scholarly journals Cumulus Cell Expansion, Its Role in Oocyte Biology and Perspectives of Measurement: A Review

2015 ◽  
Vol 45 (4) ◽  
pp. 212-225 ◽  
Author(s):  
J. Nevoral ◽  
M. Orsák ◽  
P. Klein ◽  
J. Petr ◽  
M. Dvořáková ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hyaluronic Acid ◽  
High Performance ◽  
Uv Light ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cumulus Expansion

Abstract Cumulus expansion of the cumulus-oocyte complex is necessary for meiotic maturation and acquiring developmental competence. Cumulus expansion is based on extracellular matrix synthesis by cumulus cells. Hyaluronic acid is the most abundant component of this extracellular matrix. Cumulus expansion takes place during meiotic oocyte maturation under in vivo and in vitro conditions. Quantification and measurement of cumulus expansion intensity is one possible method of determining oocyte quality and optimizing conditions for in vitro cultivation. Currently, subjective methods of expanded area and more exact cumulus expansion measurement by hyaluronic acid assessment are available. Among the methods of hyaluronic acid measurement is the use of radioactively labelled synthesis precursors. Alternatively, immunological and analytical methods, including enzyme-linked immunosorbent assay (ELISA), spectrophotometry, and high-performance liquid chromatography (HPLC) in UV light, could be utilized. The high sensitivity of these methods could provide a precise analysis of cumulus expansion without the use of radioisotopes. Therefore, the aim of this review is to summarize and compare available approaches of cumulus expansion measurement, respecting special biological features of expanded cumuli, and to suggest possible solutions for exact cumulus expansion analysis.

scholarly journals Characterisation of the cellular and molecular responses of ovine oocytes and their supporting somatic cells to pre-ovulatory levels of LH and FSH during in vitro maturation

Reproduction ◽  
2012 ◽  
Vol 144 (2) ◽  
pp. 195-207 ◽  
Author(s):  
Matthew Cotterill ◽  
Sally L Catt ◽  
Helen M Picton
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Cumulus Expansion ◽  
Serum Free ◽  
Blastocyst Rate ◽  
The Impact

The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100 ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30 min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.

scholarly journals α-Tocopherol modifies the expression of genes related to oxidative stress and apoptosis during in vitro maturation and enhances the developmental competence of rabbit oocytes

2018 ◽  
Vol 30 (12) ◽  
pp. 1728 ◽  
Author(s):  
M. Arias-Álvarez ◽  
R. M. García-García ◽  
J. López-Tello ◽  
P. G. Rebollar ◽  
A. Gutiérrez-Adán ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Rabbit Model ◽  
Developmental Competence ◽  
Antioxidant Agents ◽  
Junction Protein

The developmental competence of in vitro maturation (IVM) oocytes can be enhanced by antioxidant agents. The present study investigated, for the first time in the rabbit model, the effect of adding α-tocopherol (0, 100, 200 and 400 µM) during IVM on putative transcripts involved in antioxidant defence (superoxide dismutase 2, mitochondrial (SOD2), glutathione peroxidase 1 (GPX1), catalase (CAT)), cell cycle regulation and apoptosis cascade (apoptosis tumour protein 53 (TP53), caspase 3, apoptosis-related cysteine protease (CASP3)), cell cycle progression (cellular cycle V-Akt murine thymoma viral oncogene homologue 1 (AKT1)), cumulus expansion (gap junction protein, alpha 1, 43 kDa (GJA1) and prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclo-oxygenase) (PTGS2)) and metabolism (glucose-6-phosphate dehydrogenase (G6PD)). Meiotic progression, mitochondrial reallocation, cumulus cell apoptosis and the developmental competence of oocytes after IVF were also assessed. Expression of SOD2, CAT, TP53, CASP3 and GJA1 was downregulated in cumulus–oocyte complexes (COCs) after IVM with 100 μM α-tocopherol compared with the group without the antioxidant. The apoptotic rate and the percentage of a non-migrated mitochondrial pattern were lower in COCs cultured with 100 μM α-tocopherol, consistent with better-quality oocytes. In fact, early embryo development was improved when 100 μM α-tocopherol was included in the IVM medium, but remained low compared with in vivo-matured oocytes. In conclusion, the addition of 100 μM α-tocopherol to the maturation medium is a suitable approach to manage oxidative stress and apoptosis, as well as for increasing the in vitro developmental competence of rabbit oocytes.

