Biomedical subjects

E Wolf

Publications and source records attributed to E Wolf.

At least 127 records · Page 7Linked to original sources

PubMed1999

Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells.

Porcine primordial germ cell (PGC) derived cell lines of WAPhGH-transgenic pigs have been established that were able to contribute to chimeras. PGCs were isolated from day 25 to 28 genital ridges of more than 30 individual transgenic fetuses in order to have an easy to follow marker gene. To support undifferentiated growth, cell lines were derived and stable maintained on STO no. 8 feeder cells, a murine embryonic fibroblast cell line expressing recombinant, membrane-bound porcine stem cell factor (SCF). Fifteen lines proliferated in an undifferentiated state up to passage 13; two lines were maintained for more than 23 passages. Cell staining experiments for differentiation markers in several cell lines, indicated the presence of pluripotent cells in prolonged cultures. Further characterization using karyotyping revealed a normal, euploid set of chromosomes in cells of passages 15 and higher. Pluripotency of freshly isolated, short-term (up to 24 hr before injection) and long-term cultured, frozen/thawed cells was tested by injection into day 6 recipient blastocysts to give rise to chimeric piglets. The injected embryos (n = 209) were endoscopically transferred into the uterine horns of 11 recipient gilts. Tissue analysis from 49 fetuses and eighteen liveborn piglets for PGC contribution in chimeras was carried out using PCR analysis for the presence of the marker transgene. Thirty-two fetuses showed detectable chimerism in up to five out of 12 tissues analyzed. Skin samples from eight piglets were positive for the transgene, four of them displayed coat colour chimerism.

Animals↗

PubMed1999

Adult cloning in cattle: potential of nuclei from a permanent cell line and from primary cultures.

Nuclear transfer was used to evaluate the developmental potential of nuclei from a spontaneously immortalized bovine mammary gland epithelial cell line (MECL) and from primary cultures of mammary gland cells (PMGC) and ear skin fibroblasts (PESF) established from 3-year-old cows. Cell proliferation was investigated by incorporation and detection of 5-bromo-2'-deoxyuridine (BrdU). The proportion of cells in S-phase was significantly (P < 0.05) higher for MECL cells than for PMGC and PESF, both in the presence of serum (90% vs. 28% and 15%) and following serum starvation (27% vs. 6% and 3%). Nuclei from PESF supported the development of reconstructed embryos to the blastocyst stage significantly better than those of PMGC (60% vs. 26%; P < 0.05). Embryos reconstructed with cells from MECL failed to develop to blastocysts. After transfer of embryos derived from PMGC and PESF, respectively, 2/2 and 5/12 recipients were pregnant on day 42. On day 90, the corresponding pregnancy rates were 2/2 and 3/12. One live calf derived from a PMGC was born at day 287 of gestation. Another live PESF-derived calf was delivered by caesarean section at day 286 of gestation. Our study suggests that nuclei from primary cultures of adult cells can be successfully reprogrammed by nuclear transfer, whereas nuclei from a permanent cell line failed to support the development of nuclear transfer embryos.

Animals↗

PubMed1999

[Cytomegalovirus (CMV) retinitis in AIDS. Gancilovir implantation in comparison with systemic therapy].

INTRODUCTION: Untreated CMV retinitis with AIDS leads to blindness; therefore, a life-long virostatic treatment is required. It can either be administered systemically or locally, as there are different advantages and disadvantages. When treating patients we aim at therapy that preserves vision without diminishing quality of life. It should induce as few drug-induced side effects as possible and not shorten the patient's life expectation. METHODS AND RESULTS: A total of 111 patients (150 eyes) with systemic maintenance treatment were compared retrospectively with 33 patients (62 eyes) that received a ganciclovir implant only as maintainance therapy and no additional systemic treatment. Patients with an implant showed a prolonged interval of nonprogression of retinitis than patients receiving systemic treatment. Patients with unilateral retinitis are at higher risk of developing bilateral disease in the implant group than in the systemically treated group. Manifestation of extraocular disease was equal in both groups. Local treatment with the implant does not shorten patient survival time. CONCLUSION: Local treatment with the ganciclovir implant means quality of life for patients and also safe protection of the affected eye. Extraocular disease and survival time are not influenced adversely by local treatment. However, primarily unilateral involved patients show higher risk for bilateral disease in the implant group than in the systemically treated group.

