During folliculogenesis, the oocyte and surrounding cumulus cells form an ensemble called the cumulus-oocyte complex (COC). Due to their interdependence, research on the COC has been a hot issue in the past few decades. A growing body of literature has revealed that intercellular communication is critical in determining oocyte quality and ovulation. This review provides an update on the current knowledge of COC intercellular communication, morphology, and functions. Transzonal projections (TZPs) and gap junctions are the most described structures of the COC. They provide basic metabolic and nutrient support, and abundant molecules for signaling pathways and regulations. Oocyte-secreted factors (OSFs) such as growth differentiation factor 9 and bone morphogenetic protein 15 have been linked with follicular homeostasis, suggesting that the communications are bidirectional. Using advanced techniques, new evidence has highlighted the existence of other structures that participate in intercellular communication. Extracellular vesicles can carry transcripts and signaling molecules. Microvilli on the oocyte can induce the formation of TZPs and secrete OSFs. Cell membrane fusion between the oocyte and cumulus cells can lead to sharing of cytoplasm, in a way making the COC a true whole. These findings give us new insights into related reproductive diseases like polycystic ovary syndrome and primary ovarian insufficiency and how to improve the outcomes of assisted reproduction.

Intercellular communication in the cumulus–oocyte complex during folliculogenesis: A review

Extracellular vesicles (EV) are membrane vesicles surrounded by a lipid bilayer membrane and include microvesicles, apoptotic bodies, exosomes, and exomeres. Exosome-encapsulated microRNAs (miRNAs) released from cancer cells are involved in the proliferation and metastasis of tumor cells via angiogenesis. On the other hand, mesenchymal stem cell (MSC) therapy, which is being employed in regenerative medicine owing to the ability of MSCs to differentiate into various cells, is due to humoral factors, including messenger RNA (mRNA), miRNAs, proteins, and lipids, which are encapsulated in exosomes derived from transplanted cells. New treatments that advocate cell-free therapy using MSC-derived exosomes will significantly improve clinical practice. Therefore, using highly purified exosomes that perform their original functions is desirable. In this review, we summarized advances in the purification, modification, and application of EVs as novel strategies to treat some diseases.

/pdf/advances-in-purification-modification-and-application-of-3jb5v2if.pdf

Advances in Purification, Modification, and Application of Extracellular Vesicles for Novel Clinical Treatments

Follicular fluid (FF) is the microenvironment where a growing oocyte develops. Intrafollicular communication ensures oocyte competence and is carried out through paracrine signaling, the exchange of molecules via gap junctions, and the trafficking of extracellular vesicles (EVs). The study of FF-derived EVs is important for both translational and fundamental research in the female reproductive field. This study aimed to compare the efficacy and purity of two EV isolation methods: size-exclusion chromatography (SEC) and ultracentrifugation (UC). EVs isolated using SEC and UC were compared regarding their size and concentration using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA); protein contamination was assessed with microBCA; specific EV markers were detected with Western blot, and EV morphology was studied with transmission electron microscopy (TEM). Our results show that although both techniques isolated small EVs, a significantly increased yield in particle number was clear with UC compared with SEC. On the other hand, SEC generated purer EVs with fewer protein contaminants and aggregates. In conclusion, the selection of the most suited approach to isolate EVs must be conducted considering the degree of recovery, purity, and downstream application of the isolated EVs.

/pdf/isolation-of-extracellular-vesicles-from-human-follicular-2m5h67su.pdf

Isolation of Extracellular Vesicles from Human Follicular Fluid: Size-Exclusion Chromatography versus Ultracentrifugation

Gold nanoclusters (Au NCs) are attractive luminescent nanoprobes for biomedical applications. In vivo biosensing and bioimaging requires the delivery of the Au NCs into subcellular compartments. In this view, we explore here the possible encapsulation of ultra-small-sized red and blue emitting Au NCs into liposomes of various sizes and chemical compositions. Different methods were investigated to prepare vesicles containing Au NCs in their lumen. The efficiency of the process was correlated to the structural and morphological aspect of the Au NCs’ encapsulating vesicles thanks to complementary analyses by SAXS, cryo-TEM, and confocal microscopy techniques. Cell-like-sized vesicles (GUVs) encapsulating red or blue Au NCs were successfully obtained by an innovative method using emulsion phase transfer. Furthermore, exosome-like-sized vesicles (LUVs) containing Au NCs were obtained with an encapsulation yield of 40%, as estimated from ICP-MS.

/pdf/encapsulation-of-luminescent-gold-nanoclusters-into-12d5km8s.pdf

Encapsulation of Luminescent Gold Nanoclusters into Synthetic Vesicles

Abstract Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin. 

