Follicular Fluid Extracellular Vesicles Research Articles

Study of the uptake mechanism of two small extracellular vesicle subtypes by granulosa cells

As a new mechanism of intercellular communication, the uptake of extracellular vesicles (EVs) by receptor cells has become a hot topic in the field. Previously, research on the uptake of EVs has focused on the mechanism of small EVs (sEVs, also known as exosomes). As sEVs represent a mixed heterogeneous population, the issue of whether there are different uptake mechanisms for different subsets of sEVs by recipient cells urgently need to be addressed. There are EVs in follicular fluid, which play an important role in the communication between follicular cells and the development of oocytes. Previously, we isolated two subtypes of sEVs in follicular fluid: low density-sEVs (LD-sEVs) and high density-sEVs (HD-sEVs). The current study aimed to explore the uptake characteristics of these two subtypes of sEVs by granulosa cells. First, PKH67 was used to label the two sEVs subtypes, and we observed their uptake by granulosa cells using confocal microscopy and flow cytometry. We then explored the specific mechanisms underlying uptake of these two sEV subtypes by granulosa cells using specific inhibitors and RNA interference. The results showed that granulosa cells took up both kinds of sEVs through a clathrin-independent pathway. In addition to requiring caveolin, cholesterol, and Na+/H+ exchange, the uptake of HD-sEVs also depended on the activity of tyrosine kinase and phosphoinositide 3-kinase. A better understanding of the mechanism of granulosa cell uptake of different subtypes of sEVs in follicular fluid is of considerable significance leading to more accurate use of EVs for targeted treatment of infertility and other related diseases.

Exosomal or follicular FNDC3A decreases FOLR1 mRNA abundance and progesterone and lactate synthesis in bovine granulosa cells.

Dairy cattle experience a period of infertility postpartum that is caused in part by the development of IGF1/insulin resistance. This study suggests that an adipokine, FNDC3A, reduces IGF1-dependent glycolysis and may contribute to postpartum infertility. Dairy cows go through a period of subfertility after parturition, triggered in part by a disruption of energy homeostasis. The mobilization of body fat alters the secretion of adipokines, which have been shown to impact ovarian function. Fibronectin type III domain-containing 3A (FNDC3A) is a recently discovered adipokine-myokine, and FNDC3A mRNA abundance in subcutaneous adipose tissue is increased postpartum in cattle. In this study, we hypothesized that FNDC3A may compromise granulosa cell function in cattle and investigated this using a well-established in vitro cell culture model. Here, we demonstrate the presence of FNDC3A protein associated with extracellular vesicles in follicular fluid and in plasma, suggesting an endocrine role for this adipokine. FNDC3A protein and mRNA was also detected in the bovine ovary (cortex, granulosa and theca cells, cumulus, oocyte and corpus luteum). Abundance of FNDC3A mRNA in granulosa cells from small follicles was increased by in vitro treatment with the adipokines leptin and TNF but not by visfatin, resistin, adiponectin, chemerin or IGF1. Addition of recombinant FNDC3A at physiological doses (10 ng/mL) to granulosa cells decreased IGF1-dependent progesterone but not estradiol secretion and IGF1-dependent lactate secretion and abundance of GLUT3 and GLUT4 mRNA. This concentration of FNDC3A increased cell viability, abundance of mRNA encoding a putative receptor FOLR1, and increased phosphorylation of Akt. Collectively, these data suggest that FNDC3A may regulate folliculogenesis in cattle by modulating IGF1-dependent granulosa cell steroidogenesis and glucose metabolism.

