Urea changes oocyte competence and gene expression in resultant bovine embryo in vitro

Zygote ◽  
2018 ◽  
Vol 26 (3) ◽  
pp. 207-219 ◽  
Author(s):  
Rasoul Kowsar ◽  
Fatemeh Izadi ◽  
Nima Sadeghi ◽  
Ahmad Riasi ◽  
Faezeh Ghazvini Zadegan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dairy Cows ◽  
Binding Activity ◽  
Bovine Embryo ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
Elevated Blood Urea Nitrogen ◽  
High Dietary Protein ◽  
Hatching Rates

SummaryNutrition influences the microenvironment in the proximity of oocyte and affects early embryonic development. Elevated blood urea nitrogen, even in healthy dairy cows, is associated with reduced fertility and there is high correlation between blood urea levels and follicular fluid urea levels. Using a docking calculation (in silico), urea showed a favorable binding activity towards the ZP-N domain of ZP3, that of ZP2, and towards the predicted full-length sperm receptor ZP3. Supplementation of oocyte maturation medium with nutrition-related levels of urea (20 or 40 mg/dl as seen in healthy dairy cows fed on low or high dietary protein, respectively) dose-dependently increased: (i) the proportion of oocytes that remained uncleaved; and (ii) oocyte degeneration; and reduced cleavage, blastocyst and hatching rates. High levels of urea induced shrinkage in oocytes, visualised using scanning electron microscopy. Urea downregulated NANOG while dose-dependently upregulating OCT4, DNMT1, and BCL2 expression. Urea at 20 mg/dl induced BAX expression. Using mathematical modelling, the rate of oocyte degeneration was sensitive to urea levels; while cleavage, blastocyst and hatching rates exhibited negative sensitivity. The present data imply a novel role for urea in reducing oocyte competence and changing gene expression in the resultant embryos.

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).

Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1

1991 ◽  
Vol 11 (3) ◽  
pp. 1547-1552
Author(s):  
D Leshkowitz ◽  
M D Walker
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Activity ◽  
Insulin Gene ◽  
Hybrid Cells ◽  
Dna Binding Activity ◽  
Insulin Producing Cells ◽  
Insulin Gene Expression ◽  
Insulinoma Cells

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

scholarly journals A shorter equilibration period in the VitTrans in-straw bovine embryo vitrification method improves post-warming outcomes

2020 ◽  
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario-Hidalgo ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Direct Transfer ◽  
Treatment Groups ◽  
Cell Counts ◽  
Hatching Rates

Abstract Background VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression. Results While survival rates at 3 h and 24 h post-warming were reduced (P < 0.05) after vitrification, hatching rates of D7 embryos vitrified after SE were similar to those obtained in fresh non-vitrified blastocysts. Hatching rates of vitrified D8 blastocysts were lower (P < 0.05) than of fresh controls, regardless of treatment. Total cell counts, and inner cell mass and trophectoderm cell numbers were similar in hatched blastocysts derived from D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values, regardless of treatment. The rate of apoptotic cells was significantly higher in both treatment groups when compared to fresh controls, although apoptosis rates were lower using the SE than LE protocol. No differences emerged in expression of the genes BAX, AQP3, CX43 and IFNτ between blastocysts vitrified after SE or LE, whereas a significantly higher abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE compared to LE. Conclusions The VitTrans device combined with a shorter exposure to the equilibration medium improves vitrification/warming outcomes facilitating the direct transfer of vitrified embryos under field conditions.

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

53 EFFECT OF ADDITION OF L-CARNITINE AND CONJUGATED LINOLEIC ACID DURING BOVINE EMBRYO CULTURE ON CRYOSURVIVAL, LIPID CONTENT, AND GENE EXPRESSION

2015 ◽  
Vol 27 (1) ◽  
pp. 119
Author(s):  
A. Ruiz ◽  
P. J. Hansen ◽  
J. Block
Keyword(s):  
Gene Expression ◽  
Lipid Metabolism ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Lipid Content ◽  
Embryo Culture ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Bovine Embryo

