Extracellular vesicles would be involved in the release and delivery of seminal TGF-β isoforms in pigs

Abstract

IntroductionPig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor β (TGF-β), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-βs are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs).MethodsSource of TGF-βs was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP® technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs.ResultsAll three TGF-β isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-β isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-β isoforms and demonstrated that a substantial portion of them is associated with sEVs.DiscussionSeminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-β isoforms and in their safe transport from the male to the female reproductive tract.

Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma

“…In fact, most conventional flow cytometers are unable to detect and immunophenotype the vast majority of EVs because the reduced surface area of EVs results in low expression of surface proteins that can be masked by background noise [ 39 , 41 ]. The Cytoflex S flow cytometer used in this study has been shown to be useful for EV analysis [ 13 , 42 – 45 ] and has been successfully used for surface protein identification on porcine sEVs [ 24 , 46 – 48 ].…”

“…Efficient EV-labeling dyes should be used, and several lipid-, protein- and nucleic acid-binding fluorescent dyes have been proposed, including CFSE, BODIPY, long-chain dialkylcarbocyanines (DiD, Dil and DiO), PKH67, PKH26, among others [ 27 , 52 ]. In our study, sEVs were labeled using CFSE, a fluorescent membrane-permeable amine-reactive dye that is widely used for EV labeling [ 27 , 53 – 55 ], even for those isolated from porcine SP [ 24 , 46 – 48 ]. CFSE was chosen because it does not promote aggregate formation and therefore does not alter the typical light-scattering pattern of EVs [ 27 ].…”

The Cation/Calcium Channel of Sperm (CatSper): A Common Role Played Despite Inter-Species Variation?

“…[92][93][94]. The SEVs are crucial extracellular structures that interact with sperm cells and even provide the latter with their membrane after SEV-sperm fusing, thus incorporating into the sperm cells a plethora of channels and receptors with key roles in sperm function [92,[94][95][96][97][98]. Further implications of SEVs in sperm function and membrane exchanges are expected to be elucidated by future research.…”