Small extracellular vesicles in follicular fluids for predicting reproductive outcomes in assisted reproductive technology

BackgroundAssisted reproductive technology accounts for an increasing proportion of infertility treatments, and assessments to predict clinical pregnancy outcomes are desired. Extracellular vesicles exist in follicular fluid, and small non coding RNAs in extracellular vesicles underline the possibility of reflecting pregnancy potential.MethodsFollicular fluid samples are collected from 20 ovarian follicles of 15 infertile patients undergoing assisted reproductive technology. Extracellular vesicles are isolated by serial centrifugation and small RNA sequencing is performed to investigate the profiles of microRNAs and P-element-induced wimpy testis-interacting RNAs.ResultsSmall extracellular vesicles with a size range of approximately 100 nm are successfully isolated, and the small non coding RNA profiles of pregnant samples (n = 8) are different from those of non-pregnant samples (n = 12). Fourteen dysregulated small non coding RNAs are selected to identify the independent candidates [mean read count >100, area under the curve >0.8]. Among them, we find that a specific combination of small non coding RNAs (miR-16-2-3p, miR-378a-3p, and miR-483-5p) can predict the pregnant samples more precisely using a receiver operating characteristics curves analysis (area under the curve: 0.96). Furthermore, even in the same patients, the three microRNAs are differentially expressed between pregnant and non-pregnant samples.ConclusionsOur results demonstrate that small non coding RNAs derived from small extracellular vesicles in follicular fluid can be potential non-invasive biomarkers for predicting pregnancy, leading to their probable application in assisted reproductive technology. Further large-scale studies are required to validate the clinical usefulness of these small non coding RNAs.

O-233 Follicular fluid extracellular vesicles as a potential tool for human spermatozoa selection in vitro

Abstract Study question What characteristics and features of follicular fluid (FF) extracellular vesicles (EVs) of women of different ages can affect the functional characteristics of spermatozoa? Summary answer The size, lipid composition, and progesterone levels of the FF EVs change with a woman's age and affect the fertilizing ability of spermatozoa. What is known already Aging influences on molecular composition of the FF, reducing fertilization processes occurring in the fallopian tubes. FF is an important factor in activating spermatozoa during oocyte fertilization, enhancing the process of sperm hyperactivation and capacitation. FF progesterone is one of the main chemoattractants in the female genital tract, attracting sperm to the oocyte and starting the fertilization processes. Our previous studies have shown a significant effect of FF EVs of young women compared to advanced maternal age women (AMA) on the hyperactivation and motility of spermatozoa. The binding of FF EVs to the acrosoma of the sperm also was detected. Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 10*6/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Next steps: immunofluorescent staining, test for acrosomal reaction of spermatozoa after incubation, NTA analysis, the lipid profile and progesterone measuring of FF EVs of women of different ages. Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 21) aged 28-36. FF EVs were obtained from young (n = 14) aged 22-30 and AMA patients (n = 14) aged 42-47. The methods used in this work also include immunofluorescence microscopy, nanoparticle tracking analysis, flow cytometry, liquid chromatography–mass spectrometry, statistical data analysis. Lipids were extracted from EVs using a modified Folch extraction protocol. Main results and the role of chance Progesterone levels were significantly higher in FF EVs samples from young women compared to FF EVs samples from AMA women and controls (3951000 a.u., 598500 a.u., 6701 a.u.). This may explain the increase in sperm hyperactivation and motility after incubation with FF EVs. The difference in size (144.78 nm ± 9.92 VS 127.03 nm ± 17.17) and lipid composition of young women's FF EVs may explain the more significant functional and therapeutic effect on spermatozoa compared to AMA women FF EVs. A total of 389 vesicle lipids were identified. The level of cholesterol esters, cholesterol, lysophosphatidylcholines, phosphatidylcholines was 1,5 times higher in FF EVs of young women. The level of di- and triglycerides, ceramides, oxidized forms of phosphatidylcholines was higher in FF EVs of AMA women. The level of spontaneous acrosomal reaction was almost 3 times lower in sperm samples after incubation with FF EVs compared to the control (11,21% VS 32,88%). This supports the hypothesis that FF EVs may restrain premature acrosomal reaction in the fallopian tubes. Age-related changes are reflected in reproductive important biological processes, which leads to a change in the composition and functions of FF and affect the functional role of FF EVs. Limitations, reasons for caution Small amount of data. To be completely confident in the importance of the participation of FF EVs in the activation of spermatozoa, it is necessary to perform proteomic analyses of FF EVs to discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics. Wider implications of the findings Further studies may help not only to complement the fundamental understanding of the processes of fertilization and the impact on spermatozoa morphofunctional characteristics, but also to develop a tool for improving ART outcomes in couples with male infertility. The solutions could be the use of FF EVs from young donors. Trial registration number not applicable

