Fertilization

Multiple photon excitation fluorescence imaging of live bovine zona pellucida- free fertilization. Ejaculated bull sperm acrosome-reacted with PC-12 treatment and labeled with Hoechst 33342 (5 μg/mL) were exposed to zona pellucida-free oocytes and observed with transmitted light and three-photon optical sectioning fluorescence microscopy. The time course of events suggests that sperm head decondensation can be observed with Z-series collections and that cortical rearrangement occur with sperm incorporation. The exact molecules involved in these processes are not identified, but one of them may be actin. The use of this microscopy technique offers researchers the opportunity to perform observations in real time, provided appropriate excitable probes exist. Images originally published in: Tengowski, MW (2004) Microscopic techniques for studying sperm-oocyte interaction during fertilization and early embryonic development. Methods in Molecular Biology (Clifton, N.J.), 253, 165-99.

Cytochalasin-treated zona pellucida-free oocytes exposed to acrosome-reacted bull sperm display markedly different incorporation. Cytochalasin treatment alters the morphology of the oocyte microvilli, producing areas devoid of microvilli and microvilli with short, flat, club-like processes. After either 30 min (A), 60 min (B), 120 min (C), or 240 min (D) of sperm exposure with the oocytes in the presence of cytochalasin, the remaining oocyte microvilli are seen contacting the sperm head in various locations, yet the posterior sperm head, implantation fossa, and sperm tail with mitochondria remain outside the ooplasm, suggesting that oocyte microfilament assembly is important for successful sperm entry in the bovine system. Images originally published in: Tengowski, MW (2004) Microscopic techniques for studying sperm-oocyte interaction during fertilization and early embryonic development. Methods in Molecular Biology (Clifton, N.J.), 253, 165-99.