Biomedical subjects

A K Tarkowski

Publications and source records attributed to A K Tarkowski.

At least 19 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.

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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.

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PubMed1999

Delayed sperm incorporation into parthenogenetic mouse eggs: sperm nucleus transformation and development of resulting embryos.

In this study we examined the effect of experimentally induced asynchrony between male and female pronuclei on male pronucleus formation and developmental potential of the resulting mouse embryos. We demonstrate that when the interval between oocyte activation and sperm incorporation is up to 1.5-2 hr, the spermatozoa transform into normal pronuclei. These male pronuclei can replicate their chromosomes during the first embryonic cell cycle and are transcriptionally competent. During the first cleavage these "delayed" male pronuclei condense into discrete mitotic chromosomes and when resulting embryos are transplanted into oviducts of pregnant females at least some of them can develop to term. In contrast, when sperm nuclei are introduced into parthenogenetic eggs 3 hr or more after activation, their transformation into pronuclei is significantly impaired, and they neither replicate nor transcribe. During the first mitosis they form a group of condensed chromatin, which is displaced into one of the resulting blastomeres leading to formation of haploid/diploid mosaic embryos. These mosaic embryos have poor developmental potential: only a few can reach blastocyst stage in vitro and no full-term development of such embryos was observed after transfer into pregnant females. We conclude that the cytoplasmic factors that make possible the transformation of a sperm nucleus into a functional male pronucleus exhaust within 1.5-2 hr after fertilization and that the male genome which had skipped the first cell cycle cannot become a functional partner in the embryonic genome.

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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.

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PubMed1998

Autonomous activation of histone H1 kinase, cortical activity and microtubule organization in one- and two-cell mouse embryos.

The activation of M-phase promoting factor (MPF) in one-cell mouse embryo is independent from the nucleus. Other autonomous phenomena include the cortical activity observed at the end of the first cell cycle and the reorganization of the microtubule network. Here, we observed that the autonomous control of MPF activation is present also in two-cell mouse embryos (H1 kinase activity being higher in the first than in the second cell cycle). Moreover, the disappearance of the cortical activity in anucleated halves is observed when MPF activation takes place. The rounding up of the cytoplast and the mitotic reorganization of the microtubule network correlates with the maximum activity of H1 kinase in anucleated halves from one-cell embryos. In anucleated halves of two-cell stage blastomeres neither the cortical activity nor the microtubule reorganization were observed. The degree of activation of histone H1 kinase, and, as a consequence, the cortical activity and the microtubule reorganization, does not depend on the distribution of cyclin B. Finally, the level of cyclin B synthesis is similar in anucleated and nucleated halves derived from both one- and two-cell embryos.

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PubMed1998

Mouse chimaeras revisited: recollections and reflections.

This very personal and subjective article briefly describes the evolution of techniques used for obtaining mouse chimaeras from various sources (embryos, EC, ES and EG cells), summarizes studies on inter-specific chimaeras, mentions some of other applications ('rescuing' chimaeras), presents the contribution of Ralph Brinster to this area and tries to estimate whether the expectations I expressed in 1961 as to the usefulness of making and studying chimaeras turned out to be correct. Tribute is paid mainly to those, who as the first, contributed to various aspects of these studies.

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PubMed1997

Haploid maternal genome derived from early diplotene oocytes can substitute for the female pronucleus in preimplantation mouse development.

