Name: exploring x chromosomal aberrations in ovarian cells by using fluorescence in situ hybridization
Uploaded: 2023-04-07T13:40:00
Description: Watch this Scientific Journal Video about Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization at JoVE.com

作者感谢Marjo van Brakel，Dominique Smeets，Guillaume van de Zande，Patricia van Cleef和Milan Intezar的专业知识和技术援助。资金来源：默克雪兰诺（A16-1395），Goodlife和Ferring。

特纳生育研究方案已获得涉及人类受试者研究的中央委员会（NL57738.000.16）的批准。本研究共获取93例TS女性卵巢皮层组织。需要安全预防措施的材料列于 表1中。
表 1：安全预防措施。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>材料</td>
			<td colspan="3">危险</td>
		</tr><tr><td>醋酸</td>
			<td c...

<ol>
	<li>Vander Borght, M., Wyns, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+and+infertility:+Definition+and+epidemiology.">Fertility and infertility: Definition and epidemiology.</a> Clinical Biochemistry. 62, 2-10 (2018).</li><li>Yatsenko, S. A., Rajkovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+human+female+infertility.">Genetics of human female infertility.</a> Biology of Reproduction. 101 (3), 549-566 (2019).</li><li>Yahaya, T. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+abnormalities+predisposing+to+infertility,+testing,+and+management:+a+narrative+review.">Chromosomal abnormalities predisposing to infertility, testing, and management: a narrative review.</a> Bulletin of the National Research Centre. 45 (1), 65 (2021).</li><li>Foresta, C., Ferlin, A., Gianaroli, L., Dallapiccola, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+appropriate+use+of+genetic+tests+in+infertile+couples.">Guidelines for the appropriate use of genetic tests in infertile couples.</a> European Journal of Human Genetics. 10 (5), 303-312 (2002).</li><li>Heard, E., Turner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sex+chromosomes+in+mammalian+fertility.">Function of the sex chromosomes in mammalian fertility.</a> Cold Spring Harbor Perspectives in Biology. 3 (10), 002675 (2011).</li><li>Reynaud, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Number+of+ovarian+follicles+in+human+fetuses+with+the+45,X+karyotype.">Number of ovarian follicles in human fetuses with the 45,X karyotype.</a> Fertility and Sterility. 81 (4), 1112-1119 (2004).</li><li>Otter, M., Schrander-Stumpel, C. T., Curfs, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triple+X+syndrome:+a+review+of+the+literature.">Triple X syndrome: a review of the literature.</a> European Journal of Human Genetics. 18 (3), 265-271 (2010).</li><li>Modi, D. N., Sane, S., Bhartiya, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+germ+cell+apoptosis+in+sex+chromosome+aneuploid+fetal+human+gonads.">Accelerated germ cell apoptosis in sex chromosome aneuploid fetal human gonads.</a> Molecular Human Reproduction. 9 (4), 219-225 (2003).</li><li>Hassold, T., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nature Reviews Genetics. 2 (4), 280-291 (2001).</li><li>Huber, D., von Voithenberg, L. V., Kaigala, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+(FISH):+History,+limitations+and+what+to+expect+from+micro-scale+FISH.">Fluorescence in situ hybridization (FISH): History, limitations and what to expect from micro-scale FISH.</a> Micro and Nano Engineering. 1, 15-24 (2018).</li><li>Hu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+(FISH):+an+increasingly+demanded+tool+for+biomarker+research+and+personalized+medicine.">Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine.</a> Biomarker Research. 2 (1), 3 (2014).</li><li>Hwang, K., Weedin, J. W., Lamb, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+in+situ+hybridization+in+male+infertility.">The use of fluorescent in situ hybridization in male infertility.</a> Therapeutic Advances in Urology. 2 (4), 157-169 (2010).</li><li>Ramasamy, R., Besada, S., Lamb, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+of+human+sperm:+diagnostics,+indications,+and+therapeutic+implications.">Fluorescent in situ hybridization of human sperm: diagnostics, indications, and therapeutic implications.</a> Fertility and Sterility. 102 (6), 1534-1539 (2014).</li><li>Chatziparasidou, A., Christoforidis, N., Samolada, G., Nijs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+aneuploidy+in+infertile+male+patients:+a+systematic+review+of+the+literature.">Sperm aneuploidy in infertile male patients: a systematic review of the literature.</a> Andrologia. 47 (8), 847-860 (2015).</li><li>Schleedoorn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TurnerFertility+trial:+PROTOCOL+for+an+observational+cohort+study+to+describe+the+efficacy+of+ovarian+tissue+cryopreservation+for+fertility+preservation+in+females+with+Turner+syndrome.">TurnerFertility trial: PROTOCOL for an observational cohort study to describe the efficacy of ovarian tissue cryopreservation for fertility preservation in females with Turner syndrome.</a> BMJ Open. 9 (12), 030855 (2019).</li><li>Peek, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+follicles+of+young+patients+with+Turner's+syndrome+contain+normal+oocytes+but+monosomic+45,X+granulosa+cells.">Ovarian follicles of young patients with Turner's syndrome contain normal oocytes but monosomic 45,X granulosa cells.</a> Human Reproduction. 34 (9), 1686-1696 (2019).</li><li>Nadesapillai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+are+some+patients+with+45,X+Turner+syndrome+fertile?+A+young+girl+with+classical+45,X+Turner+syndrome+and+a+cryptic+mosaicism+in+the+ovary.">Why are some patients with 45,X Turner syndrome fertile? A young girl with classical 45,X Turner syndrome and a cryptic mosaicism in the ovary.</a> Fertility and Sterility. 115 (5), 1280-1287 (2021).</li><li>Dolmans, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reimplantation+of+cryopreserved+ovarian+tissue+from+patients+with+acute+lymphoblastic+leukemia+is+potentially+unsafe.">Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe.</a> Blood. 116 (16), 2908-2914 (2010).</li><li>Dath, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+human+ovarian+tissue+to+nude+mice:+comparison+between+four+grafting+sites.">Xenotransplantation of human ovarian tissue to nude mice: comparison between four grafting sites.</a> Human Reproduction. 25 (7), 1734-1743 (2010).</li><li>Cacciottola, L., Donnez, J., Dolmans, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+tissue+damage+after+grafting:+systematic+review+of+strategies+to+improve+follicle+outcomes.">Ovarian tissue damage after grafting: systematic review of strategies to improve follicle outcomes.</a> Reproductive BioMedicine Online. 43 (3), 351-369 (2021).</li><li>Bishop, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+fluorescence+in+situ+hybridization+(FISH)+in+detecting+genetic+aberrations+of+medical+significance.">Applications of fluorescence in situ hybridization (FISH) in detecting genetic aberrations of medical significance.</a> Bioscience Horizons. 3 (1), 85-95 (2010).</li><li>Burgoyne, P. S., Mahadevaiah, S. K., Turner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+consequences+of+asynapsis+for+mammalian+meiosis.">The consequences of asynapsis for mammalian meiosis.</a> Nature Reviews Genetics. 10 (3), 207-216 (2009).</li></ol>

