scholarly journals Dysregulated Gene Expression of Imprinted and X-Linked Genes: A Link to Poor Development of Bovine Haploid Androgenetic Embryos

2021 ◽  
Vol 9 ◽  
Author(s):  
Luis Aguila ◽  
Joao Suzuki ◽  
Amanda B. T. Hill ◽  
Mónica García ◽  
Karine de Mattos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genomic Imprinting ◽  
X Chromosome ◽  
De Novo ◽  
Chromosomal Abnormalities ◽  
Methylation Status ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Androgenetic Embryos

Mammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genomes to early embryonic development. In this study, we analyzed different methods for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. Results indicate that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although androgenetic haploidy does not disturb early development up to around the 8-cell stage, androgenetic development is disturbed after the time of zygote genome activation and hAE that reach the morula stage are less capable to reach the blastocyst stage of development. Karyotypic comparisons to parthenogenetic- and ICSI-derived embryos excluded chromosomal segregation errors as causes of the developmental constraints of hAE. However, analysis of gene expression indicated abnormal levels of transcripts for key long non-coding RNAs involved in X chromosome inactivation and genomic imprinting of the KCNQ1 locus, suggesting an association with X chromosome and some imprinted loci. Moreover, transcript levels of methyltransferase 3B were significantly downregulated, suggesting potential anomalies in hAE establishing de novo methylation. Finally, the methylation status of imprinted control regions for XIST and KCNQ1OT1 genes remained hypomethylated in hAE at the morula and blastocyst stages, confirming their origin from spermatozoa. Thus, our results exclude micromanipulation and chromosomal abnormalities as major factors disturbing the normal development of bovine haploid androgenotes. In addition, although the cause of the arrest remains unclear, we have shown that the inefficient development of haploid androgenetic bovine embryos to develop to the blastocyst stage is associated with abnormal expression of key factors involved in X chromosome activity and genomic imprinting.

scholarly journals Dysregulated gene expression of imprinted and X-linked genes: a link to poor development of bovine haploid androgenetic embryos

2020 ◽  
Author(s):  
Luis Aguila ◽  
Jacinthe Therrien ◽  
Joao Suzuki ◽  
Mónica García ◽  
Amanda Trindade ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromosomal Abnormalities ◽  
Methylation Status ◽  
Mitotic Division ◽  
Developmental Potential ◽  
Maternal Genome ◽  
Morula Stage ◽  
Androgenetic Embryos ◽  
Zygote Genome Activation ◽  
Major Factors

AbstractMammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genome to early embryonic development. In this study, we analyzed different methodologies for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. The results showed that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although haploidy does not disturb early development up to around the 3rd mitotic division, androgenetic development is disturbed after the time of zygote genome activation those that reach the morula stage are less capable to become a blastocyst. Analysis of gene expression indicated abnormal levels of methyltransferase 3B and key long non-coding RNAs involved in X-chromosome inactivation and genomic imprinting of the KCNQ1 locus, which is associated to the methylation status of imprinted control regions of XIST and KCNQ1OT1. Thus, our results seem to exclude micromanipulation consequences and chromosomal abnormalities as major factors in developmental restriction, suggesting that their early developmental constraint is regulated at an epigenetic level.

scholarly journals Infection with Human Immunodeficiency Virus Type 1 Upregulates DNA Methyltransferase, Resulting in De Novo Methylation of the Gamma Interferon (IFN-γ) Promoter and Subsequent Downregulation of IFN-γ Production

1998 ◽  
Vol 18 (9) ◽  
pp. 5166-5177 ◽  
Author(s):  
Judy A. Mikovits ◽  
Howard A. Young ◽  
Paula Vertino ◽  
Jean-Pierre J. Issa ◽  
Paula M. Pitha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Methylation Status ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv 1 ◽  
De Novo Methylation

ABSTRACT The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-γ), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-γ, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-γ gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-γ gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-γ gene, and increased IFN-γ production, demonstrating a direct link between methyltransferase and IFN-γ gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-γ gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.

