Journal articles on the topic 'Cumulus oocyte complex (COC)'
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Lisle, R. S., K. Anthony, M. A. Randall, and F. J. Diaz. "Oocyte–cumulus cell interactions regulate free intracellular zinc in mouse oocytes." REPRODUCTION 145, no. 4 (April 2013): 381–90. http://dx.doi.org/10.1530/rep-12-0338.
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Zinc increases in the oocyte during maturation and is required for progression and completion of meiosis. The objective of this study was to determine whether cumulus cells regulate the levels of free intracellular zinc in the oocyte during maturation. In the cumulus–oocyte complex (COC) the relative level of free intracellular zinc was almost fourfold higher in cumulus cells compared with the resident germinal vesicle-stage oocyte. Removal of cumulus cells caused a fourfold increase in intracellular zinc in the oocyte by 1 h after cumulus cell removal, but subsequent coculture of denuded oocytes with COC decreased free intracellular zinc in the oocyte by 65%. Thus, cumulus cells suppress free intracellular zinc in the oocyte. The mRNA transcripts for the zinc transporter proteins Slc39a6, Slc39a8, Slc39a9, Slc39a10, Slc39a12, Slc30a2, Slc30a4, Slc30a5 and Slc30a8 mRNAs were higher in oocytes, while Slc39a1, Slc39a7, Slc39a13, Slc39a14, Slc30a6, Slc30a7 and Slc30a9 mRNAs were higher in cumulus cells. Thus a complex zinc transport network is present in the COC. Pretreatment with epidermal growth factor for 4 h abolished the ability of COCs to restrict free intracellular zinc in denuded oocytes. Coculture of denuded metaphase II oocytes with COC lowers free intracellular zinc in mature oocytes. Oocytes matured in vivo or oocytes from older mice had lower levels of free intracellular zinc than oocytes matured in vitro or from younger mice. Thus, a precise mechanism for regulating oocyte zinc homeostasis has been uncovered in the COC that is disrupted with increasing age or by removal of cumulus cells.
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Alvino, E. R., R. L. Robker, and D. L. Russell. "155. CD44 SIGNALLING IN THE CUMULUS OOCYTE COMPLEX DURING OVULATION." Reproduction, Fertility and Development 21, no. 9 (2009): 73. http://dx.doi.org/10.1071/srb09abs155.
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Oocytes develop within ovarian follicles that nurture and regulate oocyte maturation. The LH surge induces a cascade of gene expression leading to formation of the hyaluronan (HA) rich cumulus matrix around the oocyte. This cumulus oocyte complex (COC) is composed of high concentrations of HA cross-linked by several HA-binding proteins. Null mutation of several COC matrix genes results in ovulation defects demonstrating the importance of the composition and structure of the COC; but the mechanisms by which the matrix promotes ovulation are unknown. We hypothesised that HA, via activation of its receptor CD44 on cumulus cells, regulates cytoskeletal function, cell adhesion and migration, and that acquired cumulus cell motility facilitates ovulation. We investigated cellular signaling and cellular phenotypes occurring in response to the formation of the HA-rich COC matrix. Expression of CD44 was upregulated 5 to 6-fold in cumulus cells following 6 or 12h hCG (LH analog) stimulation. Signal transducers of CD44 action; Tiam1, a guanine exchange factor, and Rac1, an actin cytoskeleton remodelling Rho-family GTPase, were present in cumulus cells but not regulated by hCG. Induction of migratory and invasive capacity of cumulus cells by hCG was demonstrated using transwell migration and ECM invasion assays. Cumulus cell migration increased 8-fold 10h after hCG compared with cumulus cells from untreated mice. These cumulus cells also showed the capacity to invade through a matrigel barrier. Inhibitors of the CD44-assembled cell migration complex demonstrated the importance of this pathway in the migratory and invasive phenotype of cumulus cells. These results demonstrate that CD44 is a key factor in the assembly of a macromolecular complex facillitating cell motility in cumulus cells at the time of ovulation, and suggest that cumulus cells in the expanded COC undergo epithelial-mesenchymal transition to become invasive motile cells which may play a key role mediating ovulation.
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Su, You-Qiang, Koji Sugiura, Qinglei Li, Karen Wigglesworth, Martin M. Matzuk, and John J. Eppig. "Mouse Oocytes Enable LH-Induced Maturation of the Cumulus-Oocyte Complex via Promoting EGF Receptor-Dependent Signaling." Molecular Endocrinology 24, no. 6 (June 1, 2010): 1230–39. http://dx.doi.org/10.1210/me.2009-0497.
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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.
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Richani, Dulama, Kylie R. Dunning, Jeremy G. Thompson, and Robert B. Gilchrist. "Metabolic co-dependence of the oocyte and cumulus cells: essential role in determining oocyte developmental competence." Human Reproduction Update 27, no. 1 (October 6, 2020): 27–47. http://dx.doi.org/10.1093/humupd/dmaa043.
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Abstract BACKGROUND Within the antral follicle, the oocyte is reliant on metabolic support from its surrounding somatic cells. Metabolism plays a critical role in oocyte developmental competence (oocyte quality). In the last decade, there has been significant progress in understanding the metabolism of the cumulus–oocyte complex (COC) during its final stages of growth and maturation in the follicle. Certain metabolic conditions (e.g. obesity) or ART (e.g. IVM) perturb COC metabolism, providing insights into metabolic regulation of oocyte quality. OBJECTIVE AND RATIONALE This review provides an update on the progress made in our understanding of COC metabolism, and the metabolic conditions that influence both meiotic and developmental competence of the oocyte. SEARCH METHODS The PubMed database was used to search for peer-reviewed original and review articles. Searches were performed adopting the main terms ‘oocyte metabolism’, ‘cumulus cell metabolism’, ‘oocyte maturation’, ‘oocyte mitochondria’, ‘oocyte metabolism’, ‘oocyte developmental competence’ and ‘oocyte IVM’. OUTCOMES Metabolism is a major determinant of oocyte quality. Glucose is an essential requirement for both meiotic and cytoplasmic maturation of the COC. Glucose is the driver of cumulus cell metabolism and is essential for energy production, extracellular matrix formation and supply of pyruvate to the oocyte for ATP production. Mitochondria are the primary source of ATP production within the oocyte. Recent advances in real-time live cell imaging reveal dynamic fluctuations in ATP demand throughout oocyte maturation. Cumulus cells have been shown to play a central role in maintaining adequate oocyte ATP levels by providing metabolic support through gap junctional communication. New insights have highlighted the importance of oocyte lipid metabolism for oocyte oxidative phosphorylation for ATP production, meiotic progression and developmental competence. Within the last decade, several new strategies for improving the developmental competence of oocytes undergoing IVM have emerged, including modulation of cyclic nucleotides, the addition of precursors for the antioxidant glutathione or endogenous maturation mediators such as epidermal growth factor-like peptides and growth differentiation factor 9/bone morphogenetic protein 15. These IVM additives positively alter COC metabolic endpoints commonly associated with oocyte competence. There remain significant challenges in the study of COC metabolism. Owing to the paucity in non-invasive or in situ techniques to assess metabolism, most work to date has used in vitro or ex vivo models. Additionally, the difficulty of measuring oocyte and cumulus cell metabolism separately while still in a complex has led to the frequent use of denuded oocytes, the results from which should be interpreted with caution since the oocyte and cumulus cell compartments are metabolically interdependent, and oocytes do not naturally exist in a naked state until after fertilization. There are emerging tools, including live fluorescence imaging and photonics probes, which may provide ways to measure the dynamic nature of metabolism in a single oocyte, potentially while in situ. WIDER IMPLICATIONS There is an association between oocyte metabolism and oocyte developmental competence. Advancing our understanding of basic cellular and biochemical mechanisms regulating oocyte metabolism may identify new avenues to augment oocyte quality and assess developmental potential in assisted reproduction.
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Lee, S. H., H. J. Oh, G. A. Kim, M. J. Kim, Y. B. Choi, Y. Kwang Jo, E. Nugraha Setyawan, and B. C. Lee. "212 EFFECT OF CANINE OVIDUCT CELLS AND CUMULUS CELLS CO-CULTURE ON IN VITRO MATURATION OF PORCINE OOCYTES AND EMBRYO DEVELOPMENT." Reproduction, Fertility and Development 28, no. 2 (2016): 237. http://dx.doi.org/10.1071/rdv28n2ab212.
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In oestrus stage, canine oocytes surrounded by cumulus cells undergo maturation in oviduct for 3 days after ovulation. We hypothesised that canine cumulus cells (cCC) and canine oviduct cells (cOC) in oestrus stage might affect the maturation of oocyte and embryo development. Therefore, the present study was aimed to compare the effects of cCC and cOC co-culture system on oocyte in vitro maturation and embryo in vitro development. cCC were separated from cumulus‐oocyte complex (COC) in ovary from bitches in oestrus phase. cOC were collected from oviduct flushing of bitches in oestrus phase. Both cCC and cOC were cultured and cryopreserved until use for co-culture. In the first experiment, the effect of co-culture using cCC and cOC on porcine oocyte in vitro maturation (IVM) were investigated. The porcine COC were randomly cultured in different co-culture groups as follows: 1) co-culturing with cCC for 42 h, 2) co-culturing with cOC for 42 h, and 3) culturing in absence of cCC or cOC. After IVM, extrusion of the first polar body was observed under a microscope. In the second experiment, the matured oocytes with the first polar body derived from each group were activated with electrical stimulus. Parthenotes were cultured in porcine zygote medium-5 (PZM-5) for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The embryo developmental competence was estimated by assessing the in vitro development under microscope. The third experiment was to evaluate the reactive oxygen species (ROS) levels in each supernatant medium obtained from cCC and cOC co-culture group after IVM using a OxiselectTM ROS ELISA Assay kit. Last, analysis of genes (MAPK1/3, SMAD2/3, GDF9 and BMP15) expression in cCC and cOC co-cultured with porcine COC using real-time PCR is in progress. As results, IVM rate of cOC group (91.19 ± 0.45%) was significantly higher than that of cCC and control group (86.50 ± 0.61% and 79.81 ± 0.82%; P < 0.05). Also, cOC groups expressed the highest efficiency in cleavage rate, blastocyst formation rate, and the total cell number in blastocyst (P < 0.05). In ROS levels, cOC group (555 ± 7.77 nM) were significantly lower than cCC and control groups (596.8 ± 8.52 nM and 657.8 ± 11.34 nM). The present study demonstrated that co-culture with cOC improved the in vitro oocyte maturation and the in vitro development rate of porcine embryos. The ROS level decreased in cOC co-culture would have beneficial influence on oocytes maturation. For further study, we will investigate the relation between gene expression related to oocyte maturation and the co-culture results. This research was supported by a global PhD Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), RDA (#PJ010928032015), IPET (#311011–05–4-SB010, #311062–04–3-SB010), Research Institute for Veterinary Science, and the BK21 plus program.
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Gupta, S. C., A. Pandey, and N. Gupta. "245 CONNEXIN 43 mRNA EXPRESSION AT DIFFERENT TIME POINT IN IN VITRO MATURATION OF BUFFALO OOCYTES." Reproduction, Fertility and Development 21, no. 1 (2009): 220. http://dx.doi.org/10.1071/rdv21n1ab245.
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In advanced technologies of ART, the basic requirement is the production of in vitro-matured oocytes, and embryo production efficiency depends on healthy, matured oocytes. Oocyte growth and development depends on the ability of oocytes and their surrounding cumulus granulosa cells (Eppig et al. 1979 J. Exp. Zool. 208, 111–120). Cumulus cells provide carbohydrate precursors, amino acids, and nucleotides to the oocytes (Brower and Schultz 1982 Dev. Biol. 90, 144–153). Oocytes and cumulus cell gap junctions are required for the coordination of cytoplasmic and nuclear maturation (Carabatsos et al. 2002 Dev. Biol. 226, 167–179). In bovine COC, functional gap junctions are required for the progression of oocyte maturation. Gap junctions allow for metabolic coupling between adjacent granulosa cells. Disruption in the integrity of the gap junction inhibits oocyte maturation (Anderson and Albertini 1976 J. Cell Biol. 71, 680–686). The aim of this study was to analyze the trend of Cx43 mRNA transcript in in vitro-matured oocytes at different times of maturation in the Indian water buffalo to estimate the correlation with expression level. Oocytes collected from slaughterhouse ovaries were matured in TCM-199 medium supplemented with 2.5 mm pyruvate, gentamycin sulfate (10 mg mL–1), β-estradiol (1000 ng mL–1), FSH (500 ng mL–1), LH (500 ng mL–1), and 10% FBS at 38.5°C in 5% CO2 in air. Cumulus–oocyte complexes were used after 0, 6, 12, 18, and 24 h of maturation for the cDNA preparation with cells of a cDNA II Kit. Expression of the Cx43 gene was quantified at different time intervals for maturation with real-time PCR. Statistical analysis was performed with one-way ANOVA, followed by Duncan’s multiple pair-wise comparison. Our results showed that Cx43 mRNA abundance was affected by time of maturation. The expression of Cx43 was significantly higher at 6 h than at 18 and 24 h, whereas the 12-h value was intermediate. Our results are in agreement with decreased Cx43 protein contents in the outer cumulus layers of COC at maturation time points (Calder et al. 2003 Reprod. Biol. Endocrinol. 1, 14) and the expression of Cx43 in oocyte development regulation (Granot et al. 2002 Biol. Reprod. 66, 568–573). When Cx43 expression was compared among immature oocytes, denuded oocytes, cumulus cells, and COC at 6 h, there was no significant difference. However, 6-h-matured COC showed significantly higher expression than other groups. Further, our study supported the role of cumulus cells in COC in Cx43-mediated communication (Vozzi et al. 2001 Reproduction 122, 619–628). Differential expression of Cx43 mRNA among varying COC classes indicates that this gene may be a useful marker for oocyte quality to improve in vitro production or somatic cell nuclear transfer rates. Marker genes that predict developmental competence could be used in the optimization of maturation and culture conditions. Understanding the molecular mechanism involved in in vitro oocyte maturation would be an additional advantage in analyzing this complex biological phenomenon to improve embryo production.