AIDS-Related Opportunistic Infections↗

PubMed1999

A non-destructive technique for 3-D microstructural phenotypic characterisation of bones in genetically altered mice: preliminary data in growth hormone transgenic animals and normal controls.

A non-destructive, three-dimensional technique for microstructural phenotypic characterisation of skeletal elements in genetically altered mice is presented. Preliminary data in bovine growth-hormone transgenic animals and control littermates are shown. The technique is based on microcomputed tomography (microCT) and digital postprocessing and allows for a differential quantitative analysis of the cortical and trabecular bone compartments in the axial and peripheral skeleton. The distal femora and the first lumbar vertebral bodies of six animals were CT scanned in the axial plane with an isotropic resolution of 20 microm. The periostal surface and the marrow spaces were segmented fully automatically, and the trabecular and cortical compartments were separated interactively. After 3-D reconstruction, various regions of interest (diaphyseal, metaphyseal and epiphyseal) were selected for the analysis. The femora and vertebrae of the transgenic animals showed obvious differences in size, shape, and trabecular arrangement compared with the control animals. The total bone mass was increased by a factor of two to three, but the trabecular bone was increased much more (up to 12 times) than the cortical bone. The transgenic animals showed an increased ratio of trabecular vs cortical bone (0.90 to 1.27 vs 0.14 to 0.36 in the femoral diaphysis) and an elevated trabecular bone volume fraction (49% to 73% vs 18% to 43% in the femoral metaphysis). The mean 3-D cortical thickness was similar in the normal and transgenic animals (values between 93 microm and 232 microm in the dia- and metaphyses), but the minimal cortical thickness was lower in the transgenic animals (22 to 31 microm vs 54 microm to 110 microm in the diaphysis). The technique presented is suitable for phenotypic characterisation of bone structure in genetically altered mice.

Animals↗

PubMed1999

Factors affecting proliferation and dedifferentiation of primary bovine oviduct epithelial cells in vitro.

The oviduct is the physiological site for key events in reproduction, such as capacitation of spermatozoa, fertilization and early embryonic development. Interactions between oviduct epithelial cells and gametes or embryos cannot sufficiently be studied in vivo. Therefore, model systems are needed which mimic in vivo conditions most closely. In this study we optimised the method for isolating bovine oviduct cells and compared different cell support materials as well as two culture systems (perfusion vs static culture) for their ability to maintain characteristic morphological and functional features of oviduct cells. Out of nine different cell support materials tested, cellulose nitrate (0.45 micron pore size) was the most suitable to maintain cells in a manner similar to freshly isolated oviduct epithelial cells. Comparing static vs perfusion culture by electron microscopy, morphological differences of the cells were insignificant in the first days of culture, while they became more evident after 8 days. The cells in the static system lost typical characteristics such as columnar shape, cilia and secretory protrusions, while these features were still present in perfusion culture. In addition, intense ciliogenesis and cytoplasmic organelles for protein synthesis were found under perfusion conditions. These findings were underlined by differences in expression of the oviduct-specific oestrus-associated glycoprotein 85-97 kDa (GP 85-97) gene as revealed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The RNA levels of this specific gene were significantly higher in perfusion compared to the static culture system. Our data show clear advantages of perfusion vs static culture for primary bovine oviduct epithelial cells.

Actins↗

PubMed1999

Insulin-like growth factors and IGF-binding proteins in bovine seminal plasma.