scholarly journals Hyaluronic acid-functionalized gelatin hydrogels reveal extracellular matrix signals temper the efficacy of erlotinib against patient-derived glioblastoma specimens

2019 ◽  
Author(s):  
Sara Pedron ◽  
Gabrielle L. Wolter ◽  
Jee-Wei E. Chen ◽  
Sarah E. Laken ◽  
Jann N. Sarkaria ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Hyaluronic Acid ◽  
Tumor Microenvironment ◽  
Kinase Inhibitor ◽  
Egfr Mutations ◽  
Primary Glioblastoma ◽  
Stat3 Phosphorylation

AbstractTherapeutic options to treat primary glioblastoma (GBM) tumors are scarce. GBM tumors with epidermal growth factor receptor (EGFR) mutations, in particular a constitutively active EGFRvIII mutant, have extremely poor clinical outcomes. GBM tumors with concurrent EGFR amplification and active phosphatase and tensin homolog (PTEN) are sensitive to the tyrosine kinase inhibitor erlotinib, but the effect is not durable. A persistent challenge to improved treatment is the poorly understood role of cellular, metabolic, and biophysical signals from the GBM tumor microenvironment on therapeutic efficacy and acquired resistance. The intractable nature of studying GBM cell in vivo motivates tissue engineering approaches to replicate aspects of the complex GBM tumor microenvironment. Here, we profile the effect of erlotinib on two patient-derived GBM specimens: EGFR+ GBM12 and EGFRvIII GBM6. We use a three-dimensional gelatin hydrogel to present brain-mimetic hyaluronic acid (HA) and evaluate the coordinated influence of extracellular matrix signals and EGFR mutation status on GBM cell migration, survival and proliferation, as well as signaling pathway activation in response to cyclic erlotinib exposure. Comparable to results observed in vivo for xenograft tumors, erlotinib exposure is not cytotoxic for GBM6 EGFRvIII specimens. We also identify a role of extracellular HA (via CD44) in altering the effect of erlotinib in GBM EGFR+ cells by modifying STAT3 phosphorylation status. Taken together, we report an in vitro tissue engineered platform to monitor signaling associated with poor response to targeted inhibitors in GBM.

In Vivo Engineering of the Vocal Fold Extracellular Matrix with Injectable Hyaluronic Acid Hydrogels: Early Effects on Tissue Repair and Biomechanics in a Rabbit Model

2005 ◽  
Vol 114 (9) ◽  
pp. 662-670 ◽  
Author(s):  
Jennifer K. Hansen ◽  
Susan L. Thibeault ◽  
Jennifer F. Walsh ◽  
Xiao Zheng Shu ◽  
Glenn D. Prestwich
Keyword(s):  
Extracellular Matrix ◽  
Hyaluronic Acid ◽  
Vocal Fold ◽  
Wound Repair ◽  
Rabbit Model ◽  
Vocal Folds ◽  
Biomechanical Properties ◽  
Enzyme Linked Immunosorbent Assay ◽  
Early Effects

Objectives: A prospective, controlled animal study was performed to determine whether the use of injectable, chemically modified hyaluronic acid (HA) derivatives at the time of intentional vocal fold resection might facilitate wound repair and preserve the unique viscoelastic properties of the vocal fold extracellular matrix. Methods: We performed bilateral vocal fold biopsies on 33 rabbits. Two groups of rabbits were unilaterally treated with 2 different HA derivatives — Carbylan-SX and HA-DTPH-PEGDA — at the time of resection. Saline was injected as a control into the contralateral fold. The animals were painlessly sacrificed 3 weeks after biopsy and injection. The outcomes measured included histologic fibrosis level, tissue HA level, and tissue viscosity and elasticity. Results: The Carbylan-SX—treated vocal folds were found to have significantly less fibrosis than the saline-treated controls. The levels of HA in the treated vocal folds were not significantly different from those in the controls at 3 weeks as measured by enzyme-linked immunosorbent assay. The Carbylan-SX—treated vocal folds had significantly improved biomechanical properties of elasticity and viscosity. The HA-DTPH-PEGDA injections yielded significantly improved viscosity, but not elasticity. Conclusions: Prophylactic in vivo manipulation of the extracellular matrix with an injectable Carbylan-SX hydrogel appears to induce vocal fold tissue regeneration to yield optimal tissue composition and biomechanical properties favorable for phonation.

