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Biomedical subjects

J Fulka

Publications and source records attributed to J Fulka.

At least 73 records · Page 4Linked to original sources

Pregnancies after transfer of sheep embryos produced from oocytes matured and fertilized in vitro.

The experiments describe simple and effective methods used for the production of sheep embryos in vitro. The oocytes isolated from the ovarian follicles, 2-5 mm in diameter, were matured in culture for 22-24 h. After mixing with ram spermatozoa pretreated with heparin, 77-100% of fertilized oocytes were monospermic. Some of them (60-68.3%) cleaved in culture after 24-50 h to 2-4-cell stage. The transfer of cleaved embryos to the oviducts of 4 synchronized recipients resulted in pregnancies and 4 normal lambs were born at term.

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Penetration of intact bovine ova with ram sperm in vitro.

In culture, mature bovine ovarian oocytes were fertilized in vitro with freshly ejaculated ram spermatozoa treated with heparin. The zona pellucida does not prevent penetration of ram spermatozoa. The penetration rate varied between 10 and 84%, and in most instances, after 24 hr of culture, two normal-looking pronuclei and sperm tail were present in the cytoplasm. These results suggest that the zona pellucida of bovine oocytes does not represent a barrier for the penetration of ram spermatozoa.

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Ultrastructural localization of silver-staining nuclear proteins at the onset of transcription in early bovine embryos.

Argyrophilic nuclear proteins, known to be functionally associated with ribosomal genes, were localized, in four-, eight-, and 16-cell bovine embryo blastomere nuclei using two different silver-staining procedures. Within the eight-cell cleavage stage by the process of embryonal nucleologenesis in the cow embryo the full-capacity ribosome-producing machinery is established. In the four-cell embryo, many patches and islands of argyrophilic (Ag+) material were detected in the nucleoplasm. The nucleolus-precursor bodies (NPBs), composed uniformly of a homogeneous compact mass, were completely devoid of any silver staining. On the other hand, clear-cut localization of argyrophilic proteins was detected during the eight-cell stage either inside the transforming NPBs or in the close vicinity, or in the already differentiated nucleolus. In compact, nonvacuolated NPB, an intensive Ag+ area was detected, in the form of a lenticle, at the periphery of the NPB. During and following vacuolation of the NPB, no Ag+ was detected inside these vacuoles. It was seen, however, in the dense fibrillar nucleolar component surrounding the smaller vacuoles formed at the time of the establishment of nucleolar structure. Ag+ areas were seen repeatedly in the vicinity of NPBs, probably a part of the nucleolus-associated chromatin or, alternatively, representing the extranucleolar bodies. In blastomere nuclei of 16-cell embryos, already possessing reticulated nucleoli known from intensively synthesizing somatic cells, the silver-staining pattern corresponded to the usual situation in differentiated cells: slight staining of fibrillar centers, heavy labelling in the dense fibrillar component, and absence of silver deposits in the granular component.(ABSTRACT TRUNCATED AT 250 WORDS)

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Vitrification of mouse 8-cell embryos with glycerol as a cryoporotectant.

Glycerol at a concentration of 6.85 M has been used for cryopreservation of 8-cell mouse embryos with the aim to induce formation of ice-free glass after plunging into liquid nitrogen. Before treatment with this concentration at approximately 0 degrees C, embryos were pre-equilibrated in 1.37 M glycerol at ambient temperature. It was found that the main source of damage to embryos is due to treatment with a high concentration of glycerol and osmotic events during its dilution. Cooling and warming of embryos per se induce little or no harm to their capacity to form expanded blastocysts after 48 h in vitro. Best results (together 169/198, 85.4%) were obtained, when both time (not more than 15 min) and temperature (approximately 0 degrees C) of exposure of embryos to vitrification media were controlled properly.

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Nucleologenesis and the onset of transcription in the eight-cell bovine embryo: fine-structural autoradiographic study.

