利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态
Summary
本手稿提出了一个详细的方法生成X-染色体臂探针和执行荧光原位杂交 (鱼), 以检查中期和中期我被捕的姐妹染色单体的内聚力状态果蝇卵母细胞。本协议适用于确定不同基因型的减数分裂手臂的内聚力是否完好或中断。
Abstract
在人类中, 卵母细胞的染色体分离错误是导致大多数流产和先天缺陷的原因。此外, 随着妇女年龄的增长, 她们受孕体胎儿的风险急剧增加, 这种现象被称为孕产妇年龄效应。在减数分裂的过程中, 准确的染色体分离的一个要求是在卵母细胞经历的延长的前期阶段保持姐妹染色单体的内聚力。人类和模型生物体的细胞学证据表明, 在衰老过程中, 减数分裂的凝聚力会恶化。此外, 人类卵母细胞中的偏聚错误在减数分裂 I 中最为普遍, 与过早失去手臂的内聚力一致。模型生物体的使用对于解开依赖于年龄的丧失凝聚力的机制至关重要。果蝇为研究卵母细胞减数分裂凝聚的调节提供了一些优势。然而, 直到最近, 在不同基因型的卵母细胞或不同的实验条件下, 只有遗传试验才能检测到手臂的内聚力丧失。在这里, 提供了一个详细的协议, 使用荧光原位杂交 (fish) 直接可视化缺陷的手臂凝聚力在中期 i 和中期我逮捕了果蝇卵母细胞。通过生成一个鱼探针, hybridizes 到X染色体的远端臂并收集共焦 Z 栈, 研究人员可以在三维度中可视化单个鱼信号的数量, 并确定姊妹染色单体的手臂是否分离.这一过程使人们有可能在数以百计的果蝇卵母细胞中量化手臂的内聚力缺陷。因此, 这种方法提供了一个重要的工具, 以调查的机制, 促进凝聚力的维护, 以及导致其消亡的因素在老化过程中。
Introduction
在有丝分裂和减数分裂过程中适当的染色体分离需要建立、维护和以协调的方式释放姊妹染色单体的内聚力1,2。衔接是在 S 阶段建立的, 由内聚力复合体介导, 形成物理联系, 把姐妹染色在一起。在减数分裂, 衔接远端的交叉也功能举行重组同系物一起, 这个物理协会有助于确保正确定位的二价在中期 I 轴 (图 1)3,4, 5。在后期 I 释放手臂的内聚力允许同系物分离到相反的主轴杆。然而, 如果手臂的凝聚力过早丢失, 重组同系物将失去他们的物理连接和随机隔离, 这可能导致体配子 (图 1)。
在人类卵母细胞中, 染色体分离的错误是导致流产和先天缺陷的主要原因, 如唐氏综合症6, 其发病率随着产妇年龄的增加呈指数级的7。胎卵母细胞中建立了姊妹染色单体的内聚力, 在出生前完成减数分裂重组。卵母细胞, 然后在中期前逮捕我, 直到排卵和在此逮捕期间, 重组同系物的持续物理关联依赖于姐妹染色单体的内聚力。因此, 在减数分裂和正常妊娠结局中准确的分离需要五年的内聚力保持完好。
在人类卵母细胞长期减数分裂的过程中, 过早失去凝聚力已被建议为产妇年龄效应和多行证据支持这一假设8,9。然而, 考虑到研究人类卵母细胞减数分裂凝聚的挑战, 我们对这种现象的理解大多依赖于模型生物体的使用5,10,11,12, 13,14,15。
果蝇卵母细胞为研究减数分裂凝聚和染色体分离提供了许多优势。一个简单的基因分析允许一个人从体配子中恢复后代, 并在大规模的11、16、17中测量X-染色体隔离的保真度。此外, 你也可以确定是否出现染色体偏析错误, 因为重组同系物 missegregate 在减数分裂 I, 一个表型, 是一致的过早失去手臂的凝聚力11,18, 19. 在果蝇卵母细胞中的减数分裂状态的直接观察也可以使用荧光原位杂交 (fish)。虽然荧光寡核苷酸, 杂交重复的卫星序列已经使用了十几年来监测 pericentromeric 凝聚在成熟的果蝇卵母细胞4,20, 分析手臂凝聚力更具挑战性。手臂内聚力状态的可视化需要一个探针, 它横跨一个大区域的单拷贝序列, 并且足够明亮, 可以在缺乏手臂内聚力的情况下为单个的姐妹染色产生可见的信号。此外, 卵母细胞的固定条件和大小的标记 dna 必须促进渗透21到大成熟的果蝇卵母细胞 (200 µm 宽500µm 长)。最近, 一个手臂探针成功地用于可视化果蝇卵母细胞染色在后期 i, 但作者说, 他们无法检测信号在中期或中期我逮捕了卵母细胞22。在这里, 我们提供了一个详细的协议, 以生成X-染色体臂鱼探针和卵母细胞的制备条件, 使我们能够检测早期丢失的姊妹染色单体的内聚力在中期 i 和中期 i 卵母细胞。这些技术, 使我们能够识别基因产品, 这是需要维护的减数分裂的凝聚力, 将允许其他人的姐妹染色单体内聚力缺陷的成熟的果蝇卵母细胞不同基因型。
Protocol
1. 准备工作 准备荧光原位杂交 (fish) 的解决方案。使用超纯水准备所有解决方案。 准备5x 修改的罗布的缓冲器: 275 毫米乙酸钾, 200 毫米醋酸钠, 500 毫米蔗糖, 50 毫米葡萄糖, 6 毫米氯化镁, 5 毫米氯化钙, 500 毫米 HEPES pH = 7.4。使用 10 N 氢氧化钠将 pH 值降低到7.4。过滤消毒, 贮存在-20 ° c。根据需要解冻和稀释到 1x, 在-20 ° c 时储存1x 等分。 使用1.3 米氯化钠,…
Representative Results
图 5显示了在X染色体上 hybridizes 到细胞学区域 6 e-7 b 的臂探针中获得的图像。该探针的结果是与 DAPI 的本地化的信号, 很容易区分与背景, 并已成功地用于量化的手臂凝聚力缺陷的不同基因型的19。粘聚缺陷的量化仅限于中期 i. 期和中期 i 阶段;核包膜破裂之前的卵母细胞被排除在分析19之外。一个完整的核信…
Discussion
