Gene expression profile of cumulus cells derived from cumulus - oocyte complexes matured either in vivo or in vitro

2009 ◽  
Vol 21 (3) ◽  
pp. 451 ◽  
Author(s):  
Dawit Tesfaye ◽  
Nasser Ghanem ◽  
Fiona Carter ◽  
Trudee Fair ◽  
Marc-André Sirard ◽  
...  
Keyword(s):  
Transcript Abundance ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Lh Surge ◽  
Expected Time ◽  
Custom Made ◽  
Developmental Capacity ◽  
Transcript Profiles

Although it is well established that maturation conditions have a clear influence on oocyte developmental competence, it is not known whether this could be due to downstream effects of perturbation of the transcript profile of the oocyte’s adjacent cumulus cells. Therefore, the aim of the present study was to compare the transcript profiles of cumulus cells derived from cumulus–oocyte complexes (COCs) matured in vitro or in vivo. Using a previously validated combined synchronisation and superstimulation protocol, COCs were recovered from beef heifer ovaries just before the expected time of the LH surge and matured in vitro, while in vivo-matured COCs were recovered just before ovulation (20 h after the LH surge). A custom-made cDNA microarray containing 2278 granulosa/cumulus transcripts was used for target and dye-swap hybridisations. In all, 64 genes were differentially expressed between the two groups. Transcript abundance of key genes associated with cumulus expansion (TNFAIP6) and regulation of oocyte maturation (INHBA and FST) were upregulated in in vivo-derived cumulus cells. However, cumulus cells derived from IVM COCs were enriched with genes involved in response to stress (HSPA5 and HSP90AB1). Quantitative real-time polymerase chain reaction confirmed the array results for eight of 10 genes selected for validation. The data presented here reveal that differences in oocyte developmental capacity after maturation in vitro or in vivo are accompanied by distinct differences in transcript abundance of the surrounding cumulus cells.

scholarly journals Features of the preparation of biological material for genome editing in cattle

2019 ◽  
Vol 191 (12) ◽  
pp. 40-44
Author(s):  
A. Barkova ◽  
M. Modorov ◽  
G. Isaeva ◽  
A. Krivonogova
Keyword(s):  
Genome Editing ◽  
Biological Material ◽  
Genetic Material ◽  
Cumulus Cells ◽  
Vitro Fertilization ◽  
Expected Time ◽  
Donor Material ◽  
Material Transportation

Abstract. To carry out genome editing in cattle, an effective and well-functioning system for obtaining gametes, fertilizing eggs and their cryopreservation is necessary. Aim of the work: review and research of present-day existing methods of obtaining, insemination and cryopreservation of donor material, in order to provide genome editing in cows. Methods and materials. The work is completed according to the theme No. 0532-2019-0001 “Development of complex technology of marker-based genome selection of agricultural animals” within State Order of Ministry of Education and Science of the Russian Federation. The analysis of open scientific literature on the issues of in vitro fertilization in animals, cryopreservation of oocytes and embryons, sperm preparation and methods of insemination of cows’ oocytes, and cryopreservation of oocytes and embryons of animals is done. Features of the preparation of biological material of cattle for genome editing by microinjection into ooplasm are described. Results of research and duscussion. At present time there are two ways to obtain donor material from cattle: from live animals and taking ovaries after slaughtering cows. Material transportation is carried out at a temperature of 30–37 °C depending on the distance to the laboratory and expected time period of transportation. Oocyte-cumulus complexes can be removed by ovarian dissection and aspiration of visible follicles. In both cases, immature eggs are predominantly obtained. Subsequent ripening is carried out in vitro using special media in a CO2 incubator. The culture medium for oocyte maturation should contain hormones that mimic the peak of LH (luteinizing hormone), which occurs in vivo during the maturation of oocytes before ovulation. To accumulate a certain number of eggs at the stage of MII, it is recommended to carry out their cryopreservation by the method of vitrification, having previously released the oocyte from the cumulus cells. After thawing, oocytes need to be incubated for 2–3 hours 38.5 °C in 5–6.5% CO2 to restore the spindle. In order to make editing more effective, the introduction of genetic material is recommended to be carried out in parallel with the fertilization method “icsi”. In humans, mice and rabbits, an injection of sperm into the cytoplasm is sufficient to activate the oocyte, however, in cattle, just micro-injection of the sperm is not enough and often the male pronucleus does not form. To solve the problem, various methods are used, including freezing-thawing of sperm, resulting in damage of a membrane, or addition of heparin-glutathione into the medium that increases decondensation of the sperm DNA.

