89 METHYLATION STATUS OF IGF2/H19 DMR3 REGION AFFECTS IN VITRO BLASTOCYST PRODUCTION IN GOAT (CAPRA HIRCUS)

2017 ◽  
Vol 29 (1) ◽  
pp. 152
Author(s):  
M. Tiwari ◽  
N. Rawat ◽  
P. Vats ◽  
D. Nagoorvali ◽  
M. Mahajan ◽  
...  
Keyword(s):  
Methylation Status ◽  
Calcium Ionophore ◽  
Methylation Pattern ◽  
Capra Hircus ◽  
Paternal Allele ◽  
Placental Development ◽  
Embryo Production ◽  
Cpg Sites ◽  
H19 Gene

Parthenogenesis has been observed in lower animals but no known instance has been reported in mammals because both maternal and paternal genomes are a fundamental prerequisite for embryogenesis. A major reason for developmental failure of uniparental zygotes is expression of certain genes in a parent-of-origin-specific manner, i.e. genomic imprinting of genes. Out of many imprinted genes identified so far, IGF2/H19 have been extensively studied and known to play an important role in fetal and placental development. Gene IGF2 is expressed by the paternal allele, H19 is transcribed from the maternal allele, and the reciprocal expression of both genes is regulated by the DMR3 region placed upstream of the H19 gene. In the present study we compared the methylation status of IGF2/H19 DMR in parthenogenetic activated (PA) and IVF goat (Capra hircus) blastocyst through bisulphite sequencing. For this, immature oocytes of usable quality were subjected to in vitro maturation and subsequently used for embryo production through parthenogenesis (n = 993) (by calcium ionophore and 6-DMAP activation) and IVF (n = 1096). It was found that embryo production rate at all the embryonic stages (2-cell, 4-cell, 8–16-cell, morula, and blastocyst) was significantly higher (P < 0.05) in parthenogenesis (74.66 ± 3.35%, 61.90 ± 2.73%, 47.83 ± 2.95%, 38.13 ± 5.28%, and 21.11 ± 2.51%, respectively) as compared with IVF (55.21 ± 2.02%, 38.12 ± 2.48%, 28.53 ± 1.67%, 21.57 ± 1.59%, and 8.23 ± 1.02%, respectively). When blastocysts (n = 6 each) were subjected to TUNEL, it was found that PA blastocyst showed significantly higher (P < 0.05) total cell number (217.83 ± 18.80 v. 159.67 ± 13.94) and significantly low (P < 0.05) apoptotic index (2.04 ± 0.25 v. 4.03 ± 0.29) as compared with IVF blastocysts. For the methylation pattern study, we analysed 17 CpG sites on the DMR3 region of the IGF2/H19 gene. Variable methylation pattern was observed within these CpG sites in different clones (n = 15) of PA and IVF blastocyst. The DMR3 region of the IGF2/H19 gene was significantly hypermethylated (P < 0.05) in PA blastocysts as compared with IVF blastocysts (80.39 ± 2.96, 32.55 ± 4.37, respectively), which suggests higher expression of IGF2 in parthenotes. The result suggests IGF2 might play different roles in different species; the same expression pattern of IGF2 is observed in ovine, but a contrary result is found in porcine species. Our results signify the hypermethylation of IGF2/H19 DMR3, which leads to higher expression of IGF2 to support embryonic development at the blastocyst stage. This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.

scholarly journals Deoxyribonucleic Acid Methylation Controls Cell Type-Specific Expression of Steroidogenic Factor 1

Endocrinology ◽  
2008 ◽  
Vol 149 (11) ◽  
pp. 5599-5609 ◽  
Author(s):  
Erling A. Hoivik ◽  
Linda Aumo ◽  
Reidun Aesoy ◽  
Haldis Lillefosse ◽  
Aurélia E. Lewis ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase Ii ◽  
Methylation Status ◽  
Endocrine System ◽  
Methylation Pattern ◽  
Proximal Promoter ◽  
Specific Expression ◽  
Steroidogenic Factor 1 ◽  
In Cells

Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.

