Tubal transfer of goat embryos using endoscopy.
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Animals↗
Biomedical subjects
G Brem
Publications and source records attributed to G Brem.
Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes.
Cumulus cell-enclosed bovine oocytes in germinal vesicle (GV) and in metaphase II (MII) stages were cryopreserved. Different concentrations (1 M; 1.5 M) of various cryoprotectants (glycerol, PROH, DMSO) were tested. After thawing, the oocytes were exposed to various carbohydrates (sucrose, lactose, trehalose) at a concentration of 0.1 M and 0.25 M for cryoprotectant removal. Developmental capacity of the frozen-thawed oocytes was studied by in vitro maturation, fertilization and culture. We found no difference in subsequent development using glycerol or PROH for GV and MII oocytes. The DMSO treatment led to significantly better cleavage and development up to 4-cell stage in MII oocytes. Development beyond the 8-cell stage was obtained only when unmatured oocytes were frozen. No difference in the efficiency of the 3 cryoprotectants was detected in MII oocytes. However, in GV oocytes, glycerol and PROH yielded significantly better cleavage and 4-cell rate compared to DMSO (P<0.001). Influence of the concentration of a cryoprotectant on development was not observed in GV or MII oocytes. Among the 3 cryoprotectants, DMSO was less suitable, at both concentrations, than PROH and glycerol for the development of 6- to 8-cell stage embryos in the GV group. In the MII group, 1.5 M DMSO was as efficient as PROH and as glycerol at a 1.5-M concentration, and it was more efficient than 1 M glycerol. The use of carbohydrates during rehydration did not render a beneficial effect at either of the 2 concentrations, and when no carbohydrates were used in the MII group the oocytes cleaved better than GV oocytes.
Journal Article↗
Laparoscopy through the vaginal fornix of cows for the repeated aspiration of follicular oocytes.
A simple method is described for the repeated laparoscopic examination of the internal reproductive organs of cows and heifers through the vaginal fornix. It can be performed in a simple crush in less than 15 minutes, does not require surgery and can be used under field conditions. The method has been used for aspirating oocytes from follicles which were at least 2 mm in diameter in animals under sedation and epidural anaesthesia. In a preliminary study 11 cows and eight heifers were allocated into two groups: 12 animals were treated weekly with 500 iu pregnant mare's serum gonadotrophin and seven animals were not stimulated with gonadotrophin. The mean numbers of oocytes collected from the treated cows (6.3) and heifers (3.3) did not differ significantly from the numbers collected from the stimulated cows (5.5) and heifers (4.0). After the procedure had been established a mean oocyte collection rate of up to 75 per cent of follicles aspirated was obtained in 12 unstimulated heifers. When follicles were aspirated twice instead of once a week, the mean number of follicles observed (16.2 vs 7.0) and the mean number of oocytes collected per week (12.2 vs 5.2) were significantly higher (P < 0.05).
Animals↗
A new familial glomerulonephropathy in Bernese mountain dogs.
Between 1989 and 1992, 22 Bernese mountain dogs (18 females and four males) aged between two and seven years, which had been suffering for some weeks from weight loss, anorexia, apathy, vomiting, polydipsia and polyuria, were examined. All of them had high blood urea nitrogen and serum creatinine concentrations, and many had hyperphosphataemia, hypercholesterolaemia, hypoproteinaemia and nonregenerative anaemia. All the dogs had very high protein: creatinine ratios in the urine, and macroproteinuria was identified by sodium dodecyl sulphate gel electrophoresis. The immunofluorescent titres against Borrelia burgdorferi, measured in 19 of the dogs, ranged between 256 and 32,768. In all cases, membrano-proliferative glomerulonephritis with concomitant interstitial nephritis was diagnosed. From an analysis of the dogs' pedigree it was concluded that the glomerulonephritis of these Bernese mountain dogs was inherited as an autosomal recessive trait and that its expression was influenced by a second gene locus with a sex-linked dominance exchange.
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Negative regulatory element in the mammary specific whey acidic protein promoter.