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Patel, O. V., A. Bettegowda, J. J. Ireland, and G. W. Smith. "261 IDENTIFICATION OF OOCYTE- AND CUMULUS-DERIVED CO-REGULATED AND DIFFERENTIALLY REGULATED TRANSCRIPTS ASSOCIATED WITH BOVINE MEIOTIC MATURATION." Reproduction, Fertility and Development 18, no. 2 (2006): 238. http://dx.doi.org/10.1071/rdv18n2ab261.
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Understanding the process of oocyte maturation is critical for efficient application of biotechnologies such as in vitro embryo production and nuclear transfer/cloning. Intercellular communication between the oocyte and the encompassing somatic (cumulus) cells is pivotal for successful growth of ovarian follicles and oocyte maturation. Therefore, we utilized global gene expression profiling to determine changes in the transcriptome of oocytes and their adjacent cumulus cells during meiotic maturation in vitro to identify both co-regulated and differentially regulated transcripts within the two cell compartments of the cumulus oocyte complex (COC). Germinal vesicle (GV) and in vitro matured metaphase II (MII) COC (n = 5 pools of 5 COC per group) were denuded and separated into oocytes and cumulus cells. RNA was extracted from the oocytes and cumulus cells and subjected separately to microarray analysis using a bovine cDNA array containing expressed sequence tags (ESTs) representing 15 500 unique genes. A combined total of 1045 genes displaying greater mRNA abundance in GV oocytes and associated cumulus cells compared to MII samples were detected (P < 0.05; false discovery rate (FDR) = 5%). A combined total of 711 genes displaying greater mRNA abundance in MII oocytes and enclosing cumulus cells compared to GV samples were detected (P < 0.05; FDR = 5%). Fourteen transcripts were identified that were co-regulated and of greater abundance in GV or MII oocytes and in their matching cumulus cells (P < 0.05; FDR = 5%). The co-regulated transcripts identified are implicated in metabolism (e.g. heme oxygenase-2, leukotriene B4 12-hydroxydehydrogenase), signal transduction (e.g. caveolin 1, ring finger protein 31), and cell growth (e.g. BTG family member 2, myosin regulatory light chain 2). In contrast, thirteen transcripts differentially regulated in the GV oocyte versus MII cumulus cells were identified (P < 0.05; FDR = 5%). Similarly, nine transcripts differentially regulated in the MII oocyte versus GV cumulus cells were identified (P < 0.05; FDR = 5%). Some of the identified differentially regulated transcripts encode for genes associated with the cytoskeleton (e.g. tropomyosin 1), apoptotic activity (e.g. death effector domain containing protein 2) and DNA replication (e.g. epsilon polymerase). The results provide novel insights into the identity of transcripts whose abundance is co-regulated or differentially regulated between the oocyte and cumulus cells during the transition of a COC from the GV to the MII stage. Characterization of the signaling pathways driving changes in transcript abundance for co-regulated and differentially regulated genes in oocytes versus associated cumulus cells may lead to a better understanding of regulation of meiotic maturation and potential cross-talk between germ cells and somatic cells during the oocyte maturation cascade. This work was supported by the Rackham Foundation and the MI Agriculture Experiment Station.
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Gutnisky, Cynthia, Gabriel C. Dalvit, Laura N. Pintos, Jeremy G. Thompson, Martha T. Beconi, and Pablo D. Cetica. "Influence of hyaluronic acid synthesis and cumulus mucification on bovine oocyte in vitro maturation, fertilisation and embryo development." Reproduction, Fertility and Development 19, no. 3 (2007): 488. http://dx.doi.org/10.1071/rd06134.
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During cumulus–oocyte complex (COC) maturation, cumulus expansion involves the deposition of mucoelastic compounds, especially hyaluronic acid, synthesised from glucose via the hexosamine biosynthesis pathway. The aim of the present study was to determine the effects of uridine monophosphate (UMP) and 6-diazo-5-oxo-l-norleucine (DON), inhibitors of hyaluronic acid synthesis, during bovine oocyte in vitro maturation (IVM) on cumulus expansion, glucose uptake, protein synthesis, cumulus cell number, meiotic maturation, cleavage rate and subsequent embryo development. A further aim of the study was to examine the effect of hyaluronic acid on sperm capacitation and acrosome reaction in relation to the capacity of COCs to be fertilised in vitro. A low correlation between glucose uptake and degree of cumulus expansion was observed. Total and partial inhibition of cumulus expansion was observed with DON and UMP, respectively, and was accompanied by a decrease in glucose uptake with DON. Total protein content and cumulus cell number per COC increased during IVM, but was unaffected by the presence of DON or UMP, as was oocyte meiotic maturation. Rates of cleavage and blastocyst development decreased in oocytes matured with DON and UMP, although this inhibition was reversed when the in vitro fertilisation (IVF) medium contained heparin. Hyaluronic acid induced capacitation and the acrosome reaction, and in IVF medium prevented the inhibition of cleavage and blastocyst development by DON in a similar fashion to heparin. Hyaluronic acid synthesis during cumulus mucification contributes to the penetration and fertilisation of bovine oocytes, most likely by facilitating the processes of capacitation and acrosome reaction. Mucification during IVM is independent of cumulus cell proliferation, COC protein content, oocyte meiotic maturation and subsequent developmental competence once fertilised.
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Alvino, E. R., R. L. Robker, and D. L. Russell. "269. CD44 signaling in mouse ovulatory cumulus - oocyte complexes." Reproduction, Fertility and Development 20, no. 9 (2008): 69. http://dx.doi.org/10.1071/srb08abs269.
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Oocytes grow and develop within ovarian follicles, providing a nurturing environment before their release (ovulation) into the oviduct for fertilisation. For ovulation to occur the ovarian follicle responds to LH from the pituitary, leading to a cascade of regulated gene expression and formation of the hyaluronan rich cumulus matrix around the oocyte. This COC (cumulus oocyte complex) matrix is composed of high concentrations of the ECM glycosaminoglycan hyaluronan (HA) cross-linked by several HA-binding proteins. Several of the COC matrix components are essential for ovulation, since null gene mutations in mice lead to ovulation defects. Mechanisms by which the COC matrix controls ovulation however, are unknown. We have investigated cellular signalling and cellular phenotypes that occur as part of the formation of the COC matrix. The transmembrane HA receptor CD44 was significantly upregulated in cumulus cells from 6 h after hCG (LH analogue) treatment (9.8 ± 1.5 fold) until ovulation at 12 h post hCG (11.8 ± 2.9-fold). In many cell types CD44 activates the intracellular Rho-family GTPase Rac1 and its activator, the guanine exchange factor Tiam1, pleiotropic regulators of cytoskeletal function, cell-cell adhesion and migration. We found both Rac1 and Tiam1 were strongly detected in cumulus cells, but not regulated by hCG. These observations show that at the time of ovulation a macro-molecular complex associated with cell motility is assembled through the extracellular interaction of the COC matrix and cell surface proteins. We investigated the migratory and invasive activity of COCs from hormonally stimulated mice. Migration of cumulus cells from hCG treated mice was significantly increased compared with untreated COCs. Furthermore the hCG-stimulated cumulus cells were able to invade a range of ECM substrates including collagen and laminin. These results suggest the cumulus cells in the expanded COC transition to a motile cell phenotype that may play a key role in promoting ovulation.
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Uhde, K., L. T. A. van Tol, T. A. E. Stout, and B. A. J. Roelen. "79 microRNA EXPRESSION IN BOVINE CUMULUS CELLS." Reproduction, Fertility and Development 27, no. 1 (2015): 133. http://dx.doi.org/10.1071/rdv27n1ab79.
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A mammalian oocyte within an ovarian follicle is surrounded by cumulus cells, together this structure is known as the cumulus-oocyte complex (COC). Cumulus cells are important for the development of the oocyte, they support the maturation process of the oocyte within the ovary and aid in sperm recognition. Because it is known that a Dicer knockout leads to infertility, microRNAs (miRNA) are focused to have an important role in oocyte development. MiRNAs are small noncoding RNA sequences that act as transcriptional regulators. Little is known about the expression of miRNA in cumulus cells or how cumulus-derived miRNA may regulate or be used to indicate the developmental competence of the maturing oocyte. Our aim was to investigate miRNA expression in oocytes and to identify and establish how specific miRNA influence the acquisition of developmental competence by bovine oocytes. Normalization of qPCR data requires stable reference genes. To this end, we tested the expression of various miRNA with respect to their ability to be used as reference miRNA for bovine cumulus cells; these included miR-103, miR-93, miR-26, let-7a, miR-191, and the small noncoding nuclear RNA U6. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered cows and matured in vitro. Small samples of cumulus cells were collected from these COC before and after maturation. From the cumulus cell groups recovered at different stages, small RNA were extracted and cDNA was synthesised, followed by qRT-PCR. To identify the optimal combination of reference genes, the geNorm algorithm was used. MiR-26a and let-7a were identified as the most stably expressed miRNAs, whereas U6 showed the most variable expression levels. Future investigations are planned to identify miRNA in cumulus cells that can be used as markers for oocyte developmental competence. Using a single oocyte-embryo culture system will enable us to retrospectively relate cumulus miRNA expression to the developmental capacity of the oocyte.This work was supported by EU FP7 EpiHealthNet (N°317146).
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Uhde, K., H. T. A. van Tol, T. A. E. Stout, and B. A. J. Roelen. "66 CUMULUS-OOCYTE-COMPLEX SECRETIONS FROM THE FIRST 8 HOURS OF IN VITRO MATURATION AFFECT OOCYTE DEVELOPMENTAL COMPETENCE." Reproduction, Fertility and Development 28, no. 2 (2016): 163. http://dx.doi.org/10.1071/rdv28n2ab66.
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Mammalian oocytes are surrounded by cumulus cells, forming a structure known as the cumulus-oocyte complex (COC). Cumulus cells play important protective functions during oocyte maturation, for example, protecting the oocyte against reactive oxygen species. However, it is not yet fully understood how the cumulus complex modulates the developmental competence of the enclosed oocyte. It was investigated whether direct contact between an oocyte and its cumulus cells is essential throughout the maturation process. To this end, bovine oocytes aspirated from ovarian follicles were matured in vitro. Eight hours after the onset of maturation the cumulus cells were removed, and the oocytes either placed back in the original medium or cultured further in fresh maturation medium. In all experiments, COCs/oocytes were matured for 23 h in M199 supplemented with 0.05 IU FSH and penicillin/streptomycin. All experiments were performed in triplicate, with 35 to 45 COCs per group. Student’s t-test was used for a paired comparison. Denudation after 8 h and return to the same maturation medium had no effect on the cleavage rate (93%) compared with culture without denudation (90.7%). Only if the oocytes were transferred to fresh medium did the cleavage rate decrease slightly (75.4%; P = 0.038). By contrast, blastocyst formation was reduced nearly four times if COCs were denuded before being returned to the medium, compared with controls (14% v. 50.8%; P < 0.001). If the oocytes were transferred to fresh medium after denudation, very few blastocysts resulted (0.9%; P < 0.001). In a second study, oocytes denuded immediately after removal from the follicle were matured in the absence or presence of cumulus cells in a Corning® Transwell® system. Culturing denuded oocytes in the presence of cumulus cells resulted in similar cleavage rates (83.5%) to control conditions (84.8%). However, blastocyst formation was markedly lower (4.3%) than for controls (29.6%; P = 0.003). We conclude that COCs secrete substances during the first 8 h of maturation that are beneficial for oocyte acquisition of developmental competence. Moreover, intimate contact between the cumulus cells and oocyte is essential. This work was supported by EU FP7 EpiHealthNet, PITN-GA-2012–317146.
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Antosik, P., B. Kempisty, M. Jackowska, D. Bukowska, M. Lianeri, KP Brussow, and M. Wozna. "The morphology of porcine oocytes is associated with zona pellucida glycoprotein 3 and integrin beta 2 protein levels--." Veterinární Medicína 55, No. 4 (May 19, 2010): 154–62. http://dx.doi.org/10.17221/38/2010-vetmed.
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The morphology and quality of oocytes is set during oo- and folliculo-genesis, and is completed during final maturation. Early embryonic development is associated with the morphology of the cumulus-oocyte-complex (COC). However, current knowledge of the possible relationships between oocyte morphology and the level of proteins within the oocyte, which may reflect fertilization ability, is insufficient. Using western-blot analysis and confocal microscopic observation, we determined the levels of integrin beta-2 (integrin β2) and glycoprotein 3 (pZP3) protein levels in the porcine zona pellucida of four morphologically different types of oocytes, graded according to their cytoplasmic composition and cumulus structure. The level of integrin β2 protein was increased in grade I and II oocytes as compared to other grades (P < 0.05). Moreover, the level of pZP3 protein was 3–4 fold higher in grade I oocytes (P < 0.01). We suggest that COC morphology may be associated with oocyte fertilization ability with respect to its sperm-oocyte interaction gene expression, which is the result of increased accumulation of specific proteins prior to fertilization in higher quality oocytes. Higher quality oocytes may also reflect better fertilization ability.
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Tetsuka, Masafumi, and Misato Tanakadate. "Activation of HSD11B1 in the bovine cumulus-oocyte complex during IVM and IVF." Endocrine Connections 8, no. 7 (July 2019): 1029–39. http://dx.doi.org/10.1530/ec-19-0188.
