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D J Wolgemuth

Publications and source records attributed to D J Wolgemuth.

At least 55 records · Page 3Linked to original sources
PubMed1996

Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues.

The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

Animals↗
PubMed1996

Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain.

We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

Animals↗
PubMed1996

A distinct cyclin A is expressed in germ cells in the mouse.

In this paper, the existence of two A-type cyclins in the mouse is demonstrated. In the adult mouse, the expression of cyclin A1, which has greatest sequence identity with Xenopus cyclin A1, is restricted to germ cells. In contrast cyclin A2, which has greatest sequence identity with human cyclin A and Xenopus cyclin A2, is expressed in all tissues analysed. In order to explore the function of cyclin A1 in germ cells, its expression during the meiotic cell cycle and its associated kinase subunits have been characterised in the testis. The levels of cyclin A1 mRNA rise dramatically in late pachytene spermatocytes and become undetectable soon after completion of the meiotic divisions; thus its expression is cell cycle regulated. In lysates of germ cells from adult testes, cyclin A1 is present in p13suc1 precipitates, and cyclin A1 immunoprecipitates possess histone H1 kinase activity. Three kinase partners of cyclin A1 were identified: p34cdc2, a polypeptide of 39 x 10(3) M(r) that is related to p33cdk2 and, in lesser quantities, p33cdk2. Cyclin A1 was also detected in oocytes; in metaphase I and metaphase II oocytes, a proportion of the cyclin A1 colocalises with the spindle, possibly suggestive of a functional interaction. These data indicate that mammalian germ cells contain cyclin A1-dependent kinases that either act as a substitute for, or in addition to, the cyclin A2-dependent kinases characterised in somatic tissues.

Amino Acid Sequence↗
PubMed1995

Compound mutants for the paralogous hoxa-4, hoxb-4, and hoxd-4 genes show more complete homeotic transformations and a dose-dependent increase in the number of vertebrae transformed.

The Hox gene products are transcription factors involved in specifying regional identity along the anteroposterior body axis. In the mouse, several single mutants for Hox genes show variably penetrant, partial homeotic transformations of vertebrae at their anterior limits of expression, suggesting that compound Hox mutants might show more complete transformations with greater penetrance than the single Hox mutants. Compound mutants for the paralogous group 3 genes, hoxa-3 and hoxd-3, show deletion of a cervical vertebrae, which is not readily interpretable in terms of an alteration in regional identity. Here, we report the skeletal phenotypes of compound mutants in the group 4 Hox genes, hoxa-4, hoxb-4, and hoxd-4. Mice mutant for each of these genes were intercrossed to generate the three possible double mutant combinations and the triple mutant. In contrast to the hoxa-3, hoxd-3 double mutants, group 4 Hox compound mutants displayed clear alterations in regional identity, including a nearly complete transformation of the second cervical vertebrae toward the morphology of the first cervical vertebra in one double mutant combination. In comparing the types of homeotic transformations observed, different double mutant combinations showed different degrees of synergism. These results suggest a certain degree of functional redundancy among paralogous genes in specifying regional identity. Furthermore, there was a remarkable dose-dependent increase in the number of vertebrae transformed to a first cervical vertebra identity, including the second through the fifth cervical vertebrae in the triple mutant. Thus, these genes are required in a larger anteroposterior domain than is revealed by the single mutant phenotypes alone, such that multiple mutations in these genes result in transformations of vertebrae that are not at their anterior limit of expression.

Animals↗
PubMed1995

Cdk family genes are expressed not only in dividing but also in terminally differentiated mouse germ cells, suggesting their possible function during both cell division and differentiation.