Expression of the whey acidic protein (WAP) gene is tightly regulated in a tissue and developmental stage specific manner, in that the WAP gene is exclusively expressed in the mammary gland during pregnancy and lactation. Using both deletion and competition analyses, evidence is provided for the existence of a negative regulatory element (NRE) in the WAP promoter located between -413 and -93 with respect to the WAP transcriptional initiation site. This NRE dramatically decreases transcription from linked heterologous promoter-reporter gene constructs. The activity of NRE requires WAP promoter sequences that are 230 bp apart since subfragments of the NRE fail to inhibit transcription of adjoining reporter genes. Nuclear extracts from different cell types, in which the WAP gene is not active, contain a protein or complex that specifically interacts with the entire NRE but not with subfragments of it. The contact points between this protein (NRE binding factor [NBF]) and the NRE element have been partially determined. Mutation of the implicated nucleotides severely reduces the ability of NBF to bind, and such mutated promoter fragments fail to alleviate transcriptional repression in competition experiments. This suggests that NBF binding to the NRE is at least in part responsible for the negative regulation of the WAP promoter. Since NBF is not detectable in the lactating mammary gland, where the WAP gene is expressed, we speculate that it may be a determinant of the expression spectrum of the WAP gene.
Animals↗
Sensitive detection of human growth hormone mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues by non-isotopic in situ hybridization.
A sensitive technique of non-isotopic in situ hybridization (NISH) is presented, which permits the detection of human growth hormone (hGH) mRNA in routinely formalin-fixed, paraffin-embedded transgenic mouse tissues and human post mortem pituitaries; the latter were used as positive tissue controls in this study. In addition, a double staining procedure combining NISH and immunohistochemistry for the visualization of both hGH and hGH mRNA in the same paraffin section is described. Digoxigenin-labelled antisense hGH RNA was used for NISH of hGH mRNA. The NISH protocol was based upon an established radioactive method. Alkaline phosphatase and horseradish peroxidase-based immunoenzymatic procedures for the detection of digoxigenin-labelled RNA probes using different chromogens [4-nitro blue tetrazolium chloride (NBT), Fast Blue BB, New Fuchsin, and 3,3'-diaminobenzidine tetrahydrochloride (DAB) with or without intensification of the DAB staining] were compared. The proteolytic tissue pretreatment and the detection procedure were found to be the most critical steps for successful visualization of hGH mRNA. After optimization of the permeabilization conditions, hGH mRNA could be visualized in each case studied when alkaline phosphatase/NBT-based detection was employed. The NISH technique presented here, performed either separately or in combination with immunohistochemistry, permits retrospective analyses, of hGH (trans)gene expression in archival, paraffin-embedded specimens.
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Efficient generation of chimaeric mice using embryonic stem cells after long-term culture in the presence of ciliary neurotrophic factor.
The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate their in vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.
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DNase I hypersensitive sites far upstream of the rat tryptophan oxygenase gene direct developmentally regulated transcription in livers of transgenic mice.
Expression of the gene coding for tryptophan oxygenase (TO) is switched on in rat liver about two weeks after birth. We identified two clusters of DNaseI hypersensitive (HS) sites in the TO gene upstream region; one near the promoter, the other at a distant upstream location (-8.5 kb). Hypersensitivity of upstream sites was present in adult and in 7 day old rat liver, but absent in kidney. To investigate their role in transcriptional regulation, a reporter gene controlled by both HS site regions was used to generate transgenic mice. In these animals the transgene followed the cell specific and developmental regulation of the endogenous gene: inactive after birth and active in adult liver. Transgenes containing only the promoter proximal HS site were non-functional.
Aging↗
Secretory pattern of metabolic hormones in the lactating sow.
Two experiments were conducted to monitor hormonal changes during lactation in crossbred sows (Pietrain x German Landrace). Sows were fed twice daily without weighing the remaining food. Number of piglets was not standardized. Plasma concentrations of growth hormone (GH), prolactin (PRL), insulin-like growth factor-1 (IGF-1), IGF-2, insulin (INS), triiodothyronine (T3), thyroxin (T4), free thyroxin (FT4), non esterified fatty acids (NEFA) and glucose (GLUC) were determined by RIA, EIA or enzymatically. In exp. A (n = 5 sows), blood samples were taken via permanent jugular cannula in weekly 24 h windows at 20 min intervals and additionally once daily for 6 weeks during lactation and for 3 days after weaning. In exp. B (n = 24 sows), blood was collected by needle puncture of the ear vein 2 and 1 week before parturition, the 1st and 3rd-4th week of lactation and 1 and 2 weeks after weaning. GH (0.8 ng/ml) and PRL (10.2 ng/ml) increased with onset of lactation (3.3 resp. 91.5 ng/ml), remained at high levels (2.5-2.8 resp. 39-41 ng/ml) during the 2nd and 3rd week, declined slowly thereafter and considerably after weaning to concentrations of 0.7 resp. 2.7 ng/ml. During lactation in 4 of 5 sows in exp. A, the typical episodic secretory pattern of GH and PRL was lost due to frequent suckling. Basal values, as known from non lactating sows, were not reached and number of pulses was elevated during lactation for both pituitary hormones. Insulin levels showed a high individual variation.(ABSTRACT TRUNCATED AT 250 WORDS)
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Transgenic strategies to increase disease resistance in livestock.
