261 INFLUENCE OF IN VITRO MATURATION ON EPIGENETIC MARKS AND GENE EXPRESSION IN BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 228
Author(s):  
J. Heinzmann ◽  
T. Hansmann ◽  
C. Wrenzycki ◽  
U. Zechner ◽  
T. Haaf ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Embryonic Development ◽  
Quantitative Pcr ◽  
Assisted Reproduction ◽  
In Vitro Maturation ◽  
Imprinted Genes ◽  
Assisted Reproduction Technology ◽  
Bovine Oocytes

In cattle, in vitro maturation (IVM) of oocytes is an integral part of assisted reproduction technology. However, only 30% of in vitro matured bovine oocytes develop to the blastocyst stage after fertilization (compared with 60% for in vivo matured oocytes), indicating critical involvement of maturation conditions in the developmental competence of oocytes. Oocytes for IVF and intracytoplasmic sperm injection in humans are typically allowed to mature in vivo after superovulation because IVM is not considered to be a safe medical procedure. Several studies have shown that assisted reproduction technology involving prolonged in vitro culture of human and ruminant embryos can be associated with increased risk of fetal or placental abnormalities due to aberrant DNA methylation of imprinted and non-imprinted genes. Similarities between the bovine large offspring syndrome and imprinting-related human Beckwith–Wiedemann syndrome and the general similarity of bovine and human pre-implantation development make bovine oocyte maturation and embryonic development an increasingly accepted model of human development. Differentially methylated regions and imprinting control regions for the bovine paternally imprinted gene H19 and the maternally imprinted genes PEG3 and SNRPN were identified and characterised in this study. The DNA methylation profiles of bovine oocytes could be determined by bisulfite treatment of DNA from pools of 10 oocytes, but no significant differences were observed between IVM in TCM medium with 20% O2, in SOF medium with 5% O2, or after in vivo maturation. In contrast, quantitative PCR analysis of single oocyte preparations (n ≥ 8) revealed significant differences between these groups in the expression of the 3 genes. We then investigated the expression of genes involved in other critical processes in the developing oocyte and early embryo by quantitative PCR, including SLC2A8 (glucose transport), GDF9 (growth factor), PRDX1 (antioxidant and intercellular communication), DNMT1a/b (maintenance of methylation), and DNMT3a/b (de novo methylation). We also studied IGF2R, an imprinted gene implicated in large offspring syndrome. We observed significant differences in the expression of several of these genes. Interestingly, the expression of DNMT3a and DNMT3b was significantly higher in in vitro matured oocytes than in in vivo matured oocytes and could result in the above-mentioned aberrant methylation patterns established later in embryonic development. This work was funded by DFG (FOR1041).

114 Effect of in vivo heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 183
Author(s):  
F. A. Diaz ◽  
E. J. Gutierrez ◽  
B. A. Foster ◽  
P. T. Hardin ◽  
K. R. Bondioli
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Dna Hydroxymethylation ◽  
Oocyte Collection ◽  
Grade 3 ◽  
Bovine Oocytes

