The effect of multiple simple Robertsonian heterozygosity on chromosome pairing and fertility of wild-stock house mice (Mus musculus domesticus).

The influence of Robertsonian (Rb) heterozygosity on fertility has been the subject of much study in the house mouse. However, these studies have been largely directed at single simple heterozygotes (heterozygous for a single Rb metacentric) or complex heterozygotes (heterozygous for several to many metacentrics which share common chromosome arms). In this paper we describe studies on male multiple simple heterozygotes, specifically the F(1) products of crosses between wild-stock mice homozygous for four or seven metacentrics and wild-stock mice with a standard all-acrocentric karyotype; these F(1) products were characterized by four and seven trivalents at meiosis I, respectively. Mice with the same karyotype, but two different genetic backgrounds were examined. Although a range of meiotic and fertility studies were conducted, particular emphasis was paid to analysis of chromosome pairing, previously not well-described in multiple simple heterozygous mice. The progression of spermatocytes through prophase I was followed by electron microscopy of surface spread material. As previously shown for single simple Rb heterozygotes, the trivalents that characterize multiple simple heterozygotes initially showed delayed pairing of the centromeric region and later showed side arm formation, resulting from non-homologous pairing by the centromeric ends of the acrocentric chromosomes. In the four trivalent groups of mice, 15 and 32% of trivalents showed unpairing in the centromeric region at mid pachytene; equivalent values were 29 and 39% for the seven trivalent groups. Pairing abnormalities (largely attachments and interlocks between trivalents and between a trivalent and the XY configuration) were observed in 18 and 23% of mid pachytene cells in the four trivalent groups and 36 and 49% of cells in the seven trivalent groups. The greater level of pachytene irregularity (unpairing and pairing abnormalities) in seven versus four trivalent heterozygotes was mirrored in terms of higher anaphase I nondisjunction frequency and lower germ cell counts. However, while pachytene irregularities appear to contribute to germ cell death, examples of male sterility in our material undoubtedly also involve genic incompatibilities.

Three-dimensional reconstruction of tetraploid<-->diploid chimaeric mouse blastocysts.

Studies of tetraploid<-->diploid (4n<-->2n) mouse chimaeras have demonstrated unequal contributions of 4n cells to different tissues of the midgestation conceptus. Such a pattern has also been reported in chimaeras as early as E3.5d, which show an enhanced contribution of 4n cells to the mural trophectoderm (Everett & West, 1996). In this study, sectioned 4n<-->2n and 2n<-->2n control chimaeric blastocysts were digitised and reconstructed in 3 dimensions (3-D). The 3-D images revealed only limited mixing of cells from the 2 contributing embryos of individual blastocysts in both chimaera groups. Consequently, the distribution pattern of the 2 cell types was dependent on the spatial relationship between the orientation of the blastocyst and the boundary between the 2 clusters of cells. The distribution patterns observed were not strikingly different for 4n<-->2n and 2n<-->2n chimaeras, each showing some transgenic positive cell contribution in all 3 identifiable developmental lineages. It was notable, however, that in all 4n<-->2n blastocysts at least some 4n cells were located adjacent to the blastocyst cavity. Such a consistent pattern was not evident in 2n<-->2n chimaeras. This study has demonstrated the value of 3-D reconstructions for the analysis of spatial relationships of 2 cell populations in chimaeric mouse blastocysts.

Failure of founder transgenic male mice to transmit an attenuated HSV thymidine kinase transgene results from mosaicism and sperm competition.