Extracellular vesicle-microRNAs mediated response of bovine ovaries to seasonal environmental changes

BackgroundAmong the various seasonal environmental changes, elevated ambient temperature during the summer season is a main cause of stress in dairy and beef cows, leading to impaired reproductive function and fertility. Follicular fluid extracellular vesicles (FF-EVs) play an important role in intrafollicular cellular communication by, in part, mediating the deleterious effects of heat stress (HS). Here we aimed to investigate the changes in FF-EV miRNA cargoes in beef cows in response to seasonal changes: summer (SUM) compared to the winter (WIN) season using high throughput sequencing of FF-EV-coupled miRNAs. In addition to their biological relevance, the potential mechanisms involved in the packaging and release of those miRNAs as a response to environmental HS were elucidated.ResultsSequencing analysis revealed that an average of 6.6% of the EV-RNA mapped reads were annotated to bovine miRNAs. Interestingly, miR-148a, miR-99a-5p, miR-10b, and miR-143 were the top four miRNAs in both groups accounting for approximately 52 and 62% of the total miRNA sequence reads in the SUM and WIN groups, respectively. A group of 16 miRNAs was up-regulated and 8 miRNAs were down-regulated in the SUM compared to the WIN group. Five DE-miRNAs (miR-10a, miR-10b, miR-26a, let-7f, and miR-1246) were among the top 20 expressed miRNA lists. Sequence motif analysis revealed the appearance of two specific motifs in 13 out of the 16 upregulated miRNAs under HS conditions. Both motifs were found to be potentially bonded by specific RNA binding proteins including Y-box binding proteins (YBX1 and YBX2) and RBM42.ConclusionOur findings indicate that FF EV-coupled miRNA profile varies under seasonal changes. These miRNAs could be a good indicator of the cellular mechanism in mediating HS response and the potential interplay between miRNA motifs and RNA binding proteins can be one of the mechanisms governing the packaging and release of miRNAs via EVs to facilitate cellular survival.

Progress in the effect of microRNA carried by extracellular vesicles in follicular fluid on follicular atresia

Extracellular vesicles (EVs) are membrane-bound particles actively released by cells. In prokaryotes and eukaryotes, EVs are effective bridges for communication between cells. EVs carry biological macromolecules, including proteins, lipids and nucleic acid, which affects different physiological functions of parent cells and recipient cells. Among them, the microRNA carried by EVs is the most reported and plays an important role in physiological function of organisms. During the development of follicles, only a few follicles can fully develop and ovulate, whereas most of them undergo atresia at different stages of development. In the whole process of follicular development, the changes at each stage and the regulation mechanism of follicular atresia are not completely understood. In this paper, we introduced the types, characteristics, isolation methods and uses of EVs, and emphasized how microRNA carried by EVs in follicular fluid regulated follicular atresia from the aspects of different cytokines and hormones. Additionally, the application prospect of microRNA carried by EVs in follicular fluid in reproductive regulation and reproductive disease diagnosis was discussed. This paper is significant for studying the regulation of follicular development and the effective utilization of oocytes.

P-188 The effect of follicular fluid extracellular vesicles on motility and hyperactivation of human spermatozoa and differential miRNA levels depend on the woman's age

