Ca 2+ Responses and Oocyte Activation Related Phenomena are Triggered in Human Oocytes After Photolytic Release of Injected Inositol 1,4,5-Trisphosphate (InsP 3) and Are Blocked by a Specific Antibody Directed to the Type-I Receptor

Abstract

Objective: Global waveform transients in the cytosolic Ca2+ occuring during mammalian fertilization are vital for meiosis exit and embryo development. The secondary messenger InsP3 and its receptors (InsP3R) may be actively involved in operating these Ca2+ changes. The InsP3R are expressed in human oocytes and are redistributed during maturation. In this study, we explore the role of the type I InsP3R in human oocytes using a specific function blocking monoclonal antibody (18A10, Science, 1992; 257:251–255). Design: Intracellular Ca2+ responses were recorded in oocytes after photolytic release of microinjected caged InsP3 in 3 experimental sets: (a) oocytes were loaded with a Ca2+ sensitive fluorescent dye, injected with caged InsP3 and subjected to a 1 s flash; (b) sibling oocytes were either sham injected or injected with 18A10 in addition to caged InsP3 and subjected to a 2 s flash; (c) oocytes were flashed and subsequently fixed and processed for staining the cortical granules and chromatin. Materials and Methods: In vitro matured M II oocytes were injected with 10–15 pl caged InsP3 (Calbiochem) dissolved in an intracellular buffer (5 mM) and incubated for 1 h in a medium containing 10 μM fluo-3-AM (Molecular Probes). The oocytes were then transferred to a fluorescence microscope equipped to perform Ca2+ imaging (37°C) and exposed to UV flash focused to a small spot. The Ca2+ responses were video-recorded and later analyzed. In (b) and (c), the oocytes were additionally injected with 18A10 (∼150 μg/ml, test group) or a corresponding volume of vehicle (control group) and in (c), the oocytes were further processed for staining with rhodamine-conjugated lens culinaris agglutinin and DAPI (Vector Laboratories) and subjected to confocal laser scanning microscopy. Results: In (a), flash photolysis (1 s) triggered a Ca2+ response from ∼100 nM to 220 nM in all oocytes (mean±SEM, n=8) and was followed by an exponential recovery (34±9 s). Application of the flash at the oocyte border demonstrated a Ca2+ wave spreading from the stimulation point to the opposite pole at 19.3±1.5 μm/s (n=7, 37°C). In (b), sibling oocytes (n=20) were divided into control (n=11, 9 survived injections) and test (n=9, 7 survived injections) groups. Flash photolysis (2 s) triggered a Ca2+ response in all the control oocytes [Ca2+ increase from ∼100 nM to ∼300 nM followed by recovery (n=9)]. In 3/9 oocytes, one or more spontaneous Ca2+ transients followed. No Ca2+ responses were seen in 7/7 surviving test oocytes. In (c), all control oocytes exhibited cortical granule exocytosis as well as anaphase chromosome configurations. In the test group, cortical granules were intact and the chromosomes were in metaphase. Conclusions: These experiments provide a conclusive proof to the functional presence of type I InsP3R operated Ca2+ channels in human oocytes and further suggest an active role of InsP3 and its receptor in triggering the Ca2+ rise and secondary activation phenomena at fertilization. (Support: Grant awarded by the University of Ghent, Belgium).