Cattle under the effect of heat stress have reduced fertility, with negative effects on the oocyte observed at the morphological, biochemical, transcriptional and developmental levels. There are no studies evaluating the effect of heat stress on the epigenetic profile of bovine oocytes, which plays a fundamental role in the regulation of gamete development. The objective of this study was to evaluate the effect of in vivo heat stress during the spring to summer transition on DNA methylation and DNA hydroxymethylation of bovine oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Ten Bos taurus crossbred nonlactating beef cows located at Saint Gabriel, Louisiana, USA (30°16′11.1″ N, 91°06′12.1″ W), were used for oocyte collection once monthly from April to August. Dominant follicle removal was performed 5-7 days before oocyte collection. Cumulus-oocyte complexes were collected through ovum pick-up from follicles >2mm. Germinal vesicle (GV)-stage oocytes (50% of total obtained per cow) were subjected to a standard bovine in vitro maturation protocol to obtain metaphase II (MII) stage oocytes. The DNA methylation and DNA hydroxymethylation of GV and MII oocytes was assessed by fluorescence immunohistochemistry utilising primary antibodies against 5′-methylcytosine and 5′-hydromethylcytosine. Secondary antibodies utilised were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG. Oocytes were visualised utilising a fluorescence deconvolution microscope and immunofluorescence data were expressed as corrected relative fluorescence per nucleus. The polar body was not included for fluorescence quantification when evaluating MII stage oocytes. Results (least squares means±standard error) were evaluated as cold months (April and May) and hot months (June, July, and August). Results were analysed by the type III test of fixed effects and Tukey media separation utilising Proc Glimmix of SAS 9.4 (P<0.05; SAS Institute Inc., Cary, NC, USA). Maturation rates and percent of grade 1, grade 2, and grade 3 oocytes were square root arcsine transformed for statistical analysis. The number of total oocytes obtained per cow was higher in cold compared to hot months (21.88±2.34 and 14.23±2.17, respectively). Percent of grade-1 oocytes was higher in cold compared to hot months (38.25±3.69 and 27.59±3.09, respectively). There was no difference in percent of grade-2 oocytes between cold and hot months (21.80±2.44 and 22.60±2.20, respectively). There was a lower percent of grade-3 oocytes in cold compared to hot months (39.82±4.54 and 55.87±3.98, respectively). Maturation rate (in vitro maturation) was not different between cold and hot months (81.92±4.04 and 91.11±3.36, respectively). There was no difference between cold and hot months in DNA methylation (417,218.90±71,793.86 and 313,819.88±55,528.01, respectively) and DNA hydroxymethylation (444,931.10±67,920.78 and 352,254.68±56,425.96, respectively) of GV-stage oocytes. There was no difference between cold and hot months in DNA methylation (87,122.36±14,449.47 and 89,807.26±11,303.72 AU, respectively) and DNA hydroxymethylation (102,933.83±15,517.70 and 137,622.45±11,826.86 AU, respectively) of MII-stage oocytes.

In Vitro Maturation of Bovine Oocytes with Bovine Herpesvirus 5 Does Not Compromise the Subsequent Embryonic Development.

2009 ◽  
Vol 81 (Suppl_1) ◽  
pp. 227-227
Author(s):  
Camila Silva ◽  
Alicio Martins ◽  
Renata Sanches Calegari ◽  
Marcelo Carnelli Frade ◽  
Diego Gouvea Souza ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Bovine Herpesvirus ◽  
Bovine Oocytes

270 IN VITRO MATURATION ALTERS THE EXPRESSION OF SPECIFIC PUTATIVELY IMPRINTED GENES IN BOVINE MII OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 251
Author(s):  
M. G. Katz-Jaffe ◽  
B. R. McCallie ◽  
K. Preis ◽  
G. E. Seidel ◽  
D. K. Gardner
Keyword(s):  
Genomic Imprinting ◽  
In Vitro Maturation ◽  
Expression Profiles ◽  
Housekeeping Gene ◽  
Control Individual ◽  
Imprinted Genes ◽  
Parental Allele ◽  
Regulation Of Growth

