Biomedical subjects

Stefan Hiendleder

Publications and source records attributed to Stefan Hiendleder.

13 recordsLinked to original sources

PubMed2007

Mitochondrial DNA inheritance after SCNT.

Mitochondrial biogenesis and function is under dual genetic control and requires extensive interaction between biparentally inherited nuclear genes and maternally inherited mitochondrial genes. Standard SCNT procedures deprive an oocytes' mitochondrial DNA (mtDNA) of the corresponding maternal nuclear DNA and require it to interact with an entirely foreign nucleus that is again interacting with foreign somatic mitochondria. As a result, most SCNT embryos, -fetuses, and -offspring carry somatic cell mtDNA in addition to recipient oocyte mtDNA, a condition termed heteroplasmy. It is thus evident that somatic cell mtDNA can escape the selective mechanism that targets and eliminates intraspecific sperm mitochondria in the fertilized oocyte to maintain homoplasmy. However, the factors responsible for the large intra- and interindividual differences in heteroplasmy level remain elusive. Furthermore, heteroplasmy is probably confounded with mtDNA recombination. Considering the essential roles of mitochondria in cellular metabolism, cell signalling, and programmed cell death, future experiments will need to assess the true extent and impact of unorthodox mtDNA transmission on various aspects of SCNT success.

Animals↗

PubMed2006

Tissue-specific effects of in vitro fertilization procedures on genomic cytosine methylation levels in overgrown and normal sized bovine fetuses.

Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo-fertilized controls (n=14) indicated an apparently normal phenotype for IVF1 fetuses (n=5), but IVF2 fetuses (n=10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (-16.1% and -9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

5-Methylcytosine↗

PubMed2006

A bovine oviduct epithelial cell suspension culture system suitable for studying embryo-maternal interactions: morphological and functional characterization.

We established a short-term (24 h) culture system for bovine oviduct epithelial cells (BOECs), obtained on day 3.5 of the estrous cycle and evaluated the cells with respect to morphological criteria, marker gene expression, and hormone responsiveness. BOEC sheets were isolated mechanically from the ampulla with similar yields from oviducts ipsi- and contralateral to the ovulation site (57.9 +/- 4.6 and 56.4 +/- 8.0 x 10(6) cells). BOECs showed > 95% purity and cells cultured for 24 h maintained morphological characteristics present in vivo, as determined by light microscopy, scanning electron microscopy, and transmission electron microscopy. Both secretory cells with numerous secretory granules and ciliated cells with long, well-developed, and vigorously beating kinocilia were visible. Quantitative real-time PCR failed to detect significant differences in transcript levels between ipsi-and contralateral BOECs for the majority of marker genes (estrogen receptors alpha and beta (ESR1 and ESR2), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), oviductal glycoprotein 1 (OVGP1), progesterone receptor (PGR), and tumor rejection antigen 1 (TRA1)) throughout the 24 h culture period. However, the combined data of all time points for glutathione peroxidase 4 (GPX4), a gene previously shown to be expressed at higher levels in the ipsilateral oviduct in vivo, also indicated significantly different mRNA levels in vitro. The expression of marker genes remained stable after 6 h cell culture, indicating only a short adaptation period. Western blot analysis confirmed ESR1 and PGR protein expression throughout the culture period. In agreement with cyclic differences in vivo, estradiol-17beta stimulation increased PGR transcript abundance in BOECs. Our novel culture system provides functional BOECs in sufficient quantities for holistic transcriptome and proteome studies, e.g. for deciphering early embryo-maternal communication.

Animals↗

PubMed2005

Mitochondria and the success of somatic cell nuclear transfer cloning: from nuclear-mitochondrial interactions to mitochondrial complementation and mitochondrial DNA recombination.

The overall success of somatic cell nuclear transfer (SCNT) cloning is rather unsatisfactory, both in terms of efficacy and from an animal health and welfare point of view. Most research activities have concentrated on epigenetic reprogramming problems as one major cause of SCNT failure. The present review addresses the limited success of mammalian SCNT from yet another viewpoint, the mitochondrial perspective. Mitochondria have a broad range of critical functions in cellular energy supply, cell signalling and programmed cell death and, thus, affect embryonic and fetal development, suggesting that inadequate or perturbed mitochondrial functions may adversely affect SCNT success. A survey of perinatal clinical data from human subjects with deficient mitochondrial respiratory chain activity has revealed a plethora of phenotypes that have striking similarities with abnormalities commonly encountered in SCNT fetuses and offspring. We discuss the limited experimental data on nuclear-mitochondrial interaction effects in SCNT and explore the potential effects in the context of new findings about the biology of mitochondria. These include mitochondrial fusion/fission, mitochondrial complementation and mitochondrial DNA recombination, processes that are likely to be affected by and impact on SCNT cloning. Furthermore, we indicate pathways that could link epigenetic reprogramming and mitochondria effects in SCNT and address questions and perspectives for future research.

