scholarly journals Overexpressing sperm surface beta 1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm-egg interactions.

1994 ◽  
Vol 126 (6) ◽  
pp. 1573-1583 ◽  
Author(s):  
A Youakim ◽  
H J Hathaway ◽  
D J Miller ◽  
X Gong ◽  
B D Shur
Keyword(s):  
Transgenic Mice ◽  
Zona Pellucida ◽  
Transgenic Animals ◽  
Wild Type ◽  
Sperm Surface ◽  
Receptor Aggregation ◽  
Excess Surface ◽  
Before And After ◽  
Mechanistic Basis ◽  
Egg Coat

Sperm surface beta 1,4-galactosyltransferase (GalTase) mediates fertilization in mice by binding to specific O-linked oligosaccharide ligands on the egg coat glycoprotein ZP3. Before binding the egg, sperm GalTase is masked by epididymally derived glycosides that are shed from the sperm surface during capacitation. After binding the egg, sperm-bound oligosaccharides on ZP3 induce the acrosome reaction by receptor aggregation, presumably involving GalTase. In this study, we asked how increasing the levels of sperm surface GalTase would affect sperm-egg interactions using transgenic mice that overexpress GalTase under the control of a heterologous promoter. GalTase expression was elevated in many tissues in adult transgenic animals, including testis. Sperm from transgenic males had approximately six times the wild-type level of surface GalTase protein, which was localized appropriately on the sperm head as revealed by indirect immunofluorescence. As expected, sperm from transgenic mice bound more radiolabeled ZP3 than did wild-type sperm. However, sperm from transgenic animals were relatively unable to bind eggs, as compared to sperm from wild-type animals. The mechanistic basis for the reduced egg-binding ability of transgenic sperm was attributed to alterations in two GalTase-dependent events. First, transgenic sperm that overexpress surface GalTase bound more epididymal glycoside substrates than did sperm from wild-type mice, thus masking GalTase and preventing it from interacting with its zona pellucida ligand. Second, those sperm from transgenic mice that were able to bind the zona pellucida were hypersensitive to ZP3, such that they underwent precocious acrosome reactions and bound to eggs more tenuously than did wild-type sperm. These results demonstrate that sperm-egg binding requires an optimal, rather than maximal, level of surface GalTase expression, since increasing this level decreases sperm reproductive efficiency both before and after egg binding. Although sperm GalTase is required for fertilization by serving as a receptor for the egg zona pellucida, excess surface GalTase is counterproductive to successful sperm-egg binding.

Sperm from beta 1,4-galactosyltransferase-null mice are refractory to ZP3-induced acrosome reactions and penetrate the zona pellucida poorly

Development ◽  
1997 ◽  
Vol 124 (20) ◽  
pp. 4121-4131 ◽  
Author(s):  
Q. Lu ◽  
B.D. Shur
Keyword(s):  
Acrosome Reaction ◽  
Direct Evidence ◽  
Calcium Ionophore ◽  
Wild Type ◽  
Sperm Surface ◽  
Gamete Recognition ◽  
Dependent Signal ◽  
Relative Inability ◽  
Egg Coat

A variety of sperm surface components have been suggested to mediate gamete recognition by binding to glycoside ligands on the egg coat glycoprotein ZP3. The function of each of these candidate receptors is based upon varying degrees of circumstantial and direct evidence; however, the effects on fertilization of targeted mutations in any of these candidate receptors have not yet been reported. In this paper, we describe the effects of targeted mutations in beta1,4-galactosyltransferase, the best studied of the candidate receptors for ZP3. Surprisingly, galactosyltransferase-null (gt[−/−]) males are fertile; however, sperm from gt(−/−) males bind less radiolabeled ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either ZP3 or anti-galactosyltransferase antibodies, as do wild-type sperm. In contrast, gt(−/−) sperm undergo the acrosome reaction normally in response to calcium ionophore, which bypasses the requirement for ZP3 binding. The inability of gt(−/−) sperm to undergo a ZP3-induced acrosome reaction renders them physiologically inferior to wild-type sperm, as assayed by their relative inability to penetrate the egg coat and fertilize the oocyte in vitro. Thus, although ZP3 binding and subsequent induction of the acrosome reaction are dispensable for fertilization, they impart a physiological advantage to the fertilizing sperm. A second strain of mice was created that is characterized by a loss of of the long galactosyltransferase isoform responsible for ZP3-dependent signal transduction, but which maintains normal levels of Golgi galactosylation. Sperm from these mice show that the defective sperm-egg interactions in gt(−/−) mice are due directly to a loss of the long galactosyltransferase isoform from the sperm surface and are independent of the state of intracellular galactosylation during spermatogenesis.