Genomic imprinting is an epigenetic form of gene regulation resulting in only one parental allele being expressed. Imprinted genes have diverse functions including the regulation of growth and development in mammals. Errors in genomic imprinting have been associated with human disease (e.g. Beckwith-Wiedemann Syndrome) and large offspring syndrome of in vitro-produced ruminant fetuses. The aim of this study was to investigate the effect of in vitro maturation (IVM) on the expression of specific putatively imprinted genes in the bovine oocyte. Cumulus-enclosed immature oocytes were collected from cattle after 6 injections of 50 mg FSH with transvaginal aspiration (TVA) performed 48 h post-final FSH injection. The oocytes were cultured in groups of 10 for 23 h in a defined maturation medium (G-Mat) with 5 mg of HSA and 100 ng mL-1 of epidermal growth factor (EGF; group A) or with 20% serum and 100 ng mL-1 of EGF (group B) at 38.5�C in 6% CO2 in air. In vivo-matured oocytes (group C) were also collected via TVA after the administration of 6 FSH injections (50 mg), prostaglandin (PG) with last FSH injection, and GnRH 37 h post-PG. Total RNA was extracted from individual MII oocytes in all 3 groups, and expression profiles of putatively imprinted genes (Igf2r, Peg3, and Snrpn) were assayed by real-time PCR (Roche Applied Biosciences, Indianapolis, IN, USA), relative to the housekeeping gene GAPDH. Statistical analysis of expression profiles was performed using REST software. Expression of the Igf2r and Peg3 putatively imprinted genes was significantly up-regulated in individual in vitro-matured MII oocytes (groups A and B, n = 5 replicates per group) when compared with control, individual in vivo-matured MII oocytes (group C, n = 5 replicates; P < 0.05). Gene expression did not differ between in vitro- and in vivo-matured MII oocytes for the putatively imprinted gene, Snrpn. In conclusion, following in vitro maturation of bovine oocytes, the putatively imprinted genes Igf2r and Peg3 were aberrantly expressed in individual oocytes relative to in vivo controls. Both of these putatively imprinted genes have been implicated in the regulation of growth and apoptotic pathways during mammalian development. Analysis of such putatively imprinted genes will facilitate the development of more suitable oocyte maturation conditions. This research was supported by the Serono Research Institute.

scholarly journals Differential gene expression between in vivo and in vitro maturation: a comparative study with bovine oocytes derived from the same donor pool

2019 ◽  
Author(s):  
Luiz Sergio Almeida Camargo ◽  
Michele Munk ◽  
Jose Nelio Sales ◽  
Sabine Wohlres-Viana ◽  
Carolina Capobiango Romano Quintão ◽  
...  
Keyword(s):  
Gene Expression ◽  
Comparative Study ◽  
Differential Gene Expression ◽  
In Vitro Maturation ◽  
Donor Pool ◽  
Differential Gene ◽  
Bovine Oocytes

scholarly journals IN VITRO MATURATION OF BOVINE OOCYTES FOR IN VITRO FERTILIZATION

2009 ◽  
Vol 15 (1) ◽  
pp. 16-19
Author(s):  
Ioan GROZA ◽  
Simona CIUPE ◽  
Mihai CENARIU ◽  
Emoke PALL ◽  
Anamaria PETREAN
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
In Vitro Maturation ◽  
Cultivation Conditions ◽  
Vitro Fertilization ◽  
Morphological Evaluation ◽  
17Β Estradiol ◽  
Bovine Oocytes

The objective of the present study was to asses the quality of various cultivation media used for the maturation of bovine oocytes that are prepared for IVF. Upon collection from slaughtered bovine ovaries and after morphological evaluation, a total number of 513 viable oocytes have been selected for cultivation, being divided into 3 batches, 171 oocytes / batch. The oocytes belonging to batch 1 were cultivated in TCM 199 NaHCO3 + 10% FCS + FSH 20 μl/ml. The oocytes belonging to batch 2 were cultivated in TCM 199 NaHCO3 + 10% FCS + HCG 2.3 x 103 UI/ml + FSH 8 μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml. The oocytes belonging to batch 3 were cultivated in TCM 199 NaHCO3 + 10% FCS + 17β estradiol 1 μl/ml + FSH 20 μl/ml. The cultivation conditions, for all three batches, were: 24 hours at 39°C, 5% CO2. Spermatozoa have been prepared using the Percoll method and IVF of the matured oocytes has been performed. Embryonic development has been assessed 72 hours and then up to 10 days after IVF. The results showed the superior quality of the oocytes belonging to batch 2 and matured using TCM 199 NaHCO3 + 10% FCS + HCG 2.3x103 UI/ml + FSH 8 μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml, as their use for IVF yielded the highest number of viable embryos.

scholarly journals Early embryonic development of bovine oocytes challenged with LPS in vitro or in vivo

