Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal

The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p < 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 µsec (19 ± 2% vs. 77 ± 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 µsec, although this was still lower than the rate of fusion in the CCCs (33 ± 1% vs. 80 ± 2%). The rates of cleavage (57 ± 5%) and blastocyst formation (1 ± 1%) in the DCC-derived embryos did not differ from those (55 ± 6% and 3 ± 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 ± 2%) showed higher levels of blastocyst formation (p < 0.01) than CCC-derived autologous SCNT embryos (1 ± 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts

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 (~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.Stefan Hiendleder, Valeri Zakhartchenko, Hendrik Wenigerkind, Horst-Dieter Reichenbach, Katja Brüggerhoff, Katja Prelle, Gottfried Brem, Miodrag Stojkovic, and Eckhard Wol

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.Stefan Hiendleder, Katja Prelle, Katja Brüggerhoff, Horst-Dieter Reichenbach, Hendrik Wenigerkind, Daniela Bebbere, Miodrag Stojkovic, Sigrid Müller, Gottfried Brem, Valeri Zakhartchenko, and Eckhard Wol

Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors

Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell’s mitochondrial DNA (mtDNA) in favour of the recipient oocyte’s genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.1460.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (,0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 mM 29,39- dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P,0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.9962.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos

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.Katja Brüggerhoff, Valeri Zakhartchenko, Hendrik Wenigerkind, Horst-Dieter Reichenbach, Katja Prelle, Wolfgang Schernthaner, Ramiro Alberio, Helmut Küchenhoff, Miodrag Stojkovic, Gottfried Brem, Stefan Hiendleder, and Eckhard Wol

Effects of Air Pollution on Materials and Cultural Heritage: ICP Materials Celebrates 25 Years of Research

An overview is given of all results from the International Co-operative Programme on Effects on Materials including Historic and Cultural Monuments (ICP Materials), which was launched in 1985. Since then, about twenty different materials have been exposed repeatedly in a network of test sites consisting of more than twenty sites with an extensive environmental characterisation and more than sixty official reports have been issued. Recent results on trends in corrosion, soiling, and pollution show that corrosion of carbon steel, zinc, and limestone is today substantially lower than 25 years ago, but while corrosion of carbon steel has decreased until today, corrosion of zinc and limestone has remained more or less constant since the turn of the century. Unique data are given on measured HNO3 concentrations from 2002-2003, 2005-2006, and 2008-2009, and the relative average decrease was about the same from 2002-2003 to 2005-2006 as it was from 2005-2006 to 2008-2009