WO2005060740A2 - An improved method for embryo and animal production - Google Patents

Abstract

An improved oocyte with a stabilised or increased activity of one or more protein kinases. The improved oocyte preferably also has one or more improved qualities which give it improved a competency for development following fertilisation, intracytoplasmic sperm injection (ICSI), parthenogenetic activation or embryo production by nuclear transfer.

Description

AN IMPROVED METHOD FOR EMBRYO AND ANIMAL PRODUCTION

The present invention relates to an improved method for the production of (human and non-human) embryos and animals and in particular to the production of embryos and animals using assisted conception techniques.

BACKGROUND

The production of embryos by assisted conception techniques including but not limited to; in vitro fertilisation (INF), intracytoplasmic sperm injection (ICSI) , cytoplasmic transfer, germinal vesicle transfer (GN transfer) , nuclear transfer (NT otherwise referred to as cell nuclear replacement (CNR)) , chimera production by embryo aggregation or blastocyst injection using embryo derived blastomeres or embryonic stem (ES) cells, tetraploid aggregation techniques using blastomeres or ES cells, the production of haploid or diploid parthenotes by activation and the production of transpecies embryos by NT, has many uses in the fields of animal and human reproduction, animal biotechnology including but not limited to the production of transgenic animals (including gene additions, gene removal, gene replacements or gene modifications), the production of cloned animals, in human cell therapies and animal biotechnology for the production of ES cells. All of these techniques require the use of matured metaphase II (Mil) oocytes otherwise referred to as unfertilized eggs.

Mil oocytes can be obtained from a variety of sources including but not limited to maturation in vitro following removal of immature oocytes from live animals or following slaughter, maturation in vivo or a combination of both in vitro and in vivo maturation. However, for efficient production of embryos by the techniques described above the matured oocyte must be of a sufficient quality to support development.

Nuclear Transfer.

The technique of nuclear transfer allows the production of offspring by the reconstruction of an embryo (which throughout this text include all concepts of an animal embryo such as an oocyte, egg, zygote or early embryo). Genetic material from a donor cell (karyoplast) is transferred to a suitable recipient cell from which the nuclear or genomic genetic material has been removed. In the first demonstrations of this technique successful development was only obtained when the donor genetic material was taken from undifferentiated cells or blastomeres from early embryos. Subsequently development has been obtained using donor genetic material from differentiated cells maintained in culture and isolated from embryonic (Campbell, K. H. S.et al., (1996) Nature. 380 , 64-66.) , foetal and adult tissues (Wilmut, Let al., (1997) Nature. 385 , 810-813, WO 97/07669 and WO 97/07668) . Subsequently live offspring have been obtained in the mouse using quiescent cell populations derived directly ex vivo as nuclear donors (Wakayama, T.et al., (1998) Nature. 394 , 369-374.) . The successful use of differentiated cells has now been particularly demonstrated in sheep (Campbell, K. H. S.et al., (1996) Nature. 380 , 64-66. ; McCreath, K. Let al., (2000) Nature. 405 , 1066- 1069. ; Schnieke, A. E.et al., (1997) Science. 278 , 2130-2133. ; Wilmut, Let al., (1997) Nature. 385 , 810-813.) , cattle (Cibelli, J. B.et al., (1998) Science. 280 , 1256-1258. ; Wells D Net al., (1999) Biol.Reprod. 60 , 996-1005.) , mice (Wakayama, T.et al., (1998) Nature. 394 , 369- 374.) , pigs (Polejaeva, I. A.et al., (2000) Nature. 407 , 86-90.) , rabbits (Chesne, P.et al., (2002) Nat.Biotechnol. 20 , 366-369.) , rats, cats (Shin, T.et al., (2002) Nature. 415 , 859-) and horses (Galli, C.et al., (2003) Nature. 424 , 635-) . The use of nuclear transfer technology has many benefits and uses in the production of mammalian embryos, foetuses and offspring. These include but are not limited to:

1. the ability to carry out precise genetic modification of cultured cells to be used as nuclear donors prior to embryo reconstruction. Such modifications include, but are not limited to, random gene addition, addition of multiple copies of a transgene, addition of a transgene at a precise location (targeted addition or knockin), gene removal (knockout), gene inactivation by targeted insertion, gene replacement, modification of any gene or its control sequences and gene multiplication; 2. the ability to carry out multiple genetic modifications in a single animal either by multiple genetic modifications of a cell population in culture or by sequential genetic modification, nuclear transfer and re-isolation of a cell population from the embryo, foetus or animal so produced;

3. the ability to increase the lifespan of cultured cell populations to be used for genetic modification by nuclear transfer and re-isolation of a cell population from the embryo, foetus, juvenile or adult animal so produced;

4. the ability to produce multiple copies of an animal from a genetically modified selected and cloned cell population;

5. the ability to produce multiple copies of any embryo, foetus, juvenile or adult animal by nuclear transfer from cells taken directly ex vivo or cell populations derived from any tissues taken from any of these stages with or without culture in vitro;

6. the ability to produce true clones by utilising oocytes from the maternal line of the cell donor as cytoplast recipients for embryo reconstruction;

7. the ability to store intact genomes for long periods (e.g. by freezing cell populations in liquid N₂) and to subsequently use these stored cells for the production of offspring by nuclear transfer;

8. the ability to dedifferentiate somatic nuclei and to produce undifferentiated cells that may be used for production of chimeric embryos, foetuses and adult animals by embryo aggregation or injection or to produce embryonic stem or embryonic germ cell populations; and

9. the ability to dedifferentiate any somatic cell type by nuclear transfer and to isolate from the embryo so produced embryonic stem cells, germ cells or any other desired specialised or unspecialised cell type e.g. neurones.