FISH 分析是一种众所周知的技术，用于检测男性和女性淋巴细胞或颊细胞中的 X 染色体畸变10.一些研究已经描述了FISH对X染色体畸变的男性配子的影响，但FISH获得的关于X染色体畸变女性卵巢细胞的详细信息仍然缺乏14。本文提出了基于FISH的新方法，以确定X染色体畸变女性的非移植和移植卵巢皮层组织的卵巢细胞中是否存在非整倍性。
在非?...

不孕症是男性或女性生殖系统的健康问题，影响全世界约1.86亿育龄人1。在至少35%的不孕夫妇中，不孕症是由女性生殖系统疾病引起的2。导致女性不孕的因素很多，如遗传因素、生殖道异常、内分泌功能障碍、炎症性疾病、医源性治疗等3。
遗传异常存在于大约 10% 的不育女性中4，5。在所有遗传异常中，X染色体畸变是卵巢发育不全的最常见原因2。一些研究报告，患有特纳综合征 （TS） 或三重 X 综合征的女性的 X 染色体畸变与生殖细胞加速丢失或卵子生成受损导致的卵巢早衰有关6，7，8。
X染色体畸变可分为：1）数值畸变，其中X染色体数量不同但X染色体完整;2）结构畸变，其中X染色体获得或失去遗传物质3，9。X染色体的数值畸变比结构异常更常见，通常是由细胞分裂过程中的自发错误引起的3，9。当减数分裂期间发生这种错误时，可能会导致非整倍体配子，并最终导致所有细胞中染色体畸变的后代。当体细胞中由于个体发生早期有丝分裂过程中发生的错误而导致体细胞中出现染色体缺陷时，可能会导致嵌合体。在这些个体中，存在具有正常X染色体含量的细胞和具有X染色体畸变的细胞。
在 1980 年代，开发了一种称为荧光 原位 杂交 （FISH） 的细胞遗传学技术，用于可视化和定位中期和间期染色体10，11 上的特定核酸序列。该技术使用荧光标记的DNA探针与染色体中的特定序列结合，然后可以使用荧光显微镜将其可视化。
如今，FISH被广泛用作临床诊断工具，被认为是检测染色体畸变的金标准10。在生殖医学领域，对精子的FISH分析已被用于深入了解体细胞中具有数字或结构染色体畸变的男性精子的X染色体含量12，13，14。这些研究表明，与具有正常核型12，13，14的男性相比，染色体畸变的男性更有可能在其精液中出现更高的非整倍体精子的频率。
与精子相反，对染色体畸变个体卵巢细胞（包括卵母细胞、颗粒/膜细胞和基质细胞）的 X 染色体含量知之甚少，以及这些细胞的非整倍性对其生殖潜力的可能后果。与精子相比，卵巢细胞核型信息稀缺的一个重要原因是女性必须接受侵入性手术，例如卵泡穿刺或手术以获得卵母细胞或卵巢皮层组织。因此，很难获得用于研究目的的雌配子。
目前，荷兰正在进行一项观察性干预研究，以探索卵巢组织冷冻保存对TS15年轻女性的疗效。患者的卵巢皮层组织的一个片段可用于鉴定卵巢细胞的X染色体含量16，17。作为研究的一部分，开发了一种基于解离卵巢皮层组织的FISH的新方法，以确定在非卵巢体细胞（如淋巴细胞或颊细胞）中携带染色体畸变的女性卵巢细胞中是否存在染色体畸变。此外，还确定了卵巢细胞中非整倍性对卵泡生成的影响。为此，修改了一个已建立的FISH方案，以便在免疫功能低下的小鼠长期异种移植期间人工诱导的卵泡生成后分析卵巢皮层组织的组织学切片。在这项研究中，我们提出了两种基于FISH的方法来确定X染色体畸变女性非移植和移植卵巢皮层组织中卵巢细胞中的X染色体含量，旨在提高该主题的基础科学。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Biosolve BV</td>
			<td>0001070602BS</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 1200</td>
			<td>Hettich Universal</td>
			<td>4140</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Sigma</td>
			<td>131470</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>VWR</td>
			<td>0631-0146</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vector</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline&#160;</td>
			<td>Lonza</td>
			<td>BE17-513Q</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Merck</td>
			<td>108421</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin-Y</td>
			<td>Sigma</td>
			<td>1159350100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMSURE</td>
			<td>1009832500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life technology</td>
			<td>10100147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope for sections DM4 B</td>
			<td>Leica Microsystems&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope scope A1</td>
			<td>Zeiss AXIO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent labeled probes for dissociated cells</td>
			<td>Abbott Diagnostics</td>
			<td>CEPX (DXZ1) 05J1023 
			CEP18 (D18Z1) 05J0818</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent labeled probes for tissue sections</td>
			<td>Abbott Diagnostics</td>
			<td>CEP X (DXZ1 05J08-023 
			CEP 18 (D18Z1)&#160; 05J10-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma</td>
			<td>252549</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>108337</td>
			<td></td>
		</tr>
