Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development

Abstract

Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3-6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (<200 nm) were isolated for further analysis. Additionally, small-extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3-6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3-6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3-6 mm follicles) in embryos. Interestingly, the addition of EVs from 3-6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3-6 mm follicles during oocyte maturation and early embryo development can partially modify metabolic and developmental related genes as well as miRNA and global DNA methylation and hydroxymethylation, suggesting that EVs play an important role during oocyte maturation and early embryo development in vitro.

Fig 1. Characterization of small-extracellular vesicles (EVs) from 3–6 mm ovarian follicles.

(A) The size of the EVs isolated from a pooled follicular fluid was evaluated by blockage of electrical current analysis, which demonstrated the presence of small size vesicles between 30-200nm. Different colors represent different replicates. (B) Transmission electron microscopy of isolated EVs demonstrated the presence of a homogenous population of small vesicles at 80kV (Scale bar 500nm). (C) EVs-RNA size distribution according to electropherogram of RNA molecules present in EVs from follicular fluid demonstrated the absence of 18s and 28s bands, as well as the presence of small RNA molecules. (D) Immunoblotting analysis of proteins in EVs and follicular cells. Herein we identified the presence of ALIX and CD63 two EVs markers, present in follicular fluid (FF), FF without EVs (FF-EV), follicular cells (FC), and EVs, isolated from 3-6mm ovarian follicles. We also identified the presence of PABP, an RNA binding protein, in FF, FF without EVs, follicular cells, and EVs. ACTB, a structural protein, was determined in the preparations, and demonstrated not to be enriched in EVs. Additionally, we verified the presence of Cytochrome C (CYT C), a mitochondrial protein, which serves as a negative control for cell contamination. EVs = extracellular vesicles <200 nm; ALIX = Programmed cell death 6-interacting protein; CD63 = CD63 molecule; PABP = Polyadenylate-binding protein 1; ACTB = Beta-actin; CYT C = Cytochrome c-1.

Fig 2. Presence of selected mRNAs involved in epigenetic chromatin modification and mRNA translation in small-extracellular vesicles from 3–6 mm ovarian follicles (EVs3-6mm).

DNMT1, DNMT3A, EHMT1 and HDAC2 are genes involved in epigenetic chromatin modifications, while EIF4B and EIF4E are involved in mRNA stabilization during translation. Data are presented as mean of 2^{^-ΔCt} ± s.e.m.

Fig 3. Uptake of PKH67 labeled small-extracellular vesicles (EVs) by follicular cells.

(A) Granulosa cells exposed to EVs labeled with PKH67 (green) for 24h in vitro, granulosa cell nucleus (blue) with a 40x magnification. (B) Cumulus cells exposed to PKH67 labeled EVs for 9h during maturation in vitro with a 63X magnification. (C) Intact cumulus-oocyte-complex exposed to PKH67 labeled EVs for 9h during maturation in vitro with 100x magnification. Uptake of PKH67 labeled EVs by the different follicular cells (arrows). The enlarged white box shows the presence of EVs (green dots) inside the zona pellucida. GC = granulosa cells; CC = cumulus cells; OO = oocyte; ZP = zona pellucida.

Fig 4. The small-extracellular vesicles (EVs) uptake by an embryo in in vitro culture.

(A) Blastocysts stained with DAPI (nuclear staining) with a 40x magnification. (B) Blastocysts stained with DAPI (nuclear staining) with a 40x magnification. C) Blastocysts exposed to PKH-67 labeled follicular fluid EVs during embryo culture (D1-D7) presenting a punctuated staining due to labeled small-extracellular vesicles, with a 40x magnification. D) Blastocysts exposed to PKH67 labeled PBS 1x during embryo culture (D1-D7) presenting a non-specific green staining with a 40x magnification. Blue = DAPI (nuclear staining); Green dots = PKH67 labeled small-extracellular vesicles (arrow).

Fig 5. Transcription levels of genes involved in chromatin remodeling and embryo development.

(A) Relative transcript levels in blastocysts treated during in vitro culture (IVC) with different EVs supplementation conditions. (B) Transcription levels of selected genes in extracellular vesicles (EVs) from 3–6 mm or pre-ovulatory follicles. (C) Transcription levels of ACSL6, CDH1, REST and FADS2 in embryos treated during IVC with different EVs supplementation conditions. EVsfree-FCS (EVs-depleted FCS), EVs3-6mm (EVs from 3–6 mm in size follicles) and EVsPreOv (EVs from pre-ovulatory follicles). Different letters indicate p<0.05. ND = not detected. Data are presented as mean of 2^{^-ΔCt} ± s.e.m.

Fig 6. MiRNA-631 levels in in vitro produced 4-cell embryos.

Levels of miR-631 in pooled 4-cell embryos (n = 3) after treatment with small-extracellular vesicles (EVs) during oocyte maturation and embryo culture. Different letters indicate p<0.05. EVsfree-FCS (EVs-depleted FCS), EVs3-6mm (EVs from 3–6 mm in size follicles). Data are presented as mean of 2^{^-ΔCt} ± s.e.m.

Fig 7. In vitro embryo developmental rates.

(A) Blastocyst rates following small-extracellular vesicles (EVs) treatment during oocyte in vitro maturation (IVM) and embryo culture (IVC). Results are based on five independent in vitro production (IVP) routines in total ~979 cumulus-oocyte-complexes (COCs) (EVsfree-FCS = 318, EVs3-6mm = 318, EVsPreOv = 343). EVsfree-FCS (EVs-depleted FCS), EVs3-6mm (EVs from 3–6 mm in size follicles) and EVsPreOv (EVs from pre-ovulatory follicles). Different letters indicate statistical difference P<0.05.

Fig 8. Global DNA methylation and hydroxymethylation levels in blastocysts after supplementation with small-extracellular vesicles (EVs).

(A) Global DNA methylation levels in D7 blastocysts. (B) Global DNA hydroxymethylation levels in D7 blastocysts. (C) Confocal images of embryos that underwent different treatments during oocyte maturation and embryo development (40x objective). Different capital letters (A and B, A’ and B’ or A” and B”) indicate differences (p<0.05) among IVM groups within a given IVC group. Different small letters (a and b, a’ and b’, or a” and b”) indicate differences (p<0.05) among IVC groups within a given IVM group. Control (normal FCS), EVsfree-FCS (EVs-depleted FCS), EVs3-6mm (EVs from 3–6 mm in size follicles). Data are presented as mean ± s.e.m.