264 ANALYSIS OF DNA METHYLATION PATTERN IN PRE-IMPLANTATION-STAGE EMBRYOS DERIVED FROM NUCLEAR TRANSFER USING PORCINE EMBRYONIC GERM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 248
Author(s):  
D.-H. Choi ◽  
C.-H. Park ◽  
S.-G. Lee ◽  
H.-S. Kim ◽  
H.-Y. Son ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Nuclear Transfer ◽  
Perinatal Death ◽  
Epigenetic Modification ◽  
Methylation Pattern ◽  
Paternal Allele ◽  
High Birth Weight ◽  
Aberrant Methylation

Somatic cell nuclear transfer (SCNT) has been successfully used to produce live cloned offspring in various mammals. However, some studies had reported that cloned embryos by SCNT had many problems in reprogramming or epigenetic modification, such as DNA methylation. DNA methylation is an essential process in epigenetic modification for development, and aberrant methylation in cloned embryos gives rise to abortion, high birth weight, and perinatal death. In this study, embryonic germ (EG) cells were used as donor cells for nuclear transfer. EG cells may have less reprogramming or demethylation than SCNT because these are already in erased status. However, little is known about methylation state or developmental capacity of the EG cell as a donor. The objective of this study was to analyze the methylation pattern of pre-implantation embryos cloned from porcine EG cells. Two regions, PRE-1 and microsatellite (MS), were analyzed for methylation patterns of cloned embryos from porcine EG cells and compared with the pattern of mature oocytes and in vitro-fertilized (IVF) embryos as a control. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries and matured in vitro for 44 h, followed by use for IVF and NT with porcine EG cells. The porcine EG cells were prepared from 28-day-old fetuses after mating; genital ridges were isolated from fetuses, and then transferred into a culture medium on a feeder layer. The number of embryos for analysis was 300 for matured oocytes, 50–80 for 4–8 cell embryos, 30–40 for morulae, and 20–30 for blastocysts. The genomic DNA was isolated from the embryos and treated with bisulfite solution. PCR was performed for the amplification of PRE-1 and MS regions. The PCR products were sequenced by using an automatic DNA sequencer. The methylation rates of the PRE-1 and MS regions in IVF embryos showed that the demethylation process had occurred during the pre-implantation stage, which is a typical phenomenon of in vivo counterparts (Kang et al. 2001 J. Biol. Chem. 276, 39 980). However, compared to IVF embryos, embryos derived from NT using EG cells showed differences at the morula (PRE-1) and blastocyst (MS) stage. These results indicate that porcine EG cells also have problems in reprogramming during NT. For detailed and reliable results, the methylation pattern analysis of the imprinting region, for example, H19 in maternal allele and Igf2 in paternal allele, must be examined. Table 1.Methylation of PRE-1 and MS regions in embryos derived from IVF and NT using porcine EG cells

90 OVIDUCTAL EPITHELIAL CELLS Co-CULTURE PROMOTES GOAT (CAPRA HIRCUS) IN VITRO PARTHENOGENETIC EMBRYO DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 152
Author(s):  
M. Mahajan ◽  
D. Nagoorvali ◽  
N. Rawat ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Calcium Ionophore ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Capra Hircus ◽  
Fetal Bovine

