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Biomedical subjects

M A Handel

Publications and source records attributed to M A Handel.

At least 19 recordsLinked to original sources
PubMed1998

Chromatin configuration during meiosis I prophase of spermatogenesis.

During the pachytene stage of meiotic prophase in male mammals, the X and Y chromosomes become transcriptionally inactive and establish a chromatin domain, the sex body, that is visually distinct from the transcriptionally active autosomes. We used objective criteria to assess these chromatin differences by DNase I sensitivity (DS) of sex chromosome and autosomal sequences at both the cytological and molecular levels. For cytological studies, in situ nick translation techniques were used on air-dried preparations of testicular cells. For molecular studies, nuclei from pachytene spermatocytes were subjected to nuclease sensitivity assays. Both sex-linked and autosomal sequences were assessed, including some gene sequences that are expressed and some that are not expressed in pachytene spermatocytes. There was a wide range of DS in different genomic sequences; however, the sex-linked sequences generally were less nuclease sensitive than were autosomal sequences. Interestingly, a hot spot of recombination (within the Eb gene) showed a high level of nuclease sensitivity, while a cold spot of recombination (centromeric satellite region) exhibited lower sensitivity, more similar to that of sex-linked sequences. We also examined the nuclease sensitivity of a tyrosinase transgene insert, TyBS. In one line of mice, the transgene insert is X-linked, whereas in another, it is autosomal. The transgene was less nuclease sensitive when X-linked than as an autosomal insert. These results support the hypothesis that in pachytene spermatocytes the XY chromosome pair is more condensed and inaccessible to enzymatic digest, whereas the autosomal chromatin is in a more open configuration. In addition, we examined the nuclease sensitivity of some of the same genes in the earlier leptotene/zygotene prophase stage, when the sex chromatin is not maximally condensed. We found that while autosomal gene nuclease sensitivity was equivalent to that at the pachytene stage, X-linked sequences were more nuclease sensitive. Overall, these differences in chromatin nuclease sensitivity correlate with differences in meiotic recombination activity and may be mechanistically related.

Animals↗
PubMed1998

Germ cell loss in the XXY male mouse: altered X-chromosome dosage affects prenatal development.

Male mammals with two X chromosomes are sterile due to the demise of virtually all germ cells; however, the underlying reasons for the germ cell loss remain unclear. The use of a breeding scheme for the production of XXY male mice has allowed us to experimentally address the question of when and why germ cells die in the XXY testis and whether the defect is due to the presence of an additional X chromosome in the soma, the germ cells themselves, or both. Our studies demonstrate that altered X-chromosome dosage acts to impair germ cell development in the testis at a much earlier stage than suggested by previous studies of XX sex-reversed males or XX/XY chimeras. Specifically, we noted significantly reduced germ cell numbers in the XXY testis during the period of germ cell proliferation in the early stages of testis differentiation. Although the somatic development of the XXY testis is morphologically and temporally normal, our studies indicate that germ cell demise reflects a defect in somatic/germ cell communication, since, in an in vitro system, the proliferative potential of fetal germ cells from XXY males is indistinguishable from that of normal males.

Animals↗
PubMed1998

Differential expression of the zinc finger gene Zfp105 during spermatogenesis.

We report the isolation of Zfp105, the mouse homolog of the human ZNF35 zinc finger gene. Zfp105 and ZNF35 are highly conserved at the protein and nucleotide level, and Zfp105 maps to a region of mouse Chromosome (Chr) 9 that is homologous to the human region containing ZNF35. Zpf105 is highly expressed in the testis, especially in pachytene spermatocytes and round spermatids. The possible role of this gene product in maintaining an ordered germ cell differentiation process is discussed.

Amino Acid Sequence↗
PubMed1998

Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for Dmc1, a germline-specific RecA homolog.

DMC1 is a meiosis-specific gene first discovered in yeast that encodes a protein with homology to RecA and may be component of recombination nodules. Yeast dmc1 mutants are defective in crossing over and synaptonemal complex (SC) formation, and arrest in late prophase of meiosis I. We have generated a null mutation in the Dmc1 gene in mice and show that homozygous mutant males and females are sterile with arrest of gametogenesis in the first meiotic prophase. Chromosomes in mutant spermatocytes fail to synapse, despite the formation of axial elements that are the precursor to the SC. The strong similarity of phenotypes in Dmc1-deficient mice and yeast suggests that meiotic mechanisms have been highly conserved through evolution.

Adenosine Triphosphatases↗
PubMed1997

Analysis of expression and function of topoisomerase I and II during meiosis in male mice.

