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J M Trasler

Publications and source records attributed to J M Trasler.

At least 19 recordsLinked to original sources
PubMed2006

A unique configuration of genome-wide DNA methylation patterns in the testis.

In the mammalian lifecycle, the two periods of genome-wide epigenetic reprogramming are in the early embryo, when somatic patterns are set, and during germ cell development. Although some differences between the reprogrammed states of somatic and germ cells have been reported, overall patterns of genomic methylation are considered to be similar. Using restriction landmark genomic scanning to examine approximately 2,600 loci distributed randomly throughout the genome, we find that the methylation status of testicular DNA is highly distinct, displaying eightfold the number of hypomethylated loci relative to somatic tissues. Identification and analysis of >300 loci show that these regions are generally located within nonrepetitive sequences that are away from CpG islands and 5' regions of genes. We show that a contributing factor for these differences is that the methylation state of non-CpG-island DNA is correlated with the regional level of GC content within chromosomes and that this relationship is inverted between the testis and somatic tissues. We also show that in Dnmt3L-deficient mice, which exhibit infertility associated with abnormal chromosomal structures in germ cells, this unique testicular DNA methylation pattern is not established. These special properties of testicular DNA point to a broad, distinct epigenetic state that may be involved in maintaining a unique chromosomal structure in male germ cells.

Animals↗
PubMed2006

Sex-specific promoters regulate Dnmt3L expression in mouse germ cells.

BACKGROUND: Dnmt3L, a member of the DNA methyltransferase 3 family, lacks enzymatic activity but is required for de-novo methylation of imprinted genes in oocytes and for transposon repression in male germ cells. METHODS: We used northern blots, RT-PCR, 5' rapid amplification of complementary DNA (cDNA) ends (RACE), RNase H mapping, real-time/quantitative RT-PCR and in situ hybridization to identify and characterize Dnmt3L transcripts produced during germ cell development. RESULTS: Mouse Dnmt3L uses three sex-specific promoters, not the single promoter previously thought. A promoter active in prospermatogonia drives transcription of an mRNA encoding the full-length protein in perinatal testis, where de-novo methylation occurs. Late pachytene spermatocytes activate a second promoter in intron 9 of the Dnmt3L gene. After this stage, the predominant transcripts are three truncated mRNAs, which appear to be non-coding. We could also detect similar adult testis transcripts in humans. In the mouse ovary, an oocyte-specific promoter located in an intron of the neighbouring autoimmune regulator (Aire) gene produces a transcript with the full open reading frame (ORF). This is the only Dnmt3L transcript found in growing oocytes and is absent in the oocytes of Dnmt3L-/- females. CONCLUSIONS: Sex-specific promoters control Dnmt3L expression in the mouse germ line, mirroring the situation at the Dnmt1 and Dnmt3A loci.

Animals↗
PubMed2004

Reproductive epigenetics.

Epigenetics refers to covalent modifications of DNA and core histones that regulate gene activity without altering DNA sequence. To date, the best-characterized DNA modification associated with the modulation of gene activity is methylation of cytosine residues within CpG dinucleotides. Human disorders associated with epigenetic abnormalities include rare imprinting diseases, molar pregnancies, and childhood cancers. Germ cell development and early embryo development are critical times when epigenetic patterns are initiated or maintained. This review focuses on the epigenetic modification DNA methylation and discusses recent progress that has been made in understanding when and how epigenetic patterns are differentially established in the male and female germlines, the mouse, and human disorders associated with abnormalities in epigenetic programming in germ cells and early embryos, as well as genetic and other modulators (e.g. nutrition and drugs) of reproductive epigenetic events.

Animals↗
PubMed2001

Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene.

Maintenance of genomic methylation patterns in mammalian somatic cells depends on DNA methyltransferase-1 (Dnmt1). Mouse oocytes and preimplantation embryos lack Dnmt1 but express a variant of this protein called Dnmt1o. We eliminated Dnmt1o by deletion of the oocyte-specific promoter and first exon from the Dnmt1 locus. Homozygous animals were normal, but most heterozygous fetuses of homozygous females died during the last third of gestation. Although genomic methylation patterns were established normally in Dnmt1o-deficient oocytes, embryos derived from such oocytes showed a loss of allele-specific expression and methylation at certain imprinted loci. Transient nuclear localization of Dnmt1o in 8-cell embryos suggests that this variant of Dnmt1 provides maintenance methyltransferase activity specifically at imprinted loci during the fourth embryonic S phase.