Oocyte Induction of EGF Responsiveness in Somatic Cells Is Associated With the Acquisition of Porcine Oocyte Developmental Competence

Endocrinology ◽  
2015 ◽  
Vol 156 (6) ◽  
pp. 2299-2312 ◽  
Author(s):  
Lesley J. Ritter ◽  
Satoshi Sugimura ◽  
Robert B. Gilchrist
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Egf Receptor ◽  
Somatic Cells ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Cumulus Expansion ◽  
Cell Functions ◽  
Oocyte Developmental Competence

Abstract Oocytes progressively acquire the competence to support embryo development as oogenesis proceeds with ovarian folliculogenesis. The objectives of this study were to investigate oocyte-secreted factor (OSF) participation in the development of somatic cell epidermal growth factor (EGF) responsiveness associated with oocyte developmental competence. A well-established porcine model was employed using oocytes from small (&lt;4 mm) vs medium sized (&gt;4 mm) antral follicles, representing low vs moderate developmental competence, respectively. Cumulus-oocyte complexes (COCs) were treated in vitro with inducers of oocyte maturation, and cumulus cell functions and oocyte developmental competence were assessed. COCs from small follicles responded to FSH but, unlike COCs from larger follicles, were incapable of responding to EGF family growth factors known to mediate oocyte maturation in vivo, exhibiting perturbed cumulus expansion and expression of associated transcripts (HAS2 and TNFAIP6). Low and moderate competence COCs expressed equivalent levels of EGF receptor (EGFR) mRNA; however, the former had less total EGFR protein leading to failed activation of phospho-EGFR and phospho-ERK1/2, despite equivalent total ERK1/2 protein levels. Native OSFs from moderate, but not from low, competence oocytes established EGF responsiveness in low competence COCs. Four candidate recombinant OSFs failed to mimic the actions of native OSFs in regulating cumulus expansion. Treatment with OSFs and EGF enhanced oocyte competence but only of the low competence COCs. These data suggest that developmental acquisition by the oocyte of capacity to regulate EGF responsiveness in the oocyte's somatic cells is a major milestone in the oocyte's developmental program and contributes to coordinated oocyte and somatic cell development.

428. Normal follicle growth and maturation in a three-dimensional in vitro culture system: follicular environment manipulation and assessment of oocyte outcomes

2008 ◽  
Vol 20 (9) ◽  
pp. 108
Author(s):  
K. R. Dunning ◽  
L. K. Akison ◽  
D. L. Russell ◽  
R. J. Norman ◽  
R. L. Robker
Keyword(s):  
Culture System ◽  
Cumulus Cell ◽  
Three Dimensional ◽  
Oocyte Quality ◽  
Metabolic Effects ◽  
Follicle Growth ◽  
Cumulus Expansion ◽  
Follicle Diameter

In vivo, the oocyte matures in a niche environment surrounded by somatic cells, and later in ovarian follicular development, by follicular fluid. Maternal diet influences the environment in which an oocyte matures but the mechanisms by which an altered metabolic profile, such as hyperinsulinemia, affects oocyte quality are not known. We investigated the use of a three dimensional follicle culture system allowing direct manipulation of the follicular environment thus circumventing systemic hormonal and metabolic effects. Secondary follicles (113.4 ± 1.02µm, n = 54) were isolated from mice at d12, encapsulated individually in 2µl of alginate matrix, and cultured in aMEM/5%FCS/10 mIU/mL LH/100 mIU FSH at 37°C/5%CO2, with media sampling and replacement every second day. Following 12 days of culture there was a significant 3-fold increase in follicle diameter (320 ± 10.1µm, n = 51). Histological analysis showed normal follicular morphology and antrum formation. Analysis of oestradiol (15.0ng/mL), androstenedione (7.8ng/mL) and progesterone (23.7ng/mL) in the media at d12 confirmed normal steroidogenesis and differentiation. Treatment of follicles with an ovulatory stimulus (1.5IU/mL hCG/5ng/mL Egf), resulted in cumulus expansion and hyaluronan localising to the cumulus oocyte complex (COC) and follicular basement membrane. These analyses were consistent with follicle growth and induction of ovulation in vivo. Further, COCs isolated from follicles and matured in vitro (IVM) in the presence of Egf and FSH, underwent cumulus expansion (CEI 2.8 ± 0.2) and were capable of fertilisation and blastocyst development. LH did not induce IVM COC expansion (CEI 1.36 ± 0.2), reflecting the normal in vivo differentiation process. However, culturing follicles in high insulin (5ug/mL) led to a significant increase in the degree of IVM cumulus expansion in response to LH (CEI 2.1 ± 0.3) indicating inappropriate cumulus cell differentiation, which may lead to poorer oocyte quality. These results demonstrate that this technique recapitulates normal in vivo folliculogenesis and is useful for manipulation of the follicular environment and assessment of oocyte outcomes.

