|DOI : http://dx.doi.org/10.12750/JET.2012.27.4.193|
1Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju 600-701, Republic of Korea
3Institute of Agriculture & Life Science, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju 660-701, Korea
2Animal Developmental and Biotechnology Group, Department of Animal Science, School of Biosciences, The University of Nottingham, Sutton Bonington, Loughborough, Leicestershire, LE12 5RD, United Kingdom
|received: 2012. 10. 14, revised: 2012. 10. 15, accepted: 2012. 11. 6|
|The development of embryos reconstructed by nuclear transfer is dependent upon numerous factors including the type of recipient cell, method of enucleation, the type of donor cell, method of reconstruction, activation, the cell cycle stage of both the donor nucleus and the recipient cytoplasm and the method of culture of the reconstructed embryos. Many of these points which have been reviewed extensively elsewhere (Sun and Moor, 1995; Colman, 1999; Oback and Wells, 2002; Renard et al., 2002; Galli et al., 2003b), here we will concentrate on main area, the production of suitable cytoplast and nuclear donor, nuclear-cytoplasmic coordination, oocyte activation, culture of reconstructed embryos, and the effects that this may have on development.|
The concept of nuclear transfer (NT) was first proposed in 1938 by Spemann, who suggested the transfer of a single nucleus into an enucleated oocyte as a method to study cell differentiation and nuclear equivalence. However, due to his death and to inadequate technical skills the first successful NT experiments were not reported until 1952 when Briggs and King demonstrated the production of swimming tadpoles after transplantation of blastula nuclei into enucleated frog eggs. Experiments in amphibians continued and in 1962, John Gurdon reported the production of adult toads (Xenopus laevis) after the transfer of nuclei from tadpole intestinal epithelial cells (Gurdon, 1962a; Gurdon, 1962b). This was the first de-monstration that the nucleus from a differentiated cell could successfully support development, however, in subsequent ex-periments using nuclei derived from adult keratinocytes, al-though swimming tadpoles were produced no adult animals were obtained (Gurdon et al., 1975).
In mammals, due to the size of the oocyte and the require-ment for more specialised equipment NT was not reported until 1975 when Bromhall attempted NT using rabbit eggs. In the early 1980s McGrath and Solter reported the first successful NT in mammals, live offspring were obtained after the swapping of pronuclei between fertilised zygotes demonstrating that embryo development could occur after micromanipulation (Mc-Grath and Solter, 1983), however, when nuclei from later de-velopmental stages were transferred into enucleated zygotes no live offspring were obtained (McGrath and Solter, 1984). Using a modification of the technique reported by McGrath and Solter, Willadsen reported in 1986 the birth of live lambs after the production of embryos by NT using early embryonic blas-tomeres from 8～16 cell embryos as nuclear donors and enu-cleated Metaphase II (MII) oocytes as cytoplast recipients (Willadsen, 1986). The use of early embryos as nuclear donors and enucleated MII oocytes as recipients continued in other species and both cattle (Robl et al., 1987) and pigs (Prather et al., 1989) were subsequently cloned. The use of early em-bryos as nuclear donors and the low frequency of development obtained severely limited the application of this technology and efforts were focused on the development of NT techniques from cultured cell populations which would allow the produc-tion of large number of genetically identical animals and pro-vide a route for precise genetic modification.
In 1994, Sims and First reported the production of live calves from inner cell mass cells which had been maintained in culture, however, under the conditions employed these cells grew very slowly, if at all, and were of limited use. In 1996, Campbell and colleagues (Campbell et al., 1996b) reported the birth of live lambs from cultured cells which were established from a blastocyst stage embryo, these cells had in fact diffe-rentiated in culture and this report paved the way for the sub-sequent development of offspring using cells derived from foetal and adult tissues (Wilmut et al., 1997). Since this time offspring have been reported in a range of species including cattle (Cibelli et al., 1998), mice (Wakayama et al. 1998), goats (Baguisi et al., 1999), pigs (Polejaeva et al., 2000), cats (Shin et al., 2002), rabbits (Chesne et al., 2002), mules (Woods et al,. 2003), rats (Zhou et al., 2003) and horses (Galli et al., 2003a) and from a variety of cell types derived from embryos, foetuses, juvenile and adult animals.
Studies in mammalian NT have utilised a range of embryo-nic cells as cytoplast recipients including oocytes, zygotes and early cleavage stage embryos with varying success. Enucleated zygotes of both mouse (McGrath and Solter, 1983; McGrath and Solter, 1984; Kwon and Kono, 1996), cattle (Prather and First, 1990), and pig (Prather et al., 1989) resulted in limited development of the reconstructed embryos. Although studies in the mouse demonstrated that enucleated 2-cell embryos could support development from early blastomere nuclei (Tsunoda et al., 1987), there are no reports of successful development from later stage nuclear donors. The use of matured oocytes (also termed unfertilised eggs) arrested at metaphase of the second meiotic division (MII) has resulted in successful development from a range of cell types in a variety of species and have commonly become the cytoplast of choice. MII oocytes for use as cytoplast recipients can be obtained by a variety of means including; in vivo maturation by flushing from the ovi-duct (Willadsen, 1986), by in vitro maturation of oocytes as-pirated from antral follicles of living animals or by in vitro maturation of oocytes recovered from slaughtered animals. Although the use of in vivo matured oocytes may have bene-ficial effects on early embryo and foetal development (Wells et al., 1997), in vitro matured oocytes are commonly used for NT in farm animal species according to cost effectiveness (Farin et al., 2001). MII oocytes have been used as cytoplast recipients for NT utilising a number of protocols, differences in these methods including the method of enucleation, the ti-ming of enucleation, the method and timing of nuclear transfer (fusion or injection) and activation may all effect the develop-ment of the reconstructed embryos. Some of these differences will be discussed in more detail with relation to biological differences in the resultant cytoplast and the possible effects on development.
