91 Effect of heat stress during mice germ-cell DNA methylation programming on oocyte developmental competence: A preliminary study

2021 ◽  
Vol 33 (2) ◽  
pp. 153
Author(s):  
M. T. Moura ◽  
C. A. I. Carvalho ◽  
F. R. O. Barros ◽  
F. Mossa ◽  
D. Bebbere ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Heat Stress ◽  
Germ Cell ◽  
Cell Function ◽  
De Novo ◽  
Chorionic Gonadotrophin ◽  
Developmental Competence ◽  
Attractive Alternative ◽  
Oocyte Growth ◽  
Female Mice

Heat stress (HS) is characterised by an elevation in body temperature that ultimately undermines organism physiology. Most livestock production occurs in tropical regions under potential HS conditions that diminish productive and reproductive potential. Despite extensive evidence of HS-mediated effects in cell function, stage-specific detrimental effects of HS during oogenesis remain elusive. Mouse models represent an attractive alternative for faster interrogation of stage-specific phenomena during oogenesis. Therefore, the aim of the study was to determine the effects of HS exposure during the major window of female mice germ-cell DNA methylation programming. CD1/Swiss female mice with litters (F0 progeny) at postnatal Day 10 (P10) were randomly allocated to HS (35°C/12-h light; 21°C/12-h dark) or control (CTL: 21°C/24h) for 11 days. The F0 progeny were weaned at P21 and superovulated after reaching puberty at P35. F0 females were superovulated by intraperitoneal injections with 5.0IU of equine chorionic gonadotrophin (PMSG) followed by 5.0IU of human chorionic gonadotrophin (hCG) within a 48-h interval. Pre-implantation embryos were harvested at Day E3.5 in M2 medium under a stereomicroscope. One F0 female per litter was randomly mated to control mice when it reached 6 weeks of age. Data were subjected to least-squares analysis of variance using the General Linear Models procedure of SAS (SAS Institute Inc.). The experiment was replicated twice (CTL: n=4 F0 females and HS: n=4 F0 females). Preliminary results are given as LSM±s.e.m. There was no effect of heat stress on the number of embryos collected per female (CTL: 9.75±4.87 vs. HS: 11.25±4.81) or the percentage of non-viable embryos (CTL: 25.0±0.23% vs. HS: 42.5±0.25%). However, heat stress tended (P=0.07) to reduce the percentage of embryos that reached the morula stage from 63.5±0.08% for CTL to 35.1±0.09% for HS. The percentage of blastocysts collected (CTL: 11.45±0.18% vs. HS: 22.32±0.19%) and litter size of F0 females (CTL: 7.47±1.76 vs. HS: 7.66±1.47) was not affected by treatment. In conclusion, exposure of female mice to 11-day HS during the major wave of de novo DNA methylation during oocyte growth tended to reduce subsequent pre-implantation embryonic development, although it did not affect full-term development after natural mating.

scholarly journals Brown Fat Dnmt3b Deficiency Ameliorates Obesity in Female Mice

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1325
Author(s):  
Fenfen Li ◽  
Xin Cui ◽  
Jia Jing ◽  
Shirong Wang ◽  
Huidong Shi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Energy Expenditure ◽  
Knockout Mice ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Brown Fat ◽  
Chow Diet ◽  
Female Mice ◽  
Diet Induced Obesity

Obesity results from a chronic energy imbalance due to energy intake exceeding energy expenditure. Activation of brown fat thermogenesis has been shown to combat obesity. Epigenetic regulation, including DNA methylation, has emerged as a key regulator of brown fat thermogenic function. Here we aimed to study the role of Dnmt3b, a DNA methyltransferase involved in de novo DNA methylation, in the regulation of brown fat thermogenesis and obesity. We found that the specific deletion of Dnmt3b in brown fat promotes the thermogenic and mitochondrial program in brown fat, enhances energy expenditure, and decreases adiposity in female mice fed a regular chow diet. With a lean phenotype, the female knockout mice also exhibit increased insulin sensitivity. In addition, Dnmt3b deficiency in brown fat also prevents diet-induced obesity and insulin resistance in female mice. Interestingly, our RNA-seq analysis revealed an upregulation of the PI3K-Akt pathway in the brown fat of female Dnmt3b knockout mice. However, male Dnmt3b knockout mice have no change in their body weight, suggesting the existence of sexual dimorphism in the brown fat Dnmt3b knockout model. Our data demonstrate that Dnmt3b plays an important role in the regulation of brown fat function, energy metabolism and obesity in female mice.