Animals↗
PubMed2000

Deoxyribonucleic acid hypomethylation of male germ cells by mitotic and meiotic exposure to 5-azacytidine is associated with altered testicular histology.

Genomic methylation patterns originate during gametogenesis and are postulated to be involved in important developmental events, including gene regulation, embryogenesis, and genomic imprinting. In previous work, treatment of male rats with 5-azacytidine, a drug that blocks DNA methylation, resulted in abnormal embryo development when germ cells were exposed throughout spermatogenesis, encompassing mitotic, meiotic, and postmeiotic development, but not if they were only exposed postmeiotically. To explore the mechanisms underlying the effects of 5-azacytidine on sperm function, we determined the effects of the drug on testicular morphology, assessed whether exposure of meiotic spermatocytes resulted in abnormal pregnancy outcome, and examined the role of germ cell genomic demethylation in mediating the effects of 5-azacytidine on spermatogonia and spermatocytes. Male Sprague Dawley rats were treated three times a week with saline or 5-azacytidine (2.5 and 4.0 mg/kg) for 6 weeks (meiotic and postmeiotic germ cell exposure) and 11 weeks (mitotic, meiotic, and postmeiotic exposure). Six weeks of paternal treatment with the highest dose of 5-azacytidine resulted in an increase in preimplantation loss (corpora lutea minus implantation sites) without affecting testicular morphology or altering sperm DNA methylation levels. Eleven weeks of 5-azacytidine treatment at doses that cause preimplantation loss resulted in severe abnormalities of the seminiferous tubules, such as degeneration and loss of germ cells, atrophy of seminiferous tubules, presence of multinuclear giant cells, and sloughing of immature germ cells into the lumen, and a 22-29% decrease in genomic methylation levels in epididymal sperm. On closer evaluation of testicular histology using terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling detection in situ, both 6 and 11 weeks of 5-azacytidine treatment resulted in an increase over the control value in the number of apoptotic germ cells in the seminiferous tubules. Analysis of DNA methylation levels in isolated germ cells of treated males indicated that spermatogonia were more susceptible to the hypomethylating effects of 5-azacytidine than were spermatocytes. These studies provide evidence of an association between demethylation of germ cell DNA and alterations in testicular histology.

Animals↗
PubMed1999

Acquisition of the H19 methylation imprint occurs differentially on the parental alleles during spermatogenesis.

The imprinted mouse H19 gene is hypomethylated on the expressed maternal allele and hypermethylated on the silent paternal allele. A 2-kb region of differential methylation located from -2 to -4 kb relative to the H19 transcriptional start site has been proposed to act as the imprinting mark since hypermethylation in this region is inherited from sperm and retained on the paternal allele throughout development. Here, we describe a temporal analysis of the methylation patterns at the H19 locus during postnatal male germ cell development. The 2-kb region is methylated on the paternal allele throughout spermatogenesis, suggesting that methylation is acquired in this region prior to the resumption of mitosis in postnatal male mice. Likewise, more than half of the maternal alleles are hypermethylated prior to the resumption of mitosis. However, the remaining maternal alleles are not hypermethylated until the completion of meiosis I, indicating that de novo methylation in this region is a continuous process. Sequences proximal to the H19 promoter, which are methylated in spermatozoa and on the paternal allele in somatic cells, are differentially methylated in diploid, mitotic spermatogonia. The maternal allele becomes hypermethylated in this region during meiotic prophase. Thus, the parental H19 alleles acquire methylation differentially in the male germline.

Alleles↗
PubMed1999

Sialidase-mediated depletion of GM2 ganglioside in Tay-Sachs neuroglia cells.

Tay-Sachs disease is a severe, inherited disease of the nervous system caused by accumulation of the brain lipid GM2 ganglioside. Mouse models of Tay-Sachs disease have revealed a metabolic bypass of the genetic defect based on the more potent activity of the enzyme sialidase towards GM2. To determine whether increasing the level of sialidase would produce a similar effect in human Tay-Sachs cells, we introduced a human sialidase cDNA into neuroglia cells derived from a Tay-Sachs fetus and demonstrated a dramatic reduction in the accumulated GM2. This outcome confirmed the reversibility of GM2 accumulation and opens the way to pharmacological induction or activation of sialidase for the treatment of human Tay-Sachs disease.

Base Sequence↗
PubMed1999

Cellular immunolocalization of occludin during embryonic and postnatal development of the mouse testis and epididymis.

