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Biomedical subjects

X Vignon

Publications and source records attributed to X Vignon.

At least 19 recordsLinked to original sources

Large offspring or large placenta syndrome? Morphometric analysis of late gestation bovine placentomes from somatic nuclear transfer pregnancies complicated by hydrallantois.

Somatic nuclear transfer (NT) in cattle is often complicated by fetal oversize (i.e., large offspring syndrome), hydrallantois, and placentomegaly in late gestation. The aims of this work were to obtain data on the placentome structure in NT-recipient cows with hydrallantois (NTH) and to relate these with fetal and placental weights to better understand the abnormalities observed in NTH pregnancies during the third trimester. Pregnant cows were slaughtered between Gestation Days 180 and 280. The fetuses were weighed, and the placentomes were numbered and weighed. Placentomes were examined by histologic and stereological techniques. Macroscopic data showed that placental overgrowth preceded fetal overgrowth, and the ratio of the fetal to the total placentome weight in the NTH group was lower than that in controls after Gestation Day 220. This suggests that placental overgrowth is due to placental default rather than due to fetal overgrowth, as shown also by stereological analysis showing primary deregulation of the growth of cotyledonary tissues. Observed alterations, such as thinning of the maternal epithelium within placentomes and increased trophoblastic surface, could be secondary adaptations. Thus, placental growth deregulations would be due to modifications of the expression of placental factors. Various examples of placental deficiency were observed, suggesting that some fetal abnormalities observed in NTH calves, such as enlarged heart, enlarged umbilical cord, and abdominal ascites, are consequences of placental dysfunction. Therefore, the condition described by the term "large offspring syndrome" might better be described by "large placenta syndrome," because this syndrome affects an average of 50% of late-gestation NT pregnancies. No conclusion can be drawn from this work on apparently normal pregnancies.

Allantois↗

Zootechnical performance of cloned cattle and offspring: preliminary results.

This paper presents information on the evolution of sets of cloned heifers of Holstein breed in comparison to that of control heifers derived from artificial insemination (AI) in the same farm, as well as data on a set of cloned bulls and their semen characteristics. Preliminary observations on a group of calves sired by a cloned bull and offspring of cloned females are reported. Mean birth weight in the clone group (50 females) was statistically higher than that of 68 contemporary female controls obtained by AI (49.27 +/- 10.98 vs. 40.57 +/- 5.55 kg, respectively, p < 0.05). Growth rate was within normal values for Holstein heifers (from 0.7 to 0.8 kg/day) and daily gain was not influenced by the high or low birth weight of clones. Within animals of the same clone, variability of daily gain was reduced compared to their control counterparts. Semen production from three cloned bulls was within the parameters expected for young bull of the same age. A direct comparison of morphological analysis was made between the frozen thawed semen of the donor bull and of his three clones collected at the same age. The overall semen picture appeared within acceptable limits and the clones presented similar percentages of sperm abnormalities (80% of morphologically normal spermatozoa) as the donor. These preliminary results suggest no deleterious effect of cloning on the semen picture of cloned sires. Frozen semen from one clone bull was used for an AI trial, resulting in 65% pregnancies, 25 live calves were naturally delivered. Concerning the offspring of both female and male clones, the phenotypical and clinical observation of the calves in the first week of age did not reveal any clinical abnormality, suggesting that the deviations observed in clones are not transmitted to the progeny.

Animals↗

Nuclear transfer technologies: between successes and doubts.

Cloning of mammals by nuclear transfer can lead to the birth of healthy adult animals but more often compromises the development of the reconstructed embryos. A high incidence of fetal and postnatal losses has been observed in several species, revealing the existence of long-lasting effects induced by the nuclear transfer procedures. Remodeling of donor chromatin by the recipient cytoplasm after nuclear transfer is frequently associated with the deregulation of specific genes, and recent observations point to the potential importance of time-dependent DNA methylation events in the occurrence of these alterations. Screening strategies to design nuclear transfer procedures that would mimic the epigenetic remodeling occurring in normal embryos are being designed, and improvement in the efficiency of procedures could imply a pre-conditioning of donor cells. Early mammalian development appears to be rather tolerant to epigenetic abnormalities, raising the possibility that even a fully functional reprogrammed genome may have been subjected to some epigenetic alterations. Bringing nuclear transfer to routine practice requires greater knowledge and understanding of the basic biological processes underlying epigenetic controls of nuclear activities. An important issue at present is to limit the production of those aberrant phenotypes that may result in significant insult to the nature and welfare of animals.

