-Paso específica Clasificación de Ratón Espermátidas por Citometría de Flujo
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
La diferenciación de espermátidas de ratón es uno proceso crítico para la producción de un gameto masculino funcional con un genoma intacto para ser transmitido a la siguiente generación. Hasta ahora, los estudios moleculares de esta transición morfológica se han visto obstaculizados por la falta de un método que permite una separación adecuada de estos pasos importantes de la diferenciación espermátide para análisis posteriores. Los anteriores intentos de la selección adecuada de estas células usando citometría de flujo pueden haber sido difícil debido a un aumento en la fluorescencia peculiar ADN en espermátidas sometidos a remodelación de la cromatina. Sobre la base de esta observación, proporcionamos los detalles de un flujo sencillo citometría de esquema, lo que permite la purificación reproducible de cuatro poblaciones de espermátidas de ratón fijados con etanol, cada uno representando un estado diferente en el proceso de remodelación nuclear. Enriquecimiento de Población se confirma el uso de marcadores de pasos específicos y criterios morfológicos. Las espermátidas purificadas se pueden utilizar para genómico y proteomic analiza.
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
Células de espermatogénesis siempre han sido difíciles de estudiar debido a la complejidad del epitelio seminífero, así como el éxito limitado de cultivo in vitro. A través de los años, muchos enfoques para purificar se desarrollaron las células germinales a partir de diversas especies. Utilizando técnicas de sedimentación de purificación de la gravedad con Percoll o gradientes de albúmina de suero bovino por lo general proporcionan un buen rendimiento de células germinales intactas, pero carecen …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Los autores desean agradecer al Dr. Leonid Volkov y Éric Bouchard por su asesoramiento técnico en relación con la microscopía de epifluorescencia.
Apoyo financiero
Financiado por los Institutos Canadienses de Investigación en Salud (subvención # MOP-93781) de GB
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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