Insulin-like growth factor-I (IGF-I) is an important factor for germ cell development and maturation of spermatozoa. Actions of IGFs are modulated by IGF-binding proteins (IGFBPs) that may, depending on their concentration and site of expression, inhibit or enhance effects of IGF-I. We characterized IGFs and IGFBPs in seminal plasma from bulls routinely used for artificial insemination (AI) and from bulls producing poor-quality semen (low mass and individual motility of spermatozoa). IGFs were measured by specific radioimmunoassay in 22 samples of seminal plasma from nine different AI bulls with high (> 76.8%), average (72.8-73.4%), or low (< 69.5%) nonreturn rate (NRR). IGF-I and IGF-II levels were 144 +/- 9 ng/ml (mean +/- SE; range, 79-238 ng/ml) and 144 +/- 10 ng/ml (range, 55-221 ng/ml), respectively, and did not correlate with NRRs. IGF-I concentrations in seminal plasma from bulls producing poor-quality semen (n = 10) were significantly (P < 0.05) greater (194 +/- 26 ng/ml; range, 94-370 ng/ml), whereas IGF-II levels were significantly (P < 0.05) lower (93 +/- 17 ng/ml; range, 38-183 ng/ml) than in AI bulls. Ligand blot analysis of seminal plasma for IGFBPs revealed the presence of a 38-/45-kDa doublet band and a 30-kDa IGFBP. These IGFBPs were identified as IGFBP-3 and IGFBP-5, respectively, by immunoprecipitation using specific antibodies. In addition, a low amount of IGFBP-4 was detected in bovine seminal plasma by immunoprecipitation. There was a marked difference in the activity of IGFBPs between individual bulls, with a relatively small within-bull variance. The differences in IGFBP activities did not correlate with the fertilization capacity of the bulls in vivo or in vitro nor with immunoreactive IGF-I and IGF-II levels in seminal plasma. Our results demonstrate the presence of IGFBPs in bovine seminal plasma. In contrast to human seminal plasma, high activity of IGFBP-3 was detected in seminal plasma of some bulls, suggesting species-specific regulation of IGFBP activity by proteases.

Animals↗

PubMed1999

[Sex-specific analysis of bone mass in normal and growth hormone transgenic mice using dual energy x-ray absorptiometry (DXA)].

The aim of the present study was the non-invasive, sex-specific measurement of bone mass in bovine growth hormone (bGH) transgenic mice and normal controls with dual energy X-ray absorptiometry (DXA). The transgenic mouse constitutes a suitable animal model to study the influence of growth hormone on the skeletal system. We analysed 28 animals, aged 12 weeks (14 transgenic, 14 controls, 7 male and 7 female, respectively), using a peripheral DXA scanner that had been adapted to the measurement of small animals. At a measurement time of 20 min, the precision (RMS average CV%) was 4.4% for bone mass (BMC), 2.5% for areal bone density (BMD), 0.86% for total body weight and 4.5% for the percentage BMC (relative to body weight). While the absolute bone mass was not significantly different between male and female animals, we found a higher percentage of the BMC relative to the total bone mass in females (+21% in controls, +31% in transgenics; p < 0.01). The absolute bone mass was higher in the transgenic animals (+71% in females and +62% in males; p < 0.01), but relative to the body weight the transgenic females yielded similar and the transgenic males lower values (-7.2%; p < 0.05). Using DXA it is possible to non-invasively determine the mass of mineralised tissue in the mouse with relatively high precision and to effectively discriminate between different groups. Although a strong influence of growth hormone on the absolute bone mass is observed, the results show that this increase is not higher than that of the total body mass.

Absorptiometry, Photon↗

PubMed1999

Maintenance treatment with medroxyprogesterone acetate in patients with advanced breast cancer responding to chemotherapy: results of a randomized trial. Essen Breast Cancer Study Group.