Approaches to modify disease resistance or susceptibility by transgenic means will be a major asset to animal welfare as well as to the economics of animal production. Candidates for gene transfer experiments include all genes known to influence non-specific and specific host defence mechanisms against infectious pathogens. Additional strategies such as 'intracellular immunization', 'genetic and congenital immunization', antisense RNA approaches and targeted disruption of disease susceptibility genes promise to gain importance in conferring increased disease resistance. The cytokine network regulates cellular viability, growth and differentiation in physiological and pathophysiological states. Detailed understanding of cytokine signal transduction pathways and transcriptional activators will provide not only new target molecules for modulating the immune response but will also facilitate the elucidation of host-pathogen interactions.
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Familial nephropathy in Bernese mountain dogs.
Between January 1988 and March 1992 nephropathies were frequently diagnosed in Bernese Mountain Dogs. During this period, 20 animals (16 females, four males), ages 2-5 years (average age at time of diagnosis = 3.3 years) presented with clinically renal insufficiency. Morphologic diagnosis of the renal lesions was identical in all cases, i.e., membranoproliferative glomerulonephritis (MPGN) with concomitant interstitial nephritis. Deposits of immunoglobulin-M (IgM) and of the third complement component were regularly demonstrated immunohistochemically in the glomeruli; deposits of immunoglobulin-A (IgA) and immunoglobulin-G (IgG) were found only in isolated cases. Reduplication of glomerular basement membranes, mesangial interposition, and subendothelial deposits of the immunocomplex type were also detected by electron microscopy. A pedigree analysis indicated that the MPGN in these 20 Bernese Mountain Dogs of approximately the same age was of hereditary genesis. Thus, MPGN should be allocated to the group of familial nephropathies. Serologically, high IgG titers against Borrelia burgdorferi were found in 17 dogs. These findings are discussed in relation to familial nephropathies in humans.
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Consequences of postnatally elevated insulin-like growth factor-II in transgenic mice: endocrine changes and effects on body and organ growth.
Insulin-like growth factor-II (IGF-II) is an important regulator of embryonic growth and differentiation, but its function in postnatal life is unclear. To address this point, we generated transgenic mice harboring fusion genes in which a human IGF-II complementary DNA is placed under the transcriptional control of the rat phosphoenolpyruvate carboxykinase promoter. Transgene-specific messenger RNA was detected in liver, kidney, and several parts of the gut. Serum IGF-II levels in transgenic mice were 2-3 times higher than those in controls and increased after starvation. Circulating IGF-I correlated negatively and IGF-binding protein-2 (IGFBP-2) positively with IGF-II levels, suggesting that IGF-I is displaced from IGFBPs by IGF-II and that IGF-II is a major regulator of IGFBP-2. Serum levels of IGFBP-3 and IGFBP-4 tended to be higher in phosphoenolpyruvate carboxykinase-IGF-II transgenic mice than in controls, as evaluated by ligand blot analysis. Starvation reduced serum IGF-I, but increased IGFBP-2 in transgenic mice more markedly than in controls. Fasting insulin levels were significantly reduced in transgenic mice, whereas glucose levels were not influenced by elevated IGF-II. The body growth of 4- and 12-week-old mice was not significantly influenced by elevated IGF-II, but transgenic mice displayed increased kidney and testis weight at the age of 4 weeks, and increased adrenal weight at the age of 12 weeks. Our results demonstrate that elevated IGF-II in postnatal life has multiple endocrine consequences and subtle time-specific effects on organ growth.
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Characterization of a cardiac-selective and developmentally upregulated promoter in transgenic mice.
