Miodrag Stojkovic

Epigenetic Marking Correlates with Developmental Potential in Cloned Bovine Preimplantation Embryos

During differentiation, somatic nuclei acquire highly specialized DNA and chromatin modifications, which are thought to result in cellular memory of the differentiated state. Upon somatic nuclear transfer into oocytes, the donor nucleus may have to undergo reprogramming of these epigenetic marks in order to achieve totipotency. This may involve changes in epigenetic features similar to those that occur in normal embryos during early development. However, there is accumulating evidence that epigenetic reprogramming is severely deficient in cloned embryos. Several reports reveal inefficient demethylation and inappropriate reestablishment of DNA methylation in quantitative and qualitative patterns on somatic nuclear transfer. Here we examine histone H3 lysine 9 (H3-K9) methylation and acetylation in normal embryos and in those created by somatic nuclear transfer. We find that H3-K9 methylation is reprogrammed in parallel with DNA methylation in normal embryos. However, the majority of cloned embryos exhibit H3-K9 hypermethylation associated with DNA hypermethylation, suggesting a genome-wide failure of reprogramming. Strikingly, the precise epigenotype in cloned embryos depends on the donor cell type, and the proportion of embryos with normal epigenotypes correlates closely with the proportion developing to the blastocyst stage. These results suggest a mechanistic link between DNA and histone methylation in the mammalian embryo and reveal an association between epigenetic marks and developmental potential of cloned embryos.

Mitochondrial Distribution and Adenosine Triphosphate Content of Bovine Oocytes Before and After In Vitro Maturation: Correlation with Morphological Criteria and Developmental Capacity After In Vitro Fertilization and Culture

Abstract In this study, we evaluated mitochondrial distribution and ATP content of individual bovine oocytes before and after in vitro maturation (IVM). Cumulus-oocyte complexes were classified according to morphological criteria: category 1, homogeneous oocyte cytoplasm, compact multilayered cumulus oophorus; category 2, cytoplasm with small inhomogeneous areas, more than five layers of compact cumulus; category 3, heterogeneous/vacuolated cytoplasm, three to five layers of cumulus including small areas of denuded zona pellucida; category 4, heterogeneous cytoplasm, completely or in great part denuded. In immature oocytes, staining with MitoTracker green revealed mitochondrial clumps in the periphery of the cytoplasm, with a strong homogenous signal in category 1 oocytes, a weaker staining in category 2 oocytes, allocation of mitochondria around vacuoles in category 3 oocytes, and poor staining of mitochondria in category 4 oocytes. After IVM, mitochondrial clumps were allocated more toward the center, became larger, and stained more intensive in category 1 and 2 oocytes. This was also true for category 3 oocytes; however, mitochondria maintained their perivacuolar distribution. No mitochondrial reorganization was seen for category 4 oocytes. Before IVM, the average ATP content of category 1 oocytes (1.8 pmol) tended to be higher than that of category 2 oocytes (1.6 pmol) and was significantly (P < 0.01) higher than in category 3 (1.4 pmol) and 4 oocytes (0.9 pmol). The IVM resulted in a significant (P < 0.01) increase in the average ATP content of all oocyte categories, with no difference between oocytes extruding versus nonextruding a polar body. After in vitro fertilization (IVF) and culture, significantly (P < 0.05) more category 1 and 2 than category 3 and 4 oocytes developed to the morula or blastocyst stage (determined 168 h after IVF). Total cell numbers of expanded blastocysts derived from category 1 and 2 oocytes were significantly (P < 0.05) higher than of those originating from category 3 and 4 oocytes. These data indicate that mitochondrial reorganization and ATP levels are different between morphologically good and poor oocytes and may be responsible for their different developmental capacity after IVF.

Efficient transgenesis in farm animals by lentiviral vectors.

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV‐PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV‐PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ‐line. Tissue‐specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV‐K14). LV‐K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV‐PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.

Downregulation of multiple stress defense mechanisms during differentiation of human embryonic stem cells

Evolutionary theory predicts that cellular maintenance, stress defense, and DNA repair mechanisms should be most active in germ line cells, including embryonic stem cells that can differentiate into germ line cells, whereas it would be energetically unfavorable to keep these up in mortal somatic cells. We tested this hypothesis by examining telomere maintenance, oxidative stress generation, and genes involved in antioxidant defense and DNA repair during spontaneous differentiation of two human embryonic stem cell lines. Telomerase activity was quickly downregulated during differentiation, probably due to deacetylation of histones H3 and H4 at the hTERT promoter and deacetylation of histone H3 at hTR promoter. Telomere length decreased accordingly. Mitochondrial superoxide production and cellular levels of reactive oxygen species increased as result of increased mitochondrial biogenesis. The expression of major antioxidant genes was downregulated despite this increased oxidative stress. DNA damage levels increased during differentiation, whereas expression of genes involved in different types of DNA repair decreased. These results confirm earlier data obtained during mouse embryonic stem cell differentiation and are in accordance with evolutionary predictions.

Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning

Mammals have been cloned from adult donor cells. Here we report the first cases of mitochondrial DNA (mtDNA) heteroplasmy in adult mammalian clones generated from fetal and adult donor cells. The heteroplasmic clones included a healthy cattle equivalent of the sheep Dolly, for which a lack of heteroplasmy was reported.

Adult cloning in cattle: potential of nuclei from a permanent cell line and from primary cultures.

