Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis

Abstract

Many hermaphroditic organisms possess a self-incompatibility system to avoid self-fertilization. Recently, we identified the genes responsible for self-sterility in a hermaphroditic primitive chordate (ascidian), Ciona intestinalis: sperm-side polycystin 1-like receptors s-Themis-A/B and egg-side fibrinogen-like ligands on the vitelline coat (VC) v-Themis-A/B. Here, we investigated the sperm behavior and intracellular Ca 2+ concentration ([Ca 2+ ] i ) in response to self/ nonself-recognition. We found that sperm motility markedly decreased within 5 min after attachment to the VC of self-eggs but not after attachment to the VC of nonself-eggs and that the apparent decrease in sperm motility was suppressed in low Ca 2+ seawater. High-speed video analysis revealed that sperm detached from the self-VC or stopped motility within 5 min after binding to the self-VC. Because s-Themis-B contains a cation channel domain in its C terminus, we monitored sperm [Ca 2+ ] i by real-time [Ca 2+ ] i imaging using Fluo-8H-AM (AAT Bioquest, Inc.). Interestingly, we found that sperm [Ca 2+ ] i rapidly and dramatically increased and was maintained at a high level in the head and flagellar regions when sperm interacted with the self-VC but not when the sperm interacted with the nonself-VC. The increase in [Ca 2+ ] i was also suppressed by low-Ca 2+ seawater. These results indicate that the sperm self-recognition signal triggers [Ca 2+ ] i increase and/or Ca 2+ influx, which elicits a self-incompatibility response to reject selffertilization in C. intestinalis. Sexual reproduction is an essential process to elicit genetic diversity in the next generation. Therefore, many hermaphrodite species have acquired a self-incompatibility (SI) system that prevents inbreeding. In flowering plants, SI systems depend on S proteins, which are encoded in an SI specificity-determining locus (1). The invertebrate chordate Ciona intestinalis is a hermaphroditic animal that releases sperm and eggs nearly simultaneously and exhibits strict self-sterility. These features were reported by Tomas Hunt Morgan about a century ago (2). Rosati and De Santis (3) and Kawamura et al. (4) later clarified that a self/nonself-discrimination site resides on the vitelline coat (VC), an acellular matrix surrounding the egg, and that the VC shows higher affinity to allogeneic (nonself) sperm than to autologous (self) sperm (3, 4). Recently, we revealed that the SI system in C. intestinalis is controlled by two multiallelic independent loci, loci A and B. Each of the loci contains a pair of SI candidate genes, egg-side v-Themis-A/B and sperm-side sThemis-A/B. All four proteins show high polymorphisms among individuals. Comprehensive proteome analysis of the isolated VC revealed that both v-Themis-A and v-Themis-B exist in the VC (5, 6). Interestingly, v-Themis-A/B genes reside in the cDNA strand of the first intron of s-Themis-A/B genes, respectively, suggesting that these egg-and sperm-side SI factors would be hardly segregated by homologous recombinatio...

Allorecognition and Lysin Systems During Ascidian Fertilization

“…As s-Themis-B contains a cation-channel domain in its C terminus, we monitored ] i imaging using Fluo-8H-AM, a cell-permeable Ca 2+ indicator. Interestingly, we found that sperm [Ca 2+ ] i rapidly and dramatically increased and was maintained at a high level in the head and fl agellar regions when sperm interacted with the self-VC but not when the sperm interacted with the nonself VC (Saito et al 2012 ). The increase in [Ca 2+ ] i was also suppressed by low-Ca 2+ seawater (Saito et al 2012 ).…”

“…Interestingly, we found that sperm [Ca 2+ ] i rapidly and dramatically increased and was maintained at a high level in the head and fl agellar regions when sperm interacted with the self-VC but not when the sperm interacted with the nonself VC (Saito et al 2012 ). The increase in [Ca 2+ ] i was also suppressed by low-Ca 2+ seawater (Saito et al 2012 ). These results indicate that the sperm self-recognition signal triggers [Ca 2+ ] i increase or Ca 2+ infl ux, which induces an SI response to reject self-fertilization.…”

“…We found that sperm motility markedly decreased within 5 min after attachment to the VC of self-eggs but not after attachment to the VC of non-self eggs and that the apparent decrease in sperm motility was suppressed in low-Ca 2+ seawater (Saito et al 2012 ). It was also revealed that sperm detach from the self-VC or stop motility within 5 min after binding to the self-VC.…”

In silico identification of the genes for sperm-egg interaction in the internal fertilization of the newt Cynops pyrrhogaster

“…2) with stronger expression of a1C, H, and S in spermatogenic testes. Elongation factor (EF) 1 gene expression was detected at similar levels in both testes, and a spermatidspecific gene, protamine 1 (Yoshinobu et al, 1997) Darszon et al, 2006;Kirichok and Lishko, 2011), and sperm binding to the egg envelope (Saito et al, 2012). Spermatozoa possess multiple types of cation channels in the plasma membrane and Ca 2+ stores, which are critical for increasing cytoplasmic Ca 2+ in every step of sperm-egg interaction (Darszon et al, 2006;Marquez et al, 2007).…”

Structure, Function, and Phylogenetic Consideration of Calaxin

“…Sperm with this waveform show cane-shaped "quiescence," which is observed by electric or mechanical stimulation of sperm fl agella in the sea urchin (Shingyoji and Takahashi 1995 ;Kambara et al 2011 ). On the self-nonself recognition of sperm and egg in Ciona , sperm show quiescence with a straight fl agellum in response to the increase in intracellular Ca 2+ (Saito et al 2012 ). The basic regulation of fl agellar waveform by Ca 2+ is thought to be performed by the specifi c activation of dynein arms, which results in the changes in fl agellar waveforms (Brokaw 1979 ;Lindemann and Goltz 1988 ).…”