HPRT activity in embryos of a South American opossum Monodelphis domestica

1994 ◽  
Vol 6 (4) ◽  
pp. 529 ◽  
Author(s):  
PG Johnston ◽  
D Dean ◽  
JL VandeBerg ◽  
ES Robinson
Keyword(s):  
X Chromosome ◽  
Blastocyst Stage ◽  
Monodelphis Domestica ◽  
X Inactivation ◽  
Hypoxanthine Phosphoribosyl Transferase ◽  
South American ◽  
Cell Stage ◽  
Phosphoribosyl Transferase ◽  
Stage Of Development ◽  
American Opossum

Marsupial females show preferential paternal X-inactivation. However, the time at which X-inactivation occurs in early development has not yet been determined. A double microassay which measures the activities of X-linked hypoxanthine phosphoribosyl transferase (HPRT) and the autosomally-coded adenine phosphoribosyl transferase (APRT) from the same sample was performed on a collection of embryos from a South American opossum Monodelphis domestica. The embryos ranged in age from the 2-cell stage to the bilaminar blastocyst stage. The results indicate that their embryonic HPRT and APRT are not expressed until just before the unilaminar blastocyst stage in M. domestica. This is at a later stage of development than that in the mouse where embryonic HPRT and APRT expression first occurs at the 4-8-cell stage. It is concluded that HPRT is an uniformative enzyme for assessing X chromosome activity in cleaving embryos of M. domestica. The widespread distribution of HPRT:APRT ratios after the unilaminar blastocyst stage also makes it difficult to draw conclusions about the state of X chromosome activity in early marsupial development.

139 SEX-SPECIFIC GENE EXPRESSION IN PORCINE PRE-IMPLANTATION EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 199
Author(s):  
D. Kradolfer ◽  
J. Knubben ◽  
V. Flöter ◽  
J. Bick ◽  
S. Bauersachs ◽  
...  
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
Current Knowledge ◽  
Critical Time ◽  
Blastocyst Stage ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Specific Gene ◽  
Embryo Survival ◽  
Specific Gene Expression

X-Chromosome inactivation in female mammals starts during early blastocyst stage with expression of the X-inactive specific transcript (XIST), which coats and silences the inactive X chromosome. However, this compensation is not complete in blastocysts, as a large number of X-linked transcripts are more highly expressed in female embryos than in males. Furthermore, the process of X chromosome inactivation is altered in IVF and cloned porcine embryos, possibly explaining problems of embryo survival with these techniques. The aim of this study was to gain more insights into the transcriptional dynamics of the porcine pre-implantation embryo, with a particular focus on sex-specific differences. RNA sequencing (RNA-Seq) was performed for individual blastocysts at 8, 10, and 12 days after ovulation, and the temporal development of sex-specific transcripts was analysed. German Landrace sows were cycle synchronized and inseminated with sperm of the same Pietrain boar. On Days 8, 10, and 12 post-insemination, sows were slaughtered and embryos were removed from the uterus using 10 mL of PBS (pH 7.4) per horn. Single embryos were shock frozen in liquid nitrogen and stored at –80°C until the extraction of RNA and DNA (AllPrep DNA/RNA Micro Kit, Qiagen, Valencia, CA, USA). Using the isolated DNA, the sex of the embryos was determined and 5 female and male embryos, respectively, were analysed per stage. Illumina TruSeq Stranded mRNA libraries (Illumina Inc., San Diego, CA, USA) were sequenced on a HiSEqn 2500 (Illumina Inc.), and 15 to 25 million 100-bp single-end reads were generated per sample. Reads were filtered and processed using Trimmomatic and mapped to the porcine genome assembly Sscrofa10.2 with TopHat2. Mapped reads were counted by the use of QuasR qCount based on the current National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) GFF3 annotation file. Statistical analysis of count data was performed with the BioConductor R (https://www.bioconductor.org/) package DESEqn 2. At all 3 stages, we found 7 Y-linked transcripts that were highly expressed in male embryos (EIF2S3, EIF1AY, LOC100624590, LOC100625207, LOC100624329, LOC102162178, LOC100624937). On the other hand, 47 X-linked transcripts showed increased expression in female blastocysts, most of them at all 3 time points. However, a small number of genes (DDX3X, LAMP2, and RPS6KA3) were more highly expressed in females at Days 8 and 10 but more highly expressed in males at Day 12. Three X-linked genes (OFD1, KAL1, and LOC100525092) were more highly expressed in male embryos, although only at a low fold change of 1.2 to 1.4. Furthermore, expression of 8 transcripts located on autosomes was higher in females. In conclusion, our study expands the current knowledge of sex-specific gene expression in 8- to 12-day-old porcine blastocysts, a critical time period during pre-implantation embryo development.

