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J Fulka

Publications and source records attributed to J Fulka.

At least 19 recordsLinked to original sources
PubMed2005

Mammalian oocyte therapies.

In assisted human reproduction, the cytoplasm of oocytes recovered from follicles is often abnormal. Its lower quality, especially in older patients, may be responsible for certain chromosomal abnormalities or developmental arrest. Thus, the deficiency of some vital molecules, which are necessary for oocyte maturation, can be the cause of infertility in women. Moreover, mutated mitochondrial DNA (mtDNA) that is located in the oocyte cytoplasm might be transmitted to offspring. With the advance of new micromanipulation techniques like the oocyte nucleus replacement or cytoplasmic transfer, some of these abnormalities could be theoretically eliminated. In this review, we briefly discuss some of these approaches and their potential use in assisted human reproduction.

Animals↗
PubMed2005

Effect of protein supplement source on porcine oocyte maturation and subsequent embryonic development after parthenogenetic activation.

The aim of this study was to compare the effect of purified GPBoS and commonly used FCS on porcine oocyte maturation and subsequent embryonic development after their parthenogenetic activation. COCs were obtained from dissected follicles and cultured for 18, 24, 30, 36, 42 and 48 h in M-199 medium either with GPBoS or FCS. After 24 h with GPBoS, 91% of oocytes reached MI stage while in the medium supplemented with FCS, only 29% of oocytes reached the same stage (P < 0.05). The majority of oocytes from the FCS group (61%) reached MI stage approximately 6 h later. In the time periods between 36 to 48 h both groups of oocytes reached the same stage of maturation. After 48 h of culture the oocytes with extruded polar bodies were activated by a single electric pulse and then cultured with 4 mM 6-DMAP. Activated oocytes were cultured in PZM-3 medium supplemented with 3 mg/ml of BSA. After 7 days, the development and the quality of embryos were evaluated. The results showed that the maturation of oocytes in the presence of GPBoS significantly increased their subsequent developmental ability when compared with FCS supplementation (27% vs. 19% of blastocysts, P < 0.05). However, differential staining revealed that once blastocysts were formed in either group, they had the same total cell number (40 vs. 41) and also the ICM/total cell ratio (0.27 vs. 0.29).

Animals↗
PubMed2005

An attempt to reduce polyspermic penetration in lamb oocytes.

The incidence of polyspermy in lamb oocytes matured and fertilized in vitro is very high and this results in a reduced developmental potential of embryos arising from them. We have attempted to produce oocytes more resistant to this fertilization anomaly. The oocytes from prepubertal lambs 7-12 weeks old were matured in a medium supplemented with various blood sera and oviductal fluid and fertilized in vitro. Significantly higher monospermic penetration was found in a medium supplemented with BSA--3 mg/ml (63.9%) and OF--20% concentration (55.8%). Lower monospermy was recorded in the presence of 10% LS (44.6%) or 10% SS (40.8%), and particularly in a medium with 10% FCS (26.9%). In contrast, high monospermy (78.7%) was observed in oocytes from adult donors matured and fertilized in an identical system. In another set of experiments we estimated whether polyspermy can be reduced by improvement of the cytoplasmic maturation of prepubertal oocytes using a two-step maturation protocol. After artificial arrest of the maturation for 24 h with a specific cdk inhibitor--BL-I, 50 miocroM--more than 80% oocytes from prepubertal and adult donors did not resume meiosis. When incubated thereafter in a drug-free medium for another 24 h, the oocytes of both categories progressed to MII in the rate comparable with control (80% to 90% MII). However, after fertilization no significant differences in the level of monospermic penetration was recorded between the arrested group (59.8%) and control (58.8%), both matured in the presence BSA, and 46.6% and 52.3% after treatment with OF. Also, no significant difference was observed between the arrested and control oocytes from adult donors (72.6% and 84.8%, respectively). These results suggest that high polyspermy in prepubertal oocytes is caused by developmental imperfection and can't be fully eliminated either by modifying the composition of culture media or by prolongation of the culture interval.

Animals↗
PubMed2004

[Cloning of mammals--biological aspects].

Cloning by somatic cell nucleus transfer into enucleated oocytes represents a very straightforward approach. The nucleus is simply transferred into the oocyte from which its nuclear genetic material (chromosomes) has been earlier removed. By using this approach clones of sheep, cattle, horse, rabbit, goat, rat, mule, pig, cat and interspecies-specific clone mouflon x sheep were obtained. The success of cloning is, however, disappointingly low and only 1-3% of reconstructed embryos develop to term. Some cloned animals are abnormal and die prematurely. This clearly speaks against the use of cloning in humans. On the other hand, so called therapeutic cloning, when the nucleus is transferred into an oocyte and the developing embryo is then used for the production of embryonic stem cells is generally well accepted. This promising technique may be probably used in some as yet untreatable diseases and in certain serious injuries.

Animals↗
PubMed2003

Enucleolation of porcine oocytes.

