A novel role for milk fat globule‐EGF factor 8 protein (MFGE8) in the mediation of mouse sperm–extracellular vesicle interactions

Abstract

Spermatozoa transition to functional maturity as they are conveyed through the epididymis, a highly specialized region of the male excurrent duct system. Owing to their transcriptionally and translationally inert state, this transformation into fertilization competent cells is driven by complex mechanisms of intercellular communication with the secretory epithelium that delineates the epididymal tubule. Chief among these mechanisms are the release of extracellular vesicles (EV), which have been implicated in the exchange of varied macromolecular cargo with spermatozoa. Here, we describe the optimization of a tractable cell culture model to study the mechanistic basis of sperm-extracellular vesicle interactions. In tandem with receptor inhibition strategies, our data demonstrate the importance of milk fat globule-EGF factor 8 (MFGE8) protein in mediating the efficient exchange of macromolecular EV cargo with mouse spermatozoa; with the MFGE8 integrin-binding Arg-Gly-Asp (RGD) tripeptide motif identified as being of particular importance. Specifically, complementary strategies involving MFGE8 RGD domain ablation, competitive RGD-peptide inhibition and antibody-masking of alpha V integrin receptors, all significantly inhibited the uptake and redistribution of EV-delivered proteins into immature mouse spermatozoa. These collective data implicate the MFGE8 ligand and its cognate integrin receptor in the mediation of the EV interactions that underpin sperm maturation.

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

“…It is generally accepted in the field that somatic-to-germline transmission of paternal experience likely involves delivery of experience-encoding cargo to sperm that then may deliver some aspect of this cargo to the oocyte upon fertilization. Groups have visualized bulk labeled cargo (e.g., biotinylated EV proteins or metabolically labeled RNA) delivery to sperm in vitro, however, in using an exogenous labeling strategy of isolated EV preparations, questions as to the differences in the behavior of specific subsets of EVs (of different cell type or biosynthetic origin) remain 9 , 14 – 16 . In addition, the delivery of epididymal EV cargo to oocytes at fertilization to shape the embryonic epigenome and development has only been inferred from assessments of offspring outcomes following experimental manipulations of the epididymal epithelium 26 , 27 , 31 .…”

“…Sperm exiting the seminiferous tubules of the testes are functionally immature and require interaction with epididymal EVs to undertake their basic physiological function of traversing the female reproductive tract and fertilizing an oocyte. These sperm-EV interactions actively shape the protein, lipid, and sncRNA content associated with sperm 7 , 9 – 16 . In particular, sperm-associated sncRNA profiles display considerable plasticity, being substantially remodeled as the cells transit the epididymis 7 , 11 – 14 , 17 .…”

Proteomic analysis of koala (phascolarctos cinereus) spermatozoa and prostatic bodies

“…Taken together, such findings support the tenet that prostatic bodies may share generalized functional roles with that of eutherian seminal plasma EVs. If this were to be the case, then the physical characteristics that distinguish prostatic bodies, and in particular their abundance and large size, may engender them as an ideal model with which to study the mechanisms of sperm -EV interactions [67,68], as well as the functional consequences that arise from such interactions. In any case, the fact that prostatic bodies are replete with molecular chaperones is of considerable interest owing to the role of this family of proteins in the augmentation of sperm activity [55].…”

Exosomes: New regulators of reproductive development

“…Molecules bound to the zona pellucida are transported to the plasma membrane and coat the acrosome [ 41 ]. The fusion of epididymosomes with sperm and the redistribution of inclusions are mediated by milk fat globule-EGF Factor 8 (MFGE8) [ 42 ].…”