The roles of the cyclin dependent kinase (Cdk) family in murine germ cell development have been examined by studying the expression of five Cdk family genes (Cdc2, Cdk2, Cdk4, Pctaire-1, and Pctaire-3) in mouse reproductive organs. Northern blot and in situ hybridization analyses revealed distinctive expression patterns of these genes with striking cellular, lineage, and developmental stage specificity. We observed Cdk expression in cell types with proliferative activity: Cdc2 and Cdk2 expression in premeiotic spermatocytes in the testis, and Cdc2, Cdk2, and Cdk4 expression in granulosa cells of ovarian follicles. Cdc2 transcripts were most abundant in late pachytene to diplotene spermatocytes, soon to undergo meiosis. Surprisingly, we also observed expression of Cdk family genes in non-proliferating cell types. All five Cdk family genes examined were expressed in Sertoli cells of the adult testis, which are no longer mitotically active. With regard to Pctaire-1 and Pctaire-3, the highest levels of expression were observed in postmeiotic spermatids. Immunoblot analysis also revealed the presence of high levels of Pctaire-1 in postmeiotic germ cells. These results suggest that Cdk family kinases may exhibit various functions in germinal and somatic cells during gametogenesis, not only in the cell cycle but also in other regulatory processes, including differentiation.

Animals↗
PubMed1995

Distinct patterns of expression of the D-type cyclins during testicular development in the mouse.

The three D-type cyclins have been shown to be differentially expressed in a number of isolated cell types and cell lines, suggesting distinct roles in cell cycle regulation in particular cell lineages. The testis provides unique opportunities to study genes involved in cell cycle regulation, since it contains cells in both mitosis and meiosis as well as differentiated cells with little proliferation activity. Major transcripts of 4.2 kb, 6.8 kb, and 2.3 kb were detected in the adult mouse testis by Northern hybridization analyses for cyclin D1, cyclin D2, and cyclin D3, respectively. Additional transcripts of 1.8 and 2.7 kb were detected by Northern hybridization for cyclin D3 in the testis, but not in other tissues, and these transcripts were limited to germ cells. Northern and in situ hybridization analyses of normal and germ cell-deficient testes showed the surprising result that cyclin D1 was expressed in a pattern consistent with expression in the non-dividing Sertoli cells. Cyclin D2 levels appeared slightly enriched in germ cell-deficient testes as compared to intact testis, but in situ hybridization analysis did not reveal any distinct cellular localization. Also surprising was the observation that cyclin D3 expression was highest in the non-dividing, haploid, round spermatids. The possible roles of these cyclins in the events of spermatogenesis are discussed.

Animals↗
PubMed1995

The distinct and developmentally regulated patterns of expression of members of the mouse Cdc25 gene family suggest differential functions during gametogenesis.

Cdc25 genes encode protein threonine/tyrosine phosphatases that activate cyclin-dependent kinases. A cDNA encoding the murine homologue of the human Cdc25C gene was isolated in studies designed to identify those genes involved in regulating the mitotic and meiotic cell cycles of developing mouse germ cells. Northern blot and in situ hybridization along with immunoblot analyses were performed to determine the patterns of expression of Cdc25C compared to those of a second Cdc25 family member, Cdc25B. Murine Cdc25C transcripts of 2.1 kb were detected in midgestation embryos and in several adult tissues, including testis and ovary. The highest levels of Cdc25C transcripts were detected in the testis, with low levels of the 2.1-kb transcript and abundant levels of a 1.9-kb transcript that was not detected in the other tissues examined. In the testis, Cdc25C expression was localized in germ cells, specifically in late pachytene-diplotene spermatocytes and round spermatids, whereas Cdc25B expression was most readily detected in the somatic cells. In the ovary, Cdc25C expression was apparent in cumulus granulosa cells, whereas the expression of Cdc25B was detected in both growing oocytes and somatic cells, including the granulosa cells. These results not only indicate that the expression of the murine Cdc25 genes is lineage- and developmental stage-specific in the mouse testis and ovary but also suggest that the Cdc25 genes may have different functions in the germinal and somatic compartments of the ovary and the testis, only some of which are involved in cellular proliferation.

