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A Rybouchkin

Publications and source records attributed to A Rybouchkin.

13 recordsLinked to original sources
PubMed2002

Developmental potential of cloned mouse embryos reconstructed by a conventional technique of nuclear injection.

The fact that most of the advances in mouse cloning by nuclear transfer originate from research in a limited number of laboratories demonstrates the complexity of the reported technologies. The development of alternative and more simple techniques of nuclear transfer may therefore be of interest. Furthermore, the preimplantation biology of cloned mouse embryos originating from somatic cells has not yet been studied in detail. In the present study, a modified conventional injection (mCI) technique for cloning mice from somatic cells is described. The preimplantation development and morphology of the resulting nuclear transfer embryos in comparison with parthenogenetic embryos and embryos obtained by intracytoplasmic sperm injection (ICSI) under comparable conditions was also studied. Finally, the capacity of nuclear transfer embryos for full-term development was investigated. Eighty-nine per cent of oocytes injected with cumulus cell nuclei under mCI conditions survived and formed zygotes. However, the rate of development of these zygotes to the blastocyst stage was significantly lower (29%) than that of ICSI or parthenogenetic zygotes (95 and 92%, respectively). Cloned blastocysts had a significantly lower mean number of cells in the inner cell mass (9) and trophectoderm (52) and a lower inner cell mass:total cell ratio (14%) than did their counterparts (31, 143 and 18% for ICSI and 21, 92 and 18% for parthenogenetic blastocysts, respectively). This correlated with a significantly higher proportion of dead cells in the cloned blastocysts. The poor quality of cloned blastocysts may explain the low rate of full-term fetal development of somatic mouse clones.

Animals↗
PubMed2001

Effect of culture media on in vitro development of cloned mouse embryos.

The effect of simple and sequential embryo culture media on the preimplantation development of mouse nuclear transfer (NT) embryos reconstructed with cumulus cell nuclei using a mechanical NT technique was studied. Blastocyst formation rate was evaluated using CZB medium and the sequential media G1/G2 and KSOM/G2. Arrested two- and three-cell NT embryos were Hoechst-stained to check for nuclear abnormalities. Nonmanipulated and sham-manipulated parthenogenetic embryos served as controls for, respectively, the medium and the handling technique. Rates of blastocyst formation for medium and handling control embryos were similar in CZB (58% and 61%), in G1/G2 (94% and 85%), and in KSOM/G2 (88% and 84%). Development of NT embryos was significantly impaired from the two-cell stage onwards, reaching the blastocyst stage at a rate of 5% in CZB, 14% in G1/G2, and 28% in KSOM/G2. Arrested two- and three-cell stage NT embryos showed a high rate of binucleation. These data demonstrate not only that NT embryos are more sensitive to in vitro culture conditions than parthenogenetic control embryos but also that selection of culture media can influence the preimplantation development of NT embryos.

Animals↗
PubMed1997

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.

Animals↗
PubMed1997

Disintegration of chromosomes in dead sperm cells as revealed by injection into mouse oocytes.

Intracytoplasmic sperm injection of immotile (dead) ejaculated human spermatozoa has been carried out by several centres for the treatment of infertility caused by severe asthenozoospermia (necrozoospermia). No healthy pregnancies have been reported as yet, suggesting irreversible damage to sperm DNA, centrioles and/or other important structures. We investigated this hypothesis by injection of immotile human spermatozoa obtained from several male infertility patients into mouse oocytes and analysis of the oocyte activation rate and sperm chromosome integrity. Motile spermatozoa of the same sample were used as a control. The proportion of living spermatozoa among the immotile was also assessed in each sample and was related to the results of the mouse oocyte injection test. The oocyte activation rate after injection of immotile human spermatozoa into mouse oocytes was the same or only slightly lower than after injection with initially motile spermatozoa (87-100% versus 100% respectively). The rate of normal sperm chromosome spreads correlated significantly (r = 0.90, P < 0.05) with the proportion of living immotile spermatozoa in a given sample. It varied from 4 to 48% for samples containing respectively 8 and 40% of living spermatozoa. Most of the mouse oocytes injected and activated with immotile human spermatozoa were arrested during a prolonged period of time at the interphase of the first cell cycle (from 22 to 60%). Others underwent a delayed nuclear envelope breakdown but showed signs of abnormal structure of both male and female or only the male pronuclear chromosomes. Our data demonstrate an irreversible damage of chromosomes in dead ejaculated human spermatozoa and provide an experimental basis for recommending the use of testicular or epididymal spermatozoa for treatment of male infertility due to necrozoospermia.