scholarly journals Role for cumulus cell-produced EGF-like ligands during primate oocyte maturation in vitro

2009 ◽  
Vol 296 (5) ◽  
pp. E1049-E1058 ◽  
Author(s):  
Jenna K. Nyholt de Prada ◽  
Young S. Lee ◽  
Keith E. Latham ◽  
Charles L. Chaffin ◽  
Catherine A. VandeVoort
Keyword(s):  
Rhesus Macaque ◽  
Oocyte Maturation ◽  
Egf Receptor ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Lower Percentage ◽  
Egfr Activation

The developmental competence of in vitro-matured (IVM) rhesus macaque cumulus oocyte complexes (COCs) is deficient compared with in vivo-matured (IVM) oocytes. To improve oocyte quality and subsequent embryo development following IVM, culture conditions must be optimized. A series of experiments was undertaken to determine the role of epidermal growth factor (EGF) during IVM of rhesus macaque COCs. The addition of Tyrphostin AG-1478 (a selective inhibitor of the EGF receptor EGFR) to the IVM medium yielded fewer oocytes maturing to metaphase II of meiosis II (MII), decreased cumulus expansion, and a lower percentage of embryos that developed to the blastocyst stage compared with untreated IVM controls, indicating that EGFR activation is important for IVM maturation in the rhesus macaque. However, the addition of recombinant human EGF (r-hEGF) to the IVM medium did not enhance outcome. The expression of mRNAs encoding the EGF-like factors amphiregulin, epiregulin, and betacellulin in cumulus cells indicates that these factors produced by cumulus cells may be responsible for maximal EGFR activation during oocyte maturation, precluding any further effect of exogenous r-hEGF. Additionally, these results illustrate the potential futility of exogenous supplementation of IVM medium without prior knowledge of pathway activity.

scholarly journals Failure to launch: aberrant cumulus gene expression during oocyte in vitro maturation

Reproduction ◽  
2017 ◽  
Vol 153 (3) ◽  
pp. R109-R120 ◽  
Author(s):  
Hannah M Brown ◽  
Kylie R Dunning ◽  
Melanie Sutton-McDowall ◽  
Robert B Gilchrist ◽  
Jeremy G Thompson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Metabolism ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Oocyte Competence ◽  
Oocyte Developmental Competence ◽  
Growth Factor Signalling

In vitro maturation (IVM) offers significant benefits for human infertility treatment and animal breeding, but this potential is yet to be fully realised due to reduced oocyte developmental competence in comparison with in vivo matured oocytes. Cumulus cells occupy an essential position in determining oocyte developmental competence. Here we have examined the areas of deficient gene expression, as determined within microarrays primarily from cumulus cells of mouse COCs, but also other species, between in vivo matured and in vitro matured oocytes. By retrospectively analysing the literature, directed by focussing on downregulated genes, we provide an insight as to why the in vitro cumulus cells fail to support full oocyte potential and dissect molecular pathways that have important roles in oocyte competence. We conclude that the roles of epidermal growth factor signalling, the expanded extracellular matrix, cumulus cell metabolism and the immune system are critical deficiencies in cumulus cells of IVM COCs.

256 ROLE OFTHE BOVINE OOCYTE-SPECIFIC PROTEIN JY-1 IN REGULATION OF MESSENGER RNA ABUNDANCE FOR GENES LINKED TO CUMULUS EXPANSION

2010 ◽  
Vol 22 (1) ◽  
pp. 285 ◽  
Author(s):  
G. Wee ◽  
K. B. Lee ◽  
J. J. Ireland ◽  
G. W. Smith
Keyword(s):  
Extracellular Matrix ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Specific Protein ◽  
Negative Control ◽  
Mrna Abundance ◽  
Cumulus Expansion ◽  
Vitro Fertilization ◽  
Maturation Medium