Previously we found that male mice carrying either of two attenuated herpes simplex virus thymidine kinase reporter transgenes displayed low level ectopic expression of the reporter gene in the testis and, although fertile, exhibited reduced fecundity. In contrast to males of later generations, many of the founder males failed to transmit the transgene to their progeny. This led to the suggestion that these fertile non-transmitting males are mosaic, with the sperm developing from the non-transgenic lineage outperforming those from the heterozygous transgenic lineage. Here we present the results of artificial insemination (AI) and in vitro fertilization (IVF) experiments designed to test this hypothesis. Albino CF(1) hybrid females were inseminated with mixtures of equal numbers of sperm from heterozygous transgenic (HT) males (equivalent to C57BL/6 x CBAF(2)) and CF(1) males. Similar mixed inseminations were carried out in parallel with sperm from non-transgenic (NT) siblings of the HT mice and 13-day fetuses were scored by eye color to determine their paternity. The pooled data from five experiments gave ratios of CF(1) to HT and CF(1) to NT offspring of 8.13 and 0.22 respectively, implying a calculated HT to NT ratio of 0.027. This indicates that, in competition with each other, the NT sperm would be almost 40 times more successful in fertilization than the HT sperm. Smaller differences were observed between HT and NT when AI was performed with unmixed sperm, consistent with the fertility of HT non-founder males. However, in five IVF experiments carried out with unmixed sperm, 142/212 oocytes exposed to NT sperm were activated and divided, while only 8/226 oocytes treated with HT sperm reached the two-cell stage. This confirms that HT sperm are defective and indicates that the IVF method employed amplified these deficiencies, which may have only a small effect upon natural reproduction when the HT sperm are not in competition with normal sperm.

Evidence for selection against tetraploid cells in tetraploid<-->diploid mouse chimaeras before the late blastocyst stage.

Tetraploid (4n) cells do not contribute equally to all tissues of midgestation mouse chimaeras and mosaics. Our previous studies of early blastocysts showed that 4n cells are preferentially allocated to the mural trophectoderm of the early blastocyst and this may contribute to the restricted distribution pattern seen at later stages. In this study of later-stage blastocysts we found evidence for selection against 4n cells. The contribution of 4n cells to 4n<-->2n chimaeric blastocysts decreased between E3.5 and E4.5 days, whereas the composition of 2n<-->2n controls changed little over this period. These results suggest that, prior to implantation, blastocysts have already lost some tetraploid cells from their embryonic and extra-embryonic lineages due to a combination of preferential allocation of 4n cells to the mural trophectoderm and selection against 4n cells throughout the embryo.

A study of meiotic pairing, nondisjunction and germ cell death in laboratory mice carrying Robertsonian translocations.

Frequencies of anaphase I nondisjunction, germ cell death and pairing abnormalities at pachytene were assessed in male mice singly heterozygous and homozygous for the Robertsonian (Rb) translocations: Rb (1.3)1Bnr, Rb(11.13)4Bnr and Rb(10.11)8Bnr. Rb homozygotes showed low frequencies of nondisjunction but substantial germ cell death. This germ cell death could not be attributed to problems at pachytene as Rb homozygotes showed no increase in pairing abnormalities over the (C3H/HeH x 101/H)F1 controls. Instead genic factors are involved. Rb heterozygotes showed substantial frequencies of nondisjunction and even greater germ cell death than found in the homozygotes. Pachytene pairing abnormalities were observed and it appears that these, together with genic factors, cause physiological perturbation of meiocytes, thereby promoting germ cell death, with nondisjunction of the trivalent as a sublethal response.

The influence of ploidy on the distribution of cells in chimaeric mouse blastocysts.

Previous studies of tetraploid<-->diploid mouse chimaeras and mosaics have revealed that tetraploid cells do not contribute equally to all tissues of the conceptus. In this study we have shown that, within 30 h of aggregating cleavage stage embryos, tetraploid cells were non-randomly distributed among different tissues of the early blastocyst. They were preferentially allocated to the mural trophectoderm regardless of cell size at the time of aggregation. This early effect may underlie the restricted distribution of tetraploid cells at later stages. We have demonstrated for the first time that ploidy can influence the relative position of blastomeres in the preimplantation embryo.

Pattern and frequency of nocodazole induced meiotic nondisjunction in oocytes of mice carrying the 'tobacco mouse' metacentric Rb(16.17)7Bnr.