Cellular junctions in the testis and epididymis play crucial roles for the development and maturation of spermatozoa. In the testis, tight junctions between Sertoli cells form a functional blood testis barrier between 10 and 16 days of age, whereas the tight junctional blood epididymal barrier between adjacent epithelial cells is formed between days 18 and 21. In the present study, occludin, a constituent integral membrane protein of tight junctions, was localized by immunofluorescent confocal microscopy in embryonic (days 13.5-18.5), postnatal (days 5-23) and adult (day 70) mouse testes and epididymides to correlate its expression with the onset of tight junctions and eventual formation of these barriers. At embryonic days 13.5 and 16.5, low diffuse cytoplasmic levels of occludin were observed in cells of the testicular cords. By embryonic day 18.5, the level of occludin was still low but appeared as a filiform-like network streaming toward the center of the cord. At postnatal days 5 and 7 immunostaining became more intense and appeared to outline the periphery of Sertoli cells of seminiferous tubules. Postnatal day 14 marked the appearance of an intense, focal band-like localization of occludin at the base of the tubules, correlating with the appearance of a functional blood-testis barrier. By day 23 and in adults, expression of occludin was noted at the base of the tubule appearing as intense, wavy, discontinuous bands similar in appearance irrespective of the stage of the seminiferous epithelium cycle. In the developing epididymis, intense cytoplasmic immunostaining was present in epithelial cells of many epididymal tubules at embryonic day 13.5. By embryonic day 16.5, intense occludin immunostaining appeared along the lateral plasma membranes of epithelial cells, whereas at embryonic day 18.5, immunostaining was punctate and apically located, suggesting the presence of tight junctions by this age; similar immunostaining was noted at postnatal days 5 and 7. In the adult epididymis, distinct punctate apical staining was observed between adjacent principal cells of all epididymal regions except the proximal initial segment, where occludin was found only in association with narrow cells. These results indicate that in the epididymis, the appearance of occludin at apical sites between adjacent epithelial cells occurs during embryonic development suggesting that tight junctions form earlier than in the testis. While occludin was expressed in a similar pattern between Sertoli cells at all stages of the cycle in the adult testis, its expression in the adult epididymis was cell- and region-specific. Taken together these data suggest that different factors regulate occludin expression in the testis and epididymis.

Aging↗
PubMed1998

Origin and roles of genomic methylation patterns in male germ cells.

The epigenetic modification of DNA by methylation at cytosine residues is initiated in the germ line and is required for normal embryonic development in mammals. Marked differences in genomic methylation between male and female gametes arise during gametogenesis and have been implicated in genomic imprinting. While DNA methylation patterns for different types of gene sequences are known to change during spermatogenesis, the precise role(s) of DNA methylation in the normal development of male germ cells is poorly understood. The expression of the one known active form of DNA methyltransferase is highly regulated during spermatogenesis and may provide insight into mechanisms underlying the establishment of methylation patterns in germ cells.

Animals↗
PubMed1998

Cloning of the cDNA and gene encoding mouse lysosomal sialidase and correction of sialidase deficiency in human sialidosis and mouse SM/J fibroblasts.

Lysosomal sialidase occurs in a multienzyme complex that also contains beta-galactosidase and cathepsin A. We previously cloned the human lysosomal sialidase cDNA and characterized mutations in human sialidosis patients. Here, we report the cloning and expression of the mouse lysosomal sialidase cDNA and gene. The 1.77 kb cDNA encodes an open reading frame of 408 amino acids which shows high homology to the human lysosomal sialidase (80%), the rat cytosolic sialidase (65%) and viral and bacterial sialidases (50-55%). The sialidase gene is approximately 4 kb long and contains six exons. The five introns range in size from 96 to 1200 bp. Northern blot analysis revealed high expression of multiple sialidase transcripts in kidney and epididymis, moderate levels in brain and spinal cord, and low levels in adrenal, heart, liver, lung and spleen. Transient expression of the cDNA clone in sialidase-deficient SM/J mouse fibroblasts and human sialidosis fibroblasts restored normal levels of sialidase activities in both cell types. Immunocytochemically expressed sialidase co-localized with a lysosomal marker, LAMP2, confirming its lysosomal nature. Since sialidase activity requires its association with beta-galactosidase and cathepsin A, the expression of mouse sialidase within human sialidosis cells underlines the structural similarity between mouse and human enzymes and suggests that the mechanism for complex formation and function is highly conserved.