Topoisomerases are nuclear enzymes that remove torsional stress in DNA. Their function is important for replication, transcription, chromosome condensation, and chromosome segregation during mitosis and meiosis. The goal of this work is to analyze both expression and function of topoisomerases during the meiotic stages of mammalian spermatogenesis. The patterns of expression of topoisomerase I and topoisomerase II alpha genes were followed on Northern blots of RNA from testes of mice of different ages and from specific germ cell populations. The transcript of the topoisomerase I gene was highest in somatic cells of the testis and in the mitotically proliferating spermatogonia and meiotic prophase spermatocytes, with the level of transcript decreasing dramatically in postmeiotic spermatids. In contrast, the levels of topoisomerase II alpha transcript were negligible in germ-cell free testes and highest in late meiotic prophase cells and round spermatids. Enzyme activity for both topoisomerase I and topoisomerase II was detected in both pachytene spermatocytes and in round spermatids; topoisomerase II exhibited a higher level of activity in meiotic spermatocytes than in round spermatids. In cultured cells, camptothecin, an inhibitor of topoisomerase I, caused some abnormalities of paired meiotic homologs, but did not inhibit the transition to metaphase. In contrast, teniposide and ICRF-193, inhibitors of topoisomerase II, dramatically inhibited the formation of metaphase chromosomes in cells induced to progress from prophase to metaphase. However, the disassembly of the synaptonemal complex was not inhibited, indicating that this process could be uncoupled from condensation of chromatin to form chromosomes. These studies constitute evidence for a functional requirement for topoisomerase II activity in the transition from meiotic prophase to meiotic metaphase I in mammalian spermatocytes.

Animals↗
PubMed1996

A genetic strategy for differential screening of meiotic germ-cell cDNA libraries.

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis.

Animals↗
PubMed1996

Mouse ret finger protein (rfp) proto-oncogene is expressed at specific stages of mouse spermatogenesis.

Many proteins involved in the regulation of cell growth and differentiation possess structural motifs that participate in specific molecular interactions. The human rfp (ret finger protein) has a tripartite motif, consisting of two novel zinc fingers (the RING linger and the B box) and a coiled-coil domain, and belongs to the B box zinc finger protein family. Rfp becomes oncogenic when its tripartite motif is recombined with the tyrosine kinase domain from the c-ret proto-oncogene. To further understand the function of rfp during normal development and cellular differentiation, we cloned the mouse rfp cDNA and analyzed its pattern of expression and subcellular distribution. We found that the mouse rfp cDNA shared a 98.4% homology with the human sequence. The gene mapped to human chromosome 6 and mouse chromosome 13 indicating that it was linked to a several other genes encoding proteins that possess common domains. rfp transcripts and protein were ubiquitous in day 10.5-13.5 mouse embryos, however, they were restricted in adult mice, with the highest level of expression in pachytene spermatocytes and round spermatids of differentiating sperm. The rfp protein was detected within cell nuclei as nuclear bodies similar to the PODs (PML oncogenic domains) observed with another B box family member, PML (promyelocytic leukemia protein). These results suggest that rfp may function in the regulation of cell growth and differentiation during mouse embryogenesis and sperm differentiation.

Amino Acid Sequence↗
PubMed1996

A new mouse mutation causing male sterility and histoincompatibility.

Male sterility and histoincompatibility, mshi, is an autosomal recessive mutation in BALB/cBy mice that causes reduced testis size and sterility in homozygous males. The testes of homozygous mutants are highly disorganized and appear to have a block in the regulation of male germ cell proliferation. No heterozygous effect is detectable. Reproduction is unaffected in females carrying the mutation. The mutation also affects histocompatibility; most homozygous males and females reject sex-matched skin grafts from BALB/cBy mice. We used an intercross between BALB/cBy and CAST/Ei to map the mshi mutation to the proximal end of Chromosome (Chr) 10. The most likely gene order places the mutation between D10Mit80 and D10Mit16, near the interferon gamma receptor locus, Ifgr, which may be a candidate gene for this mutation.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Culture of pachytene spermatocytes for analysis of meiosis.

