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D J Dix

Publications and source records attributed to D J Dix.

At least 19 recordsLinked to original sources
PubMed2001

Development of a 950-gene DNA array for examining gene expression patterns in mouse testis.

BACKGROUND: Over the past five years, interest in and use of DNA array technology has increased dramatically, and there has been a surge in demand for different types of arrays. Although manufacturers offer a number of pre-made arrays, these are generally of utilitarian design and often cannot accommodate the specific requirements of focused research, such as a particular set of genes from a particular tissue. We found that suppliers did not provide an array to suit our particular interest in testicular toxicology, and therefore elected to design and produce our own. RESULTS: We describe the procedures used by members of the US Environmental Protection Agency MicroArray Consortium (EPAMAC) to produce a mouse testis expression array on both filter and glass-slide formats. The approaches used in the selection and assembly of a pertinent, nonredundant list of testis-expressed genes are detailed. Hybridization of the filter arrays with normal and bromochloroacetic acid-treated mouse testicular RNAs demonstrated that all the selected genes on the array were expressed in mouse testes. CONCLUSION: We have assembled two lists of mouse (950) and human (960) genes expressed in the mouse and/or human adult testis, essentially all of which are available as sequence-verified clones from public sources. Of these, 764 are homologous and will therefore enable close comparison of gene expression between murine models and human clinical testicular samples.

Adult↗
PubMed2001

Recovering filter-based microarray data for pathways analysis using a multipoint alignment strategy.

The use of commercial microarrays is rapidly becoming the method of choice for profiling gene expression and assessing various disease states. Research Genetics has provided a series of biological and software tools to the research community for these analyses. The fidelity of data analysis using these tools is dependent on a series of well-defined reference control points in the array. During the course of our investigations, it became apparent that in some instances the reference control points that are required for analysis became lost in background noise. This effectively halted the analysis and the recovery of any information contained within that experiment. To recover this data and to increase analytical veracity, the simple strategy of superimposing a template of reference control points onto the experimental array was developed. The utility of this tool is established in this communication.

Filtration↗
PubMed2000

DNA arrays: technology, options and toxicological applications.

The human genome contains an estimated 3 billion bases of DNA making up some 100000 genes, and the variation within this genome accounts for human diversity and, in many cases, disease. Defining and understanding the expression profile of given genotypes is essential to understanding adverse effects from acute or chronic exposure to environmental toxicants or other stimuli. DNA array technology could help researchers understand how organisms function in response to exposure by elucidating the molecular mechanisms that underlie them. DNA arrays have been developed and refined over the past 5 years and matured into a relatively accessible and affordable technology. They vary in design from membrane-based filters with a few hundred cDNAs, to glass-based 'chips' with tens of thousands of genetic elements. Mammalian DNA arrays will soon allow expression analysis on a genome-wide scale, similar to that already accomplished in some lower organisms (e.g. S. cerevisiae, E. coli). These whole-genome arrays will be powerful tools for identifying and characterizing toxicants in environmental and pharmaceutical science. This review discusses the technology behind the production of DNA arrays, the options available to those interested in applying them to their own research, and the possible toxicological applications of this exciting new technology.

Animals↗
PubMed1999

Completion of meiosis is not always required for acrosome formation in HSP70-2 null mice.

Hsp70-2 is a unique member of the mouse 70-kDa heat shock protein family that is synthesized during meiosis in spermatogenic cells. Germ cells in male mice homozygous for a targeted mutation in the Hsp70-2 gene (Hsp70-2(-/-)) arrest in development and undergo apoptosis at the end of the pachytene spermatocyte stage of meiotic prophase. However, cells with a putative acrosome were present occasionally in histological sections of the testes of juvenile and adult Hsp70-2(-/-) mice. This study verified that acrosomes were present and investigated the relationship between acrosome formation and the process of meiosis. Histochemistry with the periodic acid-Schiff procedure and immunostaining with monoclonal antibody MN7 verified that acrosomes were present in Hsp70-2(-/-) mice, and electron microscopy showed that some of these cells had condensing nuclei characteristic of step 8-9 spermatids. The frequency of acrosome-containing cells in Hsp70-2(-/-) mice was less than 0.01% of that in wild-type mice. Propidium iodide staining and cytophotometry indicated that the average DNA content of nuclei in MN7-positive cells in Hsp70-2(-/-) mice was usually about twice, or occasionally the same as, that of nuclei in round spermatids of wild-type mice. Meiotic metaphase I and II chromosome spreads were observed in spermatogenic cells from Hsp70-2(-/-) mice but at a much lower frequency than in wild-type mice. These results indicate that not all pachytene spermatocytes in Hsp70-2(-/-) mice arrest in meiosis, but they may divide once or sometimes twice and begin acrosome formation and nuclear condensation. This demonstrates that some aspects of spermatid development can occur without the completion of meiosis in mice, as has been reported recently for Drosophila.

