Effects of oocyte‐derived paracrine factors on release of extracellular vesicles by murine mural granulosa cells in vitro

Abstract LH triggers the maturation of the cumulus-oocyte complex (COC), which is followed by ovulation. These ovarian follicular responses to LH are mediated by epidermal growth factor (EGF)-like growth factors produced by granulosa cells and require the participation of oocyte-derived paracrine factors. However, it is not clear how oocytes coordinate with the EGF receptor (EGFR) signaling to achieve COC maturation. The aim of the present study was to test the hypothesis that oocytes promote the expression of EGFR by cumulus cells, thus enabling them to respond to the LH-induced EGF-like peptides. Egfr mRNA and protein expression were dramatically reduced in cumulus cells of mutant mice deficient in the production of the oocyte-derived paracrine factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). Moreover, microsurgical removal of oocytes from wild-type COCs dramatically reduced expression of Egfr mRNA and protein, and these levels were restored by either coculture with oocytes or treatment with recombinant GDF9 or GDF9 plus recombinant BMP15. Blocking Sma- and Mad-related protein (SMAD)2/3 phosphorylation in vitro inhibited Egfr expression in wild-type COCs and in GDF9-treated wild-type cumulus cells, and conditional deletion of Smad2 and Smad3 genes in granulosa cells in vivo resulted in the reduction of Egfr mRNA in cumulus cells. These results indicate that oocytes promote expression of Egfr in cumulus cells, and a SMAD2/3-dependent pathway is involved in this process. At least two oocyte-derived growth factors, GDF9 and BMP15, are required for EGFR expression by cumulus cells.

Download Full-text

Emerging role of extracellular vesicles in communication of preimplantation embryos in vitro

Reproduction Fertility and Development ◽

10.1071/rd16318 ◽

2017 ◽

Vol 29 (1) ◽

pp. 66 ◽

Cited By ~ 9

Author(s):

Krishna C. Pavani ◽

Carmen Alminana ◽

Eline Wydooghe ◽

Maaike Catteeuw ◽

Miguel A. Ramírez ◽

...

Keyword(s):

Embryo Development ◽

Extracellular Vesicles ◽

Molecular Mechanisms ◽

Culture Media ◽

Somatic Cells ◽

Indirect Evidence ◽

Preimplantation Embryos ◽

Limited Information ◽

Paracrine Factors

In vitro, efficient communication between mammalian embryos in groups or between embryos and cocultured somatic cells implies that there is a sender, a message and a receiver that is able to decode the message. Embryos secrete a variety of autocrine and paracrine factors and, of these, extracellular vesicles have recently been implicated as putative messengers in embryo–embryo communication, as well as in communication of the embryo with the maternal tract. Extracellular vesicles (EVs) are membrane-bound vesicles that are found in biofluids and in culture media conditioned by the presence of embryos or cells. EVs carry and transfer regulatory molecules, such as microRNAs, mRNAs, lipids and proteins. We conducted a systematic search of the literature to review and present the currently available evidence regarding the possible roles of EVs in in vitro embryo communication and embryo development. It is important to note that there is limited information available on the molecular mechanisms and many of the biologically plausible functions of EVs in embryo communication have not yet been substantiated by conclusive experimental evidence. However, indirect evidence, such as the use of media conditioned by embryos or by somatic cells with improved embryo development as a result, may indicate that EVs can be an important asset for the development of tailor-made media, allowing better embryo development in vitro, even for single embryo culture.

Download Full-text

Cartilage Protective and Immunomodulatory Features of Osteoarthritis Synovial Fluid-Treated Adipose-Derived Mesenchymal Stem Cells Secreted Factors and Extracellular Vesicles-Embedded miRNAs

Cells ◽

10.3390/cells10051072 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1072

Author(s):

Enrico Ragni ◽

Alessandra Colombini ◽

Marco Viganò ◽

Francesca Libonati ◽

Carlotta Perucca Orfei ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Synovial Fluid ◽

