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.

Oxygen tension modulates extracellular vesicles and its miRNA contents in bovine embryo culture medium

“…Grade I and II COCs presenting compact cumulus and homogeneous cytoplasm were selected and matured in vitro in TCM199 culture medium, to which we added 26 mM sodium bicarbonate, 22 μg/ml sodium pyruvate, 50 μg/ml gentamicin, 0.5 μg/ml follicle stimulating hormone (Folltropin®; Bioniche Animal Health, Belleville, ON, Canada), 50 μg/ml human chorionic gonadotropin (hCG; Vetecor® 5000 U.I. Intervet, São Paulo, SP, Brazil) plus 10% custom made EV‐depleted fetal bovine serum (FBS), previously described in other experiments (da Silveira et al, ). The COCs were matured at 38.5°C and 5% CO 2 for 22 hr.…”

Composing the Early Embryonic Microenvironment: Physiology and Regulation of Oviductal Secretions

“…Extracellular vesicles from the preovulatory follicle may also enter the oviduct at ovulation. In cattle, EVs from FF were reported to be internalized by developing embryos in vitro and to modulate metabolic and developmental-related genes as well as miRNA and global DNA methylation [14].…”

In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

“…Prematuration and maturation of oocytes from antral follicles also increased the expression of cMOS , CCNB1 , and H1FOO . Although IVM rates (IVM) are relatively high, approximately 90% (Lonergan & Fair, ), low rates of embryonic development are generally attributed to the inability of oocytes to support cell multiplication to the blastocyst stage (Camargo et al, ; Meirelles, Chiaratti, Ferreira, Meirelles, & Bressan, ), suggesting that factors necessary for the proper development of the embryo are absent in the culture media (Da Silveira et al, ). During IVM, oocytes need to undergo nuclear and cytoplasmic maturation (Ferreira et al, ).…”