使用鱼探针评估中期 i 和中期 i果蝇卵母细胞的臂部凝聚状态是在果蝇减数分裂领域的一个重要进展。从历史上看,果蝇研究人员已经被限制在成熟的卵母细胞中推断出过早丧失手臂的凝聚力11,18,19。现在, 用这里所提出的方法, 可以直接使用鱼来测定手臂的内聚力状态。获得身体证据的能力过早失去了手臂的…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 NIH 授予 GM59354 颁发给沙龙 e. Bickel 的支持。我们感谢 Huy, 阮协助开发的协议, 产生荧光手臂探头, Ann Lavanway 的帮助与共焦显微镜, j. 因苏阿芦苇技术援助。我们还感谢果蝇社区的许多同事提供有益的讨论和建议。
Materials
| Kits | |||
| Midi Prep kit | Qiagen | 12143 | Prep BAC clone DNA |
| GenomePlex Complete Whole Genome Amplification (WGA) Kit | Sigma | WGA2 | Amplify BAC clone DNA |
| ARES Alexa Fluor 647 DNA labeling kit | Invitrogen | A21676 | Label BAC clone DNA |
| PCR purification kit | Qiagen | 28104 | Remove non-conjugated dye following labeling of BAC clone DNA |
| Name | Company | Catalog Number | Comments |
| Chemicals & Solutions | |||
| Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization. | |||
| Bovine serum albumin (BSA) | Fisher Scientific | BP1600-100 | Prepare 10% stock Freeze aliquots |
| Calcium chloride | Fisher Scientific | C75-500 | |
| DAPI (4’, 6-Diamidino-2-Phenylindole, Dihydrochloride) | Invitrogen | D1306 | Toxic: wear appropriate protection. Prepare 100µg/ml stock in 100% ethanol, store in aliquots at -20 °C. Prepare 1 µg/ml solution in 2X SSCT before use. |
| Dextran sulfate | Sigma | D-8906 | |
| Drierite | Drierite Company | 23001 | |
| Dithiothreitol (DTT) | Invitrogen | 15508-013 | Prepare 10 mM stock |
| dTTP (10 µmol, 100 µl) | Boehringer Mannheim | 1277049 | Prepare 1 mM stock |
| EDTA (Disodium ethylenediamine tetraacetic acid) | Fisher Scientific | S311-500 | Prepare 250 mM stock |
| 100% ethanol (molecular grade, 200 proof) | Decon Laboratories | 2716 | |
| Tris (Ultra Pure) | Invitrogen | 15504-020 | |
| EGTA (Ethylenebis(oxyethylenenitrilo)tetraacetic acid) | Sigma | E-3889 | |
| 16% formaldehyde | Ted Pella, Inc. | 18505 | Toxic: wear appropriate protection |
| Formamide | Invitrogen | AM9342 | Toxic: wear appropriate protection |
| Glucose | Fisher Scientific | D16-1 | |
| Glycogen | Roche | 901393 | |
| HEPES | Boehringer Mannheim | 737-151 | |
| Heptane | Fisher Scientific | H350-4 | Toxic: wear appropriate protection. |
| Hydrochloric acid | Millipore | HX0603-4 | Toxic: wear appropriate protection. |
| Hydroxylamine | Sigma | 438227 | Prepare 3 M stock |
| 4.9 M Magnesium chloride | Sigma | 104-20 | |
| Na2HPO4 Ÿ 7H2O | Fisher Scientific | S373-500 | |
| NaH2PO4 Ÿ 2H2O | Fisher Scientific | S369-500 | |
| Poly-L-lysine (0.1mg/ml) | Sigma | P8920-100 | |
| Potassium acetate | Fisher Scientific | BP364-500 | |
| Sodium acetate | Fisher Scientific | S209-500 | Prepare 3M stock |
| Sodium cacodylate | Polysciences, Inc. | 1131 | Toxic: wear appropriate protection. Prepare 400mM stock |