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The bovine cumulus-oocyte complex (COC) is capable of converting cortisone, an inert glucocorticoid to active cortisol. This mechanism is mediated by 11β-hydroxysteroid oxidoreductase type 1 (HSD11B1), whose expression dramatically increases in the mature COC. In this study, we investigate the time course expression of HSD11B1 and the enzyme activity in the bovine COC undergoing maturation and fertilization in relation to key events taking place in the COC. Bovine COCs were subjected to in vitro maturation (IVM) and fertilization (IVF). The activities of HSD11B1 and HSD11B2, which mediates the opposite reaction, were measured using a radiometric conversion assay. In parallel studies, cumulus expansion, P4 production and the expression of genes associated with ovulation were measured. The reductive activity of HSD11B1 increased in the latter half of IVM and remained high during IVF, whereas the oxidative activity of HSD11B2 remained unchanged over both periods. Consequently, the net glucocorticoid metabolism in the bovine COC shifted from inactivation to activation around the time of ovulation and fertilization. The increase in HSD11B1 expression lagged behind that of P4 increase and cumulus expansion but ahead of the expressions of genes responsible for PGE2 synthesis. The reductive activity of HSD11B1 was well correlated with the cumulus expansion rate. This outcome indicates that the ability of the cumulus to activate glucocorticoids is related to its ability to synthesize hyaluronan. These results also indicate that the activation of HSD11B1 is an integral part of the sequential events taking place at the ovulation and fertilization in the bovine COC.
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Dunning, K. R., L. N. Watson, J. G. Thompson, R. L. Robker, and D. L. Russell. "146. MOLECULAR FILTRATION PROPERTIES OF THE EXPANDED CUMULUS MATRIX: CONTROLLED SUPPLY OF METABOLITES AND EXTRACELLULAR SIGNALS TO CUMULUS CELLS AND THE OOCYTE." Reproduction, Fertility and Development 22, no. 9 (2010): 64. http://dx.doi.org/10.1071/srb10abs146.
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Cumulus matrix genes are positively correlated with oocyte competence [1]. Formation of the expanded cumulus matrix during oocyte maturation is well described; however its function remains elusive. We investigated whether cumulus matrix acts as a molecular filter, based on recognised filtration properties of analogous matrices. We found that cumulus matrix controls metabolite supply to the oocyte and retains prostaglandin E2 (PGE2), which is critical in oocyte maturation. The uptake of fluorescently labelled hydrophilic and hydrophobic metabolites showed that cumulus matrix formation significantly impeded diffusion to the oocyte. Expanded in vivo matured cumulus oocyte complexes (COCs, eCG+hCG16h) resisted uptake of glucose and cholesterol compared to unexpanded (eCG44h, P < 0.05), as assessed by confocal microscopy and spatial quantitation of fluorescence (P < 0.05). In vitro maturation (IVM) results in pronounced compositional deficiency of cumulus matrix proteins [2] and poor oocyte quality. Glucose and cholesterol were transported more readily into cumulus cells and the oocyte of IVM COCs (matured in αMEM/5% FCS/50 mIU/mL FSH, 16 h) compared to in vivo matured COCs (P < 0.05 and P = 0.08, respectively). Taking the inverse approach we found that PGE2 synthesised by cumulus cells is retained within the matrix compartment of in vivo matured COCs but IVM COCs did not retain PGE2 and secreted 4.3-fold more into the media. The relationship of retained to secreted PGE2 was significantly higher after in vivo maturation vs IVM COCs (P < 0.0001). This property of the COC matrix reveals a potential mechanism whereby the prostaglandin signal intensifies through a physicochemical mechanism rather than gene regulation. This is the first demonstration that cumulus matrix regulates diffusion toward and secretion from the COC, thus excluding glucose, known to negatively affect oocyte quality, and trapping factors, including PGE2, with critical roles in oocyte maturation and fertilisation. Thus, IVM may reduce oocyte quality due to poor trafficking of metabolites and signalling molecules. (1) McKenzie LJ, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod 2004; 19: 2869–2874.(2) Dunning KR, et al. Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes. Hum Reprod 2007; 22: 2842–2850.
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Rodrigues, Thais A., Kubra M. Tuna, Abdel A. Alli, P. Tribulo, P. J. Hansen, Jin Koh, and F. F. Paula-Lopes. "Follicular fluid exosomes act on the bovine oocyte to improve oocyte competence to support development and survival to heat shock." Reproduction, Fertility and Development 31, no. 5 (2019): 888. http://dx.doi.org/10.1071/rd18450.
Full textAbstract:
Addition of follicular fluid to oocyte maturation medium can affect cumulus cell function, increase competence of the oocytes to be fertilised and develop to the blastocyst stage and protect the oocyte from heat shock. Here, it was tested whether exosomes in follicular fluid are responsible for the effects of follicular fluid on the function of the cumulus–oocyte complex (COC). This was accomplished by culturing COCs during oocyte maturation at 38.5°C (body temperature of the cow) or 41°C (heat shock) with follicular fluid or exosomes derived from follicular fluid and evaluating various aspects of function of the oocyte and the embryo derived from it. Negative effects of heat shock on cleavage and blastocyst development, but not cumulus expansion, were reduced by follicular fluid and exosomes. The results support the idea that exosomes in follicular fluid play important roles during oocyte maturation to enhance oocyte function and protect it from stress.
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Liu, Zhilin, Daniel G. de Matos, Heng-Yu Fan, Masayuki Shimada, Stephen Palmer, and JoAnne S. Richards. "Interleukin-6: An Autocrine Regulator of the Mouse Cumulus Cell-Oocyte Complex Expansion Process." Endocrinology 150, no. 7 (March 19, 2009): 3360–68. http://dx.doi.org/10.1210/en.2008-1532.
Full textAbstract:
Ovulation has long been regarded as a process resembling an inflammatory response. Recent studies indicate that genes associated with innate immune responses were also expressed during the ovulation process. Because the innate immune genes are induced in cumulus cell oocyte complexes (COCs) later than the inflammation-associated genes, we hypothesize that COC expansion is dependent on specific sequential changes in cumulus cells. Because IL-6 is a potent mediator of immune responses, we sought to determine what factors regulate the induction of Il6 mRNA in COCs and what impact IL-6 alone would have on COC expansion. We found that the levels of Il6 mRNA increased dramatically during COC expansion, both in vivo and in vitro. Moreover, IL-6, together with its soluble receptor (IL-6SR), could bypass the need for either amphiregulin and/or prostaglandin E2 to induce the expansion of COCs. This ability of IL-6/IL-6SR to induce COC expansion was blocked by the inhibitors to p38MAPK, MAPK kinase 1/2, and Janus kinase. More importantly, when COCs were in vitro maturated in the presence of IL-6, they had a significantly higher embryo transfer rate than the ones without IL-6 and comparable with in vivo matured oocytes. IL-6/IL-6SR activated multiple signaling pathways (Janus kinase/signal transducer and activator of transcription, ERK1/2, p38MAPK, and AKT) and progressively induced genes known to impact COC expansion, genes related to inflammation and immune responses, and some transcription factors. Collectively, these data indicate that IL-6 alone can act as a potent autocrine regulator of ovarian cumulus cell function, COC expansion, and oocyte competence.
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O'Donnell, John B., Julia L. Hill, and David J. Gross. "Epidermal growth factor activates cytosolic [Ca2+] elevations and subsequent membrane permeabilization in mouse cumulus–oocyte complexes." Reproduction 127, no. 2 (February 2004): 207–20. http://dx.doi.org/10.1530/rep.1.00027.
Full textAbstract:
The role of epidermal growth factor (EGF) in the maturation of mammalian oocytes is well known but not well characterized. It is known that EGF enhances oocyte maturationin vitroand that EGF stimulation of cumulus–oocyte complexes (COCs) induces pulsatile Ca2+efflux from the cell complex. By use of quantitative Fura-2 imaging, EGF-stimulated changes in intracellular [Ca2+] in germinal vesicle stage murine COCs are shown to occur in a subpopulation of cumulus cells that interact cooperatively within individual COCs. Oocytes fail to respond to EGF stimulus. In many of the cumulus cells responding with a rise in intracellular [Ca2+], a concomitant permeabilization of the plasma membrane is found. Neither cumulus cells of control COCs nor those that show a rise in intracellular [Ca2+] in response to calcium ionophore treatment display a similar membrane permeabilization, although those cells responding with a prolonged [Ca2+] increase in response to thimerosal or thapsigargin do display plasma membrane permeabilization. Thus, EGF stimulation of mammalian COCs activates release of Ca2+from intracellular stores of cumulus cells, the depletion of which activates permeabilization of the plasma membrane. This membrane permeabilization leads to loss of cell contents and presumptive cumulus cell death. This catastrophic EGF-induced plasma membrane permeabilization of individual cumulus cells within a COC leads to pulsatile Ca2+efflux as previously seen, and may lead to improved cumulus cell expansion during COC maturation.
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Dragovic, R. A., L. J. Ritter, S. J. Schulz, D. T. Armstrong, and R. B. Gilchrist. "231. Mouse oocyte paracrine signalling to cumulus cells by TGF-β superfamily molecules is indispensable for cumulus expansion." Reproduction, Fertility and Development 17, no. 9 (2005): 90. http://dx.doi.org/10.1071/srb05abs231.
Full textAbstract:
Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex (COC), which is necessary for ovulation. Members of the transforming growth factor-β (TGF-β) superfamily are prime candidates for the mouse cumulus expansion-enabling factor (CEEF), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. This study was conducted to examine TGF-β superfamily processes regulating cumulus expansion. COCs were collected from eCG-primed mice and the oocyte microsurgically removed to generate oocytectomised (OOX) complexes. An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P < 0.05) induced by either GDF9 (score: mean ± SEM, 3.7 ± 0.1), activin A (2.6 ± 0.1), or co-culture with oocytes (3.2 ± 0.2). The type-I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively. We tested the ability of the ALK4/5/7 kinase inhibitor, SB431542, to neutralise cumulus expansion. SB431542 completely neutralised (P < 0.05) the response of OOX complexes to GDF9, activin and oocyte-induced cumulus expansion. SB431542 also neutralised (P < 0.05) COC expansion in a dose dependent manner. Follistatin, an activin antagonist was effective at neutralising the response of OOX complexes to activin (score: 0), but had no significant effect (P > 0.05) on the expansion of OOX complexes co-cultured with oocytes (score: 2.7 ± 0.2). This study provides evidence that activin is not the sole CEEF, but signalling through the ALK4/5/7 pathway is indispensable for mouse cumulus expansion.
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Salustri, Antonietta, Antonella Camaioni, and Cristina D'Alessandris. "Endocrine and paracrine regulation of cumulus expansion." Zygote 4, no. 04 (November 1996): 313–15. http://dx.doi.org/10.1017/s0967199400003312.
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In a Graafian follicle, granulosa cells are classified into two principal cell subpopulations: cumulus cells, which are closely associated with the oocyte to form the cumulus cell-oocyte complex (COC), and mural granulosa cells, which are organised as a stratified epithelium at the periphery of the follicle. Following the preovulatory gonadotropin surge, cumulus cells lose contact with mural granulosa cells and start to synthesise and secrete a large amount of hyaluronan (HA), a glycosaminoglycan with high molecular weight and large hydrodynamic domains (Salustriet al., 1992). Proteins derived from serum (Chenet al., 1992, 1994) and synthesised by cumulus cells (Camaioniet al., 1993, 1996) organise the strands of HA into an intercellular elastic network that traps the cumulus cells and the oocyte in a unit which can not be mechanically dissociated – a process also referred to as cumulus expansion. At ovulation, the expanded COC is released through the ruptured follicle wall and transferred to the oviduct. The matrix in the expanded COC facilitates its extrusion from the follicle and its capture by oviductal fimbria, and provides, together with the cumulus cells, a suitable microenvironment for sperm penetration and fertilisation (for references see Salustriet al., 1993).
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Tetsuka, Masafumi, Ryo Takagi, Nobuhiro Ambo, Thet Su Myat, Yuta Zempo, and Asuka Onuma. "Glucocorticoid metabolism in the bovine cumulus–oocyte complex matured in vitro." REPRODUCTION 151, no. 1 (January 2016): 73–82. http://dx.doi.org/10.1530/rep-15-0363.
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Glucocorticoid action in target organs is regulated by relative activities of 11β-HSD type 1 (HSD11B1) that mainly converts cortisone to active cortisol and type 2 (HSD11B2) that inactivates cortisol to cortisone. HSD11Bs have been shown to be expressed in the ovary of various species. However, little is known about the expression and activity of HSD11Bs in the bovine cumulus–oocyte complex (COC). In the present study, we investigated the expression and activities of HSD11Bs in in vitro-matured (IVM) bovine COCs. Bovine COCs were matured in M199 supplemented with or without FSH and FCS. The expression of HSD11B1 and HSD11B2 was measured by using quantitative RT-PCR in denuded oocytes (DO) and cumulus cells (CC). Reductive and oxidative activities of HSD11Bs were determined by radiometric conversion assay using labeled cortisol, cortisone or dexamethasone in intact COCs, DO or CC in the presence or absence of 11-keto-progesterone (11kP), a selective inhibitor of HSD11B2. The presence of HSD11Bs in the oocyte was examined by immunofluorescence microscopy. Oocytes exclusively expressed HSD11B2 and its expression and activity were largely unchanged during IVM. CC, on the other hand, exclusively expressed HSD11B1 and its expression and activity were upregulated as IVM progressed. As a result, the net glucocorticoid metabolism shifted from inactivation to activation towards the end of IVM. These results indicate that the bovine COC is capable of modulating local glucocorticoid concentration and, by doing so, may create an environment that is favorable to ovulating oocyte for maturation, fertilization and subsequent development.
