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M A Hultén

Publications and source records attributed to M A Hultén.

At least 19 recordsLinked to original sources
PubMed2004

The initiation of homologous chromosome synapsis in mouse fetal oocytes is not directly driven by centromere and telomere clustering in the bouquet.

We investigated the behaviour of centromeres and distal telomeres during the initial phases of female meiosis in mice. In particular, we wished to determine whether clustering of centromeres and telomeres (bouquet formation) played the same crucial role in homologous chromosome pairing in female meiosis as it does in the male. We found that synapsis (intimate homologous chromosome pairing) is most frequently initiated in the interstitial regions of homologous chromosomes, apparently ahead of the distal regions. The proximal ends of the chromosomes appear to be disfavoured for synaptic initiation. Moreover, initiation of synapsis occurred in oocytes that showed little or no evidence of bouquet formation. A bouquet was present in a substantial proportion of cells at mid to late zygotene, and was still present in some pachytene oocytes. This pattern of bouquet formation and pairing initiation is in stark contrast to that previously described in the male mouse. We propose that although dynamic movements of centromeres and telomeres to form clusters may facilitate alignment of homologues or homologous chromosome segments during zygotene, in the female mouse positional control of synaptic initiation is dependent on some other mechanism.

Animals↗
PubMed2004

Inter-sex variation in synaptonemal complex lengths largely determine the different recombination rates in male and female germ cells.

Meiotic chromosomes in human oocytes are packaged differently than in spermatocytes at the pachytene stage of meiosis I, when crossing-over takes place. Thus the meiosis-specific pairing structure, the synaptonemal complex (SC), is considerably longer in oocytes in comparison to spermatocytes. The aim of the present study was to examine the influence of this length factor on meiotic recombination in male and female human germ cells. The positions of crossovers were identified by the DNA mismatch repair protein MLH1. Spermatocytes have approximately 50 crossovers per cell in comparison to more than 70 in oocytes. Analyses of inter-crossover distances (and presumptively crossover interference) along SCs suggested that while there might be inter-individual variation, there was no consistent difference between sexes. Thus the higher rate of recombination in human oocytes is not a consequence of more closely spaced crossovers along the SCs. The rate of recombination per unit length of SC is higher in spermatocytes than oocytes. However, when the so-called obligate chiasma is excluded from the analysis, then the rates of recombination per unit length of SC are essentially identical in the two sexes. Our analyses indicate that the inter-sex difference in recombination is largely a consequence of the difference in meiotic chromosome architecture in the two sexes. We propose that SC length per se, and therefore the size of the physical platform for crossing-over (and not the DNA content) is the principal factor determining the difference in rate of recombination in male and female germ cells. A preliminary investigation of SC loop size by fluorescence in situ hybridization (FISH) indicated loops may be shorter in oocytes than in spermatocytes.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Growth factors promote meiosis in mouse fetal ovaries in vitro.

Mouse fetal ovaries were cultured to investigate germ cell development in the presence of a combination of the growth factors (GFs) stem cell factor, insulin-like growth factor-1 and leukaemia inhibitory factor. Ovaries were isolated from fetal mice at 13 and 14 days post-coitum (dpc) and cultured to the equivalent of 17 dpc. Culture conditions comprised minimal essential medium-alpha plus 5% fetal calf serum, with or without GFs. Oocytes were assessed using immunofluorescence to illustrate synaptonemal complexes and recombination foci. The proportions of pachytene cells in freshly isolated 13, 14 and 17 dpc ovaries were 0, 8 and 74% respectively. There was a significant (P < 0.0001) increase in the number of pachytene cells after 4 days culture with GFs, with 24% of germ cells from 13 dpc ovaries reaching pachytene. In contrast, no pachytene cells were detected in cultures of 13 dpc ovaries without GFs. After 3 days in culture with GFs, 38% of germ cells from 14 dpc ovaries were at pachytene compared with 19% without GFs. In conclusion, we have demonstrated positive effects of GFs upon oocyte formation by meiosis in vitro. The observed results could be explained by an increased survival of premeiotic oogonia entering meiosis, or by effects on oocytes already in early meiosis.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Meiotic chromosome pairing in fetal oocytes of trisomy 21 human females.