The purpose of this randomized phase III trial was to study whether medroxyprogesterone acetate (MPA) maintenance treatment prolongs the time to progression in advanced breast cancer patients responding to an induction chemotherapy. Patients with progressive advanced breast cancer previously untreated with anthracylines and progestins were given epirubicin (30 mg/m2) and ifosfamide (2 g/m2) on days 1 and 8 at 3-weekly intervals. Patients without disease progression after 6 cycles of chemotherapy were randomly assigned to receive, until progression, either no treatment or MPA at a daily total dose of 500 mg. Ninety patients were randomized: 46 to the MPA arm and 44 to the observation arm. Median time to progression was longer in the MPA arm: 4.9 months versus 3.7 months in the intent-to-treat analysis (p = 0.02), and 4.9 months versus 3.0 months in the secondary efficacy analysis (p = 0.012). Seven patients were removed from MPA due to side effects. The changes in patient-rated quality of life scores were similar in both groups. The median length of survival from randomization was 17.4 months for patients receiving MPA and 18.3 months for patients randomized to observation (p = 0.39). In conclusion, in patients with advanced breast cancer achieving remission or non-progression with 6 cycles of epirubicin and ifosfamide chemotherapy, MPA maintenance treatment led to a significant, though modest, prolongation of the time to progression without affecting overall survival of the study patients.

Aged↗

PubMed1999

Pregnancy rate after ultrasound-guided follicle aspiration in nonlactating cows from different breeds.

Thirty-six cows (21 Simmental, 5 Holstein-Frisian, 5 Brown Swiss and 5 Charolais) with high genetic superiority were punctured by ultrasound-guided follicle aspiration within the last 18 months for 503 times under equal conditions. Follicle aspiration was done twice per week. Most of the donor cows suffered from several disturbances of fertility. On average, 5 oocytes per session were collected. After in vitro maturation (IVM), fertilisation (IVF) and culture (IVC), 0.8 embryos per puncture session were transferred. After evaluation of the embryos by morphological criteria, these embryos were transferred to heifers that were oestrus synchronised (2.0 ml Estrumate i.m.) seven days after onset of oestrus. On day 21 after onset of oestrus the progesterone level in plasma was determined by radioimmunoassay. The pregnancy control was performed by ultrasound on day 35. After transfer of 397 embryos (to synchronised heifers), 125 pregnancies were established. Comparison of the different breeds and donor cows revealed significant differences in the number of oocytes recovered, embryos produced and pregnancies established. Transfer of embryos with insufficient morphological quality resulted in increased rates of embryonic loss between day 21 and day 35.

Animals↗

PubMed1999

Overgrowth of skin in growth hormone transgenic mice depends on the presence of male gonads.

Growth hormone has been shown to possess stimulatory effects on various connective tissues. We observed that skin growth in male rat phosphoenolpyruvate carboxykinase-bovine growth hormone transgenic mice (serum growth hormone levels: 740-1940 ng per ml) is progressive with age, resulting in an "oversized coat" phenotype with a marked increase in absolute and relative skin weight and surface area, and in thickness of the dermis. Histologic changes include severe dermal fibrosis and replacement of subdermal adipose tissue by fibrous tissue. Apart from an increase in skin surface area, these changes were not noted in female transgenic mice, arguing for a specific interaction of growth hormone with male sex hormones. To clarify this point, 6 wk old male transgenic mice and control mice were castrated and compared with their noncastrated counterparts in parameters of skin growth at an age of 8 mo. The skin weight of castrated transgenic mice was smaller (p < 0.01) than that of intact transgenic mice both absolutely and relative to body weight. The relative skin weight of castrated transgenic mice was in the same range as in intact and castrated control mice. Absolute and relative skin area of castrated transgenic mice was greater (p < 0. 001 and p < 0.05) than in controls but lower than in intact transgenic mice (p < 0.001 and p < 0.05). When compared with control mice, intact transgenic mice displayed an increase (p < 0.01) in the thickness of dermis. In castrated transgenic mice the thickness of the dermis was in the same range as in control mice. Our findings demonstrate a specific interaction of growth hormone with male sex hormones resulting in a marked stimulation of skin growth.