scholarly journals Bi-transgenic Mice Reveal that K-rasVal12 Augments a p53-independent Apoptosis When Small Intestinal Villus Enterocytes Reenter the Cell Cycle

1997 ◽  
Vol 138 (1) ◽  
pp. 167-179 ◽  
Author(s):  
Craig M. Coopersmith ◽  
Chitra Chandrasekaran ◽  
M. Shane McNevin ◽  
Jeffrey I. Gordon
Keyword(s):  
Cell Cycle ◽  
Transgenic Mice ◽  
Transgenic Animals ◽  
Wild Type ◽  
Activating Mutations ◽  
Cell Cycle Reentry ◽  
Cell Matrix ◽  
Small Intestinal ◽  
Small Intestinal Villus ◽  
Cell Matrix Interactions

Studies in cell culture systems have indicated that oncogenic forms of Ras can affect apoptosis. Activating mutations of Ras occur in ∼30% of all human tumors and 50% of colorectal carcinomas. Since these mutations appear at early or intermediate stages in multistep journeys to neoplasia, an effect on apoptosis may help determine whether initiated cells progress towards a more neoplastic state. We have tested the effects of K-rasVal12 on apoptosis in transgenic mice. A lineage-specific promoter was used to direct expression of human K-rasVal12, with or without wild-type (wt) or mutant SV-40 T antigens (TAg), in postmitotic villus enterocytes, the principal cell type of the small intestinal epithelium. Enterocytes can be induced to reenter the cell cycle by TAgWt. Reentry is dependent upon the ability of TAg to bind pRB and is associated with a p53-independent apoptosis. Analyses of K-rasVal12 × TAgWt bi-transgenic animals indicated that K-rasVal12 can enhance this apoptosis threefold but only in cycling cells; increased apoptosis does not occur when K-rasVal12 is expressed alone or with a TAg containing Glu107,108→ Lys107,108 substitutions that block its ability to bind pRB. Analysis of bi-transgenic K-rasVal12 × TAgWt mice homozygous for wild-type or null p53 alleles established that the enhancement of apoptosis occurs through a p53-independent mechanism, is not attributable to augmented proliferation or to an increase in abortive cell cycle reentry (compared to TAgWt mice), and is not associated with detectable changes in the crypt–villus patterns of expression of apoptotic regulators (Bcl-2, Bcl-xL, Bak, and Bax) or mediators of epithelial cell–matrix interactions and survival (e.g., α5β1 integrin and its ligand, fibronectin). Coexpression of K-rasVal12 and TAgWt produces dysplasia. The K-rasVal12-augmented apoptosis is unrelated to this dysplasia; enhanced apoptosis is also observed in cycling nondysplastic enterocytes that produce K-rasVal12 and a TAg with a COOH-terminal truncation. The dysplastic epithelium of K-rasVal12 × TAgWt mice does not develop neoplasms. Our results are consistent with this finding: (a) When expressed in initiated enterocytes with a proliferative abnormality, K-rasVal12 facilitates progression to a dysplastic phenotype; (b) by diminishing cell survival on the villus, the oncoprotein may impede further progression; and (c) additional mutations may be needed to suppress this proapoptotic response to K-rasVal12.

