步特定老鼠的精子细胞分选流式细胞仪
Summary
We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.
Abstract
小鼠精子细胞分化是用于生产的官能雄性配子具有完整基因组中的一个关键的工艺要发送给下一代。到目前为止，这种形态转变的分子研究受到阻碍，缺乏一种方法允许精子细胞分化为后续分析这些重要步骤，充分分离。因为在精子细胞进行染色质重塑一个奇特的增加，DNA荧光在这些细胞使用流式细胞仪的正常浇注较早尝试可能是困难的。基于这一观察，我们提供了一个简单的流式细胞术方案细节，让四个种群的小鼠精子细胞固定用乙醇可重放纯化，每个代表在核重塑过程不同的状态。富集的人口使用步骤的具体指标和形态准则确认。纯化的精子细胞，可用于基因组和蛋白质组IC分析。
Introduction
Haploid round spermatids differentiate into spermatozoa by a process called spermiogenesis. This involves many different steps including the acquisition of a flagellum, chromatin and cytoskeleton remodeling, condensation of the nucleus as well as the loss of most of the cytoplasm. These unique cellular events must be finely regulated in order to produce a mature functional gamete with an intact genome suitable for fertilization. Spermiogenesis can hardly be studied in vitro since no reliable cell culture system has so far been able to support progression through the different steps of the process. Moreover, actual in vitro techniques lead to a poor yield1,2. In vivo, proper transitions through the different steps of spermiogenesis are crucial for the natural functional integrity of the male gamete. Successful purification of spermatids according to their differentiation steps has never been accomplished with a level of enrichment sufficient to allow molecular characterization of spermiogenesis. For instance, purification of key steps of the spermatidal differentiation would be especially useful to study the developing acrosome, formation of the midpiece3, cell junction dynamics4, RNA dynamics5, chromatin remodeling process6,7 or genomic stability8. Purification of spermatids has been hampered by their progressive morphological transformation, the lack of known stage-specific external biomarkers, and their peculiar shape and size.
Although most male germ cells display a direct relationship between DNA staining and ploidy (DNA content), we noticed that such positive correlation is no longer applicable to spermatids. This stems from our early observation that seminiferous tubule sections show variable intensity of DNA staining throughout the different spermiogenesis steps. Although DNA staining is consistent with their haploid set of chromosomes from spermiogenesis steps 1 to 7 (round spermatids), we observed a sharp increase in fluorescence intensity with DAPI or SYTO 16 around the onset of nuclear reorganization and chromatin remodeling (spermiogenesis step 8) reaching a peak at the onset of nuclear condensation (spermiogenesis steps 11-12). Following condensation of the nucleus, DNA staining intensity decreases until spermiation (spermiogenesis step 16). We surmised that this was likely associated with the formation of their peculiar chromatin structure transition where histones are replaced by protamines. We therefore developed a reliable flow cytometry method that allows the separation of spermatids using the variation of DNA intensity of spermatids as a main selection parameter.
A simple flow cytometry approach is described to separate mouse spermatids with high purity (95-100%) based on their apparent DNA content (SYTO16 staining), size and granulosity. Spermatids are separated into four populations; spermiogenesis steps 1-9, 10-12, 13-14 and 15-16. Purified spermatids are suitable for genetic/genomic analysis, as well as proteomic applications as described in a recent publication from our group9.
Protocol
Representative Results
Discussion
生精细胞始终是具有挑战性的研究给出的生精上皮的复杂性， 以及在体外培养的有限的成功。多年来，许多方法来净化来自不同物种生殖细胞被开发。利用重力纯化用的Percoll或牛血清白蛋白梯度沉降技术通常提供完整生发细胞的良好的产率，但缺乏某些类型的细胞之间的适当定义如减数分裂四倍体细胞和精子细胞10。而且，这些技术需要特殊的装置（通常自制的）可能不容?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢列昂尼德·沃尔科夫博士和Eric布沙尔他们关于荧光显微镜技术咨询。
经济支持
由健康研究（批＃MOP-93781），以GB的加拿大学院资助
Materials
| Isoflurane | ABBOT | 05260-05 | For mouse anesthesia before euthanasia |
| Fetal bovine serum | Wisent | 90150 | For tube coating |
| 1X PBS | |||
| EDTA | BioShop | EDT | For sorting buffer preparation |
| HEPES | Sigma | H | For sorting buffer preparation |
| 100 % Ethanol | Les alcools de commerce | 092-09-11N | For cell fixation |
| SYTO 16 | Life Technologies | S7578 | DNA staining |
| 5 ml polypropylene round bottom tubes | BD Falcon | 352063 | Sorted cells collection |
| 15 ml polypropylene conical bottom tubes | PROgene | 1500 | |
| 50 ml polypropylene conical bottom tubes | PROgene | 5000 | |
| TEC4 anaesthetic vaporizer | Ohmeda | 1160526 | For mouse euthanasia |
| CO2 gas tank | Praxair | C799117902 | For mouse euthanasia |
| O2 gas tank | Praxair | O254130501 | For mouse euthanasia |
| Homemade mouse gas chamber | For mouse euthanasia | ||
| 40 µm Falcon cell strainer | Corning Incorporated | 352340 | |
| 50-micron sample line filters | BD Biosciences | 649049 | |
| Vortex mixer | Labnet international, inc. | S0200 | For cell fixation |
| Dynac centrifuge | Clay Adams | 101 | |
| Celltrics 50 µm filters | Partec | 04-004-2327 | |
| 488 nm laser-euipped cell sorter | BD Biosciences | FACSAria III | |
| Accudop Fluorescent Beads | BD Biosciences | 345249 | |
| Sorting Buffer: 1X PBS, 1mM EDTA pH 8.0, 25mM HEPES pH 7.0, 1%FBS | FBS is heat-inactivated. Make fresh solution, 0.22 μm filtered and keep at 4°C. |
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