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D J Wolgemuth

Publications and source records attributed to D J Wolgemuth.

At least 73 records · Page 4Linked to original sources
PubMed1994

[Genetic control of cellular differentiation and proliferation during gametogenesis in mammals].

Understanding the genetic program that controls the regulation of cells progressing through the mitotic and meiotic cell cycles, whether in response to growth factors, other extracellular signals, or intrinsic programs, is critical to problems in infertility and in gonadal neoplasias. In this study, we present a consideration of important stages of cell cycle control in mammalian germ cells and data illustrating the experimental approaches that can be used to determine the pattern of such genes which may be involved in these events.

Animals↗
PubMed1994

Expression of proliferating cell nuclear antigen in the mouse germ line and surrounding somatic cells suggests both proliferation-dependent and -independent modes of function.

The distribution of proliferating cell nuclear antigen (PCNA) in specific somatic and germ cells of the adult mouse ovary and testis was assessed using immunocytochemical staining and immunoblot analysis and was correlated with cellular proliferation and differentiation. In the adult ovary, immunocytochemical staining for PCNA within follicular cells varied depending on the stage of follicular growth. Since PCNA staining has proven to be a useful indicator of cells involved in DNA synthesis and repair, the pattern of PCNA staining in the ovary was compared to previous studies which used tritiated thymidine labeling as a marker for DNA synthesis. In the testis, PCNA was detected in the mitotically proliferating spermatogonia, but not in spermatocytes which had just entered meiosis. PCNA staining was again observed in spermatogenic cells in later stages of meiotic prophase, in particular zygotene and pachytene spermatocytes. As these cells are undergoing meiotic recombination, the presence of PCNA in these meiotic prophase cells could reflect a second function of PCNA, that of DNA excision repair.

Animals↗
PubMed1993

Structural abnormalities associated with congenital megacolon in transgenic mice that overexpress the Hoxa-4 gene.

Congenital megacolon develops in transgenic mice that overexpress the homeobox-containing gene, Hoxa-4. The current study was done to identify abnormalities of the terminal colon that might account for the phenotype. The terminal bowel of transgenic mice was compared with that of control and lethal spotted (ls/ls) mice, a strain in which megacolon also develops. The terminal colon of the transgenic mice contained fewer ganglia than that of controls, but was hypoganglionic, rather than aganglionic like that of ls/ls mice. The neurons present in the adult transgenic colon were significantly increased in size and a subset of very large neurons (> 40 microns in maximum diameter) were observed. Electron microscopic studies of young adult transgenic mice revealed that the ganglia and nerves of the myenteric plexus had the ultrastructure of extraenteric peripheral nerve rather than that of the enteric nervous system (ENS). The myenteric ganglia in the transgenic animals contained Schwann cells associated with a basal lamina that enveloped axons completely and individually, instead of glia. Although collagen is excluded from the ganglia and thin nerve fibers of the normal ENS, a collagen-containing endoneurium surrounded each of the axon-Schwann cell units of the abnormal nerve fibers of the transgenic colon. Some of the neurons of the transgenic mice were located in these nerve bundles rather than in ganglia. There were also smooth muscle abnormalities in the terminal bowel of the transgenic mice. Wide gaps were present in the longitudinal muscle of the transgenic mice; these gaps contained ganglia that were in contact with the adventitia. These longitudinal smooth muscle cells were more irregular than those of controls and they contained fewer puncta adherens; moreover, a larger proportion of the volume of the cytoplasm of transgenic smooth muscle cells was occupied by organelles. Finally, an extensive thickening and reduplication of the basal lamina surrounding the smooth muscle cells of the muscularis mucosa was observed in the transgenic colon and resembled that found in ls/ls mice. These data suggest that both smooth muscle and the innervation of the terminal bowel of neonatal Hoxa-4 transgenic mice are structurally abnormal. Although some of the abnormalities seen in Hoxa-4 transgenic mice are similar to those which arise in ls/ls mice, the two conditions are not identical. In both animals, the data are consistent with the hypothesis that the defects arise as a result of a defective interaction between the precursors of enteric neurons and smooth muscle.

