Effect of follicle size on mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify marker genes for developmental competence

2009 ◽  
Vol 21 (5) ◽  
pp. 655 ◽  
Author(s):  
Ester Siqueira Caixeta ◽  
Paula Ripamonte ◽  
Maurício Machaim Franco ◽  
José Buratini Junior ◽  
Margot Alves Nunes Dode
Keyword(s):  
Gene Expression ◽  
Bos Indicus ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Marker Genes ◽  
Oocyte Competence ◽  
Follicular Size ◽  
Follicle Size

To identify the genes related to oocyte competence, we quantified transcripts for candidate genes in oocytes (H1Foo, H2A, H3A, GHR, GDF9, BMP15, OOSP1) and cumulus cells (FSHR, EGFR, GHR, PTX3, IGFII) using the follicle size model to select oocytes of better developmental quality. Follicles were dissected and distributed into four groups according to diameter as follows: 1.0–3.0, 3.1–6.0, 6.1–8.0 and ≥8.1 mm. Cumulus–oocyte complexes (COCs) were released, classified morphologically, matured, fertilised and cultured in vitro or denuded for measurement of diameter and determination of gene expression. Denuded germinal vesicle oocytes and their cumulus cells were used for gene expression analysis by reverse transcription–polymerase chain reaction. The blastocyst rate was highest for oocytes recovered from follicles >6 mm in diameter. In the oocyte, expression of the H2A transcript only increased gradually according to follicle size, being greater (P < 0.05) in oocytes from follicles ≥8.1 mm in diameter than in oocytes from follicles <6.0 mm in diameter. In cumulus cells, expression of FSHR, EGFR and GHR mRNA increased with follicular size. In conclusion, we confirmed the importance of H2A for developmental competence and identified important genes in cumulus cells that may be associated with oocyte competence.

scholarly journals Effects of C-type natriuretic peptide on meiotic arrest and developmental competence of bovine oocyte derived from small and medium follicles

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Zhenwei Jia ◽  
Xueli Wang
Keyword(s):  
Natriuretic Peptide ◽  
Basal Medium ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
The Novel ◽  
Bovine Oocyte ◽  
Meiotic Arrest ◽  
Follicle Size

Abstract The present study aimed to evaluate the effects of C-type natriuretic peptide (CNP) on meiotic arrest and developmental competence of bovine oocyte derived from follicles of different sizes. Collected immature cumulus-oocyte complexes from small follicles (< 3 mm) and medium follicles (3–8 mm) were cultured for 6 h in basal medium supplementated without or with 200 nM CNP. We observed that CNP effectively sustained meiotic arrest at germinal vesicle stage in in vitro cultured bovine oocytes from follicles of different sizes. Moreover, CNP treatment significantly improved the levels of cGMP in both cumulus cells and oocytes, as well as the levels of cAMP in oocytes regardless of follicle size. Based on the above results, we tested the effect of a novel in vitro maturation (IVM) system based on CNP-pretreatment, including a pre-IVM phase for 6 h using 200 nM CNP, followed by a extended IVM phase for 28 h, on developmental competence of bovine oocyte derived from small follicles (< 3 mm) and medium follicles (3–8 mm) compared to standard IVM system. The results showed that athough the novel IVM system based on CNP-pretreatment enhanced the developmental potencial of oocytes obtained from large follicles, but had no effect on the developmental comptence of oocytes obtained from small follicles.

Impact of oocyte-secreted factors on its developmental competence in buffalo

Zygote ◽  
2017 ◽  
Vol 25 (3) ◽  
pp. 313-320 ◽  
Author(s):  
Swati Gupta ◽  
Sriti Pandey ◽  
Mehtab S. Parmar ◽  
Anjali Somal ◽  
Avishek Paul ◽  
...  
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Marker Genes ◽  
Enabling Factors ◽  
Secreted Factors ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Follicle Size

SummaryOocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus–oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, <6 mm) were collected and matured in vitro either in the presence of GDF9 or BMP15, or both, or with the denuded oocytes (DOs) as a source of native OSFs. Cleavage and blastocyst rates were significantly (P < 0.05) higher in LF-derived than SF-derived oocytes. Cleavage and blastocyst rates were significantly higher (P < 0.05) in the DOs and the combination groups compared with the control, GDF9 alone and BMP15 alone groups, both in LF-derived and SF-derived oocytes, although the cleavage and blastocyst rates did not differ significantly (P > 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.

