Department of Pharmacology and Center for Epigenetic Research in Child Health and Brain Development, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
Department of Psychiatry, University of Colorado School of Medicine, CU Anschutz Medical Campus, 12800 E. 19th Avenue, Aurora, CO, 80045, USA.
Sci Rep. 2023 Jan 13;13(1):707. doi: 10.1038/s41598-023-27898-5.
Extracellular vesicles (EVs) are a unique mode of intercellular communication capable of specificity in transmitting signals and cargo to coordinate local and distant cellular functions. A key example of this is the essential role that EVs secreted by epithelial cells lining the lumen of the male reproductive tract play in post-spermatogenic sperm maturation. We recently showed in a preclinical mouse model that this fundamental process had a causal role in somatic-to-germline transmission of biological information regarding prior stress experience capable of altering the rate of fetal development. However, critical mechanistic questions remain unanswered as to the processes by which signaling occurs between EVs and sperm, and whether EVs or their cargo are delivered at conception and are detectable in the early embryo. Unfortunately, notable methodological limitations shared across EV biology, particularly in the isolation and labeling of EVs, complicate efforts to answer these important questions as well as questions on EV targeting specificity and mechanisms. In our current studies, we developed a novel approach to track EVs using a conditional transgenic construct designed to label EVs via conditional Cre-induced hemagglutinin (HA) tagging of the EV endogenous tetraspanin, CD63. In our exhaustive validation steps, this internal small molecular weight tag did not affect EV secretion or functionality, a common problem found in the previous design of EV tags using larger molecular weight proteins, including fluorescent proteins. Utilizing a stably transfected immortalized epididymal epithelial cell line, we first validated key parameters of the conditional HA-tagged protein packaged into secreted EVs. Importantly, we systematically confirmed that expression of the CD63-HA had no impact on the production, size distribution, or surface charge of secreted EVs, nor did it alter the tetraspanin or miRNA composition of these EVs. We also utilized the CD63-HA EVs to verify physical interactions with sperm. Finally, using in vitro fertilization we produced some of the first images confirming sperm delivered EV cargo at conception and still detectable in the early-stage embryo. As such, this construct serves as a methodological advance and as a valuable tool, with applications in the study of EV function across biomedical research areas.
细胞外囊泡（EVs）是一种独特的细胞间通讯方式，能够特异性地传递信号和货物，以协调局部和远处的细胞功能。一个关键的例子是，男性生殖道管腔衬里的上皮细胞分泌的 EV 在精子发生后的精子成熟中发挥着至关重要的作用。我们最近在一个临床前小鼠模型中表明，这个基本过程在体细胞到生殖细胞传递关于先前应激经历的生物信息方面起着因果作用，这种信息能够改变胎儿发育的速度。然而，关于 EV 与精子之间信号传递的过程，以及 EV 或其货物是否在受孕时传递并可在早期胚胎中检测到，仍然存在关键的机制问题尚未得到解答。不幸的是，EV 生物学中存在着显著的方法学限制，特别是在 EV 的分离和标记方面，这使得回答这些重要问题以及关于 EV 靶向特异性和机制的问题变得复杂。在我们目前的研究中，我们开发了一种使用条件性转基因构建体来追踪 EV 的新方法，该方法旨在通过条件性 Cre 诱导的 EV 内四跨膜蛋白 CD63 的血凝素（HA）标记来标记 EV。在我们详尽的验证步骤中，这种内部小分子标签不会影响 EV 的分泌或功能，这是以前使用较大分子量蛋白质（包括荧光蛋白）设计 EV 标签时常见的问题。我们利用稳定转染的永生化附睾上皮细胞系，首先验证了包装在分泌性 EV 中的条件性 HA 标记蛋白的关键参数。重要的是，我们系统地证实了 CD63-HA 的表达对分泌性 EV 的产生、大小分布或表面电荷没有影响，也没有改变这些 EV 的四跨膜蛋白或 miRNA 组成。我们还利用 CD63-HA EV 来验证与精子的物理相互作用。最后，我们利用体外受精产生了一些首批图像，证实了精子在受孕时传递了 EV 货物，并仍可在早期胚胎中检测到。因此，这种构建体是一种方法学上的进步，也是一种有价值的工具，可应用于生物医学研究领域中 EV 功能的研究。