The influence of trisomy on meiotic chromosome association and synapsis was studied in oocytes of two trisomy 21 fetuses. The patterns of association of the three chromosomes 21 were determined by analysis of late zygotene to early diplotene fetal oocytes after immunofluorescent staining of synaptonemal complexes. The identity of chromosome 21 was confirmed using FISH with either a whole chromosome 21 paint or an alpha-satellite DNA repeat probe. In both fetuses, a wide variety of configurations was present at pachytene. The most common configurations were a trivalent (35.5% and 51.6% of analyzable cells) and a bivalent plus univalent (62.9% and 45.2%). These different frequencies between the fetuses were not significant. Trivalents showed either triple synapsis or double synapsis with pairing-partner switches. The extent of triple synapsis varied from a short segment, either terminal or interstitial, to the whole chromosome length. Through use of immunofluorescent staining of the centromeres, we identified novel types of abnormal chromosome behavior in trisomy 21 fetal oocytes. Thus, we found that 6/41 trivalents had one of the chromosomes associated "out of register," i.e., in a nonhomologous fashion, with its two homologs. Likewise, we found three cells with bivalent plus univalent configurations, in which the univalent showed self-synapsis. The presence of three copies of chromosome 21 therefore results not only in the formation of complex and highly variable synaptic associations but also causes a significant increase in the occurrence of nonhomologous synapsis in human fetal oocytes.

Abortion, Induced↗
PubMed2001

Automatic signal classification in fluorescence in situ hybridization images.

BACKGROUND: Previous systems for dot (signal) counting in fluorescence in situ hybridization (FISH) images have relied on an auto-focusing method for obtaining a clearly defined image. Because signals are distributed in three dimensions within the nucleus and artifacts such as debris and background fluorescence can attract the focusing method, valid signals can be left unfocused or unseen. This leads to dot counting errors, which increase with the number of probes. METHODS: The approach described here dispenses with auto-focusing, and instead relies on a neural network (NN) classifier that discriminates between in and out-of-focus images taken at different focal planes of the same field of view. Discrimination is performed by the NN, which classifies signals of each image as valid data or artifacts (due to out of focusing). The image that contains no artifacts is the in-focus image selected for dot count proportion estimation. RESULTS: Using an NN classifier and a set of features to represent signals improves upon previous discrimination schemes that are based on nonadaptable decision boundaries and single-feature signal representation. Moreover, the classifier is not limited by the number of probes. Three classification strategies, two of them hierarchical, have been examined and found to achieve each between 83% and 87% accuracy on unseen data. Screening, while performing dot counting, of in and out-of-focus images based on signal classification suggests an accurate and efficient alternative to that obtained using an auto-focusing mechanism.

Amniotic Fluid↗
PubMed2001

GELFISH--graphical environment for labelling fluorescence in-situ hybridization images.

Signal (dot) counting in fluorescence in-situ hybridization (FISH) images that relies on an automatic focusing method for obtaining clearly defined images is a time-consuming procedure prone to errors. Our recently developed system has dispensed with automatic focusing, and instead relies on a neural network classifying focused and unfocused signals into valid and artefact data, respectively, and thereby discriminating between in- and out-of-focus images. However, to train the classifier accurate labelling of the image signals is required. GELFISH is a Graphical Environment for Labelling FISH images that enables the rejection of unanalysable nuclei and labelling of FISH signals simply and rapidly. GELFISH is flexible and can be modified easily for additional FISH applications. Also, implemented using popular software, the environment can be employed on any computer by any user. Finally, GELFISH is proposed in controlling a classifier-based dot counting system.

Artifacts↗
PubMed2000

Meiotic studies of a human male carrier of the common translocation, t(11;22), suggests postzygotic selection rather than preferential 3:1 MI segregation as the cause of liveborn offspring with an unbalanced translocation.