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

Changes in DNA methylation during mouse embryonic development in relation to X-chromosome activity and imprinting

1990 ◽  
Vol 326 (1235) ◽  
pp. 299-312 ◽  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Embryonic Development ◽  
X Chromosome ◽  
De Novo ◽  
Germ Line ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Chromatin Configuration ◽  
Methylation Patterns

Changing DNA methylation patterns during embryonic development are discussed in relation to differential gene expression, changes in X-chromosome activity and genomic imprinting. Sperm DNA is more methylated than oocyte DNA, both overall and for specific sequences. The methylation difference between the gametes could be one of the mechanisms (along with chromatin structure) regulating initial differences in expression of parental alleles in early development. There is a loss of methylation during development from the morula to the blastocyst and a marked decrease in methylase activity. De novo methylation becomes apparent around the time of implantation and occurs to a lesser extent in extra-embryonic tissue DNA. In embryonic DNA, de novo methylation begins at the time of random X-chromosome inactivation but it continues to occur after X-chromosome inactivation and may be a mechanism that irreversibly fixes specific patterns of gene expression and X-chromosome inactivity in the female. The germ line is probably delineated before extensive de novo methylation and hence escapes this process. The marked undermethylation of the germ line DNA may be a prerequisite for X-chromosome reactivation. The process underlying reactivation and removal of parent-specific patterns of gene expression may be changes in chromatin configuration associated with meiosis and a general reprogramming of the germ line to developmental totipotency.

scholarly journals Comparative analysis of differential gene expression indicates divergence in ontogenetic strategies of leaves in two conifer genera

2021 ◽  
Author(s):  
Cynthia Webster ◽  
Laura Figueroa-Corona ◽  
Iván Méndez-González ◽  
Lluvia Soto-Álvarez ◽  
David Neale ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Life Stage ◽  
Expression Patterns ◽  
Adult Life ◽  
Gene Interaction ◽  
Gene Families ◽  
Interaction Patterns ◽  
Gene Interaction Network ◽  
Stage Of Development

In land plants, heteroblasty broadly refers to a drastic change in morphology during growth through ontogeny. Juniperus flaccida and Pinus cembroides are conifers of independent lineages known to exhibit leaf heteroblasty between the juvenile and adult life stage of development. Juvenile leaves of P. cembroides develop spirally on the main stem and appear decurrent, flattened and needle-like; whereas, adult photosynthetic leaves are triangular or semi-circular needle-like, and grow in whorls on secondary or tertiary compact dwarf shoots. By comparison, J. flaccida juvenile leaves are decurrent and needle-like, and adult leaves are compact, short and scale-like. Comparative analyses were performed to evaluate differences in anatomy and gene expression patterns between developmental phases in both species. RNA from twelve samples was sequenced and analyzed with available software. They were assembled de novo from the RNA-Seq reads. Following assembly, 63,741 high quality transcripts were functionally annotated in P. cembroides and 69,448 in J. flaccida. Evaluation of the orthologous groups yielded 4,140 shared gene families among the four references (adult and juvenile from each species). Activities related to cell division and development were more abundant in juveniles than adults in P. cembroides, and more abundant in adults than juveniles in J. flaccida. Overall, there were 509 up-regulated and 81 down-regulated genes in the juvenile condition of P. cembroides and 18 up-regulated and 20 down-regulated in J. flaccida. Gene interaction network analysis showed evidence of co-expression and co-localization of up-regulated genes involved in cell wall and cuticle formation, development, and phenylpropanoid pathway, in juvenile P. cembroides leaves. Whereas in J. flaccida, differential expression and gene interaction patterns were detected in genes involved in photosynthesis and chloroplast biogenesis. Although J. flaccida and P. cembroides both exhibit leaf heteroblastic development, little overlap was detected and unique genes and pathways were highlighted in this study.

