Due to the lack of an efficient in vitro spermatogenesis system, studies on mammalian spermatogenesis require the isolation of specific germ cell populations for further analyses. BSA gradient and elutriation have been used for several decades to purify testicular germ cells; more recently, flow cytometric cell sorting has become popular. Although each method has its advantages and disadvantages and is used depending on the purpose of the experiment, reliance on flow cytometric cell sorting is expected to be more prevalent because fewer cells can be managed. However, the currently used flow cytometric cell sorting method for testicular germ cells relies on karyotypic differences via DNA staining. Thus, it remains challenging to separate post-meiotic haploid cells (spermatids) according to their differentiation stage despite significant variations in morphology and chromatin state. In this study, we developed a method for finely separating testicular germ cells using VC mice carrying fluorescently tagged histones. This method enables the separation of spermatogonia, spermatocytes, and spermatids based on the intensity of histone fluorescence and cell size. Combined with a DNA staining dye, this method separates spermatids after elongation according to each spermiogenic stage. Although the necessity for a specific transgenic mouse line is less versatile, this method is expected to be helpful for the isolation of testicular germ cell populations because it is highly reproducible and independent of complex cell sorter settings and staining conditions.

中文翻译：

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通过生精过程中动态组蛋白的掺入和去除分离阶段特异性生精细胞

由于缺乏有效的体外精子发生系统，哺乳动物精子发生的研究需要分离特定的生殖细胞群进行进一步分析。BSA 梯度和淘析已用于纯化睾丸生殖细胞数十年；最近，流式细胞术细胞分选变得流行。尽管每种方法都有其优点和缺点，并且根据实验目的使用，但由于可以管理的细胞较少，预计对流式细胞术细胞分选的依赖将更加普遍。然而，目前使用的睾丸生殖细胞流式细胞术细胞分选方法依赖于通过 DNA 染色的核型差异。因此，尽管形态和染色质状态存在显着变化，但根据其分化阶段分离减数分裂后单倍体细胞（精子细胞）仍然具有挑战性。在这项研究中，我们开发了一种使用携带荧光标记组蛋白的 VC 小鼠精细分离睾丸生殖细胞的方法。该方法能够根据组蛋白荧光强度和细胞大小分离精原细胞、精母细胞和精子细胞。该方法与 DNA 染色染料相结合，根据每个精子发生阶段在伸长后分离精子细胞。尽管对特定转基因小鼠系的需求不太通用，但该方法预计将有助于睾丸生殖细胞群的分离，因为它具有高度可重复性并且独立于复杂的细胞分选仪设置和染色条件。