Genomic expression profiles in cumulus cells derived from germinal vesicle and MII mouse oocytes

2016 ◽  
Vol 28 (11) ◽  
pp. 1798 ◽  
Author(s):  
Li Shao ◽  
Ri-Cheng Chian ◽  
Yixin Xu ◽  
Zhengjie Yan ◽  
Yihui Zhang ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Expression Patterns ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Genomic Expression ◽  
Oocyte Competence ◽  
Before And After

Cumulus cells (CCs) are distinct from other granulosa cells and the mutual communication between CCs and oocytes is essential for the establishment of oocyte competence. In the present study we assessed genomic expression profiles in mouse CCs before and after oocyte maturation in vitro. Microarray analysis revealed significant changes in gene expression in CCs between the germinal vesicle (GV) and metaphase II (MII) stages, with 2615 upregulated and 2808 downregulated genes. Genes related to epidermal growth factor, extracellular matrix (Ptgs2, Ereg, Tnfaip6 and Efemp1), mitochondrial metabolism (Fdx1 and Aifm2), gap junctions and the cell cycle (Gja1, Gja4, Ccnd2, Ccna2 and Ccnb2) were highlighted as being differentially expressed between the two development stages. Real-time polymerase chain reaction confirmed the validity and reproducibility of the results for the selected differentially expressed genes. Similar expression patterns were identified by western blot analysis for some functional proteins, including EFEMP1, FDX1, GJA1 and CCND2, followed by immunofluorescence localisation. These genes may be potential biomarkers for oocyte developmental competence following fertilisation and will be investigated further in future studies.

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

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

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

192 9-cis-RETINOIC ACID AFFECTS OOCYTE COMPETENCE MARKER GENE EXPRESSION IN THE BOVINE IN VITRO-MATURED OOCYTES AND CUMULUS CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 208
Author(s):  
G. K. Deb ◽  
S. R. Dey ◽  
J. I. Bang ◽  
I. K. Kong
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Embryo Development ◽  
Reference Genes ◽  
Cumulus Cells ◽  
Receptor Expression ◽  
Marker Genes ◽  
Oocyte Competence ◽  
Maturation Medium

Oocyte competence is the ability of an oocyte to undergo pre- and post-implantation development and to deliver a healthy offspring. A close association between the oocyte and the cumulus cells (CC) affects oocyte competence. Expression of several genes in the CC, known as oocyte competence markers, correlates with subsequent embryo development and quality. Addition of 9-cis-retinoic acid (9cisRA) to maturation medium increases oocyte competence through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factors signaling, oxidative-stress protection, or decreasing oocyte TNF-α gene expression. However, the effect of 9cisRA on the expression of oocyte competence markers in the oocytes and CC has not been determined. Therefore, the present study evaluated the effect of 9cisRA on the expression of oocyte competence marker genes in the oocytes and in the CC. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of 0 or 5 nM 9cisRA in the maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 1 μg mL–1 of β-oestradiol, 10 μg mL–1 of follicle stimulating hormone, 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, expression of target transcripts was quantified in CC and zona-free oocytes by SYBER green real-time PCR. The expression was normalized against a minimum of 2 out of 4 reference genes analyzed each time with target genes. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (BioRad) based on M-value during the analysis of gene expression data. A minimum of 5 biological replicates (50–60 oocytes/replicate) were performed for statistical analysis using a Student's t-test. Results indicated that 9cisRA increased (P < 0.05 to P < 0.001) expression of gremlin 1, prostaglandin G/H synthase 2, pentraxin 3, midkine, CD9 and thioredoxin mRNA in oocytes (3.0-, 2.8-, 3.1-, 2.0-, 4.0- and 2.4-fold) and CC (4.0-, 3.2-, 1.9-, 1.7-, 4.0- and 2.4-fold) compared to controls. In contrast, the aldose reductase 1b1 mRNA was down-regulated both in oocytes (1.0- vs 2.0-fold; P < 0.05) and in CC (1.0- vs 1.9-fold; P < 0.04) compared to the respective controls. In conclusion, the present study indicates that 9cisRA influences mRNA expression of oocytes and cumulus cells. This might be another explanation of the improved embryo development and quality in response to 9cisRA during in vitro maturation. This work was partly supported by a scholarship from the BK21 program, the KRF (KRF-2008-211-F00011), the Next-generation BioGreen 21 Program (No. PJ007990012011), IPET (110020-3 and 109016-3) and the KOSEF (10525010001-05N2501-00110).

