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Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.

Gymnures are an ancient group of small insectivorous mammals and are characterized by a controversial taxonomic status and the lack of a description of karyotypes for certain species. In this study, conventional cytogenetic techniques (Giemsa, CBG- and GTG-banding, Ag-NOR), CMA3-DAPI staining, and fluorescent in situ hybridization (FISH) with telomeric DNA probes were used to examine for the first time the karyotypes of lesser gymnures of group Hylomyssuillus Müller, 1840 from northern and southern Vietnam. All studied specimens had karyotypes with 2n=48, NFa=64. C-positive heterochromatic blocks existed in centromeric regions of 7 bi-armed autosomes and the submetacentric X chromosome. The Y chromosome is a C-positive and dot-like. The nucleolus organizer regions resided terminally on the short arms of 2 small bi-armed pairs. Positive signals at the telomeres of all chromosomes were revealed by FISH. CMA3-positive blocks were localized on the telomeric and pericentric regions of most bi-armed and acrocentric chromosomes. Despite the large genetic distances between Hylomys Müller, 1840, lesser gymnures from H.suillus-group from northern and southern Vietnam have similar karyotypic characteristics.

Session : Reproduction; Cryoconservation du sperme de salmonidés : évolution des pratiques et amélioration des fécondances. 6. Journées de la Recherche Filière Piscicole

International audience; Cryopreservation of genetic resources is of major interest for the security of biodiversity and the sustainability of the agronomic industry. Cryopreservation of somatic cells is one means of preserving the genome of both parents, but regeneration of functional breeders requires the mastering of somatic cell NT (SCNT). In mammals, SCNT consists of injecting the somatic nuclei into enucleated recipient oocytes to obtain clones that bear the genome of the donor. Cloning in fish presents some advantages over cloning in mammals: oocytes are produced by the hundreds, and development is external. Moreover, embryonic genome activation takes place after 10 mitoses only. These features are favourable to a better understanding of cellular reprogramming of the somatic cell. Nonenucleated oocytes are often used for SCNT, which allows the production of 2n clones bearing only the donor genome when the maternal chromatin is spontaneously removed by an unknown mechanism (25% of cases). This ability can help us understand the fate of the maternal chromatin and the role of its surrounding factors on the cellular behaviour of the somatic cell during the first developmental steps of embryonic development: meiosis resumption (MR) and mitosis. In this study, fin somatic cells and mature oocytes were obtained from a 2-year-old goldfish (Carassius auratus). For SCNT, the whole fin cell was injected into the sperm entry site, and the oocyte was activated by water contact after 30min to begin the development. Somatic and maternal chromatin behaviour during MR and early mitosis were characterised by immunofluorescence (Hoechst/Vybrant labelling for chromatin identification and α-tubulin for spindle organisation). Variability in chromatin condensation was observed after the somatic cell injection before the activation. Clone analysis (n=16) revealed that some of them presented a condensed somatic nuclei (71%) and others presented a decondensed nuclei (29%). After activation, most clones underwent normal polar body extrusion of the maternal chromatin (66%, n=69/95 clones v. 96%, n=96/100 controls oocytes) without extrusion of a somatic polar body. Afterward, clones that presented a first symmetric division (n=14/35) were analysed at the 2-cells stage and compared with fertilized embryos (n=7). The maternal chromatin was observed as fragmented on the cleavage furrow (79%), where it cannot contribute to the development. Spindle defects were observed in 50% of clone cells (v. 0% in controls), such as multicentrosomal spindles, chromosome misalignment, abnormal segregation, and DNA fragmentation. To conclude, those clone (2n or 3n) defects are probably due to the chromatin condensation observed before activation. The fish oocyte volume did not allow the MR of the condensed somatic chromatin, and that may induce an abnormal anaphase and the following clone developmental defects.

