Preparation of Meiotic Chromosome Spreads from Mouse Oocytes for Assessment of Synapsis and Recombination

Abstract

Three critical and interdependent processes define meiotic prophase I: homologous chromosomes must pair together, a proteinaceous structure called the synaptonemal complex forms to tether homologs together (synapsis), and homologs undergo recombination, reciprocally exchanging genetic material to form crossovers (COs). Errors in these processes can result in premature ovarian insufficiency, aneuploidy, and ultimately, pregnancy loss and infertility. Meiotic recombination is particularly error-prone in oocytes, with over 7% of human oocytes containing at least one chromosome pair without a crossover, and between 20%-80% of eggs versus 2.5%-7% of sperm are aneuploid. However, it remains unclear why chromosomal errors show such striking sex disparities. The present protocol describes methods for the preparation and analysis of oocyte prophase I and metaphase I chromosome spreads from fetal and juvenile mice, respectively. To trace critical chromosome dynamics throughout meiotic prophase I, this protocol employs immunofluorescence staining of common markers for meiotic recombination (RAD51 for double strand breaks, MSH4 for CO intermediates, and MLH1/MLH3 to identify most COs) and synapsis (SYCP3 for chromosome axes, SYCP1 for synapsed regions, and HORMAD1 for asynapsed regions). Further, this protocol uses in vitro maturation of oocytes collected to assess the number of paired chromosomes (bivalents) and the total number of crossovers (chiasmata) in metaphase I. Together, these techniques provide a comprehensive and quantitative framework to examine mechanisms regulating early chromosome dynamics in female meiosis.