Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle

2014 ◽  
Vol 26 (8) ◽  
pp. 1107 ◽  
Author(s):  
Mohammad Bozlur Rahman ◽  
Md. Mostofa Kamal ◽  
Tom Rijsselaere ◽  
Leen Vandaele ◽  
Mohammed Shamsuddin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Chromatin Condensation ◽  
Dna Demethylation ◽  
In Vitro Fertilisation ◽  
Paternal Genome ◽  
Time Points ◽  
Fertilisation Rate ◽  
De Novo Methylation

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n = 1239) were collected at three different time points (12, 18 and 24 h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P < 0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.

6 ALTERED CHROMATIN CONFORMATION IN BOVINE SPERMATOZOA PERTURBS DYNAMIC DNA METHYLATION IN THE MALE PRONUCLEUS AFTER FERTILIZATION IN VITRO

2013 ◽  
Vol 25 (1) ◽  
pp. 150 ◽  
Author(s):  
M. B. Rahman ◽  
M. M. Kamal ◽  
T. Rijsselaere ◽  
L. Vandaele ◽  
M. Shamsuddin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
De Novo ◽  
Chromatin Condensation ◽  
Sperm Chromatin ◽  
Chromatin Conformation ◽  
Epigenetic Marks ◽  
Time Points ◽  
Male Pronucleus ◽  
De Novo Methylation

Soon after fertilization, mammalian zygotes need proper DNA methylation reprogramming, at which time the epigenetic marks that the oocyte and sperm have acquired during gametogenesis are erased to allow totipotent zygotic development. Aberrant epigenetic marks in the paternal genome are thought to be associated with altered chromatin condensation in spermatozoa of suboptimal quality. We have recently reported that heat stress on bulls during germ cell development, especially at the spermiogenesis stage, altered sperm chromatin condensation. The objective of this study was to investigate dynamic DNA methylation reprogramming in the male pronucleus after fertilization of oocytes with sperm known to have altered chromatin conformation. To evaluate dynamic DNA methylation reprogramming, zygotes collected at 3 different time points [i.e. 12, 18, and 24 h post-insemination (hpi)] were immunocytochemically investigated using an antibody against 5-methylcytosine (5mC). The total fluorescence intensity of the male pronuclei (n = 89, ≥25 in each group) was measured by ImageJ and data were analyzed by ANOVA. The DNA methylation pattern in male pronuclei when oocytes were fertilized with heat-stressed sperm did not change between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. The results of this study indicated that dynamic DNA methylation reprogramming patterns such as DNA demethylation followed by de novo methylation in the male pronucleus soon after fertilization were altered when oocytes were fertilized with heat-stressed sperm. In conclusion, altered sperm chromatin conformation due to heat stress perturbs dynamic DNA methylation reprogramming in the male pronucleus, which may hamper nuclear totipotency and embryo survival.

139 PERTURBATION OF THE DYNAMICS OF DNA METHYLATION IN THE PATERNAL GENOME FOLLOWING IN VITRO FERTILIZATION IN CATTLE: THE SECRETS OF HEAT STRESS EFFECTS

2014 ◽  
Vol 26 (1) ◽  
pp. 183
Author(s):  
M. B. Rahman ◽  
T. Rijsselaere ◽  
A. Van Soom
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
In Vitro Fertilization ◽  
Chromatin Condensation ◽  
Histone Variants ◽  
Dna Demethylation ◽  
Paternal Genome ◽  
Vitro Fertilization ◽  
Fourier Harmonic

Ejaculates collected from breeding bulls during the summer season under tropical or subtropical conditions are often reported to have a lower fertilization potential. We recently reported that sperm cells at post-meiotic stages of development were more susceptible to heat stress (scrotal insulation technique). In post-meiotic stages of sperm cell development, extensive incorporation of histone variants and hyperacetylation confirms unstable chromatin. The unstable forms of chromatin are more vulnerable to heat stress and affect sperm DNA-protamine condensation. By using a special stain and nuclear morphometric assessment, Chromomycin A3 and Fourier harmonic analysis, respectively, we observed protamine-deficient spermatozoa with altered chromatin condensation in the heat-stressed ejaculates. In a recent study, we investigated the patterns of global DNA methylation in pronuclear development and fertilization potential of such altered chromatin condensed spermatozoa. To this end, 1239 in vitro-matured oocytes were fertilized with spermatozoa of 3 groups (i.e. standard IVF control, non-heat-stressed control, and heat-stressed spermatozoa). Data were analysed by means of ANOVA. The results showed that heat-stressed spermatozoa with altered chromatin condensation perturb the dynamics of DNA methylation reprogramming in the paternal pronucleus (i.e. disordered the active demethylation followed by a de novo methylation pattern during one cell stage). In addition, there was a tendency for a decrease in the size of both paternal and maternal pronuclei developed after fertilization with heat-stressed spermatozoa in comparison with standard IVF control spermatozoa, leading to lower fertilization rates (54 v. 82%). Future studies should focus on the mechanisms of perturbed DNA demethylation in the paternal genome after in vitro fertilization of oocytes with spermatozoa having altered chromatin condensation.

