Biomedical subjects

M A Handel

Publications and source records attributed to M A Handel.

At least 19 recordsLinked to original sources

PubMed2006

Impact of trisomy on fertility and meiosis in male mice.

BACKGROUND: Chromosomal abnormalities frequently are associated with impairment or arrest of spermatogenesis in mammals but are compatible with fertility in female carriers of the same anomaly. In the case of trisomy, mice have extra genomic DNA as well as the chromosomal abnormality, usually present as an extra, unpaired chromosome. Thus, impairment of spermatogenesis in trisomic males could be due to the presence of extra genomic material (i.e. triplicated genes) or due to the chromosomal abnormality and presence of an unpaired chromosome in meiosis. METHODS: In this study, fertility and chromosomal pairing configurations during meiotic prophase were analysed in male mice trisomic for different segments of the genome. Four have an extra segmental or tertiary trisomic chromosome--Ts(17(16))65Dn, Ts(10(16))232Dn, Ts(12(17))4Rk and Ts(4(17))2Lws--and one has the triplicated segment attached to another chromosome--Ts(16C-tel)1Cje. Ts(17(16))65Dn and Ts(16C-tel)1Cje have similar gene content triplication and differ primarily in whether the extra DNA is in an extra chromosome or not. RESULTS: The presence of an intact extra chromosome, rather than trisomy per se, is associated with male sterility. Additionally, sterility is correlated with a high frequency of association of the unpaired chromosome with the XY body, which contains the largely unpaired X and Y chromosomes. CONCLUSIONS: Intact extra chromosomes disrupt spermatogenesis, and unpaired chromosomes establish a unique chromatin territory within meiotic nuclei.

Animals↗

PubMed2006

Mutagenesis-generated mouse models of human infertility with abnormal sperm.

BACKGROUND: The aetiology of human male fertility, with impairment of sperm number, motility and morphology (oligoasthenoteratozoospermia), has been difficult to understand, partly for lack of animal models. METHODS: An ethylnitrosourea (ENU) mutagenesis strategy has been successful in producing heritable gene mutations with phenotypes similar to human male infertility, and here, we describe three independent ENU-induced mutations that cause a phenotype of oligoasthenoteratozoospermia in mice. RESULTS: The loci identified by these three mutations are designated swm2, repro2 and repro3. All mutant males were characterized by low sperm concentration, poor sperm morphology and negligible motility, but the infertile males were apparently normal in other respects. Sperm from mutant males failed to fertilize oocytes in vitro. Ultrastructural analyses revealed varied abnormalities apparent in both testicular spermatids and epididymal sperm. Genetic mapping placed the swm2 gene on chromosome 7, the repro2 gene on chromosome 5 and the repro3 gene on chromosome 10. CONCLUSION: The single-gene mutations caused complex and non-specific sperm pathologies, a point with important implications for managing cases of human male infertility. The ultimate identification of the loci for the mutations causing these phenotypes will clarify aetiology of complex syndromes of infertility with sperm abnormalities consistent with oligoasthenoteratozoospermia.

Animals↗

PubMed2004

New mouse genetic models for human contraceptive development.

Genetic strategies for the post-genomic sequence age will be designed to provide information about gene function in a myriad of physiological processes. Here an ENU mutagenesis program (http://reprogenomics.jax.org) is described that is generating a large resource of mutant mouse models of infertility; male and female mutants with defects in a wide range of reproductive processes are being recovered. Identification of the genes responsible for these defects, and the pathways in which these genes function, will advance the fields of reproduction research and medicine. Importantly, this program has potential to reveal novel human contraceptive targets.

Animals↗

PubMed2003

Temporal expression of cell cycle-related proteins during spermatogenesis: establishing a timeline for onset of the meiotic divisions.