Androgens↗

PubMed1999

Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.

Coenzyme Q(10) (CoQ(10)) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ(10) is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ(10) is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ(10), inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 microM CoQ(10) (66.5 +/- 0.8% and 68.7 +/- 1.1%, respectively) and lowest in 10 microM CoQ(10) (55.3 +/- 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 +/- 0.6% and 25.2 +/- 0.3% in medium supplemented with 10 microM and 30 microM CoQ(10), respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 +/- 0.4% and 11.3 +/- 0.4%). In the presence of 30 microM CoQ(10), significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 +/- 1.3 vs. 8.4 +/- 2.2). Expanded blastocysts produced in the presence of 30 microM CoQ(10) had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer. Our results show that noncrystalline CoQ(10) in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system.

Adenosine Triphosphate↗

PubMed1999

Phase I-II study of 5-fluorouracil, recombinant interferon alpha2a, and cisplatin in combination with external beam radiation therapy followed by surgery in patients with locally advanced carcinoma of the esophagus.

Multimodality therapy has been demonstrated to be superior to external beam radiation therapy and possibly surgery alone for the treatment of carcinoma of the esophagus. The combination of 5-fluorouracil (5-FU), cisplatin, and recombinant interferon alpha2a (IFN) has yielded 65% response rates in metastatic and regionally advanced carcinoma of the esophagus. A phase I-II study was performed to assess the feasibility of combining 5-FU, IFN, and cisplatin with external beam radiation therapy followed by surgery in potentially resectable patients. Eligibility included biopsy-proven stage II-III squamous cell or adenocarcinoma of the esophagus with no prior therapy. External beam radiation therapy was administered concurrently with chemotherapy beginning on day 1, 5 days per week, twice a day with 1.5 Gy/fraction to a total dose of 45 Gy. 5-FU was administered at 750 mg/m2 on days 1, 8, 15, 22, and 29 after the administration of IFN and cisplatin. IFN was given at a dose of 6 million units subcutaneously three times per week beginning on day 1. Dose levels I, II, and III of cisplatin were 25, 30, and 35 mg/m2 administered on days 1, 8, 15, 22, and 29. The sequence of administration was IFN followed by cisplatin followed immediately by 5-FU. Dose escalation between patient cohorts occurred if 0/3 or < or = 1/6 patients had dose-limiting toxicity, i.e., grade II-III toxicity attributable to cisplatin. A phase II trial was planned using the maximum tolerated dose of cisplatin determined from the phase I trial. Patients who successfully completed therapy underwent thoracic exploration to resect residual disease. Twelve patients were enrolled; all were eligible. The demographics of the population were median age, 60 years (range, 44-77); nine male and three female patients; nine squamous cell carcinoma, one adenocarcinoma, and two adenosquamous histology; stage II:III, 2:10. Grade 3-4 toxicities included granulocytopenia (12 patients), thrombocytopenia (six), anemia (three), infection (six), diarrhea (two), mucositis (two), and renal and hepatic toxicities (one). Five patients had a clinical complete response, among whom four underwent surgery. At surgery, one patient had no evidence of residual disease and three patients had microscopic disease only. Two patients had progressive disease and five could not complete the therapy because of toxicities. Two patients are alive and disease free at 25 and 23 months, respectively. This regimen, though active, demonstrated an unfavorable toxicity profile and cannot be recommended for further study.

Adenocarcinoma↗

PubMed1999

Combinatorial codons: a computer program to approximate amino acid probabilities with biased nucleotide usage.

