Julia Heinzmann

Epigenetic profile of developmentally important genes in bovine oocytes.

Assisted reproductive technologies are associated with an increased incidence of epigenetic aberrations, specifically in imprinted genes. Here, we used the bovine oocyte as a model to determine putative epigenetic mutations at three imprinted gene loci caused by the type of maturation, either in vitro maturation (IVM) in Tissue Culture Medium 199 (TCM) or modified synthetic oviduct fluid (mSOF) medium, or in vivo maturation. We applied a limiting dilution approach and direct bisulfite sequencing to analyze the methylation profiles of individual alleles (DNA molecules) for H19/IGF2, PEG3, and SNRPN, which are each associated with imprinting defects in humans and/or the mouse model, and are known to be differentially methylated in bovine embryos. Altogether, we obtained the methylation patterns of 203 alleles containing 4,512 CpG sites from immature oocytes, 213 alleles with 4,779 CpG sites from TCM‐matured oocytes, 215 alleles/4,725 CpGs in mSOF‐matured oocytes, and 78 alleles/1,672 CpGs from in vivo‐matured oocytes. The total rate of individual CpGs and entire allele methylation errors did not differ significantly between the two IVM and the in vivo group, indicating that current IVM protocols have no or only marginal effects on these critical epigenetic marks. Furthermore, the mRNA expression profiles of the three imprinted genes and a panel of eight other genes indicative of oocyte competence were determined by quantitative real‐time PCR. We found different mRNA expression profiles between in vivo‐matured oocytes versus their in vitro‐matured counterparts, suggesting an influence on regulatory mechanisms other than DNA methylation. Mol. Reprod. Dev. 78:188–201, 2011.

Desorption Electrospray Ionization Mass Spectrometry Reveals Lipid Metabolism of Individual Oocytes and Embryos.

Alteration of maternal lipid metabolism early in development has been shown to trigger obesity, insulin resistance, type 2 diabetes and cardiovascular diseases later in life in humans and animal models. Here, we set out to determine (i) lipid composition dynamics in single oocytes and preimplantation embryos by high mass resolution desorption electrospray ionization mass spectrometry (DESI-MS), using the bovine species as biological model, (ii) the metabolically most relevant lipid compounds by multivariate data analysis and (iii) lipid upstream metabolism by quantitative real-time PCR (qRT-PCR) analysis of several target genes (ACAT1, CPT 1b, FASN, SREBP1 and SCAP). Bovine oocytes and blastocysts were individually analyzed by DESI-MS in both positive and negative ion modes, without lipid extraction and under ambient conditions, and were profiled for free fatty acids (FFA), phospholipids (PL), cholesterol-related molecules, and triacylglycerols (TAG). Principal component analysis (PCA) and linear discriminant analysis (LDA), performed for the first time on DESI-MS fused data, allowed unequivocal discrimination between oocytes and blastocysts based on specific lipid profiles. This analytical approach resulted in broad and detailed lipid annotation of single oocytes and blastocysts. Results of DESI-MS and transcript regulation analysis demonstrate that blastocysts produced in vitro and their in vivo counterparts differed significantly in the homeostasis of cholesterol and FFA metabolism. These results should assist in the production of viable and healthy embryos by elucidating in vivo embryonic lipid metabolism.

Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes.

To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.

Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality.

Cyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

Characterization of Differentially Methylated Regions in 3 Bovine Imprinted Genes: A Model for Studying Human Germ-Cell and Embryo Development

Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie –6 kb to –3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.

Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency.

Extended in vitro maturation affects gene expression and DNA methylation in bovine oocytes

To mimic post-ovulatory ageing, we have extended the in vitro maturation (IVM) phase to 48 h and examined effects on (i) developmental potential, (ii) expression of a panel of developmentally important genes and (iii) gene-specific epigenetic marks. Results were compared with the 24 h IVM protocol (control) usually employed for bovine oocytes. Cleavage rates and blastocyst yields were significantly reduced in oocytes after extended IVM. No significant differences were observed in the methylation of entire alleles in oocytes for the genes bH19, bSNRPN, bZAR1, bOct4 and bDNMT3A. However, we found differentially methylated CpG sites in the bDNMT3Ls locus in oocytes after extended IVM and in embryos derived from them compared with controls. Moreover, embryos derived from the 48 h matured oocyte group were significantly less methylated at CpG5 and CpG7 compared with the 24 h group. CpG7 was significantly hypermethylated in embryos produced from the control oocytes, but not in oocytes matured for 48 h. Furthermore, methylation for CpG5-CpG8 of bDNMT3Ls was significantly lower in oocytes of the 24 h group compared with embryos derived therefrom, whereas no such difference was found for oocytes and embryos of the in vitro aged group. Expression of most of the selected genes was not affected by duration of IVM. However, transcript abundance for the imprinted gene bIGF2R was significantly reduced in oocytes analyzed after extended IVM compared with control oocytes. Transcript levels for bPRDX1, bDNMT3A and bBCLXL were significantly reduced in 4- to 8-cell embryos derived from in vitro aged oocytes. These results indicate that extended IVM leads to ageing-like alterations and demonstrate that epigenetic mechanisms are critically involved in ageing of bovine oocytes, which warrants further studies into epigenetic mechanisms involved in ageing of female germ cells, including humans.

