Screening of genes involved in chromosome segregation in meiosis I

Prophase I of male meiosis involves dynamic chromosome segregation processes during early spermatogenesis, including synapsis, meiotic recombination, and cohesion. Genetic defects in genes participating in these processes consistently cause reproduction failure in mice. To identify candidate genes responsible for infertility in humans, we performed expression profiling of mouse spermatogenic cells undergoing meiotic prophase I. Cell fractions enriched in spermatogonia, leptotene/zygotene spermatocytes, or pachytene spermatocytes were separately isolated from mouse testes for RNA extraction. To minimize the contamination of other cell types, we fractionated the testicular cells undergoing the first round of spermatogenesis using Percoll gradient procedure. The cell fractions were characterized by morphological analysis by phase contrast microscopy and Nomarski interference microscopy, and then expression of cell lineage- and spermatogenesis stage-specific genes were examined by RT-PCR. The most enriched fractions for spermatogonia (fraction2 from day8), leptotene/zygotene spermatocytes (fraction5 from day12), and pachytene spermatocytes (fraction8 from day15) were subjected to hybridization on Affymetrix microarrays.雄性减数分裂前期I是精子发生早期中动态的染色体分离过程，涵盖联会、减数分裂重组与染色体黏连等环节。参与上述过程的基因若发生遗传缺陷，会持续导致小鼠生殖失败。为鉴定与人类不育相关的候选基因，我们对处于减数分裂前期I的小鼠生精细胞开展了表达谱分析。我们分别从小鼠睾丸中分离得到富集了精原细胞、细线期/偶线期精母细胞以及粗线期精母细胞的细胞组分，用于RNA提取。为尽可能降低其他细胞类型的污染，我们采用Percoll密度梯度离心法，对处于第一轮精子发生过程中的睾丸细胞进行组分分离。随后通过相差显微镜与Nomarski干涉显微镜开展形态学分析以鉴定各细胞组分的特征，并借助RT-PCR（逆转录聚合酶链式反应，Reverse Transcription-Polymerase Chain Reaction）检测细胞谱系特异性及精子发生阶段特异性基因的表达水平。最终，富集程度最高的精原细胞组分（第8天小鼠样本的组分2）、细线期/偶线期精母细胞组分（第12天小鼠样本的组分5）以及粗线期精母细胞组分（第15天小鼠样本的组分8），均在Affymetrix基因芯片上完成了杂交实验。