RESUMO
The responses of the egg to insemination in a modified Fish Ringer's solution (FRS) were examined in eggs of the zebrafish (Brachydanio rerio) primarily by scanning electron microscopy. FRS is a physiological saline which temporarily inhibits parthenogenetic activation of the egg for 5-8 min. Spermatozoa were collected in a small volume of water and pipetted over eggs in FRS. Eggs inseminated in FRS typically incorporated the fertilizing sperm within 3-4 min. Inseminated cells showed an absence of a fertilization cone and no cortical granule exocytosis. The deep conical depression in the egg surface beneath the micropyle remained unaltered. Control eggs inseminated in tank water developed a large fertilization cone during sperm incorporation. Occasionally, eggs inseminated in water were observed to incorporate the entire sperm head prior to egg activation. Our results corroborate earlier findings showing that in the zebrafish, cortical granule exocytosis, fertilization cone formation and elevation of the sperm entry site are not triggered by the fertilizing sperm in experimental conditions (18, 19). Furthermore, sperm incorporation requires neither egg activation nor formation of a fertilization cone in this fish.
RESUMO
The effects of the divalent ionophore A23187 upon unfertilized eggs of the freshwater teleost fish, Brachydanio rerio, have been examined by light, scanning (SEM) and transmission (TEM) electron microscopy. Treatment of eggs with micromolar amounts (1 µM, 10 µM) of A23187 triggers cortical granule exocytosis and elevation of the chorion. However, the exocytosis of cortical granules in ionophore-activated eggs is explosive and occurs more rapidly than in eggs naturally activated in conditioned tap water. Eggs treated with A23187 in a medium lacking extra-cellular calcium also show cortical granule exocytosis, suggesting strongly that egg activation in Brachydanio results from release of calcium primarily from intracellular stores; however, there is a distinct delay in the onset of cortical granule breakdown. Unfertilized eggs exposed to A23187 for 1-5 min show noticeable disturbances in cell surface topography, including loss of microplicae and the appearance of prominent membrane-limited blebs.To determine if cortical granule exocytosis is self-propagating once initiated, A23187 was applied to a localized portion of the unfertilized egg surface, using either a G-50 sephadex gel bead or a 1 mm glass capillary tube. Eggs placed in continuous contact for 15 min with a bead coated with 10 µM A23187 show neither exocytosis of cortical granules nor elevation of the chorion. All eggs exhibit exocytosis when positioned against a glass rod coated with 1 µM A23187. The cortical granule breakdown is partial and restricted to less than 50% of the egg surface in most cells. The complete exocytosis of cortical granules in the zebra danio egg appears to require the stimulation and release of calcium from multiple sites over the cortex.