Genetic modification of animals and the production of stem cell and differentiated cell populations by nuclear transfer technology have numerous uses in the fields of human medicine, agriculture, genetic preservation and research. These include but are not limited to the production of human therapeutic proteins in the bodily fluids, disease prevention, increasing required production traits, cell based therapies, cell based delivery systems for genetic therapy, tissue and organ transplantation. The uses of such technology have been previously discussed in WO 98/30683 and WO 98/39416. In general, oocytes arrested at metaphase of the second meiotic division have been used as cytoplast recipients in nuclear transfer protocols. The donor genetic material has been introduced into the recipient cell cytoplasm by the processes of (i) cell fusion and (ii) injection of intact cells, lysed cells or nuclei. Transfer of genetic material may occur either at the time of activation, preceding activation (see patent applications WO 97/07669 and WO 97/07668) or following activation (Campbell, K. H. S.et al., (1993) Biol.Reprod. 49, 933-942.) (Campbell, K. H. S.et al, (1994) Biol.Reprod. 50, 1385-1393.) . Additionally, a double nuclear transfer procedure may be used to produce the resultant embryo (Polejaeva, I. A.et al, (2000) Nature. 407, 86-90 and WO 00/42174). In each of these instances the ploidy of the reconstructed embryo must be maintained by the use of donor genetic material at an appropriate stage of the cell cycle (for reviews see Campbell, K. H. S.et al., (1996) Rev Reprod. 1, 40-46.; Campbell, K. H. S.et al, (2003) Reproduction Supplement. 61, 477-494.) . This process may be coupled with genetic manipulation techniques for the production of transgenic offspring (McCreath, K. J.et al., (2000) Nature. 405, 1066-1069. ; Schnieke, A. E.et al, (1997) Science. 278, 2130-2133 and WO 98/37183) . The use of nuclear transfer coupled to genetic modification of cells in culture and their selection prior to animal production has a number of advantages including; 1. the production of non-mosaic animals ensuring germ line transmission of the genetic modification/s;

2. an increased efficiency in the production of such genetically modified animals; 3. the production of multiple copies of the offspring thereby reducing the generation interval to produce flocks or herds for production purposes or increasing the numbers of animals for dissemination of genetic modification into the population as a whole;

4. the production of animals containing multiple genetic modifications; and

5. the production of transgenic animals with superior expression characteristics by utilisation of the pre-selection of the integration site of the transgene.

The process of embryo reconstruction and production of viable offspring by nuclear transfer is a multistep procedure; each of these will now be described in more detail prior to a description of the present invention.

The Recipient Cell or Cytoplast.

Oocytes, fertilised zygotes and two cell embryos have been used as cytoplast recipients for nuclear transfer. In general oocytes arrested at metaphase of the second meiotic division (also termed unfertilised eggs) have become the cytoplast of choice. At this point in oocyte development the genetic material is arranged upon the meiotic spindle and is easily removed using mechanical means. Several reports have demonstrated that during maturation i.e. between the germinal vesicle stage (prophase of the first meiotic division) and arrest at metaphase of the second meiotic division genomic DNA can be removed and the resulting cytoplast used for NT (Kato, Y.et al, (1993) Mol Reprod.Dev. 36 , 276-278.) . The use of fertilised zygotes as cytoplast recipients has been reported in mouse (Kwon, O. Y.et al, (1996) Proc.Natl.Acad.Sci.U.S.A. 93 , 13010-

13013.) , cattle (Prather, R. S.et al, (1990) J Reprod Fertil Suppl. 41 , 125-134.), and pigs (Prather, R. S.et al, (1989) Biol.Reprod. 41 , 414- 418.) . In cattle and pigs development of embryos reconstructed using zygotes as cytoplast recipients is low and on the whole restricted to the exchange of pronuclei suggesting that factors essential for successful development are removed with the pronuclei.

Preparation of a Cytoplast Recipient by Removal of the Genomic Genetic Material

This process has in general been termed enucleation. In the majority of recipients utilised the genomic DNA is not enclosed within a nuclear membrane at the time of removal. The removal of the genetic material as described by the term 'enucleation' does not require that genomic DNA is present in a nuclear membrane (it may or may not be, or may partially be) . Enucleation may be achieved physically by actual removal of the nucleus, pronuclei, anaphase, metaphase or telophase of meiosis I or II (depending upon the recipient cell) , or functionally, such as by the application of ultra-violet radiation or other enucleating influence. Removal of the genetic material is possible by physical and/or chemical means. In the early reports of nuclear transfer, Mil oocytes were simply cut in half on the basis that one half would contain the genetic material and the other would not. Modifications to this approach have been made in order to reduce the volume of cytoplasm which was removed. This may be achieved by aspiration of a small amount of cytoplasm from directly beneath the l^sl polar body using glass micropipettes or by using a knife to cut away that part of the oocyte beneath the polar body. To facilitate plasticity of the oocyte it may be pre-treated with the microtubule inhibitor Cytochalasin B or other such agent that disrupts the cytoskeleton. In contrast to physical enucleation, chemical treatment has been demonstrated to cause complete removal of the genetic material in the mouse. Treatment of maturing oocytes with chemicals such as topoisomerase inhibitor ectoposide (Elsheikh, A. S.et al., (1998) Jpn.J Net. Res. 45, 217-220) , or cytochalsin D (Baguisi, Aet al., (2000) Theriogenology. 53, 209-209) results in the expulsion of all genomic material with the 1^st polar body. Centrifugation of Mil oocytes combined with Cytochalasin B treatment has been reported to cause enucleation in hamster and cattle oocytes (Tatham, B. G.et al, (1995) Biol.Reprod. 53 , 1088-1094) . In principle any method which results in the removal of the oocyte genetic material prior to or following nuclear transfer and at any time during the maturation process can be used.

