The role of small extracellular vesicle-miRNAs in endometriosis.

Endometriosis is defined by the presence of extrauterine endometrial-like tissue, which can cause pain and infertility in 10% of reproductive-age women. To date, the pathogenesis is poorly understood resulting in significant diagnostic delays and poor therapeutic outcomes in many women. Small extracellular vesicles (sEVs) (<200 nm) are cell-derived vesicles containing molecules that can influence gene expression and behaviour in target cells. One such cargo are microRNAs (miRNAs), which are short, non-coding RNAs mostly 19-25 nucleotides in length that regulate post-transcriptional gene expression. This mini-review focuses on the role of sEV-miRNAs, which are conceivably better biomarkers for endometriosis than free miRNAs, which reflect the true pathophysiological state in the body, as sEV-encapsulated miRNAs are protected from degradation compared to free miRNA and provide direct cell-to-cell communication via sEV surface proteins. sEV-miRNAs have been implicated in the immunomodulation of macrophages, the proliferation, migration and invasion of endometrial cells, and angiogenesis, all hallmarks of endometriosis. The diagnostic potential of sEV-miRNA was investigated in one study that reported the sensitivity and specificity of two sEV-miRNAs (hsa-miR-22-3p and hsa-miR-320a-3p) in distinguishing endometriosis from non-endometriosis cases. Only three studies have explored the therapeutic potential of sEV-miRNAs in vivo in mice-two looked into the role of sEV-hsa-miR-214-3p in decreasing fibrosis, and one investigated sEV-hsa-miR-30c-5p in suppressing the invasive and migratory potential of endometriotic lesions. While early results are encouraging, studies need to further address the potential influence of factors such as the menstrual cycle as well as the location and extent of endometriotic lesions on miRNA expression in sEVs. Given these findings, and extrapolating from other conditions such as cancer, diabetes, and pre-eclampsia, sEV-miRNAs could present an attractive and urgently needed future diagnostic and therapeutic target for millions of women suffering from endometriosis. However, research in this area is hampered by lack of adherence to the International Society for Extracellular Vesicles 2018 guideline in separating and characterising sEVs, as well as the World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonisation Project protocols.

Macrophages and small extracellular vesicle mediated-intracellular communication in the peritoneal microenvironment: Impact on endometriosis development.

Endometriosis is an inflammatory disease that is defined as the growth of endometrium-like tissue outside the uterus, commonly on the lining of the pelvic cavity, visceral organs and in the ovaries. It affects around 190 million women of reproductive age worldwide and is associated with chronic pelvic pain and infertility, which greatly impairs health-related life quality. The symptoms of the disease are variable, this combined with a lack of diagnostic biomarkers and necessity of surgical visualisation to confirm disease, the prognosis can take an average timespan of 6-8 years. Accurate non-invasive diagnostic tests and the identification of effective therapeutic targets are essential for disease management. To achieve this, one of the priorities is to define the underlying pathophysiological mechanisms that contribute to endometriosis. Recently, immune dysregulation in the peritoneal cavity has been linked to endometriosis progression. Macrophages account for over 50% of immune cells in the peritoneal fluid and are critical for lesion growth, angiogenesis, innervation and immune regulation. Apart from the secretion of soluble factors like cytokines and chemokines, macrophages can communicate with other cells and prime disease microenvironments, such as the tumour microenvironment, via the secretion of small extracellular vesicles (sEVs). The sEV-mediated intracellular communication pathways between macrophages and other cells within the peritoneal microenvironment in endometriosis remain unclear. Here, we give an overview of peritoneal macrophage (pMΦ) phenotypes in endometriosis and discuss the role of sEVs in the intracellular communication within disease microenvironments and the impact they may have on endometriosis progression.

Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro.