Eight-cell cow embryos were isolated and cultured in vitro in a medium enriched with 200 microCi of [5-3H]uridine for 20 min. Epon ultrathin sections of the embryos were investigated for the nucleolar morphology and for the appearance and localization of the sites of [5-3H]uridine incorporation by means of electron microscopic autoradiography. In addition to this, a general pattern of replicated embryonal DNA distribution was revealed by [methyl-3H]thymidine incorporation and light microscopic autoradiography. The essential phases of the transformation of the small nucleolus precursor body (NPB) into a vast, functionally fully active nucleolus, characterized by typical nucleolar substructural components, are taking place within the eight-cell stage. This process differed in its morphology from the nucleologenetic process in early embryogenesis of other mammals, especially of that in the mouse. The first sign of NPB transformation was the appearance of a large central vacuole followed later on by perinucleolar chromatin penetration into NPB, documented by both morphology and [3H]thymidine autoradiography. In some cases, concentration of dense fibrillar material forming clumps or stalks was seen in the central vacuole. The following rapid nucleolar development was characterized by the formation of secondary vacuoles concomitant with the onset of [5-3H]uridine incorporation into the dense fibrillar component and with the appearance of the first granules in the otherwise fibrillar structure of the nucleolus. During the late eight-cell stage, the still-rounded nucleolus developed features of a reticulated nucleolus known from somatic cells intensively synthesizing rRNA: a dense fibrillar component with associated labeling encircling fibrillar centers and a well-developed granular component. The labeled dense fibrillar component was observed mostly in the central area of the nucleolus; early embryonic NPB dense fibrous material not involved in transcription was disappearing rapidly. At the transition to the 16-cell stage the nucleoli lost their rounded shape because of the accumulation of a large amount of granular component, and they occupied a considerable part of the nucleus. In conclusion, the appearance of the nucleolar vacuole in eight-cell cow embryo is the starting point for following morphogenetic events linked with the onset of transcription.

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Activity of maturation promoting factor in pig oocytes after microinjection and serial transfer of maturing cytoplasm.

Microinjection of 120 pl homologous or heterologous (mouse) maturing cytoplasm into immature pig oocytes resulted in germinal vesicle breakdown (GVBD) and chromosome condensation (CC) after 8 h of culture. In contrast, nearly all control oocytes remained at the germinal vesicle stage. Immature cytoplasm did not induce nuclear maturation of recipient oocytes when injected. The cytoplasm of the oocytes injected with maturing cytoplasm preserved the capacity to cause GVBD in the second recipients. These results suggest that the level of injected maturation promoting factor (MPF) must rise before GVBD can occur. When the pig oocytes injected with 120 pl of mouse maturing cytoplasm were cultured in cycloheximide for 8 h, the presence of GVBD was dramatically reduced. Thus, in pig oocytes the increase in active MPF up to the effective concentration requires protein synthesis.

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Effect of cycloheximide upon maturation of bovine oocytes.

Germinal vesicle breakdown (GVBD) of bovine oocytes was completely blocked by cycloheximide added to culture medium at concentrations of 1-20 micrograms/ml. Nevertheless, under such conditions a certain degree of chromatin condensation inside the germinal vesicle was observed. The inhibitory effect was not influenced by the presence or absence of cumulus cells and was fully reversible; but the process of GVBD was then significantly accelerated. The critical period in which the proteins necessary for GVBD are synthesized lasts approximately the first 5 h of culture. When germinal vesicle-arrested oocytes are fused to maturing bovine oocytes containing condensed chromosomes, GVBD of immature oocytes occurs within 3 h, even in the presence of cycloheximide. In the mouse, GVBD cannot be inhibited by protein synthesis inhibitors. When immature mouse oocytes are fused with immature bovine oocytes and the giant cells are then cultured in cycloheximide-supplemented medium, both GVs are observed, or only mouse GVBD occurs in common cytoplasm after 8 h of culture. We conclude that protein synthesis is necessary for GVBD of bovine oocytes. Our results also suggest that maturation-promoting factor (MPF) is not autocatalytically amplified in mammalian oocytes.

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Quantitative inhibitory influence of porcine cumulus cells upon the maturation of pig and cattle oocytes in vitro.