Acetylcholinesterase↗
PubMed1993

HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis.

This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells.

Animals↗
PubMed1993

Protein product of the somatic-type transcript of the Hoxa-4 (Hox-1.4) gene binds to homeobox consensus binding sites in its promoter and intron.

The murine Hoxa-4 gene encodes a protein with a homeodomain closely related to those produced by the Antennapedia-like class of Drosophila genes. Drosophila homeodomain proteins can function as transcription factors, binding to several specific DNA sequences. One sequence that is frequently encountered contains a core ATTA motif within a larger consensus sequence, such as CAATTAA. The in vitro synthesized protein product of Hoxa-4 was shown to bind to a subset of restriction fragments of the Hoxa-4 gene itself as determined by gel retardation experiments. Direct examination of the sequences of the fragments bound by Hoxa-4 protein revealed the presence of four regions containing the core ATTA motif. Two regions contained sequences of the CAATTAA class and were located approximately 1 kb upstream from the putative somatic Hoxa-4 promoter and within the intron. Two additional binding sites containing the consensus target sequence involved in autoregulation of Drosophila Deformed gene were identified: one immediately downstream of the putative embryonic transcription start site and one within the intron, respectively. Specific binding of the in vitro produced Hoxa-4 protein to oligonucleotides corresponding to these sequences was observed in gel retardation assays. The same results were obtained with Hoxa-4 protein produced in a Baculovirus expression system. Experiments using oligonucleotides containing base substitutions in positions 1, 3, 4, and 5 in the sequence CAATTAA showed severely reduced binding. The use of truncated mutant Hoxa-4 proteins in gel retardation assays and in transient co-transfection experiments revealed that the intact homeodomain was required for the binding. These results also suggested that the Hoxa-4 gene has the potential to auto-regulate its expression by interacting with the homeodomain binding sites present in the promoter as well as in the intron.

Amino Acid Sequence↗
PubMed1993

Members of the raf gene family exhibit segment-specific patterns of expression in mouse epididymis.

The proto-oncogene c-raf-1 and the related genes A-raf and B-raf encode serine/threonine protein kinases thought to be involved in regulating gene expression by acting as part of second-messenger signaling pathways within the cell. Among the tissues in which A-raf and c-raf-1 have been shown to be expressed was mouse epididymis. The present studies were undertaken to determine if the raf family genes exhibited specificity in their pattern of expression that might be indicative of specific function in the epididymis. Northern and in situ hybridization analyses demonstrated that c-raf-1 mRNA was expressed as a 3.1 kb transcript at uniform levels throughout the length of the epididymis in all types of epididymal epithelial cells. Neither the germ cell-specific testicular transcripts nor the somatic transcripts of B-raf were detected by either Northern or in situ hybridization analysis in any region of the epididymis. A-raf, expressed as two transcripts of 2.6 and 4.3 kb, was the only gene examined which exhibited a segment-specific pattern of expression, being highest in the principal epithelial cells of the proximal caput epididymis and decreasing progressively in more distal regions of the tubule. These studies indicate that each raf gene exhibits a characteristic pattern of expression in the epididymis; A-raf in particular may play a unique regulatory role in the regionalized functions of the epididymis.

Animals↗
PubMed1993

Conservation and divergence of patterns of expression and lineage-specific transcripts in orthologues and paralogues of the mouse Hox-1.4 gene.