Aim: To investigate exosomes as a noninvasive delivery tool for mammalian sperm. Materials & Methods: Exosomes were isolated from HEK293T cells and co-incubated with boar sperm in vitro. Results: Internalized exosomes were detected within 10 min of co-incubation. Computer-assisted sperm analysis and flow cytometry demonstrated that even after 5-h of exposure to exosomes, there were no significant deleterious effects with regard to sperm motility, viability, membrane integrity and mitochondrial membrane potential (p > 0.05), thus indicating that exosomes did not interfere with basic sperm function. Conclusion: HEK293T-derived exosomes interacted with boar sperm without affecting sperm function. Exosomes represent a versatile and promising research tool for studying sperm biology and provide new options for the diagnosis and treatment of male infertility.

Characterization of exosomes in peritoneal fluid of endometriosis patients.

OBJECTIVE: To demonstrate the feasibility of studying exosomes directly from peritoneal fluid, we isolated exosomes from endometriosis patient samples and from controls, and characterized their cargo. DESIGN: Case-control experimental study. SETTING: Academic clinical center. PATIENT (S): Women with and without endometriosis who underwent laparoscopic surgery (n = 28 in total). INTERVENTION (S): None. MAIN OUTCOME MEASURE (S): Concentration of exosomes within peritoneal fluid and protein content of the isolated exosomes. RESULT (S): Peritoneal fluid samples were pooled according to the cycle phase and disease stage to form six experimental groups, from which the exosomes were isolated. Exosomes were successfully isolated from peritoneal fluid in all the study groups. The concentration varied with cycle phase and disease stage. Proteomic analysis showed specific proteins in the exosomes derived from endometriosis patients that were absent in the controls. Five proteins were found exclusively in the endometriosis groups: PRDX1, H2A type 2-C, ANXA2, ITIH4, and the tubulin α-chain. CONCLUSION (S): Exosomes are present in peritoneal fluid. The characterization of endometriosis-specific exosomes opens up new avenues for the diagnosis and investigation of endometriosis.

In vitro decidualisation of human endometrial stromal cells is enhanced by seminal fluid extracellular vesicles.

Extracellular vesicles are highly abundant in seminal fluids and have a known role enhancing sperm function. Clinical pregnancy rates after IVF treatment are improved after female exposure to seminal fluid. Seminal fluid extracellular vesicles (SF-EVs) are candidate enhancers, however, whether SF-EVs interact with cells from the endometrium and modulate the implantation processes is unknown. Here, we investigated whether SF-EVs interact with endometrial stromal cells (ESCs) and enhance decidualisation, a requisite for implantation. SF-EVs, isolated from human seminal fluid (n = 11) by ultracentrifugation, were characterised by nanoparticle tracking analysis and Western blotting, and purified using size exclusion chromatography. Non-decidualised and decidualised primary ESCs (n = 5) were then treated with SF-EVs. Binding of bio-maleimide-labelled SF-EVs was detected by flow cytometry and fluorescence microscopy. Prolactin and IGFBP-1 protein levels in culture media were also analysed after single and multiple SF-EV exposure. SF-EVs size ranged from 50 to 300 nm, and they expressed exosomal markers (ALIX, SYNTENIN-1, CD9 and CD81). SF-EVs bound to non-decidualised and decidualised ESCs at similar levels. ESCs prolactin secretion was increased after single (p = 0.0044) and multiple (p = 0.0021) SF-EV exposure. No differences were found in IGFBP-1 protein levels. In conclusion, SF-EVs enhance in vitro ESC decidualisation and increase secretion of prolactin, an essential hormone in implantation. This elucidates a novel role of SF-EVs on endometrial receptivity. Abbreviations: ECACC: European Collection of Authenticated Cell Cultures; ESCs: endometrial stromal cells; EVs: extracellular vesicles; FCS: foetal calf serum; HRP: horse-radish peroxidase; IFNγ: interferon-gamma; IGF: insulin-like growth factor; IGFBP-1: insulin-like growth factor binding protein 1; IVF: in vitro fertilisation; MVB: multivesicular bodies; NTA: nanoparticle tracking analysis; PRLR-/-: homozygous prolactin receptor knockout; RT: room temperature; SF-EVs: seminal fluid extracellular vesicles; STR: short tandem repeat; TGFß: transforming growth factor ß; uNK: uterine natural killer.