Embryonic blastomeres from early stage embryos were first used as nuclear donors. When it was established that co-ordi-nation of the cell cycle between donor and recipient cells was essential to ensure normal development several laboratories started to investigate the possibility of using differentiated cell types on specific cell cycle stages for somatic cell nuclear transfer (SCNT). After the production of the first mammals from cultured embryonic (Campbell et al., 1996) foetal and adult cell lines (Wilmut et al., 1997) numerous studies provided ex-tensive evidence that somatic cells from different tissues and ages of animals can be used for SCNT (Shiga et al., 1999; Zakhartchenko et al., 1999; Kato et al., 2000). Embryonic stem cells have been used for SCNT and better development was reported in some studies (Zhou et al., 2001; Eggan et al., 2002), although other reports indicate widespread epigenetic instability in ES cloned mice (Humpherys et al., 2001). In an-other study somatic cells clones showed normal expression of imprinted genes after SCNT (Inoue et al., 2002) although this contradicted a report indicating altered gene expression pattern in clones derived from ES and cumulus cells (Humpherys et al., 2001; Humpherys et al., 2002). The differences between groups could be related to variation in the ES cells used in each study and also may be affected by the manipulation and culture systems used in each study. Unfortunately no conclu-sion can be made on what is the most appropriate cell type for SCNT. However, what is certain is that cells derived from early embryos, foetuses, adult differentiated and postmitotic cells (Eggan et al., 2004) have successfully been employed for the generation of cloned animals.
During a single cell cycle a cell must duplicate all of its components and give rise to two daughter cells which are iden-tical to each other and identical to the cell at birth (Mitchison, 1971). The events occurring during a cell cycle can be divided into those involving cell growth and those involving the nu-cleus. These two major groups of events are intimately linked, however, for simplicity I will only describe the events occu-rring in the nuclear division cycle.
The cell cycle of eukaryotic cells is divided into four dis-tinct phases: G1, S, G2 and M. The discrete period of DNA synthesis (S phase) is preceded by a pre-DNA synthesis period (G1), and followed by a post-DNA synthetic period (G2). The replicated genetic material is equally segregated to the two daughter cells during mitosis (M-phase). The nuclear division cycle involves two major events, DNA replication (S-phase) and segregation of the duplicated genetic material (M phase or mitosis). During a single cell cycle all chromosomal DNA must be replicated once. The mechanisms by which a cell co-ordinates DNA replication and prevents re-replication of pre-viously replicated DNA are unclear however; central to this control is maintenance of an intact nuclear envelope (Blow and Laskey, 1988).
Early studies in NT reconstructed embryos demonstrated the importance of cell cycle coordination between the donor nu-cleus and the recipient cytoplast in order to prevent DNA damage and maintain ploidy of the reconstructed embryos (for reviews see (Campbell et al., 1996a; Campbell, 2002; Camp-bell and Alberio, 2003)). It is now accepted that two major types of recipient oocytes (enucleated metaphase II (MII) oocytes and pre-activated enucleated MII oocytes) are suitable for development to term after single nuclear transfer. One of the major differences between these recipient oocytes is the levels of protein kinase activities present in the cytoplasm. MII arrested oocytes contain high levels of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities, cytoplasmic protein kinases responsible for the changes in nuclear and chromatin structure during both meiotic and mitotic cell cycles cell cycle (Campbell et al., 1996a). The pre-sence or absence of these protein kinases can have beneficial or deleterious effects on the fate of the transferred nucleus in NT embryos.
Matured oocytes typically become arrested at metaphase of the second meiotic division (MII) and contain high levels of MPF activity. When an interphase nucleus is transferred into MII oocyte, the presence of high levels of MPF in the cyto-plasm induces the transferred nucleus to enter a mitotic divi-sion precociously and causes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC) and dispersion of nucleoli which may be essential for nuclear reprogramming (Collas et al., 1992a). The patterns of NEBD and PCC are dependent upon the stage of the donor nucleus at the time of transfer (Campbell et al., 1996a; Campbell and Alberio, 2003). All nuclei that undergo NEBD, regardless of their cell cycle undergo DNA synthesis following the decline of MPF activity, reformation of the nuclear envelope and nuclear swelling. NEBD and PCC have no apparent deleterious effect on either G1 or G2 nuclei, forming single or double chromatids respectively. However, S-phase chromatin has a typical pulverised appea-rance thought to be associated with high levels of DNA da-mage (Collas et al., 1992b) when mitotic chromosomes are transferred the chromosomes remain condensed (Alberio et al., 2000b) (Fig. 1).
Fig. 1. Effects of nuclear transfer of karyoplasts at defined stages of the cell cycle into cytotplasts with high MPF activity on chromatin fate, DNA replication and ploidy of resultant daughter cells. M: mitosis, MPF: maturation promoting fac-tor, NEBD: nuclear envelop breakdown, PCC: premature condensation, PB: polar body.