The t(11;22)(q23;q11) translocation is the only non-Robertsonian rearrangement for which there are a large number of unrelated families, apparently with the same breakpoints. These families most often have been ascertained through an abnormal child with the karyotype 47,XX or XY, +der(22) t(11;22)(q23;q11). To explain the high incidence of 3:1 segregants, rarely seen in offspring of carriers of other reciprocal translocations, a number of theoretical models have been suggested. We have used both electron microscope analysis of the synaptonemal complex (SC) and dual-color FISH to investigate the meiotic chromosome behavior in a male carrier of the translocation who has the karyotype 46,XY, t(11;22)(q23;q11). Chromosome synapsis, first-meiotic chiasma configuration, and segregation behavior of this translocation have been analyzed directly. Examination of SCs by electron microscopy showed pachytene-cross formation in 49/50 nuclei. Approximately 50% (26/50) revealed a classical fully synapsed quadrivalent. A proportion of these (10/26), however, showed some central asymmetry, suggesting heterologous synapsis. The remaining cells appeared to have incomplete synapsis. FISH analysis showed only quadrivalents in all 100 metaphase I nuclei. The chiasma frequency was increased within the interstitial segments, in comparison with the same region in normal bivalents. All types of segregation category were found in metaphase II nuclei. There was no indication of preferential 3:1 anaphase I segregation. We conclude that the +der(22) constitution in offspring of carriers of t(11;22)(q23;q11) is not likely to be due to meiotic 3:1 segregation being especially common. Rather, the +der(22) constitution is more likely to be the result of postzygotic selection against other unbalanced karyotypes.

Cell Nucleus↗
PubMed1999

Immunocytogenetic detection of normal and abnormal oocytes in human fetal ovarian tissue in culture.

This study aimed to: (i) determine whether oocytes are present in cultures of human fetal ovary; (ii) identify whether meiotic anomalies are evident; and (iii) assess whether preparation or culture conditions influence oocyte survival and meiotic progression. Ovaries were collected from fetuses after termination at 13-16 weeks. Oocyte assessment utilized antibodies specific for synaptonemal complex proteins (associated with chromosomes only during meiosis), and antibodies to centromeric proteins. Fragments of tissue were cultured in minimal essential medium + 10% serum +/- follicle stimulating hormone (100 mIU/ml). The sera were fetal calf serum (FCS), FCS for embryonic stem cells (ES-FCS) and human female serum. The numbers and stages of oocytes were assessed after 7-40 days, and particular arrangements of chromosome synapsis identified. Results in fresh tissue included oocytes at leptotene, zygotene, pachytene and diplotene in three of five samples. Four specimens remained viable in vitro, and three had detectable oocytes after culture. The numbers of oocytes and the proportions of zygotene and pachytene cells increased with time in culture. The proportion of degenerate cells in culture was initially higher than in fresh samples, but declined subsequently. More oocytes were detected in ES-FCS and human serum than in FCS. We conclude that human oocytes survive in culture and that progression through prophase I continues.

Cell Count↗
PubMed1998

Combined immunocytogenetic and molecular cytogenetic analysis of meiosis I oocytes from normal human females.

The microspread oocytes of three fetuses, two of 16 weeks gestation and one of 15 weeks gestation, were labelled with a combination of anti-lateral element antiserum and a human centromere labelling auto-immune serum. The anti-lateral element serum was found to label both asynapsed axial elements and synapsed lateral elements strongly. Nuclei were found from leptotene to diplotene in all three fetuses. The use of the human auto-immune serum led to the observation of 'staggered centromeres' and 'centromeric associations' as well as tightly clustered centromeres in 'stellar nuclei'. Nuclei displaying various aberrant features were detected. The use of antibody-labelled microspread oocytes as substrates for fluorescence in situ hybridisation (FISH) was found to be reliably successful only with repetitive (centromeric and telomeric) probes.

Cell Nucleus↗
PubMed1997

Distribution of the Rad51 recombinase in human and mouse spermatocytes.