Animals↗
PubMed1995

Genetic control of mitosis, meiosis and cellular differentiation during mammalian spermatogenesis.

Gametogenesis in both the male and female mammal represents a specialized and highly regulated series of cell cycle events, involving both mitosis and meiosis as well as subsequent differentiation. Recent advances in our understanding of the genetic control of the eukaryotic cell cycle have underscored the evolutionarily-conserved nature of these regulatory processes. However, most of the data have been obtained from yeast model systems and mammalian cell lines. Furthermore, most of the observations focus on regulation of mitotic cell cycles. In the present paper: (i) aspects of gametogenesis in mammals that represent unique cell-cycle control points are highlighted; (ii) current knowledge on the regulation of the germ cell cycle, in the context of what is known in yeast and other model eukaryotic systems, is summarized; and (iii) strategies that can be used to identify additional cell cycle regulating genes are outlined.

Animals↗
PubMed1995

The segment-specific pattern of A-raf expression in the mouse epididymis is regulated by testicular factors.

The protooncogene homolog A-raf is expressed at high levels in a variety of steroid-responsive tissues and exhibits a highly regionalized pattern of expression in morphologically and functionally distinct segments of the mouse epididymis: the highest levels occur in the proximal caput, lower levels occur in the initial segment, and intermediate levels are found in the distal caput. Castration abolished detectable A-raf expression throughout the epididymis. The administration of exogenous androgens restored expression to precastrate levels, indicating that circulating, rather than intraluminal, levels of androgens are sufficient to maintain expression. Although overall levels of A-raf were maintained in androgen-replaced animals, the segment-specific pattern of expression was altered. Elevated levels of A-raf were expressed in the initial segment relative to those in controls. Coincident with this, the morphology of the epithelial cells in the initial segment of androgen-replaced castrates was altered to more closely resemble the morphology of proximal caput epithelial cells in controls. Similar increases in A-raf expression and changes in epithelial cell morphology relative to those in controls were observed in the initial segments of the castrate-side epididymis of hemicastrate mice, in epididymis from a germ cell-deficient strain of mutant mice, and in epididymis from intact mice supplemented with exogenous testosterone. These studies indicated that testicular products, such as spermatozoa, or substances that require germ cells for their production in the testis modulate the androgen-dependent expression of A-raf in the initial segment of the caput epididymis.

Androgen-Binding Protein↗
PubMed1995

Models and molecular approaches to assessing the effects of the microgravity environment on vertebrate development.

The extent to which gravity, and especially the lack thereof, can affect normal development in higher organisms is poorly understood. Underlying this question is the assumption that normal development depends on the embryo's ability to maintain a programmed temporal and spatial coordination of morphogenetic events. There are several reports documenting the apparently normal development of several vertebrate species, including mammals, under conditions of exposure to space flight during various periods of the development process. Evidence to the contrary also exists and it is therefore likely that some alterations in morphology do occur in a microgravity environment. Although subsequent development may appear overtly normal, more subtle abnormalities result. In all studies, the evaluation is restricted by the few numbers of specimens that can be examined and the relatively insensitive techniques for assessing potentially subtle effects. In the present discussion, we summarize some observations of mammalian development made in microgravity and consider which stages might be expected to be differentially sensitive to altered gravity conditions. While we emphasize mammalian development, we discuss the suitability of another model system for examining such effects in a cross-species context. Furthermore, we consider recent developments in our understanding of the molecular genetic program regulating embryogenesis that could serve as markers for assessing perturbations of development.

Animals↗
PubMed1994

Homeotic transformation of cervical vertebrae in Hoxa-4 mutant mice.