Animals↗

PubMed2005

Nucleotide sequence and variation of IGF2 gene exon 6 in Bos taurus and Bos indicus cattle.

The major assumption of this study is that polymorphism of a gene could be used to investigate its allele-specific expression as well as its methylation and imprinting status. Therefore, the aim of this study was to analyze the polymorphism of the coding region of the bovine IGF2 gene and to determine the sequence of its gene exon 6 in Bos taurus and Bos indicus cattle. A single nucleotide "C" deletion/insertion polymorphism was found in both cattle subspecies and a G/T transversion (RFLP-MboII) in the Bos indicus IGF2 gene. A 407-bp fragment of bovine IGF2 exon 6 was sequenced and the sequences (including variable nucleotides) were deposited in the GenBank database. A comparative analysis was performed for this fragment from different species; 99.5% identity was found between Bos taurus and Bos indicus cattle.

Animals↗

PubMed2004

Tissue-specific elevated genomic cytosine methylation levels are associated with an overgrowth phenotype of bovine fetuses derived by in vitro techniques.

Epigenetic perturbations are assumed to be responsible for abnormalities observed in fetuses and offspring derived by in vitro techniques. We have designed an experiment with bovine Day 80 fetuses generated by somatic cell nuclear transfer (SCNT), in vitro fertilization (IVF), and artificial insemination (AI) to determine the relationship between fetal phenotype and genome-wide 5-methylcytosine (5mC) content. When compared with AI controls, SCNT and IVF fetuses displayed significantly increased body weight (61% and 28%), liver weight (100% and 36%), and thorax circumference (20% and 11%). A reduced crown-rump length:thorax circumference ratio (1.175 +/- 0.017 in SCNT and 1.292 +/- 0.018 in IVF vs. 1.390 +/- 0.018 in AI, P < 0.001 and P < 0.002) was the external hallmark of this disproportionate overgrowth phenotype. The SCNT fetuses showed significant hypermethylation of liver DNA in comparison with AI controls (3.46% +/- 0.08% vs. 3.17% +/- 0.09% 5mC, P < 0.03), and the cytosine methylation levels for IVF fetuses (3.34% +/- 0.09%) were, as observed for phenotypic parameters, intermediate to the other groups. Regressions of fetal body and liver weight and thorax circumference on 5mC content of liver DNA were positive (P < 0.073-0.079). Furthermore, a significant negative regression (P < 0.021) of the crown-rump length:thorax circumference ratio on liver 5mC was observed. The 5mC content of placental cotyledon DNA was 46% lower than in liver DNA (P < 0.0001) but did not differ among groups. These data are in striking contrast with the recently reported hypomethylation of DNA from SCNT fetuses and indicate that hypermethylation of fetal tissue, but not placenta, is linked to the overgrowth phenotype in bovine SCNT and IVF fetuses.

5-Methylcytosine↗

PubMed2004

Maternal-fetal transplacental leakage of mitochondrial DNA in bovine nuclear transfer pregnancies: potential implications for offspring and recipients.

The synepitheliochorial placenta of ruminants is constructed of multiple tissue layers that separate maternal and fetal blood. In nuclear transfer cloned ruminants, however, placental anomalies such as abnormal vascular development and hemorrhagic cotyledons have been reported. We have investigated the possible exchange of genetic material between somatic cell nuclear transfer cloned (SCNT) bovine fetuses and recipients at day 80 of gestation using mitochondrial DNA (mtDNA) as a marker. Twenty-three recovered SCNT-fetuses and their recipients were screened for divergent and thus informative mtDNA combinations. Twenty-one fetuses generated by in vitro fertilization (IVF) or multiple ovulation embryo transfer (MOET) and the corresponding recipients served as controls. A search for recipient mtDNA haplotype in DNA extracts from fetal blood by PCR-RFLP analysis revealed three cases of chimerism (two SCNT, one IVF) among a total of 19 informative fetus-recipient pairs (eight SCNT, seven IVF, four MOET). Placental anomalies have also been observed in some IVF fetuses and the present data therefore suggests transplacental leakage of cell components or cells from the recipient into some fetuses generated by in vitro techniques. Further studies are necessary to determine (i) the nature of leaked material, (ii) whether there is bi-directional leakage, and (iii) whether leaked material is present in recipients and calves after parturition, i.e. whether leakage takes place in vivo. If recipients were chimeric for DNA or cells derived from genetically modified SCNT (or IVF) embryos, their subsequent utilization might be affected.

Animals↗

PubMed2003

Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype, and cellular metabolism of bovine nuclear transfer fetuses.