In vitro, the human Rad51 protein (hRad51) promotes homologous pairing and strand exchange reactions suggestive of a key role in genetic recombination. To analyse its role in this process, polyclonal antibodies raised against hRad51 were used to study the distribution of Rad51 in human and mouse spermatocytes during meiosis I. In human spermatocytes, hRad51 was found to form discrete nuclear foci from early zygotene to late pachytene. The foci always co-localized with lateral element proteins, components of the synaptonemal complex (SC). During zygotene, the largest foci were present in regions undergoing synapsis, suggesting that Rad51 is a component of early recombination nodules. Pachytene nuclei showed a greatly reduced level of Rad51 labelling, with the exceptions of any asynapsed autosomes and XY segments, which were intensely labelled. The distribution of Rad51 in mouse spermatocytes was similar to that found in human spermatocytes, except that in this case Rad51 was detectable at leptotene. From these results, we conclude that the Rad51 protein has a role in the interhomologue interactions that occur during meiotic recombination. These interactions are spatially and temporally associated with synapsis during meiotic prophase I.

Animals↗
PubMed1997

Different strategies of X-inactivation in germinal and somatic cells: histone H4 underacetylation does not mark the inactive X chromosome in the mouse male germline.

It has previously been shown by immunocytochemistry that the inactive X chromosome (Xi) in somatic cells of human and mouse females is marked by underacetylation of histone H4. It has been suggested that this may be important for transcriptional silencing of genes on Xi. We have now investigated X-inactivation in meiotic cells of the male germline. In these cells the single X chromosome is transcriptionally inactive and expresses XIST, a gene that in somatic cells is transcribed only from Xi. By immunostaining with antibodies to H4 acetylated at lysines 5, 8, 12, or 16, we demonstrate that histone H4 on the male X is not underacetylated. We conclude that there is a differential germline strategy for maintenance of X-inactivation and that H4 underacetylation, though associated with the long-term marking of inactive X chromosomes in the female soma, is not always essential for the transcriptional down-regulation of X-linked genes.

Acetylation↗
PubMed1997

Study of Alu sequences at the hypoxanthine phosphoribosyltransferase (hprt) encoding region of man.

The hypoxanthine phosphoribosyltransferase (hprt) encoding region of man is considered rich in Alu sequences: with 49 sequences present within 57 kilobases. Subfamily classification of the Alu sequences and identification of flanking direct repeats has been carried out to detect past rearrangements associated with their insertion into the region. Members of the Alu-J and three Alu-S subfamilies are present, along with the existence of free left arm sequences. Using available data, a comparison is made of the Alu subfamilies present at different gene regions. The heterogeneity in the number of each subfamily present at different genes shows that no one particular subfamily attained saturation in the genome. Several adjacent insertions of Alu sequences are seen at the hprt region. Furthermore two novel sequences are described, there is an incident where one Alu sequence has inserted into the middle poly(A) tract of an existing sequence at the hprt region; while another result from an Alu/Alu cross-over event elsewhere in the genome, before insertion into the hprt region. Once inserted, the Alu sequences are rarely subject to loss or rearrangement.

Base Sequence↗
PubMed1997

Analysis of proximal X chromosome pairing in early female mouse meiosis.

The initiation and progression of homologous chromosome pairing at meiosis were investigated in female mice. The proximal end of the X chromosome was identified in fetal oocytes using fluorescence in situ hybridisation with the repeat copy probe 70-38. The X centromeres appeared to be randomly positioned in the nuclei from pre-meiotic interphase to leptotene. The observations indicated no pre-synaptic association for the proximal end of the X chromosome. There was a significant increase in the number of paired X centromeres from mid-zygotene to late zygotene. The proximal end of the X chromosome is therefore a generally late pairing region with no significant association seen before mid-zygotene. The centromeric heterochromatin of all chromosomes could be seen to associate into varying numbers of clusters during pre-leptotene through to pachytene. These clusters do not seem to be directly involved in bringing homologues together, as X centromeres did not consistently localise to the same cluster.

Animals↗
PubMed1997

Sequential immunocytogenetics, molecular cytogenetics and transmission electron microscopy of microspread meiosis I oocytes from a human fetal carrier of an unbalanced translocation.