When using zygotes the genetic material may be removed by mechanical aspiration of both pronuclei. Dependent upon species, in order to facilitate visualisation of the pronuclei the zygotes may be centrifuged prior to enucleation.

Introduction of Genetic Material (Embryo Reconstruction).

Having prepared a suitable recipient cell or cytoplast, the donor genetic material must be introduced. Various techniques have been reported including:

1 cell fusion induced by chemical, viral or electrical means; 2 injection of an intact cell by any method; 3 injection of a lysed or damaged cell; and 4 injection of a nucleus. 5 injection of an intact genome devoid of a nuclear envelope such as mitotic chromosomes or condensed chromatin. Any of these methods may be used in any species with some modifications of individual protocols known to those skilled in the art.

Activation of the Reconstructed Embryo

In addition to the transfer of donor genetic material from the karyoplast to the cytoplast, the cytoplast must be stimulated to initiate development. When using a fertilised zygote as a cytoplast recipient, development has already been initiated by sperm entry at fertilisation. When using Mil oocytes as cytoplast recipients the oocyte must be activated by other stimuli. Various treatments have been reported to induce oocyte activation and promote early embryonic development including but not limited to; application of a DC electric stimulus, treatment with ethanol, ionomycin, inositol tris-phosphate (IP₃) , calcium ionophore A23187, treatment with extracts of sperm or any other treatment which induces calcium entry into the oocyte or release of internal calcium stores and results in initiation of development. In addition, any of these treatments in combination, their application at the same or different times or in combination with inhibitors of protein synthesis (i.e. cycloheximide or puromycin) or inhibitors of serine threonine protein kinases (i.e. 6-DMAP) , MPF or MAPK kinases may be applied alone or in combination.

Nuclear transfer reconstructed embryos may be cultured in vitro to a stage suitable for transfer to a final recipient using any suitable culture medium or culture process. 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 recipient is reached. Embryos from cattle, sheep and other species may be cultured in a trans species recipient, for simplicity a sheep provides a suitable recipient for bovine, ovine and porcine species. In order to prevent mechanical damage or attack by macrophages to the reconstructed embryos whilst in the oviduct of the temporary recipient it is usual to embed the embryos in a protective layer of agar or similar material.

Nuclear transfer using somatic cells as donors of genetic material is now a well established but inefficient technique in a range of species including sheep (Campbell, K. H. S.et al, (1996) Nature. 380 , 64-66), cattle (Wells, D. N.et al., (1999) Biol.Reprod. 60 , 996-1005) , mice (Wakayama, T.et al, (1998) Nature. 394 , 369-374) , goats (Baguisi, A.et al, (1999) Nat Biotechnol. 17 , 456-461) , pigs (Polejaeva, I. A.et al, (2000) Nature. 407 , 86-90) , cats, rabbits, goats, horses and rats. For development to occur cell cycle coordination of the donor (karyoplast) and the recipient (cytoplast) are essential in order to prevent DNA damage and maintain ploidy (for reviews see Campbell, K. H. S.et al., (1996) Rev Reprod. 1 , 40-46; and Campbell, K. H. S.et al, (2003) Reproduction Supplement. 61 , 477-494) . In addition double nuclear transfer procedures which involve production of a zygote by nuclear transfer followed by transfer of the pronucleus or pseudopronucleus to an enucleated zygote require cell cycle coordination (Polejaeva, I. A.et al, (2000) Nature. 407 , 86-90 and WO 00/42174) . Although cell cycle coordination is essential for development, typically approximately only 1- 2% of reconstructed embryos are able to develop to term and produce live offspring (Gurdon, J. B.et al, (1999) Nature. 402, 743-746.) . In addition, many of these offspring suffer from a range of abnormalities and many die within the first few months after birth. The development to term of embryos reconstructed by nuclear transfer was originally restricted to the use of embryonic blastomeres as donors of genetic material. In different species a relationship exists between the onset of zygotic transcription (MZT) and the latest stage at which development could be obtained. In the mouse the MZT occurs at the 1-2 cell stage and development was restricted to donor nuclei from 4-cell embryos for a significant period. In contrast in cattle and sheep the MZT occurs at the 8-16 cell stage and development was obtained from 32-64 cell stage embryos (Prather, R. S.et al, (1987) Biol.Reprod. 37 , 859-866) and inner cell mass cells (Smith, L. C et al, (1989) Biol.Reprod. 40 , 1027- 1035) , whilst in Xenopus the MZT occurs at 4,000 cells and development was obtained from differentiated tadpole intestinal cells (Gurdon, J. B.et al, (1966) Nature. 210 , 1240-1241) .

Maturation/M-Phase Promoting Factor (MPF)

Maturation/M-phase promoting factor (MPF) induces M-phase in all eukaryotic cells, including oocytes. MPF activity is activated at the G2-M phase transition, is maximal at metaphase of the mitotic/meiotic division, followed by a rapid decline. Matured oocytes typically become arrested at metaphase of the second meiotic division (Mil) . In Mil oocytes, MPF activity is controlled by cytostatic factor, the product of the c-mos proto- oncogene (O'Keefe, S. J.et al, (1991) Proc Natl Acad Sci U S A. 88 , 7869-7872. ; Sagata, N.et al, (1989) Nature. 342 , 512-518) . Regulation of MPF activity depends upon the association of its catalytic subunit, p34cdc2 kinase, with its regulatory subunit, cyclin B and the subsequent phosphoylation state at key tyrosine-15 (Y15) and threonine-14 (T14) residues. In general, p34cdc2 is phosphorylated at T14 and Y15 by the Mytl and Weel kinases after it's association with cyclin B, this inactive form termed pre-MPF accumulates during the G2-phase of the cell cycle. Activation, at the G2-M phase transition depends upon dephosphorylation by the cdc25 phosphatase (Kikuchi, K.et al, (2000) Biol.Reprod. 63 , 715-722) .

