Extracellular Vesicles and the Oviduct Function

Abstract

In mammals, the oviduct (or the Fallopian tube in humans) can be divided into the infundibulum (responsible for oocyte pick-up), ampulla (site of fertilization), isthmus (where preimplantation embryos develop), and uterotubal junction (where embryos transit to the uterus). The oviductal fluid, as well as extracellular vesicles produced from the oviduct epithelial cells, referred to as oEVs, have been shown to improve the fertilization process, prevent polyspermy, and aid in embryo development. oEVs contain molecular cargos (such as miRNAs, mRNAs, proteins, and lipids) that can be delivered and fuse to recipient cells. oEVs produced from the ampulla appear to be functionally distinct from those produced from the isthmus. In multiple species including mice, cats, dogs, pigs, and cows, oEVs can be incorporated into the oocytes, sperm, and embryos. In this review, we show the positive impact of oEVs on gamete function as well as blastocyst development and how they may improve embryo quality in in vitro conditions in an assisted reproductive technology setting for rodents, domestic animals, farm animals, and humans.

Keywords: egg; embryo; exosome; extracellular vesicle; fallopian tube; microvesicle; oocyte; oviduct; oviductosome; sperm.

Figure 1

Transmission electron microscopy images of extracellular vesicles present in the oviductal fluid from different species, including rodents, domestic animals, farm animals, reptiles, and humans. (A) Mouse: exosome (*) and microvesicles (**). Reprint with permission from [23]. (B) Cat: extracellular vesicles (*). Reprint with permission from [28]. (C) Dog: oEVs. Reprint with permission from [25]. (D) Pig: exosomes (*) and microvesicles (arrows). Reprint with permission from [26]. (E) Cow: exosomes (blue arrows) and microvesicles (red arrows) isolated from in vivo oviduct. Reprint with permission from [24]. (F) Soft-shelled turtle: extracellular vesicles (arrows) in the lumen of the oviduct from the isthmus region. Reprint with permission from [27]. C; cilia, Ex; exosome, Sr; oviduct secretions, Sv; secretory vesicles. (G,H) Human: exosomes (*) and microvesicles (**) with or without immunogold labeling. Reprint with permission from [19].

Figure 2

EVs from the oviduct are transferred to the oocytes in dogs (left panel) and pigs (right panel). (Left) Canine oEV incorporation in the canine cumulus-oocyte increases over time. (A,B) negative control, (C,D) 24 h, (E,F) 48 h, and (G,H) 72 h after incubation with (PKH67)-labeled canine oEVs. (I) Incorporation of exosomes as measured by fluorescent intensity over time. Bright field images (A,C,E,G), green fluorescent (PKH67) images (B,D,F,H). Length of scale bars was not indicated in the original study. Reprint with permission from [58]. (Right) Images of oocytes from pigs after 20 h of incubation in the presence of (A,C) negative control or (B,D) 0.4 μg/μL of green florescent (PKH67)-labelled porcine oEVs. (A,B) porcine denuded mature oocytes. (C,D) cumulus-oocyte complex. ZP; zona pellucida, PM; plasma membrane, blue; Hoechst staining, green; PKH67-EVs staining. Scale bars; 50 μm. Reprint with permission from [59].

Figure 3

EVs from the oviduct are transferred to the sperm using fusogenic mechanism. (A–C) Mouse sperm. (A) Pmca4^+/+ and (B) Pmca4^−/− sperm incubated with FM4–64FX-labeled oviductosome (red fluorescent signal) and PMCA4 antibody (green fluorescent signal). Yellow arrows; acrosome, white arrows; sperm midpiece, blue arrows; posterior head, red arrows; neck, *; distal midpiece; aqua bar, intense staining of FM4–64FX-labeled oviductosomes. Sperm nuclei were stained with DRAQ5 (blue). (C) Fusion of microvesicles on the sperm membrane from WT mice. Green arrow; fusion of FM4–64FX-labeled microvesicles over the acrosome. Transmission electron microscopy (TEM) analysis indicates the fusion (green arrow). Inset shows transfer of gold particles (PMCA4, yellow arrow) from OVS to the sperm membrane over the acrosome. Reprint with permission from [23]. (D) Uptake of oEVs by sperm in domestic cats: (a) cat sperm incubated with red-fluorescent labeled oEVs, arrow indicates that EVs bind to sperm acrosome and mid-piece (dashed box). TEM images (b–e) of sperm incubated with EVs (black arrows), indicating that EVs bound to sperm head (b,c) and mid-piece (d,e). Abbreviations: N; nucleus, A; acrosome, Ax; axoneme, and M; mitochondria. Scale bar: yellow; 15 μm, black; 200 nm. Reprint with permission from [28]. (E) Incorporation of porcine oviductal EVs labeled with PKH67 (a green fluorescent dye) at different regions of pig sperm including (A) head, (B) head and intermediate piece, and (C) head, intermediate piece, and flagella. Reprint with permission from [59].

Figure 4

Incorporation of bovine oEVs into the blastocyst. (A–C) oEVs from bovine oviducts labeled with green fluorescent dye (PKH67) are incorporated into cytoplasmic contents in the bovine blastocyst. (C) High magnification image shows that in vivo-derived oEVs attach to the blastocyst plasma membrane and are internalized into the cytoplasm. Hoechst 3342 staining; nucleus. Reprint with permission from [24].