Porcine cumulus oocyte complexes (COCs) were cultured together in 10-microliters droplets of culture medium. When 10 COCs were cultured for 24 h, germinal vesicle breakdown (GVBD) occurred in 81% of them. When more COCs (20 or 40) were put into the same volume of medium the frequency of GVBD gradually decreased. This inhibition was not observed in denuded oocytes. The process of GVBD was adversely influenced when 10 COCs were cultured in cumulus-preconditioned medium. It is concluded that porcine cumulus cells produced a factor inhibiting GVBD. After removing the inhibitory block and extensive washing, GVBD of arrested oocytes was significantly accelerated. The addition of LH or heparin only partially overcame the inhibitory action. This factor produced by porcine cumulus cells negatively influenced maturation of bovine oocytes; however, a similar effect was not demonstrated in the mouse. Our results suggest that a high concentration of porcine cumulus cells exerts a quantitative inhibitory effect upon GVBD of porcine and cattle oocytes cultured in vitro.

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Fusion of mammalian oocytes: SEM observations of surface changes.

Mouse oocytes at the germinal vesicle (GV) stage were fused with maturing oocytes in which GVs were no longer visible. The fused cells were fixed at different time-intervals after the initiation of fusion and prepared for scanning electron microscope (SEM) observation. Concomitantly, some fused cells were prepared for light microscope evaluation. Our SEM observations showed no significant differences in surface morphology between immature and maturing oocytes. However, immediately after fusion was initiated, dramatic changes occurred on the surface of the maturing oocytes. The microvilli were shortened or disappeared locally and the plasma membrane was deeply ruffled. One hour after fusion, when the giant cells were nearly spherical, the microvilli reappeared and the ruffling gradually disappeared. In some areas, the microvilli were extremely long. Three hours after fusion, the fused cells were perfectly round and their surfaces were generally covered with microvilli of equal length. No further ruffling was observed. It is suggested that cytoplasmic mechanisms regulate the surface morphology of the oocytes during fusion.

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RNA and protein synthesis requirements for the resumption of meiosis in rabbit oocytes: the role of cumulus cells.

In vitro maturation of rabbit cumulus-enclosed oocytes was fully inhibited in alpha-amanitin- (100 micrograms/ml) and cycloheximide- (5 micrograms/ml) supplemented media. The inhibition was reversible and substantially reduced by delaying the addition of alpha-amanitin (2h) or cycloheximide (3 h). In contrast, both drugs did not inhibit germinal vesicle breakdown in denuded oocytes. Co-culture of granulosa cells (1 x 10(6)/ml) with denuded oocytes did not substitute for an intact cumulus. The data presented here suggest that the resumption of meiosis in rabbit cumulus-enclosed oocytes is dependent upon early transcriptional and translational events which probably occur within the cumulus cells.

Amanitins↗

In vitro techniques of bovine oocyte maturation, fertilization and embryo culture resulting in the birth of a calf.

Oocyte cumulus complexes were aspirated from 3 to 5 mm follicles of cows prestimulated with 2.000 IU PMSG 24 h before slaughter. Oocytes matured in culture were fertilized in vitro by heparinized freshly ejaculated or epididymal spermatozoa. The cultivation procedure for fertilized eggs was the same as that used for cultivation of oocytes. From 163 matured oocytes, 109 cleaved to the 2-cell stage 24 h after fertilization and after 6 days of cultivation, 18 developed to the late morula and 18 to the blastocyst stages. Eleven blastocyts and 1 late morula were transferred surgically to the uteri of 7 recipient heifers. Two heifers became pregnant: one delivered a bull-calf at term, while the other pregnancy resulted in abortion at the 3rd month. The examination of some embryos by transmission electron microscopy showed an almost normal morphology for most cells. The degenerated cells contained mostly electron-dense residual bodies of unknown origin.

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Localization of replicated DNA-containing sites in preimplantation bovine embryo in relation to the onset of RNA synthesis.

