Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.     

Improved efficiency of bovine cloning by autologous somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) has been used for the cloning of various mammals. However, the rates of successful, healthy birth are generally poor. To improve cloning efficiency, we report the utilization of an 'autologous SCNT' cloning technique in which the somatic nucleus of a female bovine donor is transferred to its own enucleated oocyte recovered by ovum pick up, in contrast to the routine 'allogeneic SCNT' procedure using oocytes from unrelated females. Our results showed that embryos derived from autologous SCNThave significantly higher developmental competence than those derived from allogeneic SCNT, especiallyat the eight-cell (60 vs 44%), morula (45 vs 36%), and blastocyst (38 vs 23%) stages. The pregnancy and birth rates were also higher for the autologous (39 and 23%), compared to the allogeneic (22 and 6%) SCNT groups. Genome-wide histone3-lysine9 methylation profiles reveal that autologous SCNTembryos have less epigenetic defects than the allogeneic SCNTembryos. This study indicates that autologous SCNT can improve the efficiency of bovine cloning with less reprogramming deficiency.     

Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning

The birth of 'Dolly', the first mammal cloned from an adult donor cell, has sparked a flurry of research activities to improve cloning technology and to understand the underlying mechanism of epigenetic reprogramming of the transferred somatic cell nucleus. Especially in ruminants, somatic cell nuclear transfer (SCNT) is frequently associated with pathological changes in the foetal and placental phenotype and has significant consequences for development both before and after birth. The most critical factor is epigenetic reprogramming of the transferred somatic cell nucleus from its differentiated status into the totipotent state of the early embryo. This involves an erasure of the gene expression program of the respective donor cell and the establishment of the well-orchestrated sequence of expression of an estimated number of 10 000-12 000 genes regulating embryonic and foetal development. The following article reviews the present knowledge on the epigenetic reprogramming of the transferred somatic cell nucleus, with emphasis on DNA methylation, imprinting, X-chromosome inactivation and telomere length restoration in bovine development. Additionally, we briefly discuss other approaches towards epigenetic nuclear reprogramming, including the fusion of somatic and embryonic stem cells and the overexpression of genes crucial in the formation and maintenance of the pluripotent status. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realising the great potential of SCNT for basic biological research and for various agricultural and biomedical applications.     

ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows

Aberrant epigenetic nuclear reprogramming, especially imprinting pattern disorders, is one of the major causes of failure of clone development from somatic cell nuclear transfer (SCNT). Previous studies showed that ZFP57 is a key protein required for imprint maintenance after fertilization. In this study, we found that imprinting control regions in several imprinted genes were significantly hypomethylated in cloned embryos compared with in vitro fertilization embryos, indicating a loss of imprinted gene methylation. The ZFP57 expression was capable of maintaining the correct degree of methylation at several imprinting control regions and correcting abnormal hypomethylation. Moreover, we successfully obtained bovine fetal fibroblasts overexpressing ZFP57, which were used as donors for SCNT. Our results demonstrated that overexpression of ZFP57 increased total and trophectoderm cell numbers and the ratio of inner cell mass to total cells, reduced the apoptosis rate and significantly enhanced the development of SCNT blastocysts in vitro, ultimately achieving a degree of methylation similar to that in in vitro fertilization embryos. We concluded that overexpression of ZFP57 in donor cells provided an effective method for enhancing nuclear reprogramming and developmental potential in SCNT embryos. The ZFP57 protein played a key role in maintaining the methylation of imprinted genes during early embryonic development, which may be effective for enhanced SCNT in cattle.     

Unexpected nuclear localization of Cdc25C in bovine oocytes, early embryos, and nuclear-transferred embryos

It is clear from a wide range of studies that the nuclear/cytoplasmic distribution of Cdc25C has important functional consequences for cell cycle control. It is now admitted that in somatic cells, the localization of Cdc25C in the cytoplasm is required to maintain the cell in an interphasic state and that Cdc25C has to translocate to the nucleus just before M-phase to induce mitotic events. We characterized the expression and localization of Cdc25C during oocyte maturation, the first embryo mitosis, and the first steps of somatic cell nuclear transfer (SCNT) in cattle. We demonstrated that Cdc25C was expressed throughout the maturation process and the early development. We clearly showed that Cdc25C was localized in the nucleus at the germinal vesicle stage and during the early development until the blastocyst stage. However, the signal change in blastocyst and Cdc25C became cytoplasmic as is the case in somatic cells. Thus, oocytes and early embryonic cells presented a specific nuclear Cdc25C localization different from the one observed in somatic cells, suggesting that Cdc25C could have a particular localization/regulation in undifferentiated cells. Following SCNT, Cdc25C became nuclear as soon as the nucleus swelled, and this localization persisted until the blastocyst stage, as is the case in in vitro fertilized embryos. The Cdc25C nuclear localization appeared to constitute a major change, which could be associated with the reorganization of the somatic nucleus upon nuclear transfer.     