scholarly journals Isolation of Glomerular Podocytes by Cationic Colloidal Silica-coated Ferromagnetic Nanoparticles

2016 ◽  
Vol 9 (1) ◽  
pp. 67-87
Author(s):  
Andreas Blutke
Keyword(s):  
Transgenic Mice ◽  
Transgenic Animals ◽  
Dominant Negative ◽  
Ferromagnetic Nanoparticles ◽  
Wild Type ◽  
Isolated Cells ◽  
Specific Expression ◽  
Specific Antibodies ◽  
Isolated Glomeruli ◽  
Fluorescent Markers

Background: Podocyte homeostasis plays a crucial role for the maintenance of physiological glomerular function and podocyte injury is regarded as a major determinant of development and progression of renal disease. Objective: Investigation of podocytes requires appropriate methods for their isolation. Previously reported methods use podocyte specific antibodies or transgenic mice with podocyte specific expression of fluorescent markers for isolation of podocytes by magnetic or fluorescence activated cell sorting. Method: Here, a novel, antibody-free method for isolation of podocyte protein and RNA from mouse glomeruli is described. Preparations of isolated glomeruli were added to a suspension of cationic silica-coated colloidal ferromagnetic nanoparticles. The nanoparticles bound to the negatively charged cell surfaces of podocytes residing on the outer surface of the isolated glomeruli. After enzymatic and mechanical dissociation of glomerular cells, nanoparticle-coated podocytes were isolated in a magnetic field. The method was tested in adult wild-type mice without renal lesions and in mice of two nephropathy models (Growth hormone (GH)-transgenic mice and transgenic mice expressing a dominant negative receptor for the glucose dependent insulinotropic polypeptide, GIPRdn) displaying albuminuria, glomerular hypertrophy and evidence for a reduced negative cell surface charge of podocytes. Results: The isolated cells displayed typical morphological and ultrastructural properties of podocytes. On average, 182,000 ± 37,000 cells were counted in the podocyte isolates harvested from ~10,000-12,000 glomeruli per mouse. On the average, the purity of podocyte isolates of these mice accounted for ~63 ± 18 % and the podocyte isolates displayed high mRNA and protein expression abundances of podocyte markers (nephrin and WT1), whereas the expression of endothelial (Cd31) and mesangial markers (Serpinb7) was significantly decreased in podocyte isolates, as compared to samples of isolated glomeruli. The numbers of cells isolated from GH- transgenic and GIPRdn-transgenic mice were not markedly different from that of wild-type mice. Conclusion: The described method represents an alternative for podocyte isolation, particularly in experiments where podocyte specific antibodies or transgenic animals with podocyte specific expression of fluorescent markers are not applicable.

Impaired pericyte recruitment and abnormal retinal angiogenesis as a result of angiopoietin-2 overexpression

2007 ◽  
Vol 97 (01) ◽  
pp. 99-108 ◽  
Author(s):  
Yuxi Feng ◽  
Franziska vom Hagen ◽  
Frederick Pfister ◽  
Snezana Djokic ◽  
Sigrid Hoffmann ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Transgenic Animals ◽  
Mouse Retina ◽  
Vascular Density ◽  
Wild Type ◽  
Transgenic Mouse Line ◽  
Oxygen Induced Retinopathy ◽  
Genes Encoding ◽  
Angiopoietin 2 ◽  
The Impact

