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M H Kaufman

Publications and source records attributed to M H Kaufman.

At least 91 records · Page 5Linked to original sources
PubMed1991

Cleavage rates of diploid and tetraploid mouse embryos during the preimplantation period.

Despite the fact that spontaneous tetraploidy is a rare phenomenon in mice, such embryos may be produced experimentally by a variety of means, though only a very limited degree of postimplantation development has been achieved. Despite this apparent limitation, much data on the rate of development of preimplantation tetraploid embryos has been published. However, the findings from these studies has often been conflicting. In the light of the recent successful achievement of advanced postimplantation tetraploid development in our laboratory, we decided it was an opportune time to re-evaluate the preimplantation development of these embryos in as near to optimal conditions as we could achieve. Three groups were studied, namely 1) control (diploid) embryos developing in vivo, 2) control (diploid) embryos that had been isolated at the 2-cell stage, briefly retained in culture, then transferred to the oviducts of pseudopregnant recipients, and 3) tetraploid embryos produced by electrofusion of blastomeres at the 2-cell stage, then transferred to the oviducts of pseudopregnant recipients. Embryos were isolated from females from each group at specific times after the HCG injection to induce ovulation. The total cell number of each embryo was established and the log mean values were plotted against time. From the gradients of the lines it was possible to establish that there was a significant difference between the cell doubling time of the transferred controls (group 2) compared to the in vivo controls (group 1) with cell doubling times of 15.86 +/- 1.45 h and 10.27 +/- 0.24 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Developmental potential and chromosome constitution of strontium-induced mouse parthenogenones.

The brief exposure of recently ovulated mouse oocytes to M16 embryo culture medium supplemented with strontium chloride (M16 Sr2+) for 2-10 min was observed to induce a high incidence of parthenogenesis. A lower incidence of activation and a significant rate of oocyte degeneration was observed when oocytes were incubated in M16 Sr2+ medium for 20-60 min. The majority of the oocytes exposed to this agent for 2-10 min developed as single-pronuclear haploid parthenogenones. The incidence of this parthenogenetic class was reduced as the duration of exposure to M16 Sr2+ was increased from 2 to 30 min. Under these conditions a greater proportion of the activated oocytes developed as two-pronuclear diploid parthenogenones, due to failure of second polar body extrusion. The activation frequency and the proportionate incidence of the pathways of parthenogenetic development observed following the exposure of ovulated oocytes to calcium-free M16 medium differed significantly from that induced by exposure to M16 Sr2+. Cytogenetic analysis of the single-pronuclear haploid class of Sr(2+)-induced parthenogenones at metaphase of the first-cleavage mitosis has shown that this agent did not induce a significant increase in the incidence of chromosome segregation errors during the completion of the second meiotic division. Analysis of the developmental potential of the two-pronuclear class of diploid Sr(2+)-induced parthenogenones during the preimplantation stages of embryogenesis revealed that their cell number and rate of cell division were less than those of fertilised embryos retained either in vivo or in vitro. The novel methods of activating oocytes indicated in this study present new opportunities to improve the efficiency of embryo cloning techniques with the ruminant species.

Animals↗
PubMed1991

New insights into triploidy and tetraploidy, from an analysis of model systems for these conditions.

While triploids and tetraploids together probably account for approximately 20% of all spontaneous abortions with a numerical chromosomal defect, and consequently give rise to a significant proportion of human pregnancy wastage, relatively little is known about their early embryogenesis and phenotypic features. We have therefore studied the early post-implantation stages of development of spontaneously occurring digynic triploid mouse embryos and experimentally induced diandric and digynic triploids, as well as diandric heterozygous diploids and homozygous tetraploid embryos. This material has been examined morphologically and cytogenetically, in order to investigate whether any relationship exists between their phenotype, genotype and developmental potential. Attention is drawn to the fact that the detailed analysis of this material has already shed important new light on the genetic factors that influence early mammalian development. In addition, it appears likely that it has the potential to provide new insights into pattern formation, in particular in relation to the craniofacial region, the heart and the post-cranial vertebral axis.

Abortion, Spontaneous↗
PubMed1991

Histochemical identification of primordial germ cells and differentiation of the gonads in homozygous tetraploid mouse embryos.