Cryogenic electron microscopy reveals morphologically distinct subtypes of extracellular vesicles among porcine ejaculate fractions

The oocyte microenvironment constituted by the follicular fluid (FF) is a key for the optimal development of female gametes. Its composition reflects the physiological state of the ovarian follicle. The particularity of FF is to contain a huge diversity of extracellular vesicles specific to women, in the same way as seminal plasma in men. Here, we described and compared morphological aspects of broad subcategories of human FF-related Extracellular Vesicles (EVs). EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. EVs isolated from FF are involved in different biological functions related to follicular growth, oocyte maturation, and embryo development. However, knowledge on the morphology of FF-derived EVs is limited, mainly due to their sub-micrometer size and to intrinsic limitations in methods applied for their characterization. The aim of this study was to provide a comprehensive morphological description of EVs from FF of healthy subjects and quantification. EVs separation was realized by centrifugation, with comparison of the EV yield obtained from differential centrifugation and one-step ultracentrifugation. Cryo-Transmission Electron Microscopy was used to reveal the morphology, size, and phenotype of EVs. Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) were used to quantify and analyze the size distribution for each centrifugation step. We performed a comprehensive inventory of human follicular fluid EVs. We show that human FF contains a huge diversity of EVs. This study brings novel insights on EVs from normal FF and provides a reference for further studies of EVs in ovarian diseases.

/pdf/diversity-of-extracellular-vesicles-in-human-follicular-2wvt63xa.pdf

Diversity of Extracellular Vesicles in Human Follicular Fluid: Morphological Analysis and Quantification

According to their high electron density and ultrasmall size, gold nanoclusters (AuNCs) have unique luminescence and photoelectrochemical properties that make them very attractive for various biomedical fields. These applications require a clear understanding of their interaction with biological membranes. Here we demonstrate the ability of the AuNCs as markers for lipidic bilayer structures such as synthetic liposomes and biological extracellular vesicles (EVs). The AuNCs can selectively interact with liposomes or EVs through an attractive electrostatic interaction as demonstrated by zetametry and fluorescence microscopy. According to the ratio of nanoclusters to vesicles, the lipidic membranes can be fluorescently labeled without altering their thickness until charge reversion, the AuNCs being located at the level of the phosphate headgroups. In presence of an excess of AuNCs, the vesicles tend to adhere and aggregate. The strong adsorption of AuNCs results in the formation of a lamellar phase as demonstrated by cryo-transmission electron microscopy and small-angle X-ray scattering techniques.

https://hal.science/hal-03773450/document

Luminescent Gold Nanoclusters Interacting with Synthetic and Biological Vesicles.


 
 
 Our aim is to analyze the morphology of follicular fluid extracellular vesicles (EVs) according to ovarian pathology by labeling the vesicles with luminescent gold nanoclusters.
 
 
 
 Cryo-TEM analysis reveals that EVs from the follicular fluid of patients presenting with polycystic ovary syndrome have a particularly complex morphology compared to normo-ovulatory women.
 
 
 
 EVs are particles enclosed by a lipid membrane and secreted by cells. Previously considered as cellular debris, they are in fact involved in a multitude of biological functions and are potential biomarkers. Development of an optical biosensor based on luminescent nanoparticles would make it possible to analyze them in biological fluids.
 
 
 
 Samples were provided by GERMETHEQUE, Biobank dedicated to human fertility. Samples from 32 patients presenting with ovarian pathology (ovarian failure, polycystic ovary syndrome) and 32 normo-ovulatory patients were analyzed.
 
 
 
 Luminescent gold nanoclusters (1 to 2 nm in diameter) have been synthesized and functionalized in order to interact with extracellular vesicles. Analysis of the morphology of EVs was carried out by cryoTEM. Qualitative results were analyzed by Chi2 or Fisher tests. The quantitative results were analyzed by the Kruskal Wallis test.
 
 
 
 The interactions between luminescent gold nanoclusters and lipid membranes show that it is possible to induce electrostatically and in a controlled manner either significant structural changes in the membranes or membrane labeling. Patients presenting with ovarian pathology have more oval vesicles (p = 0.0014). Differences are especially significant in polycystic ovary syndrome showing that simple vesicles are less abundant than controls (p < 0.0001) and large tubules more frequent (p = 0.0023).
 
 
 
 The first results concerning the interactions between luminescent nanoclusters and vesicles demonstrate their potential for labeling these membrane structures
 
 
 
 The characterization of these anomalies could allow a better understanding of ovarian pathology and open up new therapeutic fields. An efficient method of encapsulation in liposomes of gold nanoclusters has been developed.
 
 
 
 GERM 20220705


https://academic.oup.com/humrep/article-pdf/38/Supplement_1/dead093.1009/50655764/dead093.1009.pdf

P-685 Extracellular vesicles of human follicular fluid as biomarkers of ovarian disease

Extracellular Vesicles From Follicular Fluid in Infertile Women: Size, Morphology and miRNA Content Analysis

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Papers

Diversity of Extracellular Vesicles in Human Follicular Fluid: Morphological Analysis and Quantification

Luminescent Gold Nanoclusters Interacting with Synthetic and Biological Vesicles.

P-685 Extracellular vesicles of human follicular fluid as biomarkers of ovarian disease

Extracellular Vesicles From Follicular Fluid in Infertile Women: Size, Morphology and miRNA Content Analysis