HA-tag CD63 is a novel conditional transgenic approach to track extracellular vesicle interactions with sperm and their transfer at conception

Abstract

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.

miR‐125b‐5p impacts extracellular vesicle biogenesis, trafficking, and EV subpopulation release in the porcine trophoblast by regulating ESCRT‐dependent pathway

“…Next, we verified the involvement of miR‐125b‐5p in the modulation of EV trafficking, focusing on the localization of EV‐related proteins in pTr cells. VPS36 (miR‐125b‐5p target and marker of ESCRT‐II complex present in endosomes/MVBs 47 ) and CD63 (marker of EVs 48 ) were selected based on our recent studies pointing to embryonic signals as triggers of EV trafficking in endometrial luminal epithelial cells 26 . Regions of interest (ROIs) were set based on the overlapping localization of both proteins present in the highly visible and well‐shaped cell borders (white) in comparison to the whole cell (Figure 4A).…”

When Dad’s Stress Gets under Kid’s Skin—Impacts of Stress on Germline Cargo and Embryonic Development

“…This sentiment was corroborated by a different group where changes in sperm miRNA were much more prevalent 12 weeks after four weeks of chronic randomised psychological stress as opposed to one week after, concluding that they had been taken up during epididymal transit, which requires two weeks in mouse [49]. Tracking EVs using a novel labelling approach based on an HAtagged tetraspanin endogenous to EVs, the authors showed EV content to be taken up and present within sperm heads [50]. Conversely, a recent study proposed that the majority of non-coding RNA in sperm is acquired from cytoplasmic droplets, transient organelles storing RNA specifically in testicular and epididymal sperm [51].…”

miR-125b-5p impacts extracellular vesicle biogenesis, trafficking, and EV subpopulation release in the porcine trophoblast by regulating ESCRT-dependent pathway

“…4.4 CD63 and VPS36 showed spatial-dependent colocalization and correlation after miRNA delivery Next, we verified the involvement of miR-125b-5p in the modulation of EV trafficking, focusing on the localization of EV-related proteins in pTr cells. VPS36 (miR-125b-5p target and marker of ESCRT-II complex present in endosomes/MVBs [47]) and CD63 (marker of EVs [48]) were selected based on our recent studies pointing to embryonic signals as triggers of EV trafficking in endometrial luminal epithelial cells [26]. Regions of interest (ROIs) were set based on the overlapping localization of both proteins present in the highly visible and well-shaped cell borders (white) in comparison to the whole cell (Fig.…”