Co-culture of pre-implantation embryos with oviducal epithelial cells mimics the in vivo conditions, thus, playing a crucial role in embryo metabolism and gene expression and finally supporting embryonic developmental competence in several ways. Hence, the objective of the present study was to evaluate the effect of goat oviducal epithelial cells (GOEC) co-culture on goat parthenogenetic embryonic development, quality, and relative mRNA abundance of genes related to developmental competence and oxidative stress. The GOEC were obtained from goat oviducts by squeezing and thorough washing with TCM-199 + 10% fetal bovine serum. Goat cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured in TCM-199 + 10% fetal bovine serum supplemented with 5 μg mL−1 of FSH, 10 μg mL−1 of LH, and 1 μg mL−1 of β-oestradiol for 27 h in CO2 incubator with 5% CO2 and at 38.5°C with >95% RH. In vitro matured cumulus–oocyte complexes were denuded and activated with 5 μM calcium ionophore and 2 mM 6-DMAP. Following activation, embryos were co-cultured with and without GOEC (control) in mCR2aa media. The blastocyst development rate was significantly (P < 0.05) higher (23.00 ± 1.15% v. 17.33 ± 1.45%) in the media cultured with GOEC than in control. The total cell number of blastocysts (n = 4) was also found to be significantly more (167.25 ± 17.51 v. 110.25 ± 12.02) than that of control (P < 0.05). However, the apoptotic index (3.76 ± 0.23% v. 7.97 ± 1.99%) was not significantly different in both groups. Further, RNA was isolated from both groups (20 each) of blastocysts on Day 8, and cDNA was prepared. Analysis by qPCR revealed that the relative mRNA abundance of development related genes, i.e. VEGF, BMP4, and CCNB1, showed significantly high (P < 0.05) expression, whereas the expression of CRABP1 was significantly low (P < 0.05) in GOEC co-culture than control. Oxidative stress related genes GPX-1 and SOD2 had comparable expression in both the culture systems, whereas a nonsignificant (P < 0.05) increase in expression of PRDX1 was observed in GOEC co-culture group. In conclusion, co-culture of embryos with GOEC in the simple culture media like mCR2aa helps in improving developmental competence and quality of parthenogenetic embryos. This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.

scholarly journals Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA

Open Biology ◽  
2015 ◽  
Vol 5 (10) ◽  
pp. 150128 ◽  
Author(s):  
Akihisa Osakabe ◽  
Fumiya Adachi ◽  
Yasuhiro Arimura ◽  
Kazumitsu Maehara ◽  
Yasuyuki Ohkawa ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Methylation Status ◽  
Nucleosome Positioning ◽  
Pericentric Heterochromatin ◽  
Micrococcal Nuclease ◽  
Chromosome 1 ◽  
Thermal Stabilities ◽  
Heterochromatin Formation ◽  
Cpg Sites

DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methylation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells.

80 EFFECT OF BOVINE OVIDUCTAL FLUID ON DNA METHYLATION OF BOVINE BLASTOCYSTS PRODUCED IN VITRO

2017 ◽  
Vol 29 (1) ◽  
pp. 147
Author(s):  
A. D. Barrera ◽  
E. V. García ◽  
M. Hamdi ◽  
M. J. Sánchez-Calabuig ◽  
D. Rizos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Environmental Influence ◽  
Methylation Status ◽  
Methylation Pattern ◽  
Early Embryo ◽  
Sequencing Analysis ◽  
Bisulfite Conversion ◽  
Cleavage Rate ◽  
Time Points