Differential reprogramming of somatic cell nuclei after transfer into mouse cleavage stage blastomeres

Mammalian somatic cell cloning requires factors specific to the oocyte for reprogramming to succeed. This does not exclude that reprogramming continues during the zygote and cleavage stages. The capacity or role of zygotic and cleavage stages to reprogram somatic cell nuclei is difficult to assess due to the limited development of somatic cell nuclei transplanted into cytoplasts of these stages. Alternatively, tetraploid embryos have been used to study reprogramming and can be assessed for their contribution to extra-embryonic lineages. When mouse cumulus cell nuclei transgenic for Oct4-green fluorescent protein (GFP) were injected into intact two- and four-cell stage blastomeres, manipulated embryos developed into blastocysts with expression of Oct4-GFP as observed in embryos produced by nuclear transfer into metaphase II oocytes. However, only the latter contributed to extra-embryonic tissues in day 10.5 conceptuses, with the exclusion of the somatic genome in cells originating from transfer into blastomeres already at 5.5 days post conception. Somatic nuclei transferred into cleavage stage blastomeres reinitiated expression of an embyronic-specific transgene, but lacked the extent of reprogramming required for contribution to postimplantation development, even when complemented by an embryonic genome.     

Epigenetic alteration of the donor cells does not recapitulate the reprogramming of DNA methylation in cloned embryos

Epigenetic reprogramming is a prerequisite process during mammalian development that is aberrant in cloned embryos. However, mechanisms that evolve abnormal epigenetic reprogramming during preimplantation development are unclear. To trace the molecular event of an epigenetic mark such as DNA methylation, bovine fibroblasts were epigeneticallyaltered by treatment with trichostatin A (TSA) and then individually transferred into enucleated bovine oocytes. In the TSA-treated cells, expression levels of histone deacetylases and DNA methyltransferases were reduced, but the expression level of histone acetyltransferases such as Tip60 and histone acetyltransferase 1 (HAT1) did not change compared with normal cells. DNA methylation levels of non-treated (normal) and TSA-treated cells were 64.0 and 48.9% in the satellite I sequence (P < 0.05) respectively, and 71.6 and 61.9% in the alpha-satellite sequence respectively. DNA methylation levels of nuclear transfer (NT) and TSA-NT blastocysts in the satellite I sequence were 67.2 and 42.2% (P < 0.05) respectively, which was approximately similar to those of normal and TSA-treated cells. In the alpha-satellite sequence, NT and TSA-NT embryos were substantially demethylated at the blastocyst stage as IVF-derived embryos were demethylated. The in vitro developmental rate (46.6%) of TSA-NT embryos that were individually transferred with TSA-treated cells was higher than that (31.7%) of NT embryos with non-treated cells (P < 0.05). Our findings suggest that the chromatin of a donor cell is unyielding to the reprogramming of DNA methylation during preimplantation development, and that alteration of the epigenetic state of donor cells may improve in vitro developmental competence of cloned embryos.     

Nuclear-cytoplasmic incompatibility and inefficient development of pig-mouse cytoplasmic hybrid embryos

Inter-species somatic cell nuclear transfer (iSCNT) embryos usually fail to develop to the blastocyst stage and beyond due to incomplete reprogramming of donor cell. We evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all iSCNT embryos, irrespective of karyoplast origin, were arrested during early development. We hypothesized that nuclear-cytoplasmic incompatibility could be another reason for failure of embryonic development from iSCNT. We used pig-mouse cytoplasmic hybrids as a model to address nuclear-cytoplasmic incompatibility in iSCNT embryos. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/10 (small) and 1/5 (large) total volume of mouse zygote). The presence of pig cytoplasm significantly reduced the development of mouse zygotes to the blastocyst stage compared with control embryos at 120?h post-human chorionic gondotropin (41 vs 6 vs 94%, P<0.05; 1/10, 1/5, control respectively). While mitochondrial DNA copy numbers remained relatively unchanged, expression of several important genes namely Tfam, Polg, Polg2, Mfn2, Slc2a3 (Glut3), Slc2a1 (Glut1), Bcl2, Hspb1, Pou5f1 (Oct4), Nanog, Cdx2, Gata3, Tcfap2c, mt-Cox1 and mt-Cox2 was significantly reduced in cytoplasmic hybrids compared with control embryos. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of inter-species nuclear transfer embryos.     