Animals↗

Novel approaches and hurdles to somatic cloning in cattle.

The overall efficiency of somatic cloning in cattle is still low. Many factors are necessary for successful birth of live offspring. Among them, the source of donor cells reveals the importance of the donor genotype but also the influence of the cell line itself. The cell cycle stage has been intensively investigated, and recent results indicate that, in cattle, the G0 stage of the donor nuclei is not a prerequisite for reprogramming, as highly proliferating cultured fibroblasts also result in live offspring after nuclear transfer. A technical approach using direct microinjection of fibroblast nuclei, instead of fusion of the whole cell, has proved to result in high in vitro development rates in cattle. However, full-term development of somatic cloned embryos is still limited by long-lasting effects and a high incidence of losses at periimplantation time (as well as in late gestation and around calving).

Animals↗

Frequency and occurrence of late-gestation losses from cattle cloned embryos.

Nuclear transfer from somatic cells still has limited efficiency in terms of live calves born due to high fetal loss after transfer. In this study, we addressed the type of donor cells used for cloning in in vivo development. We used a combination of repeated ultrasonography and maternal pregnancy serum protein (PSP60) assays to monitor the evolution of pregnancy after somatic cloning in order to detect the occurrence of late-gestation losses and their frequency, compared with embryo cloning or in vitro fertilization (IVF). Incidence of loss between Day 90 of gestation and calving was 43.7% for adult somatic clones and 33.3% for fetal somatic clones, compared with 4.3% after embryo cloning and 0% in the control IVF group. Using PSP60 levels in maternal blood as a criterion for placental function, we observed that after somatic cloning, recipients that lost their pregnancy before Day 100 showed significantly higher PSP60 levels by Day 50 than those that maintained pregnancy (7.77 +/- 3.3 ng/ml vs. 2.45 +/- 0.27 ng/ml for normal pregnancies, P < 0.05). At later stages of gestation, between 4 mo and calving, mean PSP60 concentrations were significantly increased in pathologic pregnancy after somatic cloning compared with other groups (P < 0.05 by Day 150, P < 0.001 by Day 180, and P < 0.01 by Day 210). In those situations, and confirmed by ultrasonographic measurements, recipients developed severe hydroallantois together with larger placentome size. Our findings suggest that assessing placental development with PSP60 and ultrasonography will lead to better care of recipient animals in bovine somatic cloning.

Animals↗

Nucleolar changes in bovine nucleotransferred embryos.

This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1-2 h (early 1-cell stage), 10-15 h (late 1-cell stage), 22-24 h (2-cell stage), 37-38 h (4-cell stage), 40-41 h (early 8-cell stage), 47-48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT embryos in comparison with the embryonic NT group. It is concluded that complete reprogramming of rRNA gene expression is part of the general nuclear reprogramming necessary for development after NT.

Animals↗

Clinical, hormonal, and hematologic characteristics of bovine calves derived from nuclei from somatic cells.

