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The biochemical and ultrastructural changes in the envelope of the Xenopus laevis egg that occur during oviposition and fertilization have been thoroughly studied (Hedrick, J. L., and Nishihara, D. M., Methods Cell Biol. 36, 231-247, 1991; Larabell, C. A., and Chandler, D. E., J. Electron Microsc. Tech. 17, 294-318, 1991). However, the biological significance of these changes with respect to gamete interaction has been unclear. In the current study, it was found that changes in the envelope are directly responsible for regulating sperm-egg adhesion, an initial step of fertilization. As a result of these transformations, sperm bind only to unfertilized oviposited eggs, not to oocytes or coelomic eggs. In addition, they do not bind to fertilized eggs. The molecular and cellular basis of the regulation of the sperm binding process was investigated in the context of our recent findings that two structurally related envelope glycoproteins, gp69/64, serve as sperm receptors during fertilization (Tian, J.-D., Gong, H., Thomsen, G. H., and Lennarz, W. J., J. Cell Biol. 136, 1099-1108, 1997). Although the purified gp69/64 glycoproteins isolated from the oocyte or coelomic egg envelopes exhibited sperm binding activity, when these proteins are part of the intact oocyte or coelomic egg envelopes, they are not accessible to either anti-gp69/64 antibodies or to sperm. During the conversion from the coelomic to the vitelline envelope, the gp69/64sperm receptors become exposed on the surface, an event that correlates with proteolytic cleavage of gp43 and accompanying ultrastructural alterations in the envelope. Conversely, after fertilization, when the vitelline envelope of the egg is converted to the fertilization envelope of the zygote, limited proteolytic cleavage of the sperm receptor results in loss of sperm binding activity. In addition, formation of a fertilization layer on top of the structurally altered VE adds another physical block to sperm binding. These results provide new insights into structure-function relationships between envelope components of the anuran egg, and provide further evidence supporting the key role of gp69/64 as sperm receptors during X. laevis fertilization.
FIG. 1. Measurement of the number of sperm bound to one hemi-sphere of an oocyte, coelomic egg, dejellied oviposited egg, or dejel-lied fertilized egg (0.5 hr after fertilization). The number of bound sperm per hemisphere was determined by focusing stepwise through the depth of the top half of the egg, and counting sperm heads. (For details of the assay see Tian et al., 1997.) Bars indicate the standard deviation (SD) with n 15 eggs.
FIG. 2. Western blot showing the presence of gp69/64 glycoproteins in the OE, CE, and VE, and the modified forms, gp66/61, in the FE. Total envelope glycoproteins (1.5 mg) purified from different sources were separated by 7.5% SDS–PAGE and electroblotted onto a nitro- cellulose membrane. Polyclonal anti-gp69/64 was used as primary antibody for the immunoblot. See Materials and Methods for details.
FIG. 3. Inhibitory effects of envelope glycoproteins on sperm–egg binding. (A) Purified gp69/64 glycoproteins (5 mg/ml) from OE, CE,
VE, and gp66/61 from FE were used as competitors in the sperm binding competition assays. (B) Heat-dissolved total envelope proteins
(50 mg/ml) isolated from OE, CE, VE, or FE were used as competitors. The sperm binding level in the presence of equal concentration of
BSA (5 mg/ml in A or 50 mg/ml in B) was used as control and designated as 100% (- - -). Bars indicate SD with n 15 eggs.
FIG. 4. (A) Fluorescence micrographs of sperm binding to an oocyte, coelomic egg, dejellied oviposited egg, and fertilized egg (0.5 hr after
fertilization). The sperm heads were stained with Hoechst 33342 (0.2 mg/ml) and appeared in the images as bright dots on the surface of
the top, darker animal hemisphere the egg. The vegetal hemisphere appeared brighter due to autofluorescence. Because of the size of the
egg (1.2–1.3 mm in diameter), not all of the sperm are in focus. Bar, 0.2 mm. (B) Fluorescence micrographs showing polyclonal anti-gp69/
64 antibody staining of an oocyte, coelomic egg, dejellied oviposited egg, and a dejellied fertilized egg. The vegetal hemisphere appears
brighter due to autofluorescence, not because of a higher level of staining by the antibody. See Materials and Methods for details.
FIG. 5. 125I surface labeling profile of the glycoproteins in the intact VE. Lane 1, VE proteins separated by 7.5% SDSâPAGE and stained with Coomassie blue. The proteins are identified on the left. Lane 2, autoradiograph of radioiodinated VE surface proteins. A minor contaminating band below gp112 (not visible in Coomassie blue-stained gel) was also labeled.
FIG. 6. (A) Effects of F-layer or hydrolysis of gp69/64 on sperm binding. The experimental design was described in the text. Eggs were
activated by addition of 2 mM (final concentration) of ionophore A23187 under different conditions. Thirty minutes after activation, the
eggs were washed with 0.31 MR (pH 7.8) and tested for sperm binding activity. Bars indicate relative sperm binding levels to: 1, unactivated,
mature, dejellied eggs; the average number of sperm bound to each egg in this group (1300 { 180) represents approximately the normal
binding level and is set as 100%. The relative levels of sperm binding to other groups was obtained by comparison to this number; 2,
eggs with intact jelly preincubated in 1 1 DB (pH 7.2) for 0.5 hr and then activated and dejellied; 3, 4, and 5, dejellied eggs activated in
11 DB, 2 mM of chymostatin in 0.051 DB, or 0.05 1 DB (without protease inhibitor), respectively. Bracketed lines represent SD with n
20 eggs. (B) SDS–PAGE analysis of total envelope proteins from the same samples in (A). The gel was stained by silver.
FIG. 7. Kinetics of detachment of sperm from dejellied eggs after
egg activation. Sperm (107 sperm/ml) were incubated with dejellied
eggs in 0.31 MR (pH 7.8) for 20 min. Then, the eggs were divided
into two groups: one group was washed with 11 DB, pH 7.2 (open
circles); the other was washed with 0.051 DB (filled circles), to
remove unbound sperm. After the wash, the egg in both groups
simultaneously activated by addition of 2 mM of ionophore
A23187. At different time points, an aliquot of eggs was removed
to determine the number of sperm remaining bound on the egg was
surface (see text). The average number of sperm bound on the dejel-
lied eggs (1500 { 200 sperm/egg) immediately before activation was
normalized as 100%. Bracketed lines represent SD with n =
15 eggs.