This study was undertaken to establish whether primordial germ cells are differentiated by homozygous tetraploid mouse embryos produced by the technique of electrofusion, and to study the morphological features of their gonads. Tetraploid embryos were transferred to the oviducts of pseudopregnant recipients, and these were autopsied either on day 11 or days 15 or 16 of gestation. In the developmentally less advanced group, embryos in which cytogenetic analysis of their extraembryonic membranes confirmed that they had a tetraploid chromosome constitution were analysed histochemically in order to demonstrate the presence of intracellular alkaline phosphatase enzyme activity. This enabled the presence or absence of germ cells to be established. Out of a total of 9 early limb-bud stage embryos studied, all contained primordial germ cells. The latter were mostly located in association with the hindgut, though some germ cells were still present at the base of the allantois. The sex ratio in this group was close to unity. In the 2nd group in which recipients were autopsied on either day 15 or 16 of gestation, a total of 7 healthy tetraploid embryos were recovered. All displayed the characteristic craniofacial features seen in tetraploid embryos. Four of these embryos had a normal postcranial axial morphology, and their crown-rump lengths were only slightly less (81-91%) than those of developmentally matched control diploid embryos. Three of the tetraploid embryos had an abnormal postcranial axis associated with a body wall closure defect involving the anterior abdominal and lower thoracic region. In all 7 of these embryos, gonadal differentiation was consistent with their developmental age.

Animals↗
PubMed1990

Effect of maternal age on the incidence of digynic triploidy in LT/Sv strain mice: implications for the ovulation of primary and secondary oocytes in this strain.

LT/Sv strain mice ovulate both primary and secondary oocytes, which are fertilisable, giving rise to digynic triploid and normal diploid fertilised conceptuses, respectively. Since the proportion of primary and secondary oocytes ovulated in our earlier studies varied widely among females, we investigated whether the proportion of primary oocytes ovulated in LT/Sv strain mice was influenced by maternal age. Females 6, 12, 18, 24, and 30 weeks old were mated with F1 hybrid males. The females were autopsied on the 10th day of gestation, and the intact conceptuses or extraembryonic membranes analysed cytogenetically. Since no selective loss of the triploids occurs up to the 10th day of gestation, analysis at this time also provides indirect information on the proportion of primary and secondary oocytes ovulated. We observed 1) that the overall incidence of triploidy decreased from 55% in the 6-week-old females to 6% in the 30-week-old group; 2) that the number of females from which both triploid and diploid embryos were recovered decreased with increased maternal age; 3) that a substantial decrease in the proportion of triploid embryos was observed in those females in which both triploid and diploid embryos were recovered, in relation to increased maternal age; 4) that there was no overall decrease in the total number of implants with increasing age; and 5) that there was no increase in the incidence of resorptions with increasing maternal age. We believe that no comparable relationship between the ovulation of primary and secondary oocytes and maternal age has previously been reported.

Animals↗
PubMed1990

Histochemical identification of primordial germ cells in diandric and digynic triploid mouse embryos.

Diandric and digynic triploid mouse embryos were isolated in the morning on day 10 of gestation. The embryos were separated from their extraembryonic membranes, and the latter were analysed cytogenetically by G-banding to establish the ploidy and sex chromosome constitution of these embryos. The diandric triploid embryos were produced by the technique of nuclear micromanipulation. Females were mated with male mice with a morphologically distinguishable "marker" chromosome to confirm the diandric status of these embryos. Digynic triploid and normal diploid embryos were isolated from LT/Sv strain females. These females spontaneously ovulate both primary and secondary oocytes, which are fertilisable and give rise to digynic triploid and normal diploid embryos, respectively. All the embryos were serially sectioned and processed in order to demonstrate the presence of alkaline phosphatase enzyme activity. This histochemical technique allowed primordial germ cells to be readily recognised, due to their characteristic location, cellular morphology, and staining appearance. Primordial germ cells were found in all the embryos studied, being located within the visceral yolk sac, at the base of the allantois, and/or in association with the wall or mesentery of the hindgut. The total number of germ cells present was established in nine diandric triploids and in five digynic triploids. The findings presented here represent the first demonstration that primordial germ cells can differentiate in either diandric or digynic triploid mammalian embryos.

Animals↗
PubMed1990

Sex-chromosome constitution of postimplantation tetraploid mouse embryos.