84 MARKERS OF DNA METHYLATION IN OVINE UTERO–PLACENTAL TISSUES DURING EARLY PREGNANCY: EFFECTS OF ASSISTED REPRODUCTIVE TECHNOLOGY

2012 ◽  
Vol 24 (1) ◽  
pp. 154
Author(s):  
A. T. Grazul-Bilska ◽  
M. L. Johnson ◽  
P. P. Borowicz ◽  
D. A. Redmer ◽  
L. P. Reynolds
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technology ◽  
Early Pregnancy ◽  
De Novo ◽  
Reproductive Technology ◽  
The Other ◽  
Gravid Uterus ◽  
Natural Breeding ◽  
De Novo Methylation

Compromised pregnancies can be caused by genetic, epigenetic, environmental and/or other factors. Assisted reproductive technology (ART) may have profound effects on placental and fetal development, leading eventually to compromised pregnancy. DNA methylation, regulated by DNA methyltransferases (Dnmt) and other factors, plays an important role during embryonic, including placental, development. Altered DNA methylation in the trophoblast and, subsequently, the placenta has been reported for compromised pregnancies and may contribute to embryonic/fetal loss. Little is known, however, about DNA methylation processes in placental tissues during early stages of normal or compromised pregnancies in any species. Thus, we hypothesised that ART would affect the expression of 5 methylcytosine (5mC; a marker of global methylation) and mRNA for Dnmt1, 3a and 3b in utero-placental tissues during early pregnancy in sheep. Pregnancies (n = 7 per group) were achieved through natural breeding (NAT, control), or transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF) or in vitro activation (IVA; parthenogenetic clones). On Day 22 of pregnancy, caruncle (CAR; maternal placenta) and fetal membranes (FM; fetal placenta) were snap-frozen separately for RNA extraction followed by quantitative real-time PCR. In addition, cross sections of gravid uterus were fixed and then used for immunohistochemical detection and image analysis of 5 mC in FM. In FM, expression of mRNA for Dnmt3a was ∼2-fold greater (P < 0.01) in IVA compared with the other groups and was similar in NAT, NAT-ET and IVF groups. Expression of 5 mC was ∼2- to 3-fold greater (P < 0.02) in IVF and IVA than in NAT. In CAR, mRNA expression for Dnmt1 was ∼1.5-fold greater (P < 0.04) in IVA compared with the other groups, but Dnmt3a expression was less (P < 0.04) in NAT-ET and IVA than NAT. Expression of mRNA for Dnmt1 in FM and 3b in FM and CAR was similar in all groups. In IVA and/or IVF pregnancy, increased expression of Dnmt3a mRNA and/or 5 mC in FM may indicate de novo methylation in the fetal placenta. Furthermore, in pregnancies created through ART, decreased expression of Dnmt3a in CAR may indicate reduced de novo methylation in maternal placenta. Thus, in sheep, ART may have specific effects on growth and function of utero-placental and fetal tissues through regulation of DNA methylation and likely other mechanisms. These data provide a foundation for determining the basis for altered DNA methylation of specific genes in placental and embryonic tissues in compromised pregnancies. In addition, these data will help us to better understand placental regulatory mechanisms in compromised pregnancies and to identify strategies for rescuing such pregnancies. Supported by Hatch Project ND01712; USDA grant 2007-01215 to LPR and ATGB, NIH grant HL64141 to LPR and DAR and NSF-MRI-ARRA grant to ATGB.