Oocyte maturation involves the transition from prophase of the I^s' meiotic division (MI) to metaphase of the second meiotic division (Mil) . The timing of these events is species dependent however, in general at this point the oocytes are able to be fertilised or activated parthenogenetically. Activation or fertilisation releases the oocyte from the Mil block, cytostatic factor (CSF) the product of the c-mos proto-oncogene is destroyed and inactivation of p34cdc2 kinase occurs . In porcine and ovine oocytes inactivation of p34cdc2 has been related to the degradation of cyclin B. However, when oocytes remain at Mil in the absence of activation or fertilisation (here termed ageing) the level of active MPF activity declines and pre-MPF accumulates. This ageing of Mil oocytes is characterised by an increase in spontaneous activation, a decrease in the time required to form pronuclei and an increase in fragmentation (Kikuchi, K.et al, (2000) Biol.Reprod. 63 , 715-722.) . The ageing of porcine oocytes has been associated with a gradual increase in pre-MPF, which is a p34cdc2 and cyclin B complex inactivated by phosphorylation at the inhibitory phosphorylation site of p34cdc2. This is in contrast to the decrease in pre-MPF observed after fertilisation, indicating the degradation of cyclin B.

Mitogen-Activated Protein (MAP) Kinase

Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase (MPF) and mitogen- activated protein

(MAP) kinase. Activation of both kinases occurs in the majority of species prior to germinal vesicle breakdown (GVBD) but the activation of MAP kinase is not necessary for entry into meiosis I. However, its activation is essential to ensure that oocytes enter meiosis II and do not enter S-phase (Greaves, S. (2000) Nat. Cell Biol. 2 , E125-). MAP kinase has been implicated as a key regulator of the mitotic spindle (Home, M.

M.et al. , (2003) J Cell Biol. 161 , 1021-1028.) and during meiosis a correlation between chromatin configuration and histone Hl/MAP-kinase activities occurs (Torner, H.et al. , (2001) Theriogenology. 55 , 885- 899.) . Following activation, both MPF and MAP kinase activities decline, however, the decline in MAP kinase occurs more slowly than MPF.

STATEMENT OF INVENTION

According to a first aspect the invention provides an oocyte with a stabilised or an increased activity of one or more protein kinases.

Preferably, the one or more protein kinases is selected from the group comprising maturation/M-phase promoting factor (MPF) and mitogen-activated protein (MAP) kinase.

Surprisingly, it has been found that by increasing or stabilising MPF and/or MAP kinase activity in an oocyte, oocyte ageing can be reduced significantly, a rejuvenation of oocyte quality is observed and there is an increased 'reprogramming' ability. This has the advantage that the oocyte can be more readily and successfully used in assisted conception techniques, such as nuclear transfer and ICSI.

Reprogramming may be simply defined as alterations in chromatin structure which affects gene expression but does not involve alterations in the DNA sequence. Such modifications may include, but are not limited to, changes in histone acetylation, phosphorylation, ubiquitination or poly ADP ribosylation or changes in DNA methylation. Such modifications are preferably referred to as 'epigenetic' .

Preferably, oocytes according to the invention have an increased competency for development following fertilisation, intracytoplasmic sperm injection (ICSI) , parthenogenetic activation or embryo production by nuclear transfer. The increased competency of the oocyte may arise due to an increased ability to 'reprogramme' the nuclear genetic material from somatic cells; preferably the nuclear genetic material of the somatic cell has been transferred into the oocyte. The resultant embryos may have benefits for assisted conception, for the multiplication of genotypes and transgenic genotypes by the production of foetuses and offspring, and for the production of isogenic embryonic stem (ES) cells including human isogenic ES cells. The production of genetically engineered or transgenic mammalian embryos, foetuses and offspring may be facilitated by the use of genetically modified donor cells or sperm obtained from genetically modified animals or modified in culture. Preferably the oocytes according to the invention are useful for cloning techniques .

Preferably the oocyte has been treated with a protein kinase stabilising or increasing agent to cause the stabilisation or increase in protein kinase activity in the oocyte.

Typically, in an untreated oocyte the protein kinase activity will decrease over time. If the protein kinase activity in the oocyte has been stabilised, then the protein kinase activity will be substantially the same as it would have been in an untreated oocyte, and the decrease in activity, which would occur in an untreated oocyte over time, is reduced. If the protein kinase activity in the oocyte is increased, then the protein kinase activity in the treated oocyte will be greater than the activity in an untreated oocyte. Preferably, the protein kinase activity in the oocyte is increased by at least about 20%. Preferably the protein kinase activity in the oocyte is increased by at least about 50%. More preferably the activity of protein kinase in the oocyte is increased by at least about 75%. The activity of protein kinase in the oocyte may be increased by at least about 100%.