Reprogramming somatic cells into stem cells

Recent scientific achievements in cell and developmental biology have provided unprecedented opportunities for advances in biomedical research. The demonstration that fully differentiated cells can reverse their gene expression profile to that of a pluripotent cell, and the successful derivation and culture of human embryonic stem cells (ESCs) have fuelled hopes for applications in regenerative medicine. These advances have been put to public scrutiny raising legal, moral and ethical issues which have resulted in different levels of acceptance. Ethical issues concerning the use of cloned human embryos for the derivation of stem cells have stimulated the search for alternative methods for reversing differentiated cells into multi/pluripotent cells. In this article, we will review the present state of these reprogramming technologies and discuss their relative success. We also overview reprogramming events after somatic cell nuclear transfer (SCNT), as they may further instruct ex ovo strategies for cellular manipulation.     

Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

The processes of cellular differentiation were studied in somatic cell nuclear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients on day 6. All embryos were processed for examination by light and transmission electron microscopy or immunohistochemical labelling for alpha-1-fetoprotein and vimentin. Overall, morphological development of in vivo embryos was superior to IVC and SCNT embryos. Day 7 and particularly day 9 IVC and SCNT embryos had impaired hypoblast development, some lacking identifiable inner cell masses. On day 11, only in vivo and IVC embryos had developed an embryonic disc, and gastrulation was evident in half of in vivo embryos and one IVC embryo. By day 13, all in vivo embryos had completed gastrulation whereas IVC and SCNT embryos remained retarded. On days 17 and 19, in vivo embryos had significantly more somites and a more developed allantois than IVC and SCNT embryos. We conclude that IVC and particularly SCNT procedures cause a retardation of embryo development and cell differentiation at days 7-19 of gestation.     

Overexpression of miR-101-2 in donor cells improves the early development of Holstein cow somatic cell nuclear transfer embryos

Accumulating studies have suggested that microRNA play a part in regulating multiple cellular processes, such as cell proliferation, apoptosis, the cell cycle, and embryo development. This study explored the effects of miR-101-2 on donor cell physiological status and the development of Holstein cow somatic cell nuclear transfer (SCNT) embryos in vitro. Holstein cow bovine fetal fibroblasts (BFF) overexpressing miR-101-2 were used as donor cells to perform SCNT; then, cleavage rate, blastocyst rate, inner cell mass-to-trophectoderm ratio, and the expression of some development- and apoptosis-related genes in different groups were analyzed. The miR-101-2 suppressed the expression of inhibitor of growth protein 3 (ING3) at mRNA and protein levels, expedited cell proliferation, and decreased apoptosis in BFF, suggesting that ING3, a target gene of miR-101-2, is a potential player in this process. Moreover, by utilizing donor cells overexpressing miR-101-2, the development of bovine SCNT embryos in vitro was significantly enhanced; the apoptotic rate in SCNT blastocysts was reduced, and the inner cell mass-to-trophectoderm ratio and SOX2, POU5F1, and BCL2L1 expression significantly increased, whereas BAX and ING3 expression decreased. Collectively, these findings suggest that miR-101-2 promotes BFF proliferation and vitality, reduces their apoptosis, and improves the early development of SCNT embryos.     

Early zygotes are suitable recipients for bovine somatic nuclear transfer and result in cloned offspring

Cloning by somatic cell nuclear transfer (SCNT) subverts sperm-mediated fertilization that normally leads to physiological activation of the oocyte. Therefore, artificial activation is required and it is presently unclear what developmental consequences this has. In this study, we aimed to improve cattle cloning efficiency by utilizing a more physiological method of activating SCNT reconstructs. We carried out in vitro fertilization (IVF) of zona-intact bovine oocytes before SCNT. We removed the zona pellucida 4 h after insemination, stained the fertilized eggs with Hoechst 33342 and mechanically removed both male and female chromatin. The enucleated pre-activated cytoplasts were fused with male adult ear skin fibroblasts ("IVF-NT" group). Chemically activated SCNT embryos, produced according to our standard operating procedure for zona-free SCNT, served as controls. After 7 days, in vitro development to blastocysts of morphological grade 1-3 or grade 1-2 was very similar in both groups (39 vs 40% and 20 vs 21% respectively). However, post-implantation development was improved after sperm-mediated activation. Across four replicate runs, pregnancy establishment at day 35 was significantly higher for IVF-NT than for control SCNT embryos (30/49 = 61 vs 17/41 = 42% respectively; P < 0.05). Development into calves at term or weaning was also higher in the IVF-NT group compared with control SCNT (9/49 = 18 vs 3/41 = 7% and 6/49 = 12 vs 3/41 = 7%; P = 0.11 and 0.34 respectively).     