Tetraploid mouse embryos were produced at the two-cell stage by blastomere fusion induced by inactivated Sendai virus. The embryos were from chromosomally normal female mice that had been fertilised by homozygous Rb(1.3)1Bnr males carrying a pair of large metacentric marker chromosomes in their karyotype. These "reconstructed" one-cell tetraploid embryos were then transferred to the oviducts of pseudopregnant recipients, which were subsequently autopsied early on the 10th day of gestation. Two-cell stage embryos that did not undergo blastomere fusion after 4-5 h were transferred to a second group of recipients, which were also autopsied early on the 10th day of gestation. From a total of 153 tetraploid embryos transferred to females that subsequently became pregnant, 135 implanted. Sixty-eight implantation sites were found to contain resorptions, whereas 67 contained mostly headfold presomite-stage embryos. Four embryos possessed four to six pairs of somites. All 57 embryos that could be analysed cytogenetically were found to be tetraploid. G-banding analysis revealed that 30 of these embryos had an XXYY and 27 and XXXX sex-chromosome constitution. The presence of two marker chromosomes in all mitotic preparations from each of these tetraploid embryos confirmed that they had all been produced by duplication of their original XY or XX diploid chromosome constitution, respectively. The XXYY:XXXX sex ratio observed was not significantly different from unity. In the control series of transfers, all of the embryos recovered were at the forelimb bud stage and had a diploid chromosome constitution. The results reported here differ from human clinical findings, in which the XXYY:XXXX sex ratio of 120 human tetraploid spontaneous abortions recovered over the last 20 years is 45:75. Possible explanations for these differences are briefly discussed.

Animals↗
PubMed1990

Postimplantation development of tetraploid mouse embryos produced by electrofusion.

Despite the fact that a variety of experimental techniques have been devised over the years to induce tetraploid mammalian embryonic development, success rates to date have been limited. Apart from the early study by Snow, who obtained development to term of a limited number of cytochalasin B-induced tetraploid mouse embryos, no other researchers have achieved development of tetraploid embryos beyond the early postimplantation period. We now report advanced postimplantation development of tetraploid mouse embryos following electrofusion of blastomeres at the 2-cell stage, and subsequent transfer of these 1-cell 'fused' embryos to appropriate recipients. Cytogenetic analysis of the extraembryonic membranes of all of the postimplantation embryos encountered in the present study has provided an unequivocal means of confirming their tetraploid chromosome constitution. A preliminary morphological and histological analysis of the tetraploid embryos obtained by this technique has revealed that characteristic craniofacial abnormalities particularly involving the forebrain and eyes were consistently observed, and these features were often associated with abnormalities of the vertebral axis and heart. The most advanced viable embryo in this series was recovered on the 15th day of gestation, and its morphological features suggest that it was developmentally equivalent to a normal embryo of about 13.5-14 days p.c.

Animals↗
PubMed1989

Cytogenetic analysis of ethanol-induced parthenogenesis.

The brief exposure of recently ovulated mouse oocytes to a dilute solution of ethanol in vitro for 1, 3, or 5 min induced a uniform high incidence of parthenogenetic activation. The majority of parthenogenones developed a single haploid pronucleus after the extrusion of a second polar body. The proportionate incidence of this parthenogenetic class was significantly reduced as the duration of ethanol exposure increased from 1 min to 5 min. There was a concomitant increase in the incidence of parthenogenones that developed two haploid pronuclei following failure of extrusion of the second polar body. Cytogenetic analysis of the ethanol-induced single-pronuclear haploid parthenogenones at metaphase of the first cleavage division clearly demonstrated that a significant proportion were aneuploid. The incidence of aneuploidy observed was directly related to the duration of ethanol exposure. G-band analysis of the aneuploid metaphases revealed that the chromosomes were not randomly involved in the malsegregation events. This observation may be a reflection of the relationship of particular chromosomes to the meiotic spindle apparatus rather than on any specific property of the agent to which they were exposed. It is believed that ethanol disrupts the organisation of cytoskeletal elements and, in particular, interferes with the processes of chromosome segregation at the second meiotic division.

Aneuploidy↗
PubMed1989

Cytochalasin D-induced triploidy in the mouse.

