Abstract
BackgroundAt fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca2+ ([Ca2+]i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca2+]i oscillations through the generation of inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release by binding to IP3 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.ResultsMouse 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 [Ca2+]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 IP3 receptors.ConclusionMouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca2+]i transients, and to be activated after completion of maturation.
Highlights
At fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca2+ ([Ca2+]i)
Efficiency of intracytoplasmic sperm injection (ICSI) into maturing oocytes Injection of spermatozoon does not affect the ability of the oocytes to complete maturation; 83% of injected and 83% of control oocytes extruded PB1 and reached metaphase II (MII) stage
The proportion of oocytes, which had spermatozoa in their cytoplasm at the end of maturation (12 – 18 h after ICSI), was significantly lower (p < 0.05) than the proportion of oocytes in which the presence of the spermatozoonderived chromatin was observed 0.5 – 5 h after ICSI. This suggested that some maturing oocytes discarded the microinjected spermatozoa. This was confirmed by our finding that half (6/11) of the oocytes subjected to ICSI and lacking the spermatozoon-derived chromatin in their cytoplasm had, in addition to first polar body (PB1), the "pseudo-polar bodies" containing recondensed chromatin of the spermatozoon, and another half of the oocytes (5/11) had noticeably larger first polar body that, besides the meiotic chromosomes, contained the spermatozoon-derived chromatin
Summary
Mammalian oocytes are activated by oscillations of intracellular Ca2+ ([Ca2+]i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca2+]i oscillations through the generation of inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release by binding to IP3 receptors located on the endoplasmic reticulum (ER) of the oocyte. The increase in [Ca2+]i during fertilisation occurs in the form of [Ca2+]i oscillations, which start when the oocyte is penetrated by spermatozoon [1,2,3,4,5] and last for several hours until the formation of pronuclei [6] It has (page number not for citation purposes). It was demonstrated that in maturing mouse oocyte, ER undergoes reorganisation and in fully mature oocyte it aggregates within the cortical region [21] These changes coincide with the redistribution and increase in the number of IP3 receptors [22]
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