Effect of lanosterol on the in vitro maturation in semi-defined culture system of prepubertal ewe oocytes

Zygote ◽  
2011 ◽  
Vol 22 (1) ◽  
pp. 50-57 ◽  
Author(s):  
F. Marco-Jiménez ◽  
J.S. Vicente ◽  
M.P. Viudes-de-Castro
Keyword(s):  
Oxygen Concentration ◽  
Cell Function ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Low Oxygen ◽  
Oocyte Growth ◽  
Important Indicator ◽  
Nuclear Maturation ◽  
Defined Culture

SummaryThe choice of medium and supplements can affect meiotic regulation and may have an impact on the regulation of mammalian oocyte growth and embryonic cell function. The aim of the present study was to assess the effects of oxygen concentration and endogenous lanosterol on the in vitro maturation (IVM) media without serum and based on recombinant human chorionic gonadotrophin in prepubertal ewe oocytes. Firstly, the effect of varying oxygen concentrations (5% and 20%) during IVM in TCM-199 supplemented (4 mg/ml bovine serum albumin (BSA), 100 μM cysteamine, 0.3 mM sodium pyruvate, 0.1 UI/ml recombinant follicle-stimulating hormone (r-FSH; Gonal-F® 75 UI, Serono, Italy), 0.1 UI/ml recombinant leuteinizing hormone (r-LH; Lhadi® 75 UI, Serono, Italy) and 1 μg/ml estradiol-17β) on subsequent nuclear maturation of oocytes examined under ultraviolet light following staining with bisbenzimide (Hoechst 33342) was investigated. Secondly, two concentrations of lanosterol (0, 10 and 50 μM) were added to the IVM medium. Nuclear maturation of oocytes was examined as previously. Lipid content in oocytes, an important indicator of cytoplasmic maturity, was also measured using Nile red fluorescent stain. The results showed that low oxygen concentration affected the nuclear maturation. Similarly, a significantly higher rate of meiosis resumption was observed with 10 μM (72.3%) of lanosterol compared with the control (51.8%) or 50 μM of lanosterol (59.4%). A significantly higher content of lipids was also observed with 10 and 50 μM of lanosterol (7.3 ± 0.2 × 106 and 7.4 ± 0.2 × 106 arbitrary units of fluorescence) compared with the control (6.7 ± 0.2 × 106 arbitrary units of fluorescence). The results indicate that 10 μM lanosterol during IVM in medium without serum and based on recombinant human chorionic gonadotrophin has a positive effect on maturation of prepubertal ewe oocytes.

Dynamics in the expression of epigenetic modifiers and histone modifications in perinatal rat germ cells during de novo DNA methylation†

2020 ◽  
Author(s):  
Arlette Rwigemera ◽  
Rhizlane El omri-Charai ◽  
Laetitia L Lecante ◽  
Geraldine Delbes
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
Histone Modifications ◽  
Germ Cells ◽  
Posttranslational Modifications ◽  
Fluorescent Protein ◽  
De Novo ◽  
Expression Patterns ◽  
Dna Methyltransferases ◽  
Epigenetic Modifiers

Abstract Epigenetic reprogramming during perinatal germ cell development is essential for genomic imprinting and cell differentiation; however, the actors of this key event and their dynamics are poorly understood in rats. Our study aimed to characterize the expression patterns of epigenetic modifiers and the changes in histone modifications in rat gonocytes at the time of de novo DNA methylation. Using transgenic rats expressing Green Fluorescent Protein (GFP) specifically in germ cells, we purified male gonocytes by fluorescent activated cell sorting at various stages of perinatal development and established the transcriptomic profile of 165 epigenetic regulators. Using immunofluorescence on gonad sections, we tracked six histone modifications in rat male and female perinatal germ cells over time, including methylation of histone H3 on lysines 27, 9, and 4; ubiquitination of histone H2A on lysine119; and acetylation of histone H2B on lysine 20. The results revealed the dynamics in the expression of ten-eleven translocation enzymes and DNA methyltransferases in male gonocytes at the time of de novo DNA methylation. Moreover, our transcriptomic data indicate a decrease in histone ubiquitination and methylation coinciding with the beginning of de novo DNA methylation. Decreases in H2AK119Ub and H3K27me3 were further confirmed by immunofluorescence in the male germ cells but were not consistent for all H3 methylation sites examined. Together, our data highlighted transient chromatin remodeling involving histone modifications during de novo DNA methylation. Further studies addressing how these dynamic changes in histone posttranslational modifications could guide de novo DNA methylation will help explain the complex establishment of the male germ cell epigenome.