SummaryAngiopoietin-2 (Ang2) is among the relevant growth factors induced by hypoxia and plays an important role in the initiation of retinal neovascularizations. Ang2 is also involved in incipient diabetic retinopathy, as it may cause pericyte loss. To investigate the impact of Ang2 on developmental and hypoxia-induced angiogenesis, we used a transgenic mouse line overexpressing human Ang2 in the mouse retina. Transgenic mice displayed a reduced coverage of capillaries with pericytes (-14 %; p<0.01) and a 46% increase of vascular density of the capillary network at postnatal day 10 compared to wild type mice. In the model of oxygen-induced retinopathy (OIR), Ang2 overexpression resulted in enhanced preretinal (+103%) and intraretinal neovascularization (+29%). Newly formed intraretinal vessels in OIR were also pericyte-deficient (-26 %; p<0.01). The total expression of Ang2 in transgenic mice was seven-fold, compared with wild type controls. Ang2 modulated expression of genes encoding VEGF (+65%) and Ang1 (+79%) in transgenic animals. These data suggest that Ang2 is involved in pericyte recruitment, and modulates intraretinal, and preretinal vessel formation in the eye under physiological and pathological conditions.

Spermatozoa from a marsupial, the brushtail possum, contain β1,4-galactosyltransferase

2008 ◽  
Vol 20 (3) ◽  
pp. 402 ◽  
Author(s):  
A. G. Braundmeier ◽  
William G. Breed ◽  
D. J. Miller
Keyword(s):  
Molecular Weight ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Brushtail Possum ◽  
Laboratory Mice ◽  
Sperm Surface ◽  
Protein Functions ◽  
Sperm Acrosome Reaction ◽  
Porcine Spermatozoa ◽  
Egg Coat

β1,4-Galactosyltransferase-I (GalTase-I) is one of the key molecules on the sperm surface of eutherian mammals that is likely to be involved in binding to the egg coat, the zona pellucida, to mediate sperm–egg interaction. In laboratory mice, the species for which most data are available, this protein functions as a receptor for the zona pellucida protein ZP3 of the oocyte and, upon binding, triggers the sperm acrosome reaction. In the present study, we investigated the presence and abundance of GalTase-I in epididymal sperm extracts of a marsupial, the brushtail possum, Trichosurus vulpecula. For this, spermatozoa were collected from cauda epididymides and the amount of β1,4-galactosyltransferase activity in washed sperm extracts was compared with that of porcine spermatozoa. Overall β1,4-galactosyltransferase enzyme activity was found to be more abundant in possum sperm extracts than those from porcine spermatozoa (P < 0.05). Immunoblots with an antibody to mouse GalTase-I revealed that the molecular weight of possum spermatozoa GalTase-I was 66 kDa, which is similar to the molecular weight of GalTase-I in spermatozoa from eutherian mammals. The molecular weight of GalTase-I was the same in sperm extracts collected from the caput and cauda epididymides. These results demonstrate that GalTase-I is indeed present in possum spermatozoa and thus it may be a gamete receptor molecule on the sperm surface of marsupials as well as those of eutherian mammals.

A simple polymerase chain reaction assay for genotyping the retinal degeneration mutation (Pdebrd1) in FVB/N-derived transgenic mice

2001 ◽  
Vol 35 (2) ◽  
pp. 153-156 ◽  
Author(s):  
E. Giménez ◽  
L. Montoliu
Keyword(s):  
Polymerase Chain Reaction ◽  
Transgenic Mice ◽  
Retinal Degeneration ◽  
Inbred Mice ◽  
Transgenic Animals ◽  
Mouse Strains ◽  
Rod Photoreceptor ◽  
Wild Type ◽  
Chain Reaction ◽  
Polymerase Chain

FVB/N mice are one of the most common inbred strains for the generation of transgenic animals. This mouse strain is preferred for transgenesis because of its fertilized oocytes, which have unique pronuclei for microinjection, and its vigorous reproductive performance along with consistently large litter sizes. However, these inbred mice carry a retinal degeneration mutation caused by a proviral insertion into the Pdeb gene, encoding the β subunit of cGMP phosphodiesterase. This mutation ( Pdebrd1, formerly known as rd) results in postnatal rod photoreceptor degeneration and causes severe visual impairment, which may be relevant for behavioural and vision-related research. This deficit can be overcome by crossing these mice with other mouse strains carrying the wild-type allele at the Pdeb locus. We have devised a simple polymerase chain reaction (PCR)-based method for distinguishing between the mutant and the wild-type alleles, thus allowing the efficient monitoring of the Pdebrd1 mutation in FVB/N-derived transgenic mice prior to experimentation where visual deficit is expected to have an influence in the phenotype.

