단계 별 마우스 정자의 정렬을 유동 세포 계측법에 의해
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
마우스 정자의 분화는 그대로 게놈과 수컷 생식 기능의 제조를위한 하나의 중요한 과정이 다음 세대로 전달 될 것이다. 지금까지, 이러한 형태 학적 변화의 분자 연구는 후속 분석을위한 spermatid 분화 이러한 중요한 단계의 적절한 분리를 가능하게하는 방법의 결여에 의해 방해되어왔다. 유동 세포 계측법을 사용하여 이러한 세포의 적절한 게이트에서 이전 시도 때문에 염색질 리모델링을받은 정자의 DNA 형광의 독특한 증가로 어려웠을 수있다. 이러한 관찰에 기초하여, 우리는 에탄올로 고정 마우스 정자 네 개체군의 재현성 정화를 허용 방식 계측법 단순 흐름의 세부 사항을 제공하는, 각각의 핵 리모델링 과정에서 다른 상태를 나타내는. 인구 농축 단계 특정 마커와 형태 criterions에를 사용하여 확인할 수있다. 정자 정제 게놈과 proteom 위해 사용될 수있다IC는 분석한다.
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a direct relationship between DNA staining and ploidy (DNA content), we noticed that such positive correlation is no longer applicable to spermatids.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
정자 세포는 항상 정액을 운반하는 상피 세포의 복잡성뿐만 아니라 체외 문화의 제한된 성공을 주어 연구에 도전하고있다. 수년에 걸쳐, 많은 접근법은 다양한 종에서 생식 세포가 개발되었다 정제한다. 퍼콜 또는 소 혈청 알부민 구배로 중력을 이용하여 침전 정화 기법은 일반적으로 그대로 생식 세포의 우수한 수율을 제공하지만, 이러한 감수 배체 세포 및 정자 10 일부 세포 유?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
저자는 표면 형광 현미경에 관한 기술적 조언 박사 레오 니드 볼코프 에릭 보우 차드을 감사드립니다.
경제적 지원
GB의 건강 연구 (보조금 # 1 MOP-93781)의 캐나다 연구소에 의해 자금 지원
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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