During the transit through the oviduct, the early embryo undergoes an epigenetic reprogramming of its genome, which induces changes in DNA methylation pattern. Given that epigenetic modifications are susceptible to environmental influence, the oviducal milieu may affects DNA methylation marks in the developing embryo. The aim of this study was to evaluate whether bovine oviducal fluid (OF) exerts an effect on methylation status of genomic regions at different time points of embryo development. In vitro-produced zygotes were cultured in SOF + 3 mg mL−1 BSA (control, C) or in SOF + 1.25% OF at 3 different time points: until 98 h post-insemination (hpi) (OF1–16: 1–16 cell), 52 hpi (OF1–8: 1–8 cell), or from 52 until 98 hpi (OF8–16: 8–16 cell). The OF used was acquired from Embryocloud (Murcia, Spain) from cow oviducts at the early luteal phase (Day 1–4). After, embryo culture took place in control medium up to Day 8. For all the groups, the speed of development was considered, and normal developing embryos that reached ≥6 cells at 52 hpi and ≥16 cells at 98 hpi were selected and separately cultured from slow developing embryos. Cleavage (52 hpi) and blastocyst yield (Day 7–8) were analysed by ANOVA (8 replicates). Expanding blastocysts (Day 7–8) from the normal developing groups were collected for bisulfite sequencing analysis. The DNA bisulfite conversion was performed with a MethylEdge Bisulfite Conversion System kit (Promega, WI, USA) in groups of 20 blastocysts obtained from 5 replicates. Methylation status was analysed on regions localised in 4 developmental important genes (MTERF2, ABCA7, OLFM1, and GMDS) and within 2 LINE L1 elements located on chromosomes 9 (L9) and 29 (L29). Methylation percentages (10 sequenced clones/group) were compared using statistical z-test. No significant differences were found on cleavage rate (C: 89.7 ± 1.0, OF1–16: 84.9 ± 1.7; OF1–8: 85.4 ± 1.9; OF8–16: 89.1 ± 1.9%) and blastocyst yield between normal developing embryos (C: 36.8 ± 5.3; OF1–16: 34.7 ± 3.7; OF1–8: 41.0 ± 3.8; OF8–16: 43.9 ± 5.1%). Blastocysts derived from all OF groups showed the CpG region of MTERF2 hypomethylated compared with C group (20.0, 26.2, and 32.9% v. 56.2%, respectively; P < 0.001). The CpG sequence of ABCA7 exhibited significant hypomethylation in embryos from OF1–16 group compared with OF1–8, OF8–16, and C groups (31.1 v. 56.8, 57.9, and 65.8%, respectively; P < 0.001). Although the methylation of the CpG region within OLFM1 did not differ between OF1–16 and C groups (24.1 v. 19.4%, respectively), embryos from OF1–8 group showed a highly methylated region (47.1%) compared with OF1–16 and C groups (P < 0.001). The CpG sequence on L9 showed a high methylation level in blastocysts derived from OF1–16 group compared with OF8–16 and C groups (36.4 v. 14.5 and 20.0%, respectively; P < 0.05). There were no differences in methylation marks between groups examined for CpG regions of GMDS and L29. These results indicated that embryos exhibit a temporal sensitivity to OF at early embryonic stages, which is reflected by DNA methylation changes of specific genes at blastocyst stage. This is the first report describing that OF could modify specific epigenetic marks of the bovine embryonic genome.

Establishment of Myelodysplastic Syndrome-Specific Methylome: Implications for the Pathogenesis of Clonal Evolution and Association with Survival

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 850-850
Author(s):  
Rrebecca D ganetzky ◽  
Ying Jiang ◽  
Courtney Prince ◽  
Mikkael A. Sekeres ◽  
Yogen Saunthararajah ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Methylation Level ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Methylation Pattern ◽  
Epigenetic Silencing ◽  
Methylation Analysis ◽  
Suppressor Genes ◽  
Cpg Sites ◽  
Disease Specific

Abstract Epigenetic silencing of genes, such as tumor suppressor genes (TSG), throughaberrant promoter hypermethylation has been implicated in the pathogenesis of MDS. Epigenetic silencing may cooperate with chromosomal abnormalities to completelysilence a TSG or duplication of methylated alleles through UPD could potentially leadto complete TSG silencing. Hypomethylating agents can reverse aberrant silencing;however targeted application of epigenetic therapy is not possible, as ypermethylatedsites relevant to MDS pathogenesis remain mostly unknown. Traditional echnologieslimited methylation analysis to a small number of individual loci; as a result, there has been a lack of systematic studies on the methylation pattern in MDS. New methylationarray techniques allow for rapid polygenic analysis of methylation and the stablishmentof tissue- and disease-specific methylomes. We hypothesized that using ethylationarrays (Illumina®) aberrantly hypermethylated CpG sites and whole methylation patternspathognomic for MDS can be identified. First, we compared patients with MDS andAML (n=240) to controls (n=64) using low-density methylation arrays (1,505 CpG sites)to explore the general applicability of whole genome methylation arrays. We hen usedhigh density arrays (27,578 CpG sites) to fully explore a disease-specific ethylome in a representative sub-cohort of MDS/AML patients. We developed ananalytic algorithm that included establishment of the methylome of normal marrow as a reference and analysis of concordantly hypermethylated genes in patients, using methylation status as either a continuous or dichotomized variable. Global methylation analysis demonstrated that there was concordant hypermethylation in 25% and 50% of MDS patients in 1,199 CpG and 93 CpG sites, respectively, and in 25% and 50% of high-risk MDS patients at 1,816 and 288 CpG sites, respectively. The average methylation level was significantly higher in MDS than in controls and was associated with IPSS score (p=.06). The methylation pattern in MDS include aberrant hypermethylation of tumor suppressor genes (DCC, HIC1), and genes involved in DNA repair (OGG1, MGMT), cell cycle control (DBC1), development and differentiation (HOXA5, HOXB6) and apoptosis (ALOX12). Analysis of the most frequently aberrantly methylated genes identified several genes and we used FZD9 as a candidate TSG on chromosome 7 as an illustrative example for further analysis. Methylation level at this site was significantly predictive of survival in proportional hazards regression analysis (p=.002) and inversely correlated with expression of FZD9 mRNA. Using high density arrays to examine methylation status at locations most commonly associated with chromosomal lesions in MDS (chromosome 5, 7, 11, 13 and 20), we discovered 8 genes with functions and tissue expression patterns suggestive of involvement with MDS. Of these genes, 7 have previously been reported to be aberrantly hypermethylated in malignancy. Patients who did not exhibit hypermethylation at any of these sites were less likely to have developed AML (OR = 4.2, p = 0.074) and showed prolonged survival (p =.09). The absence of hypermethylation at one of the 8 pathognomonic sites was significantly predictive of survival (p=.05). In conclusion, genetic silencing by hypermethylation can produce molecular phenotypes identical to loss of function mutations and deletion of genetic information; however, unlike genetic lesions, epigenetic lesions are more common and are reversible by hypomethylating therapy. Development of a predictive algorithm based on methylation data will allow targeted therapy with epigenetic therapies.