Reduced placental protein 13 (PP13) in placental derived syncytiotrophoblast extracellular vesicles in preeclampsia - A novel tool to study the impaired cargo transmission of the placenta to the maternal organs.

INTRODUCTION: Placental syncytiotrophoblast (STB) release extracellular vesicles (STB-EVs) that communicate physiological and pathological placental signals to the maternal organs. STB-EV release also increases in preeclampsia (PE). Here we explored the cargo of PP13 in STB-EVs from PE versus control placentas. METHODS: Placentae were harvested following cesarean section deliveries, and dual placental lobe perfusion was used to harvest STB-EV. Maternal side perfusate was centrifuged at 10,000 × g to yield the STB microvesicles, and then at 150,000 × g to yield STB exosomes. Total STB-EVs (tSTB-EVs) were collected using a one step 150,000 × g centrifugation. Placental origin and size distribution were assessed by Western blotting and Nanoparticle Tracking Analysis, respectively. PP13 expression was determined by Western blot and ELISA. RESULTS: Placental alkaline phosphatase (PLAP; a STB specific marker) was present in all preparations. Total tSTB-EVs and STB-EXs also expressed the exosome markers such as the Apoptosis-Linked Gene 2-Interacting Protein X (Alix) and the cluster differentiation protein 9 (CD9). PP13 was localized to the outer surface and intra-vesicular compartments of all fractions. Surface to total PP13 ratios were ∼1:1 for all STB-EV preparations. In contrast to the previously reported higher circulating concentrations of soluble PP13 in PE, significantly lower levels of PP13, normalized to total vesicular protein, were observed in PE samples. PP13 reduction in all STB-EVs' sub-populations may be attributed to differences in gestational age (GA). A simple correction for GA suggested that PE may be an important influence. CONCLUSIONS: PP13 is located in and on all types of STB-EVs. Circulating PP13 may therefore be either soluble or associated with extracellular vesicles with different pathophysiological effects in the maternal circulation.

Endothelium-derived extracellular vesicles promote splenic monocyte mobilization in myocardial infarction.

Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1-positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion- and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.

Analysis of Extracellular Vesicles Using Fluorescence Nanoparticle Tracking Analysis.

Fluorescence nanoparticle tracking analysis (fl-NTA) allows for accurate sizing, counting, and phenotyping of extracellular vesicles (EV). Here, we present two protocols for the analysis of EVs using fl-NTA, highlighting the potential pitfalls and challenges. The first protocol utilizes CellMask Orange™ (CMO) as a general membrane marker to label EVs derived from plasma. The second protocol describes the use of a Qdot-conjugated antibody to identify syncytiotrophoblast (STB)-derived EVs. "Standard" preparations of STB-derived EVs enriched for either microvesicles (STBMV) or exosomes (STBEX), containing a known amount of EV positive for the STB specific antigen placental alkaline phosphatase (PLAP), were also used to optimize fl-NTA camera settings.

Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia.

Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N G-nitro-l-arginine methyl ester (eNOS inhibitor; P<0.05) but not by N-(3-(aminomethyl) bezyl) acetamidine) (inducible nitric oxide synthase inhibitor). STBEV-eNOS catalytic activity was confirmed by visualizing eNOS dimerization. STBEV-eNOS was more abundant in uterine vein compared with peripheral blood, indicating placental origin. STBEV isolated from preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P<0.05) and overall lower NO activity (STBMV, not significant; syncytiotrophoblast extracellular exosomes, P<0.05) compared with those from NP. Circulating plasma STBMV from preeclampsia women had lower STBEV-eNOS expression compared with that from NP women (P<0.01). This is the first observation of functional eNOS expressed on STBEV from NP and preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease.

Insights into pancreatic ß cell energy metabolism using rodent ß cell models.