On the other hand, if nuclei are transplanted into pre-acti-vated oocytes, in which MPF and MAPK activities have declined following activation or fertilization, no NEBD or PCC are observed. These oocytes are so called ‘permissive’ to do-nor nuclei in G1, S or G2-phases of the cell cycle with coordi-nated DNA replication occurring dependent upon the cell cycle phase of the donor nucleus due to maintenance of an intact nuclear envelope. G1 nuclei undergo a single round of replica-tion, S phase nuclei continue replication and G2 nuclei do not re-replicate (Barnes et al., 1993; Campbell et al., 1993) main-taining correct ploidy in embryos produced under these condi-tions. Therefore, all of the resultant daughter cells will be di-ploid (2n). When any of these three cell cycle stage donor cells are transferred into pre-activated oocytes, development to blastocyst can be achieved (Campbell et al., 1994). This type of oocyte has been termed “a universal recipient” (Campbell et al., 1993). When quiescent cells (G0) are used as nuclear donors, an exception to coordinated replication exists. DNA replication requires the presence of chromosome-bound factors that are thought to attach following mitosis and prior to nuclear assembly. In quiescent cells these factors are lost with time and initiation of DNA replication requires permeabilisation of the nuclear membrane (Leno and Munshi, 1994), this would occur in cytoplasts with high MPF activity. The fate of M phase donor nuclei transferred into pre-activated oocytes has not been described. However, an M phase nucleus may undergo chromatin decondensation, pronuclear formation and DNA syn-thesis, resulting in the production of a tetraploid (4n) daughter cell (Fig. 2).
Fig. 2. Effects of nuclear transfer of karyoplasts at defined stages of the cell cycle into cytotplasts with low MPF activity on chromatin fate, DNA replication and ploidy of resultant daughter cells. M: mitosis, MPF: maturation promoting factor, NEBD: nuclear envelop breakdown, PCC: prema-ture condensation, PB: polar body.
Although development to term has been obtained with both of these cytoplast recipients, improved development to both the blastocyst stage and to term has been reported when the donor chromatin is exposed to the recipient MII cytoplasm for an extended period (Wells et al., 1999; Wells et al., 2003).
Mammalian oocytes are ovulated and arrested at MII until fertilization. During oocyte maturation (progress from the G2/M stages of the first meiotic division to MII) specific reorgani-zation and redistribution of intracellular organelles occurs and the oocytes obtain a full complement of signalling molecules (Miyazaki et al., 1993; Carroll, 2001). The oocytes are re-leased from the meiotic arrest by fertilization and initiate early embryonic development by inducing a series of cellular events within the oocyte. This is referred to as “oocyte activation”. The characteristic event of oocyte activation is initiation of intracellular calcium ([Ca2+]i) oscillations, leading to other events including, resumption and completion of meiosis, cortical gra-nule exocytosis, decondensation of the sperm nucleus, recruit-ment of maternal mRNAs, formation of male and female pro-nuclei and the initiation of DNA synthesis. In NT transfer re-constructed embryos in addition to the transfer of donor genetic material from the karyoplast to the cytoplast, the cytoplast must be ‘activated’ in order to initiate development.
The fusing of the sperm to the oocyte plasma membrane induces an acute increase in cytosolic free Ca2+ concentration (Stricker, 1999) which induces oocyte activation. In all mam-malian oocytes, the initial increase in Ca2+ is followed by a se-ries of highly repetitive Ca2+ transients of high amplitude (Ca2+oscillations). This Ca2+ release originates from the point of sperm entry and subsequent Ca2+oscillations arise almost synchro-nously in the entire oocyte within a second (Miyazaki et al., 1986). These oscillations have been reported in mouse (Cuth-bertson and Cobbold, 1985; Kline and Kline, 1992), rat (Ben- Yosef et al., 1993), rabbit (Fissore and Robl, 1992), bovine (Fissore et al., 1992; Sun et al., 1994), porcine (Sun et al., 1992; Machaty et al., 1997) and human (Taylor et al., 1993; Tesarik, 1994). It is accepted that the site of Ca2+release is the endo-plasmic reticulum (ER), where ryanodine receptors or inositol 1,4,5-triphosphate (IP3) receptors are present (Kline and Kline, 1992). Although a single Ca2+ rise is sufficient for oocyte ac-tivation, sustained Ca2+oscillations require a continuous Ca2+influx to refill the endoplasmic reticulum stores (Miyazaki, 1995) for additional development events (Ozil, 1990).
The intracellular Ca2+release induces the cortical reaction (CR). The cortical granules are located within the cortical re-gion of the oocytes and contain specialized enzymes and gly-coproteins. The cortical reaction occurs after sperm-oocyte membrane fusion, and is manifested by the release of the con-tents of the cortical granules into the perivitelline space, thus establishing a block to polyspermy (Raz et al., 1998).
Fully matured mammalian oocytes can be induced to undergo activation artificially (parthenogenetic) by a variety of physical and chemical treatments in the absence of the male genome (Kaufman and Gardner, 1974). The activation stimuli are de-signed to mimic closely the events initiated by the sperm fac-tor released upon fertilization and result in a Ca2+ rise in the treated oocyte (Saunders et al., 2002). Such treatments include; Application of an electrical pulse. Short and high voltage DC electrical stimuli cause transmembrane Ca2+ influx through the formation of temporary pores in the plasma membranes, allowing an exchange of extracellular and intracellular ions and macro-molecules (Zimmermann and Vienken, 1982). Treatment with the Ca2+ ionophore (A23187) also induced cortical granule exocy-tosis, extrusion of the second polar body and pronuclear for-mation by the contribution of a Ca2+ influx to the [Ca2+]i in-crease and the release of intracellularly stored Ca2+ (Steinhardt et al., 1974; Steinhardt and Epel, 1974; Vincent et al., 1992). It was reported that ionomycin induced a biphasic change in [Ca2+]i and was used to depleted intracellular Ca2+ enhanced stores in mouse oocytes(Jones et al., 1995). Exposure of MII oocytes to 7% ethanol for 5～7 min induces pronuclear for-mation and successful development to blastocyst by promoting a rapid potentiation of InsP3-methiated Ca2+ release through stimulation of InsP3 formation at the plasma membrane (Ilyin and Parker, 1992). In porcine oocytes, intracellular injection of CaCl2 into the cytoplasm induced the exocytosis of cortical granule, decline in the histone H1 kinase activity, changes in the protein synthetic profile, pronuclear formation and subse-quent development (Machaty et al., 1996).