Abstract Study question How does the woman’s reproductive age affect the ability of follicular fluid (FF) extracellular vesicles (EV) to change the motility and hyperactivation of spermatozoa and follicular fluid microRNA profiles? Summary answer The effect of FF EVs on sperm motility and hyperactivation decreases with a woman's age. MiR-134-5p, miR-21-5p expression levels increase in older age group. What is known already Aging reduces human fertility. FF is an important factor in attracting and activating spermatozoa in the oviduct during oocyte fertilization, enhancing the process of sperm capacitation and acrosomal reaction in various mammalian species, including humans. EVs from seminal plasma and oviduct fluid carry proteins and miRNAs playing a vital role in a multi-step process including sperm motility, capacitation, acrosomal reaction, further fertilization. Results of our previous experiments showed that FF EVs from young women significantly improve the indices of sperm motility and hyperactivation. Several studies have isolated some age associated differentially expressed miRNAs (hsa-miR-424, hsa-miR-21-5p, hsa-miR-134, hsa-miR-190b, hsa-miR-99b -3p). Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds, frozen at -80 °C. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 106/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Samples were centrifuged and fixed in 2.5% glutaraldehyde in 0.1 M buffer for TEM. Sperm motility were assessed using CASA. MicroRNAs isolation (miRNeasy Serum/Plasma Kit (Qiagen)). Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 18) aged 28-36 years without severe pathozoospermia. FF EVs were obtained from young (n = 4) aged 27-31 (EVs+) and older patients (n = 4) aged 41-46 with several IVF attempts (EVs-). The methods used in this work include sperm and EVs isolation, incubation, transmission electron microscopy, RT-PCR, statistical data analysis, computer-assisted semen analysis (CASA). Main results and the role of chance Our results showed that sperm incubation with EVs+ led to a significant increase in the number of progressively motile spermatozoa (paired Student's t test, p < 0.001), indicators of general mobility (p = 0.05) and hyperactivation of spermatozoa after 1 hour. Sperm incubation with EVs- slightly changed these parameters compared to the control (p = 0.171). Results from TEM showed that EVs- bind to spermatozoa worse than EVs+ after incubation. EVs- bind predominantly to the lateral membrane of the spermatozoon, rather than to the acrosomal region, compare to EVs+, which may reflect changes in the functional composition of FF EVs in older patients group and target areas of interaction with spermatozoa. This hypothesis is also supported by slight improvements in progressive sperm motility after incubation. Age-related changes are reflected in energy, metabolic and other important biological processes, which leads to a change in the composition and functions of FF (decrease in progesterone, glucose, increase in lactate and pyruvate, increase in reactive oxygen species) and affect the functional role of FF EVs. MiR-134-5p (p = 0.008), miR-21-5p (p = 0.008) expression levels were statistically significantly higher in EVs- group compared with EV + group with a successful first attempt. The expression of other microRNAs analyzed did not differ between groups. Limitations, reasons for caution Small amount of data. This study does not characterize isolated FF EVs in any way (specific markers, EVs concentration, size). To discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics, it is necessary to perform metabolomics analyses of FF EVs. Wider implications of the findings Further studies of the influence of FF EVs on sperm motility may help to improve of ART outcomes, associated with male and female infertility by improving sperm morphofunctional characteristics and increasing their fertilizing ability. The solutions to this problem could be the use of FF EVs from young fertile donors. Trial registration number not applicable

Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24–38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART.

Comparison of MicroRNA Profiles in Extracellular Vesicles from Small and Large Goat Follicular Fluid.

Simple SummaryOvarian follicular development is associated with ovulation and is further related to litter size in goats. Extracellular vesicles (EVs) derived from miRNAs within follicular fluid undergo dynamic changes, and, together with follicle growth, may be considered as potential regulators of follicular development. However, the function and changes in EVs remain ambiguous. Here, we identified miRNA changes in EVs from small to large goat follicular fluid. Using bioinformatics tools, we demonstrated the existence of differentially expressed miRNAs in EVs from follicles of different sizes that are responsible for an altered biological effect. This study contributes to a better understanding of follicular development in goats.Extracellular vesicles (EVs), which exist in the follicular fluid of ruminant ovaries, are considered as cargo carriers for the transfer of biomolecules to recipient cells. However, the functions and changes in EVs in antral follicles remain ambiguous. In the present study, we isolated and characterized EVs from goat follicular fluid by means of differential ultracentrifugation and Western blotting of marker proteins. Bioinformatics tools were used to detect miRNA expression levels in EVs. Different miRNA expression patterns of EVs exist in small to large follicles. Thirteen differentially expressed miRNAs (seven upregulated and six downregulated) were identified and used for analysis. A total of 1948 predicted target genes of 13 miRNAs were mapped to signaling pathways, and three significantly enriched pathways (FoxO, MAPK, and PI3K-AKT signaling pathways) were involved in follicular development, as revealed by KEGG enrichment analysis. Our findings suggest that EVs in follicular fluid play biofunctional roles during follicular development in goats.