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Kind, Karen L., Kimberley K. Y. Tam, Kelly M. Banwell, Ashley D. Gauld, Darryl L. Russell, Anne M. Macpherson, Hannah M. Brown, Laura A. Frank, Daniel J. Peet, and Jeremy G. Thompson. "Oxygen-regulated gene expression in murine cumulus cells." Reproduction, Fertility and Development 27, no. 2 (2015): 407. http://dx.doi.org/10.1071/rd13249.
Full textAbstract:
Oxygen is an important component of the environment of the cumulus–oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.
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Tahir, M. Z., K. Reynaud, S. Thoumire, S. Chastant-Maillard, and M. Saint-Dizier. "121 EXPRESSION OF STEROID RECEPTORS IN THE CUMULUS - OOCYTE COMPLEX AROUND OVULATION IN THE BITCH." Reproduction, Fertility and Development 26, no. 1 (2014): 174. http://dx.doi.org/10.1071/rdv26n1ab121.
Full textAbstract:
In the bitch, oocytes are ovulated at an immature stage (prophase I) and resume meiosis in the oviduct, 3 to 4 days after ovulation while they are still surrounded by 2 to 3 layers of cumulus cells. Canine cumulus-oocyte complexes (COC) are exposed to high and changing plasma concentrations of 17β-oestradiol (E2) and progesterone (P4) during the periovulatory period. In order to explore whether oocyte maturation may be regulated by steroids in this species, the expression of E2 (ERα, ERβ) and P4 (nuclear: PR; membrane: PGRMC1, PGRMC2, mPRα, mPRβ, mPRγ) receptors was studied in COC at precise times around ovulation. Ovaries were collected from Beagle bitches during anestrus (n = 4), after the beginning of proestrus, and before the LH peak (Pre-LH, n = 7), after the LH peak and before ovulation (Pre-ov, n = 8), and at Day 1 (n = 11) and Day 4 (n = 8) post-ovulation. Anoestrus COC were recovered from follicles smaller than 1 mm in diameter. The COC at the Pre-LH and Pre-ov stages were aspirated from preovulatory follicles (4.5–6 mm in diameter). Such follicular COC were partially denuded to leave the 2 to 3 innermost cumulus layers firmly attached to the zona pellucida. Post-ovulatory COC, naturally surrounded by 2 to 3 cumulus layers, were recovered by oviductal flushing. Total RNA was extracted from 3 batches of 10 COC per stage, then reverse transcribed. The expression of steroid receptors was assessed in duplicate by qPCR (LightCycler® 480, Roche Diagnostics) using the relative standard curve method and normalized by the geometric mean value of the two most stable reference genes (BGLR and RPS5; NormFinder software) chosen among four genes previously tested. Relative amounts of mRNA levels were compared between stages by ANOVA followed, when necessary, by a Tukey test. The ERα and ERβ expression did not vary significantly with the stage. In contrast, a significant variation between stages in nuclear and 4 membrane P4 receptor expression was observed (P < 0.0001 for PR; P < 0.001 for PGRMC1 and mPRβ; P < 0.05 for PGRMC2 and mPRγ). The PR mRNA levels were significantly higher at Pre-ov than at any other stage. PGRMC1 expression was significantly higher at Pre-ov and Day 4 compared with anestrus and Pre-LH, and was at an intermediate level at Day 1. The expression of PGRMC2, mPRβ, and mPRγ remained low from anestrus to Day 1 and increased significantly at Day 4. Lastly, mRNA levels of mPRα were below the detection limit at all stages. This is the first report of steroid receptor expression in canine COC at precise times around ovulation. The stage-specific variation in expression of nuclear and of several membrane P4 receptors around ovulation suggests a role for P4 in canine oocyte maturation. The exact localisation of these receptors in cumulus cells, oocytes, or both remains to be determined.
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Pant, Disha, Lawrence P. Reynolds, Justin S. Luther, Pawel P. Borowicz, Tande M. Stenbak, Jerzy J. Bilski, Robert M. Weigl, et al. "Expression of connexin 43 and gap junctional intercellular communication in the cumulus–oocyte complex in sheep." Reproduction 129, no. 2 (February 2005): 191–200. http://dx.doi.org/10.1530/rep.1.00434.
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To evaluate the effects of FSH, LH and/or cAMP on expression of connexin 43 (Cx43) in the ovine cumulus-oocyte complex (COC) and gap junctional intercellular communication (GJIC) of cumulus cells, two experiments were carried out. In experiment 1, Cx43 was immunodetected in the COC, before or after maturation, obtained from non-treated or FSH-treated ewes. The expression of Cx43 in the COC was greater (P < 0.01) on day 16 than on day 15 of the estrous cycle. In vivo FSH treatment decreased (P < 0.02) Cx43 expression on day 16 but not on day 15 of the estrous cycle. In experiment 2, intact COCs or isolated cumulus cells obtained from small and large follicles from FSH-treated ewes were cultured with or without FSH, LH or cAMP agonist and evaluated for GJIC by laser cytometry. For large follicles, the basal rate of GJIC was greater (P < 0.01) for cumulus cells in intact COCs than for isolated cumulus cells. FSH increased (P < 0.04) GJIC in cumulus cells in intact COCs and tended to increase (P < 0.1) GJIC in isolated cumulus cells from small follicles but decreased (P < 0.01) GJIC in cumulus cells in intact COCs from large follicles. LH also increased (P < 0.01) GJIC in isolated cumulus cells from small follicles but decreased GJIC in intact COCs (P < 0.01) and isolated cumulus cells (P < 0.02) from large follicles. cAMP increased (P < 0.01) the GJIC in both intact COCs and cumulus cells from small and large follicles. These results indicate that day of estrous cycle, stage of maturation and duration of FSH treatment affect expression of Cx43 in ovine COCs. In intact COCs, GJIC in cumulus cells was enhanced, probably due to the presence of the oocyte. In addition, the effects of FSH and LH, but not cAMP, on GJIC of cumulus cells depended on the stage of follicular development and on the presence of the oocyte.
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Russell, D. L. "026. The cumulus matrix in ovulation: inert packaging or active delivery vehicle for the oocyte?" Reproduction, Fertility and Development 17, no. 9 (2005): 69. http://dx.doi.org/10.1071/srb05abs026.
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Preovulatory follicles respond to the LH-surge with a cascade of molecular events. The ovulatory signal initially impinges on the mural granulosa layers triggering rapid tissue morphogenesis and ultimately terminal differentiation of these cells. Mural granulosa cells transiently produce a suite of transcriptional regulators, EGF-like ligands as well the extracellular matrix (ECM) proteoglycan, versican and the protease ADAMTS-1. These act in concert with permissive oocyte signals to induce and organise a complex hyaluronan (HA) rich ECM surrounding the cumulus cells and oocyte. This expanded cumulus matrix is analogous in composition to an extensive form of pericellular matrices actively associated with cell migration. During ovulation the cumulus matrix becomes anti-adhesive to the intra-follicular environment but is strongly pro-adhesive for the oviductal fimbria. When the follicle apex is perforated the COC is released binds to the fimbria and transports into the oviduct where fertilisation occurs. Success of ovulation and fertilisation is sensitive to the appropriate production and assembly of cumulus matrix components that are in turn dependent on an appropriate balance of oocyte and granulosa derived signals. Production of these cumulus matrix components is thus a potential checkpoint that assures ovulation of competent oocytes. The HA matrix is cross-linked by organiser molecules and also is enriched in proteases ADAMTS-1, 4, 5. Although these have potentially redundant functionality, ADAMTS-1 null female mice are profoundly sub-fertile and have reduced ovulation rate. Specific components of the cumulus matrix are disorganised in ADAMTS-1 null mice and cleavage of versican in these cumulus complexes is reduced. Thus ADAMTS-1 and versican have unique roles in normal cumulus matrix expansion that is important for ovulation. Altered interaction of the cumulus complex with neighbouring tissues alters transport through the oviduct, while abnormal persistence of COC matrix structure after ovulation is also likely to impair sperm interaction and penetration. Evidence thus indicates that the expanded cumulus matrix plays several active roles in oocyte release, transport and sperm interaction.
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Park, S. Y., H. J. Park, J. W. Kim, J. Y. Park, S. G. Yang, J. M. Jung, M. J. Kim, and D. B. Koo. "172 THE APOPTOTIC EFFECTS OF BISPHENOL A-INDUCED MITOCHONDRIAL-DERIVED REACTIVE OXYGEN SPECIES ON MATURATION OF PORCINE OOCYTES IN VITRO." Reproduction, Fertility and Development 29, no. 1 (2017): 194. http://dx.doi.org/10.1071/rdv29n1ab172.
Full textAbstract:
Bisphenol A (BPA) is well known as oestrogen-like chemical and it is widely used in plastic products. Many studies have reported that BPA exposure has a well-known toxicity effect on reproduction function, such as reducing the number of ovulated oocytes, oocyte quality, and maturation rate. Recently, BPA induced mitochondrial-derived reactive oxygen species (mito-ROS) and disrupted mitochondrial homeostasis by increasing of superoxide anions production. In this study, we investigated how the regulation of mito-ROS production may play a critical role in meiotic maturation and expansion of cumulus cells during the in vitro maturation progression of porcine oocytes. Furthermore, we investigated the toxicity effect of BPA exposure on mitochondrial functions and mito-ROS production during porcine oocyte maturation in vitro. All results were analysed using a 1-way ANOVA followed by Bonferroni’s and Tukey’s Multiple Comparison Test and t-tests. First, porcine oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75, and 100 µM) for 44 h. Our results indicated that the rates of matured oocytes were significantly decreased by BPA exposure in a dose-dependent manner (69.4 ± 5.1, 50.9 ± 6.3, and 29.9 ± 5.8% for BPA treatments of 50, 75, and 100 μM) compared with control group (70.2 ± 7.8%; P < 0.05). Next, we confirmed the secretion functions of oocyte and cumulus cell of cumulus-oocyte complex (COC) and ROS production. Cumulus cell secretion factors (has2, tnfaip6, and cx37) mRNA expression in COC were decreased in the BPA-treated (75 µM) group. In addition, mRNA expressions of mitochondrial-specific antioxidant enzymes (sod2, P < 0.001; prdx3, P < 0.01; prdx5, P < 0.001) and mitochondrial apoptosis genes (bax and caspase-3, P < 0.01) were significantly increased in COC of the BPA-treated (75 µM) group. We measured mitochondrial membrane potential and mito-ROS production using JC-1 analysis and Mito-SOX staining, respectively. The BPA treatment caused a rapid decrease of mitochondrial membrane potential maintenance and increase of mito-ROS production in porcine COC. Moreover, mitochondrial-specific ROS scavenger, Mito-Tempo (0.1 µM) treatment was significantly increased the meiotic maturation of porcine oocytes compared with control group (78.5 ± 3.5 v. 65.8 ± 5.0%; P < 0.05). Based on these results, we first confirmed that BPA exposure reduces the meiotic maturation and cumulus cells expansion of COC by increasing mito-ROS production during porcine oocyte maturation in vitro. Therefore, controlling of mito-ROS for mitochondrial function maintenance and apoptosis plays a critical role in improving porcine oocyte maturation in vitro. This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037–04–1-HD020) through the Rural Development Administration, the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.
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Paczkowski, M., W. B. Schoolcraft, and R. L. Krisher. "Fatty acid metabolism during maturation affects glucose uptake and is essential to oocyte competence." REPRODUCTION 148, no. 4 (October 2014): 429–39. http://dx.doi.org/10.1530/rep-14-0015.
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Fatty acid β-oxidation (FAO) is essential for oocyte maturation in mice. The objective of this study was to determine the effect of etomoxir (a FAO inhibitor; 100 μM), carnitine (1 mM), and palmitic acid (1 or 100 μM) during maturation on metabolism and gene expression of the oocyte and cumulus cells, and subsequent embryo development in the mouse. Carnitine significantly increased embryo development, while there was a decrease in development following maturation with 100 μM palmitic acid or etomoxir (P<0.05) treatment. Glucose consumption per cumulus–oocyte complex (COC) was decreased after treatment with carnitine and increased following etomoxir treatment (P<0.05). Intracellular oocyte lipid content was decreased after carnitine or etomoxir exposure (P<0.05). Abundance ofSlc2a1(Glut1) was increased after etomoxir treatment in the oocyte and cumulus cells (P<0.05), suggesting stimulation of glucose transport and potentially the glycolytic pathway for energy production when FAO is inhibited. Abundance of carnitine palmitoyltransferase 2 (Cpt2) tended to increase in oocytes (P=0.1) after treatment with 100 μM palmitic acid and in cumulus cells after exposure to 1 μM palmitic acid (P=0.07). Combined with carnitine, 1 μM palmitic acid increased the abundance ofAcsl3(P<0.05) andCpt2tended to increase (P=0.07) in cumulus cells, suggesting FAO was increased during maturation in response to stimulators and fatty acids. In conclusion, fatty acid and glucose metabolism are related to the mouse COC, as inhibition of FAO increases glucose consumption. Stimulation of FAO decreases glucose consumption and lipid stores, positively affecting subsequent embryo development, while an overabundance of fatty acid or reduced FAO negatively affects oocyte quality.
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Lewis, N., K. Hinrichs, D. Brison, R. Sturmey, D. Grove-White, K. Schnauffer, and C. McGregor-Argo. "184 PRELIMINARY FINDINGS ON CARBOHYDRATE METABOLISM OF INTACT EQUINE CUMULUS-OOCYTE COMPLEXES DURING IN VITRO MATURATION." Reproduction, Fertility and Development 28, no. 2 (2016): 223. http://dx.doi.org/10.1071/rdv28n2ab184.