scholarly journals BOARD INVITED REVIEW: Post-transfer consequences of in vitro-produced embryos in cattle

2019 ◽  
Vol 97 (6) ◽  
pp. 2555-2568 ◽  
Author(s):  
Alan D Ealy ◽  
Lydia K Wooldridge ◽  
Sarah R McCoski
Keyword(s):  
Production Systems ◽  
Culture Media ◽  
Methylation Status ◽  
Culture Conditions ◽  
Retention Rates ◽  
Binucleate Cell ◽  
Low Oxygen ◽  
Embryo Production

Abstract In vitro embryo production (IVP) in cattle has gained worldwide interest in recent years, but the efficiency of using IVP embryos for calf production is far from optimal. This review will examine the pregnancy retention rates of IVP embryos and explore causes for pregnancy failures. Based on work completed over the past 25 yr, only 27% of cattle receiving IVP embryos will produce a live calf. Approximately 60% of these pregnancies fail during the first 6 wk of gestation. When compared with embryos generated by superovulation, pregnancy rates are 10% to 40% lower for cattle carrying IVP embryos, exemplifying that IVP embryos are consistently less competent than in vivo-generated embryos. Several abnormalities have been observed in the morphology of IVP conceptuses. After transfer, IVP embryos are less likely to undergo conceptus elongation, have reduced embryonic disk diameter, and have compromised yolk sac development. Marginal binucleate cell development, cotyledon development, and placental vascularization have also been documented, and these abnormalities are associated with altered fetal growth trajectories. Additionally, in vitro culture conditions increase the risk of large offspring syndrome. Further work is needed to decipher how the embryo culture environment alters post-transfer embryo development and survival. The risk of these neonatal disorders has been reduced by the use of serum-free synthetic oviductal fluid media formations and culture in low oxygen tension. However, alterations are still evident in IVP oocyte and embryo transcript abundances, timing of embryonic cleavage events and blastulation, incidence of aneuploidy, and embryonic methylation status. The inclusion of oviductal and uterine-derived embryokines in culture media is being examined as one way to improve the competency of IVP embryos. To conclude, the evidence presented herein clearly shows that bovine IVP systems still must be refined to make it an economical technology in cattle production systems. However, the current shortcomings do not negate its current value for certain embryo production needs and for investigating early embryonic development in cattle.

scholarly journals A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development.