| Sodium citrate | Fisher Scientific | BP327-1 | |
| Sodium chloride | Fisher Scientific | S271-3 | Sodium chloride |
| Sucrose | Fisher Scientific | S5-500 | |
| 10% Tween 20 | Thermo Scientific | 28320 | Surfact-Amps |
| 10% Triton X-100 | Thermo Scientific | 28314 | Surfact-Amps |
| Name | Company | Catalog Number | Comments |
| Solutions | |||
| Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization. | |||
| TE buffer | 10 mM Tris, 1 mM EDTA, pH = 8.0 | ||
| 20X SSC (Saline Sodium Citrate) | 3 M NaCl, 300 mM sodium citrate | ||
| 2X cacodylate fix solution | Toxic: wear appropriate protection. 200 mM sodium cacodylate, 200 mM sucrose, 80 mM sodium acetate, 20 mM EGTA | ||
| 1.1X Hybridization buffer | 3.3X SSC, 55% formamide, 11% dextran sulfate | ||
| Fix solution | Toxic: wear appropriate protection. 4% formaldehyde, 1X cacodylate fix solution | ||
| PBSBTx | 1X PBS, 0.5% BSA, 0.1% Trition X-100 | ||
| PBSTx | 1X PBS, 1% Trition X-100 | ||
| Extraction buffer (PBSTx + Rnase) | 1X PBS, 1% Trition X-100, 100 µg/mL RNase | ||
| 2X SSCT | 2X SSC, 0.1% Tween 20 | ||
| 2X SSCT + 20% formamide | Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 20% formamide | ||
| 2X SSCT + 40% formamide | Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 40% formamide | ||
| 2X SSCT + 50% formamide | Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 50% formamide | ||
| Name | Company | Catalog Number | Comments |
| Enzymes | |||
| AluI | New England Biolabs | R0137S | |
| HaeIII | New England Biolabs | R0108S | |
| MseI | New England Biolabs | R0525S | |
| MspI | New England Biolabs | R0106S | |
| RsaI | New England Biolabs | R0167S | |
| BfuCI | New England Biolabs | R0636S | |
| 100X BSA | New England Biolabs | Comes with NEB enzymes | |
| 10X NEB buffer #2 | New England Biolabs | Restriction enzyme digestion buffer. Comes with NEB enzymes | |
| Terminal deoxynucleotidyl transferase (TdT) 400 U/µl | Roche/Sigma | 3333566001 | |
| TdT buffer | Roche/Sigma | Comes with TdT enzyme | |
| Cobalt chloride | Roche/Sigma | Toxic: wear appropriate protection. Comes with TdT enzyme | |
| RNase A (10 mg/mL) | Thermo-Scientific | EN0531 | |
| Name | Company | Catalog Number | Comments |
| Cytology Tools etc. | |||
| Forceps | Dumont | #5 INOX, Biologie | |
| 9” Disposable glass Pasteur pipettes | Fisher | 13-678-20C | Autoclave to sterilize |
| Shallow glass dissecting dish | Custom made | ||
| Deep well dish (3 wells) | Pyrex | 7223-34 | |