Although healthy animals are born after nuclear transfer with somatic cells nuclei, the success of this procedure is generally poor (2%-10%) with high perinatal losses. Apparently normal surviving animals may have undiagnosed pathologies that could develop later in life. The gross pathology of 16 abnormal bovine fetuses produced by nuclear transfer (NT) and the clinical, endocrinologic (insulin-like growth factors I and II [IGF-I and IGF-II], IGF binding proteins, post-ACTH stimulation cortisol, leptin, glucose, and insulin levels), and biochemical characteristics of a group of 21 apparently normal cloned calves were compared with those of in vitro-produced (IVP) controls and controls resulting from artificial insemination. Oocytes used for NT or IVP were matured in vitro. NT to enucleated oocytes was performed using cultured adult or fetal skin cells. After culture, Day 7, grade 1-2 embryos were transferred (one per recipient). All placentas and fetuses from clones undergoing an abnormal pregnancy showed some degree of edema due to hydrops. Mean placentome number was lower and mean placentome weight was higher in clones than in controls (69.9 +/- 9.2 placentomes with a mean weight of 144.3 +/- 21.4 g in clones vs. 99 and 137 placentomes with a mean individual weight of 34.8 and 32.4 g in two IVP controls). Erythrocyte mean cell volume was higher at birth (P < 0.01), and body temperature and plasma leptin concentrations were higher and T4 levels were lower during the first 50 days and the first week (P < 0.05), respectively, in clones. Plasma IGF-II concentrations were higher at birth and lower at Day 15 in clones (P < 0.05). Therefore, apparently healthy cloned calves cannot be considered as physiologically normal animals until at least 50 days of age.

Adrenocorticotropic Hormone↗

Lymphoid hypoplasia and somatic cloning.

BACKGROUND: Adult somatic cloning by nuclear transfer is associated with high rate of perinatal mortality but there is still no evidence that nuclear transfer itself is responsible for these failures. We report on a longlasting defect linked to somatic cloning. METHODS: Skin cells grown from an ear biopsy specimen from a 15-day-old calf were used as a source of nuclei. The donor animal was a clone of three females obtained from embryonic cells. Clinical examination, haematological, and biochemical profiles, and echocardiography of the somatic clone were done from birth to death. FINDINGS: After 6 weeks of normal development, the somatic cloned calf had a sudden and rapid fall in lymphocyte count and a decrease in haemoglobin. The calf died on day 51 from severe anaemia. Necropsy revealed no abnormality except thymic atrophy and lymphoid hypoplasia. INTERPRETATION: Somatic cloning may be the cause of long-lasting deleterious effects. Our observation should be taken into account in debates on reproductive cloning in human beings.

Anemia↗

[Cloning: present and perspectives].

Human embryonic cells obtained through somatic cloning would allow selfgrafting for therapeutical purposes. Data available from animal research indicate that this issue should be considered with great care.

Cloning, Organism↗

Endocytosis of lutropin by Leydig cells through a pathway distinct from the high-affinity receptor.

In porcine Leydig cells in primary culture, 95% of the internalization of [125I]porcine lutropin ([125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars.

Animals↗

Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells.

Muscle and skin biopsies taken from bovine fetuses and young calves have been used as a source of donor nuclei for cloning experiments. After culture, cells were individually fused to enucleated matured oocytes and the resulting blastocysts obtained after 7 d of culture (3-8% depending on the cell type) were transferred to foster recipient heifers. Two calves, a female and a male, both originating from muscle cells were born, and four additional pregnancies have surpassed mild-term gestation. The pregnancies include one fetus established from a transgenic nucleus from a fetal skin cell, and another one resulting from a skin biopsy performed on a female calf. Our data demonstrate that nuclei from cultured bovine somatic cells obtained from differentiated tissues can be made multipotent.

Animals↗

Ultrastructural and immunocytochemical analysis of diploid germ cells isolated from fetal rabbit gonads.

Germ cells were isolated from rabbit fetal gonads between 18 and 22 days post coitum and examined morphologically, ultrastructurally and for immunocytochemical and cytochemical characteristics. Observations were compared with the information available from the corresponding cells of other mammalian species. The general morphology and ultrastructure of healthy isolated rabbit fetal germ cells were found to be very similar to those of the rabbit and mouse diploid germ cells in situ. Moreover, rabbit fetal germ cells shared common immunocytochemical characteristics with mouse undifferentiated embryonic stem cells or embryonic carcinoma cells, such as the presence of TEC-1 (SSEA-1) antigens, a peripheral network of F-actin, the absence of cytokeratins 8/18 and lamins A/C and an alkaline phosphatase activity. No difference between the sexes was observed. Morphological and physiological similarities with the migrating and cultured primordial germ cells of the mouse also suggest that diploid rabbit germ cells would be good candidates for deriving pluripotential embryonic germ cells (EG cells) if favourable culture conditions could be found. In conclusion, the rabbit may be a suitable model for investigations on EG cells in domestic mammals with delayed meiosis.

