Electron microscopic observations on sperm entry into eggs of the bitterling during cross‐fertilization

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Abstract
In the cross‐fertilization of the two bitterlings Acheilognathus lanceolata (spermatozoa) and Rhodeus ocellatus ocellatus (eggs), the morphology of the gametes and the process of sperm entry were observed using transmission and scanning electron microscopy (TEM and SEM). The spermatozoon (total length, about 30 μm) had a round head (about 1.8 μm), two centrioles, a single large mitochondrion, and a flagellum. The micropyle of the egg did not permit simultaneous passage of two spermatozoa. The chorion was mainly composed of an outer and an inner layer and progressively decreased in thickness away from the vicinity of the micropyle where it was thickest. Filamentous structures were observed in microplicae and microvilli on the egg cortex. After a fertilizing spermatozoon attached to the specialized sperm entry site, it appeared to be incorporated into the egg by membrane fusion. At this time the site swelled and plugged the micropyle. The swollen cytoplasm appeared to be a fertilization cone which did not retract in this fish. With the advance of the cortical change, the egg cytoplasm was detached from the chorion except in the area of and around the sperm entry site. About 20 min after insemination, the egg cytoplasm devoid of cortical alveoli was torn away from the other cytoplasm and adhered to the inside of the chorion. These observations showed that the process of sperm entry in cross‐fertilization was similar to that in homologous fertilization of R. ocellatus.