Biomedical subjects

W Engel

Publications and source records attributed to W Engel.

At least 37 records · Page 2Linked to original sources

PubMed2002

Mouse pelota gene (Pelo): cDNA cloning, genomic structure, and chromosomal localization.

The pelota gene of Drosophila melanogaster encodes a protein which is included in cell cycle regulation. Mutations were found to result in spermatogenic arrest, female sterility and disturbances in the patterning of the eye. We have recently isolated and characterized cDNA clones coding for the human pelota gene (PELO). Here we describe the cloning of the murine pelota cDNA and gene (Pelo) that encodes a 385-amino-acid protein. The exon-intron structure of the gene, which contains three exons, was determined. Comparison of the mouse amino acid sequences with the human and Drosophila sequences revealed an overall high identity (96% and 70%, respectively). Northern blot analysis detected a 1.7-kb transcript in all tissues studied. Southern blot analyses revealed that the pelota gene is present as a single copy in the mouse genome. The mouse pelota gene (Pelo) was mapped to the distal end of chromosome 13, in a region that is homologous with a segment of human chromosome 5q11 containing the orthologous human gene. Cloning of the mouse gene is an important step to study the function of the pelota gene in mammals and to create a mouse model for this evolutionarily conserved gene.

Amino Acid Sequence↗

PubMed2001

A 74-bp promoter of the Tnp2 gene confers testis- and spermatid-specific expression in transgenic mice.

During the final stages of spermatogenesis, round spermatids undergo several morphological, biochemical, and physiological modifications which result in the formation of mature spermatozoa. One of these is the nuclear condensation, achieved by the replacement of somatic-type and testis-specific histones by transition proteins and protamines leading to cessation of transcription several days before the completion of spermiogenesis. Therefore, a strict temporal and stage-specific gene expression is necessary for the correct differentiation of round spermatids into mature spermatozoa. In this study, the 5' regulatory region of rat Tnp2 gene was investigated by primer extension analysis and transgenic mice study. Primer extension analysis revealed a transcription start site which lies 70 bp upstream of the translation start codon. By transgenic mice studies, we demonstrated that a 147-bp 5' untranslated region corresponding to the region -74 to +73 is sufficient to confer testis- and spermatid-specific expression of rat Tnp2 gene.

3' Untranslated Regions↗

PubMed2001

Human cyritestin genes (CYRN1 and CYRN2) are non-functional.

The mouse cyritestin gene is a member of the ADAM (a disintegrin and metalloprotease) gene family and codes for a membrane-anchored sperm protein. Recently, it was shown that cyritestin is critical for male fertility in the mouse. Spermatozoa of cyritestin-deficient mice are not able to bind to the zona pellucida of the oocyte and therefore unable to fertilize the egg. However, zona-free oocytes can be fertilized and the resulting embryos show normal development. In contrast to the mouse, where only one gene for cyritestin (Cyrn) is reported, two cyritestin genes (CYRN1 and CYRN2) are known in humans. The human CYRN1 and CYRN2 genes are located on chromosomes 8 and 16, respectively. We report that 27% of fertile men are deficient for the CYRN1 gene but that all have a CYRN2 gene, suggesting that the CYRN2 gene is the orthologous mouse cyritestin gene in humans and might be involved in sperm-egg interactions. However, the characterization of CYRN2 transcripts from testicular RNA of CYRN1-deficient men demonstrated many termination codons in the synthesized cyritestin cDNA. Furthermore, Western-blot analysis with human testicular protein extracts using an anti-cyritestin antibody failed to detect any cyritestin protein. These results demonstrate clearly that both cyritestin genes are non-functional in humans.

ADAM Proteins↗

PubMed2001

Mice deficient for spermatid perinuclear RNA-binding protein show neurologic, spermatogenic, and sperm morphological abnormalities.

Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The Spnr mRNA is expressed at high levels in testis, ovary, and brain and is present in these tissues in multiple forms. We have generated a gene trap allele of the murine Spnr, named Spnr(+/GT). Spnr(GT/GT) mutants show a high rate of mortality, reduced weight, and an abnormal clutching reflex. In addition to minor anatomical abnormalities in the brain, males exhibit defects in spermatogenesis that include a thin seminiferous epithelium and disorganization of spermatogenesis. Most of the sperm from mutant males display defects in the flagellum and consequently show decreased motility and transport within the oviducts. Furthermore, sperm from mutant males achieve in vitro fertilization less frequently. Our findings suggest that SPNR plays an important role in normal spermatogenesis and sperm function. Thus, the Spnr(GT/GT) mutant male mouse provides a unique model for some human male infertility cases.