Preferably an oocyte according to the invention has one or more improved qualities; preferably the one or more improved qualities render the oocyte more useful in assisted conception techniques. The improved qualities displayed by the oocyte may include one or more of the following:

• the oocyte is more likely to produce a viable embryo; • an embryo produced from the oocyte is more likely to produce a viable-offspring; • normal age related changes in the oocyte are reduced, for example, there may be fewer apoptotic cells observed and/or the oocyte may be less susceptible to activation. In an untreated oocyte as the protein kinase activity decreases the oocytes become more susceptible to spontaneous activation, once activation has occurred the oocytes leave Mil and are no longer suitable for use in assisted conception; • the oocyte is able to produce a blastocyst with more cells, typically a blastocyst produced from an oocyte according to the invention has 20% more cells than a blastocyst from an oocyte which does not have increased or stabilised protein kinase activity, the more cells there are at the blastocyst stage the more likely it is that the embryo will develop successfully; • an embryo produced from the oocyte hatches earlier which improves its viability, hatching occurs when the fertilised oocycte breaks out of the zona pellucida to allow implantation; • the oocyte induces nuclear envelope breakdown and chromatin condensation which assists in 'reprogramming' . In some Mil oocytes the protein kinase activity may be insufficient to cause breakdown of the nuclear envelope of a donor nucleus, in which case development to an embryo or offspring is less unlikely. By increasing the kinase activity in the oocyte the quality of the oocyte may be improved such that nuclear envelope breakdown and chromatin condensation of a donor nucleus is induced; • an embryo produced from the oocyte has an increased developmental potential, that is, it is more likely to develop to an offspring;

• the oocyte displays rejuvenated developmental potential, that is, the increased or stabilised kinase activity may allow improved nuclear reprogramming, in particular of donor nuclei;

• the oocyte displays increased chromatin condensation, this may improve the chances of successful fertilization of the oocyte. The increased or stabilised protein kinase activity in the oocyte may improve reprogramming of the sperm added by ICSI or IVF, and the maternal chromatin may also be improved by increased kinase activities. Reprogramming may result in more viable embryos;

• the oocyte displays improved nuclear reprogramming. When an oocyte is at Mil with high levels of MPF and/or MAP kinase nuclear envelope breakdown and chromatin condensation occurs. Following activation, caused by physical or chemical stimuli, the kinase activity falls and the chromatin decondenses and the nucleus/pronucleus/pseudopronucleus reforms. At this time nuclear reprogramming may occur. By increasing the kinase activity and increasing chromatin condensation physical displacement of factors bound to the DNA may occur. If a donor nucleus is added to the oocyte chromatin condensation may cause the displacement of somatic factors, leaving the donor DNA more susceptible to reprogramming by the oocyte cytoplasm when it decondenses. On decondensation, typically following activation, the chromatin relaxes allowing access of cytoplasmic factors which may modify the histones or the DNA, the cytoplasmic factors may change the histones i.e substitute embryonic forms for somatic forms and may allow access of transcription factors. The transcription factors may be different to those displaced upon chromosome condensation, and may allow more normal embroyonic development with an improved chance of successful development to term. Therefore increased chromatin condensation caused by increased kinase activity may improve nuclear reprogramming; and • the oocyte has an increased window of time in which it is in a condition in which it can be fertilized, the time taken to perform the steps necessary to cause fertilisation of an oocyte can be quite lengthy and the ability to lengthen the time is useful.

Preferably the activity of one or more protein kinases in the oocyte has been stabilised or increased to a level which allows the oocyte to display one or more improved qualities over an oocyte in which the protein kinase activity is unaltered.

Preferably the oocyte of the present invention is arrested at metaphase of the second meiotic division (Mil) . Alternatively, the oocyte may be at an earlier phase of meiosis.

Preferably the oocyte is enucleated. One of the potential implications of enucleation on the quality of the resulting cytoplast is the possible removal or reduction in MPF and MAP kinase activities. This coupled with the time required to carry out the procedures of enucleation, nuclear transfer and activation and the decrease in kinase activity with the ageing process may reduce the developmental potential of the reconstructed embryo. In mouse, experiments have suggested that MPF kinase activities may be compartmentalised (Fulka, J. , Jr.et al. , (1995) Reprod.Fertil.Dev. 7, 1123-1127) with the majority of activity remaining with the metaphase plate. On oocyte ageing this compartmentalisation is highlighted by the reduction in activities associated with ageing (Czolowska, R.et al. , (1986) J Cell Sci. 84, 129-138.) . In ovine oocytes activity of MAP and MPF kinases were unaffected by enucleation, however, differences in rates of decay, ageing or absolute activity may affect the levels observed between species.

Preferably the protein kinase content is stabilised or increased by treating the oocytes with a protein phosphatase inhibitor. The protein phosphatase inhibitor may be caffeine. Caffeine is preferably used at a concentration of less than about 20mM, more preferably at a concentration of between about 5mM and about 15mM.

Alternatively, the activity of protein kinase can be increased or stabilised by any other chemical or biological agent which increases or stabilises their activity. For example, the activity of protein kinases could be increased or stabilised by injection into the oocyte of cell cytoplasm with increased activity, or injection into the oocyte of purified and/or recombination protein. Alternatively, a DNA or RNA vector which induces synthesis of a protein kinase, prevents deactivation of a protein kinase or causes activation of an inactive protein kinase may be introduced into the oocyte (this includes but is not limited to oligonucleotides, dsRNA, RNAi and plasmid expression vectors) . Alternatively the oocyte may be treated with compounds which inhibit or activate cellular factors resulting in increased kinase activities. The effects of increasing or stabilising protein kinase levels on the oocyte may be mediated through a range of mechanisms involving phosphorylation reactions .

Preferably the oocyte is derived from any species, in particular human and non-human animals. Non-human animals may include mammalian animals including livestock animals (e.g. ungulates, such as bovines, buffalo, equines, ovines, porcines and caprines) , camelids, primates (e.g. monkeys, chimpanzees, baboons and gorillas) , as well as rodents (e.g. mice, hamsters, rats and guinea pigs) , canines, felines and lagomorphs (e.g rabbits, hares) . Additionally the technology is equally applicable in ampihibia (e.g frogs, toads) urodeles (e.g. Axolotls), avians (e.g. chickens, turkeys), reptilia and fishes. The term 'mammalian' generally refers to all mammalian species including humans but may exclude the transfer of human embryos produced by cell nuclear replacement to a surrogate recipient.