Nuclear transfer consists in injecting a somatic nucleus carrying valuable genetic information into a recipient oocyte to sire a diploid offspring which bears the genome of interest. It requires that the oocyte (maternal) DNA is removed. In fish, because enucleation is difficult to achieve, non-enucleated oocytes are often used and disappearance of the maternal DNA was reported in some clones. The present work explores which cellular events explain spontaneous erasure of maternal DNA, as mastering this phenomenon would circumvent the painstaking procedure of fish oocyte enucleation. The fate of the somatic and maternal DNA during oocyte activation and first cell cycle was studied using DNA labeling and immunofluorescence in goldfish clones. Maternal DNA was always found as an intact metaphase within the oocyte, and polar body extrusion was minimally affected after oocyte activation. During the first cell cycle, only 40% of the clones displayed symmetric cleavage, and these symmetric clones contributed to 80% of those surviving at hatching. Maternal DNA was often fragmented and located under the cleavage furrow. The somatic DNA was organized either into a normal mitotic spindle or abnormal multinuclear spindle. Scenarios matching the DNA behavior and the embryo fate are proposed.

Germ stem cells transplantation in fish: an innovative biotechnology for the faithfull regeneration of cryopreserved genetic ressources collected from selected lines. Aqua 2018

Introduction: The cryopreservation of genetic resources is of major interest for the security of biodiversity and for the sustainability of agronomic industry. Cryopreservation of somatic cells, which carry the genome of both parents, is one mean to regenerate functional breeders, but this requires the mastering of nuclear transfer. In mammals, somatic cell nuclear transfer consists in injecting the somatic nuclei into an enucleated mature recipient oocyte, in order to obtain clone which bear the genome of interest from the donor animal. However, in fish species, the oocyte structure makes the enucleation step difficult to achieve (presence of the chorion and bulk nutritive reserve), so a non-enucleated oocyte is often used for nuclear transfer. To obtain a real clone and not hybrid embryos, the oocyte chromatin must therefore be spontaneously excluded from the developing embryo. This phenomenon has been described in several studies but the mechanism involved is still unknown. Deciphering this phenomenon requires a high understanding of the first developmental steps that are meiosis resumption and early mitosis. The aim of the study is to understand the interplay between the maternal chromatin and the injected one upon nuclear transfer during these entire critical steps, in order to identify and use means to improve the success rate of nuclear transfer in fish. Methods: Fin somatic cell and mature oocytes were obtained from 2 years old goldfish (Carassius auratus) females. For nuclear transfer, the whole fin cell was injected under the micropyle. The egg activation was induced 30min after cell injection. Somatic and maternal chromatin during meiosis resumption and early mitosis was characterized by immunofluorescence (hoechst and vybrant labelling for chromatin identification and α-tubulin for spindle organization). Results and Discussion: We observed that a high proportion of clone underwent a normal polar body extrusion after activation (60% vs 96 % for the controls). We hypothesize that in most cases, maternal chromatin achieved correctly its meiosis resumption and that the maternal chromatin behavior was not disturbed by nuclear transfer. However, these observations do not augur early mitosis fitness in clone. Indeed, we observed several spindle defects in clone cells: metaphase misalignment, abnormal segregation, and multicentrosomal spindle, associated with DNA fragmentation. Conclusion: Oocyte non-enucleation for SCNT procedure did not drastically alter the meiosis resumption process, but it could be involved in the perturbation of mitosis during clone early development.

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)

Le transfert nucléaire chez les poissons. Une technologie de régénération de poissons précieux. Un modèle de développement perturbé. Journées d'Animation Scientifique du département Phase (JAS Phase 2018)

Import nucléaire de facteurs ovocytaires dans les cellules somatiques en culture : une avancée prometteuse pour leur reprogrammation en vue du transfert nucléaire. Journées d'Animation Scientifique du département Phase (JAS Phase 2018)

Somatic cell nuclear transfer in rainbow trout (Onrohynchus mykiss): What makes it different from fertilization with a spermatozoa ?. 11. International Symposium on Reproductive Physiology of Fish

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

Epigenetic reprogramming after nuclear transfer in fish. 6. International Workshop on the Biology of Fish Gametes

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 2017

Optimisation et mise en oeuvre d'une technique de cryopréservation du sperme de lignées transgéniques de Médaka (Oryzias latipes) et fécondation in vitro

This study is the first demonstration of successful post-thawing development to reproduction stage of diploid cryopreserved larvae in an aquatic invertebrate. Survival, growth and reproductive performances were studied in juvenile and adult Pacific oysters grown from cryopreserved embryos. Cryopreservation was performed at three early stages: trochophore (13 +/- 2 hours post fertilization: hpf), early D-larvae (24 +/- 2 hpf) and late D-larvae (43 +/- 2 hpf). From the beginning (88 days) at the end of the ongrowing phase (195 days), no mortality was recorded and mean body weights did not differ between the thawed oysters and the control. At the end of the growing-out phase (982 days), survival of the oysters cryopreserved at 13 +/- 2 hpf and at 43 +/- 2 hpf was significantly higher (P

