Presence and dynamic redistribution of type I inositol 1,4,5-trisphosphatereceptors in human oocytes and embryos during in-vitro maturation, fertilization and early cleavage divisions

We studied the presence and distribution of the intracellular calcium chann el regulating type I inositol 1,4,5-trisphosphate receptors (IP3R) in human immature and mature oocytes, pronuclear zygotes and cleaved embryos using a specific antibody. Two approaches were used: (i) fluorescence immunocytoc hemistry using a confocal laser scanning microscope (CLSM) and (ii) Western blotting. With confocal microscopy, the receptors were found in the oocyte s, fertilized zygotes as well as cleaved embryos at all stages studied. The pattern and distribution of the receptor staining in the oocytes changed g radually from a diffuse granular patchy one at the germinal vesicle (GV) st age to a reticular and predominantly peripheral one through the metaphase a nd metaphase II (MII) stages. After fertilization, the distribution changed gradually to both, peripheral and central in the zygotes and early 2-4-cel l embryos and predominantly perinuclear in the 6-8-cell embryos. Furthermor e, an overall increase in the staining intensity was observed from GV to MI I stage oocytes and from zygotes to 6-8-cell embryos. We also studied the s patial distribution of the receptor in detail by constructing three-dimensi onal images from the serial optical sections obtained on the CLSM. Peculiar peripheral aggregates of receptor clusters were noted in the MII stage ooc ytes, zygotes and some blastomeres from early cleaved embryos. Finally, Wes tern blots performed on the extracts of 72 in-vitro matured oocytes and 50 spare cleavage stage embryos showed positive bands at similar to 260 kDa. T hese findings coincide with and thus possibly represent the dynamic changes occurring in the cellular Ca2+ release systems through oocyte maturation, fertilization and early embryogenesis. Thus, type I IP3R are likely to play a role during these stages of early development in the human.