Hartmann Sonja, Bergmann Martin, Bohle Rainer M, Weidner Wolfgang, Steger Klaus
Department of Urology and Pediatric Urology, Institute of Veterinary Anatomy, Histology and Embryology and Institute of Pathology, University of Giessen, Giessen Germany.
Mol Hum Reprod. 2006 Jun;12(6):407-11. doi: 10.1093/molehr/gal040. Epub 2006 Apr 11.
Disorders in genetic imprinting are discussed as potential genetic risk in assisted reproduction technology (ART), where most of the natural selection mechanisms are bypassed. As currently only limited information about genomic imprinting in disruptive spermatogenesis is available, we analysed the imprinting state of the paternally methylated gene H19 in various germ cell populations derived from seminiferous tubules exhibiting impaired spermatogenesis. Different germ cell types were isolated by laser microdissection from human testicular paraffin sections. Although the methylation state of the maternally imprinted gene SNRPN was investigated by methylation-specific PCR (M-PCR) to establish the isolation method, methylation of H19 was analysed by a single-strand conformation-based method. Contamination by somatic Sertoli cells was excluded because of Sertoli cell-specific vimentin immunohistochemistry before germ cell laser microdissection. We demonstrate correct genetic imprints for H19 even in spermatogonia selected from seminiferous tubules exhibiting spermatogenic arrest at the level of spermatogonia, providing no evidence for incorrect genomic imprinting in spermatozoa from infertile men used for ICSI.
基因印记紊乱被视为辅助生殖技术（ART）中的潜在遗传风险，在ART中，大多数自然选择机制都被绕过。由于目前关于破坏生精过程中基因组印记的信息有限，我们分析了来自生精功能受损的曲细精管的各种生殖细胞群体中父源甲基化基因H19的印记状态。通过激光显微切割从人睾丸石蜡切片中分离出不同的生殖细胞类型。尽管通过甲基化特异性PCR（M-PCR）研究了母源印记基因SNRPN的甲基化状态以建立分离方法，但通过基于单链构象的方法分析了H19的甲基化。在生殖细胞激光显微切割之前，由于支持细胞特异性波形蛋白免疫组织化学，排除了支持体细胞的污染。我们证明，即使在从生精功能在精原细胞水平停滞的曲细精管中选择的精原细胞中，H19的基因印记也是正确的，这没有为用于ICSI的不育男性精子中基因组印记错误提供证据。