The objective was to determine the effects of addition of l-carnitine (LC) and trans-10, cis-12 conjugated linoleic acid (CLA) during bovine embryo culture on cryosurvival, lipid content, and gene expression. For all experiments, embryos were produced in vitro using abattoir-derived oocytes. Following insemination, presumptive zygotes were randomly assigned in a 2 × 2 factorial to be cultured in SOF-BE1 supplemented with or without 3.03 mM LC and 100 μM CLA until Day 7. For Exp. 1, blastocyst- and expanded-blastocyst-stage embryos (n = 777) were slow-frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. There was no effect of LC on post-thaw re-expansion rates, but CLA reduced (P < 0.05) and tended (P < 0.08) to reduce re-expansion rate at 24 and 48 h, respectively (76.5 ± 2.5 v. 70.4 ± 2.5 and 79.5 ± 2.2 v. 76.0 ± 2.2, respectively). Whereas hatching rate at 72 h tended (P < 0.08) to be higher for embryos cultured with LC (67.8 ± 2.5 v. 74.4 ± 2.5), treatment with CLA reduced (P < 0.05) hatching rate at 48 h (62.3 ± 2.6 v. 54.9 ± 2.6). In Exp. 2, to determine lipid content, expanded blastocyst-stage embryos (n = 263) were harvested and stained using Nile Red. Embryos were examined for fluorescence using an epifluorescence microscope, and intensity of fluorescence per unit area was quantified using ImageJ software (NIH, Bethesda, MD, USA). There was a significant interaction (P < 0.01) between LC and CLA affecting embryo lipid content. Whereas addition of CLA during culture increased lipid, treatment with LC and the combination of LC and CLA reduced lipid (22.8 ± 1.1 v. 19.1 ± 1.1 v. 28.4 ± 1.1 v. 19.2 ± 1.2 for no additive, +LC, +CLA, and +LC and CLA, respectively). For Exp. 3, the effect of LC and CLA on the relative abundance of genes involved in lipid metabolism (ELOVL6, SCD1, SQLE, HMGCS1, CYP51A1, FDPS, FDFT1, LDLR, and SC4MOL) was determined. Pools of 5 expanded blastocyst-stage embryos from each treatment were collected across 5 replicates. The RNA was purified and synthesised into cDNA for RT-qPCR analysis. The SDHA, GAPDH, and YWAZ were used as housekeeping genes. Addition of LC during culture reduced (P < 0.05) the abundance of 4 of the 9 genes analysed (SQLE, HMGCS1, CYP51A1, and FDPS) and tended (P < 0.08) to reduce a fifth (FDFT1). In addition, there was a tendency (P < 0.08) for LC to increase the abundance of SCD1. Addition of CLA during culture had minimal effects on transcript abundance. In particular, CLA treatment reduced (P < 0.01) ELOVL6 and tended (P < 0.08) to increase SCD1. In contrast to previous studies, post-thaw cryosurvival was not significantly improved by treatment with LC or CLA. Results indicate that reduced embryo lipid content caused by LC treatment is due, in part, to an alteration in the abundance of genes involved in lipid metabolism. Further research is still necessary to determine whether in vivo survival following transfer of cryopreserved embryos can be enhanced by treatment with LC or CLA.Support was provided by USDA AFRI Grant 2010–85122–20623.

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Fresh Embryos ◽  
Hatching Rates

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

209 9-cis RETINOIC ACID INHIBITS EXPRESSION OF AKR1B1 TRANSCRIPT IN THE BOVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 252
Author(s):  
G. K. Deb ◽  
S. R. Dey ◽  
K. S. Huque ◽  
M. Fokruzzaman ◽  
K. L. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Embryo Implantation ◽  
Cumulus Cell ◽  
Prostaglandin F2α ◽  
Receptor Expression ◽  
Bovine Embryo