Instead of calcium-dependent mechanisms, another method of artificial activation of MII oocytes is to prevent the produc-tion of cyclin B thereby attacking a portion of the calcium- signaling pathway downstream of the initial calcium signal. Cyclin B is a component of MPF and is continuously syn-thesized in order to maintain adequate levels of active MPF. Inhibition of protein synthesis by treatment with puromycin or cycloheximide induced MII oocytes to enter the first interphase in mouse (Siracusa et al., 1978; Moses and Kline, 1995; Moos et al., 1996) and human oocytes (Balakier and Casper, 1993) but not pig oocytes (Nussbaum and Prather, 1995). Greater activation and subsequent development have been obtained when cycloheximide or puromycin treatment is used in addi-tion to a calcium transient inducing stimulus (Presicce and Yang, 1994; Nussbaum and Prather, 1995; Tanaka and Kana-gawa, 1997).
The ability to artificially activate MII arrested oocytes changes with the age of the oocyte generally determined from the initiation of maturation. Aged oocytes are easier to activate than freshly matured oocytes (Siracusa et al., 1978; Swann and Ozil, 1994; Tanaka and Kanagawa, 1997) because young oocytes continuously synthesize new CSF, which preserves MPF and maintains the meiotic arrest (Fissore and Robl, 1992; Yang et al., 1994). Young oocytes generally require the combination of a calcium stimulus with inhibition of protein synthesis or application of a kinase inhibitor (i.e. 6-dimethylaminopurine (6-DMAP)) (Susko-Parrish et al., 1994), or by inhibition of cdk activity (roscovitine, bohemine (Alberio et al., 2001a)), however, aged oocytes can be activated by a single stimulus which causes a Ca2+increase due to the inactivation of the existing CSF in the cytoplasm of the oocytes and in many cases will activate spontaneously (Plante and King, 1996; Suzuki et al., 1999).
Following artificial activation meiosis resumes and in most mammalian species the resulting parthenotes are haploid. How-ever, if extrusion of the second polar body can be prevented, the embryos become diploid and this improves subsequent de-velopment. In bovine oocytes, the combination of ionomycin and cytochalasin B resulted in completion of the second meio-tic division but prevented extrusion of the second polar body resulting in diploid embryos (Navara et al., 1994). Similarly an activation stimulus in combination with the protein kinase inhibitor 6-DMAP also prevents extrusion of the second polar body and improved development to the blastocyst stage in bovine parthenogenetic embryos (Susko-Parrish et al., 1994). 6-DMAP induced the second meiotic spindle to disintegrate, inducing oocyte entry directly into interphase with only one diploid pronucleus.
Nuclear transfer reconstructed embryos may be cultured in vitro to the blastocyst stage after which they will be transferred to a surrogate recipient for development to term. Alternatively, embryos may be cultured in vivo in the ligated oviduct of a suitable host animal (in general sheep) until a stage suitable for transfer to a final surrogate mother is reached.
A number of in vitro culture media have been developed for individual species, these have included co-culture systems uti-lizing primary oviductal cell monolayers or established cell lines (Thompson, 2000; Menezo and Herubel, 2002). Traditio-nally foetal calf serum was used as a media supplement, how-ever more recently defined culture media have been developed i.e. mSOF (synthetic oviduct fluid media) for cattle and sheep (Walker et al., 1992; Matsuyama et al., 1993), NCSU23 (North Carolina State University) for pigs (Machaty et al., 1998) and CZB (Chatot, Ziomek and Bavister) or KSOM for mice (Chatot et al., 1991). The use of low oxygen systems in the absence of co-culture has also been reported to improve development (Watson et al., 1994). The culture conditions con-sist in either one or two steps culture media, in which the requirements are adjusted for the embryo at different stages of development.
More recently an alternative strategy was reported for the culture of NT reconstructed porcine embryos (Polejaeva et al., 2000). Due to the low birth rates reported following in vitro culture of unmanipulated embryos, clones were immediately transferred into the oviduct of a synchronized recipient for de-velopment to term. Due to the low frequency of development expected, large numbers of embryos were transferred and gave rise to offspring normal. It would be desirable to develop a culture system that would promote development of embryos with high developmental potential to term. Present culture sys-tems tend to promote development to the blastocyst stage al-though the viability of those after transfer is severely compro-mised considering that only 5～20% reach term. Developing non-invasive screening methods for embryo quality, in which the whole embryo can be assessed for developmental potential, are desirable. Some methods consist in the analysis of ploidy or gene expression or single blastomeres, but the information obtained by these procedures is not entirely useful considering the high rate of mosaicism in in vitro cultured embryos, as well as differences in gene expression.
Overall the frequency of development to term of so called cloned embryos is low, although difficulties arise in comparing the results from different laboratories and in different species estimates of approximately 2～3% of fused couplets have been reported (Gurdon and Colman, 1999). Losses during early pregnancy account for up to 40% in ruminants (cattle, sheep and goats). It has been reported that failure to form a normal placenta is the main cause of abortion at this stage (Hill et al., 2000). Lack of normal placentome development and vascula-risation is also accountable for growth deficiencies as well as for the frequent observation of hydrops later in gestation (Hill et al., 1999; Heyman et al., 2002). Similar placental abnor-malities have been observed in mouse and sheep, although not in goats and pigs. Postnatal development is characterized with a higher mortality rate in the first week after delivery. This can be the result of dystocia, related to the increased body size of the foetuses, immature lungs, general weakness, predisposi-tion to infections, and weight loss (Zakhartchenko et al., 2001). Despite the high rate of losses normal cloned animals have been reported in the literature (Chavatte-Palmer et al., 2002; Cibelli et al., 2002), although some authors have shown that gene expression of all cloned mice is altered (Humpherys et al., 2002). Due to the stochastic pattern in the occurrence of abnormalities an incomplete or abnormal ‘reprogramming’ is suggested as the main reason for the altered gene expression and phenotypic aberrations. The consequences of such altera-tions are unpredictable and may be far reaching.
