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J Fulka

Publications and source records attributed to J Fulka.

At least 37 records · Page 2Linked to original sources
PubMed1998

Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation.

The requisite endpoint of mammalian oocyte maturation, whether in vivo or in vitro, is a metaphase II oocyte which is able to be fertilized and which can eventually support normal embryonic development. Oocytes which have been matured in vivo basically fulfill these criteria. On the other hand, a completely different situation exists when these cells are isolated from the ovaries and cultured in vitro. If they are too small (growing oocytes), they do not undergo maturation, or, if more advanced, will mature only to the metaphase I stage. Even in fully grown oocytes which are able to mature to metaphase II, the developmental potential after fertilization is disappointingly low, for reasons which remain unknown. The complexity of certain factors (nuclear, cytoplasmic or arising from our current culture systems) undoubtedly influences both the ability of oocytes to mature fully, as well as their developmental potential after fertilization.

Animals↗
PubMed1998

Development of parthenogenetic and cloned ovine embryos: effect of activation protocols.

Preliminary experiments carried out on ovine oocytes were designed to establish correlations between activation protocols and subsequent rates of embryonic development. The best activation protocols were thereafter used in studies on ovine parthenogenesis and cloning. The first study established that chemical activators induce pronuclear development at a slightly higher rate than physical activation (ionomycin, 96%; ethanol, 95%; electro activation, 80%). Inhibition of second polar body extrusion and one single pronucleus were observed in the majority of the oocytes (approximately 90%) treated for 3 h with 6-dimethylaminopurine (6-DMAP) following either ionomycin or ethanol activation. While over 80% of these oocytes cleaved after transfer to the oviducts of recipients, progression to the blastocyst stage was higher after ionomycin as compared with ethanol activation (58% vs. 19%). The ionomycin plus 6-DMAP activation protocol was used to produce parthenogenetic blastocysts whose subsequent development was monitored both by ultrasonography and by direct fetal examination. Over 70% of parthenogenotes were viable on Day 21 of pregnancy but dead by Day 25. The effects of 6-DMAP on nuclear remodeling and fetal development of cloned embryos was then investigated. Control cloned embryos underwent nuclear envelope breakdown (NEBD), premature chromatin condensation (PCC), and inhibition of DNA synthesis. By contrast, reconstructed embryos treated with 6-DMAP exhibited intact nuclear membranes, interphase chromatin, and no interference on DNA synthesis. Moreover, cloned embryos developed to blastocyst stage in higher percentage after 6-DMAP treatment (83% vs. 25%). We conclude that ionomycin followed by 6-DMAP incubation yields high percentages of diploid parthenogenetic embryos that develop to Day 25 before dying. Cloned embryos activated by the ionomycin-6-DMAP protocol develop readily to term.

Adenine↗
PubMed1997

Developmental failure of hybrid embryos originated after fertilization of bovine oocytes with ram spermatozoa.

The developmental ability of hybrid zygotes, produced by in vitro fertilization of in vitro matured bovine oocytes with ram sperm, was evaluated by gross morphology, autoradiographic detection of (5-3H) uridine incorporation, and fine structure morphology. Fertilization was successful in 83% of bovine oocytes inseminated with bull sperm (control embryos) compared with 67% of bovine oocytes inseminated with ram sperm (hybrid embryos) and in both cases appeared two regularly developed pronuclei. Two-cell embryos were transferred to ewe oviducts and allowed to develop to the 8-cell stage. Although the ability of hybrid embryos to reach 8-cell stage was similar to that of control embryos, in nuclei of hybrid embryos the transition from maternal to embryonic genome control assessed according to the onset of RNA synthesis indicated the differences in the frequency of labelled nuclei and intensity of their labelling. In hybrid embryos these parameters were remarkably lower and may reflect the developmental failure of hybrid embryos. These observations are consistent with delay or inefficient reactivation of the embryonic genome in the hybrid embryos.

Animals↗
PubMed1997

Induction of DNA replication in germinal vesicles and in nuclei formed in maturing mouse oocytes by 6-DMAP treatment.

Immature mouse oocytes (germinal vesicle stage, GV), oocytes at different stages during maturation (prometaphase to anaphase I) and matured oocytes (metaphase II arrested) were cultured in 6-dimethylaminopurine (6-DMAP)-supplemented medium also containing bromodeoxyuridine for the assessment of DNA replication in these cells. Immature oocytes remained arrested at the GV stage and DNA replication was never detected in them. On the other hand, oocytes at the prometaphase to anaphase-telophase I stages responded to 6-DMAP treatment by forming nuclei which synthesised DNA. Mature (metaphase II) oocytes did not respond to 6-DMAP and their chromatin remained condensed. DNA synthesis could even be induced in GV-staged oocytes, but only when they were fused to freshly activated oocytes and incubated in 6-DMAP-supplemented medium.

