Animal cloning and its contribution to veterinary research

Cloning is a technique currently used mainly for research purposes , to understand how the nucleus of a differentiated cell can, once it has been placed into the cytoplasm of an enucleated ovule, can re-acquiresthe properties of an embryonic nucleus.The rate of full term development in cloned embryos is still low, only about 5 per cent of the reconstructed embryos.However, it is now well established that clones can develop into adults with a seemingly normal physiology, as fertile animals with the same life span as animals born from sexual reproduction. In cattle, widely used as a cloning model, recent data has shown that clones’ zootechnic performances are similar, and in some instances less variable than those of animals born from a fertilised egg.In this species, cloning failures are due to early (first trimester of gestation) as well as late (last trimester of gestation) embryo and foetal mortality. Approximately 30% of clones are born with a complex lethal syndrome, known as the Large Offspring Syndrome (LOS), with a bodyweight greater than normal, placentomes in lower numbers and oedematous, and physiological disorders resulting in cardiovascular, respiratory, immune, or hepato-renal dysfunctions. Animals with this syndrome generally die within two months from birth.Current research focuses on the clinical follow-up of the gestation of clones and on embryological studies, which showed that such disorders often stem from a growth deregulation in the foetal tissues and organs.Molecular analysis of these epigenetic disorders should help understand better the ontogenesis of the body’s main functions.The study of clones shows that the programme which leads to the development of a complex living being is both robust and flexible, and that its reinitialising when introducing the somatic nucleus within the ovule cytoplasm is probably not required.Le clonage est une technique utilisée aujourd'hui essentiellement en recherche pour comprendre comment un noyau de cellules différenciées peut, une fois placé dans l'environnement cytoplasmique d'un ovule énucléé, acquérir à nouveau des propriétés de noyau embryonnaire. Le taux de développement à terme des embryons clonés est aujourd'hui toujours faible, inférieur à cinq pour cent. Il est toutefois maintenant bien établi que les clones peuvent se développer en adultes d'apparence physiologique normale, être fertiles et avoir la même durée de vie que des animaux obtenus par reproduction sexuée. Chez le bovin, largement utilisé comme modèle, des données récentes montrent que les performances zootechniques des clones sont semblables et pour certains caractères, moins variables que celles d'animaux issus de fécondation. Chez cette espèce, les échecs du clonage sont dus à des mortalités embryonnaires et foetales précoces (premier tiers de la gestation) mais aussi tardives (pendant le dernier tiers de la gestation). Environ 30 % des clones présentent à la naissance un syndrome létal complexe, le syndrome LOS, caractérisé notamment par un poids supérieur à la normale, des placentomes oedémateux en nombre réduit et des perturbations physiologiques se traduisant par des dysfonctionnements systémiques affectant soit les système cardiovasculaire, respiratoire ou immunitaire ou encore le fonctionnement hépato-rénal. Les animaux affectés par ce syndrome meurent en général dans les deux mois qui suivent la naissance. Les recherches en cours donnent une large place au suivi clinique des gestations de clones et aux études embryologiques. Elles montrent que ces perturbations ont souvent pour origine des dérégulations de la croissance des tissus et organes foetaux. L'analyse moléculaire de ces perturbations épigénétiques devrait permettre de mieux connaître l'ontogenèse des grandes fonctions de l'organisme. L'étude des clones montre que le programme de développement qui permet la construction d'un organisme vivant complexe est à la fois robuste et flexible et que sa réinitialisation au moment où l'on introduit le noyau somatique dans le cytoplasme de l'ovule n'est sans doute pas requise

Etude de la vascularisation utéro-placentaire par angiographie Doppler énergie tridimensionnelle (évaluations fondamentales de la technique sur modèles expérimentaux de brebis et lapines gestantes, et évaluations cliniques préliminaires chez la femme enceinte.)

