Paternal low protein diet and the supplementation of methyl-donors impact fetal growth and placental development in mice

IntroductionPaternal low-protein diet can alter sperm methylation status, fetal growth and program offspring ill-health, however its impact on the placenta remains poorly defined. Here we examine the influence paternal low-protein diet has on fetal and placental development and the additional impact of supplementary methyl-donors on fetoplacental physiology.MethodsMale C57BL/6J mice were fed a control normal protein diet (NPD; 18% protein), a low-protein diet (LPD; 9% protein) or LPD with methyl-donor supplementation (MD-LPD; choline chloride, betaine, methionine, folic acid, vitamin B12) for a minimum of 8 weeks. Males were mated with 8–11 week old female C57BL/6J mice and fetal and placental tissue collected on embryonic day 17.5.ResultsPaternal LPD was associated with increased fetal weights compared to NPD and MD-LPD with 22% fetuses being above the 90th centile for fetal weight. However, LPD and MD-LPD placental weights were reduced when compared to NPD. Placentas from LPD fathers demonstrated a reduced junctional zone area and reduced free-fatty acid content. MD-LPD placentas did not mirror these finding, demonstrating an increased chorion area, a reduction in junctional-specific glycogen staining and reduced placental Dnmt3b expression, none of which were apparent in either NPD or LPD placentas.DiscussionA sub-optimal paternal diet can influence fetal growth and placental development, and dietary methyl-donor supplementation alters placental morphology and gene expression differentially to that observed with LPD alone. Understanding how paternal diet and micro-nutrient supplementation influence placental development is crucial for determining connections between paternal well-being and future offspring health

Antimicrobials in a rabbit semen extender: effects on reproduction

[EN] The use of fluoroquinolone antibiotics was legally restricted by the European Commission in March 2019. Since the extender for rabbit semen Galap® contained this antibiotic, it became necessary to modify it. The purpose of this study was to search for another molecule, based on its antimicrobial activity and also on the conservation, motility and fertility of semen diluted with the new extender. Several bacterial strains were isolated from 10 poor-quality ejaculates, including Enterococcus spp., Staphylococcus aureus and Proteus spp. They were then tested for antimicrobial susceptibility. Out of 15 antibiotics evaluated, gentamicin was the one targeting such bacteria. In vivo tests were then carried out to assess the effects of this antibiotic change on sperm parameters. Up to 26 pools of good quality semen (total motility >70%) were diluted in original Galap® or in this extender with gentamicin. Ejaculates were analysed on the day (D) of collection and up to 6 d of storage at +17°C. After 24 h storage, the motility in the new extender was reduced by 7.7% compared to the original; this decrease did not worsen by storing. After 6 d of storage, no difference between the two media was detected. A total of 360 females were then inseminated with pools of 4 to 5 good quality semen diluted 1:10 in both extenders, following the routine protocol of rabbit semen processing centres. There was no difference in female fertility or prolificacy between both formulations. In conclusion, these preliminary results suggest that the addition of gentamicin to this extender is useful and also has no adverse effect on fertility or prolificacy.This study was supported by IMV TechnologiesRouillon, C.; Camugli, S.; Carion, O.; Echegaray, A.; Delhomme, G.; Schmitt, E. (2022). Antimicrobials in a rabbit semen extender: effects on reproduction. World Rabbit Science. 30(4):295-308. https://doi.org/10.4995/wrs.2022.1713229530830

Characterization of cellular defects after nuclear transfer in goldfish, and development of a new embryonic donor cell model