Adenine↗
PubMed1997

Damaged chromatin does not prevent the exit from metaphase I in fused mouse oocytes.

The presence of checkpoint mechanisms which are able to recognize damaged chromatin and thereafter to prevent exit from metaphase I has been investigated in giant mouse oocytes produced by fusion of a normal metaphase I oocyte with an equivalent oocyte with damaged chromatin. The presence of damaged chromatin did not prevent the onset of anaphase I in both sets of chromatin in the fused cells. Interestingly, fused or unfused cells containing only damaged chromatin failed to enter anaphase and persisted instead in a metaphase-like state. These results demonstrate the fragility of checkpoint controls in mammalian female germ cells.

Animals↗
PubMed1996

Oocyte-specific modulation of female pronuclear development in mice.

Cell fusion experiments were undertaken to determine both whether oocytes possess, in a stage-specific manner, suppressors of pronuclear development and whether these factors could be used to modulate female pronuclear development. The fusion of telophase II eggs obtained 2 hr after activation (A2 eggs) to germinal vesicle (GV)-staged oocytes (GV x A2) suppresses female pronuclear development. The cytoplasm of GV x A2 heterokaryons contains, at 10 hr postfusion, a micropronucleus and a GV which is significantly larger (approximately x1.6) than normal. Fusion of early pronucleate eggs (3 hr postactivation, A3) to GV-staged oocytes (GV x A3) results in the formation of a retarded half-sized pronucleus and a slightly enlarged (approximately x1.2) GV at 10 hr postactivation. Normal pronuclear formation occurs when eggs at 4 hr postactivation (A4) are fused to GV oocytes (GV x A4). Although pronuclear size is suppressed in heterokaryons formed when GV-staged oocytes are fused to eggs activated 2-3 hr earlier (A2 or A3 stage), entry into S-phase and DNA synthesis is not inhibited even in the micropronuclei. Meiotic progression in the oocyte germinal vesicle is arrested after fusion to activated eggs throughout the period during which the pronucleus is undergoing G1 and S-phase. However, at the end of S-phase in the pronucleate partner, germinal vesicle breakdown occurs, cell cycle progression is accelerated, and both nuclei reach M-phase by 12 hr postactivation. That suppressors of pronuclear development are found only in GV (G2)-staged oocytes, and not in mitotic cells at the same cell cycle stage, was demonstrated by fusing G2-staged blastomeres to A2-staged eggs (G2 mitotic x A2). By contrast to the micropronuclei formed in GV x A2 heterokaryons, normal pronuclear development occurred in G2 mitotic x A2 heterokaryons. Our results show that suppressor activity, present only in GV oocytes, restricts female pronuclear development, but only in the first 3 hr after activation. We propose to use the ability to modulate either female (this study) or male pronuclear formation in studies (i) on imprinting and (ii) on the developmental consequences of early asynchrony between male and female pronuclear development.

Animals↗
PubMed1996

Nuclear transplantation of ectodermal cells in pig oocytes: ultrastructure and radiography.

This study investigated the changes in nucleolar fine structure and the synthesis of both heterogeneous nuclear RNA (hnRNA) and ribosomal RNA (rRNA) in pig reconstructed embryos obtained by electrofusion of a single pig ectodermal cell to an enucleated metaphase-II oocyte. The nucleolar morphological changes and the pattern of transcription were examined in the ectodermal cells before fusion and in the nuclear transfer reconstructed embryos 16-18 hr after fusion. Before fusion the ectodermal cells exhibited a reticulated nucleolus with active RNA synthesis. In the reconstructed embryos, modifications of the nucleolar structure were observed, as assessed by the presence of either round-shaped, compact, dense nucleolar precursor bodies, or reticulated nucleoli. However, in both cases there was no RNA synthesis. Blebbing activity of the nuclear envelope was not observed. These results indicate that the nucleus of pig ectodermal cells exhibited either a complete or an incomplete remodelling when transferred to an enucleated metaphase-II oocyte, with no detectable RNA synthesis. Cell-cycle synchronization of ectodermal cell donor nuclei may play an important role in nuclear reprogramming after fusion.

Animals↗
PubMed1996

Nuclear transplantation in mammals: remodelling of transplanted nuclei under the influence of maturation promoting factor.