A possible correlation between the structural organization of homeobox-containing genes and their cell-specific expression was examined in studies determining similarities and differences in the expression patterns of orthologues and paralogues of the murine Hox-1.4 gene. We first compared the expression pattern of members of the Hox-1.4 subfamily, Hox-2.6 and Hox-4.2, in the adult mouse testis. Although these three evolutionarily related genes exhibited similar anterior limits of expression in the embryonic central nervous system, their cellular specificity of expression was very different in the adult testis. Hox-1.4 was abundantly expressed only in the germ cells; Hox-2.6 was expressed at very low levels in both spermatogenic cells and somatic cells; and Hox-4.2 transcripts appeared to be restricted to the somatic cells. We next analyzed the expression of several of the Hox-1.4 orthologues in the mouse testis to determine if other members of the Hox-1 cluster are involved in the male germ cell differentiation pathway. We showed that the two adjacent genes of Hox-1.4, Hox-1.3 and Hox-1.5, are expressed in the germ line but at lower levels. Further, both Hox-1.3 and Hox-1.4 produced unique, germ line-specific transcripts as compared to other adult tissues and the Day 12.5 embryo.

Animals↗
PubMed1993

Differential expression of the proto-oncogenes c-abl and c-mos in developing mouse germ cells.

C-abl and c-mos, two proto-oncogenes with unique patterns of expression in gonadal tissues, were examined by in situ hybridization with respect to their expression in the germ line. C-abl transcripts were observed to be most abundant in late round spermatids and elongating spermatids. C-mos transcripts were also expressed in round spermatids, but at much reduced levels compared to the levels of c-abl. C-abl mRNAs were also detected in mouse oocytes which have entered the growth phase. This temporal specificity of accumulation of c-abl transcripts was similar to that observed for c-mos, which also accumulates in growing and fully grown oocytes (follicle stages 4-6). Quantitative evaluation suggested that c-abl mRNA levels in oocytes are at least an order of magnitude lower than those of c-mos transcripts.

Aging↗
PubMed1993

Sequences 5' of the homeobox of the Hox-1.4 gene direct tissue-specific expression of lacZ during mouse development.

The murine homeobox-containing gene Hox-1.4 is expressed in restricted patterns during embryogenesis and in male germ cells. To begin identification of the cis-acting elements regulating this expression, transgenic mice were generated carrying a chimeric construct that contained approx. 4 kb of 5' flanking sequence and approx. 1 kb of structural gene, fused in frame to the E. coli lacZ gene. This construct directed expression of the resulting Hox-1.4,beta-galactosidase fusion protein in a pattern that reproduced virtually the complete embryonic and adult sites of expression of the endogenous gene. Embryonic expression of the fusion protein was first detected in mesoderm at day 8.0 of gestation (E 8.0). Between gestational ages E 8.5 to E 12.5, beta-gal expression was observed in the somites, the lateral walls of the posterior myelencephalon, the dorsal region and ventral wall of the spinal cord, spinal ganglia and prevertebrae and their surrounding mesenchyme, between presumptive ribs, as well as in mesenchymal layers in the lung, kidney and portions of the gut. Expression was also noted in the pancreas and in the supporting cells and sheath around subsets of peripheral nerves, sites that had not been detected previously. Adult expression was observed in testes, specifically in meiotic and post-meiotic male germ cells. In contrast, transgenic mice carrying 5' deletions of the construct which leave approx. 1.2 kb or approx. 2.0 kb of Hox-1.4 sequence 5' to the embryonic promoter, did not exhibit beta-gal staining. These deletion experiments defined at least one cis-acting control element necessary for the expression of the Hox-1.4 gene to a 2 kb region located 2 to 4 kb 5' of the embryonic transcription start site.

Animals↗
PubMed1993

Isolation of the murine cyclin B2 cDNA and characterization of the lineage and temporal specificity of expression of the B1 and B2 cyclins during oogenesis, spermatogenesis and early embryogenesis.