Amino Acid Sequence↗
PubMed1998

Transcription factor GATA-4 is expressed in a sexually dimorphic pattern during mouse gonadal development and is a potent activator of the Müllerian inhibiting substance promoter.

Mammalian gonadal development and sexual differentiation are complex processes that require the coordinated expression of a specific set of genes in a strict spatiotemporal manner. Although some of these genes have been identified, the molecular pathways, including transcription factors, that are critical for the early events of lineage commitment and sexual dimorphism, remain poorly understood. GATA-4, a member of the GATA family of transcription factors, is present in the gonads and may be a regulator of gonadal gene expression. We have analyzed the ontogeny of gonadal GATA-4 expression by immunohistochemistry. GATA-4 protein was detected as early as embryonic day 11.5 in the primitive gonads of both XX and XY mouse embryos. In both sexes, GATA-4 specifically marked the developing somatic cell lineages (Sertoli in testis and granulosa in ovary) but not primordial germ cells. Interestingly, abundant GATA-4 expression was maintained in Sertoli cells throughout embryonic development but was markedly down-regulated shortly after the histological differentiation of the ovary on embryonic day 13.5. This pattern of expression suggested that GATA-4 might be involved in early gonadal development and possibly sexual dimorphism. Consistent with this hypothesis, we found that the Müllerian inhibiting substance promoter which harbors a conserved GATA element is a downstream target for GATA-4. Thus, transcription factor GATA-4 may be a new factor in the cascade of regulators that control gonadal development and sex differentiation in mammals.

Animals↗
PubMed1998

Sex-specific exons control DNA methyltransferase in mammalian germ cells.

The spermatozoon and oocyte genomes bear sex-specific methylation patterns that are established during gametogenesis and are required for the allele-specific expression of imprinted genes in somatic tissues. The mRNA for Dnmt1, the predominant maintenance and de novo DNA (cytosine-5)-methyl transferase in mammals, is present at high levels in postmitotic murine germ cells but undergoes alternative splicing of sex-specific 5' exons, which controls the production and localization of enzyme during specific stages of gametogenesis. An oocyte-specific 5' exon is associated with the production of very large amounts of active Dnmt1 protein, which is truncated at the N terminus and sequestered in the cytoplasm during the later stages of oocyte growth, while a spermatocyte-specific 5' exon interferes with translation and prevents production of Dnmt1 during the prolonged crossing-over stage of male meiosis. During the course of postnatal oogenesis, Dnmt1 is present at high levels in nuclei only in growing dictyate oocytes, a stage during which gynogenetic developmental potential is lost and biparental developmental potential is gained.

Alternative Splicing↗
PubMed1997

beta-Hexosaminidase immunolocalization and alpha- and beta-subunit gene expression in the rat testis and epididymis.

beta-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G(M2) gangliosidoses. beta-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for beta-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the alpha- and beta-subunits of beta-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. beta-hexosaminidase alpha-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, beta-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7-91 postnatal days of age, testis levels of alpha-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, beta-subunit mRNA was expressed at low levels throughout tests development. In isolated male germ cells, beta-hexosaminidase alpha-subunit expression was most abundant in haploid round spermatids, whereas the beta-subunit mRNA was not detected in germ cells. Within the epididymis both alpha- and beta-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that beta-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, beta-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that beta-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of beta-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions.

Animals↗
PubMed1997

Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases.

Tay-Sachs and Sandhoff diseases are autosomal recessive neurodegenerative diseases resulting from the inability to catabolize GM2 ganglioside by beta-hexosaminidase A (Hex A) due to mutations of the alpha subunit (Tay-Sachs disease) or beta subunit (Sandhoff disease) of Hex A. Hex B (beta beta homodimer) is also defective in Sandhoff disease. We previously developed mouse models of both diseases and showed that Hexa-/- (Tay-Sachs) mice remain asymptomatic to at least 1 year of age while Hexb-/- (Sandhoff) mice succumb to a profound neurodegenerative disease by 4-6 months of age. Here we find that neuron death in Hexb-/- mice is associated with apoptosis occurring throughout the CNS, while Hexa-/- mice were minimally involved at the same age. Studies of autopsy samples of brain and spinal cord from human Tay-Sachs and Sandhoff diseases revealed apoptosis in both instances, in keeping with the severe expression of both diseases. We suggest that neuron death is caused by unscheduled apoptosis, implicating accumulated GM2 ganglioside or a derivative in triggering of the apoptotic cascade.