259 The Role of Extracellular Vesicles in Mitigating the Effect of Heat Stress

Abstract Environmental heat stress negatively affects the fertility of dairy cows by disrupting reproductive processes spanning from follicular development to maternal recognition of pregnancy. Investigation of cellular level responses to stress would contribute to the understanding of the mechanism behind survival responses. Extracellular vesicles (EVs), which carry biologically active signaling molecules, are reported to play a significant role in the cellular response to stress. They are produced by almost all types of cells and abundantly present in various biological fluids including follicular fluid, oviductal fluid, uterine fluids in vivo, and in spent culture media in vitro. Those EV-coupled molecular signals in biological fluids are indicative of the physiological status of the cells of their origin. This has been evidenced by the presence of EV-mediated miRNA signals in follicular fluid associated with the metabolic status of cows. Recent studies revealed the potential role of follicular fluid EVs in carrying molecular signals which can reverse or protect the damage incurred by heat stress in bovine oocytes. In addition to cellular defense responses (activation of HSP70 and HSP90, NRF2 and GRP78 &amp; 94), bovine granulosa cells exposed to heat stress in vitro released EVs enriched with selected mRNA (HSP90 and SOD1) and miRNAs. Among others, miR-1246, miR-374a, and miR-2904 were found to be enriched in EVs released from granulosa cells exposed to thermal stress. Those miRNAs were found to regulate pathways related to heat and endoplasmic reticulum stress responses. The priming of recipient bovine granulosa cells by EVs derived from heat-stressed granulosa cells induced tolerance against recurrent heat stress. Collectively, EV-mediated molecular signals would provide another layer of cell-to-cell communication and deliver protective signals against oxidative stress to recipient cells. This would provide opportunities for future potential application of EVs in tackling oxidative stress-associated fertility problems in humans and animals.

Lipid profile of extracellular vesicles and their relationship with bovine oocyte developmental competence: New players in intra follicular cell communication

Cell communication within the ovarian follicle is crucial during folliculogenesis to assure an ideal environment for the oocyte to achieve full developmental competence. Intercellular communication is facilitated by the presence of follicular fluid, which mediates the transfer of signaling molecules. Recently, extracellular vesicles (exosomes and microvesicles) containing mRNAs, miRNAs and proteins were described in mammalian follicular fluid. Besides these molecules, extracellular vesicles (EVs) can mediate the transfer of lipids that can act as signal transducers activating second messengers and modulating intracellular pathways. Our goal was to determine the lipid profile of exosomes (small extracellular vesicles) and microvesicles (large extracellular vesicles) from bovine ovarian follicles containing oocytes with different developmental capabilities to verify potential relationships to competence. Using mass spectrometry, we examined the lipid content of EVs present in the follicular fluid of follicles enclosing oocytes that were either unable to cleave (NCLEAVE), arrested at cleavage stage (CLEAVE), or developed to the blastocyst stage (BLAST) after parthenogenetic activation. Although most of the 514 lipids identified in the follicular fluid EVs were common among all groups, 10 exosome-derived lipids and 15 microvesicle-derived lipids were present exclusively in the BLAST group, suggesting a potential relationship with developmental competence. Therefore, our data indicate that the EVs present in follicular fluid of antral follicles of similar morphology contain lipids that may be used as biomarkers associated with the developmental capability of the oocyte to develop to the blastocyst stage.

Extracellular vesicles shuttle protective messages against heat stress in bovine granulosa cells

Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS.

MALDI-TOF Mass Spectrometry Revealed Significant Lipid Variations in Follicular Fluid and Somatic Follicular Cells but Not in Enclosed Oocytes between the Large Dominant and Small Subordinate Follicles in Bovine Ovary.

