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R Czołowska

Publications and source records attributed to R Czołowska.

13 recordsLinked to original sources
PubMed2001

Mouse singletons and twins developed from isolated diploid blastomeres supported with tetraploid blastomeres.

The aim of this study was to obtain mice, hopefully identical multiplets, from single diploid blastomeres isolated at the 4-cell stage, or from pairs of sister blastomeres isolated at the 8-cell stage. To this end isolated blastomeres were aggregated with one or two tetraploid carrier embryos produced by electrofusion of 2-cell embryos. Diploid embryos were albino and homozygous for the "a" allele of glucose-phosphate isomerase (GPI-1a1a) and tetraploid embryos were pigmented and GPI-1b1b. The aggregates were cultured in vitro up to the blastocyst stage. Each quartet (occasionally triplet or doublet) of chimaeric blastocysts was transplanted to the oviduct of a separate pseudopregnant recipient. Altogether 62 blastocysts were transplanted to 17 recipients. Eight full-term foetuses (two singletons and three pairs of twins) were rescued by Caesarian section on day 19, 20 or 21 of pregnancy. Three young (one singleton and twins) were successfully reared by foster mothers and proved to be normal and fertile females. All foetuses and animals were albino. In five individuals only the 1-A form of GPI (characteristic for 2n blastomere) was found. In one adult female traces of the 1-B form of GPI (characteristic for 4n carrier blastomeres) were detected in the heart and the lungs while 4 other organs contained only the 1-A form. These observations strongly suggest that the majority of foetuses/animals produced according to our experimental system are 'pure' diploids rather than 2n/4n chimaeras, and that the described method can be used in future to produce twins, triplets and quadruplets in the mouse. Our study confirms earlier work by Kelly (1975, 1977) that 'quarter' blastomeres of the mouse are still totipotent.

Animals↗
PubMed2001

How many blastomeres of the 4-cell embryo contribute cells to the mouse body?

The aim of this study was to estimate how many blastomeres of the 4-cell mouse embryo contribute cells to the embryo proper and finally to the animal. To this end, 4-cell embryos of pigmented and albino genotypes were disaggregated and single blastomeres (henceforth called '1/4' or 'quarter' blastomeres) were reaggregated in the following combinations: one 'pigmented' blastomere + three 'albino' blastomeres or vice versa (henceforth called '1+3') and two pigmented blastomeres + two albino blastomeres (henceforth called '2+2'). The aggregations were cultured in vitro and transferred as blastocysts either to the oviduct or uterus of pseudopregnant females. Recipients were allowed to litter naturally, or the foetuses were removed by Caesarian section and raised by lactating foster mothers. Chimaerism was assessed on the basis of coat (adults) or eye pigmentation (dead neonates). Among 28 '1+3' animals, there were 13 chimaeric and 15 non-chimaeric individuals. The pigmentation of non-chimaeras was always concordant with the genotype of the three 1/4 blastomeres and not with the genotype of the single blastomere in the given aggregation. These results make rather unlikely the possibility that the mouse is built of cells derived either from one or all four 1/4 blastomeres. Both two remaining options (2 or 3 1/4 blastomeres) are conceivable but the observed ratio of chimaeras to non-chimaeras among '1+3' animals (13:15) fits better the assumption of two 1/4 blastomeres contributing cells to the animal body. This assumption finds additional support in the observation that among '2+2' animals there were non-chimaeras (5 out of 7) and these would not have been expected should three 1/4 blastomeres contribute cells to the mouse body.

Animals↗
PubMed2000

Induction of DNA replication in the germinal vesicle of the growing mouse oocyte.

