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Josef Fulka

Publications and source records attributed to Josef Fulka.

17 recordsLinked to original sources
PubMed2007

Somatic cell nuclear transfer (SCNT) in mammals: the cytoplast and its reprogramming activities.

It is now more than nine years since Dolly, the world's first somatic cell cloned mammal was born, and the success of somatic cell nuclear transfer (SCNT) is still disappointingly low. Only about 3-5% of reconstructed embryos develop to term, and it is also evident that even if some clones are born, they are not necessarily fully developed and healthy. Embryonic and neonatal abnormalities of cloned offspring are probably a result of incorrect or incomplete reprogramming of the transferred donor cell nuclei. Such an incomplete reprogramming reflects the extremely low efficiency of SCNT. The key role in the process of reprogramming has been attributed to the enucleated oocyte-cytoplast into which the somatic cell nucleus is transferred. In our chapter, we will discuss the methodological approaches used for the preparation of cytoplasts and their possible reprogramming activities.

Animals↗
PubMed2007

The use of micromanipulation methods as a tool to prevention of transmission of mutated mitochondrial DNA.

The introduction of different micromanipulation techniques into reproductive and developmental biology has helped us not only to answer many essential biological questions but it is now evident that these techniques also have wide practical applications. In human-assisted reproduction, the most commonly used approach is the injection of a donor sperm into the oocyte cytoplasm--intracytoplasmic sperm injection. It is, however, speculated that with these techniques it would be also possible to improve the oocyte developmental potential especially in those cases when the quality of the cytoplasm is rather poor and thus its function is compromised. Another important application would be the elimination of mutated mitochondrial DNA (mtDNA) by transferring the nuclear material from an abnormal oocyte into a healthy donor oocyte cytoplast. Some of these techniques were already successfully tested in experimental animals, but it is evident that before their introduction into human medicine many questions must be answered, and we must be sure that these approaches are absolutely or almost absolutely safe. In our contribution, we will be specifically oriented to the nuclear (nuclear material) replacement approaches that could be potentially used to prevent the transmission of mutated mtDNA from mother to offspring. Because these techniques are very delicate, some training with oocytes from other species other than human is strongly recommended.

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PubMed2006

DNA methylation pattern in pig in vivo produced embryos.

DNA methylation/demethylation pattern, determined by 5-methylcytosine (5-MeC) immunostaining, was evaluated in porcine "in vivo" produced embryos from zygote up to the blastocyst stage. In one-cell stage embryos, only the maternal pronucleus showed a positive labeling whilst the paternal pronucleus showed almost no labeling. The intensity of labeling is high until the late morula stage. Blastocysts containing less than 100 cells showed the same intensity of labeling in both the inner cell mass (ICM) nuclei and the trophectodermal (TE) cell nuclei. Interestingly, with further cell multiplication, cells of the ICM became more intensively labeled when compared to TE cells. This distinct methylation pattern is even more profound in blastocysts containing about 200-300 cells and is not caused by the difference in the cell volume of ICM and TE cells.

Animals↗
PubMed2006

The absence of a DNA replication checkpoint in porcine zygotes.

It has been demonstrated that in the zygotes of some mammals a unique checkpoint controls the onset of DNA replication. Thus, DNA replication begins in the maternal pronucleus only after the paternal pronucleus is fully formed. In our experiments we have investigated whether this checkpoint also operates in porcine zygotes produced either by in vitro fertilization (IVF) or by intracytoplasmic sperm injection (ICSI). Our results show that the onset of DNA replication occurs in the maternal pronucleus even in the presence of an intact sperm head in zygotes produced by ICSI, as well as in polyspermic eggs where some sperm heads are intact or male pronuclei are not yet fully developed. We conclude that in porcine zygotes there is an absence of the DNA replication checkpoint that is typical for some other mammals.

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PubMed2005

Development of pig embryos after electro-activation and in vitro fertilization in PZM-3 or PZM supplemented with fetal bovine serum.