Hoxa-4 (previously known as Hox-1.4) is a mouse homeobox-containing gene that is expressed in the presumptive hindbrain and spinal cord, prevertebrae, and other tissues during embryogenesis. To understand the role of Hoxa-4 during development, we generated Hoxa-4 mutant mice. Homozygous mutants were viable and fertile. Analysis of neonatal skeletons revealed the development of ribs on the seventh cervical vertebra at variable penetrance and expressivity. A low frequency of alterations in sternal morphogenesis was also observed. In addition, we analyzed the skeletons of transgenic mice that overexpress Hoxa-4 and found that the formation of the small rib anlagen that often develop on the seventh cervical vertebra was suppressed. Analysis of adult homozygous mutant skeletons revealed that the dorsal process normally associated with the second cervical vertebra was also found on the third cervical vertebra. These results demonstrate that Hoxa-4 plays a role in conferring positional information along the anteroposterior axis to specify the identity of the third and the seventh cervical vertebrae.

Animals↗
PubMed1994

Second locus for Hirschsprung disease/Waardenburg syndrome in a large Mennonite kindred.

We have studied a large Mennonite kindred in which 20 members were affected with Hirschsprung disease (HSCR), 5 of whom had one or more manifestations of Waardenburg syndrome (WS) type II (WS2). Eleven additional relatives had signs of WS2 without HSCR. Since HSCR and WS2 each represent perturbations of neural crest migration/differentiation, this large pedigree with apparent cosegregation of HSCR and WS2 offered an opportunity to search for linkage between these loci, candidate genes, and random DNA markers, particularly in view of recent discoveries of genes for Waardenburg syndrome type I (WS1) and Hirschsprung disease (c-ret). We have examined the following possible linked markers in 69 relatives in this family: the c-ret gene (HSCR); the human PAX3 gene (HuP2) on chromosome 2q (WS1) and placental alkaline phosphatase (ALPP) on chromosome 2q (linked to WS1); argininosuccinate synthetase (ASS) on chromosome 9q, close to ABO blood groups which have shown weak linkage to WS; and the beta 1 GABA receptor gene (GABARB1) on chromosome 4q13-11, close to c-kit, deletions of which cause piebaldism. Linkage between any of these loci and HSCR/WS in this kindred was excluded, demonstrating that there is at least one further locus for HSCR other than c-ret.

Christianity↗
PubMed1994

Sequence and expression of a cDNA encoding the mouse homologue of the rat ribosomal protein L28.

A cDNA encoding the mouse homologue of the rat ribosomal protein L28 was isolated from an adult mouse total testis cDNA library. The L28 cDNA contained a single long open reading frame and exhibited a high degree of conservation to rat L28 at both the nucleotide and amino-acid levels. Northern blot hybridization analysis detected a single transcript in embryo, placenta and adult tissues.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of the developmentally regulated pattern of expression in the testis of a mouse gene exhibiting similarity to the family of phosphodiesterases.

A cDNA for a rat brain phosphodiesterase (PDE) was used to screen a mouse testis library to identify the murine PDEs which are expressed in this tissue. A clone of 981 bp, p4-6, was isolated and shown to exhibit limited identity at the amino acid level to the rat brain PDE (20%). The putative protein encoded by clone p4-6 also contains multiple potential modification sites, for phosphorylation, myristylation, and glycosylation, many of which are located at positions similar to those found for rat brain PDE. The gene identified by p4-6 yields 3 transcripts, an abundant 1.9 kb transcript, and less abundant transcripts of 3.8 and 6.7 kb. Of the nine tissues examined in this study, the expression of the corresponding gene was limited to the adult mouse testis. Furthermore, the expression in the testis was most abundant in the germ cell lineage, although low levels were detected in somatic cells of the testis as well. Analysis of RNA from testes at different stages of development suggested that the p4-6 gene is most abundantly expressed in germ cells that have completed the meiotic divisions.

Amino Acid Sequence↗
PubMed1994

Developmentally regulated expression during gametogenesis of the murine gene meg1 suggests a role in meiosis.

