Center for Reproductive Medicine, Research Institute Reproduction and Development, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.
Core Facility Cellular Imaging/LCAM-AMC, Department of Medical Biology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.
Cytometry A. 2019 Mar;95(3):309-313. doi: 10.1002/cyto.a.23698. Epub 2018 Dec 19.
Spermiogenesis is the final phase of spermatogenesis during which post-meiotic haploid round spermatids (rSpt) differentiate into elongated spermatozoa and includes several critical cell-specific processes like DNA condensation, formation of the acrosome, and production of the flagellum. Disturbances in this process will lead to complications in sperm development and subsequently cause infertility. As such, studying spermiogenesis has clinical relevance in investigating the etiology of male infertility and will improve our scientific understanding of male germ cell formation. Here, we were able to purify round spermatid and elongated spermatid fractions from a single cryopreserved human testicular tissues sample with an efficiency of 85.4% ± 4.9% and 97.6% ± 0.6%, respectively. We confirmed the cell types by morphology and immunohistochemistry for histone H4 and PNA protein expression. The purity was measured by manual counting of histone H4 positive (round) and negative (elongated) spermatids in both sorted 1 N cell fractions. This method can be applied to both human and rodent studies. Especially in studies with limited access to testicular tissue, this method provides a reliable means to simultaneously isolate these cell types with high purity. Our method allows for further investigation of germ cell development and the process of spermiogenesis in particular, as well as provides a tool to study the etiology of male infertility, including morphological and biochemical assessment of round and elongating spermatids from subfertile men. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
精子发生是精子发生的最后一个阶段，在此期间，减数分裂后的圆形精子细胞（rSpt）分化为长形精子，并包括几个关键的细胞特异性过程，如 DNA 浓缩、顶体的形成和鞭毛的产生。这个过程的紊乱会导致精子发育的并发症，进而导致不育。因此，研究精子发生在研究男性不育症的病因学方面具有临床意义，并将提高我们对男性生殖细胞形成的科学认识。在这里，我们能够从单个冷冻保存的人类睾丸组织样本中有效地纯化圆形精子细胞和伸长精子细胞分数，分别为 85.4%±4.9%和 97.6%±0.6%。我们通过形态学和组蛋白 H4 和 PNA 蛋白表达的免疫组织化学证实了细胞类型。通过手动计数两种分选 1N 细胞分数中组蛋白 H4 阳性（圆形）和阴性（伸长）精子细胞来测量纯度。该方法可应用于人类和啮齿动物研究。特别是在睾丸组织获取有限的研究中，该方法提供了一种可靠的方法，可同时分离这些具有高纯度的细胞类型。我们的方法允许进一步研究生殖细胞发育和精子发生过程，特别是为研究男性不育症的病因提供了一种工具，包括对生育能力低下的男性的圆形和伸长精子细胞进行形态和生化评估。©2018 作者。细胞仪部分 A 由 Wiley 期刊出版，代表国际细胞分析促进协会出版。
