The objective of the present study was to examine the activity changes in histone H1 kinase (also known as maturation-promoting factor [MPF]) and mitogen-activated protein kinase (MAPK) and their constituent proteins in in vitro-matured bovine oocytes after in vitro fertilization (IVF) or after parthenogenetic activation induced by calcium ionophore A23187 alone or by the ionophore followed by either 6-dimethylaminopurine (6-DMAP) or cycloheximide (CHX). Inactivation of both H1 kinase and MAPK occurred after both A23187+6-DMAP treatment and IVF; inactivation of H1 kinase preceded inactivation of MAPK. However, MAPK was inactivated much earlier in 6-DMAP-treated oocytes. Further analysis of constituent cell cycle proteins of these kinases by Western blot showed that A23187 alone could not induce changes in cdc2, cdc25, or ERK2 but induced reduction of cyclin B1. IVF and A23187+CHX induced similar changes: cyclin B1 was destroyed shortly after activation followed by accumulation of cyclin B1, phosphorylation of cdc2, and dephosphorylation of ERK2 at pronuclear formation 15 h after activation. No change in cdc25 was observed at this time. In contrast, A23187+6-DMAP treatment resulted in earlier phosphorylation of cdc2 and dephosphorylation of ERK2 at 4 h after treatment when the pronucleus formed. Moreover, accumulation of both cdc25 and cyclin B1 was detected at 15 h. Microinjection of ERK2 antibody into A23187-treated oocytes resulted in pronuclear formation. In conclusion, activation of bovine oocytes with 6-DMAP led to earlier inactivation of MAPK, while CHX induced inactivation of MAPK parallel to that following sperm-induced oocyte activation. Destruction of cyclin B is responsible for inactivation of MPF, while phosphorylation of cdc2 is likely responsible for maintaining its low activity. Inactivation of MAPK is closely associated with pronuclear development regardless of the activation protocol used.

The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase Ⅱ oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies.

Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase Ⅱ oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome.

This research was to study the in vitro and in vivo development of cloned embryos derived from adult rabbit fibroblasts following various activation protocols. Effects of serum starvation and passage number of donor cells on the efficiency of cloning were also examined. In experiment I, oocytes were activated either by electric pulses or by electric pulses followed by culture with 6-dimethylaminopurin (DMAP). For experiment Ⅱ, the best activation protocol from experiment I was employed for cloning using adult rabbit fibroblasts that were cultured for 0-15 passages. In experiment Ⅲ, the effect of serum starvation of the donor cells on cloning was examined. Finally, in experiment Ⅳ, embryo transfers were conducted. These experiments showed that combined electrical pulse and DMAP treatment resulted in superior parthenogenetic blastocyst development (up to 29%), and that activation of the cytoplast before versus after fusion was not different in supporting the in vitro development of nuclear transferred embryos (16%–18% blastocysts). Adult fibroblasts derived from nonpassaged cells were less capable of developing into blastocysts than passaged cells (6% vs. 17%). Serum starvation of donor cells improved cleavage (up to 71%) but did not improve blastocyst development (13%), and no progeny was obtained, irrespective of the treatment. Cell-cycle analysis of adult rabbit fibroblast cells showed that passage 6 and 12 cells were more likely to be in G0/G1 than passage 0 cells, which agrees with the improved embryo development in the passaged-cell groups.

Eggs must be the major locus of reproductive aging in women, because donation of eggs from younger to middle-aged women abrogates the effects of age on fertility. Oxidative stress, mitochondrial dysfunction, and apoptosis are associated with senescence. To develop an animal model of egg senescence, we treated mouse zygotes with 175uM H2O2 that induced mitochondrial dysfunction and developmental arrest, followed by delayed cell death, consistent with apoptosis. We reconstructed zygotes with nuclei and cytoplasm from treated or untreated zygotes, then followed development and apoptotic cell death in the reconstituted embryos. Pronuclear exchange between untreated, normal zygotes served as nuclear transfer controls. Rates of cleavage and development to morula and blastocysts were significantly lower (P<0.01) in zygotes reconstituted from untreated pronuclei and H2O2-stressed cytoplasts than those of nuclear transfer controls. Instead, the arrested, reconstituted zygotes displayed TUNEL staining at a similar rate to that of H2O2-treated controls, suggesting that apoptotic potential could be transferred cytoplasmically. On the other hand, rates of cleavage and development to morula and blastocyst of the reconstituted zygotes, derived from stressed pronuclei and untreated cytoplasm, were significantly increased (P<0.05), compared to those of H2O2-treated, control zygotes, indicating that healthy cytoplasm could partly rescue pronuclei from oxidative stress. Although oxidation stressed both nuclei and cytoplasm, cytoplasm was more sensitive than nuclei to oxidative stress. It is suggested that cytoplasm, most likely mitochondria, plays a central role in mediating both development and apoptotic cell death induced by oxidative stress in mouse zygotes.