Thierry Joly

Induced pluripotent stem cells derived from rabbits exhibit some characteristics of naïve pluripotency

Summary Not much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation. All rbESC lines showed molecular and functional properties typically associated with primed pluripotency. The cell cycle of rbESCs had a prolonged G1 phase and a DNA damage checkpoint before entry into the S phase, which are the 2 features typically associated with the somatic cell cycle. In contrast, the rbiPSC lines exhibited some characteristics of naïve pluripotency, including resistance to single-cell dissociation by trypsin, robust activity of the distal enhancer of the mouse Oct4 gene, and expression of naïve pluripotency-specific genes, as defined in rodents. According to gene expression profiles, rbiPSCs were closer to the rabbit inner cell mass (ICM) than rbESCs. Furthermore, rbiPSCs were capable of colonizing the ICM after aggregation with morulas. Therefore, we propose that rbiPSCs self-renew in an intermediate state between naïve and primed pluripotency, which represents a key step toward the generation of bona fide naïve PSC lines in rabbits.

STRUCTURAL, METABOLIC AND DEVELOPMENTAL EVALUATION OF OVULATED RABBIT OOCYTES BEFORE AND AFTER CRYOPRESERVATION BY VITRIFICATION AND SLOW FREEZING

The cryopreservation of oocytes is valuable for the preservation of womens fertility and might also be an interesting tool to preserve animal genetic biodiversity but it is not often used because of the very poor fertility recovered after thawing, especially in rabbit species. The objective of our study was to evaluate the effect of slow-freezing and vitrification on the structural integrity of ovulated rabbit oocytes, their ATP contents, and their developmental competence. Results show that, whatever the method is used, cryopreservation has a dramatic effect on the metabolic integrity, the structural integrity, and the developmental ability of the oocytes. Vitrification and slow freezing both impair the rabbit oocytes viability after thawing but the processes act differently. Further studies are needed to improve the cryopreservation techniques in rabbit species. Moreover, we underlined that morphology and maintenance of the structural integrity of the oocytes are not suitable enough to assess the potential for further development of cryopreserved M(II) oocytes. The assessment of ATP metabolism allows efficient evaluation of the viability of the frozen or vitrified oocytes. It should be used in addition to parthenogenesis to better assess the potential for further development.

Follicle development in cryopreserved bitch ovarian tissue grafted to immunodeficient mouse

The present study evaluated: (1) in vivo follicular development in canine ovarian tissue after slow freezing and xenotransplantation; and (2) the use of erythropoietin (EPO) as an angiogenic factor to optimise the transplantation procedure. Frozen-thawed ovarian tissue from five bitches was grafted into severe combined immunodeficient (SCID) mice (n=47) treated with or without EPO (500 IU kg(-1), once daily for 3 days) (Groups A and B, respectively) and analysed after 0, 1, 8 or 16 weeks. Follicle grade, follicle density, follicle morphology and stromal cells density were assessed by histological analysis, whereas vascularisation of the graft was quantified by immunohistochemistry with anti-α-smooth muscle actin antibody. Despite a massive loss of follicles after grafting, secondary follicle density was higher at 8 and 16 weeks than at 1 week regardless of EPO treatment. EPO significantly improved early follicle morphology and stromal cell density after 8 weeks and blood vessel density at 16 weeks after transplantation (P<0.05). Intact secondary follicles with more than three granulosa cells layers were observed 16 weeks after transplantation. The results suggest that canine ovarian tissue can be successfully preserved by our slow-freezing protocol because the tissue showed follicular growth after xenotransplantation. EPO treatment did not lessen the massive loss of follicles after transplantation.