195 ALTERED mRNA TRANSCRIPT EXPRESSION OF IN VITRO- VERSUS IN VIVO-MATURED PORCINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 210
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
K. M. Whitworth ◽  
W. G. Spollen ◽  
S. M. Blake ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Real Time ◽  
Real Time Pcr ◽  
Genetic Background ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Illumina Genome Analyzer ◽  
Similar Genetic Background

In contrast to oocytes matured in vitro, porcine embryos that result from in vivo maturation and fertilization have a high developmental competence and readily make the transition from oocyte to blastocyst. This observation led us to investigate the transcript profile differences between in vivo- and in vitro-matured porcine oocytes. For the in vivo-matured group, oviducts of 3 gilts of similar genetic background were flushed 2 days after detection of standing oestrus. MII oocytes were collected in pools of 10 and snap frozen in liquid nitrogen for RNA isolation. The in vitro-matured oocytes were obtained by euthanizing 3 gilts, again with a similar genetic background and recovering the ovaries. Follicles (2 to 8 mm in size) were aspirated and oocytes with multiple layers of cumulus cells and uniform cytoplasm were placed in M-199 supplemented with LH, FSH and epidermal growth factor for 42 h. Upon maturation, cumulus cells were stripped and the healthy MII oocytes were collected in pools of 10 and snap frozen. Total RNA was extracted from 3 pools of 10 oocytes for both treatments using an All prep DNA/RNA micro isolation kit (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized using oligo (dT′) primed reverse transcriptase with superscript III (Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was synthesized using DNA polymerase I and sequenced using Illumina Genome Analyzer II. All reads were aligned to a custom-built porcine transcriptome. There were over 18 million reads in the 2 maturation groups that tiled to the 34 433-member transcriptome: 1317 transcripts were detected with a P ≤ 0.1 (Students t-test), a minimum of 7 reads in at least 1 of the treatments and ≥2-fold difference. Real-time PCR was used on selected transcripts. Comparative CT Method was used on an IQ real-time PCR system with the Bio–Rad SYBR green mix. Statistical differences were determined using the Proc general linear model procedure of SAS (SAS Institute Inc., Cary, NC) and means separated with a l.s.d. (P ≤ 0.05). The misrepresented transcripts from the sequencing data were also characterized using the functional annotation tool DAVID. Twelve pathways were overrepresented in the in vitro-matured oocytes (the top 4 are pathways to cancer, spliceosome, cell cycle and ubiquitin-mediated proteolysis). Eight pathways were underrepresented in the in vitro-matured oocytes (the top 4 are cytoskeleton regulation, T-cell receptor signaling pathway, ubiquitin-mediated proteolysis and cell cycle). Eight transcripts were selected for real-time PCR. ZP2 was higher in the in vitro-matured oocytes as determined by both sequencing and real time. ATG4, HSP90, UBAP2 and SOX4 were not different, regardless of assay. SLC7A3, MRPS36 and PDHX2 were not different based on sequencing, but based on real-time MRPS36 and PDHX2, were higher in the in vivo group and SLC7A3 was higher in the in vitro group. In conclusion, there is an abundance of misregulated transcripts and altered pathways in in vitro-matured oocytes. This dataset is a tool that may provide clues to improve the in vitro maturation process so that in vitro-matured oocytes will be more like their in vivo-matured counterparts, thus improving developmental competence. Funded by Food for the 21st Century.