Acrosome↗
PubMed1999

Application of DNA arrays to toxicology.

DNA array technology makes it possible to rapidly genotype individuals or quantify the expression of thousands of genes on a single filter or glass slide, and holds enormous potential in toxicologic applications. This potential led to a U.S. Environmental Protection Agency-sponsored workshop titled "Application of Microarrays to Toxicology" on 7-8 January 1999 in Research Triangle Park, North Carolina. In addition to providing state-of-the-art information on the application of DNA or gene microarrays, the workshop catalyzed the formation of several collaborations, committees, and user's groups throughout the Research Triangle Park area and beyond. Potential application of microarrays to toxicologic research and risk assessment include genome-wide expression analyses to identify gene-expression networks and toxicant-specific signatures that can be used to define mode of action, for exposure assessment, and for environmental monitoring. Arrays may also prove useful for monitoring genetic variability and its relationship to toxicant susceptibility in human populations.

Environmental Exposure↗
PubMed1999

Hsp70 expression and function during embryogenesis.

This review focuses on the expression and function of 70-kDa heat shock proteins (Hsp70s) during mammalian embryogenesis, though many features of embryogenesis and the developmental expression of Hsp70s are conserved between mammals and other vertebrates. A variety of Hsp70s are expressed from the point of zygotic gene activation in cleavage-stage embryos, through blastulation, implantation, gastrulation, neurulation, organogenesis, and on throughout fetal maturation. The regulation and patterns of hsp70 gene expression and the known and putative Hsp70 protein functions vary from constitutive and metabolic housekeeping to stress-inducible and embryo-protective roles. Understanding the genetic regulation and molecular function of Hsp70s has been pursued by developmental biologists interested in the control of gene expression in early embryos as well as reproductive toxicologists and teratologists interested in how Hsp70s protect embryos from the adverse effects of environmental exposures. These efforts have also been joined by those interested in the chaperone functions of Hsp70s, and this confluence of effort has yielded many advances in our understanding of Hsp70s during critical phases of embryonic development and cellular differentiation.

Animals↗
PubMed1998

Inhibition of hsp70-1 and hsp70-3 expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic.

Mouse 70-kDa heat shock proteins Hsp70-1 and Hsp70-3 (Hsp70-1/3) are stress-inducible protein chaperones thought to protect embryonic cells and tissues from the effects of a wide range of environmental exposures. Hsp70-1/3 are expressed constitutively, and at times are stress-inducible during various stages of preimplantation embryogenesis. In order to elucidate the functions of constitutive and stress-inducible Hsp70 expression in mouse preimplantation embryos, the consequences of inhibiting expression with antisense oligonucleotides complementary to the mRNAs of hsp70-1 and hsp70-3 (A070-1/3) were evaluated. Transfection of preimplantation embryos (four-cell stage) with 2.5 microM A070-1/3 had no effect on in vitro blastocoel formation. However, transfection with 5 or 10 microM A070-1/3 reduced in vitro blastocyst development to 30% and 0%, respectively (approximately 90% control embryos developed to blastocyst). Thus constitutive expression of Hsp70-1/3 appears significant to preimplantation embryogenesis. Limiting expression of Hsp70-1/3 with 5 microM A070-1/3 also heightened embryo sensitivity to arsenic, resulting in less than 5% in vitro development to blastocyst in the presence of the subtoxic dose of 0.4 microM sodium arsenite. Whether the combined effect of A070-1/3 and arsenic is due to blocking inducible expression of the Hsp70s, or due to further reducing the amount of constitutively expressed Hsp70s available to the embryo is not known at this time. However, these results clearly indicate that some minimal amount of Hsp70-1 and/or Hsp70-3 is required for preimplantation embryogenesis, and that increasing the demand for Hsp70s by arsenic exposure heightens this requirement.