We describe the preimplantation development of mouse embryos that have received the haploid maternal genome derived from early diplotene nuclei of primordial oocytes (PO). Two generations of recipient egg-cells were used. Induction of two meiotic divisions of the PO nucleus and the reduction of the number of chromosomes to the haploid level were achieved in preovulatory oocytes (primary recipients). The developmental potential of the obtained haploid genome was examined in zygotes (secondary recipients). The nuclei of PO obtained from newborn mice were transferred by cell electrofusion to in vitro maturing (IVM) and enucleated preovulatory mouse oocytes. The reconstructed oocytes which had completed maturation, i.e., reached metaphase II, were artificially activated (8% ethanol + CHX). Activated oocytes were used as donors of haploid pronuclei of PO origin which were transferred (by karyoplast fusion) to partially enucleated zygotes containing only the male pronucleus. Thus, reconstituted zygotes were transplanted to the ligated oviducts of the cycling mice and 27% of them developed to the blastocyst stage. Our experiments demonstrate that 1) the nucleus of PO can be induced to premature meiotic divisions in an IVM enucleated preovulatory oocyte; 2) in the presence of a normal male pronucleus, the haploid pronucleus of PO origin can substitute for a female pronucleus during preimplantation development.

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PubMed1997

Transcription and DNA replication of sperm nuclei introduced into blastomeres of 2-cell mouse embryos.

The aim of this study was to investigate the behaviour of sperm nuclei in the cytoplasm of the 2-cell mouse embryo. To this end, we produced hybrids between anucleate fertilised oocyte fragments and blastomeres of the 2-cell embryos. When sperm nuclei at the stage of decondensation or recondensation were introduced into blastomeres the development of male pronuclei was usually retarded and they never reached the size of the blastomere nuclei. These abortive male pronuclei were unable to initiate transcription but they were capable of synthesising DNA. The majority of sperm nuclei introduced into blastomeres as early male pronuclei developed normally and reached the size of the blastomere nuclei. They synthesised DNA simultaneously with blastomere nuclei and were transcriptionally active. In addition they participated in the cleavage division of hybrid cells. This shows that the very early male pronucleus when transmitted from the oocyte cytoplasm to the blastomere cytoplasm can respond positively to the new cytoplasmic factors, i.e. it undertakes both DNA replication and transcription according to the time schedule characteristic of the second cell cycle.

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PubMed1997

Postimplantation development of mouse blastocysts with two separate inner cell masses.

Blastocysts with double inner cell masses (ICMs) were produced by electrofusion of two blastocysts and transplanted to pseudopregnant recipients. The implanted embryos were either examined histologically (8th day of pregnancy) or dissected and inspected in toto (11th or 12th day). In most cases both ICMs of experimental blastocysts developed into separate egg cylinders. Both cylinders were located in the common yolk sac cavity. Some cylinders were quite normal, but most of them were small and deprived of embryonic membranes. Ectoplacental cones of these cylinders were often oriented laterally or even antimesometrially. The development of cylinders seems to depend upon the position of their ectoplacental cones--cylinders with cones situated antimesometrially were handicapped in development. Among four sets of twin embryos examined on the 11th or 12th day, in one set each of the twins were equally developed, and in the three others one of the twins was more advanced.

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PubMed1996

First meiosis of early dictyate nuclei from primordial oocytes in mature and activated mouse oocytes.

Nuclei of diplotene (dictyate) primordial oocytes (PO) were transferred to metaphase II oocytes and to activated mouse oocytes using cell fusion techniques. In a metaphase II oocyte, the PO nucleus condenses within 2-3 h to bivalents which become arranged on the first meiotic spindle. After oocyte activation, homologous chromosomes segregate between the oocyte and the first polar body, and a diploid pronucleus-like nucleus reforms from the one set of dyads. This nucleus condenses in the first embryonic mitosis into 40 'somatic' chromosomes which coexist in the common metaphase plate with 20 somatic chromosomes originating from the female pronucleus. Shortening of the time between fusion and activation of about 1 h prevents bivalent differentiation. The PO nucleus condenses only partially and reforms, after oocyte activation, a pronucleus-like nucleus. This nucleus gives rise at the first embryonic mitosis to 20 bivalents which coexist with 20 somatic chromosomes originating from the female pronucleus. A PO nucleus introduced into an activated egg completes the first cell cycle as an intact interphase nucleus. It never condenses in the first embryonic mitosis into bivalents, and undergoes only initial condensation (preceding bivalent differentiation). These results indicate that: (1) condensation into bivalents, meiotic spindle formation and first meiotic division can be greatly accelerated by the introduction of an early diplotene (dictyate) oocyte nucleus into a metaphase II oocyte, and (2) depending on whether the diplotene nucleus enters the first embryonic (mitotic) cell cycle after just initiating or after completing the first meiosis, it gives rise at the first cleavage division to meiotic (bivalents) or 'somatic' chromosomes respectively.