Whilst the role of Maturation or M-phase Promoting Factor (MPF) as a universal M-phase regulator is well documented, much less attention has been paid to its role in nuclear transplantation experiments and especially to its influence upon remodelling of transplanted nuclei. There is currently wide acceptance that successful nuclear transplantation using differentiated nuclei is possible only in a cytoplasmic environment that is capable of inducing rapid nuclear de-differentiation to a pronuclear-like form. In this review our purpose is firstly, to outline the conditions under which such remodelling can be induced, and secondly, to extend the debate to include a consideration of whether complete nuclear remodelling is an absolute necessity for clonal development.

Animals↗
PubMed1996

The effect of 6-dimethylaminopurine (6-DMAP) on DNA synthesis in activated mammalian oocytes.

Mouse, sheep and bovine metaphase II oocytes were briefly preincubated in medium with 6-dimethylaminopurine (6-DMAP) and thereafter activated with ethanol before further culture in 6-DMAP supplemented medium. The presence of 6-DMAP enhances the efficiency of activation and the speed of pronuclear formation but has no effect on DNA synthesis. These results are discussed in the context of recently published data showing that the preincubation of condensed chromosomes with 6-DMAP blocks DNA synthesis when nuclei become reformed.

Adenine↗
PubMed1996

Nuclear transplantation in pigs: M-phase karyoplast to M-phase cytoplast fusion.

Porcine embryonic fibroblasts in M-phase were isolated from confluent cultures by selective detachment and fused to homologous metaphase II enucleated oocytes. The fusion products were analysed by light and electron microscopy (EM). The fusion treatment, per se, did not induce the disappearance of the maturation promoting factor (MPF) in the cloned embryos and the transferred chromatin remained condensed for up to 10 h. Spontaneous decondensation started after this time and, at about 20 h post-induction of fusion, a single large nucleus was present in the cytoplasm. The prolonged exposure of introduced chromatin to MPF resulted in a total morphological remodelling as assessed by the nucleolar morphology observed by EM in all the specimens.

Animals↗
PubMed1995

Replicating DNA does not block germinal vesicle breakdown in mouse oocytes.

Mouse germinal vesicle GV-G2 stage oocytes were fused to very early S-phase blastomeres from four cell stage embryos to test whether oocytes have a mechanism which detects replicating DNA and thereafter arrests meiosis at the GV stage. Our results show that oocytes are unable to recognize replicating DNA in transplanted nuclei and undergo germinal vesicle breakdown (GVBD). GVBD and blastomere nucleus breakdown are only blocked by inhibitors of both oocyte maturation and DNA synthesis. These results show that mouse immature oocytes do not possess a feedback control capable of detecting replicating DNA.

Animals↗
PubMed1995

UV irradiation of chromosomal DNA and its effect upon MPF and meiosis in mammalian oocytes.

Bovine oocytes were irradiated at germinal vesicle (GV) or metaphase II (MII) stage, after Hoechst staining, with chromosomally focused UV-C (254 nm) or UV-A ( > 330 nm). UV-C irradiation at GV stage did not inhibit germinal vesicle breakdown (GVBD) or chromosomal condensation; spindle formation was abolished and maturation promoting factor (MPF) levels failed to increase. UV-A irradiation at GV stage caused meiotic arrest at anaphase I; MPF levels were lower than control. UV-C irradiation at MII stage led to subsequent abnormal parthenogenetic activation when MPF levels failed to decrease. A normal male but no female pronucleus was formed at fertilization. UV-A irradiation at MII stage also caused abnormal activation; MPF levels declined normally. A normal male and abnormal female pronucleus formed at fertilization. UV-A irradiation results have implications for oocyte evaluation during development using Hoechst staining. UV-C irradiation is a potential means for oocyte enucleation in nuclear transfer.

Animals↗
PubMed1995

Mouse oocyte maturation: meiotic checkpoints.

Mouse oocytes at different stages of maturation were fused together and the ensuing cell cycle events were analyzed with the objective of identifying checkpoints in meiosis. Fusion of maturing oocytes just undergoing germinal vesicle breakdown (GVBD) induces PCC (premature chromosome condensation) but no spindle formation in immature (GV) partner oocytes. On the other hand, fusion of metaphase I (MI) oocytes containing spindles to GV oocytes induces both PCC and spindle formation in the immature partner. Thus, while molecules required for condensation are present throughout metaphase, those involved in spindle formation are absent in early M-phase. Oocytes cultured for 6 h--early metaphase I (i.e., approximately 2 h before the onset of anaphase I)--and then fused to anaphase-telophase I (A-TI) fusion partners block meiotic progression in the more advanced oocytes and induce chromatin dispersal on the spindle. By contrast, oocytes cultured for 8 h (late MI) before fusion to A-TI partners are driven into anaphase by signals from the more advanced oocytes and thereafter advance in synchrony to telophase I. When early (10 h) or late (12 h) metaphase II oocytes were fused to A-TI partners the signals generated from early MII oocytes block the anaphase to telophase I transition and induce a dispersal of A-TI chromosomes along the spindle. On the other hand, late MII oocytes respond to A-TI signals by exiting from the MII block and undergoing the A-TII transition. Moreover, the oocytes in late MI are not arrested in this stage and progress without any delay through A-TI to MII when fused to metaphase II partners. The signals from the less-developed partner force the MII oocyte through A-TII to MIII. In total, these studies demonstrate that the metaphase period is divided into at least three distinct phases and that a checkpoint in late metaphase controls the progress of meiosis in mammalian oocytes.