scholarly journals Dppa2/4 Counteract De Novo Methylation to Establish a Permissive Epigenome for Development

2020 ◽  
Author(s):  
Kristjan H. Gretarsson ◽  
Jamie A. Hackett
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Developmental Competence ◽  
Epigenetic Memory ◽  
Mammalian Development ◽  
Dna Hypomethylation ◽  
Lineage Specification ◽  
Regulatory Pathways ◽  
Genome Wide ◽  
Early Mammalian Development

ABSTRACTEarly mammalian development entails genome-wide epigenome remodeling, including DNA methylation erasure and reacquisition, which facilitates developmental competence. To uncover the mechanisms that orchestrate DNA methylation (DNAme) dynamics, we coupled a single-cell ratiometric DNAme reporter with unbiased CRISPR screening in ESC. We identify key genes and regulatory pathways that drive global DNA hypomethylation, and characterise roles for Cop1 and Dusp6. We also identify Dppa2 and Dppa4 as essential safeguards of focal epigenetic states. In their absence, developmental genes and evolutionary-young LINE1 elements, which DPPA2 specifically binds, lose H3K4me3 and gain ectopic de novo DNA methylation in pluripotent cells. Consequently, lineage-associated genes (and LINE1) acquire a repressive epigenetic memory, which renders them incompetent for activation during future lineage-specification. Dppa2/4 thereby sculpt the pluripotent epigenome by facilitating H3K4me3 and bivalency to counteract de novo methylation; a function co-opted by evolutionary young LINE1 to evade epigenetic decommissioning.

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.

De novo DNA methylation: a germ cell perspective

2012 ◽  
Vol 28 (1) ◽  
pp. 33-42 ◽  
Author(s):  
Sébastien A. Smallwood ◽  
Gavin Kelsey
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
De Novo

Meiosis and embryo technology: renaissance of the nucleolus

2005 ◽  
Vol 17 (2) ◽  
pp. 3 ◽  
Author(s):  
Poul Maddox-Hyttel ◽  
Bolette Bjerregaard ◽  
Jozef Laurincik
Keyword(s):  
Topoisomerase I ◽  
De Novo ◽  
Germinal Vesicle ◽  
Developmental Competence ◽  
Rdna Transcription ◽  
Morphological Marker ◽  
Germinal Vesicle Breakdown ◽  
Oocyte Growth ◽  
Embryonic Genome

The nucleolus is the site of rRNA and ribosome production. This organelle presents an active fibrillogranular ultrastructure in the oocyte during the growth of the gamete but, at the end of the growth phase, the nucleolus is transformed into an inactive remnant that is dissolved when meiosis is resumed at germinal vesicle breakdown. Upon meiosis, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities harbour the development of fibrillogranular nucleoli and re-establishment of nucleolar function in conjunction with the major activation of the embryonic genome. This so-called nucleologenesis occurs at a species-specific time of development and can be classified into two different models: one where nucleolus development occurs inside the NPBs (e.g. cattle) and one where the nucleolus is formed on the surface of the NPBs (e.g. pigs). A panel of nucleolar proteins with functions during rDNA transcription (topoisomerase I, RNA polymerase I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) are localised to specific compartments of the oocyte nucleolus and those engaged in late processing are, to some degree, re-used for nucleologenesis in the embryo, whereas the others require de novo embryonic transcription in order to be allocated to the developing nucleolus. In the oocyte, inactivation of the nucleolus coincides with the acquisition of full meiotic competence, a parameter that may be of importance in relation to in vitro oocyte maturation. In embryo, nucleologenesis may be affected by technological manipulations: in vitro embryo production apparently has no impact on this process in cattle, whereas in the pig this technology results in impaired nucleologenesis. In cattle, reconstruction of embryos by nuclear transfer results in profound disturbances in nucleologenesis. In conclusion, the nucleolus is an organelle of great importance for the developmental competence of oocytes and embryos and may serve as a morphological marker for the completion of oocyte growth and normality of activation of the embryonic genome.