94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
A. Velasquez ◽  
D. Veraguas ◽  
F. O. Castro ◽  
J. F. Cox ◽  
L. l. Rodriguez-Alvarez
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Gene Markers ◽  
Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

79 REACTIVE OXYGEN SPECIES LEVEL IN CULTURED PORCINE EMBRYOS AFTER HIGH HYDROSTATIC PRESSURE

2016 ◽  
Vol 28 (2) ◽  
pp. 169
Author(s):  
M. Romek ◽  
M. Kucia ◽  
B. Gajda ◽  
Z. Smorag
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrostatic Pressure ◽  
High Hydrostatic Pressure ◽  
Reactive Oxygen ◽  
Blastocyst Stage ◽  
Molecular Probes ◽  
Control Group ◽  
Oxygen Species ◽  
Developmental Potential ◽  
Stage Of Development

Our recent study (Romek et al., Proc. of 29th Scientific Meeting of A.E.T.E., 2013, 196) demonstrated that high hydrostatic pressure (HHP) decreased the potential of the inner mitochondrial membrane in porcine embryos from morula to blastocyst stage. Therefore, the aim of this study was to find out if HHP treatment of cultured porcine embryos has an effect on production of reactive oxygen species (ROS) in these cells. Gilts were superovulated and inseminated using standard methods. Then zygotes were surgically collected after flushing the oviducts of the donors gilts 22 to 24 h after insemination. Obtained zygotes were cultured in NCSU-23 (North Carolina State University-23) medium up to the blastocyst stage, in an atmosphere containing 5% CO2 in air at 39°C. In the experimental group, embryos at zygote, 2- to 4-cell, 8- to 16-cell, morula and blastocyst stages were treated with 20 MPa of hydrostatic pressure (HHP100, Cryo-Innovation Ltd., Szeged, Hungary) for 60 min at 39°C with an interval of 60 min between HHP treatment and subsequent embryo staining. For the control group of embryos at the same stage of development, the HHP treatment was omitted. An additional group of blastocysts derived after culture was analysed 4 h after the HHP treatment. ROS level was measured using 5 μM CM-H2DCFDA fluorescent dye (Molecular Probes Inc., Eugene, OR, USA). Embryos from the experimental and control groups were stained for 30 min at 39°C and then analysed under a Nikon Eclipse microscope equipped with a CCD camera. The total amount of fluorescence emitted from each individual embryo was measured. The data (in arbitrary unit) were analysed using one-way ANOVA and post-hoc Tukey test. After HHP zygote treatment, the percentage of obtained blastocysts was 67.01, whereas in control group it was 63.95 (P < 0.05). ROS level proportional to the measured amount of fluorescence (mean ± standard error of the mean) was 9.15 ± 2.70 (n = 15), 7.11 ± 2.46 (n = 18), 8.67 ± 2.4 (n = 19), 11.47 ± 1.94 (n = 29), and 54.74 ± 2.28 (n = 21) for zygote, 2- to 4-cell, 8- to 16-cell, morula, and blastocyst stage of the control group, respectively. After HHP treatment, the ROS level was 7.39 ± 2.4 (n = 19), 6.66 ± 2.28 (n = 21), 9.14 ± 2.61 (n = 16), 7.23 ± 2.28 (n = 21), 33.06 ± 2.4 (n = 19) for zygote, 2- to 4-cell, 8- to 16-cell, morula, and blastocyst stage, respectively, and 35.57 ± 2.4 (n = 10) for blastocyst 4 h after HHP treatment. In conclusion, (1) HHP treatment of porcine zygotes improve embryo developmental potential; (2) ROS level in both control and experimental groups remained unchanged up to morula stage, whereas at the blastocyst stage, after HHP treatment ROS level significantly decreased (P < 0.05) in comparison with nontreated blastocysts; (3) HHP treatment on porcine blastocysts resulted in a lowered level of ROS that remained unchanged for 4 h. These results suggest that HHP treatment could improve the quality of cultured porcine blastocysts. The project was funded by the National Science Centre based on decision number DEC-2012/07/B/NZ9/01326.

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