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

scholarly journals In vivo oocyte developmental competence is reduced in lean but not in obese superovulated dairy cows after intraovarian administration of IGF1

Reproduction ◽  
2011 ◽  
Vol 142 (1) ◽  
pp. 41-52 ◽  
Author(s):  
Miguel A Velazquez ◽  
Klaus-Gerd Hadeler ◽  
Doris Herrmann ◽  
Wilfried A Kues ◽  
Susanne Ulbrich ◽  
...  
Keyword(s):  
Protein Expression ◽  
Dairy Cows ◽  
Polycystic Ovary ◽  
Plasma Concentrations ◽  
Transcript Abundance ◽  
Developmental Competence ◽  
Obese Women ◽  
Embryo Viability

The present study investigated the role of IGF1 in lactating lean and non-lactating obese dairy cows by injecting 1 μg IGF1 into the ovaries prior to superovulation. This amount of IGF1 has been linked with pregnancy loss in women with the polycystic ovary syndrome (PCOS) and was associated with impaired bovine oocyte competence in vitro. Transcript abundance and protein expression of selected genes involved in apoptosis, glucose metabolism, and the IGF system were analyzed. Plasma concentrations of IGF1 and leptin, and IGF1 in uterine luminal fluid (ULF), were also measured. IGF1 treatment decreased embryo viability in lean cows to the levels observed in obese cows. Obese cows were not affected by IGF1 treatment and showed elevated levels of IGF1 (in both plasma and ULF) and leptin. Blastocysts from lean cows treated with IGF1 showed a higher abundance of SLC2A1 and IGFBP3 transcripts. IGF1 treatment reduced protein expression of tumor protein 53 in blastocysts of lean cows, whereas the opposite was observed in obese cows. IGF1 in plasma and ULF was correlated only in the control groups. Blastocyst transcript abundance of IGF1 receptor and IGFBP3 correlated positively with IGF1 concentrations in both plasma and ULF in lean cows. The detrimental microenvironment created by IGF1 injection in lean cows and the lack of effect in obese cows resemble to a certain extent the situation observed in PCOS patients, where IGF1 bioavailability is increased in normal-weight women but reduced in obese women, suggesting that this bovine model could be useful for studying IGF1 involvement in PCOS.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

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

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

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

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

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

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

scholarly journals Histone deacetylase inhibitor during in vitro maturation decreases developmental capacity of bovine oocytes

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0247518
Author(s):  
Thais Preisser Pontelo ◽  
Mauricio Machaim Franco ◽  
Taynan Stonoga Kawamoto ◽  
Felippe Manoel Costa Caixeta ◽  
Ligiane de Oliveira Leme ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Embryo Quality ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Nuclear Maturation ◽  
Deacetylase Inhibitor ◽  
Developmental Capacity ◽  
Bovine Oocytes

This study aimed to evaluate the effect of scriptaid during pre-maturation (PIVM) and/or maturation (IVM) on developmental competence of bovine oocytes. Cumulus-oocyte complexes (COCs) were submitted to PIVM for 6 h in the presence or absence of scriptaid. COCs were distributed into five groups: T1-IVM for 22 h, T2-PIVM for 6 h and IVM for 22 h, T3-PIVM with scriptaid for 6 h and IVM for 22 h, T4-PIVM for 6 h and IVM with scriptaid for 22 h, and T5-PIVM with scriptaid for 6 h and IVM with scriptaid for 22 h. Nuclear maturation, gene expression, cumulus cells (CCs) expansion, and embryo development and quality were evaluated. At the end of maturation, all groups presented the majority of oocytes in MII (P>0.05). Only HAT1 gene was differentially expressed (P<0.01) in oocytes with different treatments. Regarding embryo development at D7, T4 (23%) and T5 (18%) had lower blastocyst rate (P<0.05) than the other treatments (T1 = 35%, T2 = 37% and T3 = 32%). No effect was observed when scriptaid in PIVM was used in less competent oocytes (P>0.05). In conclusion, presence of scriptaid in PIVM and/or IVM did not improve developmental competence or embryo quality.