Background: Mitochondrial diabetes is primarily caused by ß-cell failure, a cell type whose unique properties are important in pathogenesis. Methods: By reducing glucose, we induced energetic stress in two rodent ß-cell models to assess effects on cellular function. Results: Culturing rat insulin-secreting INS-1 cells in low glucose conditions caused a rapid reduction in whole cell respiration, associated with elevated mitochondrial reactive oxygen species production, and an altered glucose-stimulated insulin secretion profile. Prolonged exposure to reduced glucose directly impaired mitochondrial function and reduced autophagy. Conclusions: Insulinoma cell lines have a very different bioenergetic profile to many other cell lines and provide a useful model of mechanisms affecting ß-cell mitochondrial function.

Extracellular vesicles and reproduction-promotion of successful pregnancy.

Extracellular vesicles (EVs) are membrane-bound complexes secreted from cells under both physiological and pathological conditions. They contain proteins, nucleic acids and lipids and act as messengers for cell-cell communication and signalling, particularly between immune cells. EV research is a rapidly evolving and expanding field, and it appears that all biological fluids contain very large numbers of EVs; they are produced from all cells that have been studied to date, and are known to have roles in several reproductive processes. This review analyses the evidence for the role of EVs throughout human reproduction, starting with the paternal and maternal gametes, followed by the establishment and continuation of successful pregnancies, with specific focus, where possible, on the interaction of EVs with the maternal immune system. Importantly, variations within the EV populations are identified in various reproductive disorders, such as pre-term labour and pre-eclampsia.

Multicolor flow cytometry and nanoparticle tracking analysis of extracellular vesicles in the plasma of normal pregnant and pre-eclamptic women.

Excessive release of syncytiotrophoblast extracellular vesicles (STBMs) from the placenta into the maternal circulation may contribute to the systemic inflammation that is characteristic of pre-eclampsia (PE). Other intravascular cells types (platelets, leukocytes, red blood cells [RBCs], and endothelium) may also be activated and release extracellular vesicles (EVs). We developed a multicolor flow cytometry antibody panel to enumerate and phenotype STBMs in relation to other EVs in plasma from nonpregnant (NonP) and normal pregnant (NormP) women, and women with late-onset PE. Nanoparticle tracking analysis (NTA) was used to determine EV size and concentration. In vitro-derived STBMs and EVs from platelets, leukocytes, RBCs, and endothelial cells were examined to select suitable antibodies to analyze the corresponding plasma EVs. Flow cytometry analysis of plasma from NonP, NormP, and PE showed that STBMs comprised the smallest group of circulating EVs, whereas most were derived from platelets. The next most abundant group comprised unidentified orphan EVs (which did not label with any of the antibodies in the panel), followed by EVs from RBCs and leukocytes. NTA showed that the total number of EVs in plasma was significantly elevated in NormP and late-onset PE women compared to NonP controls, and that EVs were smaller in size. In general, EVs were elevated in pregnancy plasma apart from platelet EVs, which were reduced. These studies did not show any differences in EVs between NormP and PE, probably because late-onset PE was studied.

Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis.

Nanoparticle tracking analysis (NTA) is a light-scattering technique that is useful for the rapid sizing and enumeration of extracellular vesicles (EVs). As a relatively new method, NTA has been criticised for a lack of standardisation. We propose the use of silica microspheres for the calibration of NTA measurements and describe in detail a protocol for the analysis of EVs by NTA which should minimise many of the sources of variability and imprecision associated with this technique.

Characterisation of syncytiotrophoblast vesicles in normal pregnancy and pre-eclampsia: expression of Flt-1 and endoglin.