Alleles↗

PubMed2001

Disruption of an inner arm dynein heavy chain gene results in asthenozoospermia and reduced ciliary beat frequency.

Impaired ciliary and flagellar functions resulting in male infertility and recurrent respiratory tract infections are found in patients suffering from primary ciliary dyskinesia (PCD). In most cases, axonemal defects are present, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. However, to date it is unclear whether mutations in dynein heavy chain genes are responsible for impaired flagellar and ciliary motility in mammals. To elucidate the role of the mouse dynein heavy chain 7 (MDHC7) gene, which encodes a component of the inner dynein arm, we have generated mice lacking this dynein heavy chain isoform. Both MDHC7(+/-) and MDHC7(-/-) mice are viable and show no malformations; however, homozygous males produce no offspring. In comparison to MDHC7(+/-) and wild-type mice the spermatozoa of MDHC7(-/-) mice revealed a dramatic reduced straight line velocity and progressive movement, resulting in the inability of MDHC7-deficient sperm to move from the uterus into the oviduct. Additionally, we measured the beat frequency of tracheal cilia and observed a decrease in the beat frequency of approximately 50% in MDHC7(-/-) mice. The reduction in both ciliary and flagellar motility is not correlated with any gross defects in the axonemal structure. The phenotype of MDHC7(-/-) mice is similar to that observed in some patients suffering from PCD, and our data strongly suggest that in some patients this disease could be due to mutations in the homologous human gene DNAH1 (HDHC7).

Animals↗

PubMed2001

MECP2 mutations in sporadic cases of Rett syndrome are almost exclusively of paternal origin.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that apparently is lethal in male embryos. RTT almost exclusively affects female offspring and, in 99.5% of all cases, is sporadic and due to de novo mutations in the MECP2 gene. Familial cases of RTT are rare and are due to X-chromosomal inheritance from a carrier mother. We analyzed the parental origin of MECP2 mutations in sporadic cases of RTT, by analysis of linkage between the mutation in the MECP2 gene and intronic polymorphisms in 27 families with 15 different mutations, and we found a high predominance of mutations of paternal origin in 26 of 27 cases (P<.001). The paternal origin was independent of type of mutation and was found for single-base exchanges as well as for deletions. Parents were not of especially advanced age. We conclude that de novo mutations in RTT occur almost exclusively on the paternally derived X chromosome and that this is most probably the cause for the high female:male ratio observed in patients with RTT. Affected males recently have been described in a few cases of familial inheritance. Identification of the parental origin may be useful to distinguish between the sporadic form of RTT and a potentially familial form. This distinction will allow geneticists to offer more-specific counseling and discriminate between higher (maternal origin) and lower (paternal origin) recurrence risk.

Adult↗

PubMed2001

Increase in final stages of follicular atresia and premature decay of corpora lutea in Insl3-deficient mice.

The insulin-like factor 3 (Insl3), a member of the insulin-like hormone family, is exclusively synthesized in gonads. Our recent analysis of Insl3-deficient mice revealed the regulating role of the Insl3 factor on the gubernaculum development during the transabdominal descent of the testis. Here we define the role of the Insl3 factor by histometric analysis of wild-type and Insl3(-/-) ovaries. Ovaries from 40-day-old- and 6-month-old Insl3(-/-) mice as well as from wild-type littermates were serially sectioned. Sections were stained with periodic acid Schiff reaction (PAS) for counting the number of zonae pellucidae which indicated the final stages of follicular atresia. Corpora lutea were also determined. Some sections were processed using either a modified TUNEL method for in situ detection of apoptosis or a lectin labelling technique with Griffonia simplicifolia I agglutinin (GS I) for endothelial cell occurrence. The number of zonae pellucidae was higher in Insl3-deficient ovaries of both ages than in ovaries of wild-type sisters (P < 0.05 for 40-day-old ovaries; P < 0.01 for 6-month-old ovaries). Additionally, the wild-type mice of both ages possessed threefold more corpora lutea than their Insl3 littermates (P < 0.01 for 40-day-old; P < 0.001 for 6-month-old). In general, wild-type corpora lutea looked healthy, showed GS I-positive endothelial cells and no apoptotic cells. Corpora lutea from mutants were rich in regressing GS I luteal cells, and apoptotic cells appeared. We conclude: Follicular atresia and luteolysis are accelerated in ovaries of Insl3-deficient mice probably because of increased apoptosis. The Insl3 function thus appears to rescue endocrine cells from the apoptotic pathway.