Kidney function in ANF-transgenic mice: effect of blood volume expansion

1991 ◽  
Vol 260 (1) ◽  
pp. R1-R5 ◽  
Author(s):  
L. J. Field ◽  
A. T. Veress ◽  
M. E. Steinhelper ◽  
K. Cochrane ◽  
H. Sonnenberg
Keyword(s):  
Transgenic Mice ◽  
Kidney Function ◽  
Blood Volume ◽  
Volume Expansion ◽  
Fusion Gene ◽  
Transgenic Animals ◽  
Renal Perfusion ◽  
Arterial Blood ◽  
Blood Volume Expansion ◽  
Before And After

Transgenic mice, created from inbred C3HeB/FeJ embryos, were used to overexpress selectively in the liver a fusion gene comprising mouse transthyretin (TTR) regulatory and atrial natriuretic factor (ANF) structural sequences. Animals were anesthetized, and kidney function was studied before and after blood volume expansion. Baseline urine volumes and electrolyte excretions were not significantly different from those of non-transgenic littermates, despite a markedly lower arterial blood pressure in the experimental group. A slightly lower glomerular filtration rate (GFR) in transgenics was not different statistically. Plasma ANF levels measured by radioimmunoassay were approximately 10-fold higher in the transgenic animals, compared with their nontransgenic siblings. After acute blood volume expansion, the diuretic, natriuretic, kaliuretic, and chloruretic responses were markedly enhanced in the transgenic group. Arterial pressure was increased as a result of hypervolemia, although it remained relatively depressed relative to the controls. GFR again was not different. We conclude that transgenic mice overexpressing ANF can maintain normal excretion of salt and water, possibly via ANF-induced reduction of renal perfusion pressure. After acute blood volume expansion, an increase in pressure may allow full renal expression of the chronically elevated ANF levels.

Adaptive regulation of intestinal thiamin uptake: molecular mechanism using wild-type and transgenic mice carrying hTHTR-1 and -2 promoters

2005 ◽  
Vol 288 (6) ◽  
pp. G1127-G1134 ◽  
Author(s):  
Jack C. Reidling ◽  
Hamid M. Said
Keyword(s):  
Transgenic Mice ◽  
Molecular Mechanism ◽  
Transgenic Animals ◽  
Mrna Levels ◽  
Wild Type ◽  
Cellular Functions ◽  
Thiamin Deficiency ◽  
Adaptive Regulation ◽  
Transcriptional Regulatory Mechanism ◽  
Uptake Process

Thiamin participates in metabolic pathways contributing to normal cellular functions, growth, and development. The molecular mechanism of the human intestinal thiamin absorption process involves the thiamin transporters-1 (hTHTR-1) and -2 (hTHTR-2), products of the SLC19A2 and SLC19A3 genes. Little is known about adaptive regulation of the intestinal thiamin uptake process or the molecular mechanism(s) involved during thiamin deficiency. In these studies, we addressed these issues using wild-type mice and transgenic animals carrying the promoters of the hTHTR-1 and -2. We show that, in thiamin deficiency, a significant and specific upregulation in intestinal carrier-mediated thiamin uptake occurs and that this increase is associated with an induction in protein and mRNA levels of mTHTR-2 but not mTHTR-1; in addition, an increase in the activity of the SLC19A3, but not the SLC19A2, promoter was observed in the intestine of transgenic mice. Similar findings were detected in the kidney; however, expression of both thiamin transporters and activity of both human promoters were upregulated in this organ in thiamin deficiency. We also examined the effect of thiamin deficiency on the level of expression of mTHTR-1 and mTHTR-2 messages and activity of the human promoters in the heart and brain of transgenic mice and found an increase in mTHTR-1 mRNA and a rise in activity of the SLC19A2 promoter in thiamin-deficient mice. These results show that the intestinal and renal thiamin uptake processes are adaptively upregulated during dietary thiamin deficiency, that expression of mTHTR-1 and mTHTR-2 is regulated in a tissue-specific manner, and that this upregulation is mediated via transcriptional regulatory mechanism(s).

