Dataset Information

Extracellular vesicle-coupled miRNA profiles in follicular fluid of cows with divergent post-calving metabolic status.

ABSTRACT: Most high-yielding dairy cows enter a state of negative energy balance (NEB) during early lactation. This, in turn, results in changes in the level of various metabolites in the blood and follicular fluid microenvironment which contributes to disturbed fertility. Extracellular vesicles (EVs) are evolutionarily conserved communicasomes that transport cargo of miRNA, proteins and lipids. EV-coupled miRNAs have been reported in follicular fluid. However, the association between postpartum NEB and EV-coupled miRNA signatures in follicular fluid is not yet known. Energy balance analysis in lactating cows shortly after post-calving revealed that the majority of the cows exhibited transiently negative energy balance levels, whereas the remaining cows exhibited either consistently negative or consistently positive energy levels. Metabolic status was associated with EV-coupled miRNA composition in the follicular fluid. Cows experiencing NEB showed reduced expression of a large number of miRNAs while cows with positive energy balances primarily exhibited elevated expression of EV-coupled miRNAs. The miRNAs that were suppressed under NEB were found to be involved in various metabolic pathways. This is the first study to reveal the presence of an association between EV-coupled miRNA in follicular fluid and metabolic stress in dairy cows. The involvement of differentially expressed miRNAs in various pathways associated with follicular growth and oocyte maturation suggest the potential involvement of specific follicular miRNAs in oocyte developmental competence, which may partially explain reduced fertility in cows due to post-calving metabolic stress.

SUBMITTER: Hailay T

PROVIDER: S-EPMC6731312 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Extracellular vesicle-coupled miRNA profiles in follicular fluid of cows with divergent post-calving metabolic status.

Hailay Tsige T Hoelker Michael M Poirier Mikhael M Gebremedhn Samuel S Rings Franca F Saeed-Zidane Mohammed M Salilew-Wondim Dessie D Dauben Christina C Tholen Ernst E Neuhoff Christiane C Schellander Karl K Tesfaye Dawit D

Scientific reports 20190906 1

Most high-yielding dairy cows enter a state of negative energy balance (NEB) during early lactation. This, in turn, results in changes in the level of various metabolites in the blood and follicular fluid microenvironment which contributes to disturbed fertility. Extracellular vesicles (EVs) are evolutionarily conserved communicasomes that transport cargo of miRNA, proteins and lipids. EV-coupled miRNAs have been reported in follicular fluid. However, the association between postpartum NEB and E ...[more]

PMID: 31492906

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Project description:Ovarian follicular fluid (FF) comprises a dynamic milieu whose composition alters under seasonal variation, influencing follicle development and oocyte developmental competence. Various cell subtypes (e.g. granulosa, theca, and cumulus) produce cell-secreted nanoparticles, coined extracellular vesicles (EVs), which contain bioactive regulatory molecules such as microRNAs (miRNAs) known to mediate intrafollicular communication through various forms of signaling and information transfer. Markedly, the hallmark feature of EVs transferring cellular knowledge propagates their appealing nature as direct indicators of cellular status, albeit homeostasis, dysfunction, or disease. Photoperiod alterations influencing the regulatory content of FF-EVs as downstream modifiers remain largely unknown, particularly in seasonal breeding animals. Here, we aimed to unpack the impacts of seasonal variation on the miRNA expression profiles of FF-EVs in the mare. Equine pre-ovulatory follicles were monitored and aspirated using ultrasound-guided transvaginal techniques to obtain follicular fluids during non-breeding (spring anovulatory, SAN) and breeding seasons (spring ovulatory, SOV; summer, SUM; and fall ovulatory, FOV). 97 miRNAs were found to be differentially expressed between groups. Pairwise comparisons revealed specific clusters, including six miRNAs involved in the spring transition (miR-149-200b-206-221-328-615) and many others dysregulated during that of the summer (miR-143-192451- 302b-100, and let 7c), the peak time point for breeding. Bioinformatic analyses led to unveiling significant enrichments in various biological functions, such as transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Ultimately, the miRNA content of seasonally divergent FF-EVs poses as potentially relevant indicators of intrafollicular mechanisms directing seasonal transitions and their functional regulatory roles in the pre-ovulatory stage, governing subsequent ovulation.