We have previously demonstrated a requirement of the oocyte-specific protein JY-1 for oocyte and early embryonic development in cattle. Microin- jection of JY-1 siRNA into cumulus-enclosed germinal vesicle-stage oocytes impedes progression to metaphase II and cumulus expansion during in vitro maturation and limits subsequent embryonic development following in vitro fertilization. Negative effects of siRNA-mediated reduction in JY-1 on oocyte maturation, cumulus expansion, and initial cleavage divisions following in vitro fertilization can be rescued bysupplementation with recom- binant JY-1 protein during oocyte culture. However, the mechanisms involved in JY-1 regulation of above developmental endpoints are unknown. The objective of the current study was to determine whether JY-1-induced regulation of cumulus expansion during meiotic maturation is linked to alterations in mRNA abundance for genes that promote formation and stabilization of the mucified extracellular matrix characteristic of an expanded cumulus layer. Cumulus-enclosed germinal vesicle stage-bovine oocytes were microinjected with JY-1 siRNA, subjected to negative control siRNA microinjection or served as uninjected controls (n = 10 oocytes/treatment; n = 5 replicates), and cultured for 48 h in maturation medium containing 50 μM S-roscovitine to block spontaneous germinal vesicle breakdown. Cumulus-oocyte complexes were then washed and in vitro matured for an additional 24 h in maturation medium minus S-roscovitine. Additional JY-1 siRNA injected and uninjected cumulus-enclosed oocytes were cultured as described earlier but in the presence of 1 ng/mL of recombinant JY-1 protein (n = 10 oocytes/treatment; n = 5 replicates). Dose of recombinant JY-1 protein utilized was previously shown to reverse inhibitory effects of JY-1 siRNA injection on cumulus expansion. After in vitro maturation, cumulus cells were harvested and RNA isolated and subjected to reverse transcription. Real-time PCR analysis was then conducted to determine the effect of treatments on cumulus-cell mRNA abundance for HAS2, HAS3, PTX3, and TNFAIP6. Effects of JY-1 supplementation or depletion (siRNA injection) on cumulus-cell mRNA abundance for HAS2 and HAS3 (enzymes involved in hyaluronan synthesis) were not observed. However, abundance of mRNA for TNFAIP6 and PTX3 (molecules implicated in stabilization of the hyaluronan-rich extracellular matrix) was reduced (relative to uninjected and negative control siRNA groups) in response to JY-1 siRNA injection (P < 0.05) and effects of JY-1 siRNA were rescued by JY-1 protein treatment (P < 0.05). Effects of JY-1 protein supplementation on cumulus-cell TNFAIP6 and PTX3 mRNA in uninjected controls were not observed. Results support a requirement of the oocyte-specific protein JY-1 for regulation of expression of genes functionally linked to stabilization of the hyaluronan-rich extracellular matrix and cumulus expansion. This research was supported by USDA 2008-35203-19094 to G. W. Smith.

scholarly journals Microarray analysis of mRNA from cumulus cells following in vivo or in vitro maturation of mouse cumulus–oocyte complexes

2013 ◽  
Vol 25 (2) ◽  
pp. 426 ◽  
Author(s):  
Karen L. Kind ◽  
Kelly M. Banwell ◽  
Kathryn M. Gebhardt ◽  
Anne Macpherson ◽  
Ashley Gauld ◽  
...  
Keyword(s):  
Microarray Analysis ◽  
Cell Function ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Chorionic Gonadotrophin ◽  
Developmental Competence ◽  
Rt Pcr ◽  
Oocyte Competence

The IVM of mammalian cumulus–oocyte complexes (COCs) yields reduced oocyte developmental competence compared with oocytes matured in vivo. Altered cumulus cell function during IVM is implicated as one cause for this difference. We have conducted a microarray analysis of cumulus cell mRNA following IVM or in vivo maturation (IVV). Mouse COCs were sourced from ovaries of 21-day-old CBAB6F1 mice 46 h after equine chorionic gonadotrophin (5 IU, i.p.) or from oviducts following treatment with 5 IU eCG (61 h) and 5 IU human chorionic gonadotrophin (13 h). IVM was performed in α-Minimal Essential Medium with 50 mIU FSH for 17 h. Three independent RNA samples were assessed using the Affymetrix Gene Chip Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA, USA). In total, 1593 genes were differentially expressed, with 811 genes upregulated and 782 genes downregulated in IVM compared with IVV cumulus cells; selected genes were validated by real-time reverse transcription–polymerase chain reaction (RT-PCR). Surprisingly, haemoglobin α (Hba-a1) was highly expressed in IVV relative to IVM cumulus cells, which was verified by both RT-PCR and western blot analysis. Because haemoglobin regulates O2 and/or nitric oxide availability, we postulate that it may contribute to regulation of these gases during the ovulatory period in vivo. These data will provide a useful resource to determine differences in cumulus cell function that are possibly linked to oocyte competence.

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