Nuclear transfer was used to evaluate the developmental potential of nuclei from a spontaneously immortalized bovine mammary gland epithelial cell line (MECL) and from primary cultures of mammary gland cells (PMGC) and ear skin fibroblasts (PESF) established from 3‐year‐old cows. Cell proliferation was investigated by incorporation and detection of 5‐bromo‐2′‐deoxyuridine (BrdU). The proportion of cells in S‐phase was significantly (P < 0.05) higher for MECL cells than for PMGC and PESF, both in the presence of serum (90% vs. 28% and 15%) and following serum starvation (27% vs. 6% and 3%). Nuclei from PESF supported the development of reconstructed embryos to the blastocyst stage significantly better than those of PMGC (60% vs. 26%; P < 0.05). Embryos reconstructed with cells from MECL failed to develop to blastocysts. After transfer of embryos derived from PMGC and PESF, respectively, 2/2 and 5/12 recipients were pregnant on day 42. On day 90, the corresponding pregnancy rates were 2/2 and 3/12. One live calf derived from a PMGC was born at day 287 of gestation. Another live PESF‐derived calf was delivered by caesarean section at day 286 of gestation. Our study suggests that nuclei from primary cultures of adult cells can be successfully reprogrammed by nuclear transfer, whereas nuclei from a permanent cell line failed to support the development of nuclear transfer embryos. Mol. Reprod. Dev. 54:264–272, 1999.

Transgenic Technology in Farm Animals – Progress and Perspectives

Current applications of gene transfer in farm animals include the improvement of product quality and quantity, disease resistance, the production of valuable proteins in the mammary gland or other organs, the genetic modification of pigs for xenotransplantation and the generation of new animal models in cases where rodent models are not sufficient for studying the problem under evaluation. Although DNA microinjection into pronuclei of zygotes from various farm animal species has happened since 1985, the efficiency of this method is low. Further drawbacks are related to the random integration process which may cause mosaicism, insertional mutations and varying expression due to position effects. Sperm‐mediated gene transfer is not routinely established yet, although the mechanisms of binding and internalisation of DNA by sperm cells is becoming increasingly clearer. New protocols for the use of retroviral vectors to infect metaphase II oocytes which are subsequently fertilised resulted in efficient production of transgenic cattle. In spite of extensive efforts to establish pluripotent stem cells from farm animal species, no germ‐line competent cells have been reported in mammalian species other than mouse so far. However, recent success in cloning sheep, cattle, goats and pigs from cultured cells provides an alternative route for efficient and targeted genetic modifications of farm animals.

Intracytoplasmic sperm injection in bovine: effects of oocyte activation, sperm pretreatment and injection technique.

Intracytoplasmic sperm injection (ICSI) is a very important technique for treating male subfertility and for basic research. The efficiency of ICSI in bovine is very limited because of the necessity for additional oocyte activation before or after the ICSI procedure. In this study, we compared the effects of seven different protocols on activation and fertilization rates of bovine oocytes after ICSI and on their subsequent development under in vitro conditions. The protocols include 1) different chemical activation of oocytes, 2) pretreated or nonpretreated sperm, and 3) conventional or Piezo-driven injection techniques. In all three groups, ICSI, sham-injected, and noninjected, the highest activation rates were obtained after treatment of oocytes with ionomycin followed by 6-dimethylaminopurine (6-DMAP). Using this treatment for oocyte activation, 59% of oocytes were activated and 31% of oocytes were fertilized using dithiothreitol (DTT) pretreated spermatozoa and Piezo-driven injection. Using the protocols with the same oocyte activation or activation with calcium ionophore (Ca-I) and cycloheximide (CHX), nonpretreated sperm, and conventional injection technique, early cleavage rate (79.6% and 77.6%, respectively) were significantly (P <0.01) higher when compared with all other protocols. The latter protocol resulted in 8% blastocyst and 90% of the obtained blastocysts were found to be diploid. Our results demonstrate that activation of oocytes, sperm treatment, and injection technique separately or together could improve the success of bovine ICSI.

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

Abstract 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.

Potential of fetal germ cells for nuclear transfer in cattle.

The developmental potential of bovine fetal germ cells was evaluated using nuclear transfer. Male and female germ cells at three stages of fetal development from 50‐ to 57‐, 65‐ to 76‐ or 95‐ to 105‐day‐old fetuses were fused to enucleated oocytes 2 to 4 hr prior to activation with 7% ethanol (5 min) followed by 5 hr culture in 10 μg/ml cycloheximide and 5 μg/ml cytochalasin B. The in vitro development of nuclear transfer embryos derived from germ cells was compared with those derived from embryonic cells (blastomeres from day 5 or day 6 embryos). Blastocyst rate (38%) obtained with germ cells from 50‐ to 57‐day‐old fetuses tended to be higher than when using germ cells from 65‐ to 76‐ or 95‐ to 105‐day‐old fetuses (23% and 20%, respectively). Within each stage of fetal development, the proportion of blastocysts derived from male germ cells tended to be higher than that obtained with female germ cells, but due to the high variation between individual fetuses this difference was not significant. With the post activation procedure used in this study, germ cells from 50‐ to 57‐day‐old fetuses supported the development of nuclear transfer embryos to the blastocyst stage significantly (P < 0.05) better than nuclei of embryonic cells (38% vs. 3%). After transfer of blastocysts derived from germ cells of 50‐ to 57‐ and 65‐ to 76‐day fetuses, respectively, 45% (5/11) and 50% (3/6) recipients were pregnant on day 30. The corresponding pregnancy rates on day 90 were 36% (4/11) and 17% (1/6). One live male calf was delivered by cesarean section at day 277 of gestation. Our results show that nuclei of bovine fetal germ cells may successfully be reprogrammed to support full‐term development of nuclear transfer embryos. Mol. Reprod. Dev. 52:421–426, 1999.