Small RNA profiles of follicular fluid-derived small extracellular vesicles
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ABSTRACT: Follicular fluid (FF) provides a complex and suitable environment for oocyte maturation and contains several molecules secreted from oocyte and granulosa, cumulus, and theca cells. In addition, extracellular vesicles (EV) exist in various body fluids and are known as the cargo of several mRNAs, proteins, and miRNAs to communicate from cell to cell. In this study, we investigated the miRNA profiles of FF-derived EVs.
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:<p>The metabolic profile of follicular fluid (FF) has been investigated to look for biomarkers for oocyte quality. Resolvin E1 (RvE1), a potent pro-resolving mediator, was reported to have protective action in cell function. The study aimed to examine the predictive value of RvE1 for oocyte quality and to explore the cellular mechanism of RvE1 in improving oocyte competence. Metabolic profiles of 80 FF samples showed a higher level of RvE1 in group A (blastocysts scored ≥ B3BC and B3CB according to Gardner's blastocyst scoring system, N=36) than that of group B (blastocysts scored < B3BC and B3CB, N=44, P=.0018). The receiver operating characteristic (ROC) curve analysis showed that RvE1 level in FF below 8.96 pg/ml (AUC:0.75; 95%CI: 0.64 – 0.86; P=.00012) could predict poor oocyte quality with specificity of 97.22%, suggesting RvE1 as a potential biomarker to exclude inferior oocytes. Besides, the level of RvE1 was found to be significantly lower in FF than in serum (57.49 to 17.62 pg/ml; P=.0037) and was gradually accumulated in the culture medium of cumulus cells (CCs) during cell culture, which indicated that RvE1 came from both blood exudates and local secretion. The in vitro experiment revealed the cellular mechanism of RvE1 in improving oocyte quality by decreasing the cumulus cell apoptotic rate and increasing cell viability and proliferation. It is the first time that the role of RvE1 in reproduction is explored. In conclusion, RvE1 is valuable as a potential exclusive biomarker for oocyte selection and plays a role in improving oocyte quality.</p>
Project description:Protein composition of human ovarian follicular fluid (FF) constitutes the microenvironment for oocyte development. Several proteomics studies of FF from pre-ovulatory follicles have revealed insights on oocyte maturation, however, there is a lack of knowledge on changes produced at protein levels in the FF of human small antral follicles (hSAF) related to the upcoming oocyte maturation. Using mass spectrometry-based proteomics, we evaluated the protein composition of FF that surrounds oocytes capable to reach metaphase II (MII) after IVM with the protein profile of FF that surrounds immature oocytes. The samples were collected from small antral follicles (size 6.0 ± 1.5 mm) extracted from six women, from which two or three samples were extracted. The comparison was based on both, a multivariate (sPLS-DA) and univariate analyses (t-test).
Project description:The follicular fluid (FF) fills the interior of ovarian antral follicle and provides the microenvironment for oocyte growth and acquisition of its competence to ovulate and latter support early embryo development. The FF is derived from both blood plasma and secretion of different types of follicular cells. It contains also extracellular vesicles (EVs), including exosomes, small membrane-coated EVs with 30-150 nm in diameter, which participate in cell-to cell communication and signaling by transferring their cargo of different types of RNAs, proteins and lipids into the oocyte or follicular cells. To date most studies have focused on studying the ffEVs miRNAs cargo and showing that miRNAs can influence oocyte competence and further embryo development. However, ffEVs protein cargo, which could have a direct contribution after being uptake by the oocyte or follicular cells have been less studies.
Project description:Follicles of polycystic ovaries (PCO) often become arrested in early antral stages at around 3 to 11 mm in diameter. The condition disturbs dominant follicle selection and may result in altered ovulation and anovulation. During the growth and development of human follicles, the follicular fluid (FF) constitutes the avascular microenvironment in which the oocyte develops and acts as a vehicle for hormone signaling between cues from circulation and follicular cells. Previous proteomics studies performed on FF from women with polycystic ovarian syndrome (PCOS) have revealed information on the protein changes associated with the pathophysiology of this disorder. However, these studies have been conducted on FF samples obtained in connection with oocyte pick-up during ovarian stimulation right at the time of ovulation and are limited to follicular conditions during the follicular phase of the menstrual cycle. This study aimed to detect proteomic alterations in FF from human small antral follicles (hSAF) obtained from women with PCO as compared to normal women.