Growing mouse oocytes are physiologically arrested in the G2 phase of prophase of the first meiotic division. Growing oocytes were isolated from ovaries of 9- to 12-day-old mice and fused with parthenogenetic one-cell eggs or two-cell embryos derived from fertilized eggs. Resulting hybrids were injected with Dig-11-dUTP and examined for DNA replication using immunofluorescence. Parthenogenetic one-cell eggs fused at telophase II, G1, and middle-to-late S phase, and also S-phase two-cell blastomeres, were able to trigger DNA synthesis in oocyte germinal vesicle (GV) in the majority of hybrids cultured to the end of the first cell cycle. Activation of replication in the GV occurred within 2-3 h after fusion of growing oocytes with S-phase eggs. We show indirectly that the reactivation of replication in GVs was not dependent on the breakdown of the GV envelope. Although GVs had the ability to renew DNA replication after fusion, the G2 blastomere nuclei were incapable of reinitiating DNA replication under the influence of S-phase one-cell eggs. We hypothesize that the nuclei of growing oocytes arrested in meiotic prophase are in a physiological state that is equivalent to replication-competent G1, and not G2, nuclei.

Adenine↗
PubMed1998

Nuclear envelope removal/maintenance determines the structural and functional remodelling of embryonic red blood cell nuclei in activated mouse oocytes.

Nuclei of embryonic red blood cells (e-RBC) from 12-day mouse fetuses are arrested in G0 phase of the cell cycle and have low transcriptional activity. These nuclei were transferred with help of polyethylene glycol (PEG)-mediated fusion to parthenogenetically activated mouse oocytes and heterokaryons were analysed for nuclear structure and transcriptional activity. If fusion proceeded 25-45 min after oocyte activation, e-RBC nuclei were induced to nuclear envelope breakdown and partial chromatin condensation, followed by formation of nuclei structurally identical with pronuclei. These 'pronuclei', similar to egg (female) pronuclei, remained transcriptionally silent over several hours of in vitro culture. If fusion was performed 1 h or later (up to 7 h) after activation, the nuclear envelope of e-RBC nuclei remained intact and nuclear remodelling was less spectacular (slight chromatin decondensation, formation of nucleolus precursor bodies). These nuclei, however, reinforced polymerase-II-dependent transcription within a few hours of in vitro culture. Our present experiments, together with our previous work, demonstrate that nuclear envelope breakdown/maintenance are critical events for nuclear remodelling in activated mouse oocytes and that somatic dormant nuclei can be stimulated to renew transcription at a time when the female pronucleus remains transcriptionally silent.

Animals↗
PubMed1998

Mouse oocytes and parthenogenetic eggs lose the ability to be penetrated by spermatozoa after fusion with zygotes.

Fertilised mouse eggs develop the oolemma block to sperm penetration within 1 h. This block makes zona-free eggs at the pronuclear stage (zygotes) fully resistant to sperm penetration. In this study we investigated whether this block can spread--as a result of cell fusion--to the oolemma of eggs that are competent to be penetrated by spermatozoa. Preovulatory (GV) oocytes, ovulated oocytes in metaphase II (MII) and 1-cell parthenotes were fused with zygotes and the hybrid cells inseminated at various intervals after fusion. Sperm penetration was assessed on the basis of the presence of Giemsa-positive sperm heads in the air-dried preparations. The oolemma block to sperm penetration develops in all types of hybrids although at different speeds: it develops fast (2-3 h) in oolemma derived from MII oocytes and artificially activated eggs, and slowly in oolemma derived from GV oocytes. In the GV oocyte-zygote hybrids the time of formation of the block varied: while 50% of cells lost the ability to fuse with sperm by 2 h after fusion, in the remaining cells the block must have developed some time between 5 and 18 h after fusion. How these sperm-induced modifications of the oolemma of fertilised egg spread in the hybrid cell and render the 'virgin' part of oolemma resistant to sperm penetration remains unknown.

Animals↗
PubMed1993

Differential chromatin condensation of female and male pronuclei in mouse zygotes.