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Production of equine embryos in vitro is gaining popularity, and many differences exist in composition of in vitro maturation (IVM) media. Metabolism of the cumulus-oocyte complex (COC) is essentially unknown in the horse. Here, we describe preliminary data on carbohydrate metabolism of the equine COC during IVM. COC, collected by scraping of the granulosa layer of all visible follicles of abattoir-derived ovaries, were held overnight (12–18 h) at room temperature (~20°C) and then placed in Maturation Medium (M199 with Earle’s salts, 10% FBS, with 25 μg mL–1 gentamicin and 5 mU mL–1 FSH). They were incubated singly in 10-μL droplets under mineral oil for 30 h at 38.3°C in 5% CO2 in air. Control droplets without COC were incubated in the same dish. After incubation, COC were removed and spent media kept at –80°C for later analysis. Oocytes were denuded of cumulus cells by pipetting in the presence of hyaluronidase and evaluated by light microscopy at 500×. Those with a visible polar body were classified as metaphase II (MII); oocytes with an intact oolemma and no polar body were classified as immature intact (INT) and those with an irregular or unapparent oolemma, or shrunken cytoplasm, were classed as degenerating (DEG). To adjust for variation in cumulus cell number, the stripped cumulus cells were frozen at –20°C and later analysed for DNA content using Picogreene. The spent media was analysed for depletion of glucose and appearance of lactate on a BMG Fluostar spectrophotometer using an enzyme-linked ultrafluorometric method. Data were expressed as pmol/ng DNA/hr and analysed by t-test, x2 and logistic regression. Thirty COC were cultured and analysed; 14 were classified as MII, 2 INT and 14 DEG. Seven COC (23%) depleted all the available glucose, indicating that the rate of glucose consumption in those 7 complexes was ≥1866 pmol/COC per hour. DNA content was positively correlated with glucose depletion (P = 0.02). In the COC that did not deplete available glucose, the ratio of glucose consumption:lactate production was 1.82, indicating that the major fate of exogenous glucose was production of lactate by glycolysis. In the 7 oocytes that depleted all the glucose, the ratio of glucose consumption:lactate production was 1.22. One explanation for this may be that when glucose was no longer available, it was conserved for other pathways. It was noteworthy that these COC had more cumulus cells (P < 0.01) and the maturation rate was 4/7 (57%). In the group of COC that did not deplete all of the glucose, there was no significant difference in glucose consumption (13.17 v. 12.25 pmol/ng DNA per hour; P > 0.4) or lactate production (21.48 v. 20.28 pmol/ng DNA per hour; P > 0.4) between COC in which the oocyte reached MII (10/23; 43%), and those which contained a degenerated oocyte at the end of culture, respectively. To the best of our knowledge, this is the first report documenting the metabolism of equine COC. These data underline the importance of further studies to determine optimal conditions for in vitro maturation of equine COC, especially in terms of glucose availability.
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Dragovic, R. A., L. J. Ritter, F. Amato, S. J. Scott, M. Cranfield, N. P. Groome, D. T. Armstrong, and R. B. Gilchrist. "251.Regulation of mouse cumulus expansion by oocyte-secreted growth differentiation factor-9 (GDF-9)." Reproduction, Fertility and Development 16, no. 9 (2004): 251. http://dx.doi.org/10.1071/srb04abs251.
Full textAbstract:
Oocyte paracrine signalling is necessary for mouse cumulus cell expansion, an important preovulatory process. The oocyte-secreted factor growth differentiation factor-9 (GDF-9) signals through the bone morphogenetic protein receptor-II (BMPR-II) and is currently the primary candidate molecule for the cumulus expansion enabling factor (CEEF). The present study was conducted to determine whether in the mouse GDF-9 is the CEEF. Cumulus oocyte complexes (COC) were collected from eCG-primed mice and the oocyte was microsurgically removed to generate an oocytectomised complex (OOX). An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P�<�0.05) induced by either recombinant mouse GDF-9 (score; mean +/– SEM: 2.7 +/– 0.1), recombinant TGF-μ1 (score: 2.6 +/– 0.2) or co-culture with oocytes (score: 2.3 +/– 0.2). A GDF-9 neutralising antibody mAb-53, raised against hGDF-9, was effective in neutralising the response of OOX complexes to GDF-9 (score: 0.1 +/– 0.1), but had no significant effect on the expansion of OOX complexes co-cultured with oocytes (score: 2.3 +/– 0.2). Likewise, a TGF-μ antagonist neutralised (P�<�0.05) TGF-μ-induced, but not oocyte-induced, expansion of OOX complexes. A soluble portion of the BMPR-II ectodomain, a known GDF-9 antagonist, failed to neutralise oocyte-induced cumulus expansion (P�>�0.05) at the highest dose implying that BMPR-II is not a critical receptor involved in regulating cumulus expansion. Using real-time RT-PCR, hyaluronan synthase-2 (HAS2) mRNA expression by OOXs was upregulated 6- to 7-fold by oocytes and GDF-9. The GDF-9 neutralising antibody mAb-53, partially neutralised GDF-9-induced OOX HAS2 expression, but not oocyte-induced HAS2 expression. This study provides evidence that like TGF-μ1, GDF-9 can enable FSH-induced cumulus expansion, however more importantly demonstrates that neither GDF-9 nor TGF-μ1 alone account for the crucial oocyte-secreted factor regulating cumulus expansion in the mouse.
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Jin, Jun-Xue, Jing-Tao Sun, Chao-Qian Jiang, Hong-Di Cui, Ya Bian, Sanghoon Lee, Lianjin Zhang, Byeong Chun Lee, and Zhong-Hua Liu. "Melatonin Regulates Lipid Metabolism in Porcine Cumulus–Oocyte Complexes via the Melatonin Receptor 2." Antioxidants 11, no. 4 (March 31, 2022): 687. http://dx.doi.org/10.3390/antiox11040687.
Full textAbstract:
Previous studies suggest that the inclusion of melatonin (MTn) in in vitro maturation protocols improves the developmental competence of oocytes by scavenging reactive oxygen species (ROS). However, the molecular mechanisms integrating melatonin receptor (MT)-mediated lipid metabolism and redox signaling during in vitro cumulus–oocyte complex (COC) development still remain unclear. Here, we aimed to elucidate the potential role of MTn receptors in lipid metabolic adjustments during in vitro porcine COC development. We observed that MTn-mediated Gsα–cAMP/PKA signaling facilitated lipolysis primarily through the MT2 receptor and subsequently increased fatty acid (FA) release by hydrolyzing intracellular triglycerides (TGs) in cumulus cells. Furthermore, CD36 was a critical FA transporter that transported available FAs from cumulus cells to oocytes and promoted de novo TG synthesis in the latter. In addition, MTn regulated lipogenesis and intracellular lipolysis to maintain lipid homeostasis and limit ROS production, thereby supporting oocyte cytoplasmic maturation and the subsequent embryo development. Taken together, these findings provide insight into the possible mechanism integrating MT2-mediated lipid homeostasis and redox signaling, which limits ROS production during in vitro COC development. Therefore, understanding the dynamics of the interactions between lipid homeostasis and redox signaling driven by MT2 is necessary in order to predict drug targets and the effects of therapeutics used to improve female reproductive health.
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Uzbekova, S., L. Sanchez-Lazo, A. Desmachais, V. Maillard, and S. Elis. "274 LIPOLYSIS IN CUMULUS CELLS ACCOMPANIES OOCYTE MATURATION IN BOVINE." Reproduction, Fertility and Development 27, no. 1 (2015): 226. http://dx.doi.org/10.1071/rdv27n1ab274.
Full textAbstract:
Oocyte maturation relies on energy from different nutrients, including fatty acids (FA). Cumulus cells (CC) are metabolically coupled with enclosed oocyte and active FA metabolism occurs in both compartments. Excess of lipids in oocyte environment alters its developmental competence. Lipid droplets (LD), mainly composed of triacylglycerides (TG), are formed inside of CC and in oocyte to store lipids. Liberation of free FA from TG requires lipolysis, which is catalyzed by lipases and involves FA-binding proteins (FABP) and perilipins (PLIN), which interact at the surface of LD as shown in lipogenic tissues. The objective was to elucidate the main factors involved in lipolysis in bovine cumulus-oocyte complex (COC) during oocyte maturation. Gene expression before and after maturation was analysed in CC by microarray hybridization and validated by real time RT-PCR; proteins were detected by Western blot and immunofluorescence. For statistics, ANOVA and Mann-Whitney (M-W) tests were used. In CC, adipose triglyceride lipase PNPLA2, lipoprotein lipase LPL, and monoacylglycerol lipase ABHD6 showed the highest mRNA expression level among 7 detected lipases. Both PLIN5 and PLIN2 were the most abundant perilipins, and among 8 FA-binding proteins, FABP3 and FABP5 were predominant. During in vitro maturation (IVM), expression of most of these genes increased at 6 h of IVM (P < 0.05, ANOVA) in CC. At that time, germinal vesicle breakdown occurred in enclosed oocytes and hyaluronan synthase HAS2, involved in the extra-cellular matrix formation, was upregulated in CC. The most upregulated genes after 18 h of IVM in CC were ABDH6 (48.5-fold as compared to immature, P < 0.01, M-W), FABP3 (16.6-fold, P < 0.01, M-W), and PLIN2 (5.5-fold, P < 0.05, M-W). Expression of all of these lipolysis-related genes was also detected in the oocytes. At the protein level, PLIN2 was mainly localised in the cytoplasmic LD, both in CC and in the oocyte. In CC, FABP3 was detected in the cytoplasm, whereas in oocyte it was also localised to the germinal vesicle of immature oocytes and closely to the chromosomes during the first meiotic division. In addition, active phosphorylated hormone sensitive lipase HSL was always detected in CC and in mature oocytes, but not in immature oocytes. All these data demonstrate that lipolysis occurs both in CC and in the oocyte during maturation. Lipolysis may be necessary to maintain cell energy homeostasis by regulating intracellular concentration of free FA. Moreover, CC were already described to store the excess FA from follicular fluid in order to protect the oocyte. Our data corroborate the essential role of CC in oocyte survival through controlling FA metabolism inside the COC. Active lipolysis may therefore be required to reduce lipid storages as well as to produce energy necessary for oocyte meiosis progression and extracellular matrix secretion by CC in order to prepare COC for further fertilization.This work was supported by INRA, ANR (OSCILE project) and European subvention FP7-KBBE-2012–6 (FECUND project).
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Lo, Belinda K. M., Agnes Archibong-Omon, Panayiota Ploutarchou, Anthony J. Day, Caroline M. Milner, and Suzannah A. Williams. "Oocyte-specific ablation of N- and O-glycans alters cumulus cell signalling and extracellular matrix composition." Reproduction, Fertility and Development 31, no. 3 (2019): 529. http://dx.doi.org/10.1071/rd18209.
Full textAbstract:
Cumulus–oocyte complex (COC) expansion is essential for ovulation and fertilisation and is linked to oocyte quality. Hyaluronan (HA), the major matrix constituent, is cross-linked via inter-α-inhibitor heavy chains (HCs), pentraxin 3 (PTX3) and tumour necrosis factor-stimulated gene 6 (TSG-6). All except HCs are secreted by cumulus cells in response to oocyte-secreted factors, which signal via SMAD pathways. The double mutant (DM) mouse generates oocytes lacking complex N- and O-glycans due to oocyte-specific deletion of core 1 β1,3-galactosyltransferase (C1galt1) and N-acetylglucosaminyltransferase I (Mgat1) and has modified cumulus expansion. We compared COCs before expansion (48 h-post-pregnant mare serum gonadotrophin (PMSG)) and at late-stage expansion (9 h-post-human chorionic gonadotrophin (hCG); control n=3 mice, DM n=3 per group). Using histochemistry the levels of HA, HCs, PTX3, TSG-6 and phosphorylated-SMAD1/5/8 and -SMAD2 (12–25 COCs per group) were assessed. DM COCs did not differ from Controls in cumulus size or cell density at 9 h-post-hCG; however, HA and HC levels and phosphorylated-SMAD1/5/8 were reduced. Furthermore, no correlations were found between the levels of matrix molecules and cumulus area in DM or Control samples. These data suggest that HA and HCs can support cumulus expansion provided that they are present above minimum threshold levels. We propose that oocyte-specific ablation of C1galt1 and Mgat1 may affect bone morphogenetic protein 15 synthesis or bioactivity, thereby reducing SMAD1/5/8 phosphorylation and HA production.
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Liu, Honglin, and Fugaku Aoki. "Transcriptional activity associated with meiotic competence in fully grown mouse GV oocytes." Zygote 10, no. 4 (October 31, 2002): 327–32. http://dx.doi.org/10.1017/s0967199402004069.
Full textAbstract:
The involvement of cumulus cells and chromatin organisation in transcriptional activity was investigated. In addition, the relationship between transcriptional activity and meiotic competence in fully grown mouse oocytes was surveyed. Transcriptional activity was detected in fully grown oocytes in which chromatin did not surround the nucleolus in the germinal vesicle (NSN-type oocytes), but not in oocytes in which chromatin surrounded the nucleolus (SN-type oocytes). Cumulus cells seemed to downregulate transcriptional activity in NSN-type oocytes, since transcriptional activity was 3 times greater in the denuded NSN-type oocytes free of cumulus cells (DO oocytes) than in NSN-type oocytes enclosed in cumulus cells (COC oocytes). Higher transcriptional activity corresponded to lower germinal vesicle breakdown (GVB) competence of fully grown oocytes in culture. Although GVB occurred in nearly all (99%) the SN-type oocytes, it occurred in 88% of COC/NSN-type oocytes (cumulus-oocyte complex with SN-type configuration) and in 61% of DO/NSN-type oocytes (denuded oocytes with NSN-type configuration). There was a negative correlation between transcriptional activity and the capacity of a cell to complete the progression to the second metaphase (MII). In GVB oocytes, the percentage of first polar body (PBI) extrusion differed among COC/NSN-type (81%), DO/SN-type (66%), COC/NSN-type (47%) and DO/NSN-type (29%) oocytes. After activation with 10 mM Sr2+, the frequency of parthenogenetic activation was greater in SN-type oocytes (46.9%) than in transcriptionally active NSN-type oocytes (27.5%). These results suggest that transcriptional activity has a detrimental effect on the competence of meiotic maturation and subsequent activation in fully grown GV oocytes. Alternatively, active transcription in the fully grown oocytes suggests that they are still in the process of synthesising substances required for meiotic maturation and are not yet competent for these processes.