Effects of Slow Freezing Procedure on Late Blastocyst Gene Expression and Survival Rate in Rabbit

ABSTRACT Studies of embryo cryopreservation efficiency have focused mainly on technical and embryo factors. To determine how a slow freezing process affects embryo and fetal development, we studied in vivo development ability after the freezing procedure by assessing blastocyst development at Day 6, implantation, and birth rates. A transcriptional microarray study was also performed to compare gene expression of 6-day-old rabbit embryos previously frozen and transferred into recipient rabbit females to their in vivo counterparts. Our goal was to study which alteration caused by the freezing procedure still remained in late blastocyst stage just at the time when the implantation process began. A microarray specifically designed to study rabbit gene expression profiling was used in this study. Lower implantation and birth rates were obtained in frozen embryos than in the control group (29.9% and 25.7% vs 88.5% and 70.8% for frozen and control embryos, respectively). Likewise, differences were also observed in gene expression profiles. Compared to 6-day-old in vivo-derived embryos, viable frozen embryos presented 70 differentially expressed genes, 24 upregulated and 46 downregulated. In conclusion, our findings showed that the slow freezing process affected late blastocyst development, implantation, and birth rates and that the gene expression alterations identified at late blastocyst stage could be useful in understanding the differences in developmental potential observed and the deficiencies that might hinder implantation and fetal development.

Effect of recombinant gonadotropins on embryo quality in superovulated rabbit does and immune response after repeated treatments

This study aimed first to evaluate the effect of recombinant human FSH (rhFSH) with and without recombinant human LH (rhLH) on fresh and frozen-thawed embryo development and also to analyze the immune response of rabbit does (Oryctolagus cuniculus) subjected to repeated rhFSH treatments. Nulliparous New Zealand White does were used. In Experiment 1, 120 does were superovulated with 25 IU rhFSH alone or in combination with 5% or 10% rhLH (1.25 IU or 2.50 IU rhLH). A total of 1116 embryos at the compacted morula stage were cultured at 38.5 degrees C, 5% CO(2), and saturated humidity for 48 h. The embryo development to hatching blastocyst was significantly lower for the group with 10% rhLH versus that of the control group (65.6 vs. 79.5 for rhFSH+10% rhLH vs. control, respectively). However, no significant difference was found in development to hatching blastocyst for the control, rhFSH alone, and rhFSH+5% rhLH groups. The developmental potential of frozen-thawed embryos obtained from all groups was similar, with an 83.5% in vitro development rate until the expanded blastocyst stage. To detect anti-FSH antibodies, in Experiment 2, does were subject to four superovulation treatments. The hormone administration had a significant effect on immune response in the superovulation group after two treatments (0.14+/-0.074 and 0.15+/-0.076 vs. 0.46+/-0.078 and 0.50+/-0.078 optical density for the first, second, third, and forth cycles, respectively). Nevertheless, none of the treated does had an immune response in both the first and second treatments; on the contrary, a significant increase in the antibody levels was observed in these females at the moment of the third and fourth superovulation treatments. In conclusion, rhFSH superovulation treatments increase the reproductive potential of rabbit does.

A Chemically Defined Medium for Rabbit Embryo Cryopreservation

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in rabbit embryo cryopreservation solutions. This evaluation was performed using two approaches: a thermodynamic approach using differential scanning calorimetry and a biological approach using rabbit embryo slow-freezing. During the experiment, foetal calf serum (FCS) was used as a reference. Because FCS varies widely by supplier, three different FCS were selected for the thermodynamic approach. The rabbit embryo slow-freezing solutions were made from Dulbeccos phosphate buffer saline containing 1.5 M Dimethyl Sulfoxide and 18% (v.v−1) of CRYO3 or 18% (v.v−1) of FCS. These solutions were evaluated using four characteristics: the end of melting temperature, the enthalpy of crystallisation (thermodynamic approach) and the embryo survival rates after culture and embryo transfer (biological approach). In the thermodynamic approach, the solutions containing one of the three different FCS had similar mean thermodynamic characteristics but had different variabilities in the overall data with aberrant values. The solution containing CRYO3 had similar thermodynamic properties when compared to those containing FCS. Moreover, no aberrant value was measured in the solution containing CRYO3. This solution appears to be more stable than the solutions containing a FCS. In the biological approach, the in vitro embryo survival rates obtained with the solution containing CRYO3 (73.7% and 81.3%) and with the solution containing a FCS (77.6% and 71.9%) were similar (p = 0.7). Nevertheless, during the in vivo evaluation, the implantation rate (21.8%) and the live-foetuses rate (18.8%) of the CRYO3 group were significantly higher than the implantation rate (7.1%, p = 0.0002) and the live-foetuses rate (5.3%, p = 0.0002) of the FCS group. The pregnancy rate was also higher in the CRYO3 group compared to the FCS group (81.3% and 43.8%, respectively, p = 0.066). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in rabbit embryo cryopreservation solutions.

