Induction of DNA Replication in the Germinal Vesicle of the Growing Mouse Oocyte

Abstract

Growing mouse oocytes are physiologically arrested in the G2 phase of prophase of the first meiotic division. Growing oocytes were isolated from ovaries of 9- to 12-day-old mice and fused with parthenogenetic one-cell eggs or two-cell embryos derived from fertilized eggs. Resulting hybrids were injected with Dig-11-dUTP and examined for DNA replication using immunofluorescence. Parthenogenetic one-cell eggs fused at telophase II, G1, and middle-to-late S phase, and also S-phase two-cell blastomeres, were able to trigger DNA synthesis in oocyte germinal vesicle (GV) in the majority of hybrids cultured to the end of the first cell cycle. Activation of replication in the GV occurred within 2-3 h after fusion of growing oocytes with S-phase eggs. We show indirectly that the reactivation of replication in GVs was not dependent on the breakdown of the GV envelope. Although GVs had the ability to renew DNA replication after fusion, the G2 blastomere nuclei were incapable of reinitiating DNA replication under the influence of S-phase one-cell eggs. We hypothesize that the nuclei of growing oocytes arrested in meiotic prophase are in a physiological state that is equivalent to replication-competent G1, and not G2, nuclei.

Factors engaged in reactivation of DNA replication in the nuclei of growing mouse oocytes introduced into the cytoplasm of parthenogenetic one-cell embryos

“…In contrast, activation of DNA synthesis in the nucleus of the growing oocyte in hybrid cell could occur only after initiation of replication by the female pronucleus and usually was observed 2-3 hours later than in the pronucleus. The firing of replication domains proceeded along different pattern than in the pronucleus (Czolowska and Borsuk, 2000;this work). Usually, the first signs of the incorporation of microinjected dU analogue were observed in the regions of heterochromatin surrounding the nucleolus.…”

“…Two of these proteins, Mcm2 and Mcm7, were detected in the insoluble and soluble form, while Mcm6 was found only in the soluble form. In the present study we used previously developed experimental system permitting to reactivate replication in growing mouse oocytes (Czolowska and Borsuk, 2000). In this system parthenogenetic one-cell embryo exerts stimulatory influence on DNA replication in growing oocyte.…”

“…The abundance of these replication-related proteins may partly explain why G2 nuclei of growing oocytes are able to reinitiate DNA replication stimulated by exogenous stimulus. We showed before (Czolowska and Borsuk, 2000) that such stimulus of unknown nature is provided by parthenogenetic one-cell embryos at different phases of the first cell cycle (telophase II/G1, G1/S and S). What is important, in the same experimental conditions (fusion with one-cell embryo) neither the nuclei of fully-grown mouse oocytes (Borsuk, unpub-lished data) nor the G2 nuclei of embryonic cells (blastomeres of 2-cell embryos; Czolowska and Borsuk, 2000) were capable of reinitiating DNA synthesis.…”

“…We showed before (Czolowska and Borsuk, 2000) that such stimulus of unknown nature is provided by parthenogenetic one-cell embryos at different phases of the first cell cycle (telophase II/G1, G1/S and S). What is important, in the same experimental conditions (fusion with one-cell embryo) neither the nuclei of fully-grown mouse oocytes (Borsuk, unpub-lished data) nor the G2 nuclei of embryonic cells (blastomeres of 2-cell embryos; Czolowska and Borsuk, 2000) were capable of reinitiating DNA synthesis. In the present studies we searched for the factors which may be involved in the renewal of DNA replication by G2 nuclei of growing oocytes in the hybrid cells.…”

“…Normally the nuclei of somatic and embryonic G2 cells are unable to restart DNA synthesis, even after fusion with S-phase cells Czolowska and Borsuk, 2000). New round of chromatin licensing, preceding new round of DNA replication, is possible only after nuclear envelope breakdown and the completion of mitosis with concomitant decrease in the CDK1 activity (reviews: Nurse, 1997;Nishitani and Lygerou, 2002).…”