1997 ◽  
Vol 17 (8) ◽  
pp. 4322-4329 ◽  
Author(s):  
K D Tremblay ◽  
K L Duran ◽  
M S Bartolomei
Keyword(s):  
Methylation Status ◽  
Proximal Region ◽  
Differentially Methylated Region ◽  
Paternal Allele ◽  
Genomic Sequencing ◽  
Regulatory Domain ◽  
Cpg Dinucleotides ◽  
Somatic Tissues ◽  
H19 Gene ◽  
Promoter Proximal Region

The imprinted mouse H19 gene is hypermethylated on the inactive paternal allele in somatic tissues and sperm. Previous observations from a limited analysis have suggested that methylation of a few CpG dinucleotides in the region upstream from the start of transcription may be the mark that confers parental identity to the H19 alleles. Here we exploit bisulfite mutagenesis coupled with genomic sequencing to derive the methylation status of 68 CpGs that reside in a 4-kb region 5' to the start of transcription. This method reveals a 2-kb region positioned between 2 and 4 kb upstream from the start of transcription that is strikingly differentially methylated in midgestation embryos. At least 12 of the cytosine residues in this region are exclusively methylated on the paternal allele in blastocysts. In contrast, a 350-bp promoter-proximal region is less differentially methylated in midgestation embryos and, like most of the genome, is largely devoid of methylation on both alleles in blastocysts. We also demonstrate exclusive expression of the maternal H19 allele in the embryos that exhibit paternal methylation of the upstream 2-kb region. These data suggest that the 2-kb differentially methylated region acts as a key regulatory domain for imprinted H19 expression.

236 CREATION OF PARTHENOGENETIC SHEEP EMBRYOS: EFFECTS OF SERUM AND BREEDING SEASON

2009 ◽  
Vol 21 (1) ◽  
pp. 216
Author(s):  
A. T. Grazul-Bilska ◽  
P. P. Borowicz ◽  
D. A. Redmer ◽  
J. J. Bilski ◽  
L. P. Reynolds
Keyword(s):  
Breeding Season ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Culture Conditions ◽  
Imprinted Genes ◽  
Blastocyst Formation ◽  
Placental Development ◽  
Developmentally Regulated ◽  
Paternal Genome

The monoparental embryo (with only the maternal genome, termed a parthenogenote, or with only the paternal genome, termed an androgenote) is a powerful model to study the epigenetic status of developmentally regulated genes, including imprinted genes (those expressed only when inherited from one parent). Therefore, to use monoparental embryos for future study of placental development in normal and compromised pregnancies, the objective of this study was to test, validate and optimize the methodologies necessary to create parthenogenetic sheep embryos. In Exp. 1, cumulus–oocyte complexes (COC) were collected during the breeding season from nonpregnant and early pregnant ewes (n = 18) and matured overnight in vitro. The oocytes were then activated using ionomycin (a calcium ionophore) and 6-dimethylaminopurine (DMAP; a protein kinase inhibitor) in medium with (n = 47 COC) or without (n = 112 COC) 2% serum. In Exp. 2, COC were collected from nonpregnant, seasonally anestrous (n = 7; 79 COC) and late pregnant (n = 4; 44 COC) ewes, matured in vitro and activated as in Exp. 1 in the presence of serum. In Exp. 1, the rates of activation and blastocyst formation were not affected by reproductive status (nonpregnant v. pregnant). Activation of oocytes in serum-containing v. serum-free medium resulted in 76.3% v. 4.35% (P < 0.0001) cleavage rates and 21.9% v. 8.3% (P < 0.006) blastocyst formation rates, respectively. In Exp. 2, cleavage rates were similar for nonpregnant and pregnant ewes (31.9 v. 37.5%), but blastocyst formation was 13% in nonpregnant and 0% in pregnant ewes. These data demonstrate that (1) during the breeding season, the presence of serum in activation medium enhances cleavage and blastocyst formation rates; (2) in out-of-season ewes blastocyst formation but not cleavage rates are greater for nonpregnant than pregnant ewes, and (3) cleavage and blastocyst formation rates are greater (P < 0.0001 and P < 0.04, respectively) during the breeding season than out-of-season. Thus, oocytes obtained from ewes during the breeding season and activation medium containing serum should be used for creating parthenogenetic embryos in sheep. This study also demonstrated that creation of parthenogenotes in sheep, which can be used in the future to study parentally imprinted genes, is feasible but requires specific season and culture conditions. Supported by USDA-NRI grant 2007-01215 to LPR and ATGB, and NIH grant P20 RR016741 (INBRE program of the NCRR, NIH).

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