La cryoconservation des ressources génétiques est aujourd’hui un moyen de préserver la biodiversité et la durabilité des industries agronomiques. Chez les poissons, le transfert nucléaire est un moyen de régénérer des individus à partir de tissus somatiques cryoconservés, faciles à collecter. Pour autant, les faibles taux d’obtention de clones viables limitent aujourd’hui l’usage de cette biotechnologie. L’objectif de la thèse était donc de comprendre l’origine des défauts de développement des embryons après transfert nucléaire. Nous avons focalisé nos questionnements scientifiques sur l’identification des possibles perturbations induites par la présence de l’ADN maternel, ainsi que sur le devenir de l’ADN somatique lors des étapes critiques du développement précoce : la reprise de méiose et la première mitose embryonnaire. Un résultat majeur de la thèse a été de montrer que dans certains clones, l’ADN maternel se retrouve séquestré sous le sillon de la première mitose,où il est ainsi vraisemblablement écarté de toute contribution au génome du clone. Nous avons également identifié de nombreux désordres dans les fuseaux de division, que nous expliquons par l’observation de localisations et états de condensation variables du noyau injecté. Nous avons enfin développé un nouveau modèle d’étude pour le transfert nucléaire, les cellules de corps embryonnaires. Elles possèdent des caractéristiques cellulaires compatibles avec le transfert nucléaire : petite taille, cycle ralenti, peu différenciées selon les critères épigénétiques et transcriptionnels analysés. En conclusion, notCryopreservation of genetic resources is a mean to preserve biodiversity and sustainability of the agronomic industry. In fish, nuclear transfer is a method to regenerate breeders from cryopreserved somatic tissues that are easy to collect. However, the low rates of viable clone limit the application of this biotechnology. The objective of this thesis work was to understand the origin of embryo development defects after nuclear transfer. We have focused our scientific questions on identifying the possible disturbances induced by the presence of maternal DNA, as well as on the fate of somatic DNA during critical stages of early development: meiosis resumption and first embryonic mitosis. A major result of the thesis was to demonstrate that in some clones, maternal DNA is sequestered under the first mitosis furrow, where it is probably excludedfrom any contribution to the genome of the clone. We have also identified many disorders in the division spindle, for which we propose an explanation based on the observation of variable locations and condensation states of the injected nucleus. Finally, we have developed a new model for nuclear transfer, embryoid body cells. They have cellular characteristics compatible with nuclear transfer: small size, slow cell cycle, and limited differentiation according to the epigenetic and transcriptional criteria that were analysed. For the first time in fish, the early cellular events after nuclear transfer were described. Integrating the results allowed us to suggest hypothesis for the clone development disorders. This work also allowed u

Caractérisation des défauts cellulaires après transfert nucléaire chez le poisson rouge, et mise au point d'un nouveau modèle de cellules donneuses embryonnaires

Cryopreservation of genetic resources is a mean to preserve biodiversity and sustainability of the agronomic industry. In fish, nuclear transfer is a method to regenerate breeders from cryopreserved somatic tissues that are easy to collect. However, the low rates of viable clone limit the application of this biotechnology. The objective of this thesis work was to understand the origin of embryo development defects after nuclear transfer. We have focused our scientific questions on identifying the possible disturbances induced by the presence of maternal DNA, as well as on the fate of somatic DNA during critical stages of early development: meiosis resumption and first embryonic mitosis. A major result of the thesis was to demonstrate that in some clones, maternal DNA is sequestered under the first mitosis furrow, where it is probably excludedfrom any contribution to the genome of the clone. We have also identified many disorders in the division spindle, for which we propose an explanation based on the observation of variable locations and condensation states of the injected nucleus. Finally, we have developed a new model for nuclear transfer, embryoid body cells. They have cellular characteristics compatible with nuclear transfer: small size, slow cell cycle, and limited differentiation according to the epigenetic and transcriptional criteria that were analysed. For the first time in fish, the early cellular events after nuclear transfer were described. Integrating the results allowed us to suggest hypothesis for the clone development disorders. This work also allowed usLa cryoconservation des ressources génétiques est aujourd’hui un moyen de préserver la biodiversité et la durabilité des industries agronomiques. Chez les poissons, le transfert nucléaire est un moyen de régénérer des individus à partir de tissus somatiques cryoconservés, faciles à collecter. Pour autant, les faibles taux d’obtention de clones viables limitent aujourd’hui l’usage de cette biotechnologie. L’objectif de la thèse était donc de comprendre l’origine des défauts de développement des embryons après transfert nucléaire. Nous avons focalisé nos questionnements scientifiques sur l’identification des possibles perturbations induites par la présence de l’ADN maternel, ainsi que sur le devenir de l’ADN somatique lors des étapes critiques du développement précoce : la reprise de méiose et la première mitose embryonnaire. Un résultat majeur de la thèse a été de montrer que dans certains clones, l’ADN maternel se retrouve séquestré sous le sillon de la première mitose,où il est ainsi vraisemblablement écarté de toute contribution au génome du clone. Nous avons également identifié de nombreux désordres dans les fuseaux de division, que nous expliquons par l’observation de localisations et états de condensation variables du noyau injecté. Nous avons enfin développé un nouveau modèle d’étude pour le transfert nucléaire, les cellules de corps embryonnaires. Elles possèdent des caractéristiques cellulaires compatibles avec le transfert nucléaire : petite taille, cycle ralenti, peu différenciées selon les critères épigénétiques et transcriptionnels analysés. En conclusion, no