Effects of long-term dietary supplementation with conjugated linoleic acid on bovine oocyte lipid profile

Nutritional and environmental conditions around conception and during early embryonic development may have significant effects on health and well-being in adult life. Here, a bovine heifer model was used to investigate the effects of rumen-protected fat supplementation on oocyte quality and embryo development. Holstein-Friesian heifers (n=84) received a dietary supplement consisting of rumen-protected conjugated linoleic acid (CLA) or stearic acid (SA), each on top of an isocaloric basic diet. Oocytes were collected via ultrasound-guided follicular aspiration and subjected to in vitro maturation followed by either desorption electrospray ionisation mass spectrometry (DESI-MS) for lipid profiling of individual oocytes or in vitro fertilisation and embryo culture. The type of supplement significantly affected lipid profiles of in vitro-matured oocytes. Palmitic acid and plasmalogen species were more abundant in the mass spectra of in vitro-matured oocytes after rumen-protected SA supplementation when compared with those collected from animals supplemented with CLA. Lipid concentrations in blood and follicular fluid were significantly affected by both supplements. Results show that rumen-protected fatty-acid supplementation affects oocyte lipid content and may pave the way for the establishment of a large-animal model for studies towards a better understanding of reproductive disorders associated with nutritional impairments.

DNA methylation and mRNA expression of developmentally important genes in bovine oocytes collected from donors of different age categories.

Epigenetic changes are critical for the acquisition of developmental potential by oocytes and embryos, yet these changes may be sensitive to maternal ageing. Here, we investigated the impact of maternal ageing on DNA methylation and mRNA expression in a panel of eight genes that are critically involved in oocyte and embryo development. Bovine oocytes were collected from donors of three different age categories—prepubertal (9–12 months old), mature (3–7 years old), and aged (8–11 years old)—and were analyzed for gene‐specific DNA methylation (bTERF2, bREC8, bBCL‐XL, bPISD, bBUB1, bDNMT3Lo, bH19, and bSNRPN) and mRNA expression (bTERF2, bBCL‐XL, bPISD, and bBUB1). A total of 1,044 alleles with 88,740 CpGs were amplified and sequenced from 362 bovine oocytes. Most of the detected molecules were either fully methylated or completely unmethylated. Only 9 out of 1,044 alleles (<1%) were abnormally methylated (>50% of CpGs with an aberrant methylation status), and seven of the nine abnormally methylated alleles were within only two candidate genes (bDNMT3Lo and bH19). No significant differences were detected with regard to mRNA expression between oocytes from the three groups of donors. These results suggest that genes predominantly important for early embryo development (bH19 and bDNMT3Lo) are less resistant to abnormal methylation than genes critically involved in oocyte development (bTERF2, bBCL‐XL, bPISD, bBUB1, and bSNRPN). Establishment of DNA methylation in bovine oocytes seems to be largely resistant to changes caused by maternal ageing, irrespective of whether the genes are critical to achieve developmental competence in oocytes or early embryos. Mol. Reprod. Dev. 83: 802–814, 2016

Gene-specific profiling of DNA methylation and mRNA expression in bovine oocytes derived from follicles of different size categories.

Epigenetic changes, such as DNA methylation, play an essential role in the acquisition of full developmental competence by mammalian oocytes during the late follicular growth phase. Here we used the bovine model to investigate the DNA methylation profiles of seven candidate genes (imprinted: bH19, bSNRPN; non-imprinted: bZAR1, bDNMT3A, bOCT4, bDNMT3 Lo and bDNMT3 Ls) and the mRNA expression of nine candidate genes (imprinted: bSNRPN, bPEG3, bIGF2R; non-imprinted: bPRDX1, bDNMT1B, bDNMT3A, bZAR1, bHSF1 and bNLRP9) in oocytes from antral follicles of three different size classes (≤2mm, 3-5mm, ≥6mm) to unravel the epigenetic contribution to this process. We observed an increased number of aberrantly methylated alleles in bH19, bSNRPN and bDNMT3 Lo of oocytes from small antral follicles (≤2mm), correlating with lower developmental competence. Furthermore, we detected an increased frequency of CpG sites with an unclear methylation status for DNMT3 Ls, specifically in oocytes from follicles ≥6mm, predominantly at three CpG positions (CpG2, CpG7 and CpG8), of which CpG7 is a potential regulatory site. No major differences in mRNA expression were observed, indicating that the transcriptional machinery may not yet be active during the follicular growth phase. Our results support the notion that a follicle diameter of ~2mm is a critical stage for establishing DNA methylation profiles and indicate a link between DNA methylation and the acquisition of oocyte developmental competence.