Reproduction ◽  
2019 ◽  
Vol 158 (5) ◽  
pp. 453-463
Author(s):  
Joao Alveiro Alvarado Rincón ◽  
Patricia Carvalho Gindri ◽  
Bruna Mion ◽  
Ferronato Giuliana de Ávila ◽  
Antônio Amaral Barbosa ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Lps Challenge ◽  
Bovine Oocytes

The aim of this study was to evaluate the effect of exposing bovine oocytes to lipopolysaccharides (LPS) in vivo and in vitro on early embryo development. In experiment 1, cumulus oocyte complexes (COCs, n = 700/group) were challenged with 0, 0.1, 1.0 or 5.0 μg/mL of LPS during in vitro maturation (IVM). Later, in vitro fertilization (IVF) and in vitro culture (IVC) were performed. In experiment 2, COCs (n = 200/group) matured and in vitro fertilized without LPS were subjected to IVC with the same doses of LPS from experiment 1. In experiment 3, heifers received two injections of saline solution (n = 8) or 0.5 μg/kg of LPS (n = 8) 24 h apart, and 3 days later, COCs were recovered and submitted to IVM, IVF, and IVC. In experiments 1 and 3, the expression of TLR4, TNF, AREG and EREG genes in cumulus cells was evaluated. Exposure to 1 and 5 μg/mL of LPS during IVM decreased nuclear maturation (39.4 and 39.6%, respectively) compared with control (63.6%, P < 0.05). Despite that, no effect on cleavage and blastocyst rates were observed. Exposure to LPS during IVC did not affect embryonic development. In vivo exposure to LPS decreased the in vitro cleavage rate (54.3 vs 70.2%, P = 0.032), but cleaved embryos developed normally. Number of cells per embryo and gene expression were not affected by the LPS challenge in any experiment. In conclusion, although in vitro exposure to LPS did not affect early embryo development, in vivo LPS exposure reduced cleavage rate.

115 ADDITION OF ETHANOL AT SUB-STRESS CONCENTRATIONS DURING IN VITRO MATURATION OF BOVINE OOCYTES IMPROVES BLASTOCYST CRYOTOLERANCE

2010 ◽  
Vol 22 (1) ◽  
pp. 216 ◽  
Author(s):  
M. A. M. M. Shehab-El-Deen ◽  
J. L. M. R. Leroy ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
In Vitro Maturation ◽  
Embryo Quality ◽  
Calf Serum ◽  
Ethanol Stress ◽  
Stress Concentrations ◽  
Low Concentrations ◽  
Carry Over ◽  
Bovine Oocytes

In in vitro experiments, the percentage of bovine oocytes that develops to the blastocyst stage is much lower compared with the in vivo counterparts. The quality of the oocyte is the main factor affecting blastocyst yield. Moreover, in vitro-produced bovine embryos are more sensitive to cryo-injuries than those produced in vivo. Exposure of oocytes to sub-lethal concentrations of stressors may enhance their quality through upregulation of intracellular shock proteins. We aimed to evaluate whether addition of ethanol at low concentrations (0.27 or 0.53%) during oocyte maturation could have a carry-over effect on embryo quality and could subsequently affect embryo cryotolerance. Cumulus-oocyte complexes (n = 934) were matured in serum-free TCM199 plus 20 ng mL-1 of epidermal growth factor (control), supplemented with ethanol 0.27% (treatment 1) or 0.54% (treatment 2), in 3 replicates. After fertilization, the presumptive zygotes were cultured for 6 days in modified SOF medium supplemented with 5% fetal calf serum (FCS); the number of blastocysts was recorded and classified. Then, expanded blastocysts were cryopreserved by open pulled straw vitrification using the 2-step approach described by Vajta G et al. (1998 Mol. Reprod. Dev. 51, 53-58). After 24 h, vitrified embryos were warmed and cultured in groups of <25 per 50 μL droplet of modified SOF medium with 5% FCS under mineral oil for 48 h and examined for re-expansion and hatching. Differences between the groups in blastocyst yield were analyzed by ANOVA; differences in survival rates between the groups were analyzed using logistic regression analysis. For all statistical models, group was included as fixed effect, and also the effect of replicate was included. Differences were considered to be statistically significant when P < 0.05. Addition of ethanol to in vitro maturation media had no significant effects on blastocyst yield on 7 dpi. Also, addition of ethanol at 0.27% did not affect blastocyst cryotolerance. However, addition of ethanol 0.54% to in vitro maturation media significantly increased the survival of bovine blastocysts after vitrification (P < 0.01; Table 1). The results of the present study indicate that maturation of oocytes under ethanol stress at low concentrations has carry-over effects on embryo quality, leading to improved cryotolerance. Table 1.Survival percentage (mean ± SE) of vitrified expanded bovine blastocysts matured in the presence of sub-stress concentrations of ethanol The authors thank I. Lemahieu and P. Vandamme for their excellent technical support. This research was supported by the special research fund, Ghent University (Grant, BOF/DOS No. 01W05706).