180 EXPRESSION OF CANDIDATE GENES FOR OOCYTE COMPETENCE IN BOVINE CUMULUS CELLS

2009 ◽  
Vol 21 (1) ◽  
pp. 189 ◽  
Author(s):  
E. S. Caixeta ◽  
P. R. Figueiredo ◽  
G. M. Machado ◽  
M. M. Franco ◽  
J. Buratini ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
The Other ◽  
Fsh Receptor ◽  
Pentraxin 3 ◽  
Developmental Potential ◽  
Oocyte Competence ◽  
Gh Receptor ◽  
Follicular Size ◽  
Follicle Size

Cumulus cells (CC) are closed connected to the oocytes through a gap junction network during follicular development and ovulation. This reciprocal functional interconnection is essential for oocyte growth, acquitision of competence, and complete maturation. Therefore, CC are a promising source of markers for predicting oocyte developmental potential without damaging the oocyte. There is a clear relationship between follicular size and oocyte competence, being those obtained from large follicles more developmentally competent in vitro. The aim of this study was to identify in CC genes candidates to be involved in the acquisition of competence, using the follicles size model to recovered cumulus–ooctye complexes (COCs) with various levels of competence. Follicles from slaughterhouse ovaries were dissected, selected, and distributed according to their diameter into 4 groups: (1) 1.0 to 3.0 mm, (2) 3.1 to 6.0 mm, (3) 6.1 to 8.0 mm, (4) ≥8.1 mm. The COCs were released by follicle rupture, morphologically classified, matured, fertilized, and cultured in vitro or denuded for the recovery of CC. Then, CC were frozen until gene expression analysis. The developmental potential of oocytes was evaluated by cleavage and blastocyst rates at 48 and 168 h post-insemination, respectively. Transcripts for FSH receptor, GH receptor, epidermal growth factor (EGF) receptor, pentraxin 3, and insulin-like growth factor II (IGF-II) were quantified by real-time RT-PCR and normalized by cyclophilin expression. Data from cleavage and blastocyst rates were analyzed by chi-square test. The relative abundance of mRNA for the 5 genes in CC were evaluated by ANOVA and Tukey’s test. Non-parametric test (Wilcoxon) was used when data were not normally distributed. The results are presented as mean ± SEM, and P < 0.05 was considered statistically different. Cleavage and blastocyst rates were higher in oocytes originated from groups 3 (86 and 62%) and 4 (87 and 60%) than those obtained from groups 1 (20%) and 2 (34%). The lowest level of expression for FSH receptor was observed in CC from 1.0–3.0 mm follicles (1.1 ± 0.07) and the greatest in CC from ≥8.1-mm follicles (2.1 ± 0.09), whereas the expression in the intermediary groups 2 (1.6 ± 0.1) and 3 (1.6 ± 0.2) did not show any difference from the other groups. Similar pattern of expression was observed for EFG receptor gene, except that CC from group 3 (1.7 ± 0.2) already presented a significant increase in mRNA level compared to group 1 (0.9 ± 0.1). Relative abundance of transcript for GH receptor rose gradually in CC according to follicular size, being the higher expression detected in the largest follicles and the lower in the smallest follicles, with means of 0.8 ± 0.1, 2.3 ± 0.6, 2.7 ± 0.6, and 3.9 ± 0.1 for groups 1, 2, 3, and 4, respectively. The other 2 candidates (pentraxin 3 and IGF-II) did not show any significant differences related to follicle size. The results indicate that increases in expression of receptor for FSH, EGF, and GH was coincident with the increase in follicle size and in oocyte developmental potential; therefore, they can be used as markers for bovine oocyte competence. However, additional studies are needed to confirm the markers identified. Financial support from EMBRAPA/CAPES.