Retard de croissance intra-utérin (RCIU) et prééclampsie (PE) sont des complications majeures de la grossesse humaine et sont le plus souvent due à une insuffisance de vascularisation utéro-placentaire. Notre objectif était d'évaluer l angiographie Doppler énergie tridimensionnelle (PDA) comme nouvel outil de dépistage de la PE et du RCIU et d étude de la fonction placentaire et du RCIU sur modèles animaux. La corrélation entre les indices Doppler 3D et l'écoulement de sang réel au sein de l'unité utéro-placentaires a d'abord été évaluée sur modèle de brebis gravide, ainsi que l'impact des réglages de la machine. Un degré de corrélation plus élevé a été observé pour VI et VFI (r = 0,86 et 0,82 respectivement, p <0,0001) que pour FI (r = 0,64, p <0,0001).L'intérêt de la technique a été ensuite démontré dans un modèle de RCIU chez le lapin (femelles traitées par du L-NAME).Troisièmement, la valeur prédictive du PDA comme test de dépistage du RCIU et de la PE a été démontrée par la réalisation d'une étude prospective multicentrique chez 70 femmes enceintes à bas risque (AUC 0,95, 100% VAN avec une spécificité de 85% pour un seuil de 36.784 FI placentaire).Intra-uterine growth retardation (IUGR) and preeclampsia (PE) are major complications of human pregnancy & are most often due to an insufficient utero-placental vascularization. Our aim was to evaluate the three-dimensional power Doppler angiography (PDA) as a new tool for the screening IUGR & PE & for the study placental function and IUGR in animal models. The correlation between 3D Doppler indices and the real blood flow within the utero-placental unit was first evaluated in the pregnant sheep model, as well as the impact of machine settings. A higher correlation degree was observed for VI and VFI (r = 0.86 and 0.82 respectively p<0.0001) than for FI (r = 0.64; p<0.0001).The interest of the technique was secondly demonstrated in a rabbit IUGR model (females treated with L-NAME).Thirdly, the predictive value of PDA as a screening test for IUGR & PE was demonstrated by conducting a prospective multicentric study in 70 low risk pregnant women (AUC 0.95, 100% NPV with a specificity of 85% for a 36.784 placental FI threshold).PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Discriminative imaging of maternal and fetal blood flow within the placenta using ultrafast ultrasound

Remerciements à INRA UCEA et CR2iInternational audienceBeing able to map accurately placental blood flow in clinics could have major implications in the diagnosis and follow-up of pregnancy complications such as intrauterine growth restriction (IUGR). Moreover, the impact of such an imaging modality for a better diagnosis of placental dysfunction would require to solve the unsolved problem of discriminating the strongly intricated maternal and fetal vascular networks. However, no current imaging modality allows both to achieve sufficient sensitivity and selectivity to tell these entangled flows apart. Although ultrasound imaging would be the clinical modality of choice for such a problem, conventional Doppler echography both lacks of sensibility to detect and map the placenta microvascularization and a concept to discriminate both entangled flows. In this work, we propose to use an ultrafast Doppler imaging approach both to map with an enhanced sensitivity the small vessels of the placenta (~100 μm) and to assess the variation of the Doppler frequency simultaneously in all pixels of the image within a cardiac cycle. This approach is evaluated in vivo in the placenta of pregnant rabbits: By studying the local flow pulsatility pixel per pixel, it becomes possible to separate maternal and fetal blood in 2D from their pulsatile behavior. Significance Statement: The in vivo ability to image and discriminate maternal and fetal blood flow within the placenta is an unsolved problem which could improve the diagnosis of pregnancy complications such as intrauterine growth restriction or preeclampsia. To date, no imaging modality has both sufficient sensitivity and selectivity to discriminate these intimately entangled flows. We demonstrate that Ultrafast Doppler ultrasound method with a frame rate 100x faster than conventional imaging solves this issue. It permits the mapping of small vessels of the placenta (~100 μm) in 2D with an enhanced sensitivity. By assessing pixel-per-pixel pulsatility within single cardiac cycles, it achieves maternal and fetal blood flow discrimination

Animal models of intrauterine growth retardation of vascular origin

Placental insufficiencies can complicate up to 7% of all pregnancies. They can lead to intrauterine growth retardation (IUGR) and preeclampsia, and represent a major public health concern. The pathophysiology of these placental anomalies is not yet fully understood. For obvious ethical reasons, studies in pregnant women are limited to non-invasive techniques, such as ultrasound scans and maternal blood tests. Therefore, animal models of IUGR play an important role in the study of this condition. Because the models using maternal undernutrition to induce foetal IUGR are not as appropriate to mimic the vascular IUGR observed in developed countries, we describe the animal models of vascular IUGR used currently: models based on an application of stress to the mother, genetic models, and surgical models. Finally, we describe briefly the controlled hypoperfusion model in non-anaesthetized ewes, currently developed in our laboratory.Les défauts de perfusion placentaire compliquent environ 7 % des grossesses. Ils peuvent entraîner un retard de croissance intra-utérin (RCIU) et une pré-éclampsie, et posent un problème majeur de santé publique. La physiopathologie de ces défauts n'est pas totalement connue. Les études chez la femme sont limitées à des examens non invasifs, échographiques et biologiques, pour des raisons éthiques évidentes. Il est donc important de disposer de modèles animaux expérimentaux pour appréhender cette pathologie. Nous n'abordons pas ici les modèles de sous-nutrition maternelle, qui ne nous semblent pas complètement appropriés pour reproduire les aspects physiopathologiques observés dans les pays développés. Les modèles animaux de RCIU d'origine vasculaire jusqu'ici utilisés sont décrits: modèles basés sur l'application d'un stress à la mère, modèles génétiques et modèles chirurgicaux. Enfin. Nous décrivons aussi brièvement le modèle d'hypoperfusion contrôlée chez la brebis vigile, actuellement mis au point au laboratoire

