Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor

Abstract

The ubiquitin-proteasome system is essential for intracellular protein degradation, but an extracellular role of this system has not been known until now. We have previously reported that the proteasome is secreted into the surrounding seawater from sperm of the ascidian (Urochordata) Halocynthia roretzi on sperm activation, and that the sperm proteasome plays a key role in fertilization. Here, we show that a 70-kDa component (HrVC70) of the vitelline coat is the physiological substrate for the ubiquitin-proteasome system during fertilization of H. roretzi. A cDNA clone encoding the HrVC70 precursor (HrVC120) was isolated, and a homology search revealed that HrVC120 contains 13 epidermal growth factor-like repeats and a mammalian zona pellucida glycoprotein-homologous domain. HrVC70 functions as a sperm receptor. We demonstrate that HrVC70 is ubiquitinated both in vitro and in vivo. The immunocytochemical localization of multiubiquitin chains in the vitelline coat and the inhibitory effect of monoclonal antibodies against the multiubiquitin chains on fertilization strongly support the role of the ubiquitin-proteasome system in ascidian fertilization. Taken together, these results indicate that the ubiquitin-proteasome system is responsible for extracellular degradation of the sperm receptor HrVC70 and, consequently, for sperm penetration of the vitelline coat during fertilization.fertilization ͉ vitelline coat ͉ ubiquitin F ertilization is a key event in sexual reproduction, creating a new individual with novel genomic information. In animal reproduction, species-specific binding of sperm to the proteinaceous egg coat, called the vitelline coat in marine invertebrates or the zona pellucida in mammals, is particularly important for successful fertilization. Because the egg coat is a potential barrier to sperm-egg fusion, sperm must use a lytic agent (lysin) to penetrate it (1, 2). In mammals, it has long been believed that the sperm acrosomal trypsin-like protease acrosin is a zona lysin, which digests zona pellucida proteins to enable sperm to penetrate through the zona pellucida (3). However, recent studies by using acrosin-knockout mice revealed that acrosin is not essential for fertilization or sperm penetration through the zona pellucida (4). Rather, it is currently thought that acrosin is involved in the dispersal of acrosomal proteins during acrosome reaction (5) and that a sperm protease(s) other than acrosin is the actual zona-lysin in mammalian fertilization (6).Ascidians (Urochordata) occupy a phylogenetic position between invertebrates and segmented vertebrates. They are hermaphrodites that usually release sperm and eggs simultaneously during the spawning season. Several ascidians, including Ciona intestinalis (7) and Halocynthia roretzi (8), strictly prohibit selffertilization. Because self-nonself recognition in fertilization is accomplished by interaction between the sperm and vitelline coat (7,8), the sperm-lysin system seems to be triggered after the sperm recognizes the vitelline coat as nonself...

Sperm-Surface ATP in Boar Spermatozoa is Required for Fertilization: Relevance to Sperm Proteasomal Function

“…The proteasome facilitates sperm binding and penetration through the vitelline coats of the eggs of a solitary ascidian (Halocynthia roretzi) [Sawada et al 1983[Sawada et al , 2002aSaitoh et al 1993]. The sperm proteasomes in H. roretzi are localized on the plasma membrane surface of the sperm head [Sawada et al 2002b].…”

Sperm Proteasome as a Putative Egg Coat Lysin in Mammals

“…After self/non-self recognition via interactions between the sperm and VC, the sperm-borne egg coat lysin system is activated (Sawada et al 2002a ). Data from Sawada's laboratory have provided evidence that VC penetration by ascidian sperm involves the sequential ubiquitination and proteasomal degradation of the sperm receptor protein HrVC70, a major protein component of the ascidian VC.…”

“…Studies in two ascidian species, Ciona intestinalis and Halocynthia roretzi , have shown a strict self-sterility in fertilization (Sawada et al 2002a ). After self/non-self recognition via interactions between the sperm and VC, the sperm-borne egg coat lysin system is activated (Sawada et al 2002a ).…”

“…Data from Sawada's laboratory have provided evidence that VC penetration by ascidian sperm involves the sequential ubiquitination and proteasomal degradation of the sperm receptor protein HrVC70, a major protein component of the ascidian VC. Ubiquitination of HrVC70 is accomplished by a 700-kDa ubiquitin-conjugating enzyme complex released during sperm activation (Sawada et al 2002a ;Sakai et al 2003 ). A 930-kDa proteasome (26S-like proteasome) seems to function as the egg coat lysin that directly degrades the VC.…”

“…The HrVC70 is a 70-kDa component of the VC shown to be a novel sperm receptor that bears signifi cant similarity to components of the mammalian zona pellucida, particularly the proposed mammalian/murine sperm receptor ZPC (Sawada et al 2002a ). Three N-terminal cystine/Cys residues in the ZP domain of the 120-kDa HrVC70 precursor (HrVC120) share conserved positions within mammalian and frog ZPC (Sawada et al 2002a ).…”