Germinal vesicles (GVs) in immature mammalian oocytes contain prominent nucleoli whose role in the process of oocyte maturation is not fully understood. Here we report that the microsurgical removal of nucleoli from immature fully grown porcine oocytes permits germinal vesicle breakdown and chromosome condensation and the enucleolated oocytes mature up to the second metaphase. Interestingly, the enucleolation of growing oocytes which, although unable to mature, resulted in germinal vesicle breakdown and the formation of a cluster of condensed chromatin. These results indicate that the nucleolus in fully grown oocytes is dispensable at least for nuclear maturation. On the other hand, the results obtained in growing oocytes suggest the role of the nucleolus in the cell cycle regulation.

Animals↗
PubMed2003

[Successful treatment of azoospermia diagnosed as "Sertoli cells only syndrome with maturation arrest"--developing a quality embryo].

OBJECTIVE: The transfer of good quality embryo in the program of assisted reproduction in the case of azoospermia, dg. Sertolli cells only syndrome (SCO sy) + maturation arrest (MA). Testes were assessed and found to have a high occurrence of Sertolli cells and very low occurrence of germinal cells, which were arrested at the round spermatid level. The histological evaluation was hypospermatogenesis gr. 3 (minimum 1 spermatid/sample). DESIGN: Case report. SETTING: Laboratory IVF, Iscare, a. s., Department of Biology and Biochemistry of Fertilization, Institute of Molecular Genetics, Czech Academy of Sciences, Prague. SUBJECT AND METHOD: The successful integration of three methods provides a solution for this case of azoospermia. Immunology and histology can more exactly diagnose the degree of azoospermia. Detection and visualisation of spermatids using monoclonal antibodies against sperm proteins predicts the eventual occurrence of spermatogenesis, and histological evaluation confirms these immunological findings. Using the information of both methods it is possible to use special in vitro cultivation of testicular cells and so obtain injectable spermatozoa, or precursors of sperm, for the ICSI method. CONCLUSION: The probability of acquisition of good-quality embryo in the program of assisted reproduction is higher when these three methods are applied in combination.

Adult↗
PubMed2002

Nucleus replacement in Mammalian oocytes.

Our contribution discusses the potential use of cell therapies (nucleus replacement) in mammalian oocytes. It is assumed that these approaches may be used, for example, for the elimination of mutated maternally transmitted mitochondrial DNA (mtDNA) as well as for the reconstruction of normal oocytes from oocytes that are developmentally compromised. Moreover, it is speculated that the replacement of germinal vesicles by somatic cells may result in cells of the haploid genome: the production of germ cells from somatic cells. The preliminary results obtained in our laboratories are discussed in this article.

Animals↗
PubMed2002

Somatic and embryonic cell nucleus transfer into intact and enucleated immature mouse oocytes.

BACKGROUND: The aim of our study was to evaluate the possibility of embryonic or somatic cell haploidization after fusion with intact or enucleated immature oocytes which were subsequently cultured in vitro. Embryonic or somatic cell nuclei do not undergo premature chromosome condensation when fused to intact or enucleated immature oocytes whose maturation is prevented by dibutyryl cyclic AMP (dbcAMP). The presence of dbcAMP permits, however, the completion of DNA replication in somatic cell nuclei. METHODS AND RESULTS: The chromosomes condensed when the reconstructed cells were released from the dbcAMP block. When somatic or embryonic nuclei were introduced into intact immature meiotically competent oocytes and subsequently cultured their chromosomes assembled on a common spindle with meiotic chromosomes and proceeded through the meiotic-like division, judged according to the presence of the first polar body extruded. When embryonic cell nuclei were introduced into cytoplasts obtained from immature meiotically competent oocytes, polar bodies were extruded in about 75% of reconstructed cells but the metaphase plates were abnormal in almost all cases. When somatic cell nuclei were inserted into the above cytoplasts, polar bodies were extruded only very exceptionally and in these cells chromosomes were arranged in abortive metaphase plates. CONCLUSIONS: Our results suggest that somatic cell nuclei are unable to proceed through the reduction division (haploidization) when introduced into an immature oocyte meiotic cytoplasm.

Animals↗
PubMed2001

Nucleus transfer in mammals: how the oocyte cytoplasm modifies the transferred nucleus.

Successful development of clones depends on the reprogramming of transferred nuclei in enucleated oocytes. Thus far, oocytes are the only cells that can convert nuclei, which are already differentiated, into undifferentiated stages resembling pronuclei in freshly fertilized zygotes and that can then complete development of the reconstructed embryos. However, we still don't know exactly how the enucleated oocyte (cytoplast) secures this reprogramming. Oocytes exhibit a number of cytoplasmic activities that may be involved reprogramming. We discuss how these activities may be involved in reprogramming of transferred nuclei.

Animals↗
PubMed2001

Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells.

Since the advent of procedures for cloning animals, conservation biologists have proposed using this technology to preserve endangered mammals. Here we report the successful cloning of a wild endangered animal, Ovis orientalis musimon, using oocytes collected from a closely related, domesticated species, Ovis aries. We injected enucleated sheep oocytes with granulosa cells collected from two female mouflons found dead in the pasture. Blastocyst-stage cloned embryos transferred into sheep foster mothers established two pregnancies, one of which produced an apparently normal mouflon. Our findings support the use of cloning for the expansion of critically endangered populations.

