UV irradiation of pig metaphase chromosomes: maturation-promoting factor degradation, nuclear cytology and cell cycle progression.
Experiments were designed to test two hypotheses. The first was that irradiation of pig metaphase chromosomes would block the normal sequence of cytological and molecular events associated with activation; the second postulated that damaged DNA would prevent eggs from progressing through the first mitotic cleavage cycle. The experimental protocol involved selectively irradiating the metaphase II plate of pig oocytes with highly focused 254 nm ultraviolet (UV) light, followed by activation using standard electroactivation procedures. The following assessments were made of different groups of eggs: (i) nuclear membrane reassembly; (ii) chromosomal cytology; (iii) changes in maturation-promoting factor kinase (MPF kinase) activity at 1 h intervals after activation; and (iv) mitotic progression of eggs containing damaged chromosomal fragments. UV irradiation neither prevented the reassembly of nuclear membranes required for pronuclear formation nor interfered with the normal pattern of MPF kinase degradation after egg activation. UV irradiation did induce a wide range of chromatin defects, including condensation and dispersal of DNA fragments which, in turn, resulted in the formation of micronuclei in the treated eggs and embryos. The presence of damaged DNA retarded, but did not inhibit, progression through the first mitotic cycle. No evidence was obtained that the subsequent mitotic cycle was adversely affected by the presence of UV-damaged DNA. Overall, these results indicate that early cleavage divisions in pig eggs are not blocked by the presence of damaged, hypercondensed chromatin. In this respect, pig eggs are similar to Xenopus eggs, but are different from bovine eggs. On the basis of these findings it is suggested that focused UV irradiation offers a simple and rapid technique for the non-invasive enucleation of pig oocytes provided that the residual hypercondensed chromatin does not affect later developmental stages.
Leal
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Protein Function Laboratory, Babraham Institute, Cambridge, UK.
Lee
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C
Moor
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RM
D000704:Analysis of Variance; D000818:Animals; D002453:Cell Cycle / Q000528:radiation effects; D002467:Cell Nucleus / Q000528:radiation effects / Q000648:ultrastructure; D002478:Cells, Cultured; D016009:Chi-Square Distribution; D002875:Chromosomes / Q000528:radiation effects*; D005260:Female; D008677:Metaphase; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D009865:Oocytes / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D013552:Swine; D014466:Ultraviolet Rays
D020928:Mitogen-Activated Protein Kinases