Behavioural changes induced by a conditional disruption of bone formation

2016 ◽  
Vol 27 (3) ◽  
Author(s):  
Andreas Zimmer ◽  
David-Marian Otte ◽  
Andras Bilkei-Gorzo ◽  
Samar Muhammad Armin ◽  
Itai Bab
Keyword(s):  
Bone Formation ◽  
Transgenic Mice ◽  
Bone Remodelling ◽  
Transgenic Animals ◽  
Volume Density ◽  
Wild Type ◽  
Parasympathetic Nerve ◽  
Brain Functions ◽  
Simplex Virus ◽  
The Brain

AbstractIt has been shown that the brain regulates bone remodelling through sympathetic and parasympathetic nerve fibres. However, it is unclear if signals from the skeleton also influence brain functions and animal behaviours.Bone formation was conditionally disrupted by daily injections of aciclovir (10 mg/kg) to transgenic mice expressing a herpes-simplex-virus thymidine kinase under the control of the osteoblast-specific promoter of the Bglap gene. Behavioural studies were conducted after 10 weeks of treatment.Transgenic mice receiving aciclovir injections showed a reduced number of osteoblasts with a concomitantly reduced trabecular bone volume density, when compared to wild-type controls that were treated identically. The general health of the animals was not severely affected, as indicated by a similar increase in body weight, similar activity profiles and similar social behaviours. However, transgenic mice showed significantly increased despair behaviour and increased adrenal gland weights.Specific animal behaviours can be modulated by a selective disruption of bone formation. The increased despair behaviour observed in transgenic animals indicates that these animals may be more prone to depression-related phenotypes. These findings are important in the context of the well-established clinical association between depression and reduced bone mass.

scholarly journals Chronic hepatitis, hepatocyte fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutant.

1995 ◽  
Vol 131 (5) ◽  
pp. 1303-1314 ◽  
Author(s):  
N O Ku ◽  
S Michie ◽  
R G Oshima ◽  
M B Omary
Keyword(s):  
Chronic Hepatitis ◽  
Transgenic Mice ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Relative Increase ◽  
Wild Type ◽  
Intermediate Filament Proteins ◽  
Keratin 18 ◽  
Filament Networks

The two major intermediate filament proteins in glandular epithelia are keratin polypeptides 8 and 18 (K8/18). To evaluate the function and potential disease association of K18, we examined the effects of mutating a highly conserved arginine (arg89) of K18. Expression of K18 arg89--&gt;his/cys and its normal K8 partner in cultured cells resulted in punctate staining as compared with the typical filaments obtained after expression of wild-type K8/18. Generation of transgenic mice expressing human K18 arg89--&gt;cys resulted in marked disruption of liver and pancreas keratin filament networks. The most prominent histologic abnormalities were liver inflammation and necrosis that appeared at a young age in association with hepatocyte fragility and serum transaminase elevation. These effects were caused by the mutation since transgenic mice expressing wild-type human K18 showed a normal phenotype. A relative increase in the phosphorylation and glycosylation of detergent solubilized K8/18 was also noted in vitro and in transgenic animals that express mutant K18. Our results indicate that the highly conserved arg plays an important role in glandular keratin organization and tissue fragility as already described for epidermal keratins. Phosphorylation and glycosylation alterations in the arg mutant keratins may account for some of the potential changes in the cellular function of these proteins. Mice expressing mutant K18 provide a novel animal model for human chronic hepatitis, and for studying the tissue specific function(s) of K8/18.

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