| Fisherfinest Premium microscope slides | Fisher Scientific | 22-038-104 | Used to cover deep well dishes |
| Frosted glass slides, 25 x 75 mm | VWR Scientific | 48312-002 | |
| Glass slides, 3 x 1 in, 1 mm thick | Thermo-Scientific | 3051 | |
| Coverslips, 10 x 10 mm, No. 1.5 | Thermo-Scientific | 3405 | |
| Tungsten needle | homemade | ||
| Prolong GOLD mounting media | Molecular Probes | P36930 | |
| Compressed-air in can | Various | ||
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| PCR machine | Various | ||
| Nanodrop 2000, spectrophotometer | Thermo-Scientific | microvolume spectrophotometer | |
| Vortexer | Various | ||
| Table top microfuge at room temperature | Various | ||
| Table top microfuge at 4 °C | Various | ||
| Heat block | Various | ||
| Hybridization oven or incubator with rotator | Various | ||
| Nutator | Various | ||
| A1RSi laser scanning confoal | Nikon | 40X oil Plan Fluor DIC (NA 1.3) | |
| Name | Company | Catalog Number | Comments |
| Consumables etc. | |||
| 50 mL conical tubes | Various | ||
| 15 mL conical tubes | Various | ||
| 1.5 mL microfuge tubes | Various | ||
| 500 µl microfuge tubes | Various | ||
| 200 µl PCR tubes | Various | ||
| Plastic container with tight fitting lid | Various | To hold Drierite | |
| Kimwipes | Various | disposable wipes | |
| Parafilm | Various | paraffin film | |
| Name | Company | Catalog Number | Comments |
| Outro | |||
| HPLC purified 5'-labeled oligonucleotides | Integrated DNA Technologies | Cy3-labeled probes that recognize the 359 bp satellite repeat of the X chromosome | |
| Volocity 3D Image Analysis Software | PerkinElmer | Version 6.3 |
Referências
- Peters, J. M., Nishiyama, T. Sister chromatid cohesion. CSH Perspect Biol. 4 (11), (2012).
- McNicoll, F., Stevense, M., Jessberger, R. Cohesin in gametogenesis. Curr Top Dev Biol. 102, 1-34 (2013).
- Buonomo, S. B., et al. Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin. Cell. 103 (3), 387-398 (2000).
- Bickel, S. E., Orr-Weaver, T., Balicky, E. M. The sister-chromatid cohesion protein ORD is required for chiasma maintenance in Drosophila oocytes. Curr. Biol. 12 (11), 925-929 (2002).
- Hodges, C. A., Revenkova, E., Jessberger, R., Hassold, T. J., Hunt, P. A. SMC1beta-deficient female mice provide evidence that cohesins are a missing link in age-related nondisjunction. Nat Genet. 37 (12), 1351-1355 (2005).
- Freeman, S. B., et al. The National Down Syndrome Project: design and implementation. Public Health Rep. 122 (1), 62-72 (2007).