Previous studies have shown that in adult male mice, expression of the meg1 gene is restricted to meiotic and early postmeiotic testicular germ cells. We have now analyzed the expression of meg1 during postnatal testicular development and the comparable meiotic stages in the female. The 0.75 kb transcript for meg1 begins to accumulate in testes at d8-9 of postnatal (pn) development, coincident with the entry of germ cells into meiosis, and is expressed most abundantly at pn d14 and subsequent stages, when the spermatocytes have entered pachytene. In situ hybridization analysis shows that meg1 is expressed at very low levels in leptotene cells and increases as the cells progress through zygotene and pachytene stages. In the embryonic ovary, meg1 is not detected until after day 15 of gestation when the cells have entered the pachytene stage of meiosis I. In situ hybridization analysis suggests that meg1 transcripts are expressed at higher levels in degenerating rather than in healthy pachytene stage oocytes; meg1 is not expressed in any cells of the adult ovary, regardless of the stage of follicular development. These results suggest that meg1 is indeed a meiosis-associated gene in both male and female germ cells through the pachytene stage of meiosis I and appears to exhibit sex-specific differences in its expression thereafter.

Animals↗
PubMed1994

Regulation of M-phase promoting factor activity during development of mouse male germ cells.

While the role of M-phase promoting factor (MPF) in controlling meiosis in oocytes has been well documented, very little is known about its function in male germ cells. Previous studies have localized transcripts for cyclin B1, the regulatory subunit of MPF, in male germ cells, with highest levels in postmeiotic, early round spermatids and much reduced levels in the meiotically dividing pachytene spermatocytes. The present study describes the localization of the regulatory and the catalytic subunits of MPF, CycB1 and Cdc2, respectively, to specific cell types within the testis. Immunoblotting revealed that both CycB1 and Cdc2 were present at highest levels in pachytene spermatocytes, with lower levels observed in the postmeiotic compartment. To assay for MPF activity, kinase complexes were isolated from lysates of testicular cells using p13suc1 agarose and antibodies directed against Cdc2 and CycB1. Activity of these kinase complexes was analyzed using histone H1 as an exogenous substrate. Cdc2 and CycB1-associated kinase activities were localized to the meiotically dividing pachytene spermatocytes, but not to postmeiotic spermatids.

Animals↗
PubMed1994

Proenkephalin gene expression in testicular interstitial cells is down-regulated coincident with the appearance of pachytene spermatocytes.

Two distinct forms of proenkephalin messenger RNA (mRNA) are present in the murine testis, a family of 1.7 kilobases (kb), germ cell-specific transcripts and a 1.45-kb form that is also found in somatic tissues. In situ hybridization and molecular analysis of purified spermatogenic cell types were used to characterize the cellular localization of these different transcripts during development of the mouse testis. Both forms of proenkephalin mRNA were observed in isolated germ cells by RNA gel-blot analysis, but in distinct developmental patterns; the 1.7-kb transcripts were present in cells undergoing meiosis and spermiogenesis, whereas the 1.45-kb mRNA was detected primarily in type B spermatogonia. In contrast, in situ hybridization analysis did not detect significant amounts of the 1.45-kb transcript in any spermatogenic cell type. Using transcript-specific probes, distinct patterns of developmental expression were evident for the two mRNAs. The 1.45-kb transcript was the only form detected in the prepubertal testis, where it was localized mainly in interstitial cells. In contrast, the 1.7-kb transcripts were the major mRNAs observed in the adult testis and were localized to spermatogenic cells. A transition from the prepubertal to the adult pattern occurred on or about postnatal day 21, when proenkephalin-expressing pachytene spermatocytes begin to populate the seminiferous tubules. In situ hybridization analysis further demonstrated that proenkephalin gene expression in mutant (at/at) mice, which lack germ cells, was identical to that observed in the early prepubertal testis. These results suggest that the 1.45-kb proenkephalin mRNA is developmentally down-regulated in mouse interstitial cells and that this process requires ongoing spermatogenesis.

Animals↗