Project description:Purpose: Extracellular vesicles (EVs) are nanoparticles that can be secreted by different cells, including cells found within the ovarian follicle. Currently, EVs are considered an important form of intercellular communication, since they carry biological contents. The goal of this study was to survey the effects of small EVs from follicles at different estrous cycle stage in bovine cumulus cells. Methods: We used an established model to obtain follicular fluid (FF) at early and late estrous cycle stage according to corpus luteum appearance, corresponding to low and high progesterone (P4) levels, respectively. We collected FF from 3-6 mm follicles and isolated small EVs, which were used as a supplement during in vitro maturation (IVM). Cumulus cells were collected from cumulus-oocyte complexes (COCs) pools and the RNAs were obtained and subjected to RNA sequencing. Results: The results showed that small EVs from different estrous cycle stage are capable to affect transcripts in cumulus cells and modulate different pathways and biological processes related to oocyte maturation, ovulation and immune response. Conclusion: This study demonstrated that small EVs from low and high P4 group impact the RNA profile in cumulus cells after 9 hours of in vitro maturation.
Project description:Follicular somatic cells (mural granulosa cells and cumulus cells) and the oocyte communicate through paracrine interactions and through direct gap junctions between oocyte and cumulus cells. Considering that mural and cumulus cells arise through a common developmental pathway and that their differentiation is essential to reproductive success, understanding how these cells differ is a key aspect to understanding their critical functions. Changes in global gene expression before and after an ovulatory stimulus were compared between cumulus and mural granulosa cells to test the hypothesis that mural and cumulus cells are highly differentiated at the time of an ovulatory stimulus and further differentiate during the periovulatory interval. The transcriptomes of the two cell types were markedly different (>1500 genes) before an ovulatory hCG bolus but converged after ovulation to become completely overlapping. The predominant transition was for the cumulus cells to become more like mural cells after hCG. This indicates that the differentiated phenotype of the cumulus cell is not stable and irreversibly established but may rather be an ongoing physiological response to the oocyte. We compared transcriptomes of mural granulosa cells isolated from the follicles before (PM-GC) and after (VVM-GC) an ovulatory stimulus. These data were analyzed with previously published cumulus cells data to compare transitions in granulosa cell state before and after an ovulatory stimulus with transitions in cumulus cells.
Project description:Follicular somatic cells (mural granulosa cells and cumulus cells) and the oocyte communicate through paracrine interactions and through direct gap junctions between oocyte and cumulus cells. Considering that mural and cumulus cells arise through a common developmental pathway and that their differentiation is essential to reproductive success, understanding how these cells differ is a key aspect to understanding their critical functions. Changes in global gene expression before and after an ovulatory stimulus were compared between cumulus and mural granulosa cells to test the hypothesis that mural and cumulus cells are highly differentiated at the time of an ovulatory stimulus and further differentiate during the periovulatory interval. The transcriptomes of the two cell types were markedly different (>1500 genes) before an ovulatory hCG bolus but converged after ovulation to become completely overlapping. The predominant transition was for the cumulus cells to become more like mural cells after hCG. This indicates that the differentiated phenotype of the cumulus cell is not stable and irreversibly established but may rather be an ongoing physiological response to the oocyte.
Project description:Cumulus cells are biologically distinct from other follicular cells and perform specialized roles, transmitting signals within the ovary and supporting oocyte maturation during follicular development. The bi-directional communication between the oocyte and the surrounding cumulus cells is crucial for the acquisition of oocyte competence. Using Illumina/deep-sequencing technology, we dissected the small RNAome of pooled human mature MII oocytes and cumulus cells.