Previous attempts to obtain digynic triploid mouse development in vivo have either been entirely or only marginally successful, generally with the production of heteroploid rather than triploid conceptuses. We report that when a single intraperitoneal injection of 15 micrograms of cytochalasin D is given to recently mated female mice during a restricted period following ovulation induced by exogenous gonadotrophins, between 14 and 18% of conceptuses isolated on the 10th day of gestation had a triploid chromosome constitution. Triploidy was only induced in those eggs that were exposed to cytochalasin D when they were passing through a critical phase of the second meiotic division corresponding to the time when the second polar body was about to be extruded. Exposure to this agent either before or after this critical period only results in the development of normal diploid conceptuses. When females were mated to males carrying an easily recognisable paternally derived 'marker' chromosome, convincing cytogenetic evidence was obtained that only digynic triploidy was induced. No examples of diandric triploidy were recognised when conceptuses were analysed on the 10th day of gestation. The technique described therefore represents a simple and direct means of inducing digynic triploid mouse conceptuses whose development potential may be compared directly with that of their normal diploid littermates.

Animals↗
PubMed1989

Ultrastructural analysis of abnormalities in the morphology of the second meiotic spindle in ethanol-induced parthenogenones.

A high frequency of parthenogenetic activation occurs when ovulated mouse oocytes are briefly exposed to a dilute solution of ethanol in vitro. Cytogenetic analyses of parthenogenones at metaphase of the first cleavage division have confirmed that parthenogenetic activation, per se, does not increase the incidence of chromosome segregation errors during the completion of the second meiotic division. Ethanol-induced activation, however, significantly increases the incidence of aneuploidy. The ultrastructural changes that occur in the morphology and organization of the second meiotic spindle apparatus in ethanol- and hyaluronidase-activated oocytes is reported here. Abnormalities in the arrangement of microtubule arrays and chromosome position were principally observed in ethanol-activated oocytes at anaphase and telophase of the second meiotic division, but were only rarely observed in hyaluronidase-activated oocytes. It is proposed that the abnormalities in spindle morphology and chromosome displacement observed in ethanol-activated oocytes represent the initial events that lead to chromosome segregation errors following exposure to this agent.

Animals↗
PubMed1989

Ultrastructural and histochemical changes in the murine zona pellucida during the final stages of oocyte maturation prior to ovulation.

The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi-thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra-thin sections were examined by transmission electron microscopy. A parallel series of semi-thin sections of non-osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm-zona binding and sperm-egg interactions.

Animals↗
PubMed1989

The sex-chromosome constitution and early postimplantation development of diandric triploid mouse embryos.

Diandric triploid mouse embryos were produced by standard micromanipulatory techniques, using eggs isolated from female mice with a normal chromosome constitution that had been mated to homozygous Rb(1.3)1Bnr males (which carry a large metacentric "marker" chromosome, viz., a Robertsonian translocation involving chromosomes 1 and 3). The tripronucleate embryos were transferred to the oviducts of pseudopregnant mice, which were subsequently autopsied at about midday on the 10th day of gestation. Although a relatively small number of the isolated conceptuses consisted of morphologically abnormal egg-cylinder-like structures or empty gestational sacs, most were at clearly distinguishable embryonic stages, from the primitive streak stage to embryos with about 20 pairs of somites present. These embryos all appeared to be morphologically normal but were substantially smaller than normal (diploid) fertilized embryos analyzed at similar stages of development. A total of 63 diandric triploid conceptuses were recovered and analyzed cytogenetically. They were G-banded to determine their sex-chromosome constitution and confirm their diandric triploid status. No obvious difference was observed in the developmental potential of the 58,XXX class of diandric triploids, compared to that of the 58,XXY class. The ratio of 58,XXX to 58,XXY embryos was close to the expected ratio of 1:2, assuming that unfertilized eggs have an equal chance of becoming fertilized by an X- or a Y-bearing spermatozoon and that the additional (i.e., "donor") male pronucleus also has an equal chance of having either an X or a Y sex chromosome present. However, the development of the 58,XYY class appeared to be restricted, even at the stage of gestation analyzed, in that no embryos with this genetic constitution were observed that had progressed beyond the early somite stage. The present findings are discussed in relation to the cytogenetic findings in human triploid conceptuses, the majority of which are spontaneously aborted during the first half of pregnancy. In man, the 69,XYY class (equivalent to the 58,XYY class in our study) is only rarely encountered, and it has been assumed that these triploid embryos are probably lost at a very early stage of gestation.

Animals↗
PubMed1989

Post-implantation development and cytogenetic analysis of diandric heterozygous diploid mouse embryos.