		<tr>
			<td>Glue (Fixogum)</td>
			<td>Leica Microsystems</td>
			<td>LK071A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma</td>
			<td>1159380025</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization buffer</td>
			<td>Abott Diagnostics</td>
			<td>32-804826/06J67-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Station&#160;</td>
			<td>Dako</td>
			<td>S2451</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Merck</td>
			<td>1003171000</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing software individual ovarian cortex cells (Cytovision 7.7)</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing software on paraffine sections&#160;</td>
			<td>Leica Application Suitex (3.7.5.24914)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immunohitochemistry microscope slides</td>
			<td>Dako</td>
			<td>K802021-2</td>
			<td></td>
		</tr>
		<tr>
			<td>L15</td>
			<td>Lonza</td>
			<td>12-700Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase DH</td>
			<td>Roche</td>
			<td>05 401 151 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Zeiss West Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulphate</td>
			<td>Merck</td>
			<td>A335586</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Honeywell</td>
			<td>14262-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Vectashield, Vector</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P40</td>
			<td>Sigma</td>
			<td>7385-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Poth Hile</td>
			<td>2712.20.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma</td>
			<td>P7000-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>11530546</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic pipette</td>
			<td>CooperSurgical</td>
			<td>7-72-4075/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride&#160;</td>
			<td>Merck</td>
			<td>1049361000</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Qiagen</td>
			<td>19131</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotation microtome HM 355S</td>
			<td>Thermo sceintific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Dahlhausen</td>
			<td>11.000.00.515</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide for FISH on dissociated cells</td>
			<td>Thermo scientific</td>
			<td>J1810AM1JZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>55761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Sodium Citrate (SSC)</td>
			<td>Fisher Scientific, Invitrogen</td>
			<td>10515203</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope IX 70</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Target Retrieval Solution &#160;&#160;</td>
			<td>Dako</td>
			<td>GV80511-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>ThermoFisher</td>
			<td>85113</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>BOOM</td>
			<td>760518191000</td>
			<td></td>
		</tr>
	</tbody>
</table>

exploring x chromosomal aberrations in ovarian cells by using fluorescence in situ hybridization