The oocyte may be treated in vivo to have a stabilised or increased protein kinase activity. Alternatively, the oocyte may be treated in vitro to have a stabilised or increased protein kinase activity.

An oocyte according to the invention may be used in the following processes, but is not limited thereto; 1. the generation of animals including but not being limited to genetically selected and/or modified animals; 2. the dedifferentiation of animal or human somatic cells and the production of undifferentiated embryonic cells; 3. the production of animal or human embryos by nuclear transfer including but not being limited to genetically selected and/or modified embryos; 4. the production of animal or human embryos by in vitro fertilization; 5. the production of animal or human embryos by intracytoplasmic sperm injection (ICSI) ; 6. the production of animal or human embryos by parthenogenesis; 7. the production of trans species embryos by nuclear transfer; 8. to the method of the generation of animal or human embryos; 9. the derivation of animal or human Embryonic Stem (ES) cells and isogenic ES cells from embryos produced by any of the above; 10. the prevention or reversal of animal or human oocyte ageing (oocyte rejuvenation) ; 11. the use of rejuvenated animal oocytes for the generation of animals including but not being limited to genetically selected and/or modified animals; 12. the use of rejuvenated animal or human oocytes for the generation of embryos by fertilisation; 13. the use of rejuvenated animal or human oocytes for the generation of embryos by ICSI; 14. the use of rejuvenated animal or human oocytes for the generation of embryos by parthenogenesis; 15. the use of rejuvenated animal or human oocytes for the generation of embryos by nuclear transfer including but not being limited to genetically selected and/or modified embryos and trans species embryos; 16. the use of rejuvenated oocytes for the production of ES cells and isogenic ES cells from animal or human embryos (including but not being limited to genetically selected and/or modified embryos) produced by fertilization, nuclear transfer, ICSI parthenogenesis or any other means; and 17. the use of rejuvenated oocytes for the production of animal or human ES cells and isogenic ES cells from transpecies embryos (including but not being limited to genetically selected and/or modified embryos) produced by nuclear transfer.

The applicant has demonstrated that by treating mammalian oocytes with caffeine it is possible to improve the quality of the oocyte. The improved quality of the oocyte is exemplified by having (i) an increased frequency of the development of embryos produced by nuclear transfer; (ii) an increased number of cells at the blastocyst stage of embryos produced by nuclear transfer; (iii) a reduction in oocyte ageing; and (iv) the ability for aged oocytes to be rejuvenated. The effects of caffeine may be manifested through a number of mechanisms including but not limited to the elevation of oocyte protein kinase activities which can result in increased cytoplasmic maturation and developmental potential of the oocyte and increased reprogramming of a transferred nucleus or sperm containing DNA. These observations have important implications for all aspects of assisted conception techniques in animals and humans including but not limited to IVF, ICSI, parthenogenesis, nuclear transfer, de- differentiaition of somatic cells and the production of embryonic stem (ES) cells. These applications include but are not limited to: 1. by increasing the MPF and/or MAP kinase activities in oocytes the frequency of development and quality of nuclear transfer embryos is increased; 2. increased MPF and/or MAP kinase activity will increase the development of embryos produced by ICSI; 3. manipulation of MPF and/or MAP kinases allows the production of a uniform population of cytoplast recipients for nuclear transfer by increasing and maintaining MPF and/or MAP kinase activities which otherwise decline with time as the oocyte ages. This allows control over the timing and precise cytoplasmic environment of the recipient in terms of kinase activity; 4. preventing increased susceptibility to activation associated with oocyte ageing but maintaining developmental competence of the oocyte; 5. when in vivo matured oocytes are recovered in the majority of species maturation occurs prior to ovulation. The induced increase of MPF and/or MAP kinase levels through application of the present invention to above those usually attained at Mil provides a more efficient oocyte for fertilisation, ICSI, parthenogenetic development or as a cytoplast recipient for NT; 6. oocytes that fail to fertilise can be collected and treated with agents that increases kinase levels, reversing the ageing process. This will allow these oocytes to be re-used as cytoplast recipients for NT, ICSI or fertilisation for embryo production or nuclear reprogramming prior to NT. This application may have extreme importance in humans not only for re-fertilisation for embryo production but also for parthenogenesis or NT for the production of ES cell populations; and 7. a population of oocytes, which may all be at different stages of development and thus not all in a condition suitable for fertilisation, may be treated so as to stabilise or increase the activity of MPF and/or MAP kinase thereby evening out the characteristics of the population of oocytes such that substantially all may now be in a condition for fertilisation.

According to another aspect, the invention provides the use of an oocyte according to the invention in the generation of a human or non-human animal embryo.

Preferably the embryo is produced using an assisted conception technique. The assisted conception technique may be selected from the group comprising nuclear transfer, in vitro fertilisation, intracytoplasmic sperm injection and parthogenesis.

The embryo may be generated by introducing into the oocyte a donor nucleus. Alternatively, the embryo may be generated by introducing into the oocyte a donor intact genome substantially devoid of a nuclear membrane. The intact genome substantially devoid of a nuclear membrane may comprise mitotic chromosomes or condensed chromatin. The embryo generated may be genetically selected and/or genetically modified. A genetically modified embryo may be produced by introducing a genetically modified donor nucleus or donor intact genome into the oocyte.

The embryo generated may be transpecies, this may be achieved by introducing a donor nucleus or donor intact genome from one species to an oocyte from a different species.