La congelation du sperme de salmonides a fait l'objet de nombreuses etudes rapportees dans les syntheses bibliographiques realisees par Horton et Ott (1976), Scott et Baynes (1980), Stoss (1983) et plus recemment par Leung et Jamieson (1991). La plupart des auteurs s'accordent sur la tres grande variabilite des resultats obtenus avec le sperme de salmonides congele. Les dilueurs de congelation contiennent souvent des sucres a des concentrations variables : le saccharose est employe a 125 mM chez le saumon atlantique, Salmo salar, (Mounib, 1978) et chez la truite arc-en-ciel, Oncorhynchus mykiss, (Legendre et Billard, 1980) et a 600 mM chez la truite arc-enciel (Holtz et al., 1991); le glucose est utilise a une concentration de 300 mM chez le saumon atlantique (Stoss et Refstie, 1983), chez la truite commune, Salmo trutta, et l'omble chevalier, Salvelinus alpinus, (Piironen, 1993) et 600 mM chez la truite arc-enciel (Holtz et al., 1991). Cependant il n'existe pas d'etude comparative entre les differents glucides ni d'essai systematique sur d'autres sucres alors qu'ils presentent un grand interet en cryoconservation. En effet, Anchordoguy et al. (1987) ont montre que le saccharose et le trehalose interagissent avec les phospholipides de la membrane et augmentent la stabilite de cette derniere pendant la congelation et la decongelation, et concluent que les disaccharides pourraient etre de meilleurs cryoprotecteurs que le glycerol et le dimethylsufoxyde. Dans cette etude nous avons voulu comparer l'effet cryoprotecteur de differents sucres en remplacement du saccharose dans le dilueur de Mounib (1978).

Un lot de 40 mâles d'une souche de truite arc-en-ciel (Salmo gairdneri) à reproduction printanière a été suivi. Seuls les spermes d'un volume supérieur ou égal à 6 ml ont été caractérisés et congelés en boulettes. L'analyse factorielle des correspondances entre les caractéristiques et la fécondance du sperme après 5 à 9 mois de congélation a montré en particulier que l'aptitude à la cryoconservation peut être caractérisée par l'analyse du plasma séminal : les spermes médiocres sont associés à une osmolarité plasmatique élevée (260 m-osmoles) et/ou une teneur élevée en protéine 42 KD, l'un des constituants majeur de la membrane des spermatozoïdes; les très bons spermes sont associés à l'absence de protéine 42 KD dans le liquide séminal. Ces résultats renforcent l'idée que l'aptitude à la cryoconservation du sperme de truite dépend en premier lieu de la qualité de la membrane des spermatozoïdes., Milt was obtained from 40 males of a spring-spawning strain of rainbow trout (Salmo gairdneri). Only semen with a volume greater or equal to 6 ml were characterized and frozen in pellets. Factorial analysis of correspondences between the physico-chemical characteristics and the fertility of sperm after 5 to 9 months freezing revealed that the fitness for cryopreservation can be particularly characterized by the composition of the seminal plasma: sperm with the lowest capacity to fertilize were associated with a plasma osmolality greater than 260 m-Osm and/or a high level of 42 KD protein, one of the major components of the spermatozoan membrane; sperm with the highest fertilizing ability lacked 42 KD protein in the seminal plasma. These results support the idea that the fitness for cryopreservation of trout sperm primarily depends on the condition of the spermatozoan membrane.

Biology of gametes, eggs and embryos

Several methods to control gamete availability in northern pike were investigated. Precocious induction of ovulation was achieved with a single injection of partially purified salmon gonadotropin, but the treatment should be administered soon after capture in order to avoid ovarian atresia. Ovulated oocytes should be inseminated within 24 hours after ovulation. Loss of fertility due to aging occurred 1-3 days after ovulation. High doses of progesterone induced a threefold increase of sperm release. Cryopreservation of diluted sperm at -196 C led to variable fertilizing capacities after thawing, depending on the donor male. A diluent originally tested for trout was used successfully for artificial insemination.