BACKGROUND: The placental syncytiotrophoblast releases micro and nanovesicles (STBM), into the maternal circulation in normal pregnancy and in increased amounts in pre-eclampsia (PE), which have proinflammatory and antiangiogenic activity and are implicated in PE pathophysiology. Better characterisation of STBM is essential to understand their role in PE. METHODS AND RESULTS: STBM prepared by placental lobe dual perfusion (pSTBM) and mechanical disruption (mSTBM) were analysed by four colour flow cytometry (4CFC), nanoparticle tracking analysis (NTA) and Western blotting to determine vesicle size, purity and Flt-1 and endoglin (Eng) expression. Biological activity of STBM associated Flt-1 and endoglin was assessed by the ability of VEGF, PlGF and TGFß to bind to mSTBM and inhibit mSTBM induced endothelial monolayer disruption. STBM content was consistently high (~87-95%) across the different preparations. However, surface antigen intensities differed, with significantly lower placental alkaline phosphatase (P<0.05) and Eng (P<0.05) expression on mSTBM, and Flt-1 (P<0.05) expression on pSTBM. For PE placenta derived preparations, pSTBM contained lower Eng positive STBM (P<0.05) and mSTBM Eng expression was increased (P<0.05). Western blotting revealed increased Flt-1/sFlt-1 (P<0.02) and decreased placental alkaline phosphatase (P = 0.0002) content of PE placenta pSTBM. Using NTA, perfused PE placentas released significantly larger MV (P<0.001). Finally, VEGF, PlGF and TGFß bound to mSTBM at physiologically relevant concentrations and inhibited mSTBM induced endothelial disruption (P<0.05-P<0.001). CONCLUSIONS: This study has found differences in physical and antigenic characteristics of normal and PE placenta STBM preparations produced by placental perfusion or mechanical disruption. We have also demonstrated that large quantities of biologically active STBM associated endoglin and Flt-1/sFlt-1 could contribute to the increased circulating levels measured in PE patients and add to the perturbation of the maternal vascular endothelium, normally attributed to non-membrane bound sFlt-1 and sEndoglin.

Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis.

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ≈ 50 nm and is far more sensitive than conventional flow cytometry (lower limit ≈ 300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. FROM THE CLINICAL EDITOR: The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry.

Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

INTRODUCTION: Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. MATERIALS AND METHODS: Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. RESULTS: Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. CONCLUSIONS: This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease.

New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes.

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.

Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion.

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.

Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation.

Oocytes regulate follicle growth by secreting paracrine growth factors that act on neighbouring granulosa cells (GCs). Those factors identified to date are mainly members of the transforming growth factor-beta (TGFbeta) superfamily, but little is known about which specific receptor/signalling system(s) they employ. This study was conducted to determine the requisite pathways utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded mouse oocytes are co-cultured with mural GCs. Oocytes, growth differentiation factor-9 (GDF9), TGFbeta1 and activin-A all promoted GC DNA synthesis, but bone-morphogenetic protein 6 (BMP6) did not. Subsequently, we tested the capacity of various TGFbeta superfamily receptor ectodomains (ECD) to neutralise oocyte- or specific growth factor-stimulated GC proliferation. The BMP type-II receptor (BMPR-II) ECD antagonised oocyte and GDF9 bioactivity dose-dependently, but had no or minimal effect on TGFbeta1 and activin-A bioactivity, demonstrating its specificity. The TGFbetaR-II, activinR-IIA and activinR-IIB ECDs all failed to neutralise oocyte- or GDF9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective native ligands. An activin receptor-like kinase (ALK) 4/5/7 inhibitor, SB431542, also antagonised both oocyte and GDF9 bioactivity in a dose-dependent manner. Consistent with these findings, oocytes, GDF9 and TGFbeta1 all activated SMAD2/3 reporter constructs in transfected GC, and led to phosphorylation of SMAD2 proteins in treated cells. Surprisingly, oocytes did not activate the SMAD1/5/8 pathway in transfected GCs although exogenous BMP6 did. This study indicates that oocyte paracrine factors primarily utilise a similar signalling pathway first identified for GDF9 that employs an unusual combination of TGFbeta superfamily receptors, the BMPR-II and a SMAD2/3 stimulatory ALK (4, 5 or 7), for transmitting their mitogenic actions in GC. This cell-signalling pathway may also have relevance in the hypothalamic-pituitary axis and in germ-somatic cell interactions in the testis.