Animals↗
PubMed1996

Factors affecting the role of the spindle during early response of mouse oocytes to ethanol stimulation.

Winston et al. ([1995] J. Cell Sci., 108:143-151) have shown recently that short (6 min) exposure of spindle intact oocytes from Swiss mice to 8% ethanol induced activation of most oocytes, while disruption of the spindles in these oocytes by nocodazole, before and during ethanol exposure, completely inhibited oocyte activation. We compared the activation rates (ARs) of nocodazole-treated and intact oocytes recovered from SJL and B6D2 F1 hybrid mice under the same experimental conditions. The difference between the ARs of nocodazole-treated and intact SLJ oocytes was about the same as reported for Swiss oocytes (2% vs. 82%, respectively). In contrast, this difference was minor for B6D2 oocytes (87% vs. 100%, respectively). Moreover, 41% of these oocytes underwent activation when the spindle was absent, not only before and during, but also 2 h after ethanol exposure. Shortened exposure (2 min) of B6D2 oocytes to ethanol, however, increased the difference in the ARs of nocodazole-treated and intact oocytes (18% vs. 67%, respectively). We conclude that at least two parameters affect the necessity of the presence of the spindle during ethanol exposure for the activation of mouse oocytes. They are the genotype of the oocytes and the duration of exposure to ethanol. Under one set of these parameters the presence of the spindle is absolutely necessary, while under the other the appearance of the spindle a few hours after ethanol exposure is sufficient to allow the activation of some oocytes.

Animals↗
PubMed1996

Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes.

Intracytoplasmic sperm injection (ICSI) in the human is a very effective procedure which allows the fertilization of the majority of oocytes even in cases of extreme oligoasthenoteratozoospermia. Round-headed acrosomeless human spermatozoa, however, form an exception to this rule, because in about half of the couples with globozoospermia all oocytes remain unfertilized after injection. The incapacity of the spermatozoon to activate the oocyte following injection of round-headed spermatozoa could be the underlying mechanism. To investigate this hypothesis, activation rates of mouse oocytes injected with spermatozoa from a patient with globozoospermia were compared with those obtained after injection with normal spermatozoa. Of mouse oocytes surviving the injection with donor spermatozoa, 95% underwent activation, compared to none of the 88 mouse oocytes surviving the injection with round-headed spermatozoa. After fixation, prematurely condensed sperm chromosomes were found in these oocytes. Parthenogenetic activation of mouse oocytes (8% ethanol at 40 min after injection) injected with round-headed spermatozoa led to the activation of 96% of oocytes. These oocytes developed normally to the first mitosis and were fixed for analysis of the sperm karyotypes. The incidence of chromosomal abnormalities of round-headed spermatozoa (6%) was similar to that in spermatozoa from a fertile donor (9%). These data provide further information on the basic defect in cases of globozoospermia and demonstrate that globozoospermia is not associated with sperm karyotype abnormalities.

Adult↗
PubMed1995

Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation.

In the present study we investigated the relevance of sperm immobilization prior to intracytoplasmic sperm injection (ICSI) in the fertilization process. Using supravital staining of the spermatozoa with eosin and studying sperm decondensation with 2 mM dithiothreitol (DTT) in conditions imitating sperm handling during ICSI, we demonstrated that immobilization of the spermatozoon by squeezing its tail between the glass pipette and the bottom of the dish damages the sperm plasma membrane. Polyvinylpyrrolidone (PVP), which is usually present in the drop with the spermatozoon to facilitate its handling, was found to impede the access of both eosin and DTT to the sperm nucleus. We conclude that (i) sperm immobilization prior to ICSI damages the sperm plasma membrane, that (ii) this damage is sufficient for thiol-reducing agents to gain access to the sperm nucleus, and finally that (iii) PVP possibly interferes with sperm nucleus decondensation.