- Nagaoka, S. I., Hassold, T. J., Hunt, P. A. Human aneuploidy: mechanisms and new insights into an age-old problem. Nat Rev Genet. 13 (7), 493-504 (2012).
- Angell, R. R. First-meiotic-division nondisjunction in human oocytes. Am. J. Hum. Genet. 61, 23-32 (1997).
- Tsutsumi, M., et al. Age-related decrease of meiotic cohesins in human oocytes. PLoS One. 9 (5), e96710 (2014).
- Liu, L., Keefe, D. L. Defective cohesin is associated with age-dependent misaligned chromosomes in oocytes. Reprod Biomed Online. 16 (1), 103-112 (2008).
- Subramanian, V. V., Bickel, S. E. Aging predisposes oocytes to meiotic nondisjunction when the cohesin subunit SMC1 is reduced. PLoS Genet. 4 (11), e1000263 (2008).
- Chiang, T., Duncan, F. E., Schindler, K., Schultz, R. M., Lampson, M. A. Evidence that weakened centromere cohesion is a leading cause of age-related aneuploidy in oocytes. Curr. Biol. 20 (17), 1522-1528 (2010).
- Lister, L. M., et al. Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2. Curr. Biol. 20 (17), 1511-1521 (2010).
- Duncan, F. E., et al. Chromosome cohesion decreases in human eggs with advanced maternal age. Aging Cell. 11 (6), 1121-1124 (2012).
- Yun, Y., Lane, S. I., Jones, K. T. Premature dyad separation in meiosis II is the major segregation error with maternal age in mouse oocytes. Development. 141 (1), 199-208 (2014).
- Subramanian, V. V., Bickel, S. E. Heterochromatin-mediated association of achiasmate homologues declines with age when cohesion is compromised. Genética. 181, 1207-1218 (2009).
- Jeffreys, C. A., Burrage, P. S., Bickel, S. E. A model system for increased meiotic nondisjunction in older oocytes. Curr. Biol. 13 (6), 498-503 (2003).
- Weng, K. A., Jeffreys, C. A., Bickel, S. E. Rejuvenation of Meiotic Cohesion in Oocytes during Prophase I Is Required for Chiasma Maintenance and Accurate Chromosome Segregation. PLoS Genetics. 10 (9), e1004607 (2014).
- Perkins, A. T., Das, T. M., Panzera, L. C., Bickel, S. E. Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors. Proc Natl Acad Sci U S A. 113 (44), E6823-E6830 (2016).
- Dernburg, A. F., Sedat, J. W., Hawley, R. S. Direct evidence of a role for heterochromatin in meiotic chromosome segregation. Cell. 86, 135-146 (1996).
- Dernburg, A. F., Sedat, J. W. . Method Cell Biol. 53, 187-233 (1998).
- Guo, Z., Batiha, O., Bourouh, M., Fifield, E., Swan, A. Role of Securin, Separase and Cohesins in female meiosis and polar body formation in Drosophila. J Cell Sci. 129 (3), 531-542 (2016).
- Giauque, C. C., Bickel, S. E. Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes. Genética. 203 (1), 173-189 (2016).
- Joyce, E. F., Apostolopoulos, N., Beliveau, B. J., Wu, C. T. Germline progenitors escape the widespread phenomenon of homolog pairing during Drosophila development. PLoS Genet. 9 (12), e1004013 (2013).
- Gilliland, W. D., Hughes, S. F., Vietti, D. R., Hawley, R. S. Congression of achiasmate chromosomes to the metaphase plate in Drosophila melanogaster oocytes. Dev Biol. 325 (1), 122-128 (2009).
- Gyuricza, M. R., et al. Dynamic and Stable Cohesins Regulate Synaptonemal Complex Assembly and Chromosome Segregation. Curr Biol. 26 (13), 1688-1698 (2016).
- Radford, S. J., McKim, K. S. Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes. J Vis Exp. (116), (2016).
Citar este artigo
Perkins, A. T., Bickel, S. E. Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes. J. Vis. Exp. (130), e56802, doi:10.3791/56802 (2017).