Fate of centrosomes following somatic cell nuclear transfer (SCNT) in bovine oocytes

Cloning mammalians by somatic cell nuclear transfer (SCNT) remains inefficient. A majority of clones produced by SCNT fail to develop properly and of those which do survive, some exhibit early aging, premature death, tumors, and other pathologies associated with aneuploidy. Alterations of centrosomes are linked to aberrant cell cycle progression, aneuploidy, and tumorigenesis in many cell types. It remains to be determined how centrosomes are remodeled in cloned bovine embryos. We show that abnormalities in either distribution and/or number of centrosomes were evident in approximately 50% of reconstructed embryos following SCNT. Moreover, centrosome abnormalities and failed 'pronuclear' migration which manifested during the first cell cycle coincided with errors in spindle morphogenesis, chromosome alignment, and cytokinesis. By contrast, nuclear mitotic apparatus protein (NuMA) exhibited normal expression patterns at metaphase spindle poles and in 'pronucleus' during interphase. The defects in centrosome remodeling and 'pronuclear' migration could lead to chromosome instability and developmental failures associated with embryo production by SCNT. Addressing these fundamental problems may enhance production of normal clones.     

Analysis of DNA fragmentation in bovine somatic nuclear transfer embryos using TUNEL

The production of cloned animals is an inefficient process because of early or late embryonic losses. This study focused on the DNA fragmentation that occurs during embryonic development. The occurrence of DNA fragmentation was examined in bovine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (NT) using the terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL). IVF and NT embryos at the two-cell to blastocyst stage were stained by TUNEL for the analysis of DNA-fragmented nuclei and with propidium iodide for determination of the total number of cells. DNA fragmentation was first detected in NT embryos at the four-cell stage, but in IVF embryos at the six- to eight-cell stage. The percentage of embryos with at least one DNA-fragmented nucleus increased with the advance of the developmental stage of embryos in both IVF and NT groups. The DNA-fragmented nucleus index in NT embryos that developed beyond the four-cell stage was significantly higher (P<0.01) than that of IVF embryos at the same stage. In the both IVF and NT groups, TUNEL-labelled cells were detected in almost all blastocysts and were mainly observed in presumptive inner cell mass (ICM) cells of embryos. The DNA-fragmented nucleus index was negatively correlated with the total number of cells in NT blastocysts, but this relationship was not observed in IVF blastocysts. These results suggest that the high occurrence of DNA fragmentation observed in NT embryos may be related to early embryonic loss after transfer.     

Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are not required for the global histone deacetylation observed after germinal vesicle breakdown (GVBD) in porcine oocytes

The acetylation of nuclear core histone has been suggested to work as an epigenetic mark for transmitting gene expression patterns to daughter cells. Global histone deacetylations, presumably involved in the reprogramming of the gene expression, have been observed after germinal vesicle breakdown (GVBD) in a cell cycle-dependent manner during meiotic maturation of mouse and porcine oocytes, although the regulation mechanism of histone deacetylation has not been studied well. In the present study, we examined the involvement of a crucial cell-cycle-regulator, maturation-promoting factor (MPF), and a meiosis-related kinase, mitogen-activated protein kinase (MAPK), in the global histone deacetylation during porcine oocyte maturation. In order to know whether the activities of MPF and MAPK were required, or the breakdown of GV membrane was sufficient, for the global histone deacetylation observed after GVBD, we artificially destroyed the GV membrane of the porcine immature oocytes. The artificial GV destruction (AGVD) induced histone deacetylation without the activation of MPF and MAPK. This deacetylation after AGVD was not affected by an MPF inhibitor, roscovitine, or an inhibitor of protein synthesis, cycloheximide, but was completely prevented by an inhibitor of histone deactylases (HDACs), trichostatine A. HDAC1 was present in the GV of the immature oocytes and localized on chromosomes after GVBD and AGVD. These results suggest that the MPF and MAPK activities were dispensable and the breakdown of the GV membrane was sufficient for the global histone deacetylation, which was catalyzed by HDAC activity.     