Diandric heterozygous diploid mouse embryos were produced by standard micromanipulatory techniques using eggs from female mice with a normal chromosome constitution and fertilised by homozygous Rb(1.3)1Bnr males containing a pair of large metacentric marker chromosomes in their karyotype. The constructed diandric eggs were transferred to the oviducts of pseudopregnant recipients and subsequently autopsied midday on the eighth day of gestation. From a total of 85 eggs transferred to females that subsequently became pregnant, 30 implanted. Eighteen implantation sites were found to contain resorptions, and 12 egg cylinder stage embryos were recovered. These were cytogenetically examined. In two cases, no mitoses were observed, and in a third embryo of normal size, only a single paternally-derived marker chromosome was present in its mitoses, indicating that this embryo had a normal chromosome constitution. This presumably resulted from a technical error during the micromanipulatory procedure. The remaining nine morphologically small but normal embryos were diploid, and each had two paternally-derived marker chromosomes, thus establishing their ploidy and confirming their diandric origin. G-banding analysis revealed that all of these embryos had an XY sex chromosome constitution. Since the expected XX:XY:YY ratio of 1:2:1 was not observed, it is clear that the XX class embryos were lost at some stage during the pre- or early post-implantation period, though whether they are represented by the resorption sites is not yet established. The YY class would not be expected to be recovered in any case, as these embryos are believed to be lost during early cleavage. The cytogenetic findings reported here are therefore similar to the results of the chromosomal analyses of the human complete hydatidiform moles of dispermic origin, all of which apparently have an XY karyotype. It is unclear why, both in the human and in the mouse, the XX diandric heterozygous diploid group should develop poorly compared to similar embryos with an XY karyotype.

Animals↗
PubMed1989

Analysis of the sex-chromosome constitution of digynic triploid mouse embryos.

LT/Sv strain mice regularly ovulate up to 50% of their eggs as primary oocytes, which are fertilisable and give rise to digynic triploid embryos. A similar number of eggs are ovulated as secondary oocytes and, following fertilisation, give rise to normal diploid embryos. Pregnant LT/Sv females were autopsied at about midday on day 10 of gestation, when normal diploid embryos would be expected to possess between 25 and 30 pairs of somites. While a few of the triploid embryos either consisted of disorganised embryonic masses or were resorbing, most were at readily recognisable embryonic stages. Just over half of the embryos recovered were "unturned," while the remainder had "turned" and possessed between 15 and 25 pairs of somites. The triploids were usually readily recognised, owing to their small size and because they often displayed neural tube and cardiac defects. All of the embryos recovered were analysed cytogenetically by G-banding to establish their ploidy and sex-chromosome constitution. The XY:XX sex ratio of the 105 diploid embryos recovered, all of which had "turned," was 1.06:1, while the overall XXY:XXX sex ratio of the 120 triploids was 1:1. Analysis of only the developmentally most advanced triploid embryos (i.e., the 49 that had "turned") revealed that the XXY:XXX sex ratio in this group was 1.13:1, which was not significantly different from the expected ratio of 1:1. The crown-rump lengths of the XY and XX "turned" embryos were almost identical, as were those of the XXY and XXX "turned" embryos, although the triploids were significantly smaller than the diploids. No obvious effect of sex-chromosome constitution on developmental potential was therefore observed in this study in relation to either the digynic triploid or the control diploid embryos.

Animals↗
PubMed1989

Influence of diandric and digynic triploid genotypes on early mouse embryogenesis.

Standard micromanipulatory techniques were used to produce tripronucleate diandric and digynic triploid mouse conceptuses. When these were transferred to suitable recipients, most implanted. A wide range of embryonic stages from the primitive streak to the 15- to 25-somite stage were isolated in both triploid series in otherwise identical recipients. In the diandric triploid series, all of the embryos recovered appeared to be morphologically normal, but considerably smaller than fertilized embryos analysed at similar stages of development. This contrasts with the digynic triploid conceptuses which, though also ranging from the primitive-streak stage to about the 10- to 15-somite stage at the time of their isolation, generally showed poorer embryonic development than the diandric triploids, and were invariably morphologically abnormal. Unlike the situation observed in man, where the placentas of diandric triploid conceptuses commonly display widespread trophoblastic hyperplasia and form the characteristic 'partial' or 'incomplete' type of hydatidiform moles, the extraembryonic membranes of the diandric triploid mouse conceptuses (as well as the digynic triploids) did not appear to be grossly abnormal).

Animals↗