Transcriptional regulatory mechanisms which mediate cardiac-specific gene expression have not yet been completely understood. Potential cardiac-specific promoter sequences, sharing similar protein binding motives, show either coexpression in skeletal muscle, local restriction to the atrium or late onset of expression during fetogenesis. Based on in situ hybridization studies that indicated the expression of the cardiac myosin-light-chain-2 (MLC-2) gene in ventricular myocardium and in the lower outflow tract, a model system for selective targeting of foreign genes to the heart of transgenic mice has been developed. The regulatory promoter element was derived from the rat cardiac MLC-2 gene. 2100 bp of the 5' regulatory MLC-2 sequences were found to drive constitutive cardiac expression of a firefly luciferase reporter gene from early tubular heart formation. During ventricular loop and septum formation luciferase activity was 10-fold upregulated in comparison to steady-state levels observed 10 days after birth. No luciferase activity was detectable in any other muscle or non-muscle tissue of transgenic mice. These data suggest that the 2.1 kb DNA sequences of the 5' flanking region of the cardiac MLC-2 gene contain sufficient regulatory elements for a selective gene expression in cardiac myocytes from embryogenesis. The transgenic model should aid in determining the influences of pathogenic gene products on developing and mature heart muscle to elucidate the etiology of myocardial diseases such as cardiomyopathies.
Animals↗
Disruption of the CNTF gene results in motor neuron degeneration.
CNTF is a cytosolic molecule expressed postnatally in myelinating Schwann cells and in a subpopulation of astrocytes. Although CNTF administration prevents lesion-mediated and genetically determined motor neuron degeneration, its physiological function remained elusive. Here it is reported that abolition of CNTF gene expression by homologous recombination results in a progressive atrophy and loss of motor neurons in adult mice, which is functionally reflected by a small but significant reduction in muscle strength.
Aging↗
Piglets born after transcervical transfer of embryos into recipient gilts.
A method is described for the non-surgical transfer of embryos in pigs. Embryos at the 8-cell to the hatched blastocyst stage, recovered on days 4 to 7 of the oestrous cycle by flushing the oviducts and uteri of superovulated donors, were transferred transcervically into the uterine body of anaesthetised recipient gilts using a sterile disposable plastic spiral catheter and an embryo transfer cannula. Fifty-eight non-surgical transfers have been performed and six pregnancies were established. Eight and three normal fetuses were recovered from two recipients slaughtered between 35 and 45 days after embryo transfer. Three recipients came to term and gave birth to litters of two, six and seven living piglets. One recipient aborted between 45 and 60 days of gestation.
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Inheritance and tissue-specific expression of transgenes in rabbits and pigs.
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Animals↗
Genomic organization of the porcine skeletal muscle ryanodine receptor (RYR1) gene coding region 4624 to 7929.
Excitation-contraction coupling in skeletal muscle is mediated by two calcium channels located in the membranes of the transverse tubule and the sarcoplasmic reticulum. Calcium is released from the terminal cisternae of the sarcoplasmic reticulum via the ryanodine receptor. Abnormal increases in myoplasmic free calcium caused by a defect in the ryanodine receptor have been reported in malignant hyperthermia. Malignant hyperthermia is a life-threatening pharmacogenetic disorder in a variety of species and is triggered by volatile anesthetics and depolarizing muscle relaxants. To study the genomic organization of the porcine skeletal muscle ryanodine receptor gene, we have isolated six genomic fragments spanning approximately 80 kb of chromosomal DNA. In this report, we describe the genomic organization of a 15.5-kb genomic fragment comprising 18 exons coding for region 4624 to 7929 of the porcine skeletal muscle ryanodine receptor gene.
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Effects of long-term elevated serum levels of growth hormone on life expectancy of mice: lessons from transgenic animal models.
In this study, we characterize transgenic mice carrying fusion genes, in which the genes coding for human (h) or bovine (b) growth hormone (GH) have been put under the transcriptional control of the mouse metallothionein I (MT) or the rat phosphoenolpyruvate carboxykinase (PCK) promoter as models for investigating the long-term effects of elevated GH on life expectancy. Circulating GH concentrations ranged from 3000 to 900,000 ng/ml, from 320 to 2960 ng/ml and from 34 to 1050 ng/ml in transgenic mice belonging to the MThGH, the PCKbGH and the MTbGH groups, respectively, and were high on a short-, medium-, and long-term basis. As a consequence of excess GH in their serum, GH transgenic mice exhibited drastically reduced life span which was primarily due to severe kidney lesions (glomerular hypertrophy, sclerosis and hyalinosis associated with tubulo-interstitial changes) consistently found in these animals. Alterations of the liver observed in transgenic mice included both hepatocellular megaly and various degrees of regressive, regenerative and fibrotic changes. In older MTbGH and PCKbGH transgenic mice, hepatocellular neoplasms including both adenoma and carcinoma were frequently found in addition to non-neoplastic changes. Our study points out the suitability of GH transgenic mice to evaluate the effects of various levels of GH in long-term studies without having to take antibody production against the heterologous hormone into account. Findings in GH transgenic animals suggest that the long-term benefits and risks of GH therapy should be carefully evaluated.
Aging↗