During spermatogenesis, the complex events of the first meiotic prophase and division phase bring about dramatic changes in nuclear organization. One factor frustrating mechanistic dissection of these events is lack of knowledge about precisely what events occur, in what order they occur, and how they may be interrelated by temporal sequence; in other words, a precise timeline is lacking. This temporal ordering problem can be tackled by following expression and localization in mouse spermatocytes of proteins critical to events of the meiotic cell division process. These include ones that are primarily chromosomal and related to pairing and recombination, as well as kinases and substrates that mediate the cell cycle transition. Distinct and protein-specific patterns occur with respect to expression and localization throughout meiotic prophase and division and dramatic relocalization of proteins occurs as spermatocytes enter the meiotic division phase. This information provides a foundation for a meiotic timeline that can be augmented to provide, eventually, a complete catalog of meiotic events and their temporal sequence. Such a framework can clarify mechanisms of normal meiosis as well as mutant phenotypes and aberrations of the meiotic process that lead to aneuploidy.

Animals↗

PubMed2001

Evidence for meiotic spindle checkpoint from analysis of spermatocytes from Robertsonian-chromosome heterozygous mice.

Mice heterozygous for Robertsonian centric fusion chromosomal translocations frequently produce aneuploid sperm. In this study RBJ/Dn x C57BL/6J F1 males, heterozygous for four Robertsonian translocations (2N=36), were analyzed to determine effects on germ cells of error during meiosis. Analysis of sperm by three color fluorescence in situ hybridization revealed significantly elevated aneuploidy, thus validating Robertsonian heterozygous mice as a model for production of chromosomally abnormal gametes. Primary spermatocytes from heterozygous males exhibited abnormalities of chromosome pairing in meiotic prophase and metaphase. In spite of prophase abnormalities, the prophase/metaphase transition occurred. However, an increased frequency of cells with misaligned condensed chromosomes was observed. Cytological analysis of both young and adult heterozygous mice revealed increased apoptosis in spermatocytes during meiotic metaphase I. Metaphase spermatocytes with misaligned chromosomes accounted for a significant proportion of the apoptotic spermatocytes, suggesting that a checkpoint process identifies aberrant meioses. Immunofluorescence staining revealed that kinetochores of chromosomes that failed to align on the spindle stained more intensely for kinetochore antigens CENP-E and CENP-F than did aligned chromosomes. Taken together, these observations are consistent with detection of malattached chromosomes by a meiotic spindle checkpoint mechanism that monitors attachment and/or congression of homologous chromosome pairs. However, the relatively high frequency of gametic aneuploidy suggests that the checkpoint mechanism does not efficiently eliminate all germ cells with chromosomal abnormalities.

Aneuploidy↗

PubMed1999

Characterization of the mouse Xpf DNA repair gene and differential expression during spermatogenesis.

The human XPF protein, an endonuclease subunit essential for DNA excision repair, may also function in homologous recombination. To investigate a possible link between mammalian XPF and recombination that occurs during meiosis, we isolated, characterized, and determined an expression profile for the mouse Xpf gene. The predicted mouse XPF protein, encoded by a 3.4-kb cDNA, contains 917 amino acids and is 86% identical to human XPF. Appreciable similarity also exists between mouse XPF and homologous proteins in budding yeast (Rad1), fission yeast (Rad16), and fruit fly (Mei-9), all of which have dual functions in excision repair and recombination. Sequence analysis of the 38.3-kb Xpf gene, localized to a region in proximal mouse chromosome 16, revealed greater than 72% identity to human XPF in 16 regions. Of these conserved elements, 11 were exons and 5 were noncoding sequence within introns. Xpf transcript and protein levels were specifically elevated in adult mouse testis. Moreover, increased levels of Xpf and Ercc1 mRNAs correlated with meiotic and early postmeiotic spermatogenic cells. These results support a distinct role for the XPF/ERCC1 junction-specific endonuclease during meiosis, most likely in the resolution of heteroduplex intermediates that arise during recombination.

Amino Acid Sequence↗

PubMed1999

Differential gene expression of mammalian SPO11/TOP6A homologs during meiosis.