Impact of exposure to diesel exhaust during pregnancy on mammary gland development and milk composition in the rabbit

Exposure to fine-particulate air pollution is a major global health concern because it is associated with reduced birth weight and an increased risk of cardiovascular disease. Here we have investigated the potential for exposure to diesel exhaust during pregnancy to influence mammary gland development and milk composition. Female rabbits were therefore exposed by nose-only inhalation to either diluted diesel exhaust fumes (1 mg/m3) or clean air for 2h/day, 5 days/week, from the 3rd to the 27th days of pregnancy. On Day 28 of pregnancy, mammary glands were collected from twelve females (six controls and six diesel-exposed) and assessed for morphological and functional alterations. Milk samples were collected from eighteen dams (nine controls and nine diesel-exposed) during early (days 2 to 4) and established (days 13 to 16) lactation to verify the composition of fatty acids and major proteins and leptin levels. The mammary alveolar lumina contained numerous fat globules, and stearoyl CoA reductase expression was higher in mammary epithelia from diesel exhaust-exposed rabbits, which together suggested increased mammary lipid biosynthesis. Gas chromatography analysis of the composition of milk fatty acids revealed a sharp rise in the total fatty acid content, mainly due to monounsaturated fatty acids. Liquid chromatography-mass spectrometry analysis of milk samples enabled identification and quantification of the main rabbit milk proteins and their main phosphorylated isoforms, and revealed important changes to individual casein and whey protein contents and to their most phosphorylated isoforms during early lactation. Taken together, these findings suggest that repeated daily exposure to diesel exhaust fumes during pregnancy at urban pollution levels can influence lipid metabolism in the mammary gland and the lipid and protein composition of milk. As milk may contribute to metabolic programming, such alterations affecting milk composition should be taken into account from a public health perspective

Evaluation of short‐term safety of ultrasound‐guided foetal fluid sampling in the dog (Canis lupus familiaris)

Background: In humans, analysis of amniotic fluid is widely used for diagnostic and prognostic purposes. Amniocentesis has scarcely been used in veterinary medicine to date, despite a tremendous potential for clinical and research applications in dogs. Our study aimed to establish a safe method for foetal fluid sampling in female dogs. Methods: Two transabdominal ultrasound-guided methods were assessed: the "free hand" and the needle-guided bracket sampling. In addition, through a subsequent routinely scheduled ovariohysterectomy, fluid was directly collected. Samples from 98 conceptuses were collected at day 46.7 +/- 7.5 of pregnancy. Results: The amount of fluid retrieved varied between 0.5 and 5.0 ml per collection. Macroscopic examination of the uterus and conceptuses identified 53% of the puncture sites. Neither fluid leakage nor foetal injury was detected, and six hematomas (5.8%) were visible. Ultrasound-guided foetal fluid collection was found to be potentially safe, and it can be performed by using either transabdominal method. Conclusion: Foetal fluid collection is possible with relative ease and low short-term risk, and may open paths for diagnostic, therapeutic and research purposes in dogs. The procedure can provide new insights into prenatal clinical medicine, including diagnostics of foetal deaths, early identification of heritable diseases and so on

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site

Peri-conception and first trimester diet modifies reproductive development in bulls

Nutritional perturbation during gestation alters male reproductive development in rodents and sheep. In cattle both the developmental trajectory of the feto–placental unit and its response to dietary perturbations is dissimilar to that of these species. This study examined the effects of dietary protein perturbation during the peri-conception and first trimester periods upon reproductive development in bulls. Nulliparous heifers (n = 360) were individually fed a high- or low-protein diet (HPeri and LPeri) from 60 days before conception. From 24 until 98 days post conception, half of each treatment group changed to the alternative post-conception high- or low-protein diet (HPost and LPost) yielding four treatment groups in a 2 × 2 factorial design. A subset of male fetuses (n = 25) was excised at 98 days post conception and fetal testis development was assessed. Reproductive development of singleton male progeny (n = 40) was assessed until slaughter at 598 days of age, when adult testicular cytology was evaluated. Low peri-conception diet delayed reproductive development: sperm quality was lowered during pubertal development with a concomitant delay in reaching puberty. These effects were subsequent to lower FSH concentrations at 330 and 438 days of age. In the fetus, the low peri-conception diet increased the proportion of seminiferous tubules and decreased blood vessel area in the testis, whereas low first trimester diet increased blood vessel number in the adult testis. We conclude that maternal dietary protein perturbation during conception and early gestation may alter male testis development and delay puberty in bulls

Extracellular vesicles generated by placental tissues ex vivo: A transport system for immune mediators and growth factors

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144634/1/aji12860_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144634/2/aji12860.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144634/3/aji12860-sup-0001-Supinfo.pd