Lipid metabolism in ovarian follicular cells supports the preparation of an enclosed oocyte to ovulation. We aimed to compare lipid composition of a dominant large follicle (LF) and subordinated small follicles (SFs) within the same ovaries. Mass spectrometry imaging displayed the differences in the distribution of several lipid features between the different follicles. Comparison of lipid fingerprints between LF and SF by Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight (MALDI-TOF) mass spectrometry revealed that in the oocytes, only 8 out of 468 detected lipids (1.7%) significantly changed their abundance (p < 0.05, fold change > 2). In contrast, follicular fluid (FF), granulosa, theca and cumulus cells demonstrated 55.5%, 14.9%, 5.3% and 9.8% of significantly varied features between LF and SF, respectively. In total, 25.2% of differential lipids were identified and indicated potential changes in membrane and signaling lipids. Tremendous changes in FF lipid composition were likely due to the stage specific secretions from somatic follicular cells that was in line with the differences observed from FF extracellular vesicles and gene expression of candidate genes in granulosa and theca cells between LF and SF. In addition, lipid storage in granulosa and theca cells varied in relation to follicular size and atresia. Differences in follicular cells lipid profiles between LF and SF may probably reflect follicle atresia degree and/or accumulation of appropriate lipids for post-ovulation processes as formation of corpus luteum. In contrast, the enclosed oocyte seems to be protected during final follicular growth, likely due in part to significant lipid transformations in surrounding cumulus cells. Therefore, the enclosed oocyte could likely keep lipid building blocks and energy resources to support further maturation and early embryo development.

Effect of extracellular vesicles and microRNAs in follicular fluid on follicular development

Extracellular vesicles (EVs) refer to bilayer membrane transport vesicles secreted by cells. EVs can take macromolecules from cells and transfer them to receptor cells. Among these macromolecular substances, the most studied are microRNAs (miRNAs). miRNA is non-coding RNA involved in the regulation of gene expression. It has been confirmed that there are different non-coding RNAs in mammalian follicular fluid EVs. EVs carrying miRNA can act as an alternative mechanism for autocrine and paracrine, affecting follicular development. This paper systematically introduced the kinds, characteristics and methods of isolation and identification of EVs, focusing on the effects of EVs and miRNAs on follicular development, including early follicular development, oocyte maturation, follicular dominance and effects on granulosa cell function. At the same time, the authors prospected the future research of EVs and microRNAs in follicular fluid, and provided ideas and directions for the research and application of EVs and miRNA functions in follicular fluid.

194 Extracellular vesicles derived from ampullary oviductal fluid improve developmental competence of bovine oocytes

Invivo, oocytes mature in the presence of follicular fluid (FF) and ampullary oviductal fluid (AOF), starting from premature to final maturation stages (3-6h postovulation). Extracellular vesicles (EVs) have been identified as important mediators of gamete/embryo-maternal interactions. They carry regulatory molecules, such as microRNAs and mRNAs. So far, the functionality of EVs derived from FF and AOF has not yet been determined. The aim of this study was to evaluate the effect of EVs derived from FF and AOF during invitro oocyte maturation (IVM) on the development and quality of resulting bovine embryos. Follicular fluid of a preovulatory follicle was collected by ovum pickup, and AOF was collected from the oviducts of slaughtered cows in early luteal phase. Then, EVs from FF and AOF were isolated by size exclusion chromatography and confirmed by western blot. The concentration of EVs was determined by NanoDrop and Nanoparticle tracking analysis. Integrity and size of EVs were assessed by electron microscopy. Bovine oocytes (n=1331) were allocated at random to five groups. In the control group (C), oocytes (n=347) were cultured for 22.5h in maturation medium (TCM-199 and epidermal growth factor 20ngmL−1) (MM). In the negative control group (NC), oocytes (n=331) were cultured for 18h in MM, and then transferred into fresh MM for 4.5h. In the FF group, oocytes (n=162) were cultured in MM supplemented with FF EVs (12.5µgmL−1) for 18h, and then transferred to MM for 4.5h. In the AOF group, oocytes (n=328) were cultured for 18h in EV-free MM, and then transferred to MM supplemented with AOF EVs (1.7µgmL−1) for 4.5h. In the FF+AOF group, oocytes (n=163) were cultured in MM supplemented with FF EVs for 18h, and then transferred into MM supplemented with AOF EVs for 4.5h. At 22.5h post-incubation, mature oocytes were fertilized invitro. At 21h post-insemination, presumptive zygotes were denuded and cultured in synthetic oviductal fluid with insulin-transferrin-selenium for 8 days. Cleavage rate and blastocyst rate were recorded at 48h and 8 days post-insemination, respectively. Blastocyst quality was assessed by differential apoptotic staining. The data were analysed using one-way ANOVA. Cleavage rate was not affected by the treatment, whereas blastocyst yield in the AOF group (48.75%) was significantly higher than that in the C (42.91%) and NC (37.72%) groups. In addition, embryos produced in the AOF group showed the highest number of trophectoderm cells and inner cell mass cells, and lowest number of apoptotic cells (P&amp;lt;0.05). Trophectoderm and inner cell mass cells were higher and apoptotic cells were lower in FF and FF+AOF compared with the C and NC groups. In conclusion, adding AOF EVs to oocyte MM in the last 4.5h of IVM improves quantity and quality of blastocysts. In addition, although FF EVs showed no effect on blastocyst yield, they improved blastocyst quality by increasing cell number.