Mouse zygotes or halves of zygotes, containing either a female or a male pronucleus, were fused with ovulated metaphase II oocytes. In 59.7% of the resulting hybrid cells, the pronuclei underwent premature chromosome condensation (PCC). In some of these heterokaryons the 2 pronuclei differed in the dynamics of condensation. Detectability of differential PCC of pronuclei (dPCC) depended on the type of preparation. In hybrids with PCC, produced by fusion of intact zygotes with metaphase II oocytes and processed for whole-mount preparations, one pronucleus was more advanced in the condensation process in 47% of cases. In air-dried preparations dPCC was detected in as many as 94% of hybrids. Experiments with the fusion of halves of zygotes with metaphase II oocytes have shown that the differential reaction of pronuclei to condensation factor depended on their parental origin. Maternal chromatin responded faster to the condensation factor and attained more advanced stages of PCC than paternal chromatin. Different responses of the maternal and paternal pronucleus to the condensation factor suggests that the 2 pronuclei are not identical with regard to the organization of chromatin and/or the lamin composition of the nuclear envelope.

Animals↗
PubMed1988

Remodeling of mouse thymocyte nuclei depends on the time of their transfer into activated, homologous oocytes.

The potential of parthenogenetically activated mouse oocytes to remodel somatic cell nuclei was studied by ultrastructural means using oocyte-thymocyte hybrids. Complete nuclear remodeling, initiated by nuclear envelope breakdown and chromosome condensation (which is followed by formation of pronucleus-like nucleus) is possible only during a short time gap between metaphase II and telophase of meiotic division. Maturation-promoting factor activity is high during this period. The thymocyte nucleus can follow the sequence of morphological changes only in concert with the development of the native nucleus and only after exposure of the chromatin to the ooplasm. If hybridization is effected with pronucleate oocytes, the thymocyte nucleus retains its interphase character but shows particular modifications in nucleolar morphology (identical to changes observed during reactivation of the nucleolus in stimulated lymphocyte) and in the activity of the nuclear envelope (blebbing). Thus the nucleus not exposed to maturation-promoting factor activity may be influenced by a 'programme' specific for oocyte (blebbing) and by a programme inherent in the introduced somatic cell nucleus.

Animals↗
PubMed1986

Post-fusion reaction of mouse oocyte cortex to incorporated thymocyte cells.

Ovulated alcohol-activated or inactivated mouse oocytes were fused with mouse thymocytes. Activated oocytes react to the presence of foreign nuclei by forming in the peripheral cytoplasm incorporation cones. In this region the cell membrane is smooth and a cortical layer of thin filaments underlies it. It resembles the fertilization cone. In non-activated oocytes a layer of thin cortical filaments of the same thickness is formed over the foreign chromatin but the surface protuberance (cone) is absent. These results suggest that the cortical alteration in the oocyte architecture may be a general reaction of the oocyte surface to various chromatins introduced, not only to sperm chromatin. The role of oocyte activation in the evolution of these cortical changes is discussed.

Actin Cytoskeleton↗
PubMed1986

Chromosome condensation activity in ovulated metaphase II mouse oocytes assayed by fusion with interphase blastomeres.

Fusion of large and small karyoplasts produced from metaphase II mouse oocytes with interphase blastomeres from 2-cell and 8-cell embryos (volume ratio of partners, 1:1) results in premature chromosome condensation (PCC) of the interphase nucleus in the majority of the fusion products (hybrids). Fused under the same experimental protocol, oocyte-derived cytoplasts also induce PCC of the blastomere nucleus in the fusion products (cybrids) provided they originate from recently ovulated oocytes (141/2-15 h after injection of human chorionic gonadotrophin (HCG)). In cytoplasts derived from older oocytes (16-20 h post-HCG) chromosome condensation activity gradually decreases with time as can be inferred from the increasing proportion of cybrids retaining interphase blastomere nuclei. However, even the oldest cytoplasts (19-20 h post-HCG) can induce PCC if the cytoplast volume significantly exceeds the volume of the interphase partner (7:1). We postulate that the condensation activity is predominantly bound to the nuclear apparatus (most probably to the chromosomes), and that in the cytoplasm of metaphase II mouse oocyte it decreases with post-ovulatory age.

Animals↗
PubMed1986

Remodelling of thymocyte nuclei in activated mouse oocytes: an ultrastructural study.