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Watson, L. N., M. Sasseville, R. B. Gilchrist, and D. L. Russell. "118. CHARACTERISATION OF HEPARAN SULPHATE PROTEOGLYCANS IN THE MATURING CUMULUS OOCYTE COMPLEX." Reproduction, Fertility and Development 21, no. 9 (2009): 37. http://dx.doi.org/10.1071/srb09abs118.
Full textAbstract:
Many growth factors including members of the transforming growth factor beta (TGFβ) superfamily and epidermal growth factor (Egf)-like ligands signal via interactions with heparan sulphate proteoglycans (HSPGs). Cell surface HSPGs can act by sequestering ligands at their site of action, by presenting a ligand to its signalling receptor, or by preventing ligand-receptor interaction. The oocyte secreted factors (OSF) growth differentiation factor 9 and bone morphogenetic protein 15 are members of the TGFβ superfamily that act selectively on cumulus cells. Conversely Egf-like ligands are secreted by mural granulosa cells and transmit LH-induced signals to cumulus cells. We investigated the possibility that HSPGs contribute to the spatially restricted responses these signals exert on cumulus cells. Syndecan-1 and Glypican-1 are cell surface HSPGs that are involved in numerous biological processes, including growth factor regulation, cell proliferation and differentiation. Microarray analysis showed Syndecan-1 and Glypican-1 mRNA expression induced 6-fold (P=10-9) and 3-fold (P=10-7) respectively in Egf+FSH stimulated cumulus oocyte complexes (COCs). Furthermore, Syndecan-1 and Glypican-1 mRNA were induced 27- and 16-fold respectively in COCs after hCG treatment of mice. Syndecan-1 and Glypican-1 protein was localised specifically to the COC through immunohistochemical analysis. In Vitro Maturation (IVM) of oocytes is a valuable alternative to gonadotropin mediated superovulation, but IVM COCs are less competent than those matured in vivo. Several components of the COC have been shown to be altered in IVM, including the chondroitin sulphate proteoglycan Versican. COCs from mice that underwent IVM in the presence of Egf+FSH and cilostamide for 16 hours had >16 fold reduced mRNA for Syndecan-1 when compared with In Vivo matured COCs. The lack of Syndecan-1 in IVM COCs could reduce signalling capacity of growth factors including OSFs. This may contribute to the reduced capacity of IVM oocytes to fertilise and produce a healthy embryo, and ultimately, a healthy offspring.
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Frank, L. A., M. L. Sutton-McDowall, D. L. Russell, M. Lane, R. B. Gilchrist, and J. G. Thompson. "535. THE EFFECT OF ALTERING GLUCOSE LEVELS DURING COLLECTION AND MATURATION OF MOUSE OOCYTES ON SUBSEQUENT DEVELOPMENTAL COMPETENCE." Reproduction, Fertility and Development 21, no. 9 (2009): 133. http://dx.doi.org/10.1071/srb09abs535.
Full textAbstract:
The preconception environment is known to influence oocyte developmental competence. In particular, hyperglycaemic conditions during cumulus-oocyte complex (COC) maturation result in decreased oocyte quality. This is, in part, due to perturbations in O-linked glycosylation in the cumulus cells. In embryos, even a brief exposure to glucose during early cleavage can have significant impact on O-linked glycosylation and further development. The aim of this study was to determine the effect of altering glucose concentrations during the collection and maturation phases of COCs on oocyte developmental competence. COCs were collected and matured for 17h at 37°C in 6% CO2 with 0 or 10mM glucose in a 2 x 2 factorial design. A fifth group used standard concentrations of 0.5mM and 5.55mM glucose in the collection and maturation media respectively. Following maturation, oocytes were inseminated and cultured to the blastocyst stage. The average time for collection was 1 h. COCs exposed to 0mM glucose during collection and 10mM glucose during maturation had the greatest cumulus expansion despite no change in the proportion of COCs completing nuclear maturation. However, this same treatment group resulted in significantly lower blastocyst production than the control group (8.4% vs. 25.0%, P<0.05). These results show that glucose concentration in collection medium has a significant influence on maturation indices and oocyte developmental competence, as determined by blastocyst development rates. Our data further supports the concept that the conditions used for the collection of oocytes can have profound effects on subsequent development. We intend to investigate if these effects are related to perturbations in cumulus cell O-linked glycosylation.
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Yang, X., K. R. Dunning, T. E. Hickey, R. J. Norman, X. Liang, and R. L. Robker. "511. THE EFFECTS OF HIGH FAT DIET ON LIPID LOCALISATION IN THE PERI-OVULATORY CUMULUS OOCYTE COMPLEX." Reproduction, Fertility and Development 21, no. 9 (2009): 110. http://dx.doi.org/10.1071/srb09abs511.
Full textAbstract:
Intracellular neutral lipids are stored in discrete droplets that are surrounded by lipid associated proteins, such as adipophilin and perilipin, which control cellular lipid metabolism by regulating the access of lipases. The role of lipids in oocyte maturation is unclear, although they have a potential role as an energy source for the oocyte and early embryo. To elucidate potential mechanisms controlling lipid utilisation in the peri-ovulatory cumulus-oocyte-complex (COC) we 1) localised lipid droplets by immunohistochemistry for adipophilin and perilipin and direct staining of neutral lipids with BODIPY and 2) investigated whether a high fat diet can alter oocyte lipid quantity or localisation. Ovaries were isolated from 21 day old mice before and 10h after the ovulation stimulus hCG. Adipophilin and perilipin were both detected by immunohistochemistry in peri-ovulatory follicles with similar localisation before and after hCG. In separate experiments, adult mice were fed a high fat or control diet for 4 weeks and COCs were isolated from preovulatory follicles prior to hCG or from the oviduct 13h after hCG stimulation followed by BODIPY staining and quantification with confocal microscopy. BODIPY staining showed that COCs possess low levels of lipids evenly distributed in the oocyte before hCG but increased lipid assembled as droplets in the oocyte after ovulation. In mice fed a high fat diet, intracellular lipids were markedly increased in both the cumulus cells and oocytes from preovulatory and ovulated COCs. The ubiquitous expression of lipid droplet proteins in the peri-ovulatory follicle together with the changes in neutral lipid storage concurrent with ovulation suggests that lipid metabolism play an important role in oocyte release, transport and/or developmental competence. Furthermore, the dramatic effect of dietary fat on COC lipid content may contribute to the impaired oocyte quality we have observed in obese mice as well as reduced fertility in obese women
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Ormond, C. M., P. F. Lima, D. T. Jardina-Sartor, C. A. Price, and J. Buratini. "257 EFFECTS OF FIBROBLAST GROWTH FACTOR 8 ON CUMULUS EXPANSION AND NUCLEAR MATURATION IN BOVINE CUMULUS - OOCYTE COMPLEXES." Reproduction, Fertility and Development 25, no. 1 (2013): 276. http://dx.doi.org/10.1071/rdv25n1ab257.
Full textAbstract:
Recent data indicate that fibroblast growth factor (FGF) signalling regulates oocyte developmental competence. Fibroblast growth factor 10 enhanced nuclear maturation, cumulus expansion, and embryo development in cattle (Zhang et al. 2010 Reproduction 140, 815–826). Like FGF10, FGF8 is expressed in the bovine oocyte, but whereas FGF10 activates FGF receptors (FGFR) 1B and 2B with higher affinity, FGF8 preferentially activates FGF receptor (FGFR) 2C, FGFR3C, and FGFR4. The involvement of FGF8 in the regulation of bovine cumulus–oocyte complex (COC) maturation has remained unknown. This study aimed to assess the effects of FGF8 supplementation in the in vitro maturation medium on nuclear maturation, degree of cumulus expansion, and expression of the genes necessary for expansion in bovine COC. Groups of 20 immature COC (grades 1 and 2) aspirated from 3- to 8-mm follicles were cultured in 200-µL drops of TCM-199 supplemented with FSH (1 µg mL–1), LH (10 IU mL–1), pyruvate (22 µg mL–1), amikacin (75 µg mL–1), and graded doses of recombinant human FGF8 (Peprotech, Rocky Hill, NJ, USA; 0, 1, 10, and 100 ng mL–1) for 22 h at 38.5°C and 5% CO2. After culture, COC were visually classified according to the degree of cumulus expansion (grades 1 to 3, indicating absent, moderate, and full expansion, respectively). Oocytes were mechanically separated from cumulus cells and stained with Hoechst 33342 to assess meiosis progression. Total RNA was extracted from cumulus cells using RNeasy (Qiagen, Venlo, the Netherlands), and 100 ng of RNA was reverse-transcribed using Omniscript (Qiagen). Expression levels of messenger RNA encoding genes necessary for cumulus expansion [prostaglandin endoperoxide synthase 2 (PTGS2), hyaluronan synthase 2 (HAS2), pentraxin 3 (PTX3) and tumour necrosis factor-stimulated gene-6 protein (TSG6)] were assessed by real-time PCR, with cyclophilin (CYCA) as the housekeeping gene. Data were derived from 5 replicates. Maturation and expansion data were transformed to arcsine, and gene expression data were log transformed. Effects of treatments were tested by ANOVA, and means were compared with the Tukey-Kramer honestly significant difference test. The FGF8 at 10 and 100 ng mL–1 reduced the proportion of oocytes reaching metaphase II (70, 64.8, 52.8, and 36% for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively; P = 0.005) and increased the proportion of oocytes in metaphase I at 22 h of culture (30, 35.2, 47.2, and 64% for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively; P = 0.004). Fibroblast growth factor 8 did not affect the degree of cumulus expansion as visually assessed. However, FGF8 at 10 and 100 ng mL–1 increased the messenger RNA abundance of PTGS2 [P = 0.0002; relative values (±SEM) of 0.69 ± 0.10, 0.63 ± 0.11, 1.56 ± 0.44, and 1.67 ± 0.20 for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively] and HAS2 [P = 0.0002; relative values (±SEM) of 1.38 ± 0.15, 1.37 ± 0.24, 3.58 ± 0.61, and 4.14 ± 0.27 for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively] in cumulus cells. In conclusion, the present data suggest the involvement of FGF8 in the mechanisms regulating transcription of expansion-inducing genes in cattle. In contrast with previous findings with FGF10, FGF8 inhibited nuclear maturation, suggesting different actions for different FGF in the regulation of COC maturation. Further research is needed to clarify the roles of FGF8 in the bovine COC. Supported by FAPESP.
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Yamashita, Yasuhisa, Ikkou Kawashima, Yoshiari Yanai, Masahide Nishibori, JoAnne S. Richards, and Masayuki Shimada. "Hormone-Induced Expression of Tumor Necrosis Factor α-Converting Enzyme/A Disintegrin and Metalloprotease-17 Impacts Porcine Cumulus Cell Oocyte Complex Expansion and Meiotic Maturation via Ligand Activation of the Epidermal Growth Factor Receptor." Endocrinology 148, no. 12 (December 1, 2007): 6164–75. http://dx.doi.org/10.1210/en.2007-0195.
Full textAbstract:
The epidermal growth factor (EGF)-like growth factors, amphiregulin (AREG) and epiregulin (EREG), are expressed in murine cumulus oocyte complexes (COCs) where they impact the function of cumulus cells and oocyte maturation during LH-mediated ovulation. Because TNFα-converting enzyme (TACE)/a disintegrin and metalloprotease-17 (ADAM17) is essential for ectodomain shedding of AREG and EREG from the surface of other cell types, the expression and function of TACE/ADAM17 was analyzed in a porcine COC culture system in which FSH- and LH-mediated expansion and oocyte meiotic maturation have been well characterized and shown to occur between 20 and 40 h. In this model, Areg, Ereg, and Tace/Adam17 mRNAs increased significantly with maximal levels observed between 5 and 20 h of culture with FSH plus LH. TACE/ADAM17 protein and protease activity were up-regulated markedly at 10 h and maintained to 40 h. Treatment of COCs with the TACE/ADAM17-selective inhibitor TNFα-processing inhibitor-2 (TAPI-2) significantly suppressed in a time-dependent manner downstream targets of EGF receptor activation such as ERK1/2 phosphorylation, Ptgs2, Has2, and Tnfaip6 mRNA expression, hormone-induced COC expansion, and meiotic maturation of the oocytes. Addition of EGF to COCs cultured in the presence of FSH/LH reversed the inhibitory effects of TAPI-2 on these ovulation-related processes. Gonadotropin-induced phosphorylation of ERK1/2 was also inhibited in rat granulosa cells treated with TAPI-2 or after transfection with Tace/Adam17 small interfering RNA. Induced expression of Tnfaip6 mRNA was also reduced by Tace/Adam17 small interfering RNA. Thus, TACE/ADAM17 is induced and the activity is involved in porcine COC expansion as well as oocyte meiotic maturation through the activation of EGF receptor in cumulus cells.
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Kawashima, Ikko, Zhilin Liu, Lisa K. Mullany, Toshihiro Mihara, Joanne S. Richards, and Masayuki Shimada. "EGF-Like Factors Induce Expansion of the Cumulus Cell-Oocyte Complexes by Activating Calpain-Mediated Cell Movement." Endocrinology 153, no. 8 (June 6, 2012): 3949–59. http://dx.doi.org/10.1210/en.2012-1059.