scholarly journals 135DNA METHYLATION PROFILES IN THE PREIMPLANTATION PORCINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 190
Author(s):  
S. Yeo ◽  
Y.-K. Kang ◽  
D.-B Koo ◽  
J.-S Han ◽  
W.-K Chang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Paternal Genome ◽  
Active Demethylation ◽  
Morula Stage ◽  
De Novo Methylation

DNA methylation at CpG dinucleotides is an important epigenetic regulation process, which is associated with gene expression without any change in DNA sequence. During early development of the mouse embryo, dynamic changes in DNA methylation of the genome occur. After fertilization, active demethylation occurs on the paternal genome followed by passive demethylation until morula stage and then de novo methylation at the blastocyst stage. This study was designed to investigate changes in DNA methylation of in vivo- and in vitro-fertilized (IVF) porcine embryos. DNA methylation states were observed in preimplantation porcine embryos by using an immunofluorescence method after staining with an antibody against 5-methylcytosine. In contrast to the data from mouse embryos, active demethylation of the genome from the paternal pronucleus was not observed in the porcine embryos. Also, no passive demethylation was detected in in vivo- and IVF-derived embryos until the morula stage. Moreover, differential de novo methylation was not shown on the genome of the inner cell mass. Whole genomes of inner cell mass and trophectoderm cells were fully methylated. Our results demonstrate that DNA methylation of porcine embryos is different from that of mouse embryos during preimplantation development, suggesting that the machinery to regulate DNA methylation may be species-specific in mammals.

scholarly journals DNA methylation in early mice embryogenesis under the influence of bisphenol A

2017 ◽  
Vol 15 (3) ◽  
pp. 42-53 ◽  
Author(s):  
Ekaterina M. Noniashvili ◽  
Natalia A. Grudinina ◽  
Marija E. Kustova ◽  
Van Truong Tran ◽  
Irina O. Suchkova ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisphenol A ◽  
De Novo ◽  
Blastocyst Stage ◽  
Dna Demethylation ◽  
Sensitive Period ◽  
Control Group ◽  
Blastocyst Development ◽  
Potential Threat

Background. Nonsteroid estrogen – bisphenol A (BPA) can have a detrimental effect on human health, and therefore poses a potential threat to humans. The critical window for the effect of BPA is the time of early development of the embryo, especially during the activation of the embryonic genome during development to the stage of blastocyst. Therefore, it is especially important to understand how DNA methylation is modified in embryos of the earliest developmental period under the influence of BPA. Materials and methods. Mice hybrids F1 (CBAXC57BL) were once administered 0, 8 mg of BPA per mouse and the level of DNA methylation was estimated by detection the fluorescence of antibodies against 5-MeC in nuclei of GD3 and GD9 embryos. In other series, the level of DNA methylation and the rate of blastocyst development were estimated following cultivation of one- and two cells embryos in the presence of BPA (50 or 100 µM) during 72-96 hours in vitro. Results. BPA exposure induced the decrease of the level of DNA methylation in GD3embryos received toxicant in utero, the amount of blastomeres in these embryos was decreased too. The level of DNA methylation in GD9 embryos was slightly higher than in control group. Upon cultivation of one-two cells embryos, BPA decreased the level of DNA methylation and the rate of embryos development to blastocyst stage. Conclusion. We have determined that early embryogenesis is highly sensitive period to the BPA effects. Such effect is most likely due to active reprogramming processes in this period, primarily related to DNA demethylation/methylation de novo of both the whole genome and individual genes.

scholarly journals Dnmt1 has de novo activity targeted to transposable elements

2021 ◽  
Author(s):  
Chuck Haggerty ◽  
Helene Kretzmer ◽  
Christina Riemenschneider ◽  
Abhishek Sampath Kumar ◽  
Alexandra L. Mattei ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Critical Role ◽  
Embryonic Stem ◽  
Genetically Engineered ◽  
Genomic Recruitment ◽  
Methylation Activity ◽  
De Novo Methylation

AbstractDNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

scholarly journals Androgenic-Induced Transposable Elements Dependent Sequence Variation in Barley

2021 ◽  
Vol 22 (13) ◽  
pp. 6783
Author(s):  
Renata Orłowska ◽  
Katarzyna A. Pachota ◽  
Wioletta M. Dynkowska ◽  
Agnieszka Niedziela ◽  
Piotr T. Bednarek
Keyword(s):  
Dna Methylation ◽  
Transposable Elements ◽  
Dna Sequence ◽  
Sequence Variation ◽  
Nuclear Dna ◽  
Point Mutations ◽  
Mobile Elements ◽  
Dna Demethylation ◽  
Plant Genome