The aim of present study was to optimize culture conditions for pig embryos. Initially, we evaluated three different basic culture conditions. When embryos from electro-activation (parthenotes) or in vitro fertilization (IVF-embryos) were cultured in PZM supplemented with 3 mg/ml bovine serum albumin (PZM-3) in 4-well dishes, in medium covered with oil in 4-well dishes or in droplets under oil, 0%, 33% and 20% of the parthenotes, and 11%, 23% and 20% of the IVF-embryos developed to blastocysts. Subsequently, we examined the development of embryos when they were cultured in 4-well dishes in medium covered with oil continuously for 7 days or cultured under the same conditions but with a change to fresh medium on Days 2 and 4. In this experiment, 23% (no medium change) and 34% (change) of the parthenotes developed to blastocysts, respectively. When IVF-embryos were cultured under similar conditions, 33% and 38% of the embryos developed to blastocysts. Further improvement was achieved when PZM was supplemented with FBS from Day 4. In this experiment, 47% of the parthenotes developed to blastocysts with an average cell number of 57 +/- 7.7. In IVF-embryo group, 49% of the embryos developed to blastocysts with a mean cell number of 60 +/- 6.1. These results indicate that a change to fresh medium and inclusion of FBS in the medium during the late stages of culture can generate a higher proportion of high-quality blastocysts.

Animals↗
PubMed2005

Germinal vesicle materials are requisite for male pronucleus formation but not for change in the activities of CDK1 and MAP kinase during maturation and fertilization of pig oocytes.

In amphibian oocytes, it is known that germinal vesicle (GV) materials are essential for sperm head decondensation but not for activation of MPF (CDK1 and cyclin B). However, in large animals, the role of GV materials in maturation and fertilization is not defined. In this study, we prepared enucleated pig oocytes at the GV stage and cultured them to examine the activation and inactivation of CDK1 and MAP kinase during maturation and after electro-activation. Moreover, enucleated GV-oocytes after maturation culture were inseminated or injected intracytoplasmically with spermatozoa to examine their ability to decondense the sperm chromatin. Enucleated oocytes showed similar activation/inactivation patterns of CDK1 and MAP kinase as sham-operated oocytes during maturation and after electro-stimulation or intracytoplasmic sperm injection. During the time corresponding to MI/MII transition of sham-operated oocytes, enucleated oocytes inactivated CDK1. However, penetrating sperm heads in enucleated oocytes did not decondense enough to form male pronuclei. To determine whether the factor(s) involved in sperm head decondensation remains associated with the chromatin after GV breakdown (GVBD), we did enucleation soon after GVBD (corresponding to pro-metaphase I, pMI) to remove only chromosomes. The injected sperm heads in pMI-enucleated oocytes decondensed and formed the male pronuclei. These results suggest that in pig oocytes, GV materials are not required for activation/inactivation of CDK1 and MAP kinase, but they are essential for male pronucleus formation.

Animals↗
PubMed2005

Placental abnormalities associated with post-natal mortality in sheep somatic cell clones.

We report on cloning experiments designed to explore the causes of peri- and post-natal mortality of cloned lambs. A total of 93 blastocysts obtained by nuclear transfer of somatic cells (granulosa cells) were transferred into 41 recipient ewes, and pregnancies were monitored by ultrasound scanning. In vitro derived, fertilized embryos (IVF, n=123) were also transferred to assess oocyte competence, and naturally mated ewes (n=120) were analysed as well. Cloned embryos developed to the blastocyst stage and implanted at the same rate as IVF embryos. After day 30 of gestation, however, dramatic losses occurred, and only 12 out of 93 (13%) clones reached full-term development, compared to 51 out of 123 (41.6%) lambs born from the IVF control embryos. Three full-term lamb clones were delivered stillborn, as a result of placental degeneration. A further five clone recipients developed hydroallantois. Their lambs died within 24h following delivery by caesarian section, and displayed degenerative lesions in liver and kidney resulting from the severe hydroallantois. One set of twins was delivered by assisted parturition at day 150, but died 24h later due to respiratory distress syndrome. The remaining two clone recipients underwent caesarian section, and the corresponding two lambs displayed signs of respiratory dysfunction and died at approximately 1 month of age due to a bacterial complication. Blood samples collected from the cloned lambs after birth revealed a wide range of abnormalities indicative of kidney and liver dysfunction. Macroscopical and histopathological examination of the placentae revealed a marked reduction in vascularization, particularly at the apex of the villous processes, as well as a loss of differentiation of the trophoblastic epithelium. Our results strongly suggest that post-mortality in cloned lambs is mainly caused by placental abnormalities.

