Effect of prolonged freezing of semen on exosome recovery and biologic activity

Abstract

Exosomes are important vehicles of intercellular communication that shape host responses to physiologic, tumorigenic, and pathogenic conditions. The composition and function of exosomes are dynamic and depends on the state and condition of the cellular source. In prior work, we found that semen exosomes (SE) from healthy donors who do not use illicit drugs potently inhibit HIV-1. Following semen donation, specimens are either used immediately or frozen for use at a later time. It has been shown that short-term freezing of semen has no effect on SE-mediated HIV-1 inhibition. However, the effect of illicit drugs and prolonged freezing on SE bioactivity is unknown. Here, we show preservation of SE physical properties, (morphology, concentration, intensity/size) irrespective of illicit drug use or duration of semen freezing. Interestingly, illicit drugs and prolonged freezing decreased the levels of SE-bound CD63/CD9 and acetylcholinesterase activity respectively. Furthermore, we show differential effects of illicit drug use and prolonged freezing on SE-mediated HIV-1 inhibition. Our results highlight the importance of the source of SE and condition of semen storage on SE content and function. In-depth evaluation of donor drug-use and duration of semen storage on SE cargo and bioactivity will advance our understanding of SE composition and function.

Semen exosomes inhibit HIV infection and HIV‐induced proinflammatory cytokine production independent of the activation state of primary lymphocytes

“…We conclude from these data that exposure of lymphocytes to the level of SE used in this study (100 µg·mL −1 ) does not alter basal or stimuli‐induced cell death. These results are consistent with observations in cell lines .…”

“…As shown by others, PBMC Q may represent a barrier to productive HIV infection where in vitro HIV replication is severely restricted. Consistent with previous results in pertinent cell lines [10,11,26], SE significantly reduced HIV RNA levels in PBMC TCR from six donors in a donor-dependent manner (Fig. 2F).…”

Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays

“…The variability in the cells that secret exosomes is reflected in the composition and function of exosomes. Thus, exosomal cargo composition and function are regulated by many factors including the type and condition of the originating cell (Raposo and Stoorvogel, 2013), cellular environment, and for in vivo derived exosomes; the condition of the donor (Welch et al , 2017). Released exosomes when taken up by target cells transfer their cargo, including proteins (Iglesias et al , 2012; Charrier et al , 2014), miRNA (Shtam et al , 2013; Ong et al , 2014), and mRNA (Tomasoni et al , 2013; Madison et al , 2014; Madison et al , 2015) to the target cells.…”

“…All authors reviewed the manuscript and approved the final version. Protocols described herein are adapted from our previously published works (Madison et al ., 2014 and 2015; Welch et al ., 2017). …”

Comprehensive proteomics analysis of exosomes derived from human seminal plasma

“…Although being exocytosed, the generality of prostasomes was unknown at that time. Recently, these seminal prostasomes from seminal plasma were reportedly morphologically and molecularly consistent with exosomes and were appropriately referred to as seminal exosomes (SEs) (Madison et al ., ; Vojtech et al ., ; Welch et al ., ). Due to the difficulties in harvesting sufficient amounts of normal prostatic secretions to get ‘true prostasomes’ for proteomics analysis in the present study, we used SEs purified from seminal plasma.…”