scholarly journals Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1794
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cumulus Cells ◽  
Intramolecular Interactions ◽  
Developmental Competence ◽  
Embryo Yield ◽  
Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

Approaches to oocyte meiotic arrest in vitro and impact on oocyte developmental competence

2021 ◽  
Author(s):  
Dulama Richani ◽  
Robert B Gilchrist
Keyword(s):  
Protein Synthesis ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Antral Follicle ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Protein Synthesis Inhibitors ◽  
Meiotic Arrest ◽  
Oocyte Developmental Competence

Abstract Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50 years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents which increase intra-oocyte cAMP or prevent its degradation have been predominantly used, however agents such as kinase and protein synthesis inhibitors have also been trialled. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

193 DIFFERENTIAL GENE EXPRESSION IN CUMULUS CELLS OF DEVELOPMENTALLY COMPETENT V. CHALLENGED BOVINE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 195 ◽  
Author(s):  
R. R. Payton ◽  
L. A. Rispoli ◽  
J. L. Edwards
Keyword(s):  
Gene Expression ◽  
Gene Ontology ◽  
Heat Stress ◽  
Empirical Bayes ◽  
Rna Extraction ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Oocyte Competence ◽  
Extracellular Region

It is well established that exposure of cumulus–oocyte complexes (COC) to heat stress during the first 12 h of maturation reduces blastocyst development by 42 to 65%. Previous research supports the notion that some of the effects of heat stress on oocyte competence may be cumulus-mediated. To determine the extent to which this may occur, COC were matured at 38.5°C for 24 h (control) or 41°C for the first 12 h of maturation followed by 38.5°C for remaining 12 h (heat stress). A subset of COC underwent IVF with Percoll-prepared sperm and then was cultured in KSOM containing 0.5% BSA to assess developmental competence. Remaining oocytes were denuded. Cumulus cells, kept separate by treatment, were stored in lysis buffer at –80°C until RNA extraction. Total RNA from cumulus was amplified prior to hybridization to bovine Affymetrix GeneChips (Affymetrix Inc., Santa Clara, CA, USA; n = 8 pools per treatment collected on 8 different occasions; n = 16 chips). Following pre-processing using the MAS5.0 algorithm, microarray data were subjected to linear modeling and empirical Bayes analyses (Bioconductor, Limma package). False discovery rate was controlled using the Benjamini and Hochberg method, and differentially expressed genes were selected by an adjusted P-value (P < 0.05). Functional annotation of selected genes was performed using NetAffx (Affymetrix Inc.) and Database for Annotation, Visualization and Integrated Discovery (DAVID; NIAID, NIH, Bethesda, MD, USA). Heat stress of COC reduced blastocyst development (27.2 v. 16.1% for control v. heat stress, respectively; SEM = 1.6; P < 0.002). Approximately 66 and 65% of 24 000 possible genes were called present (i.e. expressed) in RNA from cumulus of competent (control) v. challenged (heat-stressed) oocytes, respectively. In cumulus from developmentally challenged COC, increased abundance of 42 genes (36 currently annotated) was noted. Use of DAVID demonstrated enrichment of genes important for electron transport and energy generation (NOS2A, MAOB, CYP11A1, HSD11B1L, LTB4DH). Further examination of gene ontology identified genes associated with mitochondrial function (SLC25A10, MAOB, CYP11A1), cell signaling (similar to JAK-3, FSHR, CYP11A1, WNT2B), cytoskeleton (ACTA1), antioxidant activity (GSTA1), and extracellular region (FMOD). In contrast, cumulus from developmentally competent COC had increased expression of 22 genes (20 currently annotated), of which 15% were related to protein binding (CAV1, MMP9, TGFB2) according to DAVID. Further analysis using gene ontology revealed genes associated with extracellular matrix formation (MMP9, MMP19, PCOLCE2) and neural tissue (METRNL). In summary, alterations in cumulus gene expression were associated with differences in developmental competence of oocytes. Additional research is necessary to examine the extent to which identified genes account for functional differences in oocyte competence. This research was supported in part by National Research Initiative Competitive Grant no. 2004-35203-14772 from the USDA Cooperative State Research, Education, and Extension Service.

Sign in / Sign up

Export Citation Format

Share Document