Extracellular Vesicles ◽

Immune Cell ◽

Macrophage Polarization ◽

M2 Macrophage ◽

Paracrine Factors ◽

Cell Proliferation Inhibition

Intra-articular administration of adipose-derived mesenchymal stem cells (ASCs), either in vitro expanded or within adipose tissue-based products obtained at point-of-care, has gained popularity as innovative regenerative medicine approach for osteoarthritis (OA) treatment. ASCs can stimulate tissue repair and immunomodulation through paracrine factors, both soluble and extracellular vesicles (EV) embedded, collectively defining the secretome. Interaction with the degenerative/inflamed environment is a crucial factor in understanding the finely tuned molecular message but, to date, the majority of reports have described ASC-secretome features in resting conditions or under chemical stimuli far from the in vivo environment of degenerated OA joints. In this report, the secretory profile of ASCs treated with native synovial fluid from OA patients was evaluated, sifting 200 soluble factors and 754 EV-embedded miRNAs. Fifty-eight factors and 223 EV-miRNAs were identified, and discussed in the frame of cartilage and immune cell homeostasis. Bioinformatics gave a molecular basis for M2 macrophage polarization, T cell proliferation inhibition and T reg expansion enhancement, as well as cartilage protection, further confirmed in an in vitro model of OA chondrocytes. Moreover, a strong influence on immune cell chemotaxis emerged. In conclusion, obtained molecular data support the regenerative and immunomodulatory properties of ASCs when interacting with osteoarthritic joint environment.

Download Full-text

Mesenchymal Stromal Cell-Derived Extracellular Vesicles – Silver Linings for Cartilage Regeneration?

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.593386 ◽

2020 ◽

Vol 8 ◽

Author(s):

Andrea De Luna ◽

Alexander Otahal ◽

Stefan Nehrer

Keyword(s):

Extracellular Vesicles ◽

Treatment Options ◽

Cartilage Regeneration ◽

Bioactive Molecules ◽

Paracrine Factors ◽

Differentiation Potential ◽

Vast Number ◽

The Impact

As the world’s population is aging, the incidence of the degenerative disease Osteoarthritis (OA) is increasing. Current treatment options of OA focus on the alleviation of the symptoms including pain and inflammation rather than on restoration of the articular cartilage. Cell-based therapies including the application of mesenchymal stromal cells (MSCs) have been a promising tool for cartilage regeneration approaches. Due to their immunomodulatory properties, their differentiation potential into cells of the mesodermal lineage as well as the plurality of sources from which they can be isolated, MSCs have been applied in a vast number of studies focusing on the establishment of new treatment options for Osteoarthritis. Despite promising outcomes in vitro and in vivo, applications of MSCs are connected with teratoma formation, limited lifespan of differentiated cells as well as rejection of the cells after transplantation, highlighting the need for new cell free approaches harboring the beneficial properties of MSCs. It has been demonstrated that the regenerative potential of MSCs is mediated by the release of paracrine factors rather than by differentiation into cells of the desired tissue. Besides soluble factors, extracellular vesicles are the major component of a cell’s secretome. They represent novel mechanisms by which (pathogenic) signals can be communicated between cell types as they deliver bioactive molecules (nucleic acids, proteins, lipids) from the cell of origin to the target cell leading to specific biological processes upon uptake. This review will give an overview about extracellular vesicles including general characteristics, isolation methods and characterization approaches. Furthermore, the role of MSC-derived extracellular vesicles in in vitro and in vivo studies for cartilage regeneration will be summarized with special focus on transported miRNA which either favored the progression of OA or protected the cartilage from degradation. In addition, studies will be reviewed investigating the impact of MSC-derived extracellular vesicles on inflammatory arthritis. As extracellular vesicles are present in all body fluids, their application as potential biomarkers for OA will also be discussed in this review. Finally, studies exploring the combination of MSC-derived extracellular vesicles with biomaterials for tissue engineering approaches are summarized.

Download Full-text

PSX-A-19 Late-Breaking: Potential application of granulosa cell derived extracellular vesicles to mitigate the effects of heat stress in bovine oocytes

Journal of Animal Science ◽

10.1093/jas/skab235.676 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 369-369

Author(s):

Nico G Menjivar ◽

Samuel Gebremedhn ◽

Dawit Tesfaye

Keyword(s):

