Horan Tegan S A
Department of Biomedical Sciences, Cornell University; Cornell Reproductive Sciences Center, Cornell University;
J Vis Exp. 2025 Jul 18(221). doi: 10.3791/68749.
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.
减数分裂前期I由三个关键且相互依存的过程所定义：同源染色体必须配对在一起，一种名为联会复合体的蛋白质结构形成以将同源物拴在一起（联会），同源物进行重组，相互交换遗传物质以形成交叉（COs）。这些过程中的错误会导致卵巢早衰、非整倍体，最终导致流产和不孕。减数分裂重组在卵母细胞中特别容易出错，超过7%的人类卵母细胞至少有一对染色体没有交叉，并且20%-80%的卵子是非整倍体，而精子的这一比例为2.5%-7%。然而，目前尚不清楚为什么染色体错误会表现出如此显著的性别差异。本方案分别描述了从胎儿和幼年小鼠制备和分析卵母细胞前期I和中期I染色体铺片的方法。为了追踪减数分裂前期I中关键的染色体动态，本方案采用了对减数分裂重组的常见标记（双链断裂用RAD51、CO中间体用MSH4、识别大多数COs用MLH1/MLH3）和联会（染色体轴用SYCP3、联会区域用SYCP1、未联会区域用HORMAD1）进行免疫荧光染色。此外，本方案使用收集的卵母细胞进行体外成熟，以评估中期I中配对染色体（二价体）的数量和交叉（交叉结）的总数。这些技术共同提供了一个全面且定量的框架，用于研究调节雌性减数分裂早期染色体动态的机制。