Alkaline Phosphatase↗

Dietary magnesium deficiency in rats enhances free radical production in skeletal muscle.

Recent studies suggest that free radicals may be involved in tissue injuries induced by magnesium deficiency. The aim of the present study was to assess the effect of magnesium deficiency on free radical production of skeletal muscle tissue. Male Wistar rats were pair-fed from weaning for 12 d either control or Mg-deficient diets containing 960 or 40 mg magnesium/kg diet, respectively. In the Mg-deficient rats, hypomagnesemia was accompanied by significantly lower magnesium and greater calcium concentrations in skeletal muscle tissue. Electron microscopy of skeletal muscle tissue revealed ultrastructural changes, including swelling mitochondria and disorganization of the sarcoplasmic reticulum network. Using the spin-trapping technique, we showed that significantly more hydroxyl radicals were generated in muscle homogenates of Mg-deficient rats. Moreover, the amount of spin trap adducts was increased in the presence of exogenous iron in both groups. In agreement with these observations, a greater concentration of thiobarbituric acid-reactive substances and a lower concentration of thiol groups were found in skeletal muscle of the Mg-deficient group compared with controls. These results strongly support the hypothesis that free radical-mediated injury could contribute to skeletal muscle lesions resulting from magnesium deficiency.

Animals↗

Glycogen hyperaccumulation in white muscle fibres of RN- carrier pigs. A biochemical and ultrastructural study.

1. The dominant RN- gene affects meat quality of pigs by increasing the glycogen content of muscle. Glycogen localization was studied in Longissimus dorsi muscle from RN- carrier pigs (RN- pigs) and rn+ rn+ homozygous pigs (rn+ pigs). 2. Ultrastructural study showed an excess of glycogen in the sarcoplasm of white fibres from RN- pigs as compared to rn+ pigs. 3. Lysosome-enriched fractions extracted from muscles contained 6% of the tissue glycogen content in both types of pigs. The distribution of the glycogen particles between sarcoplasm and lysosomes appeared to be similar in both RN- and rn+ pig tissues. 4. White fibres from RN- pigs with an increased glycogen level showed two ultrastructural alterations: the sarcoplasmic compartment was abnormally enlarged and a large proportion of mitochondria was morphologically modified. 5. The RN- gene seems, therefore, to be associated with alterations in the glycolytic metabolism, in the distribution of proteic compartments and in the oxidative metabolism of white muscle fibres.

Acetylglucosaminidase↗

Fluidity state of lymphocyte plasma membrane in malignant hyperthermia susceptible pigs and humans.

Recent studies suggest that abnormalities occur at the lipid level in malignant hyperthermia susceptible humans and pigs. To test this hypothesis, we first investigated the physical state of plasma membranes of lymphocytes isolated from normal and malignant hyperthermia susceptible swine. In halothane-challenged pigs, malignant hyperthermia susceptibility was also assessed by ryanodine binding assay on purified sarcoplasmic reticulum membranes. The results clearly show that plasma membrane of lymphocytes from malignant hyperthermic pigs are significantly more fluid than controls. We then attempted to apply the same methodology to lymphocytes prepared from human patients previously diagnosed by the halothane and caffeine contracture test. In that case, there was no clear relationship between malignant hyperthermia susceptibility and the fluidity state of lymphocyte plasma membranes.

Animals↗

31P NMR and freeze fracture studies of sarcoplasmic reticulum membranes from normal and malignant hyperthermic pigs: effect of halothane and dantrolene.

The effects of halothane and dantrolene on sarcoplasmic reticulum membranes isolated from normal and malignant hyperthermia pig muscle have been investigated using 31P NMR and freeze fracture electron microscopy. The dynamical and structural changes are estimated from the second moment, as calculated from 31P NMR spectra. For both membranes, addition of halothane induces a similar decrease in the spectral second moment. At high concentration of halothane, freeze fracture replicas show small unilamellar vesicles or mixed micelles, uniformly sprayed in the case of malignant hyperthermia membranes but mainly aggregated for the normal ones. The effect of halothane on both membranes is partially inhibited by adding dantrolene. These results suggest that (i) the malignant hyperthermia syndrome is not directly related to the polar heads of phospholipids and (ii) dantrolene counteracts unspecifically the disturbing effect of halothane at the lipid level.