An impediment to the investigation of mammalian spermatogenic meiosis has been the lack of an appropriate system for experimental manipulation of meiotic prophase cells. We report here the use of a simple system for the short-term culture of pachytene spermatocytes. We have assayed parameters of cell function pertinent to meiotic prophase, namely chromosome pairing and synapsis. During the culture period of 24-48 hr, cells maintained typical pachytene morphology, chromatin condensation patterns, and chromosome pairing, as assessed by light and electron microscopy. Uridine incorporation, monitored by autoradiography, reflected the chromosomal distribution found in vivo in that the autosomal chromosomes were transcriptionally active, while the sex chromosomes were not. Thus features of chromosome pairing and sex chromatin inactivation are maintained in these cultures. We have conducted experiments to demonstrate that cultured pachytene spermatocytes can be useful for the analysis of agents, some of which may be suspected mutagens, that might affect chromosome structure and function during meiosis. Treatment of cells with actinomycin D revealed a differential effect on chromatin condensation in the autosomes versus the sex chromosomes. Camptothecin, a topoisomerase inhibitor, induced desynapsis of paired chromosomes. Okadaic acid, a phosphatase inhibitor, induced premature metaphase-I condensation of pachytene chromosomes. This last experiment suggests that these cultured cells may be useful for analysis of meiotic cell cycle controls. Taken together, these results demonstrate a culture system that can be useful for analysis of meiotic events as well as in screening for potential mutagenic agents that might affect meiotic chromosome structure and function.

Animals↗
PubMed1995

Chromatin structure and imprinting: developmental control of DNase-I sensitivity in the mouse insulin-like growth factor 2 gene.

The insulin-like growth factor 2 (Igf2) gene on distal mouse chromosome 7 is expressed predominantly from the paternal allele. In previous studies we identified two regions of paternal allele-specific methylation; one at approximately 3 kb upstream of promoter 1, and a second in the 3', coding portion of the gene. The 3' region is methylated in an expressing tissue (fetal liver), whereas in a non-expressing tissue (fetal brain), it is not methylated. By contrast, in the 5' region, the paternal allele is highly methylated in all tissues. Here, we have studied another characteristic of chromatin, namely, sensitivity to DNase-I and have focused our developmental analysis on the two differentially methylated regions of Igf2. In the upstream region, four clustered DNase-I hypersensitive sites (HSS) were detected in embryonic stem (ES) cells and in midgestation embryos, but not in neonatal liver or brain. In promoter 1 (P1), at approximately 0.3 kb upstream of exon 1, we detected a tissue-specific HSS that was present in neonatal liver, in which P1 is active, but was absent in ES cells, the embryo, and in neonatal brain. No DNase-I HSS were detected in the 3' differentially methylated region of Igf2. In all these regions, we did not detect differences in DNase-I sensitivity between the parental chromosomes. These results establish major developmental and tissue-specific control of chromatin in the Igf2 locus. The presence of the HSS upstream of Igf2 precedes transcriptional activation of the Igf2 gene and may be indicative of a promoter for another transcript that is transcribed in the opposite direction. The HSS in P1 is largely liver-specific; this promoter therefore is differently regulated than the more general fetal promoters P2 and P3. Whereas methylation can be allele-specific, presumably reflecting the gene imprint, the nuclease sensitivity, as detected by our assay, is not. These results, taken together with previous observations, reveal developmental and tissue-specific complexity in the expression of the parental imprint at the level of chromatin and transcription. We propose that epigenetic features of tissue-specific control and of the control of allelic expression are intricately linked.

Alleles↗
PubMed1995

Induced premature G2/M-phase transition in pachytene spermatocytes includes events unique to meiosis.

Little is known about the control of events ending the lengthy prophase of meiosis I and leading to the G2/M-phase transition in mammalian spermatocytes, primarily because the relevant late pachytene, diplotene, and MI cells are present in low numbers in the testis and it is not possible to isolate them in significant numbers. We have utilized short-term cultures of pachytene spermatocytes from the mouse to study events of the G2/M cell-cycle transition induced by the protein phosphatase inhibitor okadaic acid (OA). Treatment of cultured pachytene spermatocytes with OA induced a rapid and premature onset of events leading to the M phase, visualized cytologically by nuclear envelope breakdown and chromosome condensation. After OA treatment, condensed chromosomes were seen as bivalents, not as univalents. Treatment with OA induced disassembly of synaptonemal complexes and resolution of crossovers as cytologically visible chiasmata. Chiasmata counts were similar in treated cells and control cells. Thus, surprisingly, even though the treated cells were in the pachytene substage of meiotic prophase, events of recombination were apparently completed to the point of chiasma formation in the majority of these cells. The sex chromosomes, forming the sex body of the pachytene spermatocyte, lagged behind the autosomal chromosomes in their condensation and progression toward the M phase. Treatment with OA induced an increase in histone H1 kinase activity, generally used as an indicator of metaphase-promoting factor (MPF) activity; furthermore, the OA-induced cell-cycle transition does not require new protein synthesis. These results suggest that OA treatment overrides a cell-cycle checkpoint control that normally keeps pachytene spermatocytes in a lengthy prophase and that this control may be exerted by regulation of protein phosphorylation status.

Animals↗
PubMed1994

Clustering of six human 11p15 gene homologs within a 500-kb interval of proximal mouse chromosome 7.