We generated a clone of bovine somatic cell nuclear transfer embryos using oocyte pools from defined maternal sources to study nuclear-cytoplasmic interactions. Nucleocytoplasmic hybrids were reconstructed with Bos taurus (Brown Swiss) granulosa cells and oocytes that contained B. taurus A (Simmental), B. taurus B (Simmental), or Bos indicus (Dwarf Zebu) cytoplasm. Another set of embryos was reconstructed with randomly selected Brown Swiss (B. taurus R) oocytes. Embryo transfer resulted in nine (12.5%), nine (13.8%), three (50%), and 11 (16.7%) Day 80 fetuses, of which eight (11.1%), three (4.6%), three (50%), and 10 (15.2%) were viable, respectively. The proportion of viable fetuses was affected by cytoplasm (likelihood ratio test, P < 0.02) and was higher for embryos with B. indicus cytoplasm than for the B. taurus A (P < 0.05) and B (P < 0.01) groups. Furthermore, the proportion of surviving Day 80 fetuses was reduced for B. taurus B as compared with B. taurus A and B. taurus R cytoplasm (P < 0.05 and P < 0.02). Body weight of nucleocytoplasmic hybrid fetuses was not significantly different from Brown Swiss control fetuses produced by artificial insemination (AI), but fetuses reconstructed with random cytoplasts of the same breed as the nuclear donor exhibited overgrowth (P < 0.01) and a higher coefficient of variation in weight. Furthermore, body weight, crown rump length, thorax circumference (P < 0.05), and femur length (P < 0.01) of fetuses with B. taurus A cytoplasm differed from fetuses with B. taurus R cytoplasms. Fetal skin, heart, and liver cells with B. indicus cytoplasm showed a greater increase in number per time period (P < 0.001) and oxygen consumption rate per cell (skin and liver, P < 0.001; heart, P < 0.08) in comparison with their counterparts with B. taurus A cytoplasm. These data point to complex oocyte cytoplasm-dependent epigenetic modifications and/or nuclear DNA-mitochondrial DNA interactions with relevance to nuclear transfer and other reproductive technologies such as ooplasmic transfer in human assisted reproduction.

Animals↗

PubMed2003

Heteroplasmy in bovine fetuses produced by intra- and inter-subspecific somatic cell nuclear transfer: neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood.

Varying degrees of mitochondrial DNA (mtDNA) heteroplasmy have been observed in nuclear transfer embryos, fetuses, and offspring, but the mechanisms leading to this condition are unknown. We have generated a clone of 12 bovine somatic cell nuclear transfer fetuses, using nuclear donor cells, recipient oocytes, and recipient heifers with defined mtDNA genotypes, to study nuclear-mitochondrial interactions and the origins of mtDNA heteroplasmy. Embryos were reconstructed from granulosa cells with Bos taurus mtDNA type A and recipient oocytes collected from three different maternal lineages with B. taurus mtDNA type B, B. taurus mtDNA type C, or B. indicus mtDNA. Sequence differences in the control region (CR) of B. taurus mtDNAs ranged from 6 to 11 nucleotides and differences between B. taurus and B. indicus CRs from 45 to 50 nucleotides. Fetuses were recovered from recipient heifers with B. taurus mtDNA type B on Day 80 after nuclear transfer (eight B. taurus A/B, two B. taurus A/C, and two B. taurus A/B. indicus). Agarose gel analysis of the CR by polymerase chain reaction-based restriction fragment length polymorphism failed to detect nuclear donor mtDNA in 11 investigated tissues of 10 viable fetuses and in DNA samples of two fetuses in resorption (one B. taurus A/B and one B. taurus A/C). A more sensitive analysis of 1801 plasmid clones with CR inserts derived from tissues of a B. taurus A/B. indicus fetus detected no or very low levels of heteroplasmy (0.5-0.7%). However, the analyses detected considerable amounts ( approximately 2.5% and 5%) of recipient heifer mtDNA in blood samples from two fetuses. Our data do not suggest a replicative advantage of somatic nuclear donor cell mtDNA in bovine transmitochondrial clones produced with oocytes from domestic forms of the same or a different aurochs (B. primigenius) subspecies. Detection of mtDNA from the recipient animal in the circulation of two fetuses points to leakage of the placental barrier, mimicking heteroplasmy.

Animals↗

PubMed2002

Molecular analysis of wild and domestic sheep questions current nomenclature and provides evidence for domestication from two different subspecies.