Animals↗
PubMed1996

DNA methyltransferase in normal and Dnmtn/Dnmtn mouse embryos.

The mouse genome experiences a large decrease in net 5-methylcytosine between fertilization and implantation; de novo methylation brings 5-methylcytosine to adult somatic cell levels between implantation and gastrulation. Very little is known of the regulation of demethylation or de novo methylation. Levels of the one known form of DNA methyltransferase are very high in early embryos, but the enzyme is localized to the cytoplasm during most of preimplantation development. We show here that DNA methyltransferase is found exclusively in nuclei of the conceptus after implantation, and that nuclei of proximal decidual cells are free of detectable DNA methyltransferase. High levels of DNA methyltransferase were seen in all tissues, including the developing nervous system, of 9.5- to 12.5-day embryos. The large maternal stores of DNA methyltransferase become limiting prior to embryonic day 9.5, as shown by barely detectable immunostaining in 9.5-day embryos homozygous for a loss-of-function mutation (Dnmtn) in the DNA methyltransferase gene. These mutant embryos failed to develop past the 25-somite stage and showed evidence of developmental delay and some developmental asynchrony. Normal embryonic and extraembryonic tissues contained similar levels of DNA methyltransferase, even though severely reduced methylation levels and a loss of imprinting have previously been observed in extraembryonic tissues. These findings suggest that methylation patterns are not a simple function of the concentration of DNA methyltransferase, and that unidentified factors must be involved in the regulation of de novo methylation during early development of the mouse.

5-Methylcytosine↗
PubMed1996

Differential expression of ornithine decarboxylase, poly(ADP)ribose polymerase, and mitochondrial mRNAs following testosterone administration to hypophysectomized rats.

The mRNAs of the nuclear encoded genes, ornithine decarboxylase (ODCase) and poly(ADP)ribose polymerase (PADPRP), and the mitochondrial encoded genes, cytochrome oxidase I and II (COI and COII) and ATPase 6, are differentially expressed during spermatogenesis (Alcivar et al., 1989: Biol Reprod 41:1133; 1989: Dev Biol 135:263; 1991: Biol Reprod 46:201). In this study, we use Northern blotting to examine the steady state levels of ODCase, PADPRP, COI, COII, and ATPase 6 mRNAs in testes of hypophysectomized male rats following testosterone administration. Four weeks after hypophysectomy, rats received 24 cm subcutaneous implants of testosterone-filled polydimethylsiloxane (PDS) and were killed at 3, 7, 14, 28, and 56 days thereafter. After hypophysectomy, the steady state levels for the PADPRP, COI, COII, and ATPase 6 mRNAs were not significantly different from controls, although hypophysectomy caused a 44% loss of preleptotene spermatocytes and an 88% loss of pachytene spermatocytes, the testicular cell types expressing the highest levels of these mRNAs. In contrast, the levels of the two ODCase mRNAs were greatly decreased after hypophysectomy and mirrored the number of germinal cells present in the testis. After testosterone treatment, ODCase mRNA levels remained low 3 days after treatment and gradually increased at days 14, 28, and 56. No major hybridization signal changes in PADPRP, COI, COII, and ATPase mRNA were observed after testosterone treatment. We conclude that the steady state mRNA levels for the housekeeping ODCase gene respond differently after hypophysectomy and testosterone treatment of male rats than the PADPRP and mitochondrial DNA transcripts.

Adenosine Triphosphatases↗
PubMed1996

Dramatically different phenotypes in mouse models of human Tay-Sachs and Sandhoff diseases.

We have generated mouse models of human Tay-Sachs and Sandhoff diseases by targeted disruption of the Hexa (alpha subunit) or Hexb (beta subunit) genes, respectively, encoding lysosomal beta-hexosaminidase A (structure, alpha) and B (structure, beta beta). Both mutant mice accumulate GM2 ganglioside in brain, much more so in Hexb -/- mice, and the latter also accumulate glycolipid GA2. Hexa -/- mice suffer no obvious behavioral or neurological deficit, while Hexb -/- mice develop a fatal neurodegenerative disease, with spasticity, muscle weakness, rigidity, tremor and ataxia. The Hexb -/- but not the Hexa -/- mice have massive depletion of spinal cord axons as an apparent consequence of neuronal storage of GM2. We propose that Hexa -/- mice escape disease through partial catabolism of accumulated GM2 via GA2 (asialo-GM2) through the combined action of sialidase and beta-hexosaminidase B.

Animals↗