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PubMed2004

Cryopreservation of isolated mouse germinal vesicles.

The storage of unfertilized oocytes, either immature, maturing or mature, is still unsatisfactory. Here we describe an approach in which germinal vesicles isolated as karyoplasts from immature oocytes are vitrified by open the pulled straws (OPS) method in evacuated porcine zonae pellucidae. After thawing, their survival was almost absolute. Moreover, when thawed GV-karyoplasts were fused to immature oocyte cytoplasts the maturation of reconstructed cells resulted in the production of secondary oocytes--metaphase II.

Animals↗
PubMed2004

Nucleus transfer in mammals: noninvasive approaches for the preparation of cytoplasts.

The production of cloned animals is a difficult and complex procedure that requires two basic steps. First, the cytoplast must be prepared by the enucleation of metaphase II oocytes. Second, the nucleus is transferred either by fusion or by direct microinjection into the cytoplast. The preparation of cytoplasts is a crucial step because they must be able to reprogram the transferred nucleus and to secure the development of reconstructed embryos. Moreover, the classical mechanical enucleation of metaphase II oocytes is rather technically difficult, requiring good equipment and considerable micromanipulation skill. For this reason the simplification of this step is permanently in the centre of interest of those scientists who are involved in the production of clones.

Animals↗
PubMed2004

DNA methylation pattern in human zygotes and developing embryos.

We report on observations of the global methylation/demethylation pattern of both pronuclei in human zygotes and in early embryos up to the blastocyst stage. Our results demonstrate that in about half of the zygotes examined the paternal chromatin was less methylated than the maternal chromatin. In the other half, both pronuclei exhibited the same intensity of labeling. The nuclei in developing embryos were intensively labeled for up to the four-cell stage; thereafter, a decline of labeling intensity was detected. Remethylation in some nuclei starts in late morulae. Surprisingly, and unlike the mouse, at the blastocyst stage the inner cell mass showed a weaker intensity of labeling than the trophectodermal cells.

Animals↗
PubMed2003

Amphibian and mammal somatic-cell cloning: different species, common results?

Since the production of Dolly the sheep cloning methods for somatic cells have been thoroughly described and are becoming routine. However, the rate at which live clones are produced remains low in all mammalian species tested so far. Remarkably, irrespective of the cloning protocol or the donor-cell type, all clones display common abnormalities, particularly in the placenta. The process is also complicated by early mortality of somatic-cell clones and the founder mammalian clone, Dolly the sheep, died in February 2003 aged six years. Based on published data and on our own experience, our view is that mammalian somatic-cell cloning and the pioneer nuclear-transfer data from amphibians have much in common. We suggest that the only way to improve nuclear reprogramming is to modify the chromatin structure of somatic cells before nuclear transfer, to provide the oocyte with a chromosomal structure that is more compatible with the natural reprogramming machinery of the oocyte.

Animals↗
PubMed2003

Failure of oocyte maturation: possible mechanisms for oocyte maturation arrest.

For human IVF, the patient's ovaries are hormonally stimulated to ensure the collection of fully matured oocytes that are at the metaphase II stage. Only these oocytes can be successfully fertilized either when mixed with sperm or after ICSI. Nevertheless, in some cases immature or maturing oocytes are recovered from follicles. Surprisingly, sometimes these oocytes do not complete maturation when cultured in vitro, for unknown reasons. In this article we discuss some possible mechanisms that may be responsible for those atypical arrests.

Animals↗
PubMed2002

Nuclei of nonviable ovine somatic cells develop into lambs after nuclear transplantation.

Here we report on the successful reprogramming of nuclei from somatic cells rendered nonviable by heat treatment. Granulosa cells from adult sheep were heated to nonphysiological temperatures (55 degrees C or 75 degrees C) before their nuclei were injected into enucleated metaphase II oocytes. Reprogramming was demonstrated by the capacity of the reconstructed embryos to develop to the blastocyst stage in vitro and into fetuses and viable offspring in suitable foster mothers. To our knowledge, this is the first report of cloned mammalian offspring originating from nonviable cells. In addition, our experiments show that heat-treating donor nuclei destabilizes higher-order features of chromatin (but leaves intact its nucleosomal organization) and results in a high proportion of reconstructed embryos developing to the blastocyst stage and beyond.

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