Developmental potential of cloned mouse embryos reconstructed by a conventional technique of nuclear injection

The fact that most of the advances in mouse cloning by nuclear transfer originate from research in a limited number of laboratories demonstrates the complexity of the reported technologies. The development of alternative and more simple techniques of nuclear transfer may therefore be of interest. Furthermore, the preimplantation biology of cloned mouse embryos originating from somatic cells has not yet been studied in detail. In the present study, a modified conventional injection (mCI) technique for cloning mice from somatic cells is described. The preimplantation development and morphology of the resulting nuclear transfer embryos in comparison with parthenogenetic embryos and embryos obtained by intracytoplasmic sperm injection (ICSI) under comparable conditions was also studied. Finally, the capacity of nuclear transfer embryos for full-term development was investigated. Eighty-nine per cent of oocytes injected with cumulus cell nuclei under mCI conditions survived and formed zygotes. However, the rate of development of these zygotes to the blastocyst stage was significantly lower (29%) than that of ICSI or parthenogenetic zygotes (95 and 92%, respectively). Cloned blastocysts had a significantly lower mean number of cells in the inner cell mass (9) and trophectoderm (52) and a lower inner cell mass:total cell ratio (14%) than did their counterparts (31, 143 and 18% for ICSI and 21, 92 and 18% for parthenogenetic blastocysts, respectively). This correlated with a significantly higher proportion of dead cells in the cloned blastocysts. The poor quality of cloned blastocysts may explain the low rate of full-term fetal development of somatic mouse clones.     

Somatic cell nuclear transfer alters peri-implantation trophoblast differentiation in bovine embryos

Abnormal placental development limits success in ruminant pregnancies derived from somatic cell nuclear transfer (SCNT), due to reduction in placentome number and consequently, maternal/fetal exchange. In the primary stages of an epithelial-chorial association, the maternal/fetal interface is characterized by progressive endometrial invasion by specialized trophoblast binucleate/giant cells (TGC). We hypothesized that dysfunctional placentation in SCNT pregnancies results from aberration in expression of genes known to be necessary for trophoblast proliferation (Mash2), differentiation (Hand1), and function (IFN-tau and PAG-9). We, therefore, compared the expression of these factors in trophoblast from bovine embryos derived from artificial insemination (AI), in vitro fertilization (IVF), and SCNT prior to (day 17) and following (day 40 of gestation) implantation, as well as TGC densities and function. In preimplantation embryos, Mash2 mRNA was more abundant in SCNT embryos compared to AI, while Hand1 was highest in AI and IVF relative to SCNT embryos. IFN-tau mRNA abundance did not differ among groups. PAG-9 mRNA was undetectable in SCNT embryos, present in IVF embryos and highest in AI embryos. In postimplantation pregnancies, SCNT fetal cotyledons displayed higher Mash2 and Hand1 than AI and IVF tissues. Allelic expression of Mash2 was not different among the groups, which suggests that elevated mRNA expression was not due to altered imprinting status of Mash2. The day 40 SCNT cotyledons had the fewest number of TGC compared to IVF and AI controls. Thus, expression of genes critical to normal placental development is altered in SCNT bovine embryos, and this is expected to cause abnormal trophoblast differentiation and contribute to pregnancy loss.     

Genome of non-living cells: trash or recycle?

Reproductive technologies have been often used as a tool in research not strictly connected with developmental biology. In this study, we retrace the experimental routes that have led to the adoption of two reproductive technologies, ICSI and somatic cell nuclear transfer (SCNT), as biological assays to probe the 'functionality' of the genome from dead cells. The structural peculiarities of the spermatozoa nucleus, namely its lower water content and its compact chromatin structure, have made it the preferred cell for these experiments. The studies, primarily focused on mice, have demonstrated an unexpected stability of the spermatozoa nuclei, which retained the capacity to form pronuclei once injected into the oocytes even after severe denaturing agents like acid treatment and high-temperature exposure. These findings inspired further research culminating in the production of mice after ICSI of lyophilized spermatozoa. The demonstrated non-equivalence between cell vitality and nuclear vitality in spermatozoa prompted analogous studies on somatic cells. Somatic cells were treated with the same physical stress applied to spermatozoa and were injected into enucleated sheep oocytes. Despite the presumptive fragile nuclear structure, nuclei from non-viable cells (heat treated) directed early and post-implantation embryonic development on nuclear transfer, resulting in normal offspring. Recently, lyophilized somatic cells used for nuclear transfer have developed into normal embryos. In summary, ICSI and SCNT have been useful tools to prove that alternative strategies for storing banks of non-viable cells are realistic. Finally, the potential application of freeze-dried spermatozoa and cells is also discussed.