Doctoral Dissertations

Date of Award

12-1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Mary Ann Handel

Committee Members

Wesley Wicks, Dan Roberts, Bruce McKee, Jeff Becker

Abstract

Sexual reproduction in mammals requires the successful completion of events during the first meiotic prophase. Despite the fundamental importance of meiosis, little is known about the control of meiotic events in mammals. The experiments described in this dissertation used mouse spermatocytes as a model system for the study of the requirements for the transition from meiotic prophase to metaphase I (MI). Three experimental strategies were utilized. 1) Cultured spermatocytes were manipulated in vitro to analyze the requirements for condensing bivalent chromosomes. Okadaic acid (OA) treatment was used to induce the precocious condensation of MI chromosomes by pachytene spermatocytes. 2) Indirect immunofluorescence was used to localize proteins within sectioned testes or in isolated spermatogenic cells. 3) The meiotic phenotype of mice null for the Dmc1 gene was analyzed. After a general introduction in Part I, Part II of this dissertation describes the expression and function of the topoisomerases during meiosis. These experiments used the specific inhibitors teniposide, ICRF-193 and camptothecin to demonstrate a requirement for topoisomerase II, but not topoisomerase I activity in condensing MI chromosomes. Part in describes the localization of the TOP2A isoform of topoisomerase II and the phosphorylation of histone H3 during spermatogenesis to test the hypothesis that phosphorylation of histone H3 is a key event instigating both localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. It was found that TOP2A is localized to the centromeric heterochromatin throughout meiotic prophase, well before histone H3 is phosphorylated at the centromeric heterochromatin at diplonema. In vitro studies demonstrated that histone H3 phosphorylation was not sufficient to condense MI chromosomes. The meiotic phenotype of the sterile Dmc1 knockout mouse is analyzed in Part IV. Chromosomes from mutant spermatocytes formed axial elements and RAD51 foci but failed to synapse. Experiments in Part V investigated the timing of the acquisition of competence of spermatocytes to condense MI chromosomes in response to OA treatment. It was found that competence is acquired at mid-pachynema. Competence was correlated with the ability to activate metaphase-promoting factor and with the appearance of histone Hit and CDC25C. As summarized in Part VI, these studies define new landmarks and requirements for the passage from meiotic prophase to metaphase during murine spermatogenesis.

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