Project description:The mammalian follicular development is a well-orchestrated and complex molecular process characterized by sequences of follicular waves that yield the ovulatory follicle. It has been reported that bi-directional communication between the growing oocyte and the surrounding somatic cells is a hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) present in the follicular fluid (FF). Bioactive molecules such as the miRNAs encapsulated in the EVs mediate this communication within the follicular environment. Here, we aimed to decipher the dynamics of the miRNAs in the EVs of equine FF aspirated in vivo during different stages of follicular development. Hence, all follicles ≥6 mm were ablated on Days 10–11 of the estrous cycle (ovulation = Day 0) and the new induced wave was ultrasound-monitored daily until the largest growing follicle for at least three consecutive days was randomly allocated to one of the five groups (n = 8 follicles/group) namely: predeviation (preDEV; 18–20 mm), deviation (DEV; 22–25 mm), postdeviation (postDEV; 26–29 mm), preovulatory (preOV; 30–35 mm), and impending ovulation (IMP; ~40 mm). The FF was collected from each target group by ultrasound-guided transvaginal follicle aspiration. After removal of cells, cellular debris, and larger particles, EVs were isolated from FF groups (4 biological replicates/group; 0.5 mL FF/replicate) using high-speed ultracentrifugation (120,000xg for 70 min). Isolated EVs were characterized using western blot for EV marker proteins, NanoSight Tracking Analysis, and electron microscopy. MiRNA enriched total RNA was isolated from EVs using an Exosomal RNA Isolation kit and small-RNA library preparation and RNAseq (NextSeq500; Illumina) was performed by Novogene Co, Ltd. Approximately 180 known miRNAs were detected in each group with 148 mutually found in all groups. Among the expressed miRNAs, miR-148a, miR-21, miR-192, and miR-99a were the top highly abundant miRNAs in all groups. Cluster analysis exhibited 15 different expression patterns during follicular development. Among these patterns, a group of 23 miRNAs (including miR-106b, miR-199a-5p, and miR-125a-5p) exhibited a stable expression at the preDev until the postDev stage, a significant increase at the preOV stage, and then a significant decrease at the IMP stage. This miRNA cluster has been found to target genes involved in TGF-signaling and endocytosis pathways. Another interesting pattern with 22 miRNAs (including miR-146b-5p, miR-140, and miR-143) exhibited a sharp reduction in their expression from the preDEV until the preOV stage and was predicted to target genes associated with fatty acid biosynthesis, cell cycle, and oocyte meiosis pathways. In contrast, a group of 16 miRNAs (including miR-483, miR-128, and miR-192) gradually increased in expression from preDEV to the postDEV and then downregulated at the preOV stage. This miRNA cluster targets several signaling pathways, including ErbB, thyroid, and estrogen signaling. In conclusion, this study provides greater insights into the stage-specific expression dynamics of the FF EV-miRNAs during equine follicular development, which may play a role in folliculogenesis in mares.

Project description:Study questionAre phthalate metabolite concentrations in follicular fluid (FF) associated with the expression of extracellular vesicle microRNAs (EV-miRNAs)?Summary answerPhthalate metabolite concentrations are associated with the expression of EV-miRNA and their associated pathways in FFs.What is known alreadyPhthalate metabolites were recently detected in FF. Urinary phthalate metabolite concentrations alter the expression of EV-miRNAs in FF.Study design, size, durationProspective study including 105 women recruited between January 2014 and August 2016 in a tertiary university-affiliated hospital.Participants/materials, setting, methodsWe assessed FF concentrations of 12 phthalate metabolites. EV-miRNAs were isolated from aliquots of the same FF, and their expression profiles were measured using a human miRNA panel. Associations between EV-miRNAs that were present in >50% of the samples and phthalate metabolites that were measured in >74% of the FF samples were tested. Genes regulated by EV-miRNAs that were found to be significantly (false discovery rate q-value < 0.1) correlated with FF-phthalates were analyzed for pathways linked with female fertility using miRWalk2.0 Targetscan database, DAVID Bioinformatics Resources and Kyoto Encyclopedia of Genes and Genomes (KEGG).Main results and the role of chanceOf 12 phthalate metabolites, 11 were measured in at least one FF sample. Mono (6-COOH-2-methylheptyl) phthalate (MCOMHP), mono-2-ethyl-5-carboxypentyl phthalate (mECPP), mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBzP), mono-isobutyl phthalate (MiBP), monoethyl phthalate (MEP) and mono (7-COOH-2-methyloctyl) phthalate (MCOMOP) were detected in more than 74% of the samples. Of 754 EV-miRNAs tested, 39 were significantly associated either with MEP, MBzP, MCOMOP, MCOMHP and/or with mECPP, after adjusting for multiple testing (P < 0.05). KEGG-based pathway enrichment analysis of the genes regulated by these miRNAs showed that these EV-miRNAs may be involved in pathways related to ovary or oocyte development, maturation and fertilization.Limitations, reasons for cautionThe use of miRNA panel array limits the number of potential relevant miRNAs. Moreover, several of the phthalate metabolites examined may be biased due to internal (enzymatic activity) or external (contamination in medical interventions) causes.Wider implications of the findingsPhthalate metabolites may alter follicular EV-miRNAs profile and thus impair pathways that are involved with oocyte development, maturation and fertilization. Our results contribute to understanding of possible mechanism(s) in which endocrine disruptor chemicals interfere with female fertility.Study funding/competing interestsThis work was supported by the National Institutes of Environmental Health Sciences [Grant R21-ES024236]; and Environmental Health Fund, Israel [Grant 1301], no competing interests.Trial registration numberN/A.

Dataset Information

Extracellular vesicle-coupled miRNA profiles in follicular fluid of cows with divergent post-calving metabolic status.

Publications

Extracellular vesicle-coupled miRNA profiles in follicular fluid of cows with divergent post-calving metabolic status.

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