Sperm-egg interaction in the painted frog (Discoglossus pictus): an ultrastructural study.

The ultrastructure of sperm changes and penetration in the egg was studied in the anuran Discoglossus pictus, whose sperm have an acrosome cap with a typical tip, the apical rod. The first stage of the sperm apical rod and acrosome reaction (AR) consists in vesiculation between the plasma membrane and the outer acrosome membrane. The two components of the acrosome cap are released in sequence. The innermost component (component B) is dispersed first. The next acrosome change is the dispersal of the outermost acrosome content (component A). At 30 sec postinsemination, when the loss of component B is first observed, holes are seen in the innermost jelly coat (J1), surrounding the penetrating sperm. Therefore, this acrosome constituent might be related to penetration through the innermost egg investments. At 1 min postinsemination, during sperm penetration into the egg, a halo of finely granular material is observed around the inner acrosome membrane of the spermatozoon, suggesting a role for component A at this stage of penetration. Gamete-binding and fusion take place between D1 (the egg-specific site for sperm interaction) and the perpendicularly oriented sperm. Spermatozoa visualized at their initial interaction (15 sec postinsemination) with the oolemma are undergoing vesiculation. The first interaction is likely to occur between the D1 glycocalyx and the plasma membrane of the hybrid vesicles surrounding the apical rod. As fusion is observed between the internal acrosome membrane and the oolemma, it can be postulated that gametic interaction might be followed by fusion of the latter with the apical rod internal membrane that extends posteriorly into the inner acrosome membrane. Insemination of the outermost jelly layer (J3) dissected out of the egg, and observations of the ultrastructural changes of spermatozoa in this coat, indicate that J3 rather than the vitelline coat (VC) induces the AR. Interestingly, at the late postinsemination stage, VC fibrils are seen crosslinking the inner acrosome membrane. The role of this binding is here discussed.

The cortical endoplasmic reticulum and its possible role in activation of Discoglossus pictis (Anura) eggs.

The role of endoplasmic reticulum was investigated in the egg of Discoglossus pictus; recent findings suggest that this organelle is the source of Ca2+ sequestration and release at activation. In the egg of Discoglossus the dimple is the only site where sperm-egg fusion occurs. Microvilli containing microfilament bundles penetrate into the dimple cytoplasm and thus define a cortical layer containing cortical granules, tubular cisternae 35 nm thick and vacuoles. In the underlying cytoplasm are clusters of small cisternae and mitochondria. In the region of the egg cortex outside of the dimple, clusters of cisternae, some vacuoles and a heterogeneous population of small granules are found. In eggs activated by pricking contraction starts from the site of pricking and travels to the antipode. Sections of eggs, fixed 20 to 60 s following fertilization or pricking, show that the tubular cisternae have disappeared and the clusters of cisternae have opened to give rise to longer cisternae arranged in chains. These chains pile up below the vacuoles which are now flat and long because of multiple fusion. A network of cisternae is thus formed whose constituents lean against the microfilament bundles and within 5 min from activation approach the plasma membrane. The flattened vacuoles and the cortical granules are exocytozed. Some cisternae fuse with the microvillar membrane thus participating in microvilli elongation. The microfilament rootlets grandually rearrange and become shorter. In the cortex outside the dimple the cisternae clusters do not open, whereas exocytosis of granules occurs. In eggs treated with A23187, vacuoles and cortical granules undergo exocytosis; however a cisternal network is not formed. The possible stimuli that cause the cisternae network to form at activation only in the dimple and in a matter of seconds are discussed. Our observations, namely exocytosis, formation of cortical wave of contraction and changes in the cytoskeleton organization at activation are consistent with an increase of free Ca2+. Vacuoles and cisternae appear to be good candidates for the sequestration and release of Ca2+ as well an important source for the increase in total plasma membrane at activation.