Cytological Characterization of Self Incompatibility in Gametes of the Ascidian, Ciona intestinalis

Abstract

We have determined how many elements are involved in the regulation of self-fertilization in the solitary ascidian, Ciona intestinalis that is an incompletely self-sterile species. Animals collected in the field were repeatedly induced to spawn in order to examine their selfing ratios. About 20% of them were self-fertile, although the ratios fluctuated considerably among respective spawnings. Naturally or acid-induced self-fertile gametes required much longer time for selfing than that for crossing. Egg-suspending seawater (egg water) as such activated sperm motility, but it lowered conspicuously the self-fertilization ratio. Self-sterile spermatozoa could scarcely bind to the vitelline coat (VC) of glycerinated autologous eggs, or in case they bound well to it the sperm flagella ceased to beat within five min of the 'insemination'. The staining of sectioned gametes with DAPI, a fluorescent dye of DNA, showed that in selfing the spermatozoa could hardly penetrate the VC even though they bound well to it. The results of this study show that the block of self-fertilization can be classified into four elements from a phenomenal viewpoint, such as egg water, low affinity of sperm-VC binding, inactivation of bound sperm and difficulty in sperm penetration through the VC.

Self/nonself recognition in ascidian fertilization: Vitelline coat protein HrVC70 is a candidate allorecognition molecule

“…Many ascidian species, including Ciona intestinalis (1)(2)(3)(4)(5)(6) and Halocynthia roretzi (7), are self-sterile because of the occurrence of a self-incompatibility system, or an allogeneic recognition system, in the interaction between sperm and vitelline coat. Although several candidate molecules involved in this process have been proposed (8,9), the mechanism of this recognition in ascidian fertilization is still unclear.…”

Concanavalin A Modifies Allo‐specific Sperm‐Egg Interactions in the Ascidian, Ciona intestinalis

“…The low affinity of sperm-VC binding is one of the most important features of self incompatibility in the gametes of C. intestinalis (13,20). In this work, ConA accelerated sperm-VC binding in autologous as well as non-autologous .insemination when the follicle cells (FCs) were removed from eggs.…”

“…The time required for the fertilization of lectin-treated eggs was determined by SDS (sodium dodecyl sulphate) method, as described previously (13). In brief, the sperm-egg suspension was treated with 0.004% SDS seawater (final conc.)…”

“…The spawned eggs were washed thoroughly with filtered seawater (FSW) to remove autologous spermatozoa. The follicle cells, accessory cells attached to the outer surface of the vitelline coat (VC), were removed from eggs by gentle pipetting or serial glycerination of eggs (13,20). The spermatozoa were taken directly from the spermiduct.…”

“…As pointed out by Rosati and De Santis (20), one of the most prominent cytological features of self incompatibility is that the spermatozoa rarely bind to the vitelline coat of autologous eggs. Another important feature is the blockage of penetration of sperm through the vitelline coat (13). In an aberrant self-fertile animal of C. intestinalis, the spermatozoa had a high affinity for binding to the autologous vitelline coat.…”