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 irradi ation of pig metaphase chromosomes would block the normal sequence of cytol ogical and molecular events associated with activation; the second postulat ed that damaged DNA would prevent eggs from progressing through the first m itotic cleavage cycle. The experimental protocol involved selectively irrad iating the metaphase II plate of pig oocytes with highly focused 254 nm ult raviolet (UV) light, followed by activation using standard electroactivatio n procedures. The following assessments were made of different groups of eg gs: (i) nuclear membrane reassembly; (ii) chromosomal cytology; (iii) chang es in maturation-promoting factor kinase (MPF kinase) activity at Ih interv als after activation; and (iv) mitotic progression of eggs containing damag ed chromosomal fragments. UV irradiation neither prevented the reassembly o f nuclear membranes required for pronuclear formation nor interfered with t he normal pattern of MPF kinase degradation after egg activation. UV irradi ation 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 r etarded, but did not inhibit, progression through the first mitotic cycle. No evidence was obtained that the subsequent mitotic cycle was adversely af fected by the presence of UV-damaged DNA. Overall, these results indicate t hat early cleavage divisions in pig eggs are not blocked by the presence of damaged, hypercondensed chromatin. in this respect, pig eggs are similar t o Xenopus eggs, but are different from bovine eggs. On the basis of these f indings it is suggested that focused UV irradiation offers a simple and rap id technique for the non-invasive enucleation of pig oocytes provided that the residual hypercondensed chromatin does not affect later developmental s tages.