Animals↗
PubMed2001

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Animals↗
PubMed2000

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation.

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.

Animals↗
PubMed2000

Manipulating the human embryo: cell cycle checkpoint controls.

Micromanipulation techniques are widely used in assisted human reproduction and it is logical to assume that successes with recent animal cloning will invariably raise the question of human cloning along with its related ethical problems. However, it is often overlooked that even in animals many complications are still associated with this technique. The purpose of our article is to highlight and discuss some of these problems in the context of the eventual use of nuclear and/or cytoplasmic transfer techniques in assisted human reproduction.

Cell Cycle↗
PubMed2000

Preimplantation development of giant triploid zygotes in the mouse.

Giant oocytes or two-cell embryos have been reported in various mammalian species. They may arise during multiplication of oogonia, after fusion of two oogonia or, more probably, when nuclear division is not accompanied by cytoplasmic division. The ultimate fate of these giant embryos is not well known. In our laboratory, giant two-cell mouse embryos have been occasionally observed. Recently, we observed two giant one-cell zygotes in the same species. Both showed two female pronuclei and one male pronucleus, as well as two second polar bodies localized at opposite poles of the embryo. These two giant zygotes showed normal viability and developmental capacity. Their triploid nature was confirmed by cytogenetic analysis. In order to study this interesting phenomenon in more detail, we produced giant oocytes containing two germinal vesicles by cell fusion and cultured them in vitro. About one third of them extruded two first polar bodies; in the second group only one polar body was observed, whilst the last group was without polar bodies. When parthenogenetically activated, the consistent answer analogical to that observed in "in vivo" oocytes was only observed when oocytes with two polar bodies were activated. The implication for IVF technologies is discussed.

Animals↗
PubMed1999

Checkpoint control of the G2/M phase transition during the first mitotic cycle in mammalian eggs.

The high incidence of chromosomally abnormal human embryos is frequently assumed to be due to a lack of checkpoint controls operating during early embryogenesis. In our study we have analysed when these mechanisms first become functional. Mouse oocytes treated in late metaphase I with either of two different cyclin-dependent kinase inhibitors [butyrolactone 1 (BL1) or 6-dimethylaminopurine (6-DMAP)] form nuclei in the cytoplasm. BL1-treated eggs enter S-phase at 16-18 h post-treatment and, after completion of DNA synthesis, cleave to 2-cell stage embryos. 6-DMAP treatment results in the rapid initiation of DNA synthesis, its completion by 12 h and then arrest in the G2 phase. Thus, two different cell cycle stages can be obtained at the same time point after the initiation of treatment: G1- after BL1 and G2-staged nuclei after 6-DMAP treatment. That this approach greatly facilitates cell cycle studies has been shown by analysing checkpoint function during the first division. Whilst G2-staged eggs enter M phase within 2-3 h when 6-DMAP is washed out, the onset of M phase is delayed after their fusion to G1 (BL1) cells. Here M phase occurs only after the less advanced nucleus completes DNA replication. Our results indicate that checkpoints in mammalian eggs are functional during the first mitotic cycle.

4-Butyrolactone↗
PubMed1999

Oviduct secretion contributes to the establishment of species specific barrier preventing penetration of oocytes with foreign spermatozoa.

Ovulated oocytes can be fertilized in vivo almost exclusively with spermatozoa of its own species only, while the species specificity of zona pellucida of in vitro matured oocytes is less restrictive. Our present experiments were undertaken to determine whether estrous oviductal fluid modifies the interaction between gametes of unrelated species. After incubation of in vitro matured sheep oocytes with bull spermatozoa, the penetration rate was 75.0%, whereas when the oocytes were matured in medium supplemented with 15% sheep oviductal fluid collected using the permanent indwelling oviductal cannulae, the penetration rate decreased to 4.8 % (4/84). In reverse combination, 70.4 % (38/54) cattle oocytes matured in vitro were penetrated with ram spermatozoa. The addition of oviductal fluid caused a drop in penetration by ram sperm to 38% (19/50). In parallel experiments, no penetration was recorded when in vivo matured sheep oocytes were incubated with bull spermatozoa; high fertilization rates (79.4% - 27/34) were recorded when such eggs were incubated with ram spermatozoa, irrespective to the presence or to the absence of oviductal fluid in the medium. The results suggest that the properties of zonae pellucidae of ovulated and in vitro matured oocytes are not identical and may be modified by contact with estrous oviductal fluid.

Animals↗
PubMed1998

Cloning by somatic cell nuclear transfer.

The birth of the first cloned mammals, produced by the introduction of somatic cell nuclei into enucleated oocytes, was an impressive and surprising development. Although the ethical debate has been intense, the important scientific questions raised by this work have been inadequately discussed and are still unresolved. In this essay we address three questions about nuclear transplantation in the eggs of mice and domestic animals. First, why were the recent experiments on somatic cell cloning successful, when so many others have failed? Second, were these exceptional cases, or is somatic cloning now open to all? Third, what are the future possibilities for increasing the efficiency and wider applicability of the cloning process?

Animals↗