Mouse oocytes fertilised by ICSI during in vitro maturation retain the ability to be activated after refertilisation in metaphase II and can generate Ca(2+ )oscillations

BACKGROUND: At fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca(2+ )([Ca(2+)](i)). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca(2+)](i )oscillations through the generation of inositol 1,4,5-triphosphate (IP(3)), which causes Ca(2+ )release by binding to IP(3 )receptors located on the endoplasmic reticulum (ER) of the oocyte. Ability to respond to this activating stimulus develops during meiotic maturation of the oocyte. Here we examine how the development of this ability is perturbed when a single spermatozoon is introduced into the oocyte prematurely, i.e. during oocyte maturation. RESULTS: Mouse oocytes during maturation in vitro were fertilised by ICSI (intracytoplasmic sperm injection) 1 – 4 h after germinal vesicle break-down (GVBD) and were subsequently cultured until they reached metaphase II (MII) stage. At MII stage they were fertilised in vitro for the second time (refertilisation). We observed that refertilised oocytes underwent activation with similar frequency as control oocytes, which also went through maturation in vitro, but were fertilised only once at MII stage (87% and 93%, respectively). Refertilised MII oocytes were able to develop [Ca(2+)](i )oscillations in response to penetration by spermatozoa. We found however, that they generated a lower number of transients than control oocytes. We also showed that the oocytes, which were fertilised during maturation had a similar level of MPF activity as control oocytes, which were not subjected to ICSI during maturation, but had reduced level of IP(3 )receptors. CONCLUSION: Mouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca(2+)](i )transients, and to be activated after completion of maturation.