The oocyte and/or donor or intact genome may be is derived from any species, in particular human and non-human animals. Non-human animals may include mammalian animals including livestock animals (e.g. ungulates, such as bovines, buffalo, equines, ovines, porcines and caprines), camelids, primates (e.g. monkeys, chimpanzees, baboons and gorillas) , as well as rodents (e.g. mice, hamsters, rats and guinea pigs), canines, felines and lagomorphs (e.g rabbits, hares) . Additionally the technology is equally applicable in ampihibia (e.g frogs, toads) urodeles (e.g. Axolotls) , avians (e.g. chickens, turkeys) , reptilia and fishes.

The embryo generated may be used to provide embryonic stem cells or isogenic stem cells.

According to a still further aspect, the invention provides a human or non-human embryo, foetus or animal produced from an oocyte according to the invention.

Preferably the embryo, foetus or a human or non-human animal embryo, foetus or animal is produced by assisted conception, such as nuclear transfer, in vitro fertilisation, intracytoplasmic sperm injection or parthogenesis. Preferably the human or non-human embryo, foetus or animal is genetically selected and/or modified.

According to a further aspect, the invention provides a method of producing a human or non-human embryo comprising treating an oocyte to increase the activity of one or more protein kinases in the oocyte.

Preferably the embryo is produced using an assisted conception technique. The assisted conception technique may be selected from the group comprising nuclear transfer, in vitro fertilisation, intracytoplasmic sperm injection and parthogenesis.

According to another aspect, the invention provides a method of producing an oocyte according to the invention with improved qualities comprising treating the oocyte to increase the activity of one or more protein kinases in the oocyte.

According to a further aspect, the invention provides a method of increasing or stabilising the activity of one or more protein kinases in an oocyte comprising treating the oocyte with a protein phosphatase inhibitor.

Preferably the protein phosphatase inhibitor is caffeine.

According to a yet further aspect, the invention provides a method of therapeutic cloning comprising the step of stabilising or increasing the activity of one or more protein kinases in an oocyte.

According to another aspect, the invention provides a stem cell produced by the method of therapeutic cloning according to the preceding aspect of the invention. According to a further aspect, the invention provides a method of reproductive cloning comprising the step of stabilising or increasing the activity of one or more protein kinase in an oocyte.

In either the method of therapeutic cloning, or the method of reproductive cloning, according to the invention a genetically modified donor nuclei or donor intact genome devoid of any nuclear membrane may be introduced into an oocyte according to the invention.

According to a still further aspect, the invention provides a method for the treatment of infertility including the step of treating an oocyte to stabilise or increase activity of one or more protein kinase.

According to a still further aspect, the invention provides a method of assisted conception including the step of treating an oocyte to stabilise or increase activity of one or more protein kinase.

According to a further aspect, the invention provides a method for the production of an animal, human or non-human, embryo by intracytoplasmic sperm injection (ICSI) or by nuclear transfer (NT) comprising treating an oocyte to stabilise or increase the activity of one or more protein kinase.

According to another aspect, the invention provides the use of a protein phosphatase inhibitor to increase the activity of one or more protein kinase in an oocyte.

According to another aspect, the invention provides the a method for the prevention or reversal of human or non-human oocyte ageing comprising treating an oocyte to stabilise or increase the activity of one or more protein kinases in an oocyte.

According to a further aspect, the invention provides a method of reconstituting a human or a non-human animal embryo, the method comprising treating an oocyte to increase or stabilise the protein kinase activity.

Preferably, the method comprises transferring a donor nucleus or a donor intact genome devoid of a nuclear membrane to the oocyte.

The donor nucleus may be transferred by cell fusion or nuclear transfer techniques .

Alternatively, the donor nucleus or donor intact genome may be transferred by injection into the oocyte.

The donor nucleus or donor intact genome may be genetically modified.

Preferably the oocyte is enucleated. The oocyte and/or the donor nucleus or donor intact genome may be selected from the group comprising cow, bull, pig, goat, sheep, horse, camel, rodent, cat or dog.

The oocyte may be derived from a different species to the donor nucleus or donor intact genome.

According to another aspect, the invention provides a transpecies human or non-human embryo, foetus or animal produced according to the preceding method. According to a yet further aspect, the invention provide a method of preparing a non-human animal, the method comprising: (a) reconstituting a non-human animal embryo according to invention; (b) causing a non-human embryo to develop to term from the embryo; and (c) optionally, breeding from the non-human animal so formed.

According to a further aspect, the invention provides a method of producing a cell or cell line, the method comprising reconstituting a non- human animal embryo according to the invention and clonally expanding cells from the embryo to produce a cell or cell line.

According to another aspect, the invention provides a method for the production of a chimeric human or non-human animal embryo wherein the recipient embryo is produced according to any method of the invention, or using an oocyte according to the invention, and the embryonic stem cells or early embryo cells are obtained from an embryo produced according to any method of the invention, or using an oocyte according to the invention.

Preferably, the genetic material of the recipient embryo and/or the embryonic stem cells/early embryo cells has been genetically modified.

According to a further aspect, the invention provides a chimeric human or non-human animal embryo produced using according to the preceding method. A chimeric foetus or offspring may be produced from the embryo . The invention covers human and non-human tissue cells and where applicable, human embryos, in particular those embryos under 14 days old.

The reader will appreciate that all the preferable features of the above invention discussed with reference to only some aspects can equally be applied to all aspects of the invention.

Preferred embodiments of the present invention will now be described merely by way of example with reference to the following figures and examples.

The examples are non-limiting and are merely representative of various aspects of the invention. Concentrations of caffeine, timing of addition and duration of exposure are variable both inter and intra-species.

Figures la and lb illustrates MPF and MAP kinase activity respectively in in vitro matured ovine oocytes. MPF and MAPK activities were measured as described in Ye, J.et al, (2002) Reproduction Abstract Series. 28, Abstract 6. The abbreviations used are as follows: GV-germinal vesicle, A-anaphase I, M-metaphase II, I-intact, E-enucleated, S-sham enucleated.