174 Follicular fluid extracellular vesicles obtained from Holstein cows kept under thermoneutral or heat stress conditions modify gene expression of in vitro-matured oocytes

Heat stress (HS) has a massive impact on bovine reproduction. In cows, some of these deleterious effects involve follicular development and oocyte quality. Extracellular vesicles (EV) secreted by granulosa cells play a critical role in the intrafollicular environment by directly influencing cumulus cells and oocyte functions. The objective of this study was to investigate the effect of follicular fluid EV obtained from Holstein cows kept under thermoneutral (TN) or HS conditions, on in vitro bovine oocyte maturation. Non-lactating Holstein cows were synchronized with the Ovsynch protocol and also received an intravaginal progesterone device. From ovulation day (Day 1), cows were randomly assigned to TN (26°C, 73% humidity; n=12) or HS (36°C, 70% humidity; n=12) environments. On Day 9, 2 follicles (F1 and F2) were individually aspirated and all remained follicles were ablated. Then, on Day 14, newly formed F1 and F2 were also aspirated. Follicular fluid from all follicles from each treatment was pooled and EV were obtained according to Silveira et al. (2017 PLoS One 12, 1-25) and diluted in PBS (100μL of PBS per mL of follicular fluid centrifuged). Pools of 20 cumulus-oocyte complexes (COC) grade 1 or 2 (Stojkovic et al. Biol Reprod 200164, 904-992], predominantly from Bos indicus, were submitted to the following treatment groups: Control (n=4): matured in 90μL of TCM-199 with Eagles’ salts, glutamine, NaHCO3, pyruvate, amikacin, and FSH (base medium); TN (n=4): matured in 81μL of base medium+9μL of TN EV suspension; and HS (n=4): matured in 81μL+9μL of HS EV suspension. All treatments were carried out at 38.5°C for 24h in a humid atmosphere with 5% CO2. After 24h of maturation, COC were evaluated for meiotic progression (Hoechst 33342 stain), DNA integrity (TUNEL stain), and expression of genes related to oocyte quality (TaqMan assay, Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). Results were analysed using ANOVA followed by Tukey post hoc test (P&amp;lt;0.05). When the experimental groups were compared with the control group, there was no treatment effect on meiotic progression, DNA integrity, or gene expression of cumulus cells. In the oocytes of the TN group, the genes HSF1, IGFBP2, BMP15, GDF9, CDCA8, HAS2, RPL15, STAT3,and PFKP were less expressed. We concluded that oocytes matured in the presence of EV from follicular fluid of cows kept under TN conditions had lesser expression of genes related to oocyte quality. This study was supported by FAPESP (Grant #2012/18297-7) and CAPES Foundation of Brazil.

Extracellular vesicles in human follicular fluid do not promote coagulation

Body fluids contain extracellular vesicles expressing tissue factor on their surface and serve as an additional trigger for coagulation. During the menstrual cycle ovarian tissue restoration is mandatory and it is unknown whether follicular fluid might provide procoagulant substances. Within an observational study, follicular fluid from women undergoing IVF/intracytoplasmic sperm injection (ICSI) was analysed by fluorescence-activated cell sorting (FACS), electron microscopy, resistive pulse sensing (RPS), nanoparticle-tracking analysis (NTA) and fibrin generation tests (FGT). The presence of extracellular vesicles, especially CD9-positive extracellular vesicles in follicular fluid, was proven. However, clotting tests revealed no procoagulant properties of the detected extracellular vesicles.