134 TRANSCRIPTOME PROFILE OF BOVINE BLASTOCYSTS DERIVED FROM ALTERNATIVE IN VIVO AND IN VITRO CULTURE CONDITIONS AT SPECIFIC PHASES OF EARLY EMBRYONIC DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 179 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Vitro Fertilization

An understanding of gene expression patterns due to altered environmental conditions during different time points of the pre-implantation period would improve our knowledge on regulation of embryonic development and improve success of embryo culture. The aim of this study was to examine the effect of alternative in vivo and in vitro culture conditions at specific phases of early embryonic development on transcriptome profile of bovine blastocysts. Using nonsurgical endoscopic oviducal transfer technology, 5 different blastocyst groups were produced. The first 2 groups were matured in vitro and then either transferred after maturation or after in vitro fertilization to synchronized recipients. The other 3 groups were matured, fertilized and cultured in vitro until 4-cell, 16-cell and morula stage before transfer. Blastocysts from each group were collected by uterine flushing at Day 7 and pooled in groups of 10. Complete in vitro (IVP) and in vivo blastocysts were produced and used as controls. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group vs the in vivo control group to examine the transcriptome profile of blastocysts. Compared with the in vivo control group, clear dramatic shifts were found in the number of differentially expressed genes (DEG, fold change ≥2) at 2 different time points. The first shift occurred for blastocyst groups that were transferred after in vitro fertilization and before embryonic genome activation (EGA). The second shift occurred for blastocyst groups that were transferred after EGA, as well as for the IVP group. Ontological classification of DEG showed that the more time spent under in vitro conditions, the higher the percentage of DEG involved in cell death and apoptotic processes. Moreover, lipid metabolism was the most significant process affected in the blastocysts transferred after in vitro maturation and blastocysts transferred at 16-cell stage. Most DEG involved in this process were down-regulated. Pathway analysis revealed that signalling pathways were the dominant pathways in all groups except the group that was transferred after in vitro maturation. That group showed significant down-regulation for genes involved in retinoic acid receptors activation pathways. These results showed that fertilization and EGA were the most critical developmental stages influenced by in vitro culture conditions and subsequently affect blastocyst quality, as measured in terms of gene expression patterns. Moreover, we identified molecular mechanisms and pathways that were influenced by altered culture conditions. These findings will enable the examination of strategies for modifying in vitro culture conditions at critical stages that will allow more efficient production of developmentally competent blastocysts.

Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity

2012 ◽  
Vol 24 (4) ◽  
pp. 568 ◽  
Author(s):  
Eva Held ◽  
Eva-Maria Mertens ◽  
Abdollah Mohammadi-Sangcheshmeh ◽  
Dessie Salilew-Wondim ◽  
Urban Besenfelder ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Zona Pellucida ◽  
In Vitro Maturation ◽  
Immature Oocytes ◽  
Developmental Capacity ◽  
Bovine Oocytes ◽  
Scanning Electron

In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus–oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.

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