Animals↗

PubMed2001

Rat transition nuclear protein 2 regulatory region directs haploid expression of reporter gene in male germ cells of transgenic mice.

During spermiogenesis, the successive replacement of somatic histones by basic proteins, the transition nuclear proteins and protamines, allows normal sperm condensation. Transition nuclear protein 2 (TNP2) is transcribed postmeiotically in round spermatids. In order to determine regulatory flanking sequences responsible for stage specific expression of TNP2 gene, different transgenic mice were generated by microinjection of fertilized eggs. We demonstrate here that 525 bp of 5'- and 920 bp of 3'-flanking sequences of rat TNP2 gene could properly and efficiently direct chloramphenicol acetyltransferase gene expression to the postmeiotic male germ cells of transgenic mice. During male germ cell differentiation the first transgene transcripts were observed in round spermatids and translation started 6 days later in elongating spermatids, which is an evidence for posttranscriptional regulation of transgene expression. In contrast, transgenic mice bearing only the 525 bp 5'-flanking sequences of rat transition protein 2 gene and 3'-flanking sequences of the simian virus 40 large T antigen showed low levels of transgene expression in testis. From these results, it can be concluded that the 525 bp 5'-flanking sequences regulate the cell specific expression and the sequences located in 920 bp 3'-flanking region either enhance the transgene expression in the male germ cells or may have a posttranscriptional role in stabilizing the mRNA in addition to its function in delaying the mRNA translation. Using comparative alignment of 5'-flanking of TNP2 genes from different species, the putative regulatory sequences are identified.

3' Untranslated Regions↗

PubMed2001

In vivo studies on the metabolism of the monoterpenes S-(+)- and R-(-)-carvone in humans using the metabolism of ingestion-correlated amounts (MICA) approach.

The major in vivo metabolites of S-(+)- and R-(-)-carvone in a metabolism of ingestion correlated amounts (MICA) experiment were newly identified as alpha,4-dimethyl-5-oxo-3-cyclohexene-1-acetic acid (dihydrocarvonic acid), alpha-methylene-4-methyl-5-oxo-3-cyclohexene-1-acetic acid (carvonic acid), and 5-(1,2-dihydroxy-1-methylethyl)-2-methyl-2-cyclohexen-1-one (uroterpenolone) on the basis of mass spectral analysis in combination with syntheses and NMR experiments. Minor metabolites were identified as reduction products of carvone, namely, the alcohols carveol and dihydrocarveol. The previously identified major in vivo metabolite in rabbits, 10-hydroxycarvone, could not be detected, indicating either concentration effects or interspecies differences. Metabolic pathways for carvone in humans including oxidation of the double bond in the side chain and, to a minor extent 1,2- and 1,4 + 1,2-reduction of carvone, are discussed. No differences in metabolism between S-(+)- and R-(-)-carvone were detected.

Cyclohexane Monoterpenes↗

PubMed2001

Targeted expression of SV40 large tumour antigen (TAg) induces a transient enhancement of spermatocyte proliferation and apoptosis.

In an attempt to determine the susceptibility of spermatocytes to malignant transformation by simian virus 40 (SV40) large tumour antigen (TAg), transgenic mice harbouring a chimeric gene composed of the SV40 TAg gene fused to the 1.4 kb promoter sequence of the human phosphoglycerate kinase 2 (PGK2) gene were generated. Northern blot analysis on RNA from different tissues indicated a specific transcription of TAg in the testis of PGK2-TAg transgenic mice. Reverse transcription-polymerase chain reaction and Western blot analysis on testes at different stages of development revealed that transcription and translation of the TAg gene starts in 12-day-old testis, which coincides with the appearance of pre-leptotene spermatocytes. Germ cells of transgenic mice showed no tendency toward transformation, but in testes of both 18- and 25-day-old transgenic mice, a significantly enhanced number of spermatocytes was found. In contrast, in 42-day-old transgenic mice no differences in the number of spermatocytes and spermatids were observed. The number of Sertoli cells was determined to be equal in transgenic and wild type mice. In-situ end labelling of fragmented DNA revealed a higher rate of apoptosis in testes of 18-day-old transgenic mice as compared with wild type mice. These results indicate that germ cell homeostasis in transgenic mice is maintained by an apoptotic mechanism.

Animals↗

PubMed2001

Teratozoospermia in mice lacking the transition protein 2 (Tnp2).