Oocytes from (C3H/HeH x 101/H)F1 and Rb(16.17)7Bnr homozygous females were exposed to a range of doses of nocodazole in vitro. The spindle poison caused a dose dependent increase in metaphase I (MI) arrest and hyperploidy. A concentration of 0.03 microgram/ml was found to induce a maximum hyperploid frequency of 3.1% and 11.6% respectively without a high level of MI arrest. Between 0.03 and 0.05 microgram/ml MI arrest increased substantially and reached a frequency of approximately 90%. In a further experiment oocytes from Rb7 homozygous, heterozygous and 3H1 females were exposed to 0.03 microgram/ml nocodazole 4, 6 or 8 h after the onset of maturation. The phase at which the spindle was inhibited resulted in a specific pattern of nondisjunction which in turn was dependent on whether the female carried an Rb metacentric. 3H1 oocytes gave a normally distributed pattern of increase in aneuploid frequency (over the spontaneous value) centering around a 6 h application. This was thought to be due to the interaction of chromosomes with the microtubules of the spindle during attachment and/or alignment. In contrast both Rb homozygotes and heterozygotes gave the same biphasic response, with a high frequency of aneuploidy in the oocytes when nocodazole was applied 4 and 8 h after the onset of maturation. In Rb homozygotes we demonstrated that the Rb bivalent underwent nondisjunction more frequently than the average acrocentric, when nocodazole was administered early.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of six dominant cataract genes in the mouse.

The mapping of six mouse autosomal dominant cataract mutations that were induced by mutagenic treatment with radiation or ethylnitrosourea is described. Three, with differing phenotypes, mapped on Chromosome 1 between the loci of fuzzy (fz) and leaden (ln) and close to the locus of the gamma-crystallin gene cluster. One of these, Cat-2t, had previously been shown to be a member of a group of five allelic mutants. In addition, the previously known mutant eye lens obsolescence, Elo, maps to the same point. There are thus now eight mutants that map to this point and that may involve mutations in one of the gamma-crystallin genes. In addition, one of these mutants may be a homologue of Coppock cataract in man, which also maps close to the gamma-crystallin locus. Of the three remaining mutants, one, with the suggested symbol Cat-5, mapped to the proximal region of Chromosome 10, 23.4 +/- 4.0 cM from downless (dl), a region with homology to human 6q. A second mutant, provisionally designated Opj, mapped on Chromosome 16, 8.2 +/- 3.9 cM from the marker mahoganoid (md). Thus, it possibly has a homologue on human 22q, a region in which one of the beta-crystallin loci is sited. A third mutant, provisionally designated Npp, mapped to Chromosome 5, 1.3 +/- 0.9 cM from the locus of W, and thus probably has a homologue on human Chromosome 4.

Male meiosis and gametogenesis in wild house mice (Mus musculus domesticus) from a chromosomal hybrid zone; a comparison between "simple" Robertsonian heterozygotes and homozygotes.

Wild male house mice Mus musculus domesticus were collected from the hybrid zone between the John o'Groats race (2n = 32) and the standard race (2n = 40) in northern Scotland. Meiosis in both homozygotes (2n = 32, 36, and 40) and single Robertsonian heterozygotes (2n = 33, 35, and 37) was found to be orderly. At prophase/metaphase I in heterozygotes, a trivalent was formed from the metacentric and two homologous acrocentrics. At pachytene, this trivalent usually had a single side arm at the position of the centromeres, as a result of nonhomologous pairing of the acrocentrics. This side arm persisted into diplotene. Generally only a single chiasma was formed between each acrocentric and the metacentric. Anaphase I nondisjunction frequencies were estimated as 1.5% for the homozygotes and 2.7% for the heterozygotes. The extent of germ cell death between the pachytene and round spermatid stages was 18% greater in heterozygotes than in homozygotes. Our results concur with previous studies which indicate that single Robertsonian heterozygotes in wild house mice have near-normal fertility.

Adhesion characteristics of human melanoma cell lines of varying metastatic potential.

The adhesive behaviour of a series of human melanoma cell lines, of varying metastatic potential, to basement membrane and stromal components was investigated in vitro. Experimental metastatic propensity was assessed from the number of pulmonary nodules formed after i.v. injection of cells into BALB/c nude mice. All cell lines showed similar kinetics of attachment when tested on plastic, type-I collagen films, type-I collagen hydrated gels, fibronectin, laminin type-IV collagen substrates and bovine aortic endothelial monolayers. Fibronectin-coated plastic compared to plastic alone produced increased cell attachment and spreading to the same extent in all the cell lines. The melanoma lines attached preferentially to cryostat sections of lung compared to other organs reflecting the pattern of organ involvement of metastasis in vivo. However, no significant quantitative differences in attachment to lung sections were seen between melanoma variants of differing metastatic capacities. Cells labelled with [125I]iododeoxyuridine to determine their initial organ distribution following i.v. injection showed that tumour-cell arrest was not significantly changed enough to explain the differing metastatic capacities. Thus it appears that adhesive properties of these melanoma cells are not correlated with their capacity to form metastases in vivo.