Using techniques from optimization theory, we have developed a computer program that approximates a desired probability distribution for amino acids by imposing a probability distribution on the four nucleotides in each of the three codon positions. These base probabilities allow for the generation of biased codons for use in mutational studies and in the design of biologically encoded libraries. The dependencies between codons in the genetic code often makes the exact generation of the desired probability distribution for amino acids impossible. Compromises are often necessary. The program, therefore, not only solves for the "optimal" approximation to the desired distribution (where the definition of "optimal" is influenced by several types of parameters entered by the user), but also solves for a number of "sub-optimal" solutions that are classified into families of similar solutions. A representative of each family is presented to the program user, who can then choose the type of approximation that is best for the intended application. The Combinatorial Codons program is available for use over the web from http://www.wi.mit.edu/kim/computing.html.

Amino Acids↗

PubMed1999

Lack of absorption of didanosine after rectal administration in human immunodeficiency virus-infected patients.

The feasibility of rectal administration of didanosine (DDI) was studied in six human immunodeficiency virus-infected patients. After oral intake of a DDI solution (100 mg/m2 of body surface area) combined with an antacid (Maalox), pharmacokinetic parametric values were in accordance with previously published data; the mean +/- standard deviation for terminal half-life was 59.5 +/- 15.0 min, that for peak concentration was 5.2 +/- 3.9 mumol/liter, and that for the area under the time-concentration curve (AUC) was 494 +/- 412 min.mumol/liter. After rectal administration of a similarly prepared DDI solution (100 mg/m2 of body surface area), plasma DDI levels were below the detection limit (0.1 mumol/liter) at all time points in five of the six patients, and in the remaining patient the AUC after rectal application was only 5% of that after oral administration. We conclude that oral administration of DDI cannot be easily replaced by rectal application.

Administration, Rectal↗

PubMed1999

Establishment of pluripotent cell lines from vertebrate species--present status and future prospects.

Pluripotent embryonic stem (ES) cells are undifferentiated cell lines derived from early embryos and are capable of unlimited undifferentiated proliferation in vitro. They retain the ability to differentiate into all cell types including germ cells in chimeric animals in vivo, and can be induced to form derivatives of all three germ layers in vitro. Mouse ES cells represent one of the most important tools in genetic research. Major applications include the targeted mutation of specific genes by homologous recombination and the discovery of new genes by gene trap strategies. These applications would be of high interest for other model organisms and also for livestock species. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have been established for vertebrate species other than mouse and chicken thus far. This review summarizes the current status of deriving pluripotent embryonic stem cell lines from vertebrates and recent developments in nuclear transfer technology, which may provide an alternative tool for genetic modification of livestock animals.

Animals↗

PubMed1999

Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal body weight gain.

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) has been shown to inhibit IGF-dependent cell proliferation in a number of in vitro studies. However, no in vivo model of IGFBP-2 overexpression has been established so far. Therefore, we have generated transgenic mice, in which expression of a mouse IGFBP-2 complementary DNA is controlled by the cytomegalovirus (CMV) promoter. In two independent transgenic strains, transgene expression was highest in pancreas and stomach, followed by skeletal muscle, heart, colon, spleen, adipose tissue, brain, and kidney. Within the pancreas, IGFBP-2 expression was found in the islets but not in the exocrine part. Serum IGFBP-2 levels of CMV-IGFBP-2 transgenic mice were about 3-fold (P < 0.05) increased, compared with controls, whereas serum levels of IGF-I and IGF-II were unaffected by IGFBP-2 overexpression. Fasted serum glucose and fasted insulin levels were slightly reduced in transgenic mice, compared with controls. Postprandial serum glucose insulin levels were not affected by the genotype. At days later than 23, body weights of transgenic mice were significantly (P < 0.05) reduced in both sexes, compared with nontransgenic littermates. This reduction in body weight was mainly attributable to significantly (P < 0.05) lower carcass weights of CMV-IGFBP-2 transgenic vs. control mice. In contrast, absolute organ weights were not (or only as a tendency) reduced, except for the weight of the spleen, which was significantly (P < 0.05) lower in male transgenic than in control mice. Our data suggest that IGFBP-2 represents a negative regulator of postnatal growth in mice, potentially by reducing the bioavailability of IGF-I.

