La Salle Sophie, Sun Fengyun, Handel Mary Ann
The Jackson Laboratory, Bar Harbor, ME, USA.
Methods Mol Biol. 2009;558:279-97. doi: 10.1007/978-1-60761-103-5_17.
Understanding meiosis is facilitated by in vitro experimental approaches, but this has not been easily applicable to mammalian meiocytes. Available methods for in vitro analysis of mammalian oocytes are generally limited to experimental analysis of the late prophase period. Short-term cultures of male germ cells have been useful for analysis of earlier meiotic prophase pathways, as well as onset of the meiotic division phase, but no studies have achieved reliable spermatogenesis in vitro. Here we describe a method for preparing highly enriched pachytene spermatocytes from mouse testicular cell suspensions using cell-size fractionation by sedimentation through a bovine serum albumin gradient at unit gravity. We also provide a procedure for short-term culture of spermatocytes and the pharmacological induction of the prophase-to-division phase transition.
体外实验方法有助于对减数分裂的理解，但这在哺乳动物减数分裂细胞中并不容易应用。现有的体外分析哺乳动物卵母细胞的方法通常仅限于对减数分裂前期后期的实验分析。雄性生殖细胞的短期培养对于分析减数分裂前期早期途径以及减数分裂分裂期的开始很有用，但尚无研究在体外实现可靠的精子发生。在这里，我们描述了一种通过在单位重力下通过牛血清白蛋白梯度沉降进行细胞大小分级，从小鼠睾丸细胞悬液中制备高度富集的粗线期精母细胞的方法。我们还提供了精母细胞短期培养的程序以及前期到分裂期转变的药理学诱导方法。