Oocyte-thymocyte mouse cell hybrids were produced using polyethylene glycol (PEG) and examined at the ultrastructural level. Fusion was accomplished either before or after activation of metaphase II oocytes. In both experimental variants thymocyte nuclei undergo remodelling which comprises the following sequence of events: nuclear envelope breakdown, initial chromatin condensation, and subsequent decondensation, nuclear envelope reformation and formation of nucleoli. In hybrids produced before oocyte activation but activated within a short time and cultured for several hours the thymocyte nuclei become identical to the female pronucleus. In the second variant (fusion with activated oocytes) the degree of remodeling of thymocyte nuclei is variable. Our observations demonstrate that between metaphase II, telophase of meiosis and early female pronuclear stages the mouse oocyte contains all "factors" necessary for remodelling of differentiated somatic nuclei and their development as if they were pronuclei.

Animals↗
PubMed1984

Behaviour of thymocyte nuclei in non-activated and activated mouse oocytes.

Cells originating from the thymus of newborn mice were fused with mouse oocytes using polyethylene glycol. The behaviour of thymocyte nuclei was studied in non-activated metaphase II oocytes, and in oocytes activated in vitro with ethanol. In non-activated oocytes all thymocyte nuclei undergo premature chromosome condensation with individualization of chromosomes; the chromosomes form separate groups in the cytoplasm, or are assembled around the metaphase II spindle, or located on the extra-spindle. In activated oocytes thymocyte nuclei start to develop along a pronucleus-like pathway (decondensation, visualization of nucleoli, swelling) and increase up to 200 times in volume during 24 h culture in vitro, eventually reaching the size of a fully grown pronucleus. Activation/fusion timing seems to be critical for the full remodelling of thymocyte nuclei. Nuclei introduced before (10-30 min) or shortly after (up to 60 min) activation often grow larger than the female pronucleus. Those introduced into oocytes long before activation (greater than 30 min) undergo premature condensation with subsequent reformation of nuclei that are sometimes deficient (as indicated by the presence of micronuclei), or of hybrid character. Nuclei introduced late after activation (greater than 60 min) are mostly doomed to retarded development. The implications of the present observations for nuclear transfer experiments in mammals are discussed.

Animals↗
PubMed1984

Autonomous cortical activity in mouse eggs controlled by a cytoplasmic clock.

Mouse eggs of Swiss albino origin, both parthenogenetic and fertilized, were bisected into nucleate (NHs) and anucleate halves (AHs) and observed in vitro (semicontinuous observations) for up to 40 h for possible manifestations of cortical activity. Three experimental groups were studied: (1) Non-fertilized eggs activated 17 h after administration of hCG with a heat-shock and bisected 4 h later. (2) Non-fertilized eggs first bisected, and the resulting sister halves activated 17 h after administration of hCG with ethyl alcohol. (3) In vivo fertilized eggs bisected 27 h after administration of hCG into an AH and a binucleate half. Parthenogenetic eggs (intact, zona-free, and incompletely bisected), and fertilized eggs collected 17, 20, and 27 h after administration of hCG were also studied. In the middle of the first cell cycle the cell surface in all types of cells studied changed from smooth to slightly undulate. In nucleate cells the surface deformations lasted for several hours and disappeared shortly before the first mitosis. In contrast, in AHs the indentations of the cell surface deepened, and developed into manifold furrows, thus leading to fragmentation. However, in 20% of AHs fragmentation was partially or completely reversed. The incidence and the intensity of fragmentation were lower, and its reversibility was more common in AHs carrying the 2nd polar body. We suggest that the interphase nucleus, i.e. the pronucleus in whole eggs and NHs, and the 2nd polar body nucleus (if 2nd polary body is attached to an AH) exerts a moderating effect on cortical activity. However, the initiation of cortical activity is nucleus-independent, as shown by the behaviour of AHs separated before activation. We believe that the observed phenomena reflect autonomous cortical activity which is regulated by a cytoplasmic clock.

Animals↗