Heat Stress ◽

Thermal Stress ◽

Granulosa Cell ◽

Extracellular Vesicles ◽

Granulosa Cells ◽

Bystander Effect ◽

Thermal Conditions ◽

Developmental Competence ◽

Cumulus Expansion

Abstract Environmental heat stress negatively affects reproductive efficiency by disrupting follicular development, ultimately compromising gamete competency in cattle. Recently, outlined through the bystander effect, granulosa cell derived extracellular vesicles (EVs) were found to suppress negative effects of recurrent heat stress in recipient bovine granulosa cells. Here, we aimed to assess the effects of supplementing granulosa cell derived EVs during bovine in vitro maturation (IVM) on developmental competence following thermal stress. For this, we modeled a cell culture protocol to generate EVs from bovine granulosa cells subjected to differing ambient temperatures, 38.5°C (body temperature) vs. 42°C (heat stress). At the time of IVM, experimental cumulus oocyte complexes (COCs) were arranged in a 2 x 3 factorial design for temperature (38.5°C or 41°C) versus EV supplementation (normal EVs, stressed EVs and non-supplemented controls) at 20% of the IVM media. Following an initial 8h priming period, half the COCs were subjected to heat shock, the others remained at normal temperature to complete IVM. Results indicate that EV supplementation increased cumulus expansion and the expression of cumulus expansion genes (PTX3, PTGS2 and EGFR). Cleavage rates were increased when supplemented with normal (90.2±1.4%; P = 0.023) or stressed (89.8±2.9%; P = 0.029) EVs, compared to the non-supplemented control (80.5±1.5%) under non-thermal conditions. Similarly, exposure to recurrent thermal stress, cleavage rates were (91±0.9%) and (89±0.6%) when supplemented with normal and stressed EVs respectively, compared to the non-supplemented control (88.5±2.5%). In the absence of exposure to recurrent heat stress, blastocysts rates were (32.4±3.5%) and (31.3±2.9%) when COCs were supplemented with normal and stressed EVs, compared to the control (20.7±4.4%). Blastocysts rates were (23.3±4.7%) and (22.5±3.2%) when COCs were supplemented with normal and stressed EVs, compared to the control (15.5±4.5%) when exposed to recurrent thermal stress. In conclusion, granulosa cell derived EVs have potential to induce oocyte tolerance against recurrent thermal stress.

Download Full-text

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

Journal of Animal Science ◽

10.1093/jas/skab235.242 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 132-133

Author(s):

Dawit Tesfaye

Keyword(s):

Oxidative Stress ◽

Heat Stress ◽

Extracellular Vesicles ◽

Granulosa Cells ◽

Follicular Fluid ◽

Biological Fluids ◽

Biologically Active ◽

Molecular Signals

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 & 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.

Download Full-text

Influence of granulosa cells and of different somatic cell types on mammalian oocyte development in vitro

Zygote ◽

10.1017/s0967199400003294 ◽

1996 ◽

Vol 4 (04) ◽

pp. 305-307 ◽

Cited By ~ 13

Author(s):

Sadra Cecconi ◽

Rosella Colonna

Keyword(s):

Gap Junctions ◽

Granulosa Cells ◽

Ovarian Follicle ◽

Cell Types ◽

Paracrine Factors ◽

Oocyte Growth ◽

Major Route ◽

High Concentrations ◽

Development In Vitro

In mammals the ability of an oocyte to become fertilised is the result of a complex process occurring within the ovarian follicle which depends on the stagespecific expression of oocyte genes and the presence of granulosa cells (for a review see Buccioneet al., 1990a). The coordinated development of germinal and somatic components of the follicle is regulated by two principal systems of interaction, based on the presence of gap junctions and on the production of paracrine factors. Gap junctions link granulosa cells to each other and to the oocyte (Anderson & Albertini, 1976), and represent a major route for the transfer of small molecules involved in oocyte metabolism (for a review see Mangiaet al., 1992) and regulation of the arrest and resumption of meiosis (for a review see Eppig, 1993). The production of paracrine factors by granulosa cells has been suggested by the findings that these cells express the production of theSteellocus, the Steel factor (SLF) or kit ligand (KL; Motroet al., 1991; Manovaet al., 1993), and that this factor promotes oocyte growthin vitrowhen used at high concentrations (Packeret al., 1994). Since KL is too large to be transmitted through gap junctions, it must necessarily be released in the extracellular environment before binding to the c-kitreceptor present on oocyte membrane (Manovaet al., 1990; Horieet al., 1991).

Download Full-text

Tumor-Derived Extracellular Vesicles Induce Abnormal Angiogenesis via TRPV4 Downregulation and Subsequent Activation of YAP and VEGFR2

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.790489 ◽

2021 ◽

Vol 9 ◽

Author(s):

Brianna Guarino ◽

Venkatesh Katari ◽

Ravi Adapala ◽

Neha Bhavnani ◽

Julie Dougherty ◽

...

