13 research outputs found
Bovine ovarian hyperstimulation induced changes in expression profile of circulatory miRNA in follicular fluid and blood plasma
Circulatory noncoding small RNAs (miRNAs), which are present in various body fluids, are reported to be potentially used as biomarkers for disease and pregnancy. The present study was conducted to investigate the effect of ovarian hyperstimulation on the expression pattern of circulatory miRNA in follicular fluid and blood plasma. For this, Simmental heifers (n=12) were synchronized using a standard synchronization protocol and six of them were hyperstimulated using FSH. Following this, whole blood samples were collected at day 0 (onset of oestrous), 3 and 7, follicular fluid samples were aspirated from dominant follicles at the day 0 from all animals by ovum pickup. Total RNA including miRNA was isolated from plasma samples of both groups at day 7 and follicular fluid at day 0. Subsequent expression profiling of miRNA was performed using the human miRCURY LNATM Universal RT miRNA PCR array platform with 745 miRNA primer assays. Of the 24 miRNAs, which were differentially expressed in blood plasma between hyperstimulated and unstimulated animals, 9 miRNAs including miR-127-3p, miR-494, miR-147, miR-134 and miR-153 were down regulated and 15 miRNAs including miR-34a, miR-103, let-7g, miR-221 were found to be up regulated in the hyperstimulated animals. Similarly, 66 miRNAs were found to be differentially expressed in follicular fluid derived from hyperstimulated and unstimulated groups. Out of these, while 32 miRNAs were down regulated, 34 were up regulated in follicular fluid aspirated from hyperstimulated animals. Ingenuity pathway analysis (IPA) of potential target genes of candidate miRNAs, which are dysregulated due to ovarian hyperstimulation, revealed axonal guidance signaling and Wnt ß-catenin signaling pathways to be the dominant ones. In conclusion, this study revealed ovarian hyperstimulation resulted in changes in expression profile of circulatory miRNA in blood and follicular fluid.Zirkulierende nicht-kodierende micro RNAs (miRNAs), die in verschiedenen Körperflüssigkeiten vorhanden sind, sind möglicherweise potenzielle Biomarker für Krankheiten und Trächtigkeit. Die vorliegende Studie wurde durchgeführt, um die Wirkung einer ovarialen Überstimulation auf das Expressionsmuster von zirkulierenden miRNAs in der Follikelflüssigkeit und im Blutplasma zu untersuchen. Dazu wurden Fleckvieh-Färsen (n=12) mit einem Standard-Synchronisationsprotokoll synchronisiert und sechs von ihnen mit FSH überstimuliert. Die Probenentnahme beinhaltete Blutproben zum Zeitpunkt 0 (Beginn der Brunst), am 3. und am 7. Tag sowie die Follikelflüssigkeit von dominanten Follikel am Tag 0 von allen Tieren durch „Ovum pickup”. Die Gesamt-RNA inklusive der miRNAs wurde aus den Plasmaproben von beiden Gruppen an Tag 7 und aus der Follikelflüssigkeit am Tag 0 isoliert. Das nachfolgende Expressionprofiling der miRNAs erfolgte unter Verwendung der Humanen miRCURY LNA TM Universal-RT-PCR- miRNA-Array-Plattform mit 745 miRNA Primer-Assays. Von den 24 miRNAs, die im Blutplasma beim Vergleich zwischen hyperstimulierten und unstimulierten Tieren unterschiedlich exprimiert waren, zeigten 9 miRNAs, einschließlich miR-127-3p, miR-494, miR-147, miR-134 und miR-153, eine Runterregulation, während 15 miRNAs einschließlich miR-34a, miR-103 , let-7g, miR-221 eine erhöhte Expression in den hyperstimulierten Tieren aufwiesen. Des Weiteren, konnten beim Vergleich der Follikelflüssigkeit von hyperstimulierten und unstimulierten Tieren 66 differentiell exprimierte miRNAs identifiziert werden. Von diesen waren 32 miRNAs herunterreguliert, während 34 in der Gruppe der hyperstimulierten Tiere raufreguliert waren. Eine Ingenuity Pathway Analyse (IPA) der potentiellen Zielgene von Kandidaten miRNAs, die aufgrund von Überstimulation der Ovarien eine Fehlregulation zeigten, ergab als dominante Signalwege die axonale Führung sowie Wnt-ß-Catenin. Die Ergebnisse dieser Studie zeigte, dass eine Überstimulation der Ovarien zu Veränderungen im Expressionsprofil von zirkulierenden miRNAs im Blut und in der Follikelflüssigkeit führte
Relative abundance of extra-cellular miRNAs in bovine follicular fluid: Implication for cell–cell communication
Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma
Background: Despite its role in increasing the number of offspring during the lifetime of an individual animal, controlled ovarian hyperstimulation (COH) may have detrimental effects on oocyte development, embryo quality and endometrial receptivity. Circulating miRNAs in bio-fluids have been shown to be associated with various pathological conditions including cancers. Here we aimed to investigate the effect of COH on the level of extracellular miRNAs in bovine follicular fluid and blood plasma and elucidate their mode of circulation and potential molecular mechanisms to be affected in the reproductive tract
Summary of the number of differentially expressed miRNAs obtained from comparisons (growing vs. fully grown) in exosomal and non-exosomal fraction of follicular fluid.