It is believed that the transition proteins (Tnp1 and Tnp2) participate in the removal of the nucleohistones and in the initial condensation of the spermatid nucleus. Later in spermatogenesis, Tnp1 and Tnp2 are replaced by the protamines 1 and 2. In an effort to elucidate the physiological role of Tnp2, we have disrupted its locus by homologous recombination. Breeding of the Tnp2(-/-) males on different genetic backgrounds revealed normal fertility on the mixed background C57BL/6Jx129/Sv, but total infertility on the inbred 129/Sv background. Light and electron microscopy showed that the germ cells were capable of undergoing chromatin condensation, although many spermatozoa exhibited head abnormalities with acrosomes not attached to the nuclear envelope. Furthermore, migration of Tnp2(-/-) spermatozoa from the uterus into the oviduct was reduced. These results suggest that male infertility of the Tnp2(-/-) mice is a result of sperm head abnormalities and reduced sperm motility. The increased level of the Tnp1 transcript in testes of the Tnp2-deficient mice raises the possibility that a deficiency created through the disruption of the Tnp2 gene can be compensated for by recruitment of the Tnp1.

Acrosome↗

PubMed2001

Cytogenetic characterization of the TM4 mouse Sertoli cell line. I. Conventional banding techniques, FISH and SKY.

Permanent Sertoli cell lines provide an ideal system for the in vitro analysis of function and responsiveness to biochemical/hormonal factors of this particular cell type. In general, cytogenetic analyses of cell lines often reveal remarkable chromosomal changes that may be associated with functional characteristics. In the present study we investigated the mouse Sertoli cell line TM4 by C-banding, silver staining, FISH and spectral karyotyping (SKY). A highly increased chromosome number (average 85-95) as well as five stable marker chromosomes were detected by the conventional staining techniques. SKY identified the markers as a translocation chromosome T(1;3), isochromosomes 11 and 18 and two different-sized microchromosomes. The results show the usefulness of combining SKY and conventional banding methods for the evaluation of chromosome alterations in widely used cell lines.

Animals↗

PubMed2001

Cloning, organisation, chromosomal localization and expression analysis of the mouse Prkag1 gene.

The mammalian 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric protein consisting of alpha-, beta- and gamma-subunits. The alpha-subunit is the catalytic subunit. The non-catalytic subunits AMPK-beta and AMPK-gamma form, together with the catalytic AMPK-alpha, the active kinase complex in mammals and its homologue in yeast. The gene for AMPK-gamma-1 has been designated recently as PRKAG1. We have isolated mouse Prkag1 cDNA from testis (1623 nt) coding for 330 aa and we have shown its ubiquitous expression as a 1.8-kb transcript. A comparison between mouse, rat and human PRKAG1 cDNA and protein sequences shows that the gene is highly conserved among these species with a homology of 96% at the protein level. Southern blot analysis indicates that there is more than one gene for PRKAG in the mouse genome. Prkag1 contains 12 exons with short introns. Analysis of 50 interspecific backcross mice mapped the mouse gene to the distal region of chromosome 15.

AMP-Activated Protein Kinases↗

PubMed2001

Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization.

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.

Acrosin↗

PubMed2001

Isolation and characterization of differentially expressed genes in invasive and non-invasive immortalized murine male germ cells in vitro.

In an attempt to elucidate the potential of premeiotic male germ cells to malignant transformation both the invasiveness and the differential gene expression of several putative tumor markers of the spermatogonia-derived cell line GC-1spg and the spermatocyte-derived cell line GC-4spc were analyzed. Studies, using RT-PCR analysis, of the expression pattern of the alkaline phosphatase isoenzymes which serve as markers for testicular germ cell tumors demonstrated that the expression of the endogenous mouse embryonic alkaline phosphatase (EAP) is upregulated in the GC-1spg cell line. Additionally, after transfection of GC-1spg cells and GC-4spc cells with a GCAP-CAT construct, an increased promoter activity of the human germ cell alkaline phosphatase (GCAP), the equivalent human isoenzyme of EAP, was shown in GC-1spg. Furthermore, an in vitro Matrigel invasion assay revealed a significant higher invasive potential of GC-1spg cells as compared to GC-4spc cells. Finally, a suppression subtractive hybridization on RNA of invasive GC-1spg cells and non-invasive GC-4spc cells was performed. In total, 31 cDNA sequences were isolated and further analyzed. Among these, 18 known sequences and 13 unknown sequences were determined. Northern blot analysis revealed that one unknown gene and eight known genes, namely integrin alpha 6, L6 antigen, annexin VIII, BVL-1 retrotransposon, protective protein, replacement variant histone 3.3, alpha-catenin and LPS-binding protein, are over-expressed in invasive GC-1spg cells. Taken together, both the enhanced invasive activity of GC-1spg cells and the upregulated expression of genes involved in the process of tumor progression suggest that the immortalized spermatogonia-derived cell line GC-1spg does have a higher potential to malignant transformation than the immortalized spermatocyte-derived cell line GC-4spc.