As the initiator of DNA double-strand breaks during meiosis in Saccharomyces cerevisiae, the SPO11 protein is essential for recombination. Similarity between SPO11 and archaebacterial TOP6A proteins points to evolutionary specialization of a DNA cleavage function for meiotic recombination. To determine whether this extends to mammals, we isolated and characterized mouse and human SPO11 cDNAs. Mammalian SPO11 genes were found to be expressed at high levels only in testis, wherein mouse Spo11 transcript is restricted primarily to meiotic germ cells and is maximally expressed at midpachynema. Mouse Spo11 is located near the distal end of chromosome 2, while human SPO11 is found in the homologous position of chromosome 20q13.2-13.3, a region that is amplified in some breast cancers. Sequence homology and differential expression together support a highly conserved role for SPO11 in the enzymatic cleavage of DNA that accompanies meiotic recombination.

Amino Acid Sequence↗

PubMed1999

What are the spermatocyte's requirements for successful meiotic division?

The consequences of error during meiotic division in spermatogenesis can be serious: aneuploid spermatozoa, embryonic lethality, and developmental abnormalities. Recombination between homologs is essential to ensure normal segregation; thus the spermatocyte must time division precisely so that it occurs after recombination between chromosomes and accumulation of the cell-cycle machinery necessary to ensure an accurate segregation of chromosomes. We use two systems to investigate meiotic division during spermatogenesis in the mouse: pharmacological induction of meiotic metaphase in cultured spermatocytes and transillumination-mediated dissection of stage XII seminiferous tubule segments to monitor progress through the division phase. By these approaches we can assess timing of acquisition of competence for the meiotic division phase and the temporal order of events as division proceeds. Competence for the meiotic division arises in the mid-pachytene stage of meiotic prophase, after chromosomes have synapsed and coincident with the accumulation of the cell-cycle regulatory protein CDC25C. The activity of both MPF and topoisomerase II are required. The earliest hallmarks of the division phase are nuclear envelope breakdown, followed by phosphorylation of histone H3 and chromosome condensation. These events are likely to be monitored by checkpoint mechanisms since checkpoint proteins can be localized in nuclei and DNA-damaging agents delay entry into the meiotic division phase. Understanding how the spermatocyte regulates its entry into the meiotic division phase can help clarify the natural mechanisms ensuring accurate chromosome segregation and preventing aneuploidy. J. Exp. Zool. (Mol. Dev. Evol.) 285:243-250, 1999.

Animals↗

PubMed1999

Acquisition of competence to condense metaphase I chromosomes during spermatogenesis.

Little is known about the timing of meiotic prophase events during spermatogenesis in the mouse or how these events are related to cell-cycle progression. This work was designed to test hypotheses about the timing and biochemical correlates of developmental acquisition of competence to condense bivalent pairs of homologous chromosomes held together by chiasmata. The experimental approach takes advantage of the fact that okadaic acid (OA) treatment of pachytene spermatocytes causes precocious entry into metaphase I (MI) of meiosis. Leptotene and zygotene (L/Z) spermatocytes are not competent to respond to OA with condensation of chiasmate bivalent chromosomes. Competence for MI condensation of chiasmate bivalents is acquired by the middle of the pachytene stage of meiotic prophase, several days after homologous chromosomes become fully synapsed. The acquisition of MI competence is paralleled by the accumulation of histone H1t in the nuclei of mid-pachytene spermatocytes. Biochemical differences also exist between the incompetent L/Z spermatocytes and the competent pachytene spermatocytes. Both have the molecular components of metaphase promoting factor, CDC2 and CYCLIN B1; however, the histone H1 kinase activity of metaphase promoting factor of incompetent L/Z spermatocytes is not activated by OA, as it is in competent pachytene spermatocytes. Additionally, the CDC25C protein phosphatase is present in competent pachytene spermatocytes, but not in incompetent L/Z or early pachytene spermatocytes. Both incompetent and competent spermatocytes accumulate MPM-2 phosphoepitopes and phosphorylated histone H3 in response to OA treatment, indicating that presence of these antigens is not sufficient to promote condensation of meiotic chromosomes. These data demonstrate that meiotic competence of spermatocytes is acquired after homologous chromosome pairing is established and is coincident with first appearance of histone H1t and CDC25C protein phosphatase in spermatocytes.