The distribution of embryonic DNA in nuclei of blastomeres of early-preimplantation cow embryos was studied by autoradiography. Two-, 4-, 8-, and 16-cell embryos were cultured for 18-20 h in the presence of [methyl-3H]thymidine to ensure that all replicated embryonic DNA was labeled. In nuclei of blastomeres before the onset of transcription, taking place in the progressed 8-cell cleavage division of the cow embryo, labeled DNA was distributed excentrically at the nuclear periphery. After transcription started, the distribution of the labeled DNA was uniform throughout the nucleus. The progressive association of DNA with the nucleolus-precursor body (NPB) also correlated with the expected onset of rRNA synthesis. The penetration of the labeled DNA into NPB was seen to start contemporaneously with the formation of a big central vacuole in NPB, but remained restricted, at this stage, to a few points of contact with the adjacent nucleolus-associated chromatin. No labeled DNA was detected inside this big vacuole, still showing a uniform nucleoplasmic texture. The inside of the nucleolus was penetrated by DNA in the next step of nucleologenesis when secondary small vacuoles were formed and when the first signs of nucleolar transcription were detected. These small vacuoles contained a coarse fibrillar component which was a frequent site of labeled DNA detection. This study of shifts of DNA-containing sites during transition from maternal to embryonic genome expression correlates closely with previous evidence obtained by a study of genome reactivation which was situated in the late 8-cell cleavage division of cattle early embryogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

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Time sequence of germinal vesicle breakdown in pig oocytes after cycloheximide and P-aminobenzamidine block.

All porcine oocytes cultured 20 hr in medium with 10 micrograms/ml cycloheximide rested in the germinal vesicle (GV) stage but with the highly condensed bivalents in nucleoplasm. When these oocytes were washed and cultured in the control medium for 2, 4, and 6 hr, germinal vesicle breakdown (GVBD) was completed in 0, 86, and 100% of them, respectively. When similarly inhibited oocytes cultured successively only 2.5 hr in the control medium were given again in cycloheximide enriched medium (3.5 hr), nearly all of them reached late diakinesis stage again. It means that oocytes cultured for 20 hr and washed free of this inhibitor of protein synthesis completed GVBD rapidly (4 hr) and protein synthesis crucial for nuclear membrane disintegration occurred already during the first 2 hr after washing of inhibitor. All oocytes cultured for 20 hr in medium with 1 mM p-aminobenzamidine rested in GV with chromatin around the compact nucleolus. The successive culture in cycloheximide (20 hr) and p-aminobenzamidine (10 hr) prevented GVBD in all oocytes, too. In contrast, when the oocytes washed after cycloheximide block (20 hr) were cultured in p-aminobenzamidine enriched medium 2 and 3 hr and again for 6 hr in cycloheximide medium, the nuclear membrane dissolved in 62 and 68% of oocytes, respectively. These data suggest that inhibition of protein synthesis in pig oocytes does not prevent the high condensation of bivalents in GV. However, nuclear membrane breakdown requires the successive protein synthesis and proteolysis.

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Does autocatalytic amplification of maturation-promoting factor (MPF) exist in mammalian oocytes?

The method of polyethylene-glycol-induced fusion of mammalian oocytes was applied to study maturation-promoting factor (MPF) activity. After homologous fusions of one maturing--late diakinesis (LD), metaphase I (MI)--pig or mouse oocyte to one, two, or three immature-germinal vesicle (GV)--oocytes, giant cells were cultured in control or cycloheximide supplemented medium for 3 hours. The occurrence of germinal vesicle breakdown (GVBD) and premature chromosome condensation (PCC) served as a control of MPF activity. In giant cells composed of one maturing and one, two or three immature oocytes, GVBD and PCC were observed in all cases after cultivation in the control medium. In the presence of cycloheximide, the completion of GVBD and PCC remained high when one maturing and one immature oocyte were fused (83.7% and 95.7% of GVBD in pig and mouse, respectively). However, in giant cells composed of one maturing and up to three immature oocytes, all GVs were broken down only occasionally (4.8% and 11.7% in pig and mouse, respectively). These results suggest that in pig and mouse oocytes MPF does not amplify autocatalytically, but requires active protein synthesis for its production.

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Activity of maturation promoting factor in mammalian oocytes after its dilution by single and multiple fusions.