Animals↗

Halothane-induced membrane reorganization monitored by DSC, freeze fracture electron microscopy and 31P-NMR techniques.

The effect of the volatile anaesthetic halothane on the structure and dynamics of lipid multilayers (dimyristoyl- and dipalmitoylphosphatidylcholine, DM- and DP-PC, aqueous dispersions) was studied using Differential Scanning Calorimetry (DSC), Freeze Fracture Electron Microscopy and solid state phosphorus-31 Nuclear Magnetic Resonance (31P-NMR). The action of the drug depends upon the halothane-to-lipid molar ratio, Ri, and temperature. With DPPC lipids, three main regions can be distinguished: i) 0 less than Ri less than 0.7, ii) 0.7 less than Ri less than 2 and iii) Ri greater than 2. As Ri increases in the first region, a linear decrease in the main gel-to-fluid phase transition temperature (Tc), a broadening in the DSC transition peak and a lowering in the enthalpy variation (delta H), are observed. A minimum in delta H is reached at Ri = 0.7. In this region, 31P-NMR spectra indicate that the multibilayer structure is maintained. In the second region, Tc still decreases with the same slope, but delta H increases up to a plateau value for Ri = 2. In the lipid fluid phase, an isotropic NMR line appears superimposed on the powder pattern that corresponds to a lamellar phase. For Ri greater than 2, Tc and delta H remain almost constant. At values of temperature that are greater than Tc, a growing isotropic line occurs in 31P-NMR spectra. This means a new supramolecular structure made of lipids and halothane is stabilized. This structure has been characterized as small vesicles of about 400 A to 600 A diameter by Freeze Fracture electron microscopy observations. With DMPC and low ratios (Ri less than or equal to 2), DSC and NMR results are similar to those obtained for DPPC. However, the minimum delta H is reached at Ri = 0.2 and the decrease in Tc is faster than for DPPC when Ri increases from 0. For Ri greater than 2, while Tc and delta H remain constant as in the case of DPPC, 31P-NMR spectra of DMPC systems show a superimposition of an isotropic line and two powder patterns, which correspond to small tumbling vesicles, a possible hexagonal phase and a lamellar phase respectively. Halothane, thus acts on model membranes in two different steps: at low Ri the bilayer is disturbed but keeps its structure. Whereas for higher drug concentrations, a new organization of lipids seems to be stabilized for T greater than Tc.

1,2-Dipalmitoylphosphatidylcholine↗

Abnormal fluidity state in membranes of malignant hyperthermia pig skeletal muscle.

The fluidity state was analyzed on sarcoplasmic reticulum membranes and phospholipid vesicles prepared from normal and malignant hyperthermia susceptible pig muscle. Electron spin resonance studies were performed to determine the fluidity state at the region near the polar headgroups and in the central core of the bilayer using 5-nitroxide (5-NS) and 16-nitroxide stearic acid (16-NS), respectively. With the 5-NS label, no differences were found between normal and malignant hyperthermia sarcoplasmic reticulum (MH SR) membranes whereas with the 16-NS label, a significant increase of the activation energy was shown with MH membranes. Lower values of fluorescence anisotropy observed with DPH-labeled MH membranes as compared with normal ones, confirmed the higher abnormal fluidity state of these membranes. The fluidizing effect of halothane, a triggering agent of malignant hyperthermia syndrome, was also studied in these membranes. We show that a relatively low concentration of the drug destabilized not only the diseased sarcoplasmic reticulum membranes but also the vesicles made of total phospholipids extracted from MH skeletal muscle. Together, these findings strongly suggest that an overall increase in membrane fluidity may be implied in the MH disease, improving the general membrane defect hypothesis for this syndrome.

Animals↗