Cryopreservation of Genetic Diversity in Rabbit Species (Oryctolagus cuniculus)

After the ratification of the international convention on the biodiversity in Rio, a National Cryobank was created in France in 1999 to preserve the genetic resources of domestic animals (www.cryobanque.org). Particular attention was carried on Oryctolagus cuniculus species with the extension of the national cryobanking to the rabbit (Joly et al., 1998). Nowadays, this tool is very useful for the management of animal diversity in France.

A Panel of Embryonic Stem Cell Lines Reveals the Variety and Dynamic of Pluripotent States in Rabbits

Summary Conventional rabbit embryonic stem cell (ESC) lines are derived from the inner cell mass (ICM) of pre-implantation embryos using methods and culture conditions that are established for primate ESCs. In this study, we explored the capacity of the rabbit ICM to give rise to ESC lines using conditions similar to those utilized to generate naive ESCs in mice. On single-cell dissociation and culture in fibroblast growth factor 2 (FGF2)-free, serum-supplemented medium, rabbit ICMs gave rise to ESC lines lacking the DNA-damage checkpoint in the G1 phase like mouse ESCs, and with a pluripotency gene expression profile closer to the rabbit ICM/epiblast profiles. These cell lines can be converted to FGF2-dependent ESCs after culture in conventional conditions. They can also colonize the rabbit pre-implantation embryo. These results indicate that rabbit epiblast cells can be coaxed toward different types of pluripotent stem cells and reveal the dynamics of pluripotent states in rabbit ESCs.

Control of the estrous cycle in guinea-pig (Cavia porcellus)

The aim of this work was to look for a simple method to obtain synchronized ovulation in guinea pigs under farming conditions while respecting animal welfare. The luteolytic activity of three different prostaglandins F2alpha (PGF2α) analogs (D-cloprostenol, D,L-cloprostenol and luprostiol) and a daily treatment with oral progestagen (altrenogest) was tested successively at different stages of the estrous cycle on the same group of females during a period of 8 mo. The estrous cycle length was not modified by the administration of PGF2α analogs, whatever the stage of the estrous cycle when the treatment was initiated. Our results led us to reject the use of PGF2α analog to induce practical synchronization of the estrus in this species. In females (n = 29), given 15 days with altrenogest (0.1 mL po once a day), ovulation occurred 4.43 ± 0.13 days after the end of the treatment. Altrenogest treatment was followed by mating. No negative impacts of the treatment on the pregnancy rates, delivery rates and litter sizes were observed. This standard method of guinea-pig estrus synchronization is less stressful for the animals compared to techniques using progesterone tubing.

Application de la cryoconservation des embryons à la protection des ressources génétiques chez le lapin

Résumé L’établissement d’une cryobanque de gamètes et d’embryons peut constituer aujourd’hui une aide précieuse pour garantir le maintien de la diversité génétique animale et la protection des populations menacées d’extinction. Nous avons entrepris d’établir une telle banque chez le lapin, espèce à la fois d’intérêt zootechnique, mais aussi modèle pour la recherche biomédicale. Dans un premier temps, nous avons défini les conditions techniques pour l’établissement d’une banque d’embryons à partir de la souche en cours d’homogénéisation «hyperféconde» de l’INRA, choisie comme référence. Nous avons ensuite commencé à appliquer ces techniques à la cryoconservation de plusieurs types de populations : une souche à grand effectif et de haute valeur zootechnique, la souche INRA A 1077, sélectionnée depuis 18 générations sur la taille de portée et maintenue en population fermée; une race à effectif réduit, le «Brun Marron de Lorraine»; une lignée d’intérêt biomédical constituée par 3 familles sélectionnées sur la variabilité allotypique des gènes codant pour la chaîne lourde des immunoglobulines; enfin, une lignée mutante, le lapin dit «sauteur d’Alfort». Cette recherche devrait nous permettre de définir les choix scientifiques et économiques qui présideront à l’extension de ce travail de conservation à d’autres populations de lapin, notamment des lignées transgéniques et des clones.