Quantitative real-time PCR has enabled quality evaluation of oocyte and pre-implantation embryo through monitoring expression of several molecular markers that are involved in metabolic activity, stress response, reprogramming, and other biological events. The aldo-keto reductase family 1 member B1 (AKR1B1) transcript is potentially involved in pregnancy failure through metabolism of progesterone and synthesis of prostaglandin F2α in the bovine uterine endometrium. High expression of the transcript in blastocysts correlates inhibition of embryo implantation and/or embryo resorption. Maturation of immature oocyte in presence of 9-cis retinoic acid (9-cis RA) increases in vitro bovine embryo development rates and embryo quality. These beneficial effects of 9-cis RA are mediated through multiple mechanisms, including FSH/LH receptor expression, polyadenylation, growth factor signalling, oxidative-stress protection, or decreasing oocyte TNFα gene expression and inhibiting cumulus cell apoptosis during maturation. The present study aimed to evaluate the effect of 9-cis RA on expression pattern of AKR1B1 transcript in the oocyte matured in vitro and embryos (8-cell and Day 8 blastocyst) produced from in vitro matured oocytes in presence or absence of 9-cis RA. Bovine cumulus–oocyte complexes, isolated from ovaries collected at the abattoir, were matured in vitro in the presence of zero (control) or 5 nM 9-cis RA in the maturation medium (TCM199 + 10% fetal bovine serum + 1 µg mL–1 β-oestradiol + 10 µg mL–1 follicle stimulating hormone + 0.6 mM cystein and 0.2 mM Na-pyruvate). After maturation, the oocytes were subjected to standardized in vitro embryo production protocol or oocyte samples were collected for gene expression analysis. The expression of AKR1B1 transcript was quantified in zona-free oocytes, 8-cell embryos, and Day 8 blastocysts by real-time PCR using SYBER green. Not less than 4 biological replicates (oocytes: 50 to 60 per replicate and 8-cell embryos/day-8 blastocyst: 3 to 5 per replicate) were done for each group. The expression was normalized against a minimum of 2 out of 4 reference transcripts (18S rRNA, β-actin, glyceraldehyde-3-phosphate dehydrogenase and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) analysed each time with AKR1B1. The best combination of reference genes was automatically calculated by the CFX manager V1.1 program (Bio-Rad) based on M-value. The differences in gene expression levels were tested by Student’s t-test. Results indicated that 9-cis RA decreased expression of AKR1B1 transcript in the oocyte (1.0- v. 2.0-fold; P < 0.05), 8-cell-embryos (1.0- v. 10.1-fold; P < 0.03), and blastocyst (1.0- v. 2.1-fold; P < 0.03) compared with control. In conclusion, the present study indicates that 9-cis RA inhibits AKR1B1 transcript expression in oocytes and pre-implantation embryos.

291 NOVEL OOCYTE SHIPPING AND MATURATION MEDIUM WITHOUT CO2 GAS PHASE IMPROVES IN VITRO MATURATION AND PREGNANCY OUTCOME IN CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 234
Author(s):  
M. Barcelo-Fimbres ◽  
L. F. Campos-Chillon ◽  
N. R. Mtango ◽  
L. Bonilla ◽  
J. Verstegen
Keyword(s):  
Embryonic Development ◽  
Pregnancy Rates ◽  
Control Group ◽  
System Control ◽  
Co2 Gas ◽  
Oocyte Competence ◽  
N2 Atmosphere ◽  
Maturation Medium ◽  
Medium System

The aim of the present work was to evaluate embryonic development after shipping and maturation of bovine cumulus oocyte complexes (COC) collected by ovum pick up (OPU) in medium (SMM) that does not require CO2 gas for transport and maturation. Two experiments were conducted, experiment 1 stimulated nonlactating Holstein (n = 4), Jersey (n = 2), Angus (n = 4), and Wagyu (n = 2) donors with 6 pFSH injections (Pluset, MOFA Global LLC, Verona, WI, USA) were used. From each donor, some OPU sessions were delivered the same day (~3 h after collection) for IVM in conventional gas bicarbonate-equilibrated medium system (control), while COC from the other sessions were placed in a portable incubator at 38.5°C, delivered the next day allowing 24 h of maturation in SMM (BoviPro, Mofa Global, WI, USA). The COC were fertilized using commercial semen for each breed, and embryos were cultured in BBH7 medium (BoviPro, Mofa Global, WI, USA) at 38.5°C in 5% O2, 5% CO2, and 90% N2 atmosphere. Embryonic development was evaluated in this experiment. For experiment 2, Day 7 fresh Holstein and Jersey embryos (n = 610) from SMM (n = 550) and controls (n = 60) were transferred in synchronized virgin heifers and pregnancies were diagnosed by ultrasonography at d 35. Data were analysed by ANOVA using GLM, percentages were transformed using arcsin square root, and pregnancy rates were analysed by GenMod using SAS statistical software (Cary, NC, USA). Similar COC numbers were recovered for maturation treatments (P > 0.1; Table 1). The COC matured in SMM had higher cleavage and blastocyst rates than the control group (P < 0.01; Table 1), and this resulted in more transferable embryos per OPU session (P < 0.05; Table 1). We did not find breeds effects or interactions for any variable (P > 0.1; Table 1). After ET, SMM had similar pregnancy rates than control (53.8 v. 58.3%; P > 0.1); however, as more blastocysts were produced per OPU session in the SMM condition, more pregnancies were obtained per session (4.3 v. 2.1; P < 0.01). We conclude that COC matured in SMM had greater oocyte competence than control in commercial settings. The SMM resulted in greater embryonic development, similar pregnancy rates, but more transferable embryos and pregnancies per OPU session than the conventional maturation system. Table 1.Least squares means (± SE) of embryonic development of COC matured in SMM or control