全世界有数百万人处理有关生育的问题。生育能力下降，甚至不孕症，可能是由于许多不同的原因，包括遗传性疾病，其中染色体异常是最常见的。荧光 原 位杂交（FISH）是检测人类染色体畸变的众所周知且常用的方法。FISH主要用于分析具有数值或结构染色体畸变的雄性精子中的染色体异常。此外，该技术也经常应用于女性，以检测已知会导致卵巢发育不良的X染色体畸变。然而，仍然缺乏关于淋巴细胞和/或颊细胞中X染色体畸变的女性卵巢细胞的X染色体含量的信息。
本研究的目的是通过提出两种基于FISH的方法来鉴定卵巢细胞的X染色体含量，从而推进有关女性X染色体畸变的基础研究。首先，描述了一种测定X染色体畸变女性非移植卵巢皮层组织中分离的卵巢细胞（卵母细胞，颗粒细胞和基质细胞）的X染色体含量的方法。第二种方法旨在通过确定卵巢组织中新形成的二级和窦卵泡的卵巢细胞的X染色体含量来评估染色体畸变对卵泡发生的影响，这些卵巢组织中来自长期移植到免疫功能低下小鼠后具有X染色体畸变的雌性。这两种方法都可能有助于未来的研究，以深入了解具有X染色体畸变的女性的生殖潜力。

移植前分离卵巢细胞上的 FISH 使用具有45，X / 46，XX（患者A）或45，X / 46，XX / 47，XXX（患者B）TS的女性冷冻保存的卵巢皮层组织来说明使用该协议的结果。在患者A中，50%的淋巴细胞具有45，X核型，50%具有46，XX。在患者B中，38%的淋巴细胞为45，X，28%为46，XX，34%为47，XXX。X号染色体（绿色）和18号染色体的着丝粒特异性探针作为对照（红色）用于确定从未接受异种移植的TS患者的卵巢?...

Watch this Scientific Journal Video about Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization at JoVE.com