A cDNA encoding the murine cyclin B2 (cycB2) was isolated from an adult mouse testis cDNA library as part of studies designed to identify cyclins involved in murine germ cell development. This cycB2 cDNA was then used to examine the pattern of cycB2 expression during male and female germ cell development and in early embryogenesis, and to compare this expression with the previously characterized expression of cycB1. A single 1.7 kb cycB2 transcript was detected by northern blot hybridization analysis of total RNA isolated from midgestation embryos and various adult tissues. Northern blot and in situ hybridization analyses revealed that cycB2 expression in the testis was most abundant in the germ cells, specifically in pachytene spermatocytes. This is in contrast to the highest levels of expression of cycB1 being present in early spermatids. In situ analysis of the ovary revealed cycB2 transcripts in both germ cells and somatic cells, specifically in the oocytes and granulosa cells of growing and mature follicles. The pattern of cycB1 and cycB2 expression in ovulated and fertilized eggs was also examined. While the steady state level of cycB1 and cycB2 signal remained constant in oocytes and ovulated eggs, signal of both appeared to decrease following fertilization. In addition, both cycB1 and cycB2 transcripts were detected in the cells of the inner cell mass and the trophectoderm of the blastocyst. These results demonstrate that there are lineage- and developmental-specific differences in the pattern of the B cyclins in mammalian germ cells, in contrast to the co-expression of B cyclins in the early conceptus.

Animals↗
PubMed1993

Multiple levels of regulation exist for expression of the Hoxa-4 (Hox-1.4) gene in the mouse testis.

The experiments presented in this study show that the testis-specific expression of the murine Hoxa-4 (formerly Hox-1.4) gene is likely to be achieved through a variety of regulatory mechanisms operating on several levels. S1 mapping analysis suggested the possibility of multiple testicular transcripts arising from alternative splicing events. Analysis of cDNA and genomic sequences revealed the presence of two polyadenylation signals: the canonical AATAAA and further 3', an ATTAAA. Primer extension analysis to determine the start site of the testicular transcripts and nuclear run-on analysis suggested the use of a more upstream promoter than the one previously identified. The nuclear run-on analysis further suggested that Hoxa-4 is under posttranscriptional control. RNase H analysis showed that the size difference between the two testicular Hoxa-4 transcripts of 1.35 and 1.45 kb was due to a postmeiotic increase in poly(A) tail length. Only a proportion of the Hoxa-4 mRNA in germ cells was found to be associated with polysomes, suggesting that translational regulation may also operate as a means of control of Hoxa-4 expression in this adult lineage.

Alternative Splicing↗
PubMed1993

Developmental and cell lineage specificity of raf family gene expression in mouse testis.

The proto-oncogene c-raf-1 and the related genes A-raf and B-raf encode serine/threonine protein kinases thought to be involved in regulating gene expression by transducing extracellular signals into the cell. All three raf family genes have been shown previously to be expressed in mouse testis. Northern and in situ hybridization analyses with probes specific for each gene demonstrated that c-raf-1 mRNA is ubiquitously expressed in both somatic and germ cells as a 3.1-kb transcript. Additionally, the levels of c-raf-1 expression are developmentally regulated in the germ cells, exhibiting highest expression in early pachytene spermatocytes and decreasing progressively through later stages. A-raf is expressed predominantly in the somatic compartment as two transcripts of 2.6 and 4.3 kb. A-raf expression in Leydig cells appears to be elevated in testes undergoing spermatogenesis. In contrast, B-raf is expressed as two major transcripts of 4.0 and 2.6 kb, with the 4.0-kb transcript first expressed at low levels in pachytene spermatocytes and the more abundant 2.6-kb transcript restricted to post-meiotic spermatids. These studies indicate that each raf gene exhibits a characteristic, limited pattern of expression and suggests that the different forms may play a unique regulatory role in androgen production and/or spermatogenesis.

Age Factors↗
PubMed1992

Zfp-37, a new murine zinc finger encoding gene, is expressed in a developmentally regulated pattern in the male germ line.

To begin to examine the function in the mouse testis of genes containing the zinc finger motif, we have screened an adult mouse total testis cDNA library with probes to a conserved region of zinc fingers. We have isolated cDNAs for a new murine zinc finger encoding gene that has been designated Zfp-37. Northern blot hybridization analysis revealed Zfp-37 transcripts at high levels in the testis, the only adult tissue in which Zfp-37 expression was observed. Zfp-37 was also expressed at lower levels in the mid-gestation embryo and placenta. The major testicular transcripts are 2.3 and 2.6 kb. A 4.0 kb transcript was detected at lower levels in the testis as well as in embryo and placenta. Northern blot and in situ hybridization analysis revealed that expression of Zfp-37 was most abundant in germ cells which have completed meiosis and are undergoing the complex morphogenetic changes of spermiogenesis. The pattern of expression of Zfp-37 and the presence of the zinc finger domain suggest that Zfp-37 may have a role in regulating spermiogenesis.