Characterization and Small RNA Content of Extracellular Vesicles in Follicular Fluid of Developing Bovine Antral Follicles.

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, whether EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth remains unknown. 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.

Exosome-mediated communication in the ovarian follicle.

Cells are able to produce and release different types of vesicles, such as microvesicles and exosomes, in the extracellular microenvironment. According to the scientific community, both microvesicles and exosomes are able to take on and transfer different macromolecules from and to other cells, and in this way, they can influence the recipient cell function. Among the different macromolecule cargos, the most studied are microRNAs. MicroRNAs are a large family of non-coding RNAs involved in the regulation of gene expression. They control every cellular process and their altered regulation is involved in human diseases. Their presence in mammalian follicular fluid has been recently demonstrated, and here, they are enclosed within microvesicles and exosomes or they can also be associated to protein complexes. The presence of microvesicles and exosomes carrying microRNAs in follicular fluid could represent an alternative mechanism of autocrine and paracrine communication inside the ovarian follicle. The outcomes from these studies could be important in basic reproductive research but could also be useful for clinical application. In fact, the characterization of extracellular vesicles in follicular fluid could improve reproductive disease diagnosis and provide biomarkers of oocyte quality in ART (Assisted Reproductive Treatment).

Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation.

Extracellular vesicles (EVs) are membrane-bound vesicles, found in biofluids, that carry and transfer regulatory molecules, such as microRNAs (miRNAs) and proteins, and may mediate intercellular communication between cells and tissues. EVs have been isolated from a wide variety of biofluids, including plasma, urine, and, relevant to this review, seminal, follicular and uterine luminal fluid. We conducted a systematic search of the literature to review and present the currently available evidence on the possible roles of EVs in follicular growth, resumption of oocyte development and maturation (meiosis), sperm maturation, fertilization and embryo implantation. MEDLINE, Embase and Web of Science databases were searched using keywords pertaining to EVs, including 'extracellular vesicles', 'microvesicles', 'microparticles' and 'exosomes', combined with a range of terms associated with the period of development between fertilization and implantation, including 'oocyte', 'sperm', 'semen', 'fertilization', 'implantation', 'embryo', 'follicular fluid', 'epididymal fluid' and 'seminal fluid'. Relevant research articles published in English (both animal and human studies) were reviewed with no restrictions on publication date (i.e. from earliest database dates to July 2015). References from these articles were used to obtain additional articles. A total of 1556 records were retrieved from the three databases. After removing duplicates and irrelevant titles, we reviewed the abstracts of 201 articles, which included 92 relevant articles. Both animal and human studies unequivocally identified various types of EVs in seminal, follicular and ULFs. Several studies provided evidence for the roles of EVs in these biofluids. In men, EVs in seminal fluid were linked with post-testicular sperm maturation, including sperm motility acquisition and reduction of oxidative stress. In women, EVs in follicular fluid were shown to contain miRNAs with potential roles in follicular growth, resumption of oocyte meiosis, steroidogenesis and prevention of polyspermy after fertilization. EVs were also detected in the media of cultured embryos, suggesting that EVs released from embryos and the uterus may mediate embryo-endometrium cross-talk during implantation. It is important to note that many of the biologically plausible functions of EVs in reproduction discussed in the current literature have not yet been substantiated by conclusive experimental evidence. A detailed understanding of the contributions of EVs in the series of events from gametogenesis to fertilization and then on to implantation, in both normal and pathological cases, may enable the development of valuable tools to advance reproductive health. Because of the early stage of the field, it is unsurprising that the current literature includes not only growing experimental evidence, but also as-yet unproven hypotheses pertaining to the roles of EVs in key reproductive processes. In this review, we present a comprehensive survey of the rapidly expanding literature on this subject, highlighting both relevant findings and gaps in knowledge.