Animals↗
PubMed1998

Protective mechanisms in germ cells: stress proteins in spermatogenesis.

A wide range of environmental exposures trigger protective mechanisms in reproductive tissues which are mediated by stress or heat shock proteins (HSPs). These stress proteins maintain normal cellular functions such as protein synthesis, as well as assist in resisting and recovering from toxicant-induced cellular damage. Over the past decade a number of laboratories have examined the expression and potential functions of these stress proteins during gametogenesis (reviewed in Dix, 1997a) and in reproductive toxicology (Dix, 1997b). This paper reviews the expression of HSPs in testes, presents a detailed analysis of the function of Hsp70-2 during the meiotic phase of spermatogenesis, and concludes with a discussion of stress-inducible HSPs and putative protective mechanisms.

Animals↗
PubMed1997

Morphological analysis of germ cell apoptosis during postnatal testis development in normal and Hsp 70-2 knockout mice.

The present study examined the occurrence of apoptotic cell death in the testis of wild-type mice from postnatal days 3 to 26 and in juvenile Hsp 70-2 knockout mice. Adult Hsp 70-2 knockout males are infertile and lack spermatids and spermatozoa (Dix et al. [1996a] Proc. Natl. Acad. Sci. U.S.A. 93:3264-3268). To identify the cell types undergoing apoptosis, we also examined the relationship between the occurrence of apoptotic cell death and the expression pattern of the Hsp 70-2 gene product (heat-shock protein 70-2 [HSP70-2]; marker for spermatocytes and spermatids), germ cell nuclear antigen 1 (GCNA1;marker for spermatogonia and spermatocytes), and vimentin (marker for Sertoli cells). This study shows that during postnatal development of the wildtype mouse testis (1) the percentage of apoptotic cell death detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method is higher in mice from days 8 to 22 than in younger or older mice, (2) the majority of apoptotic cells are spermatogonia and less frequently are spermatocytes, and (3) the degenerative cell death of spermatogonia and primary spermatocytes involves apoptosis with fragmentation of DNA. The analysis of apoptotic cell death in the testes of juvenile Hsp 70-2 knockout mice showed an additional increased level of apoptosis at day 17, during the first wave of spermatogenesis, in pachytene spermatocytes.

Animals↗
PubMed1997

HSP70-2 is required for CDC2 kinase activity in meiosis I of mouse spermatocytes.

Cyclin B-dependent CDC2 kinase activity has a key role in triggering the G2/M-phase transition during the mitotic and meiotic cell cycles. The Hsp70-2 gene is expressed only in spermatogenic cells at a significant level. In Hsp70-2 gene knock-out (Hsp70-2(-/-)) mice, primary spermatocytes fail to complete meiosis I, suggesting a link between HSP70-2 heat-shock protein and CDC2 kinase activity during this phase of spermatogenesis. Members of the HSP70 protein family are molecular chaperones that mediate protein de novo folding, translocation and multimer assembly. This study used immunoprecipitation-coupled western blot and in vitro reconstitution experiments to show that HSP70-2 interacts with CDC2 in the mouse testis, appears to be a molecular chaperone for CDC2, and is required for CDC2/cyclin B1 complex formation. Previous studies reported that most CDC2 kinase activity in the mouse testis is present in pachytene spermatocytes. Although CDC2 kinase activity for histone H1 was present in the testis of wild-type mice, it was nearly absent from the testis of Hsp70-2(-/-) mice, probably due to defective CDC2/cyclin B1 complex formation. Furthermore, addition of HSP70-2 to freshly prepared extracts of testis from Hsp70-2(-/-) mice not only restored CDC2/cyclin B1 complex formation but also reconstituted CDC2 kinase activity in vitro. It appears that one cause of failure to complete meiosis I during spermatogenesis in Hsp70-2(-/-) mice is disruption of CDC2/cyclin B1 assembly in pachytene spermatocytes, thereby preventing development of the CDC2 kinase activity required to trigger G2/M-phase transition. These studies provide novel in vivo evidence for a link between an HSP70 molecular chaperone and CDC2 kinase activity essential for the meiotic cell cycle in spermatogenesis.