<p>DE: differentially expressed.</p><p>↑: Up-regulated in the growing vs. fully grown comparison.</p><p>↓: Down-regulated in the growing vs. fully grown comparison.</p
Uptake of PKH67-labeled exosomes by bovine granulosa cells <i>in vitro</i>.
<p>Exosomes, purified from follicular fluid, were labeled with PKH67 dye and added to primary culture of granulosa cells. Granulosa cells were co-cultured with labeled exosomes isolated from follicular fluid in exosome-free medium for 22 hours under optimum cell culture conditions (37°C & 5% CO<sub>2</sub>). Nuclei are stained blue (DAPI) while PKH67-labeled exosomes are stained green (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078505#pone-0078505-g007" target="_blank"><b>Figures 7</b></a><b>A, B & C</b>). Arrows indicate exosomes that were taken up by granulosa cells. Granulosa cells cultured in exosomes free medium containing PKH67-labeled sterile PBS served as a negative control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078505#pone-0078505-g007" target="_blank"><b>Figure 7</b></a><b>D</b>). Scale bar: 10,000 nm.</p
Stability of exosomal miRNAs under <i>in vitro</i> culture conditions.
<p>Exosomes, isolated using differential ultracentrifugation from follicular fluid, were incubated under <i>in vitro</i> cell culture conditions (37°C & 5% CO<sub>2</sub>) for 6 hr, 12 hr and 24 hr in a exosome-free culture medium in order to determine the stability of exosomal miRNAs by quantitative real time PCR. Non-cultured exosomes (0 hr) were used as reference controls to check the stability of exosomes coupled miRNAs in different time point. The data is presented as means and SD of three biological replicates.</p
Validation of the enrichment of candidate miRNAs in exosomes taken up by granulosa cells.
<p>Purified exosomes from follicular fluid of follicles with growing (BCB-) or fully grown (BCB+) oocytes were subjected to total RNA extraction. Then the expression level miR-654-5p and miR-640 (enriched in exosomes derived from BCB- follicular fluid) and miR-526b* and miR-373 (enriched in follicular fluid derived from follicles with BCB+ oocyte) were investigated by real time PCR. Different superscript letters (a,b) denote a significant difference between groups, such that groups not sharing a similar letter are significantly different form each other (P<0.05). The data is presented as means ± s.d. of three biological replicates.</p
Exosome mediated delivery of miRNAs in bovine granulosa cells <i>in vitro</i>.
<p>Purified exosomes from follicular fluid of follicles from growing (Exo BCB-) or fully grown (Exo BCB+) oocytes were co-cultured with bovine granulosa cell. After 24 hrs of incubation at 37°C in a humidified incubator, cells were collected and subjected to total RNA extraction. The expression levels of candidate miRNAs were investigated by real time PCR. In both cases the level of endogenous miRNAs were significantly increase compared to untreated controls. Bars with different superscript letters (a,b) are significantly different (P<0.05) from each other. The data is presented as means ± SD of three biological replicates.</p
List of differentially expressed microRNAs in exosomal and non-exosomal fraction of bovine follicular fluid obtained from growing vs. fully grown oocyte comparison.
<p>(P<0.05) with at least two-fold change.</p
Analysis of exosomal and non-exosomal miRNAs in follicular cells.
<p>Expression patterns of miR-654-5p and miR-640 (up-regulated in exosomal fraction follicular fluid derived from follicles containing BCB- oocytes) and miR-526b* and miR-373 (up-regulated in exosomal fraction of follicular fluid from BCB+ groups) were investigated in surrounding follicular cells namely: cumulus oocyte complex (COCs), granulosa cells (GC), and theca cells (TC) from the same category of follicle which were used for miRNA PCR array analysis. Four miRNAs namely: miR-19b-1*& miR-29c (enriched in non exosomal fraction of follicular fluid from BCB-) and miR-381 & miR-30e* (enriched in non exosomal fraction of follicular fluid from BCB+ group) were also investigated for their expression in surrounding follicular cells. The data is presented as relative abundance of different miRNAs in different cell types compared to their expression in COCs as a control. Error bars represents means ± SD of three biological replicates and different superscript letters (a-c) denote a significant difference between groups (P<0.05) as determined by Student’s <i>t</i> test.</p