Animals↗

PubMed1999

Meiotic events at the centromeric heterochromatin: histone H3 phosphorylation, topoisomerase II alpha localization and chromosome condensation.

Mechanisms of chromosome condensation and segregation during the first meiotic division are not well understood. Resolution of recombination events to form chiasmata is important, for it is chiasmata that hold homologous chromosomes together for their oppositional orientation on the meiotic metaphase spindle, thus ensuring their accurate segregation during anaphase I. Events at the centromere are also important in bringing about proper attachment to the spindle apparatus. This study was designed to correlate the presence and activity of two proteins at the centromeric heterochromatin, topoisomerase II alpha (TOP2A) and histone H3, with the processes of chromosome condensation and individualization of chiasmate bivalents in murine spermatocytes. We tested the hypothesis that phosphorylation of histone H3 is a key event instigating localization of TOP2A to the centromeric heterochromatin and condensation of chromosomes as spermatocytes exit prophase and progress to metaphase. Activity of topoisomerase II is required for condensation of chromatin at the end of meiotic prophase. Histone H3 becomes phosphorylated at the end of prophase, beginning with its phosphorylation at the centromeric heterochromatin in the diplotene stage. However, it cannot be involved in localization of TOP2A, since TOP2A is localized to the centromeric heterochromatin throughout most of meiotic prophase. This observation suggests a meiotic function for TOP2A in addition to its role in chromatin condensation. The use of kinase inhibitors demonstrates that phosphorylation of histone H3 can be uncoupled from meiotic chromosome condensation; therefore other proteins, such as those constituting metaphase-promoting factor, must be involved. These results define the timing of important meiotic events at the centromeric heterochromatin and provide insight into mechanisms of chromosome condensation for meiotic metaphase.

Animals↗

PubMed1999

Imprinting of a RING zinc-finger encoding gene in the mouse chromosome region homologous to the Prader-Willi syndrome genetic region.

A novel locus in the human Prader-Willi syndrome (PWS) region encodes the imprinted ZNF127 and antisense ZNF127AS genes. Here, we show that the mouse ZNF127 ortholog, Zfp127, encodes a homologous putative zinc-finger polypeptide, with a RING (C3HC4) and three C3H zinc-finger domains that suggest function as a ribonucleoprotein. By the use of RT-PCR across an in-frame hexamer tandem repeat and RNA from a Mus musculus x M.spretus F1interspecific cross, we show that Zfp127 is expressed only from the paternal allele in brain, heart and kidney. Similarly, Zfp127 is expressed in differentiated cells derived from androgenetic embryonic stem cells and normal embryos but not those from parthogenetic embryonic stem cells. We hypothesize that the gametic imprint may be set, at least in part, by the transcriptional activity of Zfp127 in pre- and post-meiotic male germ cells. Therefore, Zfp127 is a novel imprinted gene that may play a role in the imprinted phenotype of mouse models of PWS.

Amino Acid Sequence↗

PubMed1999

New gene family defined by MORC, a nuclear protein required for mouse spermatogenesis.