Complete mitochondrial DNA (mtDNA) control regions (CR) were sequenced and analysed in order to investigate wild sheep taxonomy and the origin of domestic sheep (Ovis aries). The dataset for phylogenetic analyses includes 63 unique CR sequences from wild sheep of the mouflon (O. musimon, O. orientalis), urial (O. vignei), argali (O. ammon) and bighorn (O. canadensis) groups, and from domestic sheep of Asia, Europe and New Zealand. Domestic sheep occurred in two clearly separated branches with mouflon (O. musimon) mixed into one of the domestic sheep clusters. Genetic distances and molecular datings based on O. canadensis CR and mtDNA protein-coding sequences provide strong evidence for domestications from two mouflon subspecies. Other wild sheep sequences are in two additional well-separated branches. Ovis ammon collium and O. ammon nigrimontana are joined with a specimen from the transkaspian Ust-Urt plateau currently named O. vignei arkal. Ovis ammon ammon, O. ammon darwini and O. vignei bochariensis represent a separate clade and the earliest divergence from the mouflon group. Therefore, O. musimon, O. vignei bochariensis and Ust-Urt sheep are not members of a 'moufloniform' or O. orientalis species, but belong to different clades. Furthermore, Ust-Urt sheep could be a hybrid population or an O. ammon subspecies closely related to O. ammon nigrimontana.

Animals↗

PubMed2002

Bovine somatic cell nuclear transfer using recipient oocytes recovered by ovum pick-up: effect of maternal lineage of oocyte donors.

The efficiency of bovine nuclear transfer using recipient oocytes recovered by ultrasound-guided follicle aspiration (ovum pick-up [OPU]) was investigated. Oocyte donors were selected from 2 distinct maternal lineages (A and B) differing in 11 nucleotide positions of the mitochondrial DNA control region. A total of 1342 cumulus-oocyte complexes (COCs) were recovered. The numbers of total COCs and class I/II COCs recovered from donors of lineage A were higher (P < 0.001) than those obtained from lineage B. Follicle aspiration once per week yielded a higher (P < 0.001) total number of COCs per session than aspiration twice per week, whereas the reproduction status of donors (heifer vs. cow) had no effect on OPU results. Of the 1342 oocytes recovered, 733 (55%) were successfully matured in vitro and used for nuclear transfer. Fusion was achieved in 550 (75%) karyoplast-cytoplast complexes (KCCs), resulting in 277 (50%) cleaved embryos on Day 3. On Day 7 of culture, 84 transferable embryos (15% based on fused KCCs) were obtained. After 38 transfers (10 single, 22 double, and 6 triple transfers), 9 recipients (8 double and 1 triple transfer) were diagnosed as pregnant on Day 28, corresponding to a pregnancy rate of 24%. The proportion of transferable embryos on Day 7 was significantly (P < 0.05) influenced by maternal lineage of oocyte donors and by the frequency of follicle aspiration. Our study demonstrates the feasibility of generating nuclear transfer embryos with defined cytoplasmic background. These will be valuable tools to experimentally dissect the effects of nuclear and cytoplasmic components on embryonic, fetal, and postnatal development.

Animals↗

PubMedDate not supplied

Combined analysis of data from two granddaughter designs: A simple strategy for QTL confirmation and increasing experimental power in dairy cattle.

A joint analysis of five paternal half-sib Holstein families that were part of two different granddaughter designs (ADR- or Inra-design) was carried out for five milk production traits and somatic cell score in order to conduct a QTL confirmation study and to increase the experimental power. Data were exchanged in a coded and standardised form. The combined data set (JOINT-design) consisted of on average 231 sires per grandsire. Genetic maps were calculated for 133 markers distributed over nine chromosomes. QTL analyses were performed separately for each design and each trait. The results revealed QTL for milk production on chromosome 14, for milk yield on chromosome 5, and for fat content on chromosome 19 in both the ADR- and the Inra-design (confirmed within this study). Some QTL could only be mapped in either the ADR- or in the Inra-design (not confirmed within this study). Additional QTL previously undetected in the single designs were mapped in the JOINT-design for fat yield (chromosome 19 and 26), protein yield (chromosome 26), protein content (chromosome 5), and somatic cell score (chromosome 2 and 19) with genomewide significance. This study demonstrated the potential benefits of a combined analysis of data from different granddaughter designs.

Animals↗

PubMedDate not supplied

[Methods for transcriptome and proteome research: applications for studying the biology of reproduction in cattle].

Improvements of animal health, welfare and product quality are major goals of modern animal breeding. Thus, in addition to the classical production traits, functional traits such as disease resistance, fertility and longevity moved into the center of animal breeder's interests. Due to their low heritability, the improvement of functional traits using conventional approaches of phenotypic testing and quantitative genetics is difficult. A number of studies have been conducted worldwide in various species to map quantitative trait loci (QTLs) and to identify genetic markers for health traits. This has revealed a plethora of chromosome regions which may harbor genes with relevance for animal health. Functional genome research integrates holistic investigations at the level of the genome, at the level of gene activity (transcriptome, proteome) and at various levels of phenotypic expression. The integration of all these levels of information provides the basis for the functional dissection of complex traits. This review provides an overview of the most important strategies for holistic transcriptome and proteome analyses. The successful application of these techniques is exemplified by our studies of bovine reproductive biology.

Animals↗