Animals↗

PubMed1999

Postnatal overexpression of insulin-like growth factor II in transgenic mice is associated with adrenocortical hyperplasia and enhanced steroidogenesis.

The influence of postnatal insulin-like growth factor II (IGF-II) overexpression on adrenal growth and function was investigated in 3-month-old male phosphoenolpyruvate carboxykinase (PEPCK) promoter human IGF-II transgenic mice, which are characterized by 4-to 6-fold elevated postnatal IGF-II serum levels. Plasma corticosterone levels of PEPCK-IGF-II transgenic mice were 2-fold higher than in age- and sex-matched controls, both in the morning (7.4 +/- 1.5 vs. 17.8 +/- 3.9 ng/ml, P < 0.01) and in the evening (33.3 +/- 6.5 vs. 65.3 +/- 12 ng/ml, P < 0.01). When PEPCK-IGF-II transgenic mice were subjected to an ACTH challenge, corticosterone levels were stimulated 6-fold, to 396 +/- 17 ng/ml after 60 min, compared with 230 +/- 24 ng/ml in the control group. In contrast to corticosterone, plasma ACTH levels were similar in transgenic and control mice, excluding an indirect effect of IGF-II at the hypothalamic or pituitary level. In vitro, the basal and ACTH-induced corticosterone production of adrenal glands from transgenic mice was higher (2-fold and 1.8-fold, respectively) than that of control organs. However, when normalized for adrenal weight, the in vitro corticosterone secretion was similar in both groups. At autopsy, adrenal weights of transgenic mice were significantly greater than those of control adrenal glands (3.3 +/- 0.2 vs. 2.0 +/- 0.2 mg, P < 0.01, n = 10). Furthermore, a local expression of human IGF-II could be demonstrated in transgenic adrenal glands by RT-PCR, whereas in normal adult mice, no adrenal expression of IGF-II was detected. Stereological investigation of adrenal glands from another set of PEPCK-IGF-II transgenic mice and controls (6-month-old males) demonstrated that the increase in adrenal weight in transgenic mice is mainly caused by a 50% increase in the number of zona fasciculata cells, whereas cell volume and zonation of transgenic adrenal glands remained unchanged. In conclusion, our data indicate that postnatal overexpression of IGF-II induces an increased adrenal weight and elevated corticosterone serum levels, presumably by a direct mitogenic effect of IGF-II on adrenocortical fasciculata cells.

Adrenal Glands↗

PubMed1999

Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts.

The developmental potential of bovine fetal fibroblasts was evaluated using nuclear transfer. Fibroblasts from a 37-day-old fetus were fused to enucleated oocytes before activation. Nuclei of starved (cultured for 8 days in medium containing 0.5% serum) fibroblasts supported the development of reconstructed embryos to the blastocyst stage significantly better than those of non-starved fibroblasts (39% versus 20%; P < 0.05). When nuclear transfer morulae derived from starved or non-starved fibroblasts were used for re-cloning, the proportion of blastocysts (52 and 55%, respectively) obtained with these embryonic nuclei was significantly higher than it was with fibroblast nuclei used in the first round of nuclear transfer (P < 0.05 and P < 0.001, respectively). After transfer of blastocysts derived from non-starved and starved fibroblasts, respectively, 33% (1/3) and 78% (7/9) of recipients were pregnant on day 30 as assessed by ultrasonography. On day 90, the corresponding pregnancy rates were 33% (1/3) and 63% (5/8). Two live male twin calves, derived from non-starved fibroblasts, were delivered by Caesarean section at day 281 of gestation. This study demonstrates a positive effect of serum starvation on the efficiency of nuclear transfer using bovine fetal fibroblasts. The efficiency of nuclear transfer could be further increased by recloning.

Animals↗