Animals↗

PubMed2000

Characterisation of calcitonin gene-related peptide receptors in rat atrium and vas deferens: evidence for a [Cys(Et)(2, 7)]hCGRP-preferring receptor.

The present study was performed in order to characterise calcitonin gene-related peptide (CGRP) receptor subtypes in rat left atrium and vas deferens by using [R-(R*, S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl ]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1, 4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide (BIBN4096BS), a novel CGRP receptor antagonist. When CGRP was used as an agonist, BIBN4096BS exhibited an almost 10-fold higher affinity for CGRP receptors in rat left atrium compared to those in the vas deferens, indicating that CGRP acts through different CGRP receptor subtypes in these two tissues. In addition, BIBN4096BS was almost 10-fold more potent in antagonizing [Cys(Et)(2,7)]hCGRPalpha and human adrenomedullin-induced responses than CGRP-induced responses in rat vas deferens. This might indicate receptor heterogeneity in rat vas deferens. Accordingly, the present work provides first experimental evidence that the rat vas deferens contains two CGRP-like receptor subtypes. Namely, the CGRP(2) receptor and a "novel" receptor that possesses low efficacy for CGRP and that is selectively stimulated by [Cys(Et)(2,7)]hCGRP or adrenomedullin and which can be blocked with high affinity by BIBN4096BS.

Animals↗

PubMed2000

Refinement of the expression pattern of a mouse homologue of the porcine 135-kDa alpha-d-mannosidase (MAN2B2).

In the article by Hiramoto et al. (1997) (Stage-specific expression of a mouse homologue of the porcine 135 kDa alpha-d-mannosidase (MAN2B2) in type A spermatogonia, Biochem. Biophys. Res. Commun. 241, 439-445) a new homologue to the porcine epididymis-specific 135 kDa alpha-d-mannosidase (MAN2B2) was cloned from a mouse testis cDNA library. Northern blot analysis with RNA of different tissues showed a testis-specific expression by using a mouse MAN2B2 oligonucleotide. In situ hybridization revealed that the mouse MAN2B2 transcript is localized exclusively in spermatogonia. In consequence to this it was proposed by Hiramoto et al. that the mouse MAN2B2 homologue could serve as a marker for spermatogonia. By repeating the published experiments we observed a different expression pattern of the mouse MAN2B2 gene. Northern blot analysis with either testicular RNA from prepubertal males or with testicular RNA from the W/W(v)-mutant mouse which lacks germ cells showed an expression of the MAN2B2 gene. Furthermore, Northern blot analysis with RNA from different somatic tissues revealed that the gene is ubiquitously expressed. Therefore, our refinement clearly demonstrates that the MAN2B2 mouse homologue is not specifically expressed in spermatogonia.

Aging↗

PubMed2000

Rett syndrome: analysis of MECP2 and clinical characterization of 31 patients.

UNLABELLED: Only recently have mutations in MECP2 been found to be a cause of Rett Syndrome (RTT), a neuro-developmental disorder characterized by mental retardation, loss of expressive speech, deceleration of head growth and loss of acquired skills that almost exclusively affects females. We analysed the MECP2 gene in 31 patients diagnosed with RTT. Sequencing of the coding region and the splice sites revealed mutations in 24 females (77.40%). However, no abnormalities were detected in any of the parents that were available for investigation. Eleven mutations have not been described previously. Confirming two earlier studies, we found that most mutations are truncating and only a few of them are missense mutations. Several females carrying the same mutation display different phenotypes indicating that factors other than the type or position of mutations influence the severity of RTT. Four females with RTT variants were included in the study. Three of these presented with preserved speech while the fourth patient with congenital RTT lacked the initial period of normal development. Detection of mutations in these cases reveals that they are indeed variants of RTT. They represent the mild and the severe extremes of RTT. CONCLUSIONS: mutations in MECP2 seem to be the main cause for RTT and can be expected to be found in approximately 77% of patients that fulfil the criteria for RTT. Therefore analysis of MECP2 should be performed if RTT is suspected. Three mutation hotspots (T158M, R168X and R255X) were confirmed and a further one (R270X) newly identified. We recommend screening for these mutations before analysing the coding region.

Alleles↗