The technique of nuclear transfer (NT) is now well established in a variety of species, however, despite considerable research it still remains an inefficient technique. In general across the species examined only 1～2% of reconstructed embryos are able to develop to term and produce live offspring (Gurdon and Colman, 1999). In addition, many of the resultant offspring suffer from a range of abnormalities and many die within the first few months of birth. Although a number of refinements have been introduced, including the use of defined culture media, piezzo assisted injection for transfer of the donor nu-cleus and improvements in in vitro oocye maturation, the me-thodology of NT has remained essentially unchanged since the production of the 1st live offspring using embryonic blasto-meres as nuclear donors (Willadsen, 1986). There are nume-rous factors affecting development of the reconstructed em-bryos: the type of recipient cell, method of enucleation, the type of the cell acting as nuclear donor, the cell cycle stages of both the donor nucleus and the recipient cytoplasm and the method of culture of the reconstructed embryos. Oocyte qua-lity is critical in all reproductive technologies. In particular the cell cycle stage and the quality of the oocyte to be used as a cytoplast recipient for embryo reconstruction is central to the development of embryos produced by NT.
|1.Alberio R, Kubelka M, Zakhartchenko V, Hajduch M, Wolf E and Motlik J. 2000a. Activation of bovine oocytes by spe-cific inhibition of cyclin-dependent kinases. Molecular Re-production and Development 55:422-432.
2.Alerio R, Motlik J, Stojkovic M, Wolf E and Zakhartchenko V. 2000b. Behavior of M-phase synchronized blastomeres after nuclear transfer in cattle. Molecular Reproduction and Development 57:37-47.
3.Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Mi-dura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW and Echelard Y. 1999. Production of goats by somatic cell nuclear transfer. Nature Biotechnology 17:456-461.
4.Balakier H and Casper RF. 1993. Experimentally induced parth-enogenetic activation of human oocytes. Human Reproduc-tion 8:740-743.
5.Barnes FL, Collas P, Powell R, King WA, Westhusin M and Shepherd D. 1993. Influence of recipient oocyte cell-cycle stage on DNA synthesis, nuclear envelope breakdown, chro-mosome constitution, and development in nuclear trans-plant bovine embryos. Molecular Reproduction and Deve-lopment 36:33-41.
6.Ben-Yosef D, Oron Y and Shalgi R. 1993. Prolonged, repe-titive calcium transients in rat oocytes fertilized in vitro and in vivo. FEBS Letters 331:239-242.
7.Blow JJ and Laskey RA. 1988. A role for the nuclear enve-lope in controlling DNA replication within the cell cycle. Nature 332:546-548.
8.Briggs R and King TJ. 1952. Trasplantation of living nuclei from blastula cells into enucleated frog's eggs. Proceedings of the National Academy of Science USA 38:455-461.
9.Bromhall JD. 1975. Nuclear transplantation in the rabbit egg. Nature 258:719-722.
10.Campbell KHS and Alberio R. 2003. Reprogramming the ge-nome: role of the cell cycle. Reproduction Supplement 61: 477-494.
11.Campbell KHS, Loi P, Cappai P and Wilmut I. 1994. Improved development to blastocyst of ovine nuclear transfer em-bryos reconstructed during the presumptive S-phase of enu-cleated activated oocytes. Biology of Reproduction 50:1385- 1393.
12.Campbell KHS, Loi P, Otaegui PJ and Wilmut I. 1996a. Clo-ning mammals by nuclear transfer. Co-ordinating nuclear and cytoplasmic events. Reviews in Reproduction 1:40-46.
13.Campbell KHS, McWhir J, Ritchie WA and Wilmut I. 1996b. Sheep cloned by nuclear transfer from a cultured cell line [see comments]. Nature 380:64-66.
14.Campbell KHS, Ritchie WA and Wilmut I. 1993. Nuclear- cytoplasmic interactions during the first cell cycle of nu-clear transfer reconstructed bovine embryos: implications for deoxyribonucleic acid replication and development. Bio-logy of Reproduction 49:933-942.
15.Campbell KHS. 2002. Cell cycle regulation in cloning. In: Principles of Cloning (eds Cibelli JB, Lanza RP, Campbell KHS and West MD), pp.391-399. Academic Press (USA).
16.Carroll J. 2001. The initiation and regulation of Ca2+ signalling at fertilization in mammals. Seminars in Cell and Develop-mental Biology 12:37-43.
17.Chatot CL, Ziomek CA, Bavister BA, Lewis JL and Torres I. 1991. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. Journal of Reproduction and Fertility 86:679-688.
18.Chavatte-Palmer P, Heyman Y, Richard C, Monget P, LeBour-his D, Kann G, Chilliard Y, Vignon X and Renard JP. 2002. Clinical, hormonal, and hematologic characteristics of bovine calves derived from nuclei from somatic cells. Bio-logy of Reproduction 66:1596-1603.
19.Chesne P, Adenot PG, Viglietta C, Baratte M, Boulanger L and Renard JP. 2002. Cloned rabbits produced by nuclear transfer from adult somatic cells. Nature Biotechnology 20:366-369.