Full textAbstract:
Cumulus cell-oocyte complex (COC) expansion is obligatory for LH-induced ovulation and is initiated by LH induction of the epidermal growth factor (EGF)-like factors that mediate the synthesis of the hyaluronan-rich matrix and hyaluronan-stabilizing factors. COC expansion also involves the movement of cumulus cells within the matrix by mechanisms that have not been characterized. We document herein that two proteases, calpain 2 and to a lesser extent calpain 1, are expressed in cumulus cells and that the proteolytic activity of these enzymes is rapidly and significantly increased in COC isolated from human chorionic gonadotropin-induced ovulatory follicles in vivo. Stimulation of calpain activity was associated with proteolytic degradation of paxillin and talin (two components of focal adhesion complexes), cell detachment, and the formation of cell surface bleb-like protrusions. Injection of a calpain inhibitor in vivo reduced 1) human chorionic gonadotropin-stimulated calpain enzyme activity, 2) cell detachment, 3) membrane protrusion formation, and 4) COC expansion by mechanisms that did not alter Has2 expression. During EGF-like factor induction of COC expansion in culture, calpain activity was increased by ERK1/2 and intracellular Ca2+ signaling pathways. Inhibition of calpain activity in cultured COC blocked cumulus cell detachment, protrusion formation, and the vigorous movement of cumulus cells. As a consequence, COC expansion was impaired. Collectively, these results show that two highly coordinated processes control COC expansion. One process involves the synthesis of the hyaluronan matrix, and the other mediates cumulus cell detachment and movement. The latter are controlled by calpain activation downstream of the EGF receptor activation of the Ca2+ pathway and ERK1/2 pathways.
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Ochsner, Scott A., Anthony J. Day, Marilyn S. Rugg, Richard M. Breyer, Richard H. Gomer, and Joanne S. Richards. "Disrupted Function of Tumor Necrosis Factor-α-Stimulated Gene 6 Blocks Cumulus Cell-Oocyte Complex Expansion." Endocrinology 144, no. 10 (October 1, 2003): 4376–84. http://dx.doi.org/10.1210/en.2003-0487.
Full textAbstract:
During ovulation, the oocyte and surrounding somatic cumulus cells contained within a specialized, mucoid matrix are released from the ovary. One matrix component, TNF-α-stimulated gene 6 (TSG-6), is a hyaluronan binding protein induced in cumulus cells of preovulatory follicles by the LH surge and is decreased in cumulus cells of COX-2 and prostaglandin E2 (PGE2) receptor subtype EP2 null mice that exhibit impaired ovulation and cumulus expansion. To determine if TSG-6 was hormonally induced in cumulus cells in vitro and was functional during the formation of the expanded matrix, we established a cumulus cell-oocyte complex (COC) culture system. This system was used to analyze the effects of FSH, PGE2, EP2 receptor, and selected protein kinase inhibitors on TSG-6 production as well as specific antibodies to the TSG-6 link module on TSG-6 function. We document that TSG-6 message and protein are induced by cAMP/protein kinase A/MAPK signaling pathways and that blocking these cascades prevents expansion and the production of TSG-6. FSH but not PGE2 rescued expansion and production of TSG-6 in the EP2 null COCs, indicating that generation of a cAMP signal is essential. Furthermore, disruption of the functional interactions between TSG-6, inter-α trypsin inhibitor, and hyaluronan with specific antibodies severely altered matrix formation and cumulus expansion, as recorded by time-lapse imaging. Collectively, these results indicate that TSG-6 mRNA is induced in cumulus cells in culture by cAMP and that the secreted TSG-6 protein is a key structural component of the mouse COC matrix.
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Dunning, K. R., C. X. Yeo, and D. L. Russell. "230. Altered matrix composition of cumulus oocyte complexes following in vitro maturation." Reproduction, Fertility and Development 17, no. 9 (2005): 90. http://dx.doi.org/10.1071/srb05abs230.
Full textAbstract:
The luteinizing hormone (LH) surge initiates cumulus expansion, through synthesis of hyaluronan and cross-linking proteins including versican, which stabilise the cumulus oocyte complex (COC) matrix. Versican is a substrate for the protease ADAMTS-1 and mRNA for each are localised to granulosa cells (GCs) and greatly induced following the LH surge. In humans, the use of in vitro maturation (IVM) of oocytes is an appealing option, reducing costs and risk of side effects associated with in vitro fertilisation. IVM oocytes are of poorer quality, likely resulting from altered gene expression and environmental conditions during oocyte maturation. Real-time PCR showed that IVM and immature COCs from Balb/c mice have 12 and 13 fold-reduced levels of ADAMTS-1 and versican expression respectively compared to in vivo matured COCs (PMSG+hCG 12h). Ovulated COCs (PMSG+hCG 15h) had similar low levels of ADAMTS-1 and versican. Samples isolated from F1 C57Bl/6xCBA mice showed similar reduced versican and ADAMTS-1 mRNA. Western blot analysis revealed that full length and cleaved versican, from ADAMTS-1/4 activity, was not detected in immature COCs, was present in in vivo matured COCs isolated from follicles, but strongest in ovulated COCs. IVM COCs had no detectable versican protein, supporting the mRNA data. Full-length versican was also present in GCs after PMSG+hCG 12h or 15h. ADAMTS-1 protein was most abundant in in vivo matured COCs with reduced levels seen in ovulated COCs, but was absent from IVM and immature COCs. These results indicate that ADAMTS-1 and versican are secreted products of granulosa cells that bind and incorporate into the COC matrix. The presence of versican and ADAMTS-1 is not essential for cumulus matrix expansion in vitro, but may contribute to oocyte maturation, ovulation of the COC and/or interaction with sperm during fertilisation.
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Park, Hyo-Jin, Bokyung Kim, Deog-Bon Koo, and Dong-Seok Lee. "Peroxiredoxin 1 Controls Ovulation and Ovulated Cumulus–Oocyte Complex Activity through TLR4-Derived ERK1/2 Signaling in Mice." International Journal of Molecular Sciences 22, no. 17 (August 30, 2021): 9437. http://dx.doi.org/10.3390/ijms22179437.
Full textAbstract:
Peroxiredoxins (PRDXs) are expressed in the ovary and during ovulation. PRDX1 activity related to the immuno-like response during ovulation is unknown. We investigated the roles of Prdx1 on TLR4 and ERK1/2 signaling from the ovulated cumulus–oocyte complex (COC) using Prdx1-knockout (K/O) and wild-type (WT) mice. Ovulated COCs were collected 12 and 16 h after pregnant mare serum gonadotropin/hCG injection. PRDX1 protein expression and COC secretion factors (Il-6, Tnfaip6, and Ptgs2) increased 16 h after ovulated COCs of the WT mice were obtained. We treated the ovulated COCs in mice with LPS (0.5 μg/mL) or hyaluronidase (Hya) (10 units/mL) to induce TLR4 activity. Intracellular reactive oxygen species (ROS), cumulus cell apoptosis, PRDX1, TLR4/P38/ERK1/2 protein expression, and COC secretion factors’ mRNA levels increased in LPS- and Hya-treated COCs. The ERK inhibitor (U0126) and Prdx1 siRNA affected TLR4/ERK1/2 expression. The number and cumulus expansion of ovulated COCs by ROS were impaired in Prdx1 K/O mice but not in WT ones. Prdx1 gene deletion induced TLR4/P38/ERK1/2 expression and cumulus expansion genes. These results show the controlling roles of PRDX1 for TLR4/P38/ERK1/2 signaling activity in ovulated mice and the interlink of COCs with ovulation.
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De Santis, T., M. E. Dell'Aquila, F. Maritato, V. Casavola, and P. Minoia. "280 EFFECTS OF β-ENDORPHIN AND NALOXONE ON INTRACELLULAR CALCIUM LEVELS IN CUMULUS CELLS OF EQUINE OOCYTES." Reproduction, Fertility and Development 17, no. 2 (2005): 290. http://dx.doi.org/10.1071/rdv17n2ab280.
Full textAbstract:
Changes in intracellular calcium levels in the cumulus oocyte complex (COC) have a crucial role in oocyte maturation. In previous studies we demonstrated that the μ-opioid receptor is expressed in the bovine COC and participates in the signaling associated with oocyte maturation, by inducing an intracellular calcium increase (Dell'Aquila ME et al. 2002 Mol. Reprod. Dev. 63, 210–222). In this work we evaluated modifications of intracellular calcium induced by β-endorphin (β-end) or Naloxone (Nx) in cumulus cells of equine oocytes in relation to the time of the year and cumulus morphology at retrieval. Cumulus cells, isolated by mechanical treatment from compact (Cp, n = 120) or expanded (Exp, n = 120) COCs, recovered from the ovaries of slaughtered mares (follicles <20 mm in diameter) during anestrus, breeding season, spring transition, and autumnal transition, were cultured for 24 h and loaded with 5 μM Fura2-AM for microspectrofluorometric measurements of cytoplasmic ionized calcium (Dell'Aquila et al., 2002). The changes in β-end (30 μM)- or Nx (1mM and 10 μM)-induced calcium concentration were calculated in single cells (n = 194) and are expressed as Δ fluorescence (Fmaximal effect – Fbaseline) before and after 1-min perfusion with the drugs. The use of 1 mM Nx induced a significant increase of intracellular calcium levels in cumulus cells of oocytes recovered in all periods of the year in both Cp and Exp (P < 0.01). The addition of 10 μM Nx or 30 μM β-end significantly increased intracellular calcium only in cumulus cells from oocytes recovered in anestrus (P < 0.05). These results confirm previous observations, carried out on bovine oocytes, in which Nx behaved as a μ-receptor agonist when used at high concentration (Dell'Aquila et al. 2002). The effects of β-end and Nx may be explained in terms of a binding of the two subtances at the μ-receptor with consequent intracellular calcium increases due to extracellular calcium entry or depletion of intracellular stores. These findings could be related to differential espression and/or activation status of the μ-opioid receptor in COCs retrieved in different seasons. These substances can be used to modulate intracellular calcium in the equine COCs, and consequent effects on the stimulation/inhibition of oocyte maturation in this species need to be further investigated. This work was supported by Grant MIUR COFIN PRIN 2003.
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Gadella, B. M. "Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation." Reproduction, Fertility and Development 25, no. 1 (2013): 26. http://dx.doi.org/10.1071/rd12277.
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Recent findings have refined our thinking on sperm interactions with the cumulus–oocyte complex (COC) and our understanding of how, at the molecular level, the sperm cell fertilises the oocyte. Proteomic analyses has identified a capacitation-dependent sperm surface reordering that leads to the formation of functional multiprotein complexes involved in zona–cumulus interactions in several mammalian species. During this process, multiple docking of the acrosomal membrane to the plasma membrane takes place. In contrast with the dogma that the acrosome reaction is initiated when spermatozoa bind to the zona pellucida (ZP), it has been established recently that, in mice, the fertilising spermatozoon initiates its acrosome reaction during its voyage through the cumulus before it reaches the ZP. In fact, even acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilise another ZP-intact oocyte. The oviduct appears to influence the extracellular matrix properties of the spermatozoa as well as the COC. This may influence sperm binding and penetration of the cumulus and ZP, and, in doing so, increase monospermic while decreasing polyspermic fertilisation rates. Structural analysis of the ZP has shed new light on how spermatozoa bind and penetrate this structure and how the cortical reaction blocks sperm–ZP interactions. The current understanding of sperm interactions with the cumulus and ZP layers surrounding the oocyte is reviewed with a special emphasis on the lack of comparative knowledge on this topic in humans, as well as in most farm mammals.
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Viana, J. H. M., L. S. A. Camargo, J. F. Fonseca, A. P. Oliveira, E. K. N. Arashiro, C. Freitas, and C. A. C. Fernandes. "219 RETROSPECTIVE ANALYSIS OF CUMULUS - OOCYTE COMPLEX YIELD INA GYR (BOS TAURUS INDICUS) HERD UNDERGOING TRANSVAGINAL ULTRASOUND-GUIDED FOLLICLE ASPIRATION." Reproduction, Fertility and Development 20, no. 1 (2008): 189. http://dx.doi.org/10.1071/rdv20n1ab219.
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Bovine in vitro embryo production (IVP) is extensively used in Brazil, associated with transvaginally guided follicle aspiration (TGFA). The good results obtained with the use of TGFA-IVP in this country are related to the ovarian physiology characteristics of zebu breeds, which include a lower persistence of dominant follicles and a greater number of follicles emerging in each follicular wave. There are, however, few reports of COC yield in these breeds in large-scale TGFA-IVP systems. The aim of this study was to analyze data on COC recovery from a Gyr (dairy zebu breed) herd in Brazil. Only pluriparous, nonlactating cows were used as donors. Follicle aspiration was performed with a portable ultrasound device, using disposable 19- or 20-gauge needles and a vacuum pressure of 80 mmHg. The follicle population before TGFA was recorded, and recovered oocyes were classified according cumulus cells layers and cytoplasm aspect. Data were analyzed by ANOVA, and means were compared by Tukey's test. Associations between variables were analyzed by Pearson's correlation method. A total of 761 TGFA sessions were performed in 54 donors, with the recovery of 8082 oocytes (10.62 � 0.32 per session) and 6208 viable COC (8.19 � 0.25 per session). Each donor underwent from 1 to 42 TGFA sessions. Both the number of total oocytes and viable COC were highly correlated with the number of follicles present in the ovaries (mean of 15.81 � 0.32) in the moment of TGFA (R = 0.86 and R = 0.83, respectively, P < 0.0001). There was a significant donor effect (P < 0.0001) in the mean number of follicles in the ovaries (ranging from 4.50 � 0.65 to 37.50 � 2.50), oocytes (from 0.75 � 0.48 to 30.50 � 8.50), and viable COC (from 0.75 � 0.48 to 25.67 � 6.12) recovered. The absolute maximum and minimum values for these parameters were 74, 67, and 44 v. 3, 0, and 0, respectively. The TGFA order also affected all parameters evaluated (P < 0.0001), with a linear decrease in the number of follicles punctured (y = –0.22x + 18.64, R2 = 0.65), total oocytes ( y = –0.25x + 13.81, R2 = 0.68) and viable oocytes (y = –0.22x + 11.05, R2 = 0.77) recovered. This decrease was probably associated with the cumulative damage in ovarian structure, once all parameters were affected, although from the first to the second TGFA session the mean number of oocytes (20.09 � 1.70 v. 14.08 � 1.11) and viable COC (16.37 � 1.27 v. 11.83 � 1.04), but not of aspirated follicles (22.28 � 1.60 v. 19.38 � 1.19), were reduced (P < 0.05). The decline in oocyte yield was greater ( y = –0.71x + 10.45, R2 = 0.95) when considering donors undergoing TGFA continuously (within intervals shorter than 8 days). Overall bastocyst rate was 28.48%, with a significant correlation between COC recovered and embryos produced (R = 0.64, P < 0.0001). These results shows that (1) follicular population is the main characteristic affecting the number of recovered COC, and consequently IVP; (2) the great variability in follicular population and in COC recovery among donors allows the selection of animals for IVF; and (3) repeated TGFA negatively affect ovarian follicle emergence and COC recovery.