Mammalian spermatogenesis is a complex developmental process. The analysis of mouse mutations has provided insight into biochemical pathways required for completion of this process. We previously described the autosomal recessive mouse morc TgN(Tyr)1Az(microrchidia) mutation, a serendipitous transgenic insertional mutation which causes arrest of spermatogenesis prior to the pachytene stage of meiosis prophase I. We now report the molecular characterization of the morc locus and positional cloning of a gene disrupted by the morc TgN(Tyr)1Az mutation. This gene, which we term Morc, encodes a 108 kDa protein expressed specifically in male germ cells. The transgene integrated within the first intron of Morc and was accompanied by an intragenic deletion of approximately 13 kb of genomic sequences, removing exons 2-4 and abrogating expression of the wild-type transcript. Analysis of the MORC protein sequence revealed putative nuclear localization signals, two predicted coiled-coil structural motifs and limited homology to GHL (GyraseB, Hsp90, MutL) ATPase. Epitope-tagged MORC protein expressed in COS7 cells localized to the nucleus. We also cloned the human MORC homolog and show that it too is testis-specific, but closely related human genes are transcribed in multiple somatic tissues. Homologous proteins are also present in zebrafish, nematodes, slime mold and plants. Thus, cloning of Morc defines a novel gene family whose members are likely to serve important biological functions in both meiotic and mitotic cells of multicellular organisms.

Amino Acid Sequence↗

PubMed1998

Identification of morc (microrchidia), a mutation that results in arrest of spermatogenesis at an early meiotic stage in the mouse.

The microrchidia, or morc, autosomal recessive mutation results in the arrest of spermatogenesis early in prophase I of meiosis. The morc mutation arose spontaneously during the development of a mouse strain transgenic for a tyrosinase cDNA construct. Morc -/- males are infertile and have grossly reduced testicular mass, whereas -/- females are normal, indicating that the Morc gene acts specifically during male gametogenesis. Immunofluorescence to synaptonemal complex antigens demonstrated that -/- male germ cells enter meiosis but fail to progress beyond zygotene or leptotene stage. An apoptosis assay revealed massive numbers of cells undergoing apoptosis in testes of -/- mice. No other abnormal phenotype was observed in mutant animals, with the exception of eye pigmentation caused by transgene expression in the retina. Spermatogenesis is normal in +/- males, despite significant transgene expression in germ cells. Genomic analysis of -/- animals indicates the presence of a deletion adjacent to the transgene. Identification of the gene inactivated by the transgene insertion may define a novel biochemical pathway involved in mammalian germ cell development and meiosis.

Animals↗

PubMed1998

Monitoring meiosis in gametogenesis.

Meiotic recombination is essential to hold homologous chromosomes together so that they can separate accurately in the formation of gametes, thus preventing fetal loss due to aneuploidy. How do germ cells know when they have finished genetic recombination and that it is time to enter the meiotic division phase, and what are the elements that signal the onset of the division phase? During spermatogenesis there is no arrest at the end of meiotic prophase (as there is in oogenesis) and signals for progress into the meiotic division phase may be closely related to events of chromosome pairing and recombination. Methods for culture of male germ cells have been used to show that spermatocytes become competent for some aspects of the division phase by the early pachytene stage, long before they would normally enter division. Evidence suggests that establishment of homologous chromosome pairing is one aspect of acquiring competence. Activation of the cell cycle regulator MPF also appears to be important, and there is a requirement for activity of topoisomerase II in order for spermatocytes to exit prophase and enter the meiotic division phase. Understanding how these molecular entities tie into monitoring the completion of recombination and meiotic progress will be instructive about important gametic safeguards preventing aberrant chromosome segregation and resultant aneuploidy.

Aneuploidy↗

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

Dynamics of meiotic prophase I during spermatogenesis: from pairing to division.

This review focuses on recent developments in our understanding of meiotic chromosome behavior in mammalian spermatogenesis, with a special emphasis on prophase I events in the best characterized organism, the laboratory mouse. The dynamics of chromosome movement prior to pairing and synapsis of chromosomes are complex and implicate function for both centromeres and telomeres in getting homologous chromosomes together in intimate synapsis. Likely candidates for mediating pairing and recombination include a host of proteins implicated in DNA repair and recombination, which have been shown to localize to the synaptonemal complex during meiotic prophase I. Precocious induction of meiotic metaphase in cultured pachytene spermatocytes has led to new information about requirements for MPF and topoisomerase II activity during the transition from meiotic prophase to metaphase. Together, the studies reviewed here increase our understanding of how chromosomes get together with their homologous partners and how these partners subsequently come apart.

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↗