Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

Abstract

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.

Figure 1. Western blot analysis (A & B) of exosomal and non-exosomal fractions of follicular fluid and morphological presentation of exosomes using electron microscopy (C & D).

(A) Proteins (50 µg) from exosomal fraction (lane 1 & 2) and non-exosomal fraction (lane 3 & 4) of follicular fluid isolated either by ultracentrifugation (lane 1 & 3) or Exoquick kit (lane 2 & 4) were resolved on a 12% SDS-PAGE gel to detect CD63 and Ago2 proteins. (B) Confirmation of absence of detection of CYCS (mitochondrial protein) in both exosomal and non-exosomal proteins as isolated by ultracentrifugation (lane 1 & 3) and Exoquick kit (lane 2 & 4). A known cellular origin protein was used as a positive control (lane 5). Morphological characterization of exosomes isolated from bovine follicular fluid using Exoquick kit (C) and ultracentrifugation (D) by transmission electron microscopy. The scale bar indicates 100 nm.

Figure 2. Enriched pathways predicted to be targeted by differentially expressed miRNAs in both fraction of follicular fluid.

Pathways enriched by genes predicted to be targeted by miRNAs highly abundant in exosomal and non-exosomal fraction of follicular fluid derived from follicles with growing oocytes (BCB-) compared to fully grown oocytes (BCB+) group. The bar graphs represent the –log(p-value) for each pathway, while the line diagram indicates the ratio of number of genes from the data set to the total number of genes involved in respective pathways. The common pathways for differentially expressed miRNAs in exosomal and non-exosomal fraction of follicular fluid are underlined.

Figure 3. Representative amplification plot (A) and melting curve (B) of miR-654-5p, miR-103 and U6.

The amplification plot and melting curve was generated during analysis og the expression of miR-654-5p and the two endogenous controls (miR-103 and U6) in granulosa cells co-cultured with exosomes derived from follicular fluid containing growing (BCB-) oocytes. The amplification plot shows the rise in fluorescence signal above the threshold level (with ▵Rn  =  80.00) for the respective miRNA assays during the PCR run. The signal from the negative controls (RNase-free water) remained below the threshold line. The melting curve generated at the end of each PCR run showed the specificity of amplification, which is evidenced by a single peak for each miRNA assay. The negative controls showed no peaks and the signal remained horizontal.

Figure 4. Analysis of exosomal and non-exosomal miRNAs in follicular cells.

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 t test.

Figure 5. Stability of exosomal miRNAs under in vitro culture conditions.

Exosomes, isolated using differential ultracentrifugation from follicular fluid, were incubated under in vitro cell culture conditions (37°C & 5% CO₂) 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.

Figure 6. Uptake of PKH67-labeled exosomes by bovine granulosa cells in vitro.

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% CO₂). Nuclei are stained blue (DAPI) while PKH67-labeled exosomes are stained green ( Figures 7 A, B & C). 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 ( Figure 7 D). Scale bar: 10,000 nm.

Figure 7. Validation of the enrichment of candidate miRNAs in exosomes taken up by granulosa cells.

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.

Figure 8. Exosome mediated delivery of miRNAs in bovine granulosa cells in vitro.

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.

Figure 9. Changes in expression of target genes following exosomes transfection.

Quantitative real-time PCR were carried out using gene-selective primers. miRNA target genes were selected using seed match and their involvement in important pathways. To elucidate the exosome mediated uptake of extra-cellular miRNAs can alter the abundance of target mRNAs we used the same exosome co-cultured granulosa cells which was used for miRNA abundance study. The histogram in this figure indicated that significantly lower expression of target mRNAs except VEGFA, suggesting possible involvement of transcriptional regulation of circulating miRNA. Different superscript letters (a,b) denote a significant difference between groups, such that groups not sharing a similar letter are significantly different from each other (P<0.05). The data is presented as means ± SD of three biological replicates.