20.Cibelli JB, Campbell KHS, Seidel GE, West MD and Lanza RP. 2002. The health profile of cloned animals. Nature Biotechnology 20:13-14.
21.Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce DLN and Robl JM. 1998. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280: 1256-1258.
22.Collas P, Balise JJ and Robl JM. 1992a. Influence of cell cy-cle stage of the donor nucleus on development of nuclear transplant rabbit embryos. Biology of Reproduction 46:492- 500.
23.Collas P, Pinto-Correia C, Ponce dL and Robl JM. 1992b. Effect of donor cell cycle stage on chromatin and spindle mor-phology in nuclear transplant rabbit embryos. Biology of Reproduction 46:501-511.
24.Colman A. 1999. Somatic cell nuclear transfer in mammals: progress and applications. Cloning 1:185-200.
25.Cuthbertson KSR and Cobbold PH. 1985. Phorbol ester and sperm activates mouse oocytes by inducing sustained os-cillations in cell Ca2+. Nature 316:541-542.
26.Eggan K, Baldwin K, Tackett M, Osborne J, Gogos J, Chess A, Axel R and Jaenisch R. 2004. Mice cloned from olfac-tory sensory neurons. Nature 428:44-49.
27.Eggan K, Rode A, Jentsch I, Samuel C, Hennek T, Tintrup H, Zevnik B, Erwin J, Loring J, Jackson-Grusby L, Speicher MR, Kuehn R and Jaenisch R. 2002. Male and female mice derived from the same embryonic stem cell clone by tetra-ploid embryo complementation. Nature Biotechnology 20: 455-459.
28.Farin PW, Crosier AE and Farin CE. 2001. Influence of in vitro systems on embryo survival and fetal development in cattle. Theriogenology 55:151-170.
29.Fissore R and Robl J. 1992. Intracellular Ca2+ response of rabbit oocytes to electrical stimulation. Molecular Reproduc-tion and Development 32:9-16.
30.Fissore RA, Dobrinsky JR, Balise JJ, Duby RT and Robl JM. 1992. Patterns of intracellular Ca2+ concentrations in ferti-lized bovine eggs. Biology of Reproduction 47:960-969.
31.Galli C, Lagutina I and Lazzari G. 2003b. Introduction to clo-ning by nuclear transplantation. Cloning Stem Cells 5:223- 232.
32.Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, Duchi R and Lazzari G. 2003a. Pregnancy: A cloned horse born to its dam twin. Nature 424:635.
33.Gurdon JB and Colman A. 1999. The future of cloning. Na-ture 402:743-746.
34.Gurdon JB, Laskey RA and Reeves OR. 1975. The develop-mental capacity of nuclei transplanted from keratinized skin cells of adult frogs. Journal of Embryology and Experimen-tal Morphology 34:93-112.
35.Gurdon JB. 1962a. Adult frogs derived from the nuclei of single somatic cells. Developmental Biology 4:256-273.
36.Gurdon JB. 1962b. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. Journal of Embryology and Experimental Morphology 10:622-640.
37.Heyman Y, Chavatte-Palmer P, LeBourhis D, Camous S, Vig-non X and Renard JP. 2002. Frequency and occurrence of late-gestation losses from cattle cloned embryos. Biology of Reproduction 66:6-13.
38.Hill JR, Burghardt RC, Jones K, Long CR, Looney CR, Shin T, Spencer TE, Thompson JA, Winger QA and Westhusin ME. 2000. Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biology of Reproduction 63:1787-1794.
39.Hill JR, Roussel AJ, Cibelli JB, Edwards JF, Hooper NL, Miller MW, Thompson JA, Looney CR, Westhusin ME, Robl JM and Stice SL. 1999. Clinical and pathologic fea-tures of cloned transgenic calves and fetuses (13 case studies). Theriogenology 51:1451-1465.
40.Humpherys D, Eggan K, Akutsu H, Friedman A, Hochedlinger K, Lander ES, Golub TR and Jaenisch R. 2002. Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei. Developmental Biology 99:12889-12894.
41.Humpherys D, Eggan K, Akutsu H, Hochedlinger K, Rideout WM, Biniszkiewicz D, Yanagimachi R and Jaenisch R. 2001. Epigenetic instability in ES cells and cloned mice. Science 293:95-97.
42.Ilyin V and Parker I. 1992. Effects of alcohols on responses evoked by inositol trisphosphate in Xenopus oocytes. Jour-nal of Physiology 448:339-354.
43.Inoue K, Kohda T, Lee J, Ogonuki N, Mochida K, Noguchi Y, Tanemura K, Kaneko-Ishino T, Ishino F and Ogura A. 2002. Faithful expression of imprinted genes in cloned mice. Science 295:297.
44.Jones KT, Carroll J and Whittingham DG. 1995. Ionomycin, thapsigargin, ryanodine, and sperm induced Ca2+ release increase during meiotic maturation of mouse oocytes. Jour-nal of Biological Chemistry 270:6671-6677.
45.Kato Y, Tani T and Tsunoda Y. 2000. Cloning of calves from various somatic cell types of male and female adult, new-born and fetal cows. Journal of Reproduction and Fertility 120:231-237.
46.Kaufman MH and Gardner RL. 1974. Diploid and haploid mouse parthenogenetic development following in vitro activation and embryo transfer. Journal of Embryology and Experi-mental Morphology 31:635-642.
47.Kline D and Kline JT. 1992. Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Developmental Biology 149:80-89.
48.Kwon OY and Kono T. 1996. Production of identical sextuplet mice by transferring metaphase nuclei from four-cell em-bryos. Proceedings of the National Academy of Science USA 93:13010-13013.
49.Leno GH and Munshi R. 1994. Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane. Journal of Cell Biology 127:5-14.
