Seminal extracellular vesicles subsets modulate gene expression in cumulus cells of porcine in vitro matured oocytes

Abstract

Seminal plasma (SP), a fluid composed mainly by secretions from accessory sex glands, contains a heterogenous population of extracellular vesicles (EVs), involved in several reproductive physiological processes. Seminal plasma has been found to modulate ovary function, in terms of hormone secretion and immune regulation. This study evaluated the potential effect of SP-EV-subsets on the modulation of cumulus-oocyte-complex (COCs) physiology during in vitro maturation (IVM). Two SP-EV-subsets, small-EVs (S-EVs) and large-EVs (L-EVs), were isolated from pig SP by size-exclusion-chromatography. Next, COCs were IVM in the absence (control) or presence of each SP-EV-subset to evaluate their uptake by COCs (PKH67-EVs labelling) and their effect on oocyte and cumulus cells (CCs) (gene expression, and progesterone and estradiol-17β levels). S-EVs and L-EVs were able to bind CCs but not oocytes. Supplementation with L-EVs induced changes (P ≤ 0.05) in the transcript levels of oocyte maturation- (HAS2) and steroidogenesis-related genes (CYP11A1 and HSD3B1) in CCs. No effect on nuclear oocyte maturation and progesterone and estradiol-17β levels was observed when COCs were IVM with any of the two SP-EV-subsets. In conclusion, while SP-EV-subsets can be integrated by CCs during IVM, they do not affect oocyte maturation and only L-EVs are able to modulate CCs function, mainly modifying the expression of steroidogenesis-related genes.

Figure 1

Characterisation of small- (S-EV) and large-extracellular vesicles (L-EV) isolated from seminal plasma samples (SP; n = 7) using Size Exclusion Chromatography (SEC). (A) Violin plots represent total protein concentration levels and distribution in the two SP-EV subsets (pink: S-EVs; purple: L-EVs). Dashed line represents the median and dotted lines the 25–75% quartiles. This figure was created using GraphPad Prism version 8.2.1 (GraphPad Software, Inc., La Jolla, CA, USA; https://www.graphpad.com/). (B) Particle size distribution of the two SP-EV subsets assessed by dynamic light scattering (DLS). Each curve represents an average of intensity size distributions of all samples for S-EVs (pink) and L-EVs (purple). (C) Representative histogram of particle size distribution of the two SP-EV subsets assessed by nanoparticle tracking analysis (NTA). (D) Morphology and size of SP-EV subsets using transmission electron microscopy (TEM). (E) Flow cytometry analysis of SP-EV subsets. Representative histogram of CFSE/CD44/HSP90β/ALB expression in the two EV-subsets. CFSE: Carboxyfluorescein succinimidyl ester; ALB: Albumin.

Figure 2

Uptake of seminal plasma extracellular vesicles (SP-EV) by COCs during in vitro maturation (IVM). Representative figures of the uptake of small- (S-EV) and large-EV (L-EV) subsets by COCs. SP-EVs were stained using PKH67 and added to medium for the whole IVM process. Scale bar 50 µm.

Figure 3

Violin plots representing relative expression levels of BAX1, BCL2, CCNB1, CX53, CYP11A1, CYP19A1, HAS2, HSD3B1 and SCD1 in cumulus cells in response to the presence of small- (SP-EV) or large-extracellular vesicles (L-EV) at two concentrations (Protein concentration; Low: 0.1 mg/mL; High: 0.2 mg/mL) during in vitro maturation (IVM). Data are represented as ∆∆Ct (calculated with the Livak method, using RPL19 as housekeeping gene). Dashed line represents the median and dotted lines the 25–75% quartiles. Differences are indicated as (*) for P ≤ 0.05 and (**) for P ≤ 0.001. This figure was created using GraphPad Prism version 8.2.1 (GraphPad Software, Inc., La Jolla, CA, USA; https://www.graphpad.com/).

Figure 4

Violin plots representing relative levels of progesterone (P4) and estradiol-17β (E2) production by CCS in response to small- (SP-EV) or large-extracellular vesicles (L-EV) at two concentrations (Total protein concentration; Low: 0.1 mg/mL; High: 0.2 mg/mL). Secretion of steroid hormones was evaluated in the spent IVM media after 22 h and 44 h of IVM. Dashed line represents the median and dotted lines the 25–75% quartiles. This figure was created using GraphPad Prism version 8.2.1 (GraphPad Software, Inc., La Jolla, CA, USA; https://www.graphpad.com/).

Figure 5

Experimental design. (A) Uptake of seminal extracellular vesicles (EV) by cumulus-oocyte complexes (COCs). COCs were in vitro matured (IVM) in the presence of labelled small- (S-EV) or large-extracellular vesicles (L-EV) for 44 h, and subsequently analysed using confocal microscopy. (B) Effect of SP-EV on COCs during IVM. COCs were in vitro matured in the presence of S-EV or L-EV; after 44 h, oocytes were fixed to evaluate the meiotic stage, and cumulus cells were retrieved to analyse their gene expression using quantitative real-time PCR. In addition, IVM medium was kept after 22 h and 44 h to analyse progesterone and estradiol-17β secretion using radioimmunoassay (RIA). The drawing was created with BioRender (https://biorender.com/).