Keyword(s):

Endothelial Cell ◽

Extracellular Vesicles ◽

Tumor Angiogenesis ◽

Lung Tumor ◽

Rho Kinase ◽

Paracrine Factors ◽

Human Lung Tumor ◽

Subsequent Activation

Tumor angiogenesis is initiated and maintained by the tumor microenvironment through secretion of autocrine and paracrine factors, including extracellular vesicles (EVs). Although tumor-derived EVs (t-EVs) have been implicated in tumor angiogenesis, growth and metastasis, most studies on t-EVs are focused on proangiogenic miRNAs and growth factors. We have recently demonstrated that conditioned media from human lung tumor cells (A549) downregulate TRPV4 channels and transform normal endothelial cells to a tumor endothelial cell-like phenotype and induce abnormal angiogenesis in vitro, via t-EVs. However, the underlying molecular mechanism of t-EVs on endothelial cell phenotypic transition and abnormal angiogenesis in vivo remains unknown. Here, we demonstrate that t-EVs downregulate TRPV4 expression post-translationally and induce abnormal angiogenesis by activating Rho/Rho kinase/YAP/VEGFR2 pathways. Further, we demonstrate that t-EVs induce abnormal vessel formation in subcutaneously implanted Matrigel plugs in vivo (independent of tumors), which are characterized by increased VEGFR2 expression and reduced pericyte coverage. Taken together, our findings demonstrate that t-EVs induce abnormal angiogenesis via TRPV4 downregulation-mediated activation of Rho/Rho kinase/YAP/VEGFR2 pathways and suggest t-EVs and TRPV4 as novel targets for vascular normalization and cancer therapy.

Download Full-text

A Chemically Defined, Xeno- and Blood-Free Culture Medium Sustains Increased Production of Small Extracellular Vesicles From Mesenchymal Stem Cells

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.619930 ◽

2021 ◽

Vol 9 ◽

Author(s):

Aliosha I. Figueroa-Valdés ◽

Catalina de la Fuente ◽

Yessia Hidalgo ◽

Ana María Vega-Letter ◽

Rafael Tapia-Limonchi ◽

...

Keyword(s):

Extracellular Vesicles ◽

Large Scale ◽

Culture Medium ◽

Culture Media ◽

Cell Phenotype ◽

Target Cells ◽

Scale Production ◽

Paracrine Factors

Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV’s secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV’s production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV’s characteristics and functionality and the parental cell’s phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method’s interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.

Download Full-text

Transcriptomic profile of genes encoding proteins responsible for regulation of cells differentiation and neurogenesis in vivo and in vitro – an oocyte model approach

Medical Journal of Cell Biology ◽

10.2478/acb-2020-0001 ◽

2020 ◽

Vol 8 (1) ◽

pp. 1-11

Author(s):

Lisa Moncrieff ◽

Ievgeniia Kocherova ◽

Artur Bryja ◽

Wiesława Kranc ◽

Joanna Perek ◽

...

Keyword(s):

Granulosa Cells ◽

Ovarian Follicle ◽

Cumulus Cells ◽

Paracrine Factors ◽

Gene Markers ◽

Genes Encoding ◽

Porcine Oocyte ◽

Micro Analysis

AbstractThe growth and development of the oocyte is essential for the ovarian follicle. Cumulus cells (CCs) - a population of granulosa cells - exchange metabolites, proteins and oocyte-derived paracrine factors with the oocyte through gap junctions, to contribute to the competency and health of the oocyte. This bi-directional communication of the cumulus-oocyte complex could be better understood through the micro-analysis of a porcine oocyte gene expression before in vitro maturation (IVM) and after. Additionally, the study of the somatic and gamete cells differentiation capability into neuronal lineage would be promising for future stem cell research as granulosa cells are easily accessible waste material from in vitro fertilization (IVF) procedures. Therefore, in this study, the oocytes of 45 pubertal Landrace gilts were isolated and the protein expression of the COCs were analyzed through micro-analysis techniques. Genes belonging to two ontological groups: neuron differentiation and negative regulation of cell differentiation have been identified which have roles in proliferation, migration and differentiation. Twenty identified porcine oocyte genes (VEGFA, BTG2, MCOLN3, EGR2, TGFBR3, GJA1, FST, CTNNA2, RTN4, MDGA1, KIT, RYK, NOTCH2, RORA, SMAD4, ITGB1, SEMA5A, SMARCA1, WWTR1 and APP) were found to be down-regulated after the transition of IVM compared to in vitro. These results could be applied as gene markers for the proliferation, migration and differentiation occurring in the bi-directional communication between the oocyte and CCs.Running title: Differentiation and neurogenesis in oocyte cells

Download Full-text