195 HEAT STRESS ALTERS THE TRANSCRIPTOME OF MATURING BOVINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 228
Author(s):  
L. A. Rispoli ◽  
R. R. Payton ◽  
C. Gondro ◽  
A. M. Saxton ◽  
J. L. Edwards
Keyword(s):  
Protein Synthesis ◽  
Heat Stress ◽  
Elevated Temperature ◽  
Mitochondrial Function ◽  
De Novo ◽  
Bovine Genome ◽  
Rna Extraction ◽  
Developmental Competence ◽  
The Impact ◽  
Bovine Oocytes

Direct exposure of maturing oocytes to a physiologically relevant elevated temperature reduces embryo development after fertilisation and has been coincident with reduced de novo protein synthesis. Mechanisms responsible for heat-induced reductions in protein synthesis are unknown but may be related to alterations in the transcriptome of the maturing oocyte. To determine the extent to which this may occur, the impact of heat stress on the maternal pool of RNA in bovine oocytes was assessed using microarrays. After maturation for 24 h at 38.5°C (control) or 41°C (first 12 h only, 38.5°C thereafter; heat stress) oocytes were denuded from associated cumulus cells and lysed for RNA extraction or underwent IVF to assess developmental competence. Total RNA from oocytes was amplified by 3′-poly(A) priming or a combination of 3′-poly(A) and internal priming because oocyte transcripts may or may not have a polyadenylated tail. Amplified RNA was hybridised to GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA, USA; 8 oocyte pools per treatment were collected on 7 different occasions and amplified by 2 methods; n = 32 chips). Differential transcript abundance was determined using R and Bioconductor with only probes having a P < 0.01, a fold change of at least 1.3, and called present for at least half the arrays. Functional annotation of selected transcripts was performed using Gene Ontology and KEGG annotations (Bos taurus build 4.0) and DAVID (v 6.7) with significance level set at P < 0.10. Coincident with reduced blastocyst development (28.3 v. 15.2% for control v. heat stress, respectively; SEM = 3.6; P < 0.0003), heat stress altered the abundance of 159 transcripts (22 increased, 137 decreased); 130 of these were annotated. Use of DAVID demonstrated enrichment of genes important for mitochondrial function and RNA processing. Towards validating certain findings, the relative abundance of 3 mitochondrial transcripts (NDUFC2, COQ3, ATP5O) were assessed by quantitative PCR on non-amplified RNA from the oocyte samples used for the microarray study. Gene-specific primers were designed for 5′ and 3′ ends of transcripts when possible. Exposure to elevated temperature during the first 12 h of oocyte maturation reduced transcript levels of NDUFC2 at the 5′ and 3′ ends (P < 0.0001 and P = 0.003), COQ3 at the 3′ end (P = 0.02) and ATP5O at the 5′ end (P = 0.02). In conclusion, exposure of maturing cumulus-oocyte complexes to a physiologically-relevant elevated temperature altered the transcriptome in oocytes, especially certain transcripts important for mitochondrial function. This research was supported in part by USDA National Institute of Food and Agriculture, Hatch Project No. 227701, the state of Tennessee through University of Tennessee AgResearch, Department of Animal Science, and East Tennessee Research and Education Center.

scholarly journals Microarray analysis of mRNA from cumulus cells following in vivo or in vitro maturation of mouse cumulus–oocyte complexes

2013 ◽  
Vol 25 (2) ◽  
pp. 426 ◽  
Author(s):  
Karen L. Kind ◽  
Kelly M. Banwell ◽  
Kathryn M. Gebhardt ◽  
Anne Macpherson ◽  
Ashley Gauld ◽  
...  
Keyword(s):  
Microarray Analysis ◽  
Cell Function ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Chorionic Gonadotrophin ◽  
Developmental Competence ◽  
Rt Pcr ◽  
Oocyte Competence

The IVM of mammalian cumulus–oocyte complexes (COCs) yields reduced oocyte developmental competence compared with oocytes matured in vivo. Altered cumulus cell function during IVM is implicated as one cause for this difference. We have conducted a microarray analysis of cumulus cell mRNA following IVM or in vivo maturation (IVV). Mouse COCs were sourced from ovaries of 21-day-old CBAB6F1 mice 46 h after equine chorionic gonadotrophin (5 IU, i.p.) or from oviducts following treatment with 5 IU eCG (61 h) and 5 IU human chorionic gonadotrophin (13 h). IVM was performed in α-Minimal Essential Medium with 50 mIU FSH for 17 h. Three independent RNA samples were assessed using the Affymetrix Gene Chip Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA, USA). In total, 1593 genes were differentially expressed, with 811 genes upregulated and 782 genes downregulated in IVM compared with IVV cumulus cells; selected genes were validated by real-time reverse transcription–polymerase chain reaction (RT-PCR). Surprisingly, haemoglobin α (Hba-a1) was highly expressed in IVV relative to IVM cumulus cells, which was verified by both RT-PCR and western blot analysis. Because haemoglobin regulates O2 and/or nitric oxide availability, we postulate that it may contribute to regulation of these gases during the ovulatory period in vivo. These data will provide a useful resource to determine differences in cumulus cell function that are possibly linked to oocyte competence.

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