Amino Acid Sequence↗
PubMed1992

Identification of a mouse B-type cyclin which exhibits developmentally regulated expression in the germ line.

To begin to examine the function of cyclins in mammalian germ cells, we have screened an adult mouse testis cDNA library for the presence of B-type cyclins. We have isolated cDNAs that encode a murine B-type cyclin, which has been designated cycB1. cycB1 was shown to be expressed in several adult tissues and in the midgestation mouse embryo. In the adult tissues, the highest levels of cycB1 transcripts were seen in the testis and ovary, which contain germ cells at various stages of differentiation. The major transcripts corresponding to cycB1 are 1.7 and 2.5 kb, with the 1.7 kb species being the predominant testicular transcript and the 2.5 kb species more abundant in the ovary. Examination of cDNAs corresponding to the 2.5 kb and 1.7 kb mRNAs revealed that these transcripts encode identical proteins, differing only in the polyadenylation signal used and therefore in the length of their 3' untranslated regions. Northern blot and in situ hybridization analyses revealed that the predominant sites of cycB1 expression in the testis and ovary were in the germinal compartment, particularly in early round spermatids in the testis and growing oocytes in the ovary. Thus cycB1 is expressed in both meiotic and postmeiotic cells. This pattern of cycB1 expression further suggests that cycB1 may have different functions in the two cell types, only one of which correlates with progression of the cell cycle.

Amino Acid Sequence↗
PubMed1992

Identification and characterization of the regulated pattern of expression of a novel mouse gene, meg1, during the meiotic cell cycle.

The gene designated meg1 (meiosis expressed gene) is a new mouse gene identified during a search for mammalian genes potentially involved in meiotic processes. Two classes of complementary DNAs were isolated from an adult mouse testis complementary DNA library, which shared the same 3' end including the entire putative coding region but differed in their 5' ends. Only one of these complementary DNA classes appeared to correspond to the very abundant 0.75-kilobase testicular transcript of meg1. Sequence analysis predicts a 10.8-kilodalton protein which is highly charged and lysine rich. It is also relatively rich in potential phosphoacceptor amino acids (approximately 17%), several of which are located in phosphorylation consensus sequences. The pattern of expression of meg1 was studied utilizing a combined Northern blot and in situ hybridization analysis. Of the adult tissues examined, meg1 transcripts were detected exclusively in testis. Analysis of mRNA from testes of two germ cell deficient mutant strains did not reveal significant levels of meg1 transcripts. Analysis of RNA from enriched populations of spermatogenic cells from adult testes and localization by in situ hybridization revealed that meg1 transcripts are most abundant in pachytene spermatocytes. These results suggest a role for meg1 during germ cell differentiation, possibly during meiotic prophase.

Amino Acid Sequence↗
PubMed1991

Stage and lineage-regulated expression of two hsp90 transcripts during mouse germ cell differentiation and embryogenesis.

The expression of members of the heat shock protein 90 (hsp90) gene family during testicular and embryonic development was investigated. Two different hsp90 transcripts were detected in RNA from mouse testis, approximately 3.2 kb and 2.9 kb in size, and were shown to exhibit cellular and developmental stage specificity of expression. The larger, more abundant transcript was expressed at high levels in the germinal compartment of the testis, particularly in germ cells in meiotic prophase. The smaller hsp90 transcript was expressed predominantly in the somatic compartment of the testis. Expression of the two hsp90 transcripts was observed in testes of other species, suggesting an important role for hsp90 in mammalian testicular function. In addition, expression of both hsp90 transcripts was detected in the embryonic and extra-embryonic compartments of mid-gestation embryos.

Animals↗