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Techakumphu, M., A. Promdireg, N. Phutikanit, J. Singlor, S. Thongjan, N. Onwan, and A. Na-Chiengmai. "339 ULTRASOUND GUIDED OVUM PICK UP (OPU) IN PREPUBERTAL SWAMP BUFFALO USING THREE DIFFERENT VACUUM PRESSURES." Reproduction, Fertility and Development 17, no. 2 (2005): 320. http://dx.doi.org/10.1071/rdv17n2ab339.
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Our group successfully developed ovum pick-up in prepubertal swamp buffalo, however the quality of the oocytes that were collected was poor especially those without a cumulus mass (Techakumphu et al. 2003 Theriogenology 61, 1705–1711). The vacuum pressure used for oocyte collection was one of the factors influencing oocyte quality (Bols et al. 1997 Theriogenology 47, 1221–1236). The objective of this study was to compare the effect of three vacuum pressures on both the recovery rate and oocyte quality in prepubertal swamp buffaloes. Oocyte recovery and oocyte quality were using different groups of aspiration vacuum pressures. The maturation stages of the recovered oocytes were immediately assessed by fixation and rapid staining with basic carbol fuchsin. Twelve prepubertal calves, aged 1.5 yrs were a total of 180 mg FSH given, twice a day, in divided doses over 3 d (40/40, 30/30, 20/20). The animals were randomized into 3 groups, according to the different vacumm pressures, 100 (n = 8), 80 (n = 8) and 60 mmHg (n = 8). Two sets of treatments, carried out, with a 2 month interval between them. The oocyte recovery rates using 100 and 80 mmHg, were not different at 78.4% (29/37) and 83.6% (61/73). The 60 mmHg gave a lower rate, 65.7% (23/35) which was statistically different from the 80 mmHg group (P < 0.05). The oocytes recovered per donor showed no significant difference among the groups; 5.8 ± 4.9 for 100 mmHg, 7.6 ± 8.6 for 80 mmHg and 3.3 ± 2.1 for 60 mmHg respectively. The percentage of cumulus-oocyte complex (COC), single layered+partial cumulus oocytes (S + P) and expanded cumulus oocytes (EXP) showed no differences for any of the pressures, being 79.3, 65.5 and 82% of the total recovered. The experiment showed that follicles with a size of 2–6 mm were dominant after FSH treatment, being around 80% of the total number. Furthermore, the maturation stages of these oocytes were at prophase I and metaphase I. In conclusion, the vacuum pressure used for the oocyte retrieval technique influenced the recovery rate but not the oocyte quality. This study was supported by Rajadapisek Sompoj Fund, Chulalongkorn University year 2002. AP was PhD candidate under Royal Golden Jubilee, Thailand Research Fund.
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Dunning, K. R., L. K. Akison, D. L. Russell, R. J. Norman, and R. L. Robker. "428. Normal follicle growth and maturation in a three-dimensional in vitro culture system: follicular environment manipulation and assessment of oocyte outcomes." Reproduction, Fertility and Development 20, no. 9 (2008): 108. http://dx.doi.org/10.1071/srb08abs428.
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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.
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Mastrorocco, Antonella, Ludovica Cacopardo, Daniela Lamanna, Letizia Temerario, Giacomina Brunetti, Augusto Carluccio, Domenico Robbe, and Maria Elena Dell’Aquila. "Bioengineering Approaches to Improve In Vitro Performance of Prepubertal Lamb Oocytes." Cells 10, no. 6 (June 10, 2021): 1458. http://dx.doi.org/10.3390/cells10061458.
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Juvenile in vitro embryo technology (JIVET) provides exciting opportunities in animal reproduction by reducing the generation intervals. Prepubertal oocytes are also relevant models for studies on oncofertility. However, current JIVET efficiency is still unpredictable, and further improvements are needed in order for it to be used on a large-scale level. This study applied bioengineering approaches to recreate: (1) the three-dimensional (3D) structure of the cumulus–oocyte complex (COC), by constructing—via bioprinting technologies—alginate-based microbeads (COC-microbeads) for 3D in vitro maturation (3D-IVM); (2) dynamic IVM conditions, by culturing the COC in a millifluidic bioreactor; and (3) an artificial follicular wall with basal membrane, by adding granulosa cells (GCs) and type I collagen (CI) during bioprinting. The results show that oocyte nuclear and cytoplasmic maturation, as well as blastocyst quality, were improved after 3D-IVM compared to 2D controls. The dynamic 3D-IVM did not enhance oocyte maturation, but it improved oocyte bioenergetics compared with static 3D-IVM. The computational model showed higher oxygen levels in the bioreactor with respect to the static well. Microbead enrichment with GCs and CI improved oocyte maturation and bioenergetics. In conclusion, this study demonstrated that bioengineering approaches that mimic the physiological follicle structure could be valuable tools to improve IVM and JIVET.
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Koike, Hiroshi, Miyuki Harada, Akari Kusamoto, Chisato Kunitomi, Zixin Xu, Tsurugi Tanaka, Yoko Urata, et al. "Notch Signaling Induced by Endoplasmic Reticulum Stress Regulates Cumulus-Oocyte Complex Expansion in Polycystic Ovary Syndrome." Biomolecules 12, no. 8 (July 27, 2022): 1037. http://dx.doi.org/10.3390/biom12081037.
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Endoplasmic reticulum (ER) stress activated in granulosa cells contributes to the pathophysiology of polycystic ovary syndrome (PCOS). In addition, recent studies have demonstrated that Notch signaling plays multiple roles in the ovary via cell-to-cell interactions. We hypothesized that ER stress activated in granulosa cells of antral follicles in PCOS induces Notch signaling in these cells, and that activated Notch signaling induces aberrant cumulus-oocyte complex (COC) expansion. Expression of Notch2 and Notch-target transcription factors was increased in granulosa cells of PCOS patients and model mice. ER stress increased expression of Notch2 and Notch-target transcription factors in cultured human granulosa-lutein cells (GLCs). Inhibition of Notch signaling abrogated ER stress-induced expression of genes associated with COC expansion in cultured human GLCs, as well as ER stress-enhanced expansion of cumulus cells in cultured murine COCs. Furthermore, inhibition of Notch signaling reduced the areas of COCs in PCOS model mice with activated ER stress in the ovary, indicating that Notch signaling regulates COC expansion in vivo. Our findings suggest that Notch2 signaling is activated in granulosa cells in PCOS and regulates COC expansion. It remains to be elucidated whether aberrant COC expansion induced by the ER stress-Notch pathway is associated with ovulatory dysfunction in PCOS patients.
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Pascottini, O. B., M. Catteeuw, A. Van Soom, and G. Opsomer. "167 Effect of Storage Time and Temperature of a Commercial Embryo Holding Medium on the Maturation Kinetics of Immature Bovine Oocytes." Reproduction, Fertility and Development 30, no. 1 (2018): 223. http://dx.doi.org/10.1071/rdv30n1ab167.
Full textAbstract:
The effect of holding time and temperature during storage of immature bovine oocytes in a commercial embryo holding medium (EHM; Syngro® Ltd., Livingston, United Kingdom) was evaluated. Ovaries were collected at the local slaughterhouse and processed within 2 h. Cumulus-oocyte complexes (COC) were collected and allocated to groups of 60. The COC were held in 1-mL sterile glass osmometer tubes, filled to the top with the EHM to limit the amount of air. Vials were capped and covered with parafilm to ensure a tight seal and prevent leakage. Tubes were stored for 6 h at 4°C, room temperature (RT), or 38.5°C; for 10 h at 4°C and RT; and for 14 h at RT. Next, oocytes were fixed after storage in EHM (immature holding) or fixed after being held in EHM and subsequent 22-h maturation at 38.5°C in 5% CO2 in humidified air (mature holding). Maturation medium consisted of modified bicarbonate-buffered TCM-199 supplemented with gentamycin and epidermal growth factor. During all experiments, a control group was included each time. The control consisted of groups of 60 COC immediately fixed after collection or transferred to maturation medium for 22 h and subsequently fixed. Nuclear maturation of oocytes was assessed after Hoechst 33342 staining, using a 400× magnification fluorescence microscope. A total of 3043 COC were evaluated in 3 replicates. Oocytes maturation stages were classified as (1) oocytes in germinal vesicle stage, (2) oocytes in meiotic progression (diakinesis, metaphase I, or anaphase), (3) matured (telophase I or metaphase II), and (4) degenerated (degraded chromatin). Oocytes remained at the germinal vesicle stage when held in EHM (without subsequent maturation) regardless of holding time and temperature (P > 0.05). When oocytes were held for 6 h and subsequently matured (Table 1), the number of matured oocytes was significantly lower for oocytes held at 38.5°C compared with the other groups (control, RT, and 4°C). When held for 10 h, the oocyte maturation rate was similar between the control and RT groups (P > 0.05), but it was significantly lower in oocytes held at 4°C. Last, when compared with oocytes held at RT for 14 h, the maturation rate was higher in the control group (P < 0.05). To conclude, immature bovine oocytes can be successfully held in EHM at RT for up to 10 h. Storing immature oocytes in EHM can delay oocyte maturation and concomitantly synchronize maturation. Table 1.Kinetics of cumulus-oocyte complex nuclear status after storage in embryo holding medium for different times and temperatures and subsequent 22-h maturation
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Warzych, E., A. Wolc, A. Cieslak, and D. Lechniak-Cieslak. "217 TRANSCRIPT ABUNDANCE OF CATHEPSIN GENES IN CUMULUS CELLS AS A MARKER OF CATTLE OOCYTE QUALITY." Reproduction, Fertility and Development 25, no. 1 (2013): 257. http://dx.doi.org/10.1071/rdv25n1ab217.
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Dynamics of follicular growth and atresia is closely connected with apoptosis. Cathepsins (CTS) are involved in diverse biological functions, whereas one member of this family, cathepsin B, plays a major regulatory role in the process of apoptosis. Oocyte quality is a complex trait shaped by the follicular components (e.g. cumulus cells, CC; follicular fluid, FF). A negative relationship between relative transcript abundance (RA) of CTSB, CTSS, and CTSZ genes in CC with the quality of corresponding oocytes was reported in cattle (Bettegowda et al. 2008). Fatty acid (FA) composition of the FF and mtDNA copy number in the oocyte are other markers of oocyte quality. Therefore, in this study, we analysed relations between selected traits of the 3 follicular components (FF, CC, oocyte) within the individual follicle with the focus on oocyte quality. The experiment was based on cumulus–oocyte-complexes (COC) and FF obtained from individual follicles of slaughterhouse ovaries. Each follicle was measured and assigned into 1 of 3 classes (small <6 mm; medium 6 to 8 mm; large >8 mm). The COC morphology (grades 1 to 4) was evaluated according to Stojkovic et al. (2001). The following analyses were performed: CC, mRNA abundance of CTSB, CTSS, CTSZ, and CTSK genes (real-time PCR, 100 replicates, ACTB as a reference gene); the oocyte, mtDNA copy number (real-time PCR, 93 replicates, COX1 gene); and FF and FA composition (gas chromatography). The following procedures were employed: total RNA isolation, mirVana Paris Kit (Ambion); total DNA isolation, High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN, USA); cDNA synthesis, Transcriptor High Fidelity cDNA Synthesis Kit (Roche); and the standard curve method, to analyse the qPCR data. For statistical analysis, the Kruskal-Wallis test as well as Spearman rank correlation were applied. The highest RA of CTSB gene was noted in CC from the grade 3 COC (P < 0.05), whereas that of CTSK and CTSZ genes in CC from the grade 4 COC (P < 0.01). Because grade 3 and 4 COC are not suitable for IVM, we assumed that high RA of CTS gene in CC may indicate reduced quality of the corresponding oocyte. Surprisingly, the highest RA for CTSB gene was observed in CC from the medium follicles (P < 0.05). Significant (P < 0.05) correlations were estimated between the following: RA of CTSB gene in CC and mtDNA copy number in the oocyte (r = 0.27), RA of CTSB gene in CC and C18.3 n-3 concentration in FF (r = 0.32), RA of CTSZ gene in CC and C18.3 n-3 concentration in FF (r = 0.37), as well as RA of CTSZ gene in CC and n-3 concentration in FF (r = 0.34). Although an increase in RA of the CTS genes in CC was accompanied by the inferior oocyte morphology, it was also correlated with higher mtDNA copy number in the oocyte and FA content in FF. The last 2 features have been previously attributed to oocytes of better quality, what contrasts with the high RA of the CTS genes. Thus, higher RA of CTS genes within CC may not mark the bovine oocyte of reduced quality. Funding–National Science Center, grant no. N N302 604438.