Pasqualino Loi

Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells

Since the advent of procedures for cloning animals, conservation biologists have proposed using this technology to preserve endangered mammals. Here we report the successful cloning of a wild endangered animal, Ovis orientalis musimon, using oocytes collected from a closely related, domesticated species, Ovis aries. We injected enucleated sheep oocytes with granulosa cells collected from two female mouflons found dead in the pasture. Blastocyst-stage cloned embryos transferred into sheep foster mothers established two pregnancies, one of which produced an apparently normal mouflon. Our findings support the use of cloning for the expansion of critically endangered populations.

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.

Surviv land viability of vitrified in vitro and in vivo produced ovine blastocysts

Ovine blastocysts were produced by maturation, fertilization and in vitro culture (IVM/IVF/IVC) of oocytes from slaughtered adult and prepubertal ewes and collection from superovulated and inseminated adult animals. Dulbeccos PBS supplemented with 0.3 mM Na Pyruvate and 20% FCS was used as the basic cryopreservation solution. The embryos were exposed to the vitrification solution as follows: 10% glycerol (G) for 5 min, then 10% G +20% ethylene glycol (EG) for 5 min. Embryos were placed into 25% G + 25% EG in the center of 0.25- mL straws and plunged immediately into LN2. Warming was done by placing the straws into a water bath at 37 degrees C for 20 sec, and their contents were expelled into a 0.5 M sucrose solution for 3 min; the embryos were then transferred into 0.25 M and 0.125 M sucrose solution for 3 min each. Warmed blastocysts were transferred to the culture medium for 24 h. Survival was defined as the re-expansion of the blastocoele. All surviving blastocysts were transferred to synchronized recipient ewes, and the pregnancy was allowed to go to term. Of 68 vitrified in vitro produced blastocysts, 46 re-expanded (67.6%) and 10 lambs were born (14.7%). From the 62 in vivo derived and vitrified embryos, 52 re-expanded (83.8%) and 39 lambs were born (62.9%). The lambing rate of in vitro produced fresh transfer embryos was 40% (20 lambs/50 blastocysts transferred), and of the 32 in vivo derived blastocysts and transferred fresh, 26 lambs were born (81.2%). The results indicate that in vitro produced embryos can be successfully cryopreserved by vitrification.

Ovum pick-up in sheep: efficiency of in vitro embryo production, vitrification and birth of offspring

The production of offspring involving available technologies like ovum pick-up, in vitro embryo production and cryopreservation has not been fully described in the sheep. We tested the overall efficiency of these procedures on 20 Sarda dairy ewes that were twice stimulated for recovery of follicular oocytes. In total, 415 oocytes were aspirated from 522 follicles (11.5 oocytes/ewe), and 328 of them (9.1 oocytes/ewe) were selected for in vitro embryo production procedure. Development into blastocysts occurred in 98 embryos (2.7 blastocysts/ewe), of which 64 were vitrified and 34 were transferred, in pairs, directly to recipients. The pregnancy rate, diagnosed at 80 d for fresh and vitrified embryos, did not differ significantly (47.1 vs 42.8%, respectively), but there were significant differences in lambing rates between the 2 groups (41.2 vs 23.8%, respectively). Overall, 24 lambs were born; all weighed within the range for the breed, but head deformities were observed in 2 cases. The results of this study show that with application of the above techniques, it is possible to obtain repeatedly embryos and viable offspring.

Preservation of the Wild European Mouflon: The First Example of Genetic Management Using a Complete Program of Reproductive Biotechnologies

Abstract Although the potential use of reproductive biotechnologies for safeguarding endangered wildlife species is undoubted, practical efforts have met with limited success to date. In those instances in which modern technologies have been adapted to rescuing rare or endangered species, procedures have been applied piecemeal, and no consistent breeding program based on reproductive biotechnologies has been undertaken. Here we describe for the first time the rescue of an endangered species, the European mouflon (Ovis orientalis musimon), by the application of an integrated package of reproductive biotechnologies. This genetic management extended from the initial collection of gametes, through the in vitro production of embryos and interspecific transfer, to the birth of healthy mouflon offspring. In addition, a genetic resource bank for the European mouflon was established, with cryopreserved sperm, embryos, and somatic cells.

GENOMIC IMPRINTING IN RUMINANTS : ALLELE-SPECIFIC GENE EXPRESSION IN PARTHENOGENETIC SHEEP

Abstract. Studies in the mouse have established that both parental genomes are essential for normal embryonic development. Parthenogenetic mouse embryos (which have two maternal genomes and no paternal genome), for example, are growth-retarded and die at early postimplantation stages. The distinct maternal and paternal contributions are mediated by genomic imprinting, an epigenetic mechanism by which the expression of certain genes is dependent on whether they are inherited from mother or father. Although comparative studies have established that many imprinted mouse (and rat) genes are allele-specifically expressed in humans as well (and vice versa), so far imprinting studies have not been performed in other mammalian species. When considering evolutionary theories of genomic imprinting, it would be important to know how widely it is conserved among placental mammals. We have investigated its conservation in a bovid ruminant, the domestic sheep, by comparing parthenogenetic and normal control embryos. Our study establishes that, like in the mouse, parthenogenetic development in sheep is associated with growth-retardation and does not proceed beyond early fetal stages. These developmental abnormalities are most likely caused by imprinted genes. We demonstrate that, indeed, like in mice and humans, the growth-related PEG1/MEST and Insulin-like Growth Factor 2 (IGF2) genes are expressed from the paternal chromosome in sheep. These observations suggest that genomic imprinting is conserved in a third, evolutionarily rather diverged group of placental mammals, the ruminants.