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PubMed1995

Cytostatic factor inactivation is induced by a calcium-dependent mechanism present until the second cell cycle in fertilized but not in parthenogenetically activated mouse eggs.

Cytostatic factor (CSF) is an activity responsible for the metaphase II arrest in vertebrate oocytes. This activity maintains a high level of maturation promoting factor (MPF) in the oocyte and both activities are destroyed after fertilization or parthenogenetic activation. To study some of the characteristics of the mechanism involved in MPF and CSF destruction, we constructed hybrid cells between metaphase II arrested oocytes and early embryos obtained after fertilization or artificial activation. We found that the behavior of hybrid cells differed depending upon the type of oocyte activation. Initially, the reaction of both types of hybrid cells was similar, the nuclear envelope broke down and chromatin condensation was induced. However, while metaphase II oocytes fused with parthenogenetic eggs remained arrested in M-phase, the oocytes fused with fertilized eggs underwent activation and passed into interphase. This ability of fertilized eggs to induce oocyte activation was still present at the beginning, but not at the end of the second embryonic cell cycle. Oocyte activation induced by fusion with a fertilized egg could be prevented when calcium was chelated by BAPTA. Thus, element(s) of the mechanism involved in calcium release triggered by a sperm component at fertilization remain(s) active until the second cell cycle and is (are) inactivated before the end of the 2-cell stage.

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PubMed1990

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study.

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certain maturation-linked changes were noted. Sperm apposition and sperm-oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrounded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck-piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.

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PubMed1989

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs.

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3.5, and 8 h after activation, and in series B, 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively; chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anucleate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclear growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.

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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.

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PubMed1986

Ultrastructure of cell fusion and premature chromosome condensation (PCC) of thymocyte nuclei in metaphase II mouse oocytes.

Following PEG (polyethylene glycol) treatment of ovulated metaphase II mouse oocytes aggregated with thymocytes, fusion of cell membranes occurs. Prerequisites for cell fusion are: close apposition of lectin-agglutinated (phytohemagglutinin-treated) membranes of both cells, formation of firm punctual adhesion sites, and expansion of adhesion sites over a certain area. Establishment of the firm cell-cell contact is associated with development of actin-like filaments along both of the adhering plasma membranes. Membrane fusion occurs at single or multiple sites, and is followed by internalization of thymocyte-oocyte membrane complexes decorated with actin filaments into the hybrid cell cytoplasm. A filamentous actin layer forms also along the inner surface of newly formed hybrid oocyte-thymocyte plasma membrane. Thymocyte nuclei incorporated into oocyte cytoplasm undergo nuclear envelope breakdown and premature chromosome condensation (PCC) leading, eventually, to formation of single chromatids complete with kinetochores. Concomitantly with chromatin condensation an extensive polymerization of microtubules starts in the center of the chromatin mass which leads to the formation of an apparently non-functional spindle-like structure.

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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.

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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.

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PubMed1985

Electrofusion of mouse blastomeres.

Fusion of blastomeres of 2-cell mouse embryos with an intact zona pellucida can be induced with electric pulses. Fusion was most frequent with the field strength of 1 kV/cm and direct current pulses of 100-250 microsec duration. An electrolyte solution (PBS) can be used instead of a non-electrolyte solution (0.3 M mannitol). The viability of blastomeres fused in these two types of solution is similar. Fused 2-cell blastomeres develop into tetraploid blastocysts but die after implantation. Embryos in which blastomeres failed to fuse despite the treatment (diploid controls) can develop till term. The technique can also be applied to 3- and 4-cell embryos and to zona-free oocytes and blastomeres.

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