Animals↗
PubMed1995

Response of avian nuclei to mammalian maturation promoting factor (MPF).

Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.

Animals↗
PubMed1995

Chromosome condensation activity (CCA) in bisected C57BL/6JxCBA mouse oocytes.

Chromosome condensation activity (CCA) has been analysed in C57BL/6Jx CBA mouse oocytes bisected (i) shortly after germinal vesicle breakdown (GVBD), (ii) in metaphase I (MI) and (iii) in metaphase II (MII) into two equal halves (nucleated, enucleated) which were thereafter fused to S- or G2-phase 4-cell-stage mouse blastomeres. In nucleated halves, premature chromosome condensation (PCC) in transplanted nuclei was always induced irrespective of the cell cycle stage of the blastomere, whereas in enucleated halves only G2 nuclei underwent PCC after transplantation. Premature chromosome condensation in S-phase nuclei was induced only in enucleated halves produced shortly after GVBD. Although S-phase nuclei transplanted to MI or MII enucleated halves remained intact, their capacity to synthesize DNA was invariably suppressed. When spindles were destroyed by preincubation of the oocytes in colcemid before bisection, both nucleated and enucleated halves produced at MI or MII induced PCC of both G2- or S-phase nuclei. These results demonstrate that chromosome condensation activity in mammalian oocytes is compartmentalized rather than uniformly distributed across the cell, and that the enucleation of mammalian oocytes before nuclear transplantation may, under some conditions, influence the levels of CCA and subsequent response of introduced nuclei to cytoplasmic factors.

Animals↗
PubMed1994

Sister chromatid separation and the metaphase-anaphase transition in mouse oocytes.

The paper reports on the effect of experimentally inhibiting chromatid separation on meiotic progression and maturation-promoting factor (MPF) activity in both metaphase I (Experiment 1) and metaphase II mouse oocytes (Experiment 2) subjected to combinations of inhibitors of (a) protein synthesis, (b) topoisomerase II, and (c) cytokinesis-cytoskeleton integrity. The results from Experiment 1 showed that the inhibition of protein synthesis invariably results in the extrusion of the first polar body and the formation of an interphase nucleus. Furthermore, this inhibition induces a rapid decline in MPF activity. Similarly, in Experiment 2 the exposure of metaphase II oocytes to cycloheximide initiated a rapid fall in MPF activity, progression to anaphase, the extrusion of the second polar body, and the formation of a pronucleus. While the inhibition of protein synthesis hastened progression through the meiotic cycle, the opposite effect was observed when chromatid separation was prevented by etoposide or colcemid treatment. The results in Experiment 1 demonstrated that the inhibition of chromatid separation totally blocked meiotic progression by preventing the metaphase I to anaphase I transition. These oocytes were characterized by the persistence of high MPF activity for extended periods of time (> 20 hr). This activity declined slowly in oocytes exposed both to inhibitors of chromatin separation and protein synthesis. In Experiment 2 the results showed that the prevention of chromatin separation induced changes which paralleled those observed with MI oocytes. The prevention of chromatid separation with either etoposide or colcemid converted the oocytes from being sensitive to activation stimuli to being entirely resistant to standard activation. In addition, MPF activity remained persistently elevated and declined only when protein synthesis was inhibited. The decline in intracellular MPF activity reached basal levels 6 to 10 hr after the addition of cycloheximide and was accompanied by the slow and gradual decondensation of chromatin. Our results are in accord with those from recent experiments in yeast, insects, and amphibia which suggest that chromatid separation provides an essential signal for cell cycle progression beyond M-phase. We postulate first that exist from both metaphase I and metaphase II, and the characteristic reduction in MPF activity at anaphase in mouse oocytes, are initiated by chromosome (chromatid) separation. Second, we suggest that chemically induced chromosome (chromatid) separation block prevents the anaphase to telophase transition by inhibiting MPF degradation. Third, we postulate that the slow escape from metaphase arrest in oocytes treated with both etoposide and cycloheximide reflects a gradual decrease of MPF activity due to normal protein turnover without new synthesis.

Anaphase↗