Polyvinyl alcohol as a defined substitute for serum in vitrification and warming solutions to cryopreserve ovine embryos at different stages of development

The purpose of this study was to assess the viability of ovine embryos after replacing fetal calf serum (FCS) with polyvinyl alcohol (PVA) in vitrification and warming solutions. Ovine embryos were obtained from superovulated Sardinian breed ewes at 4, 5, 6, and 7 days after insemination. All vitrification and warming solutions were prepared using buffered saline solution with 20% FCS (group a) or 0.1% PVA (group b). Embryos were vitrified in 20 microliters of glycerol 3.4 M + ethylene glycol 4.6 M and loaded into the centre of 0.25 ml straws between two columns of sucrose solution (0.5 M), and plunged immediately into liquid nitrogen. After being warmed in a water bath at 35 degrees C for 10 s, the vitrified embryos were moved to 0.25 M sucrose solution for 3 min. Embryos were cultured in TCM-199 after washing with 10% FCS and sheep oviductal epithelial cells up to hatching or re-expansion of the blastocoelic cavity. No significant difference in the viability rates was observed between embryos vitrified/warmed in PVA or FCS solutions. In both groups, the rate of in vitro viability was (P < 0.01) lower at the precompacted and compacted morula stages than at the expanded, hatching or hatched blastocyst stage. In both groups, early blastocysts were less viable than expanded (P < 0.01), hatching or hatched blastocyst (P < 0.05). There was no significant difference in survival rates at days 14 (79 and 76%) and 45 (63 and 59%) after transfer into sychronised recipients between vitrified expanded blastocysts of groups a and b, respectively. These results suggest that it is possible replace serum with PVA in vitrification and warming solutions without reducing in vivo and in vitro viability.

Freeze-dried somatic cells direct embryonic development after nuclear transfer.

The natural capacity of simple organisms to survive in a dehydrated state has long been exploited by man, with lyophylization the method of choice for the long term storage of bacterial and yeast cells. More recently, attempts have been made to apply this procedure to the long term storage of blood cells. However, despite significant progress, practical application in a clinical setting is still some way off. Conversely, to date there are no reports of attempts to lyophilize nucleated somatic cells for possible downstream applications. Here we demonstrate that lyophilised somatic cells stored for 3 years at room temperature are able to direct embryonic development following injection into enucleated oocytes. These remarkable results demonstrate that alternative systems for the long-term storage of cell lines are now possible, and open unprecedented opportunities in the fields of biomedicine and for conservation strategies.

Nuclei of Nonviable Ovine Somatic Cells Develop into Lambs after Nuclear Transplantation

Abstract Here we report on the successful reprogramming of nuclei from somatic cells rendered nonviable by heat treatment. Granulosa cells from adult sheep were heated to nonphysiological temperatures (55°C or 75°C) before their nuclei were injected into enucleated metaphase II oocytes. Reprogramming was demonstrated by the capacity of the reconstructed embryos to develop to the blastocyst stage in vitro and into fetuses and viable offspring in suitable foster mothers. To our knowledge, this is the first report of cloned mammalian offspring originating from nonviable cells. In addition, our experiments show that heat-treating donor nuclei destabilizes higher-order features of chromatin (but leaves intact its nucleosomal organization) and results in a high proportion of reconstructed embryos developing to the blastocyst stage and beyond.

Embryonic Diapause Is Conserved across Mammals

Embryonic diapause (ED) is a temporary arrest of embryo development and is characterized by delayed implantation in the uterus. ED occurs in blastocysts of less than 2% of mammalian species, including the mouse (Mus musculus). If ED were an evolutionarily conserved phenomenon, then it should be inducible in blastocysts of normally non-diapausing mammals, such as domestic species. To prove this hypothesis, we examined whether blastocysts from domestic sheep (Ovis aries) could enter into diapause following their transfer into mouse uteri in which diapause conditions were induced. Sheep blastocysts entered into diapause, as demonstrated by growth arrest, viability maintenance and their ED-specific pattern of gene expression. Seven days after transfer, diapausing ovine blastocysts were able to resume growth in vitro and, after transfer to surrogate ewe recipients, to develop into normal lambs. The finding that non-diapausing ovine embryos can enter into diapause implies that this phenomenon is phylogenetically conserved and not secondarily acquired by embryos of diapausing species. Our study questions the current model of independent evolution of ED in different mammalian orders.