Project description:Limited research has explored the associations between microRNAs (miRNA) and diminished ovarian reserve (DOR). The study aimed to identify differentially expressed miRNAs in follicular fluid exosomes from women with DOR compared to normal ovarian reserve (NOR) and investigate their role in the proliferation and apoptosis of the human ovarian granulosa tumor cell line KGN.
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: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:Direct exposure to physiologically-relevant elevated temperatures during the first half of in vitro maturation heightens cumulus-derived progesterone production, suppresses matrix metallopeptidase 9 secretion, and alters cumulus transcriptome. We hypothesized that heat-induced perturbations in cumulus cells enveloping maturing oocyte may extend to the mural granulosa of the periovulatory follicle in the heat-stressed cow to subsequently follicular fluid proteome. Lactating Holsteins were pharmacologically stimulated to produce a dominant follicle then given GnRH to induce an LH surge. Following GnRH administration, cows were maintained at ~67 temperature humidity index (THI; thermoneutral conditions) or exposed to conditions simulating an acute heat stress event (71 to 86 THI; heat stress for ~12 h). Fluid was aspirated from periovulatory follicle ~16 h after GnRH. Follicular fluid proteome from thermoneutral (n = 5) and hyperthermic (n = 5) cows was evaluated by quantitative tandem mass spectrometry (nano LC-MS/MS). We identified 35 differentially-abundant proteins. Functional annotation revealed numerous immune-related proteins. Subsequent efforts revealed an increase in levels of the proinflammatory mediator bradykinin in follicular fluid (P = 0.0456) but not in serum (P = 0.9319) of hyperthermic cows. Intrafollicular increases in transferrin (negative acute phase protein) in hyperthermic cows (P = 0.0181) coincided with a tendency for levels to be increased in the circulation (P = 0.0683). Nine out of 15 cytokines evaluated were detected in follicular fluid. Heat stress increased intrafollicular IL6 levels (P = 0.0160). Whether hyperthermia-induced changes in the heat-stressed cow’s follicular fluid milieu reflect changes in mural granulosa, cumulus, other cell types secretions, and/or transudative changes from circulation remains unclear. Regardless of origin, heat stress/hyperthermia related changes in the follicular fluid milieu may have an impact on components important for ovulation and competence of the cumulus-oocyte complex contained within the periovulatory follicle
Project description:To explore the differences of miRNAs from exosomes of granulosa cell in different groups, we extracted exosomes from mouse granulosa cells intervened by different reagents.
Project description:Exosomes and microvesicles (i.e., extracellular vesicles; EVs) have been identified within ovarian follicular fluid, and recent evidence suggests that EVs are able to elicit profound effects on ovarian cell function. While existence of miRNA within EVs has been reported, it remains unknown if EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth. Extracellular vesicles isolated from follicular fluid of small, medium and large bovine follicles were similar in size, while concentration of EVs decreased progressively as follicle size increased. Electron microscopy indicated a highly purified population of the lipid bilayer enclosed vesicles that were enriched in exosome biomarkers including CD81 and Alix. Small RNA sequencing identified a large number of known and novel miRNAs that changed in the EVs of different size follicles. Ingenuity Pathway Analysis (IPA) indicated that miRNA abundant in small follicle EV preparations were associated with cell proliferation pathways, while those miRNA abundant in large follicle preparations were related to inflammatory response pathways. These studies are the first to demonstrate that EVs change in their levels and makeup during antral follicle development and point to the potential for a unique vesicle-mediated cell-to-cell communication network within the ovarian follicle. Examination of small RNA population in bovine follicular fluid extracellular vesicles isolated from antral follicles
Project description:Exosomes have recently been shown to play a key role in cell-to-cell communication through delivery of various functional content, including microRNAs (miRNAs). We investigated the potential roles of exosomal miRNA derived intrafollicular cells in polycystic ovary syndrome (PCOS). Using microarray profiling, a total of 492 miRNAs and 220 miRNAs were found in follicular fluid-derived exosomes and serum-derived exosomes, respectively, in PCOS and non-PCOS females. By excluding miRNAs existing in serum-derived exosomes, we found 179 miRNAs which were specifically expressed in follicular fluid-derived exosomes both in PCOS and non-PCOS females. Using microarray profiling, a total of 492 miRNAs and 220 miRNAs were found in follicular fluid-derived exosomes and serum-derived exosomes, respectively, in PCOS and non-PCOS females. By excluding miRNAs existing in serum-derived exosomes, we found 179 miRNAs which were specifically expressed in follicular fluid-derived exosomes both in PCOS and non-PCOS females.
Project description:Polycystic ovary syndrome (PCOS) is a complex endocrinopathy affecting reproductive aged women, whose etiology has not been fully understood yet. The follicular growth is arrested at preantral stage leading to cyst formation, consequently resulting in anovulatory infertility in these women. As follicular fluid provides the microenvironment for the growing oocyte, molecular profiling of the fluid may provide unique information about pathophysiology associated with follicular development in PCOS. Modification with oligosaccharide chains are known to influence functions of several secreted proteins and these glycoproteins also play a role in disease pathology. The glycoproteomic profile of follicular fluid of PCOS has not been explored in PCOS yet. In the present study, we performed comparative glycoproteomic analysis by first enriching glycoproteins using three different lectins viz. concanavalin A, wheat germ agglutinin and Jacalin from follicular fluid of women with PCOS and controls undergoing in vitro fertilization. Peptides generated by trypsin digestion were labeled with isobaric tags for relative and absolute quantification reagents and analyzed by liquid chromatography tandem mass spectrometry. We identified 10 differentially expressed glycoproteins, in the follicular fluid of women with PCOS compared to control. Two important differentially expressed proteins- SERPINA1 and ITIH4, were consistently upregulated and downregulated respectively, upon validation by Western blotting in follicular fluid and real-time polymerase chain reaction in granulosa cells. These proteins play a role in angiogenesis and extracellular matrix stabilization which are vital for follicle maturation. In conclusion, comparative glycoprotein profiling of follicular fluid from women with PCOS and controls revealed altered expression of proteins which may contribute to defects in follicle development in PCOS pathophysiology.
Project description:The composition of follicular fluid reflects crucial paracrine signalling between granulosa- and theca cells and the oocyte. Dynamic changes in the protein composition of human follicular fluid across multiple time points in the period where final meiotic maturation and release of the oocyte take place are previously undescribed. Twenty-five women undergoing IVF- or ICSI treatment in a standard antagonist protocol with agonist ovulation trigger were included in this prospective cohort study. From each patient one follicle was aspirated by transvaginal ultrasound guided puncture at one of five specific time points either before ovulation induction (T=0) or 12-, 17-, 32- or 36 hours after ovulation induction (five patients/time point). Proteomics was carried out by liquid chromatography-mass spectrometry. In total, 400 proteins were identified (FDR<0.05) and 30 were significantly regulated across time points (one-way ANOVA, adjusted p<0.05). Compared to compiled human plasma proteome sets, 47 proteins were unique to follicular fluid. Enriched biological functions among differentially expressed proteins included immune functions, wound healing and functions related to blood coagulation (FDR<0.02). The most profound changes occurred shortly after ovulation induction. We confirmed parallel protein expression to known granulosa cell mRNA changes and described many hitherto unknown protein expression profiles during ovulation. Thus, the study endorsed important biological functions of some proteins and suggested additional proteins, which may be crucial to intrafollicular signalling and oocyte competence that should be further investigated.