Yilian Huang, Yize Wang, Lou Li, Fei Gong, Ge Lin, Jing Dai
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引用次数: 0
Abstract
Purpose: This study aimed to identify new genes associated with total fertilization failure (TFF) by intracytoplasmic sperm injection (ICSI).
Methods: Whole-exome sequencing (WES) was performed on a patient presenting with TFF by ICSI. To assess the spermatozoa's morphology and ultrastructure, hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM) were employed. Furthermore, immunofluorescence staining (IF) and western blot (WB) techniques were utilized to investigate the alterations in expression and localization of CABS1 following the transfection of two mutant plasmids. Co-immunoprecipitation (Co-IP) was conducted to investigate in interaction of wild-type/mutant CABS1 with another perinuclear theca protein ACTL9. IF was conducted on spermatozoa from the patient to detect the expression levels of CABS1 and PLCζ.
Results: Homozygous mutations in CABS1 were identified in a patient with TFF after ICSI. A high proportion of spermatozoa collected from this man exhibited abnormal morphology and low motility. TEM revealed an absence of the acrosome in the spermatozoa. In vitro experiments have demonstrated that the nonsense mutation in CABS1 leads to truncation of the protein and a reduction in its interaction with ACTL9. IF analysis of spermatozoa from the patient showed a weakened and a diffuse signal for CABS1, along with abnormal localization of the sperm-borne oocyte activation factor PLCζ, ultimately leading to TFF.
Conclusion: Our results suggest that CABS1 may be crucial for acrosome formation and the localization of PLCζ. Mutations in CABS1 may lead to teratozoospermia, TFF, and male infertility.
目的：本研究旨在通过胞浆内单精子注射（ICSI）鉴定与完全受精失败（TFF）相关的新基因。方法：采用ICSI对一例TFF患者进行全外显子组测序（WES）。采用苏木精和伊红（H&E）染色和透射电镜（TEM）观察精子的形态和超微结构。此外，利用免疫荧光染色（IF）和western blot （WB）技术研究了转染两种突变质粒后CABS1的表达和定位变化。采用共免疫沉淀法（Co-IP）研究野生型/突变型CABS1与另一种核周卵膜蛋白ACTL9的相互作用。对患者精子进行IF检测CABS1和PLCζ的表达水平。结果：在ICSI后TFF患者中发现了CABS1纯合突变。从该男子收集的精子中，有很大一部分表现出形态异常和低活力。透射电镜显示精子中没有顶体。体外实验表明，CABS1的无义突变导致该蛋白的截断，并减少其与ACTL9的相互作用。患者精子的IF分析显示CABS1信号减弱和弥漫性，伴随着精子携带的卵母细胞活化因子PLCζ的异常定位，最终导致TFF。结论：我们的研究结果表明，CABS1可能是顶体形成和PLCζ定位的关键。CABS1突变可能导致畸形精子症、TFF和男性不育。
期刊介绍：
The Journal of Assisted Reproduction and Genetics publishes cellular, molecular, genetic, and epigenetic discoveries advancing our understanding of the biology and underlying mechanisms from gametogenesis to offspring health. Special emphasis is placed on the practice and evolution of assisted reproduction technologies (ARTs) with reference to the diagnosis and management of diseases affecting fertility. Our goal is to educate our readership in the translation of basic and clinical discoveries made from human or relevant animal models to the safe and efficacious practice of human ARTs. The scientific rigor and ethical standards embraced by the JARG editorial team ensures a broad international base of expertise guiding the marriage of contemporary clinical research paradigms with basic science discovery. JARG publishes original papers, minireviews, case reports, and opinion pieces often combined into special topic issues that will educate clinicians and scientists with interests in the mechanisms of human development that bear on the treatment of infertility and emerging innovations in human ARTs. The guiding principles of male and female reproductive health impacting pre- and post-conceptional viability and developmental potential are emphasized within the purview of human reproductive health in current and future generations of our species.
The journal is published in cooperation with the American Society for Reproductive Medicine, an organization of more than 8,000 physicians, researchers, nurses, technicians and other professionals dedicated to advancing knowledge and expertise in reproductive biology.
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