Oviductal protease and trypsin treatment enhance sperm–envelope interaction inBufo arenarum coelomic eggs

Abstract

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro.

Anuran and pig egg zona pellucida glycoproteins in fertilization and early development

“…It has been observed that pancreatic trypsin, a general protease with peptide bond specificity for Arg and Lys residues, mimicked the action of oviductin on coelomic eggs. Trypsin and clostripain specifically hydrolyzed only ZPC preglycoprotein and rendered coelomic eggs fertilizable (Hardy and Hedrick, 1992;Llanos et al, 2006). The suggestion was made that oviductin processing of ZPC occurs at both the Nand C-terminal ends of the glycoproteins, as the Gly-Ser-Arg cleavage site for specific hydrolysis is present at both Arg 61 and Arg 373 (Kubo et al, 1999).…”

Cloning and sequence analysis of Bufo arenarum oviductin cDNA and detection of its orthologous gene expression in the mouse female reproductive tract

“…Hydrolysis of gp40–52 by oviductin in Bufo japonicus results in the concomitant increase in gp39 and appearance of gp36 (Takamune & Katagiri, 1987). PR protease activity in B. arenarum is characterized by the disappearance of two glycoproteins, gp84 and gp55, and by the relative increase in concentration of gp39 and gp42 (Llanos et al ., 2006).…”

“…In all the species studied, proteolytic activity of oviductin is thought to produce rearrangement of certain envelope components (Mariano et al ., 1984; Larabell et al ., 1989; Hedrick & Nishihara, 1991) as well as uncover sperm binding domains on the egg envelope surface (Miceli et al ., 1980; Gerton & Hedrick, 1986; Takamune et al ., 1986; Bakos et al ., 1990). Sperm–envelope binding assays showed that coelomic envelopes treated with purified oviductin exhibited a dramatic increase in the level of sperm binding (Hardy & Hedrick, 1992; Llanos et al ., 2006).…”

“…Bearing in mind the taxonomic proximity between both species, it is likely that gp39 from B. arenarum is similar to the 39 kDa glycoprotein from B. japonicus (Llanos et al ., 2006), which has been reported to be involved in sperm adhesion (Omata & Katagiri, 1996). Proteolysis of gp43 in X. laevis seems to be the only factor responsible for the increase in sperm binding.…”

Homeostasis and secretion of calcium in the oviductal mucosa of toad Rhinella arenarum

“…The first, synthesized and secreted by the epithelial secretory cells (ESC) of the PR, is an aqueous product that contains an enzyme called oviductin (Miceli and Fernández, ; Miceli et al, ; Hardy and Hedrick, ). This enzyme is a Ca 2+ ‐dependent trypsin‐like serine protease that, through mild proteolysis, modifies the oocyte vitelline envelope (VE) at the structural and molecular levels, rendering it susceptible to sperm lysin and penetrable by sperm (Miceli and Fernández, ; Bakos et al, ; Llanos et al, ; Barrera et al, ).…”