Cell Membrane↗
PubMed1995

Timing of sperm and oocyte nuclear progression after intracytoplasmic sperm injection.

We investigated the time course of human oocyte activation after intracytoplasmic sperm injection (ICSI) by observing the oocyte chromosome configuration at different times after injection. One day old human oocytes were injected with spermatozoa and subjected to cytogenetic analysis at 2, 3, 4 and 5 h after injection. We found that anaphase is initiated in the vast majority of the oocytes between 2 and 3 h after injection, and that by 4-5 h after injection most of the oocytes have reached the chromatin mass stage. Two distinguishable stages of sperm nucleus transformation were observed. The first phase-swelling-was reached within 2 h after the injection and was independent of oocyte activation. The second phase-the "brush'-like stage or decondensed chromatin stage-was found only in activated oocytes. Moreover, this stage was not reached before the chromatin mass stage (late telophase) of the oocyte. The same proportion of metaphase II oocyte chromosome configurations and unchanged sperm nuclei was found at any given time after injection. We conclude that: (i) ICSI allows users to obtain an almost synchronized population of activated oocytes; (ii) anaphase II is initiated in the majority of oocytes not later than 2-3 h after injection and telophase II is reached approximately 5 h after injection; and (iii) there are two distinguishable phases of sperm nucleus transformation after ICSI: oocyte activation-independent swelling of the sperm head and oocyte activation-dependent chromatin decondensation which is coupled to the beginning of oocyte chromosome decondensation.

Cell Nucleus↗
PubMed1995

Human oocyte activation following intracytoplasmic injection: the role of the sperm cell.

The aim of this study was to investigate whether the human spermatozoon participates in the activation of human oocytes following intracytoplasmic sperm injection (ICSI) and if so, by what mechanism. In the first series of experiments, we randomized human oocytes which had remained unfertilized after in-vitro fertilization (IVF) or ICSI, for intracytoplasmic injection with live spermatozoa, spermatozoa presumed to be dead and no spermatozoa. Secondly, unfertilized human oocytes and freshly ovulated mouse oocytes were randomized for intracytoplasmic and sub-zonal injection with human sperm cytosolic fraction (CF) before and after heat treatment. We found that oocyte injection with initially motile spermatozoa induces human oocyte activation at a significantly higher rate than injection with dead spermatozoa (61 versus 0%; P < 0.001) or injection without a spermatozoon (61 versus 14%; P < 0.001). Intracytoplasmic injection of CF activated both human and mouse oocytes at the same rate as sperm injection of human oocytes (activation rates of 70 and 65% respectively). This effect was greatly reduced by heat treatment of the CF. From these experiments we conclude firstly that the human spermatozoon injected intracytoplasmically contributes to human oocyte activation and secondly that the spermatozoon releases into the oocyte a heat-sensitive, intracellularly active factor, which is not species-specific.

Cell Death↗
PubMed1995

Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa.

When intracytoplasmic sperm injection (ICSI) is performed, it is important to know the capacity of sperm cells to activate the oocytes, although knowledge of their ability to fuse with the oocytes is not vital. Hamster oocytes are not suitable for this purpose because they are easily activated by the injection procedure Itself. We therefore investigated whether mouse oocytes could be used to assess the activation properties of human spermatozoa. Mouse oocytes were randomized for injection with initially motile spermatozoa, medium, heat-treated or salt-extracted spermatozoa, and the survival and activation rates were examined. About half of the mouse oocytes survived the intracytoplasmic injection of a human sperm cell. Unlike hamster oocytes, the rate of activation provoked by the injection procedure itself was acceptably low (20%), resembling in this respect the behaviour of human oocytes. Following the injection of initially motile human spermatozoa, all mouse oocytes were activated. The injection of heat-treated or salt-extracted human spermatozoa resulted in activation rates of 14 and 15% respectively, comparable with the results following sham ICSI. These data support the hypothesis of a sperm-associated oocyte activation factor. In most activated oocytes, the human sperm nucleus decondensed to form a male pronucleus. Cytogenetic analysis at the first metaphase revealed that human sperm chromosomes were able to undergo replication in a heterologous environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