50.Machaty Z, Day BN and Prather RS. 1998. Development of early porcine embryos in vitro and in vivo. Biology of Re-production 59:451-455.
51.Machaty Z, Funahashi H, Day BN and Prather RS. 1997. Developmental changes in the intracellular Ca2+ release me-chanisms in porcine oocytes. Biology of Reproduction 56: 921-930.
52.Machaty Z, Funahashi H, Mayes MA, Day BN and Prather RS. 1996. Effects of injecting calcium chloride into in vitro-ma-tured porcine oocytes. Biology of Reproduction 54:316- 322.
53.Matsuyama K, Miyakoshi H and Fukui Y. 1993. Effect of glu-cose levels during the in vitro culture in synthetic oviduct fluid medium on in vitro development of bovine oocytes matured and fertilized in vitro. Theriogenology 40:595-605.
54.McGrath J and Solter D. 1983. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 220:1300-1302.
55.McGrath J and Solter D. 1984. Inability of mouse blastomere nuclei transferred to enucleated zygotes to support develop-ment in vitro. Science 226:1317-1319.
56.Menezo YJ and Herubel F. 2002. Mouse and bovine models for human IVF. Reproductive Biomedicine Online 4:170- 175.
57.Mitchison JM. 1971. The Biology of the Cell Cycle. Cambridge University Press.
58.Miyazaki S, Hashimoto N, Yoshimoto Y, Kishimoto T, Igusa Y and Hiramoto Y. 1986. Temporal and spatial dynamics of the periodic increase in intracellular free calcium at fer-tilization of Golden Hamster eggs. Developmental Biology 118:259-267.
59.Miyazaki S, Shirakawa H, Nakada K and Honda Y. 1993. Essen-tial role of the inositol 1,4,5-trisphosphate receptor/Ca2+release channel in Ca2+ waves and Ca2+ oscillations at ferti-lization of mammalian eggs. Developmental Biology 158: 62-78.
60.Miyazaki S. 1995. Calcium signalling during mammalian fertili-zation. Ciba Foundation Symposium 188:235-247.
61.Moos J, Kopf GS and Schultz RM. 1996. Cycloheximide-in-duced activation of mouse eggs: effects on cdc2/cyclin B and MAP kinase activities. Journal of Cell Science 109(4): 739-748.
62.Moses RM and Kline D. 1995. Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the ab-sence of a calcium signal or microtubule assembly. Mole-cular Reproduction and Development 41:264-273.
63.Navara CS, First NL and Schatten G. 1994. Microtubule organi-zation in the cow during fertilization, polyspermy, parthe-nogenesis, and nuclear transfer: the role of the sperm aster. Developmental Biology 162:29-40.
64.Nussbaum DJ and Prather RS. 1995. Differential effects of protein synthesis inhibitors on porcine oocyte activation. Molecular Reproduction and Development 41:70-75.
65.Oback B and Wells D. 2002. Donor cells for nuclear cloning: many are called, but few are chosen. Cloning and Stem Cells 4:147-168.
66.Ozil JP. 1990. The parthenogenetic development of rabbit oocytes after repetitive pulsatile electrical stimulation. Development 109:117-127.
67.Plante L and King WA. 1996. In vitro development of sponta-neously activated bovine oocytes. Journal of Assisted Repro-ductive Genetics 13:435-446.
68.Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A and Campbell KHS. 2000. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86-90.
69.Prather RS and First NL. 1990. Cloning embryos by nuclear transfer. Journal of Reproduction and Fertility Supplement 41:125-134.
70.Prather RS, Sims MM and First NL. 1989. Nuclear transplan-tation in early pig embryos. Biology of Reproduction 41: 414-418.
71.Presicce GA and Yang X. 1994. Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide. Molecular Reproduction and Development 38:380-385.
72.Raz T, Skutelsky E, Amihai D, Hammel I and Shalgi R. 1998. Mechanisms leading to cortical reaction in the mammalian egg. Molecular Reproduction and Development 51:295-303.
73.Renard JP, Zhou Q, LeBourhis D, Chavatte-Palmer P, Hue I, Heyman Y and Vignon X. 2002. Nuclear transfer tech-nologies: between successes and doubts. Theriogenology 57:203-222.
74.Robl JM, Prather R, Barnes F, Eyestone W, Northey D, Gilligan B and First NL. 1987. Nuclear transplantation in bovine embryos. Journal of Animal Science 64:642-647.
75.Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K and Lai FA. 2002. PLC zeta: A sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development 129:3533-3544.
76.Shiga K, Fujita T, Hirose K, Sasae Y and Nagai T. 1999. Pro-duction of calves by transfer of nuclei from cultured so-matic cells obtained from Japanese black bulls. Therio-genology 52:527-535.
77.Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, Buck S, Murphy K, Lyons L and Westhusin M. 2002. Cell bio-logy: A cat cloned by nuclear transplantation. Nature 415: 859.
78.Sims M and First NL. 1994. Production of calves by transfer of nuclei from cultured inner cell mass cells. Proceedings of the National Academy of Science USA 91:6143-6147.
79.Siracusa G, Whittingham DG, Molinaro M and Vivarelli E. 1978. Parthenogenetic activation of mouse oocytes induced by inhibitors of protein synthesis. Journal of Embryology and Experimental Morphology 43:157-166.
80.Spemann H. 1938. Embryonic Development and Induction. Hafner Publishing Company, Hafner Publishing Company.
81.Steinhardt RA and Epel D. 1974. Activation of sea-urchin eggs by a calcium ionophore. Proceedings of the National Aca-demy of Science USA 71:1915-1919.
