Division of Urology, Rhode Island Hospital, Providence, RI, USA.
Department of Pathology and Laboratory Medicine, Brown University, Providence, RI, USA.
Andrology. 2018 Mar;6(2):374-383. doi: 10.1111/andr.12471. Epub 2018 Feb 22.
Sperm RNA is a sensitive monitoring endpoint for male reproductive toxicants, and a potential biomarker to assess male infertility and sperm quality. However, isolation of sperm RNA is a challenging procedure due to the heterogeneous population of cells present in the ejaculate, the low yield of RNA per spermatozoon, and the absence of 18S and 28S ribosomal RNA subunits. The unique biology of spermatozoa has created some uncertainty in the field about RNA isolation methods, indicating the need for rigorous quality control checks to ensure reproducibility of data generated from sperm RNA. Therefore, we developed a reliable and effective protocol for RNA isolation from rat and human spermatozoa that delivers highly purified and intact RNA, verified using RNA-specific electrophoretic chips and molecular biology approaches such as RT-PCR and Western blot analysis. The sperm RNA isolation technique was optimized using rat spermatozoa and then adapted to human spermatozoa. Three steps in the sperm isolation procedure, epididymal fluid collection, sperm purification, and spermatozoon RNA extraction, were evaluated and assessed. The sperm RNA extraction methodology consists of collection of rat epididymal fluid with repeated needle punctures of the epididymis, somatic cell elimination using detergent-based somatic cell lysis buffer (SCLB) and the use of RNA isolation Kit. Rat sperm heads are more resistant to disruption than human spermatozoa, necessitating the addition of mechanical lysis with microbeads and heat in the rat protocol, whereas the human sperm protocol only required lysis buffer. In conclusion, this methodology results in reliable and consistent isolation of high-quality sperm RNA. Using this technique will aid in translation of data collected from animal models, and reproducibility of clinical assessment of male factor fertility using RNA molecular biomarkers.
精子 RNA 是一种用于监测雄性生殖毒性的敏感指标，也是评估男性不育和精子质量的潜在生物标志物。然而，由于精液中存在细胞异质性、每个精子的 RNA 产量低以及缺乏 18S 和 28S 核糖体 RNA 亚基，因此分离精子 RNA 是一项具有挑战性的操作。精子独特的生物学特性给 RNA 分离方法领域带来了一些不确定性，这表明需要进行严格的质量控制检查，以确保从精子 RNA 中生成的数据具有可重复性。因此，我们开发了一种从大鼠和人精子中分离 RNA 的可靠且有效的方法，该方法可提供高度纯化和完整的 RNA，使用 RNA 特异性电泳芯片和 RT-PCR 及 Western blot 分析等分子生物学方法进行验证。该精子 RNA 分离技术在大鼠精子中进行了优化，然后适用于人精子。对精子分离过程中的三个步骤（附睾液采集、精子纯化和精子 RNA 提取）进行了评估。精子 RNA 提取方法包括用针反复穿刺附睾采集大鼠附睾液、用基于去污剂的体细胞裂解缓冲液（SCLB）去除体细胞以及使用 RNA 分离试剂盒。大鼠精子头比人精子更能抵抗破坏，因此大鼠方案需要用微珠进行机械裂解并加热，而人方案仅需要裂解缓冲液。总之，该方法可可靠且一致地分离高质量的精子 RNA。使用该技术将有助于将从动物模型中收集的数据转化，并使基于 RNA 分子生物标志物的男性生育力临床评估具有可重复性。
