spectrometry (MS)-based proteomic analyses. Over 100 proteins were enriched...

The Primary Role of Fibrinogen-Related Proteins in Invertebrates Is Defense, Not Coagulation

“…In a proteomic analysis of C. intestinalis egg coat, 2 FBG-like molecules were identified, v-Themis-A and -B [43] . Analysis of the v-Themis-A and -B proteins showed that they were polymorphic proteins that possessed a C-terminal FBG domain.…”

A Surface Glycoprotein Indispensable for Gamete Fusion in the Social Amoeba Dictyostelium discoideum

“…The crude membrane fraction was obtained by the repeated freeze-thawing of the cells (34) and dissolved in the sample loading buffer for SDS-PAGE. Proteins were separated in a 10 to 20% gradient gel, size fractionated by horizontally slicing the gel, and then digested with trypsin for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis as described previously (42). The digested peptides were analyzed using a capillary liquid chromatography system (Ultima3000; Dionex, CA) connected online to a mass spectrometer (LTQ-XL; Thermo Scientific, MA).…”

Allorecognition and Lysin Systems During Ascidian Fertilization

“…For example, it has been reported that the terminal fucose residue on VC glycoproteins (Rosati and De Santis 1980 ) and sperm-side fucosidase may make an enzyme-substrate complex, allowing interaction between sperm and the VC of eggs (Hoshi 1986 ;Matsumoto et al 2002 ). On the other hand, we reported that sperm-side CiUrabin, a GPI-anchored CRISP family protein located at the sperm head and tail, is capable of binding to CiVC57, an EGF-like-repeatcontaining major glycoprotein on the VC, which can support the interaction between sperm and the VC of eggs (Yamaguchi et al 2011 ;Yamada et al 2009 ).…”