Josef Fulka

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.

The Maternal Nucleolus Is Essential for Early Embryonic Development in Mammals

With fertilization, the paternal and maternal contributions to the zygote are not equal. The oocyte and spermatozoon are equipped with complementary arsenals of cellular structures and molecules necessary for the creation of a developmentally competent embryo. We show that the nucleolus is exclusively of maternal origin. The maternal nucleolus is not necessary for oocyte maturation; however, it is necessary for the formation of pronuclear nucleoli after fertilization or parthenogenetic activation and is essential for further embryonic development. In addition, the nucleolus in the embryo produced by somatic cell nuclear transfer originates from the oocyte, demonstrating that the maternal nucleolus supports successful embryonic development.

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.

DNA methylation pattern in pig in vivo produced embryos

DNA methylation/demethylation pattern, determined by 5-methylcytosine (5-MeC) immunostaining, was evaluated in porcine “in vivo” produced embryos from zygote up to the blastocyst stage. In one-cell stage embryos, only the maternal pronucleus showed a positive labeling whilst the paternal pronucleus showed almost no labeling. The intensity of labeling is high until the late morula stage. Blastocysts containing less than 100 cells showed the same intensity of labeling in both the inner cell mass (ICM) nuclei and the trophectodermal (TE) cell nuclei. Interestingly, with further cell multiplication, cells of the ICM became more intensively labeled when compared to TE cells. This distinct methylation pattern is even more profound in blastocysts containing about 200–300 cells and is not caused by the difference in the cell volume of ICM and TE cells.

Cloning by somatic cell nuclear transfer

The birth of the first cloned mammals, produced by the introduction of somatic cell nuclei into enucleated oocytes, was an impressive and surprising development.(1) Although the ethical debate has been intense, the important scientific questions raised by this work have been inadequately discussed and are still unresolved. In this essay we address three questions about nuclear transplantation in the eggs of mice and domestic animals. First, why were the recent experiments on somatic cell cloning successful, when so many others have failed? Second, were these exceptional cases, or is somatic cloning now open to all? Third, what are the future possibilities for increasing the efficiency and wider applicability of the cloning process? BioEssays 20:847–851, 1998.

Enucleolation of porcine oocytes.

Germinal vesicles (GVs) in immature mammalian oocytes contain prominent nucleoli whose role in the process of oocyte maturation is not fully understood. Here we report that the microsurgical removal of nucleoli from immature fully grown porcine oocytes permits germinal vesicle breakdown and chromosome condensation and the enucleolated oocytes mature up to the second metaphase. Interestingly, the enucleolation of growing oocytes which, although unable to mature, resulted in germinal vesicle breakdown and the formation of a cluster of condensed chromatin. These results indicate that the nucleolus in fully grown oocytes is dispensable at least for nuclear maturation. On the other hand, the results obtained in growing oocytes suggest the role of the nucleolus in the cell cycle regulation.

Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine-pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16-25-cell stage for pig-bovine iSCNT embryos and at the four-cell stage for bovine-pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus-cytoplasm compatibility between these animal species.

Somatic cell nuclear transfer (SCNT) in mammals : The cytoplast and its reprogramming activities

It is now more than nine years since Dolly, the worlds first somatic cell cloned mammal was born, and the success of somatic cell nuclear transfer (SCNT) is still disappointingly low. Only about 3-5% of reconstructed embryos develop to term, and it is also evident that even if some clones are born, they are not necessarily fully developed and healthy. Embryonic and neonatal abnormalities of cloned offspring are probably a result of incorrect or incomplete reprogramming of the transferred donor cell nuclei. Such an incomplete reprogramming reflects the extremely low efficiency of SCNT. The key role in the process of reprogramming has been attributed to the enucleated oocyte-cytoplast into which the somatic cell nucleus is transferred. In our chapter, we will discuss the methodological approaches used for the preparation of cytoplasts and their possible reprogramming activities.

Antral follicles confer developmental competence on oocytes.

This paper addresses the proposition, first advanced by Wilson (1925), that successful embryogenesis depends on an ordered series of events in oogenesis. It is at the completion of this varied set of intracellular changes that the oocyte finally acquires its full capacity to support fertilisation and development. Amongst the earliest nuclear events are those associated with chromosome pairing and meiotic recombination. During the growth phase cell volume increases 300-fold and the cytoplasm becomes the storage site for RNA and protein which will be mobilised during early development. Finally, a short phase of intracellular reprogramming, or maturation, completes the series of events during oogenesis that confer developmental competence upon the oocyte. Follicle cell support is an indispensable requirement for ordered oocyte development and provides the early germline cell with many of the essential nutrients and growth regulators required to ensure progression through the protracted growth phase (see contributions by Cecconi & Rosella and De Felici et al. this issue). Although different, the interactions between the full-grown oocyte and the antral follicle are no less crucial to the acquisition of competence than those involved in the earlier stages of oogenesis.