Mouse and porcine fully grown oocytes at metaphase I(MI) were fused to one or more fully grown oocytes of the same species that contained an intact germinal vesicle (GV). In fused cells containing one GV, premature chromosome condensation (PCC) was observed. In fused cells containing more than one GV, germinal vesicle breakdown (GVBD) and PCC were delayed. Fusion of an MI fully grown oocyte with a growing oocyte resulted in rapid PCC, whereas, fusion of an MI fully grown oocyte with more than one growing oocyte resulted in neither PCC nor GVBD. Moreover, MI chromosomes formed a clump of chromatin. Results of these experiments suggest that the delay in GVBD in fusions of MI oocytes with multiple GV-intact oocytes was due to dilution of maturation promoting factor (MPF) by the cytoplasm of the GV-intact oocytes and that the cytoplasm of growing oocytes can inhibit MPF present in MI oocytes.

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Cumulus oophorus mucification during resumption of meiosis in the pig. A scanning electron microscope study.

The process of cumulus mucification in pig preovulatory follicles was examined by scanning electron microscopy. The localization of extracellular material and changes in the granulosa cell surface were observed at 0, 16, 20 and 40 h after hCG injection. At 0 h the pig oocyte cumulus complex was closely attached to the parietal layer of granulosa cells. Cumulus and parietal granulosa cells had microvilli and cytoplasmic projections connecting neighbouring cells. The network of extracellular amorphous material was observed for the first time at 16 h after hCG around the cells that formed a stalk between the parietal granulosa and the cumulus oophorus. At 20 h after hCG, the intercellular matrix was thicker and extended to almost all the cumulus oophorus surface; when visible, cells were often covered by blebs and ruffling membranes. All oocytes examined by light microscopy at 16 and 20 h after hCG were at the germinal vesicle stage with condensing bivalents. Therefore, it was concluded that oocyte nuclear maturation started at the same time that a mucified peduncle was forming between the cumulus oophorus and the parietal granulosa. Shortly before ovulation (40 h after hCG) the matrix material filled all intercellular spaces and mucification had extended to the corona cell layer and the zona pellucida surface.

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Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro.

Cumulus expansion and cumulus cell-oocyte coupling during in-vivo and in-vitro maturation of pig oocytes were studied by measuring [3H]uridine uptake. In vivo, cumulus expansion started before germinal vesicle breakdown (GVBD) (16 h versus 20 h after hCG) but no significant change occurred in the coupling index until 32 h after hCG. Intercellular coupling was decreasing at 32 h after hCG in oocytes at anaphase I and telophase I. Complete uncoupling was closely correlated with corona radiata expansion. In vitro, partial uncoupling was observed in oocyte-cumulus cell complexes from prepubertal and PMSG-stimulated gilts cultured for 16 and 32 h, respectively. The addition of FSH caused cumulus expansion, and the functional coupling between the cumulus cells and the oocyte was maintained up to at least 16 h of culture in complexes from prepubertal gilts. We conclude that, under our conditions, neither hormone-free nor FSH-supplemented medium ensured the same [3H]uridine uptake and uncoupling kinetics as during in-vivo maturation.

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Effect of cycloheximide on nuclear maturation of pig and mouse oocytes.

Culture of mouse oocytes in medium with 1 or 100 micrograms cycloheximide/ml did not prevent germinal vesicle breakdown (GVBD). In contrast, GVBD in pig oocytes was absolutely blocked at concentrations of 1, 5, 10, 50 and 100 micrograms cycloheximide/ml, respectively. The inhibition of GVBD was not influenced by the presence or absence of cumulus cells and it was fully reversible. When cycloheximide treatment (5 micrograms/ml) was given after preincubation for 6, 12 and 16 h, GVBD occurred in 15, 46 and 75% of oocytes, respectively. It is concluded that proteins important for GVBD of pig oocytes were present in sufficient amounts at about 12 h of culture. The fusion of pig oocytes in metaphase I to oocytes with an intact germinal vesicle revealed that cycloheximide did not inhibit GVBD induced by maturing ooplasm. Therefore, induction of prematurely condensed chromosomes by the maturing ooplasm did not require protein synthesis. However, continuous protein synthesis was necessary to maintain metaphase I and prematurely condensed chromosomes in a typical configuration.

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