333 IN VIVO PREMATURATION AFFECTS PROTEIN SYNTHESIS AT THE BEGINNING OF IN VITRO MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 274
Author(s):  
H. M. Knijn ◽  
P. L. A. M. Vos ◽  
S. J. Dieleman ◽  
P. J. M. Hendriksen
Keyword(s):  
Protein Synthesis ◽  
Imaging System ◽  
Cumulus Cells ◽  
Ultrastructural Level ◽  
Preovulatory Follicle ◽  
Igf Binding Proteins ◽  
Lh Surge ◽  
Oocyte Competence

The cumulus–oocyte complex (COC) from a preovulatory follicle is better equipped than a COC from a small healthy follicle to develop into a blastocyst. This might be due to the presence of mRNAs and proteins in the oocyte and/or cumulus cells from which the expression is increased or induced during the prematuration period. There are several reports that beginning atresia enhances in vitro oocyte competence and the processes of atresia show similarities with prematuration on an ultrastructural level. Therefore, we investigated the effect of in vivo prematuration and atresia on the protein synthesis in bovine oocytes collected from preovulatory and small follicles during the first period of IVM. To study the follicular population of an undisturbed cycle we synchronized 14 cows with a norgestomet ear implant. We performed progesterone and LH analysis (Hendriksen et al. 2003 Biol. Reprod. 69, 2036–2044) to determine the start of luteolysis and the LH surge. At 48–62 h after the start of luteolysis, 10 cows were ovariectomized (OVX) and were considered to be shortly before the LH surge. At 1.5–3 days after the LH surge, at the beginning of a new cycle, four cows were OVX. From the ovaries collected before the LH surge the preovulatory follicle (POF) and all follicles ≥3 mm were dissected. From the ovaries collected at the start of a new cycle all follicles ≥3 mm were dissected. All follicles were classified for degree of atresia as non-atretic (NA), light atretic (LA), and atretic (A) by the proportion of apoptotic granulosa cells and levels of estradiol and progesterone, and of small IGF-binding proteins (IGFBPs) in the follicular fluid (Hendriksen et al. 2003). The COCs were individually incubated for 4 h in maturation medium containing 35S-labeled methionine/cysteine (Amersham-Pharmacia, Sweden). After separation of oocyte and cumulus cells, the proteins produced in the oocyte were analyzed by 1-D electrophoresis (SDS-PAGE, ExcelGel-12.5%, Amersham-Pharmacia). Gels were dried and digitized images were obtained by use of a Phosphor Imaging System (Molecular Imager, Bio-Rad, Hercules, CA, USA). Twenty-eight different proteins were distinguished. To compare different oocytes, the 35S incorporation per protein band was calculated as proportion of the 35S incorporation of all bands of the oocyte. The total amount of newly synthetized proteins per oocyte was normalized to the oocyte on the gel with the highest incorporation of 35S. In all POF oocytes (n = 7), the total amount of newly synthetized protein was lower than that in the oocytes from the NA and LA follicles (Table 1). This suggests that already during in vivo prematuration new proteins were synthesized in POFs and therefore less protein synthesis occurs during maturation. We can speculate that oocytes from small follicles have to synthesize many proteins during the beginning of IVM to make up for what is normally produced during the prematuration period. In atretic oocytes fewer proteins were synthesized, which suggests that these oocytes have either undergone prematuration-like processes or are less viable. Nevertheless, some proteins remain to be synthesized in POFs, such as, for example, protein number ‘p 23’. Table 1. New proteins at the onset of IVM in oocytes from small follicles and POFs

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

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

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

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