The oocytes of a 17 week human fetus carrying an unbalanced 46,XX, add(18)(p13) translocation were studied with a sequential combination of microspreading, immunocytogenetics, fluorescence in situ hybridization (FISH) and transmission electron microscopy. This combination of technologies allowed the collection of data of unique accuracy and resolution. The translocated chromosome was found to be involved in five different synaptic configurations. A consistent feature of these configurations was the involvement of a second small bivalent, presumably chromosome 21 or 22, the normal synapsis of which was often disrupted. We conclude that chromosome 21 or 22 was the source of the translocated material, which was found to be either homologously triply synapsed, heterologously synapsed or asynapsed.

Adult↗
PubMed1997

Childhood cancer and parental use of tobacco: deaths from 1953 to 1955.

Parental smoking data have been abstracted from the interview records of the case-control study that first indicated that pregnancy radiographs are a cause of childhood cancer (Oxford Survey of Childhood Cancers, deaths from 1953 to 1955). Reported smoking habits for the parents of 1549 children who died from cancer were compared with similar information for the parents of 1549 healthy controls (matched pairs analysis). There was a statistically significant positive trend between paternal daily consumption of tobacco and the risk of childhood cancer (P< 0.001). This association could not be explained by maternal smoking, social class, paternal or maternal age at the birth of the survey child, sibship position or obstetric radiography. About 15% of all childhood cancers in this series could be attributable to paternal smoking.

Case-Control Studies↗
PubMed1997

Childhood cancer and parental use of tobacco: deaths from 1971 to 1976.

Parental smoking data have been reabstracted from the interview records of the Oxford Survey of Childhood Cancers (deaths from 1971 to 1976). Reported smoking habits for the parents of 2587 children who died with cancer were compared with similar information for the parents of 2587 healthy controls (matched pairs analysis). Maternal daily consumption of cigarettes and paternal use of pipes or cigars were unimportant, but there was a statistically significant positive trend between paternal daily consumption of cigarettes and the risk of childhood cancer (P < 0.001). This association could not be explained by maternal smoking, social class, parental ages at the birth of the survey child, sibship position or obstetric radiography. Relations between maternal consumption of cigarettes and birth weights suggested that (maternal) smoking data were equally reliable for case and control subjects. About 14% of all childhood cancers in this series could be attributable to paternal smoking. These data were combined with smoking data from two previously published reports from the Oxford Survey (deaths from 1953 to 1955, deaths from 1977 to 1981) to obtain further information on risks for different types of cancer and different ages at onset of disease. Paternal cigarette smoking emerged as a potential risk factor both for the generality of childhood cancer and for all ages at onset.

Adolescent↗
PubMed1996

Combined immunocytogenetic and molecular cytogenetic analysis of meiosis I human spermatocytes.

We have used a combination of immunocytogenetic and molecular cytogenetic technology on human spermatocytes to investigate (1) meiosis I chromosome pairing, and (2) organization of synaptonemal complex (SC)-associated chromatin with respect to whole chromosome paints, unique DNA sequences and repetitive DNA of heterochromatic blocks, centromeres and telomeres. It is evident that synapsis normally starts at the termini of homologues. In general, synapsis proceeds synchronously from termini towards the centre of bivalents without any indication of interstitial initiation. Some aberrant meiosis I spermatocytes showed asynchronous pairing, demonstrating not only large differences in the degree of SC formation between bivalents, but also chromosome alignment without synapsis as well as clear interstitial synaptic initiation. It may be the case that alignment normally takes place along the entire length of homologues before synapsis occurs and that the potential for synaptic initiation exists along the length of chromosomes. Telomeric sequences were seen tightly associated with the SCs, as might be expected considering their kinetic properties in relation to the nuclear membrane. Other repetitive DNA, i.e. centromeric alpha-satellites and classical satellites of the heterochromatic blocks 1qh and 9qh, were all found to form loops that are associated with SCs only at their bases. A unique DNA cosmid probe (21q22.3) was found to produce a hybridization pattern consisting of spots located outside SC. The fluorescence in situ hybridization (FISH) signals of these spread DNA sequences have a granular appearance, probably reflecting the pattern of coiling and chromatin condensation of the target DNAs.

Animals↗