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

本文提出了两种基于荧光 原位 杂交的方法，以确定X染色体畸变女性非移植和移植卵巢皮层组织中卵巢细胞的X染色体含量。

利用荧光 原位 杂交探索卵巢细胞中的X染色体畸变

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

对患有X染色体畸变的女性卵巢细胞进行FISH分析是一种独特的技术，可以深入了解该特定组中卵巢细胞的X染色体含量。这些技术可能有助于更多地了解X染色体畸变女性的生殖潜力。值得一提的是，这两种方法都是为了促进未来的研究，而不是为了在临床实践中用作诊断工具而设计的。演示该程序的将是，我们生育实验室的高级研究员Ronald Peek和我们病理部门的分析师Milad Intezar。首先，使用手术刀将冷冻保存或解冻的卵巢皮层组织切成一毫米一毫米的小块。在37摄氏度下酶消化四升预热L15中的组织碎片75分钟。每 15 分钟上下移取一次消化混合物。通过添加四毫升补充有10%胎牛血清或FBS的冷L15来停止酶消化。用8毫升冷L15培养基以500g离心洗涤解离的组织一次，然后重新悬浮在500微升L15培养基中。将含有大部分单个基质细胞和小卵泡的细胞悬液转移到塑料培养皿中，并在体视显微镜下检查悬浮液。使用75微米的塑料移液管手动挑选小于50微米的卵泡，并将它们转移到补充有10%FBS的L15培养基的液滴中，温度为4摄氏度。然后将纯化的卵泡转移到0.06%胰蛋白酶，每毫升EDTA1毫克和每毫升葡萄糖1毫克的溶液中，并在37摄氏度下孵育20分钟。孵育后，使用75微米塑料移液管从皮质细胞悬液中获得卵巢基质细胞。对于单个卵巢细胞的荧光原位杂交或FISH分析，将处理过的带有胰蛋白酶/ EDTA /葡萄糖或基质细胞的卵巢卵泡转移到5微升0.15毫摩尔氯化钾和15微升DPBS的液滴上载玻片，并在37摄氏度下孵育20分钟。干燥并将载玻片预先固定在 300 微升 0.05 毫摩尔氯化钾、7.5% 乙酸和 22.5% 甲醇中两分钟。用三比一甲醇乙酸覆盖载玻片两分钟以完成固定。固定后，在73摄氏度下在新制备的SSC中洗涤样品两次。用100微升2%蛋白酶K盖住，并用盖玻片密封。将载玻片在37摄氏度下在杂交站中孵育10分钟。孵育后，取下盖玻片，并在室温下振荡下在DPBS中洗涤载玻片5分钟。用1%甲醛固定样品五分钟。然后在DPBS中洗涤载玻片，并以增加的乙醇浓度连续脱水两分钟。在与荧光标记的探针杂交之前，风干样品。接下来，向样品中加入1微升CEPX，1微升CEP18和18微升杂交缓冲液，并用粘在载玻片上的盖玻片密封。将载玻片转移到杂交站，在73摄氏度下变性三分钟，然后在37摄氏度下孵育过夜。杂交后，从载玻片上取下盖玻片和任何剩余的胶水。在 0.4X SSC、0.3% 吐温-20 在 72 摄氏度下洗涤载玻片两分钟，然后在 2X SSC 和 0.1% 吐温-20 中孵育一分钟。如图所示使载玻片脱水，并在黑暗中风干，然后用含有DAPI的封片剂覆盖。将载玻片置于零下20摄氏度下10分钟，然后通过荧光显微镜进行分析。要杂交石蜡切片，请在包含石蜡碳带卵泡的切片之前或之后选择新切片。将一部分安装在载玻片上，然后在 45 摄氏度的炉子上干燥 56 分钟。将切片在二甲苯中脱蜡10分钟，然后将载玻片浸入99.5%乙醇中，并在自来水中冲洗5分钟。用目标检索溶液在96摄氏度下预处理载玻片10分钟。冷却后，用蒸馏水冲洗载玻片。用0.01摩尔盐酸处理载玻片5分钟，然后在37摄氏度下消化胃蛋白酶15分钟。再次用盐酸冲洗载玻片，随后在PBS中冲洗载玻片。将载玻片固定在 1% 甲醛 PBS 中五分钟。然后在PBS中短暂冲洗载玻片，然后在99.5%乙醇中脱水之前再次在软化水中冲洗载玻片。在预处理的载玻片上涂抹五微升探针CEP18和CEPX。然后涂上盖玻片并用照片胶密封该区域。将载玻片放入杂交器中，在80摄氏度下变性10分钟，在37摄氏度下杂交过夜。第二天，在 42 摄氏度的 SSC 中冲洗载玻片两次，每次五分钟，然后在 0.3% Nonidet P-40 中以 73 摄氏度冲洗两分钟和一分钟。再次，在2X SSC中冲洗载玻片，然后在蒸馏水中冲洗，然后用99.5%乙醇脱水。风干载玻片，并将其安装在含DAPI的安装介质中。患者A的冷冻保存卵巢皮层组织显示50%的淋巴细胞，具有45，X核型，50%具有46，XX.In 患者B，38%的淋巴细胞为45，X，28%为46，XX和34%为47，XXX。还分析了周围的基质细胞的X染色体含量。卵巢皮层组织的酶消化导致大部分解离细胞的悬浮液，但留下原始卵泡，由被单层肉芽细胞包围的完整卵母细胞组成。由于颗粒细胞的聚集，小卵泡在FISH后解释信号提供了困难。在FISH之前用胰蛋白酶对卵泡进行额外消化导致单个颗粒病细胞在卵泡表面收缩成球形形态。苏木精伊红染色显示异种移植5个月后卵巢皮层组织中的二级和窦卵泡形态正常。通过FISH分析测定这些卵泡的颗粒病细胞的X染色体含量。将移植的卵巢皮层组织固定在Bouin溶液中，由于绿色雾霾，滤泡细胞中的荧光信号模糊且在很大程度上模糊。相比之下，固定在甲醛中的卵巢皮层组织提供了极好的荧光信号。该方案的主要挑战在于卵巢细胞的分离和组织的酶消化。在此过程中，偏离此协议会导致卵巢细胞大量损失，对于已经卵巢储备较低的女性，应特别避免这种情况。额外的基因表达谱可能有助于更多地了解非整倍体卵巢细胞对卵泡生成的影响，从而了解X染色体畸变女性卵巢早衰的过程。

Research

JoVE Journal

Genetics

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

科研

遗传学

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

通过多色荧光跨染色体畸变稳定的检测<em&gt;原位</em&gt;杂交（渔业部）和光谱核型分析（SKY）的照射小鼠

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

全挂载原位杂交法可视化蜱内脏微生物群

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

利用荧光 原位 杂交探索卵巢细胞中的X染色体畸变

利用荧光原位杂交探索卵巢细胞中的X染色体畸变