Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I.

Exchanges are not equally able to enhance meiotic chromosome segregation in yeast.

Homologous chromosomes pair, and then migrate to opposite poles of the spindle at meiosis I. In most eukaryotic organisms, reciprocal recombinations (crossovers) between the homologs are critical to the success of this process. Individuals with defects in meiotic recombination typically produce high levels of aneuploid gametes and exhibit low fertility or are sterile. The experiments described here were designed to test whether different crossovers are equally able to contribute to the fidelity of meiotic chromosome segregation in yeast. These experiments were performed with model chromosomes with which it was possible to control and measure the distributions of meiotic crossovers in wild-type cells. Physical and genetic approaches were used to map crossover positions on model chromosomes and to correlate crossover position with meiotic segregation behavior. The results show that crossovers at different chromosomal positions have different abilities to enhance the fidelity of meiotic segregation.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

Regulation of meiotic chromatin loop size by chromosomal position.

At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging.

Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis.

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.

Neuronal abnormalities in microtubule-associated protein 1B mutant mice.

Microtubules play an important role in establishing cellular architecture. Neuronal microtubules are considered to have a role in dendrite and axon formation. Different portions of the developing and adult brain microtubules are associated with different microtubule-associated proteins (MAPs). The roles of each of the different MAPs are not well understood. One of these proteins, MAP1B, is expressed in different portions of the brain and has been postulated to have a role in neuronal plasticity and brain development. To ascertain the role of MAP1B, we generated mice which carry an insertion in the gene by gene-targeting methods. Mice which are homozygous for the modification die during embryogenesis. The heterozygotes exhibit a spectrum of phenotypes including slower growth rates, lack of visual acuity in one or both eyes, and motor system abnormalities. Histochemical analysis of the severely affected mice revealed that their Purkinje cell dendritic processes are abnormal, do not react with MAP1B antibodies, and show reduced staining with MAP1A antibodies. Similar histological and immunochemical changes were observed in the olfactory bulb, hippocampus, and retina, providing a basis for the observed phenotypes.

Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain.

Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5.

Gene targeting was used to create mice with a null mutation of the gene encoding the common beta subunit (beta C) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (beta C-/- mice). High-affinity binding of GM-CSF was abolished in beta C-/- bone marrow cells, while cells from heterozygous animals (beta C+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3-specific beta chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of beta C-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of beta C-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. beta C-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between beta C-/- and wild-type littermates. The data establish that beta C is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.

A maximum likelihood algorithm for genome mapping of cytogenetic loci from meiotic configuration data.

Frequencies of meiotic configurations in cytogenetic stocks are dependent on chiasma frequencies in segments defined by centromeres, breakpoints, and telomeres. The expectation maximization algorithm is proposed as a general method to perform maximum likelihood estimations of the chiasma frequencies in the intervals between such locations. The estimates can be translated via mapping functions into genetic maps of cytogenetic landmarks. One set of observational data was analyzed to exemplify application of these methods, results of which were largely concordant with other comparable data. The method was also tested by Monte Carlo simulation of frequencies of meiotic configurations from a monotelodisomic translocation heterozygote, assuming six different sample sizes. The estimate averages were always close to the values given initially to the parameters. The maximum likelihood estimation procedures can be extended readily to other kinds of cytogenetic stocks and allow the pooling of diverse cytogenetic data to collectively estimate lengths of segments, arms, and chromosomes.

Pleiotropy in microdeletion syndromes: neurologic and spermatogenic abnormalities in mice homozygous for the p6H deletion are likely due to dysfunction of a single gene.

Variability and complexity of phenotypes observed in microdeletion syndromes can be due to deletion of a single gene whose product participates in several aspects of development or can be due to the deletion of a number of tightly linked genes, each adding its own effect to the syndrome. The p6H deletion in mouse chromosome 7 presents a good model with which to address this question of multigene vs. single-gene pleiotropy. Mice homozygous for the p6H deletion are diluted in pigmentation, are smaller than their littermates, and manifest a nervous jerky-gait phenotype. Male homozygotes are sterile and exhibit profound abnormalities in spermiogenesis. By using N-ethyl-N-nitrosourea (EtNU) mutagenesis and a breeding protocol designed to recover recessive mutations expressed hemizygously opposite a large p-locus deletion, we have generated three noncomplementing mutations that map to the p6H deletion. Each of these EtNU-induced mutations has adverse effects on the size, nervous behavior, and progression of spermiogenesis that characterize p6H deletion homozygotes. Because EtNU is thought to induce primarily intragenic (point) mutations in mouse stem-cell spermatogonia, we propose that the trio of phenotypes (runtiness, nervous jerky gait, and male sterility) expressed in p6H deletion homozygotes is the result of deletion of a single highly pleiotropic gene. We also predict that a homologous single locus, quite possibly tightly linked and distal to the D15S12 (P) locus in human chromosome 15q11-q13, may be associated with similar developmental abnormalities in humans.

The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2.

Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals. Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.

Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle.

High levels of mos protooncogene product are expressed during oocyte meiotic maturation and Mos has been implicated in formation of the spindle and spindle pole. Here, we show that in Swiss 3T3 cells with 4N DNA content, high levels of Mos lead to the production of binucleated cells. The Swiss 3T3 cells in mitosis, before binucleation occurs, are anastral and the spindle poles are juxtaposed to the cell membrane. These phenotypes may be related to the meiotic process of attachment of the spindle pole to the oocyte membrane during polar body formation. The production of binucleated somatic cells could result from attachment of the altered mitotic spindle pole to the cell membrane that interferes with cytokinesis but not karyokinesis. This can explain at least one form of genetic instability that leads to altered DNA content in tumor cells.

Ultrasonography of gallbladder abnormalities due to schistosomiasis

After malaria, schistosomiasis remains the most important tropical parasitic disease in large parts of the world. Schistosomiasis has recently re-emerged in Southern Europe. Intestinal schistosomiasis is caused by most Schistosoma (S.) spp. pathogenic to humans and leads to chronic inflammation and fibrosis of the colon as well as to liver fibrosis. Gallbladder abnormalities usually occur in patients with advanced hepatic portal fibrosis due to Schistosoma mansoni infection. Occasionally, gallbladder abnormalities have been seen also in children and occurring without associated overt liver abnormalities.The specific S. mansoni-induced gallbladder abnormalities detectable by ultrasound include typical hyperechogenic wall thickening with external gallbladder wall protuberances. The luminal wall surface is smooth. The condition is usually clinically silent although some cases of symptomatic cholecystitis have been described. The ultrasonographic Murphy response is negative. Gallbladder contractility is impaired but sludge and calculi occur rarely. Contrary to other trematodes such as liver flukes, S. mansoni does not obstruct the biliary tract. Advanced gallbladder fibrosis is unlikely to reverse after therapy.