Compréhension des perturbations induites par le transfert nucléaire chez le carassin: Caractérisation de la reprise de la méiose et des premières mitoses

Compréhension des perturbations induites par le transfert nucléaire chez le carassin. Caractérisation de la reprise de la méiose et des premières mitoses. Journées d'Animation Scientifique du département Phase (JAS Phase 2018

Embryonic fate after somatic cell nuclear transfer in non-enucleated goldfish oocytes is determined by first cleavages and DNA methylation patterns

International audienceReducing the variability in nuclear transfer outcome requires a better understanding of its cellular and epigenetic determinants, in order to ensure safer fish regeneration from cryobanked somatic material. In this work, clones from goldfish were obtained using cryopreserved fin cells as donor and non-enucleated oocytes as recipients. We showed that the high variability of clones survival was not correlated to spawn quality. Clones were then characterized for their first cleavages pattern in relation to their developmental fate up to hatching. The first cell cycle duration was increased in clones with abnormal first cleavage, and symmetric first two cleavages increased clone probability to reach later on 24 hand hatching-stages. At 24 h-stage, 24% of the clones were diploids and from donor genetic origin only. However, ploidy and genetic origin did not determine clones morphological quality. DNA methylation reprogramming in the promoter region of pou2, nanog, and notail marker genes was highly variable, but clones with the nicest morphologies displayed the best DNA methylation reprogramming. To conclude, non-enucleated oocytes did allow authentic clones production. The first two cell cycles were a critical determinant of the clone ability to reach hatching-stage, and DNA methylation reprogramming significantly influenced clones morphological quality

Meiosis resumption and early mitosis pattern after somatic cell nuclear transfer in goldfish

Meiosis resumption and early mitosis pattern after somatic cell nuclear transfer in goldfish. 6. International Workshop on the Biology of Fish Gamete

Epigenetic and cellular reprogramming after nuclear transfer in fish : characterization of clone defects in relation to the donor cell origin

Epigenetic and cellular reprogramming after nuclear transfer in fish : characterization of clone defects in relation to the donor cell origin. 2. Journées scientifiques GDR 3606 REPRO : ReproSciences 201

Meiosis resumption and early mitosis pattern after somatic cell nuclear transfer in goldfish

Meiosis resumption and early mitosis pattern after somatic cell nuclear transfer in goldfish. 6. International Workshop on the Biology of Fish Gamete

Characterization of a new embryonic cellular model for nuclear transfer in fish: the embryoid body

Characterization of a new embryonic cellular model for nuclear transfer in fish: the embryoid body. 7. International Workshop on the Biology of Fish Gamete