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Ascidians (primitive chordates) are hermaphroditic animals that release spermatozoa and eggs almost simultaneously, but some species, including Halocynthia roretzi, show strict self-sterility. In H. roretzi, a 70-kDa vitelline coat (VC) protein consisting of 12 EGF-like repeats, named HrVC70, appears to be a promising candidate for the self/nonself-recognition (or allorecognition) system during gamete interaction. After spermatozoon recognizes the VC as nonself, sperm 700-kDa extracellular ubiquitin-conjugating enzyme complex appears to ubiquitinate Lys234 of HrVC70, and the ubiquitinated HrVC70 is degraded by the sperm 26S proteasome that is located on the sperm head surface. This novel ubiquitin-proteasome system enables spermatozoa to penetrate through the VC. Sperm trypsin-like proteases, acrosin and spermosin, also participate in fertilization, probably as sperm-side 'movable' binding proteins to the VC.

Fertilization and gametogenesis are key events in sexual reproduction. Our recent studies, together with several reports by other authors, demonstrated that the extracellular ubiquitin-proteasome system plays a role in fertilization and gametogenesis in addition to the traditional intracellular ubiquitin-proteasome system. Here, we summarize our recent results showing the importance of the extracellular ubiquitin-proteasome system in the sperm penetration through the vitelline coat of the egg during ascidian fertilization, together with our recent reports implicating the participation of a novel proteasome-associating complex PC530 in starfish oocyte maturation. We also describe the results by other authors showing the participation of the ubiquitin system both in the elimination of defective sperm in epididymis and in the elimination of paternal mitochondria in fertilized eggs. These are evidence of non-traditional extracellular functions of the ubiquitin system.

The ubiquitin-proteasome system is essential for intracellular protein degradation, but there are few studies of this system in the extracellular milieu. Recently, we reported that a 70-kDa sperm receptor, HrVC70, on the vitelline coat is ubiquitinated and then degraded by the sperm proteasome during fertilization of the ascidian, Halocynthia roretzi. Here, we investigated the mechanism of extracellular ubiquitination. The HrVC70-ubiquitinating enzyme activity was found to be released from the activated sperm during the fertilization process. This enzyme was purified from an activated sperm exudate, by chromatography on DEAE-cellulose and ubiquitin-agarose columns, and by glycerol density gradient centrifugation. The molecular mass of the enzyme was estimated to be 700 kDa. The purified enzyme requires CaCl2 and MgATP for activity, and is active in seawater. The purified enzyme preparation, but not the crude enzyme preparation, showed narrow substrate specificity to HrVC70. Moreover, ATP and ubiquitin are released from the activated sperm to the surrounding seawater during fertilization. These results indicate that ascidian sperm release a novel extracellular ubiquitinating enzyme system together with ATP and ubiquitin during penetration of the vitelline coat of the egg, which catalyzes the ubiquitination of the HrVC70, an essential component of ascidian fertilization.

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.

The extracellular ubiquitin–proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the "sperm reaction". However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi. [ABSTRACT FROM AUTHOR]

Fertilization is an essential process in terrestrial organisms for creating a new organism with genetic diversity. Before gamete fusion, several steps are required to achieve successful fertilization. Animal spermatozoa are first activated and attracted to the eggs by egg-derived chemoattractants. During the sperm passage of the egg's extracellular matrix or upon the sperm binding to the proteinaceous egg coat, the sperm undergoes an acrosome reaction, an exocytosis of acrosome. In hermaphrodites such as ascidians, the self/nonself recognition process occurs when the sperm binds to the egg coat. The activated or acrosome-reacted spermatozoa penetrate through the proteinaceous egg coat. The extracellular ubiquitin–proteasome system, the astacin-like metalloproteases, and the trypsin-like proteases play key roles in this process in ascidians. In the present review, we summarize our current understanding and perspectives on gamete recognition and egg coat lysins in ascidians and consider the general mechanisms of fertilization in animals and plants. [ABSTRACT FROM AUTHOR]

Levels of intracellular multi-ubiquitinated proteins in heat-shocked HeLa cells were investigated using a monoclonal antibody specific to multi-ubiquitin chains. After heat-shock treatment at 42-44°C for 30 min, the level of multi-ubiquitinated, proteins increased within the first 2 h at 37 °C and returned to the initial level within the following 2 h. The accumulation of multi-ubiquitin conjugates was elevated by increasing the temperature, while the opposite was the case for the level of ubiquitinated histone H2A. Immunocytochemical analysis revealed that the amount of ubiquitin conjugates rapidly increased in the cytosol and concomitantly decreased in the nucleus under heat-shock conditions. The heat-shock treatment elicited little apparent change in the activity of the 26S proteasome, but it did induce a gradual increase in activity of the ubiquitinating enzyme system. These results strongly suggest that the level of cytoplasmic multi-ubiquitinated proteins and that of nuclear ubiquitinated histone H2A increases and decreases, respectively, in response to heat shock and that the heat-shock-induced accumulation of multiubiquitinated proteins is caused by activation of the ubiquitinating enzyme system rather than inactivation of the 26S proteasome. [ABSTRACT FROM AUTHOR]

A sandwich ELISA has been developed to measure intracellular levels of multi-ubiquitin chains. The mixture of multi-ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus 125I-ubiquitin) and lysozyme with ubiquitin-ligating enzymes and ATP, was partially purified and established as standard named the multi-ubiquitin-chain reference preparation 1 (MUCRP1). The concentration of MUCRP1 was calculated from the recovered radioactivity of 125I-ubiquitin. All measurements by the ELISA were expressed in terms of MUCRP1. The ELISA showed good sensitivity (98 pg/ml), precision (intra-assays <6%) and reproducibility (interassay <9%). In addition, there was no substantial cross-reaction with mono-, di- and tri-ubiquitin, of mono-ubiquitinated and di-ubiquitinated lysozyme in the ELISA, and large multi-ubiquitin chains (n approximately 6) may be fully reactive. These results combined with excellent results in the recovery and dilution test guarantee accurate measurement of multi-ubiquitin chains in all extracts prepared with a lysis buffer (water soluble) of the buffer supplemented 8M urea (urea soluble). The level of the water-soluble multi-ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea soluble chain level was higher in the reticulocytes. Heat-sock treatment of HeLa cells increased the urea-soluble multi-ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi-ubiquitin-conjugate turnover. [ABSTRACT FROM AUTHOR]

Abstract: We previously reported that immature starfish oocytes contain a novel 530-kDa proteasome-associating complex PC500 [previously named PC530; E. Tanaka, M. Takagi Sawada, C. Morinaga, H. Yokosawa, H. Sawada, Isolation and characterization of a novel 530-kDa protein complex (PC 530) capable of associating with the 20S proteasome from star fish oocytes, Arch. Biochem. Biophys. 374 (2000) 181–188]. In the present study, in order to obtain an insight into the biological function of this complex, we investigated the effects of anti-PC500 monoclonal antibodies on oocyte maturation of the starfish Asterina pectinifera. A monoclonal antibody 7C5 strongly inhibited germinal vesicle breakdown (GVBD) in a concentration-dependent manner. In contrast to the inhibitory effect of the 7C5 antibody on GVBD, no inhibition of egg cleavage was observed in a 7C5-antibody-microinjected single blastomere in a 2-cell stage embryo. These results indicate that PC500 plays a key role in starfish oocyte maturation in a meiosis-specific manner. [Copyright &y& Elsevier]