Homologs of genes mapping to human chromosome 11p15 are located in three distinct, widely separated regions of mouse chromosome 7 (Mmu7). To date, six genes have been localized to the most proximal HSA11p15/Mmu7 homology region, including Ldh3 (encoding lactate dehydrogenase C), Ldh1 (lactate dehydrogenase A), Myod1 (myogenic differentiation factor-1), Tph (tryptophan hydroxylase), Saa1 (serum amyloid-A-1), and Kcnc1 (encoding a Shaw-type voltage-gated potassium channel). To define the overall size and organization of this region of Mmu7, we have established a long-range physical map including the murine Ldh1, Ldh3, Saa, Tph, Kcnc1, and Myod1 genes. Our results demonstrate that these six genes are physically clustered and are distributed throughout a 500-kb interval located just proximal of the pink-eyed dilution (p) locus. These data, together with recent mapping studies within the related region of HSA11p15, demonstrate that gene content and organization within this proximal homology segment have been highly conserved throughout evolution.

Animals↗
PubMed1994

Genetic control of sex-chromosome inactivation during male meiosis.

During meiotic prophase in male mammals, the sex chromosomes are transcriptionally inactivated and form a condensed chromatin domain known as the sex body. It is not known how the assumption of this chromatin configuration is determined and regulated. We used various genetic models to test whether a complete sex-chromosome pair, effective sex-chromosome pairing, or an intact X chromosome is required for sex-body formation or transcription inactivation. The sex chromosome aberrations studied did not interfere with sex-body formation, and there is no evidence for inactivation failure or reactivation of the aberrant sex chromosomes. The results of this study suggest that control of sex-body formation is not intrinsic to the sex chromosomes and thus may be at the level of the testis.

Animals↗
PubMed1993

Sex chromosomes, recombination, and chromatin conformation.

We review what is known about the transcriptional inactivation and condensation of heteromorphic sex chromosomes in contrast to the activation of homomorphic sex chromosomes during meiotic prephase in animals. We relate these cytological and transcriptional features to the recombination status of the sex chromosomes. We propose that sex chromosome condensation is a meiotic adaptation to prevent the initiation of potentially damaging recombination events in nonhomologous regions of the X and Y chromosome.

Animals↗
PubMed1993

Expression of the Hprt gene during spermatogenesis: implications for sex-chromosome inactivation.

The goal of this study was to determine the developmental pattern of expression of the X-linked gene for hypoxanthine phosphoribosyltransferase (Hprt) during spermatogenesis and the relevance of this expression to X-chromosome inactivation during meiotic prophase. The results demonstrated that HPRT activity is maintained in mouse spermatogenic cells throughout development in spite of X-chromosome inactivation; however, specific activities of HPRT in meiotic and postmeiotic germ cells were significantly lower than in premeiotic ones. Maintenance of Hprt transcripts at all stages was also demonstrated. Interestingly, the highest level of Hprt transcripts was found in leptotene/zygotene spermatocytes, suggesting a hyperactivation of the Hprt gene and/or stabilization of Hprt transcripts in these cells. Hprt transcripts were present at very low levels in pachytene spermatocytes, and at slightly elevated levels in round spermatids. It was also found that the relative abundance of Hprt transcripts in the somatic cells of germ-cell-deficient testes was much greater than that in meiotic and postmeiotic germ cells, even though their activities of HPRT were similar. Examination of the translational status of Hprt transcripts in testicular cells revealed that while most of the transcript was translationally active in somatic cells of testes, less than half of the transcript was on polysomes in pachytene spermatocytes and round spermatids. Since no functional autosomal Hprt gene exists in the mouse, these data suggest that the germ cell maintains both transcript and protein product of the Hprt gene in spite of apparent X-chromosome inactivation.

Animals↗
PubMed1992

Sex-chromosome pairing and activity during mammalian meiosis.

Mammalian sex chromosomes exhibit marked sexual dimorphism in behavior during gametogenesis. During oogenesis, the X chromosomes pair and participate in unrestricted recombination; both are transcriptionally active. However, during spermatogenesis the X and Y chromosomes experience spatial restriction of pairing and recombination, are transcriptionally inactive, and form a chromatin domain that is markedly different from that of the autosomes. Thus the male germ cell has to contend with the potential loss of X-encoded gene products, and it appears that coping strategies have evolved. Genetic control of sex-chromosome inactivation during spermatogenesis does not involve pairing or the presence of the Y chromosome or an intact X chromosome, and may therefore be under exogenous control by the gonad. Sex-chromosome reactivation during oogenesis and inactivation during spermatogenesis probably reflect specific meiotic events such as recombination. Understanding these phenomena may help explain other sex-related differences in genetic recombination.

Animals↗