고정의 이미지 Prometaphase 및 중기 감수 분열의 I위한 기술<em> 초파리</em> 난 모세포
Summary
We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.
Abstract
Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.
Introduction
The study of meiosis is sometimes described as the “genetics of genetics”. This is because the fundamental properties of chromosome inheritance and independent assortment are carried out through the segregation of chromosomes during gamete production. An important demonstration of the chromosome theory of inheritance came in 1916 from the work of Calvin Bridges in Drosophila melanogaster1. This and other classical genetics studies in Drosophila contributed greatly to our understanding of genetics. Cytological examination of meiotic chromosomes in Drosophila oocytes, however, has been challenging. This is primarily because immunofluorescence of late-stage Drosophila oocytes, when the spindle assembles and chromosomes are oriented for segregation, is hampered by the presence of membranes that render the oocyte impenetrable to antibodies.
Despite this challenge, Drosophila oocytes remain an attractive model for the study of chromosome behavior and spindle assembly. This is because of the powerful genetic tools available in Drosophila, but also because the oocytes arrest at metaphase I, when the chromosomes are oriented and the spindle is fully formed. This facilitates the collection and examination of large numbers of oocytes at this important stage of cell division. In addition, a simple model organism that is amenable to genetic manipulation for the study of oocyte chromosome segregation can provide an important contribution to our understanding of human reproductive health. Errors in chromosome number are the leading genetic cause of miscarriage and birth defects in humans2. A majority of these errors can be traced to the oocyte and are correlated with increasing maternal age. The average age of mothers in the U.S. has been increasing, making this a major public health concern.
We describe here methods for the cytological examination of Drosophila oocytes, including a demonstration of how to remove the oocyte membranes. These methods are modifications of protocols first described by Theurkauf and Hawley3, Zou et al.4, and Dernburg et al.5. We also include methods for the enrichment of different stages of oocytes, based on a protocol first described by Gilliland et al.6. Finally, we add instructions for the drug treatment of Drosophila oocytes. Together, these methods allow the cytological investigation of oocyte chromosome segregation and spindle assembly in Drosophila.
Protocol
Representative Results
Discussion
초파리 난 모세포를 준비
긴 karyosome 자주 중기의 난 모세포에서 prometaphase 구분하기 karyosome 모양을 사용하여, prometaphase 난자에서 볼 수 있지만 문제가 될 수 있습니다. prometaphase 동안 karyosome은 원형으로의 신장을 시작하고 난 모세포는 중기 정지에 접근 라운드 형상으로 후퇴. 이것은 많은 prometaphase 난자가 긴 karyosome이없는 것을 의미한다. 돌연변이 또는 ?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Christian Lehner for providing the CENP-C antibody and Eric Joyce for recommendations on FISH. Work in the McKim lab was funded by a grant from NIH (GM101955).
Materials
| 15 mL conical tubes | Various | ||
| 16% formaldehyde | Ted Pella, Inc. | 18505 | HAZARDOUS; once opened, discard after one month |
| 250 mL beakers | Various | ||
| 5 mL tubes | Various | ||
| active dry yeast | Various | mix with water to make a paste the consistency of peanut butter | |
| anti-α-tubulin antibody conjugated to FITC | Sigma | F2168 | clone DM1A |
| Binucleine 2 | Sigma | B1186 | HAZARDOUS |
| blender | Various | ||
| bovine serum albumin | Sigma | A4161 | |
| calcium chloride | Various | ||
| colchicine | Sigma | C-9754 | HAZARDOUS |
| coverslips | VWR | 48366-227 | No. 1 1/2 |
| dextran sulfate | Various | ||
| DMSO | Various | ||
| EGTA | Various | ||
| ethanol | Various | ||
| forceps | Ted Pella, Inc. | 5622 | Dumont tweezers high precision grade style 5 |
| formamide | Sigma | 47670-250ML-F | |
| glass slides | VWR | 48312-003 | |
| glucose | Various | ||
| graduated 1.5 mL tubes | Various | ||
| HEPES | VWR | EM-5330 | available from several venders |
| Hoechst 33342 | Various | ||
| magnesium chloride | Various | ||
| methanol | Various | ||
| large mesh (~1500 µm) | VWR | AA43657-NK | variety of formats and other suppliers, 12 or 14 mesh |
| small mesh (~300 µm) | Spectrum labs | 146 424 | variety of formats, eg 146 422 or 146 486 |
| nutator | Various | ||
| Pasteur pipets | Various | ||
| potassium acetate | Various | ||
| Cacodylic acid | Sigma | C0125 | HAZARDOUS; alternatively, sodium cacodylate may be substituted |
| potassium hydroxide | Various | ||
| sodium acetate | Various | ||
| sodium chloride | Various | ||
| sodium citrate | Various | ||
| sodium hydroxide | Various | ||
| sucrose | Various | ||
| taxol (paclitaxel) | Sigma | T1912 | HAZARDOUS |
| Triton X-100 | Fisher | PI-28314 | |
| Tween 20 | Fisher | PI-28320 | |
| vortex | Various |
Riferimenti
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- Hassold, T., Hunt, P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet. 2 (4), 280-291 (2001).
- Theurkauf, W. E., Hawley, R. S. Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein. J Cell Biol. 116 (5), 1167-1180 (1992).
- Zou, J., Hallen, M. A., Yankel, C. D., Endow, S. A. A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes. J Cell Biol. 180 (3), 459-466 (2008).
- Dernburg, A. F., Sedat, J. W., Hawley, R. S. Direct evidence of a role for heterochromatin in meiotic chromosome segregation. Cell. 86 (1), 135-146 (1996).
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- Gilliland, W. D., et al. Hypoxia transiently sequesters mps1 and polo to collagenase-sensitive filaments in Drosophila prometaphase oocytes. PLoS One. 4 (10), e7544 (2009).
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- Smurnyy, Y., Toms, A. V., Hickson, G. R., Eck, M. J., Eggert, U. S. Binucleine 2, an isoform-specific inhibitor of Drosophila Aurora B kinase, provides insights into the mechanism of cytokinesis. ACS Chem Biol. 5 (11), 1015-1020 (2010).
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- Page, A. W., Orr-Weaver, T. L. Activation of the meiotic divisions in Drosophila oocytes. Dev Biol. 183 (2), 195-207 (1997).
- Tavosanis, G., Llamazares, S., Goulielmos, G., Gonzalez, C. Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila. EMBO J. 16 (8), 1809-1819 (1997).
- Endow, S. A., Komma, D. J. Spindle dynamics during meiosis in Drosophila oocytes. J Cell Biol. 137 (6), 1321-1336 (1997).
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- Colombié, N., et al. Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis. Development. 135 (19), 3239-3246 (2008).