Animals↗
PubMed1997

HSP70-2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes.

Spermatogenic cells synthesize a unique 70-kDa heat shock protein (HSP70-2) during prophase of meiosis I, and targeted disruption of the Hsp70-2 gene has shown that this protein is required for spermatogenic cell differentiation in adult mice. HSP70-2 is associated with synaptonemal complexes formed between paired homologous chromosomes during meiotic prophase. The present study focuses on the nearly synchronous first wave of spermatogenesis in 12- to 28-day old juvenile mice to determine more precisely when HSP70-2 is required and what meiotic processes are affected by its absence. Spermatogenesis in homozygous mutant mice (Hsp70-2[-/-]) proceeded normally until day 15 when increasing numbers of pachytene spermatocytes became apoptotic and differentiation of cells beyond the pachytene stage began to falter. Synaptonemal complexes assembled in Hsp70-2(-/-) mice and spermatocytes developed through the final pachytene substage. However, synaptonemal complexes failed to desynapse and normal diplotene spermatocytes were not observed. Metaphase spermatocytes were not seen in tissue sections from testes of Hsp70-2(-/-) mice, and expression of mRNAs and antigens characteristic of late pachytene spermatocytes (e.g., cyclin A1) and development of spermatids did not occur. Thus, HSP70-2 is required for synaptonemal complex desynapsis, and its absence severely impairs the transition of spermatogenic cells through the late meiotic stages and results in apoptosis beginning with the first wave of germ cell development in juvenile mice.

Animals↗
PubMed1997

Hsp70 expression and function during gametogenesis.

The dramatic transformations in nuclear content and cellular organization that occur during gametogenesis require unique regulation and execution of the mitotic and meiotic cell cycle, apoptotic cell death, DNA recombination and repair, and cellular differentiation. These processes are accompanied by the constitutive and developmentally regulated expression of a number of hsp70 genes encoding 70 kDa heat shock proteins (Hsp70), including several hsp70s whose expression is unique to male germ cells. Examining the expression and function of Hsp70s in germ cells has provided significant insights into mechanisms of hsp70 gene regulation and Hsp70 protein function, as well as the developmental processes of gametogenesis.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

Developmentally regulated expression of Hsp70-2 and a Hsp70-2/lacZ transgene during spermatogenesis.

Germ cells synthesize large amounts of HSP70-2 protein during the meiotic phase of spermatogenesis. This developmentally regulated expression of HSP70-2 contrasts with the constitutive or inducible expression of other 70-kDa heat shock proteins (HSP70s). To better understand the genetic regulation of Hsp70-2, we used mRNA primer- extension, reverse transcriptase PCR (RT-PCR), and cDNA sequencing to determine that transcription began as far as 353 bp upstream of the start codon. We also identified a previously unrecognized 239-bp intron which is spliced out of the pre-mRNA transcript to leave a 114 nt 5'-untranslated region. Transgenic mice were then produced to delimit the upstream regulatory region required for developmental expression of Hsp70-2 during spermatogenesis. Results with multiple lines of transgenic mice containing promoter-reporter transgenes with varying lengths of Hsp7-2 sequence indicate that promoter sequences up to 640 bp upstream of the start codon and 287 bp upstream of the transcription start site are required for Hsp70-2/lacZ expression in spermatocytes. Histochemical detection of transgene beta- galactosidase activity was coincident with immunohistochemical detection of HSP70-2 protein, both in the first wave of spermatogenesis in juvenile mice and in ongoing spermatogenesis of adult mice. The distribution of Hsp7O-2 and Hsp7O-2/lacZ mRNAs was determined by Northern blot, in situ hybridization, and RT-PCR, and it was found that upregulation of expression of both Hsp7O-2 and Hsp7O-2/lacZ was specific to the meiotic phase of spermatogenesis.

Animals↗
PubMed1996

HSP70-2 is part of the synaptonemal complex in mouse and hamster spermatocytes.

