In Vitro Culture Retards Spontaneous Activation of Cell Cycle Progression and Cortical Granule Exocytosis That Normally Occur in In Vivo UnfertilizedMouse Eggs1

Abstract

We have previously demonstrated that metaphase II-arrested eggs recovered from oviducts at increasing times after hCG administration display a time-dependent spontaneous entry into anaphase, as well as release of cortical granules (CGs) and the associated modifications of the zona pellucida (ZP), a decrease in histone H1 and mitogen-activated protein kinase activities, and the recruitment of maternal mRNAs [Xu et al., Biol Reprod 1997; 57:743-750). These changes are correlated with the time-dependent increase in susceptibility of these eggs to undergo parthenogenetic activation. We report here the effect of culture of ovulated eggs, retrieved 13 or 16 h post-hCG administration and cultured in vitro for various periods of time, on the aforementioned parameters of egg activation and cell cycle resumption. In contrast to extended residence of the eggs in the oviduct, culture in vitro retarded cell cycle events associated with completion of the second meiotic reduction and inhibited CG release and the associated modifications of the ZP, as well as the recruitment of maternal mRNAs. The retardation or inhibition of these changes during in vitro culture resulted in eggs that were less susceptible to parthenogenetic activation than eggs that resided in the oviduct for comparable time periods. Results of these experiments indicate that egg culture in vitro (which likely occurs under suboptimal conditions) inhibits, rather than accelerates, the progression into the interphase-like state as compared to that seen in eggs residing in the oviduct for increasing periods of time. These results also suggest that, for studies focused on in vitro fertilization or egg activation, the ovulated eggs should be placed under appropriate in vitro conditions as soon as possible.

Establishment of the mammalian membrane block to polyspermy: evidence for calcium-dependent and -independent regulation

“…3C and F). Moderate levels of CG exocytosis and low levels of cell cycle resumption (similar to those observed previously with A23187 (Abbott et al 1998)) occurred in A21387-treated eggs that were collected at 17 h post-hCG ( Fig. 3D and E), demonstrating that A23187 treatment induced egg activation responses, albeit to a lesser extent than fertilization did, in agreement with previous studies.…”

“…Little or no CG exocytosis or exit from metaphase II arrest occurred in A23187-treated eggs that had been collected at 13 h post-hCG ( Fig. 3A and B), in agreement with previous demonstrations of low responsiveness of 'young' eggs to parthenogenetic stimuli (Fulton & Whittingham 1978, Nagai 1987, Collas et al 1989, Ware et al 1989, Fissore & Robl 1992, Abbott et al 1998.…”

“…This prompted us to re-examine the effects of Ca 2C ionophore treatment on membrane block establishment. Our studies here, include the important variable of postovulatory aged eggs (omitted from previous membrane block studies) because aged eggs are more responsive to parthenogenetic stimuli (Fulton & Whittingham 1978, Nagai 1987, Collas et al 1989, Ware et al 1989, Fissore & Robl 1992, Abbott et al 1998. We find that neither freshly ovulated nor post-ovulatory aged eggs establish even a partially effective membrane block in response to ionophore treatment, despite the fact that the Ca 2C ionophore-induced transient in [Ca 2C ] cyt has similar characteristics to the transient in eggs treated with 0.5 and 1.0 mM BAPTA-AM.…”

“…While it has been reported previously that parthenogenetically activated eggs did not establish a membrane block to polyspermy, our analyses here included some important variables not included in previous studies (Wolf et al 1979, Horvath et al 1993). First, we tested the hypothesis that post-ovulatory time could affect the ability of eggs to establish a membrane block to polyspermy in response to treatment with Ca 2C ionophore, as aged eggs are more responsive to parthenogenetic stimuli (Fulton & Whittingham 1978, Nagai 1987, Collas et al 1989, Ware et al 1989, Fissore & Robl 1992, Abbott et al 1998. Previous studies of membrane block establishment in response to ionophore treatment only tested freshly ovulated eggs (Wolf et al 1979).…”

Pyruvate prevents aging of mouse oocytes

“…If not fertilized or artificially activated in time, the mature oocytes undergo a time-dependent process of aging (Yanagimachi & Chang 1961. This process of oocyte aging has been found to be always associated with a decrease in the MPF activity (Xu et al 1997, Abbott et al 1998. Furthermore, experimental regulation of the MPF activity showed that the deteriorating changes in aged oocytes such as enhanced activation and higher fragmentation rates following parthenogenetic activation could be attributed to the gradual decrease in MPF activity during oocyte aging (Kikuchi et al 2000).…”