82.Steinhardt RA, Epel D, Carroll EJ and Yanagimachi R. 1974. Is calcium ionophore a universal activator for unfertilised eggs? Nature 252:41-43.
83.Stricker SA. 1999. Comparative biology of calcium signaling during fertilization and egg activation in animals. Develop-mental Biology 211:157-176.
84.Sun FZ and Moor RM. 1995. Nuclear transplantation in mam-malian eggs and embryos. Current Topics in Developmen-tal Biology 30:147-176.
85.Sun FZ, Bradshaw JP, Galli C and Moor RM. 1994. Changes in intracellular calcium concentration in bovine oocytes following penetration by spermatozoa. Journal of Reproduc-tion and Fertility 101:713-719.
86.Sun FZ, Hoyland J, Huang X, Mason W and Moor RM. 1992. A comparison of intracellular changes in porcine eggs after fertilization and electroactivation. Development 115:947-956.
87.Susko-Parrish JL, Leibfried-Rutledge ML, Northey DL, Schutz-kus V and First NL. 1994. Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion. Developmental Biology 166:729-739.
88.Suzuki H, Liu L and Yang X. 1999. Age-dependent develop-ment and surface ultrastructural changes following electri-cal activation of bovine oocytes. Reproduction Fertility and Development 11:159-165.
89.Swann K and Ozil JP. 1994. Dynamics of the calcium signal that triggers mammalian egg activation. International Re-view of Cytology 152:183-222.
90.Tanaka H and Kanagawa H. 1997. Influence of combined acti-vation treatments on the success of bovine nuclear transfer using young or aged oocytes. Animal Reproduction Science 49:113-123.
91.Taylor CT, Lawrence YM, Kingsland CR, Biljan MM and Cuthbertson KS. 1993. Oscillations in intracellular free cal-cium induced by spermatozoa in human oocytes at fertili-zation. Human Reproduction 8:2174-2179.
92.Tesarik J. 1994. Calcium in oocyte maturation. How the sper-matozoon awakens the oocyte: lessons from intracytoplas-mic sperm injection. Human Reproduction 9:977-978.
93.Thompson JG. 2000. In vitro culture and embryo metabolism of cattle and sheep embryos - a decade of achievement. Animal Reproduction Science 60-61:263-275.
94.Tsunoda Y, Yasui T, Shioda Y, Nakamura K, Uchida T and Sugie T. 1987. Full-term development of mouse blastomere nuclei transplanted into enucleated two-cell embryos. Jour-nal of Experimental Zoology 242:147-140.
95.Vincent C, Cheek TR and Johnson MH. 1992. Cell cycle pro-gression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium. Journal of Cell Science 103(2):389-396.
96.Wakayama T, Perry AC, Zuccotti M, Johnson KR and Yanagi-machi R. 1998. Full-term development of mice from enu-cleated oocytes injected with cumulus cell nuclei. Nature 394:369-374.
97.Walker SK, Heard TM and Seamark RF. 1992. In vitro culture of sheep embryos without co-culture: Successes and pers-pectives. Theriogenology 37:111-126.
98.Watson AJ, Watson PH, Warnes D, Walker SK, Armstrong DT and Seamark RF. 1994. Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: Comparison between coculture on oviduct epithelial cell mo-nolayers and culture under low oxygen atmosphere. Bio-logy of Reproduction 50:715-724.
99.Wells DN, Laible G, Tucker FC, Miller AL, Oliver JE, Xiang T, Forsyth JT, Berg MC, Cockrem K, L'Huillier PJ, Tervit HR and Oback B. 2003. Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle. Theriogenology 59:45-59.
100.Wells DN, Misica PM and Tervit HR. 1999. Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biology of Reproduction 60:996-1005.
101.Wells DN, Misica PM, Day TA and Tervit HR. 1997. Produc-tion of cloned lambs from an established embryonic cell line: A comparison between in vivo- and in vitro-matured cytoplasts. Biology of Reproduction 57:385-393.
102.Willadsen SM. 1986. Nuclear transplantation in sheep embryos. Nature 320:63-65.
103.Wilmut I, Schnieke AE, McWhir J, Kind AJ and Campbell KHS. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813.
104.Woods GL, White KL, Vanderwall DK, Li GP, Aston KI, Bunch TD, Meerdo LN and Pate BJ. 2003. A mule cloned from fetal cells by nuclear transfer. Science 301:1063.
105.Yang X, Presicce GA, Moraghan L, Jiang S and Foote RH. 1994. Synergistic effect of ethanol and cycloheximide on activation of freshly matured bovine oocytes. Theriogeno-logy 41:395-403.
106.Zakhartchenko V, Durcova-Hills G, Stojkovic M, Schernthaner WG, Prelle K, Steinborn R, Müller M, Brem G and Wolf E. 1999. Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts. Journal of Reproduction and Fertility Supplement 115:325- 331.
107.Zakhartchenko V, Mueller S, Alberio R, Schernthaner WG, Stojkovic M, Wenigerkind H, Wanke R, Lassnig C, Mueller M, Wolf E and Brem G. 2001. Nuclear transfer in cattle with non-transfected and transfected fetal or cloned trans-genic fetal and postnatal fibroblasts. Molecular Reproduc-tion and Development 60:362-369.
108.Zhou Q, Jouneau A, Brochard V, Adenot P and Renard JP. 2001. Developmental potential of mouse embryos recons-tructed from metaphase embryonic stem cell nuclei. Bio-logy of Reproduction 65:412-419.
109.Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Cherifi Y, Fraichard A and Cozzi J. 2003. Generation of fertile cloned rats by regulating oocyte activation. Science 302:1179.
110.Zimmermann U and Vienken J. 1982. Electric field-induced cell-to-cell fusion. Journal Membrane Biology 67:165-182.