Mouse spermatogenic cells are known to express HSP70-2, a member of the HSP70 family of heat-shock proteins. The purpose of the present study was to characterize further the expression and localization of HSP70-2 in meiotic cells of mice and hamsters. After separating mouse spermatogenic cells into cytoplasmic and nuclear fractions, proteins were separated by two-dimensional gel electrophoresis and detected with HSP-specific antibodies. Of several HSP70 proteins identified in the cytoplasm, only HSC70 and HSP70-2 were also detected in the nucleus. Immunocytological analyses of spermatocyte prophase cells revealed that HSP70-2 was associated with the synaptonemal complex. Surface-spread synaptonemal complexes at pachytene and diplotene stages labeled distinctly with the antiserum to HSP70-2. Synaptonemal complexes from fetal mouse oocytes failed to show any evidence of HSP70-2. Reverse-transcriptase-polymerase chain reaction (RT-PCR) analyses of gene expression confirmed this sex specificity; Hsp70-2 mRNA was detected in mouse testes, but not ovaries. These findings are suggestive of a previously unsuspected sexual dimorphism in structure and/or function of the synaptonemal complex.

Animals↗
PubMed1993

The influence of the base-paired flanking region on structure and function of the ferritin mRNA iron regulatory element.

Ferritin and transferrin receptors are co-ordinately regulated by the same RNA-protein interaction: the conserved iron regulatory element (IRE) in mRNA and the IRE-binding protein (IRE-BP/IRP/FRP/P-90). The 28 nucleotide IRE in ferritin mRNA is a single copy, with base-paired flanking regions (FL), located near the 5' cap. In the transferrin receptor mRNA, the IRE is located in the 3' untranslated region, as five variable copies and lacking predicted base-paired flanking regions; an alternate predicted structure without IREs has similar stability. When iron is scarce, ferritin mRNA does not form polyribosomes whereas the transferrin receptor mRNA is translated; when iron is abundant, ferritin mRNA forms polyribosomes and the transferrin receptor mRNA is degraded. To investigate structures which contribute to differences in the regulation of the two mRNAs, the effect of mutation of the ferritin FL was studied. Changes in structure (changes in reactivity with RNase V1 and RNase S1. Fe-bleomycin) and changes in function (translation in rabbit reticulocyte extracts) were compared for mutant and wild-type FL sequences in ferritin mRNA. The disruption of a triplet of base-pairs in the FL had diminished regulation; a second mutation to restore the triplet base-pairs conferred wild-type translational regulation. Conformation of the mutant RNA-IRE-BP complex was also different. We show that the triplet of base-pairs is conserved; the triplet is also the location of IRE-BP-dependent conformational changes in the FL structure previously observed. Increasing FL base-pairs had no effect on function. Structural changes associated with altered function included bleomycin sites in the IRE, suggesting an alternate conformation of the hairpin, and different base-stacking (V1 sensitivity) in the FL. The function of the FL, which is altered by mutation of phylogenetically conserved triplet base-pairs, may be enhancement of formation of a particular IRE stem-loop-protein interaction.

Animals↗
PubMed1992

The iron regulatory region of ferritin mRNA is also a positive control element for iron-independent translation.

The iron regulatory element (IRE) in the 5'-untranslated region of ferritin mRNA interacts with a specific regulator protein (P-90, IRE-BP, or FRP) to block translation. High cellular iron changes the IRE/P-90 interaction to relax the translational block and allow polyribosome formation. We now show that the IRE and base-paired flanking regions also enhance translation in the absence of P-90, explaining the high translational efficiency of deregulated ferritin mRNA observed previously. The effect of the IRE on translational efficiency was examined by comparing four sets of mRNAs: (1) +/- IRE in animal (frog) ferritin, regulated translationally by iron in vivo; (2) +/- animal IRE fused with plant (soybean) ferritin, regulated transcriptionally by iron in vivo; (3) repositioned IRE in animal ferritin; (4) mutated IRE in animal ferritin with G16A substitution, which decreases P-90 binding (negative control). The IRE region increased translational efficiency of both the animal ferritin and the heterologous IRE/soybean ferritin fusion mRNAs; the effect was observed in cell-free translation systems from either plants (wheat germ) or animals (rabbit reticulocyte). Repositioning the IRE further from the 5' cap eliminated positive control of translation. The single base mutation had no effect, indicating that positive and negative translational control involves different sections of the IRE region. Thus, the IRE region in ferritin mRNA encodes both positive translational control and, when combined with the regulator protein P-90, negative translational control.

Animals↗