A plant genome usually encompasses different families of transposable elements (TEs) that may constitute up to 85% of nuclear DNA. Under stressful conditions, some of them may activate, leading to sequence variation. In vitro plant regeneration may induce either phenotypic or genetic and epigenetic changes. While DNA methylation alternations might be related, i.e., to the Yang cycle problems, DNA pattern changes, especially DNA demethylation, may activate TEs that could result in point mutations in DNA sequence changes. Thus, TEs have the highest input into sequence variation (SV). A set of barley regenerants were derived via in vitro anther culture. High Performance Liquid Chromatography (RP-HPLC), used to study the global DNA methylation of donor plants and their regenerants, showed that the level of DNA methylation increased in regenerants by 1.45% compared to the donors. The Methyl-Sensitive Transposon Display (MSTD) based on methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach demonstrated that, depending on the selected elements belonging to the TEs family analyzed, varying levels of sequence variation were evaluated. DNA sequence contexts may have a different impact on SV generated by distinct mobile elements belonged to various TE families. Based on the presented study, some of the selected mobile elements contribute differently to TE-related SV. The surrounding context of the TEs DNA sequence is possibly important here, and the study explained some part of SV related to those contexts.

scholarly journals In vitro fertilisation (IVF) versus intracytoplasmic sperm injection (ICSI) in patients without severe male factor infertility: study protocol for the randomised, controlled, multicentre trial INVICSI

BMJ Open ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. e051058
Author(s):  
Sine Berntsen ◽  
Bugge Nøhr ◽  
Marie Louise Grøndahl ◽  
Morten Rønn Petersen ◽  
Lars Franch Andersen ◽  
...  
Keyword(s):  
Live Birth ◽  
Intracytoplasmic Sperm Injection ◽  
Controlled Trial ◽  
Male Partner ◽  
In Vitro Fertilisation ◽  
Male Factor Infertility ◽  
Male Factor ◽  
Fertilisation Rate ◽  
Randomised Controlled

IntroductionOver the last decades, the use of intracytoplasmic sperm injection (ICSI) has increased, even among patients without male factor infertility. The increase has happened even though there is no evidence to support that ICSI results in higher live birth rates compared with conventional in vitro fertilisation (IVF) in cases with nonmale factor infertility. The lack of robust evidence on an advantage of using ICSI over conventional IVF in these patients is problematic since ICSI is more invasive, complex and requires additional resources, time and effort. Therefore, the primary objective of the IVF versus ICSI (INVICSI) study is to determine whether ICSI is superior to standard IVF in patients without severe male factor infertility. The primary outcome measure is first live birth from fresh and frozen-thawed transfers after one stimulated cycle. Secondary outcomes include fertilisation rate, ongoing pregnancy rate, birth weight and congenital anomalies.Methods and analysisThis is a two-armed, multicentre, randomised, controlled trial. In total, 824 couples/women with infertility without severe male factor will be recruited and allocated randomly into two groups (IVF or ICSI) in a 1:1 ratio. Participants will be randomised in variable block sizes and stratified by trial site and age. The main inclusion criteria are (1) no prior IVF/ICSI treatment, (2) male partner sperm with an expected count of minimum 2 million progressive motile spermatozoa following density gradient purification on the day of oocyte pick up and (3) age of the woman between 18 and 42 years.Ethics and disseminationThe study will be performed in accordance with the ethical principles in the Helsinki Declaration. The study is approved by the Scientific Ethical Committee of the Capital Region of Denmark. Study findings will be presented, irrespectively of results at international conferences and submitted for publication in peer-reviewed journals.Trial registration numberNCT04128904. Pre-results.

A targeted-replacement system for identification of signals for de novo methylation in Neurospora crassa

1994 ◽  
Vol 14 (11) ◽  
pp. 7059-7067
Author(s):  
V P Miao ◽  
M J Singer ◽  
M R Rountree ◽  
E U Selker
Keyword(s):  
Dna Methylation ◽  
Neurospora Crassa ◽  
De Novo ◽  
Gene Replacement ◽  
Position Effects ◽  
Efficient Gene ◽  
Bona Fide ◽  
Replacement System ◽  
Methylation Signal ◽  
De Novo Methylation

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.

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