345 GENE EXPRESSION IN OOCYTES AFTER MEIOTIC INHIBITION WITH THE CYCLIN-DEPENDENT KINASE INHIBITOR BUTYROLACTONE I

2010 ◽  
Vol 22 (1) ◽  
pp. 329
Author(s):  
C. L. V. Leal ◽  
S. Mamo ◽  
T. Fair ◽  
P. Lonergan
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Relative Abundance ◽  
Kinase Inhibitor ◽  
Expression Profiles ◽  
Cyclin Dependent Kinase ◽  
Oocyte Competence ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Bovine Oocytes

Once removed from the follicle, mammalian oocytes resume meiosis spontaneously and progress through breakdown of the germinal vesicle to the matured state at metaphase II. The ability to reversibly inhibit such meiotic resumption has been reported and is a potentially useful method for studying developmental competence acquisition in oocytes as well as in some cases allowing flexibility in an IVF system where oocytes are collected from distant locations or on different days. The aim of the present study was to determine the effect of temporary inhibition of meiotic resumption using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes. Immature bovine oocytes were recovered from the ovaries of slaughtered heifers at a commercial abattoir and assigned to 1 of 4 groups: (1) Control: immature oocytes were collected either immediately or (2) after IVM for 24 h in TCM-199 containing 10 ng mL-1 EGF and 10% (v/v) FCS, (3) Inhibited oocytes collected either 24 h after incubation in the presence of 100 μM BLI in TCM-199 with 3 mg mL-1 BSA or (4) after meiotic inhibition for 24 h followed by in vitro maturation. All cultures were carried out at 38.5°C under 5% CO2 in air and maximum humidity. For mRNA relative abundance analysis, cumulus cells were removed and pools of 10 denuded oocytes were snap frozen in liquid nitrogen and stored at -80°C until use. A total of 42 transcripts, previously reported to be related to cell cycle regulation and/or oocyte competence were evaluated by quantitative real time PCR. Differences in relative abundance were analyzed by ANOVA and Student’s t-test. The majority of transcripts were downregulated (P < 0.05) after IVM in control oocytes (23 out of 42) and the same pattern was observed in inhibited oocytes that were allowed to mature. Twelve transcripts remained stable (P > 0.05) after IVM in control oocytes; of these, only two (PTTG1 and INHBA) did not show the same pattern in inhibited and matured oocytes. Few genes (7) were upregulated after IVM in control oocytes (P < 0.05) and of these, three (PLAT1, RBP1, and INHBB) were not upregulated in inhibited oocytes after IVM. Inhibited oocytes showed similar levels of expression (P > 0.05) as immature control oocytes, except for two genes (LUM and INHBB), which were increased in these oocytes (P < 0.05). The expression profiles of cell cycle genes were mostly unaffected by the BLI treatment. The few genes affected were previously reported as competence-related and could be useful markers of oocyte competence following pretreatment. In conclusion, the changes occurring in transcript abundance during oocyte maturation in vitro were to a large extent mirrored following inhibition of meiotic resumption prior to IVM and subsequent release from inhibition and maturation. CLV Leal was supported by CNPq, Brazil (PDE 201487/2007-1); Supported by Science Foundation Ireland (07/SRC/B1156).

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