Interferon-induced alterations in metastatic capacity, class-1 antigen expression and natural killer cell sensitivity of melanoma cells.

Pre-treatment of B16 melanoma cells with recombinant interferon-gamma (IFN-gamma) markedly increased their lung-colonising capacity following i.v. injection into syngeneic mice as compared with control cells. A similar enhancement was observed following the injection of treated cells into athymic nude mice but not in athymic mice carrying the beige mutation. Pre-treatment of syngeneic mice with anti-asialo GM1 antibody effectively abrogated any interferon-induced increase in experimental metastatic activity. The same IFN-gamma treatment significantly increased resistance of B16 cells to splenic natural killer (NK) cell activity as determined by in vitro assays. IFN-alpha/beta pre-treatment of B16 cells decreased sensitivity to NK-cell-mediated lysis to a lesser extent than IFN-gamma and had no detectable effect upon the subsequent metastatic activity of the tumor cells. Class-I antigen expression was altered by these IFN treatments, with IFN-gamma causing dramatic increases in expression of H-2Db antigen, in a pattern consistent with the possibility that increased H-2 antigen expression on B16 cells led to decreased NK-cell sensitivity which was reflected by an increase in experimental metastatic capacity.

DNA methylation levels in human and murine melanoma cell lines of varying metastatic potential.

DNA methylation levels were measured in a series of murine and human melanoma cell lines consisting of matched variants of low and high experimental metastatic capacity. The percentage of cytosine residues modified to 5-methylcytosine ranged between 2.13-3.92% in these lines. Ten cell lines were established in culture from individual lung tumor nodules produced in nude mice by i.v. injection of DX-3 human melanoma cells. Upon reinjection into groups of nude mice the individual lines manifested marked diversity for lung nodule formation (median number of pulmonary tumor nodules ranging from less than 10/group-greater than 100/group). DNA methylation levels in these lines were also heterogeneous (range 1.59 +/- 0.13 (SD)-4.04 +/- 0.15%) but no correlation was detected between methylation status of the genomic DNA and metastatic capacity.

Enhanced experimental metastatic capacity of a human tumor line following treatment with 5-azacytidine.

We have studied the effect of the nucleoside analogue 5-azacytidine (5-azaC) a known activator of gene expression, on the metastatic behavior of the human melanoma cell line DX3. After exposure to 3 microM 5-azaC for 24 h cells were carried through five subcultures before being tested for their metastatic capacity. Following i.v. injection in BALB/c nude mice, cells treated with 5-azaC showed a 40-fold increase in the number of lung tumor nodules as compared to that obtained with control cells. Tumors resulting from 5-azaC-treated cells exhibited a 5-fold increase in mitotic figures, considerable cellular pleomorphism, and variation in the histological character of the individual nodules. These in vivo differences were not reflected in vitro where treated and untreated cells showed no significant differences in morphology, karyotypes, growth rates, saturation densities, or plating efficiencies. Cells populating the lung tumor nodules retained their increased metastatic capacity through successive cycles of growth in vitro followed by reinjection into nude mice and indeed the ability of the cell lines to form lung tumors was increased with the succeeding cycles in vivo. These cell lines, each established from individual tumor nodules, maintained a human karyotype and reacted positively with human-specific antiserum. Characterization of these lines showed that while there were no alterations in in vitro growth behavior the selected lines exhibited a decreased latency period for s.c. tumor formation, increased retention in the lungs following i.v. injection, and enhanced lung nodule formation (60- to 80-fold compared to control cells). Examination of genomic DNA showed that treatment with 5-azaC brought about a significant decrease (50%) in the percentage of methylated cytosine residues but this extensive hypomethylation was not maintained in the lung tumor nodule-derived cell lines.