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

摘要

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

摘要

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.

The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

引言

Infertility is a health issue of the male or female reproductive system, affecting approximately 186 million individuals of reproductive age worldwide¹. In at least 35% of infertile couples, infertility is caused by a disorder of the female reproductive system². There are many factors that can cause female infertility, such as genetic factors, genital tract abnormalities, endocrine dysfunction, inflammatory diseases, and iatrogenic treatment³.

Genetic abnormalities are present in approximately 10% of infertile females^4,

方案

The TurnerFertility study protocol has been approved by the Central Committee on Research Involving Human Subjects (NL57738.000.16). In this study, the ovarian cortex tissue of 93 females with TS was obtained. Materials that require safety precautions are listed in Table 1.

Table 1: Safety precautions.

Material	Hazard
Acetic acid	Severe skin burns and irritation of the respiratory sys....

结果

FISH on isolated ovarian cells prior to grafting
Cryopreserved ovarian cortex tissue from females with 45,X/46,XX (patient A) or 45,X/46,XX/47,XXX (patient B) TS were used to illustrate the results using this protocol. In patient A, 50% of the lymphocytes had a 45,X karyotype and 50% had 46,XX. In patient B, 38% of the lymphocytes were 45,X, 28% were 46,XX, and 34% were 47,XXX. Centromere-specific probes for chromosome X (green) and chromosome 18 as the control (red) were used to determine the X chro.......

讨论

FISH analysis is a well-known technique to detect X chromosomal aberrations in lymphocytes or buccal cells of both males and females¹⁰. Several studies have described FISH on gametes of males with X chromosomal aberrations, but detailed information obtained by FISH on ovarian cells from females with X chromosomal aberrations is still lacking¹⁴. This article presents novel methods based on FISH to determine if aneuploidy is present in the ovarian cells of non-grafted and gra.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
乙酸	Biosolve BV	0001070602BS
离心机 1200	Hettich Universal	4140
胶原酶 I	Sigma	131470
盖玻片	VWR	0631-0146
DAPI	载体	H-1200
DNase I	罗氏	10104159001
杜尔贝科s 磷酸盐缓冲盐水	龙沙	BE17-513Q
EDTA	默克	108421
曙红-Y	Sigma	1159350100
乙醇	EMSURE	1009832500
胎牛血清 （FBS）	生命技术	10100147
用于切片的荧光显微镜 DM4 B	徕卡显微系统
荧光显微镜范围 A1	蔡司 AXIO
解离细胞的荧光标记探针	雅培诊断	CEPX （DXZ1） 05J1023 CEP18 （D18Z1） 05J0818
组织切片荧光标记探针	雅培诊断	CEP X （DXZ1 05J08-023 CEP 18 （D18Z1）  05J10-028
甲醛	Sigma	252549
葡萄糖	Merck	108337
胶 （Fixogum）	徕卡显微系统	LK071A
苏木精	Sigma	1159380025
杂交缓冲液	Abott Diagnostics	32-804826/06J67-001
杂交站	Dako	S2451
盐酸	Merck	1003171000
图像处理软件 单个卵巢皮质细胞 （Cytovision 7.7）	Leica Biosystems
石蜡切片图像处理软件	Leica Application Suitex （3.7.5.24914）
免疫组织化学显微镜载玻片	Dako	K802021-2
L15	Lonza	12-700Q
Liberase DH	Roche	05 401 151 001
光学显微镜	蔡司西德
镁	默克	A335586
甲醇	霍尼韦尔	14262-1L
封固剂	Vectashield， Vector	H-1000
Nonidet P40	Sigma	7385-1L
石蜡	Poth Hile	2712.20.10
胃蛋白酶	Sigma	P7000-25G
磷酸盐缓冲盐水 （PBS）	Gibco	11530546
塑料移液管	CooperSurgical	7-72-4075/1
氯化钾	Merck	1049361000
蛋白酶 K	Qiagen	19131
旋转切片机 HM 355S	Thermo 舞台
手术刀	Dahlhausen	11.000.00.515
用于分离细胞上 FISH 的载玻片	Thermo Scientific	J1810AM1JZ
氢钠	Sigma	55761-500G
标准柠檬酸钠 （SSC）	Fisher Scientific， Invitrogen	10515203
Stereomicroscope IX 70	奥林巴斯
靶标检索解决方案	Dako	GV80511-2
蛋白酶	Sigma	T4799
吐温-20	ThermoFisher	85113
二甲苯	BOOM	760518191000