Schritt spezifische Sortierung der Maus Spermatiden durch Durchflusszytometrie
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
Die Differenzierung der Maus Spermatiden eines kritischen Verfahren zur Herstellung einer funktionellen männlichen Keimzellen mit intakter Genoms auf die nächste Generation übertragen werden. Bisher wurden molekulare Studien dieser morphologischen Übergang durch das Fehlen eines Verfahrens ermöglicht eine angemessene Trennung dieser wichtigen Schritte spermatid Differenzierung für nachfolgende Analysen behindert. Frühere Versuche zur richtigen Gating dieser Zellen mittels Durchflusszytometrie kann schwierig gewesen, weil der eine eigentümliche Steigerung der DNA-Fluoreszenz in Spermatiden unterziehen Chromatin-Remodeling. Basierend auf dieser Beobachtung, bieten wir die Details eines einfachen Durchflusszytometrie Schema, wodurch reproduzierbare Reinigung von vier Populationen mit Ethanol fixiert Maus Spermatiden, die jeweils einen anderen Zustand in der Kernumbauprozess. Bevölkerung Anreicherung mit Schritt-spezifische Marker und morphologischen Kriterien bestätigt. Die gereinigten Spermatiden für Genom- und Proteom verwendet werdenic-Analysen.
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
Spermatogenen Zellen waren schon immer eine Herausforderung, angesichts der Komplexität der Keimepithel sowie den begrenzten Erfolg der In-vitro-Kultur zu studieren. Im Laufe der Jahre sind viele Ansätze zur Reinigung von Keimzellen aus verschiedenen Spezies entwickelt. Sedimentation Techniken unter Verwendung von Schwerkraftaufbereitung mit Percoll oder Rinderserumalbumin Gradienten bieten in der Regel eine gute Ausbeute an intakten Keimzellen, aber nicht über angemessene Definition zwischen einigen Zelltyp…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Die Autoren danken Dr. Leonid Volkov und Éric Bouchard für ihre anwendungstechnische Beratung über Epifluoreszenzmikroskopie danken.
Finanzielle Unterstützung
Finanziert von der kanadischen Institutes of Health Research (Zuschuss # MOP-93781) zu 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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