The Sperm-surface glycoprotein, SGP, is necessary for fertilization in the frog, Xenopus laevis.

To identify a molecule involved in sperm-egg plasma membrane binding at fertilization, a monoclonal antibody against a sperm-surface glycoprotein (SGP) was obtained by immunizing mice with a sperm membrane fraction of the frog, Xenopus laevis, followed by screening of the culture supernatants based on their inhibitory activity against fertilization. The fertilization of both jellied and denuded eggs was effectively inhibited by pretreatment of sperm with intact anti-SGP antibody as well as its Fab fragment, indicating that the antibody recognizes a molecule on the sperm's surface that is necessary for fertilization. On Western blots, the anti-SGP antibody recognized large molecules, with molecular masses of 65-150 kDa and minor smaller molecules with masses of 20-28 kDa in the sperm membrane vesicles. SGP was distributed over nearly the entire surface of the sperm, probably as an integral membrane protein in close association with microfilaments. More membrane vesicles containing SGP bound to the surface were found in the animal hemisphere compared with the vegetal hemisphere in unfertilized eggs, but the vesicle-binding was not observed in fertilized eggs. These results indicate that SGP mediates sperm-egg membrane binding and is responsible for the establishment of fertilization in Xenopus.

Analysis of terminal sugar moieties and species-specificities of acrosome reaction-inducing substance in Xenopus (ARISX).

The acrosome reaction of Xenopus sperm is triggered by the acrosome reaction-inducing substance in Xenopus (ARISX), an oviductal pars recta-derived, sugar-rich substance decorated on the entire surface of the vitelline envelope (VE) during ovulation. Here we addressed the functional importance of the sugar moiety in ARISX. Among various lectins examined, soybean agglutinin and Dolichos biflorus agglutinin were shown to abolish the acrosome reaction-inducing activity of ARISX present in pars recta extract or on the VE, indicating the importance of the terminal alpha-N-acetylgalactosamine residue for the function of ARISX. Consistently, the acrosome reaction-inducing activity was not affected by proteinase K digestion, in spite of the simultaneous shift of ARISX to a smaller molecular weight. Indirect immunofluorescence microscopic examinations showed that ARISX was distributed as two types of structures on VE; thick fiber-like materials and thin filamentous materials, and that a new structure appeared on the fertilization envelope instead of the thin filamentous materials. Sperm from several amphibian species were subjected to an in vitro assay during induction of the acrosome reaction with ARISX. The resulting limited population of sperm from a non-Xenopus species underwent acrosome reaction, implying a weak species-specificity of ARISX.

Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization.

In a previous study, we identified Xenopus egg uroplakin III (xUPIII), a single-transmembrane protein that localized to lipid/membrane rafts and was tyrosine-phosphorylated upon fertilization. An antibody against the xUPIII extracellular domain abolishes fertilization, suggesting that xUPIII acts not only as tyrosine kinase substrate but also as a receptor for sperm. Previously, it has been shown that the protease cathepsin B can promote a transient Ca2+ release and egg activation as seen in fertilized eggs (Mizote, A., Okamoto, S., Iwao, Y., 1999. Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization. Dev. Biol. 208, 79-92). Here, we show that activation of Xenopus eggs by cathepsin B is accompanied by tyrosine phosphorylation of egg-raft-associated Src, phospholipase Cgamma, and xUPIII. Cathepsin B also promotes a partial digestion of xUPIII both in vitro and in vivo. A synthetic xUPIII-GRR peptide, which contains a potential proteolytic site, inhibits the cathepsin-B-mediated proteolysis and tyrosine phosphorylation of xUPIII and egg activation. Importantly, this peptide also inhibits sperm-induced tyrosine phosphorylation of xUPIII and egg activation. Protease activity that digests xUPIII in an xUPIII-GRR peptide-sensitive manner is present in Xenopus sperm. Several protease inhibitors, which have been identified to be inhibitory toward Xenopus fertilization, are shown to inhibit sperm-induced tyrosine phosphorylation of xUPIII. Uroplakin Ib, a tetraspanin UP member, is found to be associated with xUPIII in egg rafts. Our results highlight novel mechanisms of fertilization signaling by which xUPIII serves as a potential target for sperm protease essential for fertilization.

Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization.

Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.

Characterization of the acrosome reaction-inducing substance in Xenopus (ARISX) secreted from the oviductal pars recta onto the vitelline envelope.

We previously demonstrated that Xenopus sperm undergo an acrosome reaction on the vitelline envelope (VE) in response to the materials secreted from the oviductal pars recta [Dev. Biol. 243 (2002), 55]. A monoclonal antibody against the acrosome reaction-inducing substance in Xenopus (ARISX) was obtained by immunizing mice with pars recta extract (PRE). The acrosome reaction by PRE or on the VE was effectively inhibited by the intact anti-ARISX antibody as well as its Fab fragment, indicating that the antibody recognizes the epitopes localized on the acrosome reaction-inducing substance. On Western blots, the anti-ARISX antibody recognized a molecule with an apparent molecular mass of 300 kDa in PRE and in the VE, but this molecule was not detected in the coelomic envelope. The amount of ARISX in PRE was increased by the treatment of females with pregnant mare serum gonadotropin. Periodate oxidation of PRE completely abolished the acrosome reaction-inducing activity, indicating the involvement of the carbohydrate moieties of ARISX in inducing the acrosome reaction. On immunofluorescence observation, ARISX was localized in the epithelial cells in the posterior region of the pars recta and on the VE as fibrous structures.

Acrosome reaction in sperm of the frog, Xenopus laevis: its detection and induction by oviductal pars recta secretion.

Previous electron microscopic observations have shown that the acrosome of the sperm of the frog, Xenopus laevis, comprises a membrane-bounded vesicle covering the anterior-most position of the head. We obtained a sperm suspension from the testes and stained it with LysoSensor Green for observation under a confocal laser scanning microscope and found a bright fluorescence reflecting the presence of the acrosomes at the top of the sperm head in about 64% of the sperm, with no deterioration of their capacity to fertilize. About 40% of the sperm with an acrosome underwent an acrosome reaction in response to Ca(2+) ionophore A23187, as evidenced by a loss of LysoSensor Green stainability, accompanied by breakdown of the acrosomal vesicle. About 53% of the sperm bound to isolated vitelline envelopes underwent an acrosome reaction, whereas both jelly water and solubilized vitelline envelopes weakly induced an acrosome reaction. When the sperm were treated with an oviductal extract obtained from the pars recta, but not the pars convoluta region, about 40% of the sperm with acrosomes underwent an acrosome reaction. The substance containing acrosome reaction-inducing activity in the pars recta extract seemed to be a heat-unstable substance with a molecular weight of greater than 10 kDa. The activity was not inhibited by protease inhibitors but required extracellular Ca(2+) ions. These results indicate that the acrosome reaction occurs on the vitelline envelopes in response to the substance deposited from the pars recta during the passage of the oocytes through the oviduct.

Clinical trial of in-situ hybridization method for the rapid diagnosis of sepsis.

We evaluated the utility of in-situ hybridization (ISH) for the rapid diagnosis of sepsis. We applied this approach to polymorphonuclear neutrophil (PMN)-rich smears from patients with suspected bacterial infection. Positive results by ISH were obtained in the smears of 123 of 292 patients (42%), while only 32 of the 292 (11%) were positive by blood culture. These findings indicate that ISH is almost four times more sensitive than the culture method for the detection of sepsis. ISH results are obtained within 1 day, while 1 day to 2 weeks is required for the results of blood culture. Blood culture and ISH methods detected the same bacteria in two patients. ISH also successfully identified the same bacteria in blood and PMN-rich body fluid (bronchoalveolar lavage samples) in 6 patients. In 19 patients, ISH of blood detected the same bacteria as those found in subcultures from other sources (e.g., stool, sputum, nasal cavity). We discuss these results in comparison with blood culture results in terms of evaluating a rapid approach to the management of patients with sepsis.