Sperm incorporation and pronuclear development during fertilization in the freshwater bivalve Dreissena polymorpha

The invasive zebra mussel, Dreissena polymorpha (D. polymorpha), is proving to be a valuable model for understanding general mechanisms of fertilization, particularly regarding sperm incorporation. In the present study, we tracked the various components of the fertilizing sperm of D. polymorpha during sperm incorporation. During fertilization the sperm membrane remains associated with the egg surface as a distinct patch that disperses over time. This patch marked the site of sperm entry that occurs predominately on the CD blastomere. Taking advantage of the relatively unpigmented cytoplasm, real-time observations were made of the incorporated sperm nucleus as it decondensed and reformed as a developing pronucleus. Pronuclear enlargement occurred progressively and at rates comparable with previously reported fixed-time point observations. Sperm mitochondria were incorporated and separated from the sperm along the leading edge of the decondensing nucleus. Sperm mitochondria labeled with Mitotracker Green remained predominately associated with the CD blastomere following first cleavage and could be tracked to the 16-cell stage before the fluorescence was too faint to detect. Additionally, the demembranated sperm axoneme was incorporated, separated during nuclear decondensation, and remained visible in the egg cytoplasm up to 30 min postinsemination (PI). The present study provides one of the most complete descriptions of incorporation on multiple sperm components into the egg and coordinates fixed-time point observations with real-time observations of sperm within the remarkably transparent egg cytoplasm of zebra mussels.