Figures 2a and 2b illustrate changes of MPF and MAP kinase activities in Mil oocytes during prolonged maturation (36hpm) .

Figure 3 illustrates the effects of caffeine on the development of ovine oocytes. In vitro matured ovine oocytes were treated with caffeine for 5 hours (24-29 hours following onset of maturation) , activated (7% ethanol, 7mins) and cultured in SOFM at 39°C, 5% O₂, 5%CO₂. The results are compared to a 24 hours oocyte control. Figures 4a and 4b show that the addition of caffeine increased the activities of both MPF and MAP kinase above those observed in control oocytes . T his provides a cytoplasmic environment more able to cause nuclear membrane breakdown and induce premature chromosome condensation in transferred nuclei or sperm.

Figures 5a and 5b show the effects of caffeine on MPF and MAP kinase activities in ovine oocytes matured for 24 hours.

Figures 6a and 6b show that caffeine treatment did not affect the decline of MPF and MAP kinase activities following activation.

Oocytes to be utilised for all aspects of the invention can be obtained from a variety of sources including but not limited to: 1. in vitro maturation of slaughterhouse material, or material recovered by trans vaginal follicle puncture; and 2. in vivo matured and surgically recovered.

The methods of maturation and the timing of events are those pertinent to the relevant species.

Example 1: Modulation of MPF and MAP kinase activities in ovine oocytes.

Ovine oocytes were aspirated from 2-4 mm follicles and matured in TCM 199 supplemented with 10% FBS, 5μg/ml FSH, 5μg/ml LH, lμg/ml estradiol, 0.3mM sodium pyruvate, lOOμM cysteamine. Cumulus cells were removed in PBS containing 300 IU/ml hyaluronidase. Oocytes were enucleated at different stages (Anaphase/Telophase I (A/TI) or Metaphase II (Mil)) in HSOF containing 5μg/ml Hoechst 33342, 4mg/ml BSA and 7.5μg/ml cytochalasin B. Control oocytes were sham enucleated by removing an equal volume of cytoplasm. Oocytes were cultured in maturation medium + /- lOmM caffeine following enucleation. MPF and MAP kinase activities were measured as previously described (Ye, J.et al, (2002) Reproduction Abstract Series. 28 , Abstract 6-) .

MPF activity in ovine oocytes and the Effects of enucleation

MPF (Figure la) and MAP (Figure lb) kinase activities in ovine oocytes are not affected by enucleation.

Activities of both MPF and MAP kinase were found to be maximal at metaphase of the second meiotic division (Mil). Enucleation at anaphase/telophase of the first meiotic division or Mil did not affect kinase activities . Enucleation at AI/TI did not affect the maximal levels of MPF and MAPK being attained at a time point equivalent to MIL

Example 2: Changes of MPF and MAP kinase activities in Mil oocytes during prolonged maturation (36hpm)

Ten oocytes were cultured, denuded and then collected in 5μl lysis buffer at 2 hour intervals from 24 to 36phm. A: is a representative assay for activities of MPF and MAP kinase in Mil oocytes at 2h intervals during prolonged maturation using histone HI and MBP as kinase substrates. B: illustrates relative levels of activities of MPF (Δ) and MAPK (O) . The activities of both kinases in Mil oocytes (24hpm) represent 100%.

Example 3: Effects of exposure to caffeine on the development of ovine parthenotes.

Ovine oocytes were exposed to a range of concentrations of caffeine and then activated. Activated oocytes were cultured for 7 days in vitro. The effects on cleavage and development to morula blastocyst stages are shown (Figure 3) .

Over the range O.OmM to < lOmM caffeine, no deleterious effects were observed on oocyte development.

Example 4: Effects of Caffeine on MPF and MAP kinase activities.

The effects of caffeine on MPF and MAP kinase activities in ovine oocytes and enucleated oocytes are shown in Figures 3a and 3b. lOmM Caffeine was added to ovine oocytes and enucleated oocytes (Figure 4a and 4b) . It is clear from the results that the addition of caffeine increases MPF and MAP kinase in enucleated oocytes.

Example 5: Effects of caffeine on MPF and MAPK in ageing ovine oocytes.

The effects of caffeine on MPF and MAPK kinase activities in ovine oocytes matured for 24 hours are shown (Figures 5a and 5b) .

Maximal MPF and MAP kinase levels were observed at Mil (24hpm) . Whilst remaining at Mil the levels of both kinase activities declined with time. Treatment with caffeine prevented the decline of MPF and MAP kinases and in fact elevated the activities of both MPF and MAP kinase.

Example 6: Effects of caffeine on the decay of MPF and MAP kinase activities following activation.

Caffeine treatment did not affect the decline of MPF and MAP kinase activities following activation (Figure 6a and 6b). Example 7: Effects of caffeine on activation and development of ovine parthenotes.

Ovine oocytes aspirated from 2-4 mm follicles were matured in TCM 199 supplemented with 10% FBS, 5 μg/ml FSH, 5 μg/ml LH, 1 μg/ml estradiol, 0.3mM sodium pyruvate and lOOμM cysteamine. At 24 hours, lOmM caffeine was added to the culture medium. Oocytes were activated at 24 and 30 hours in HSOF containing 5 μg/ml calcium ionophore (A23187) , cultured in SOF with lOμg/ml of cycloheximide (CHXM) and 7.5 μg/ml cytochalasin B for 5h and then transferred to mSOFaaBSA medium, all at 5% CO₂ and 5% O₂ and 90% N₂ at 39²C. On day 2, cleavage was assessed and 5% FBS was added to the culture medium. On day 7, development to blastocyst was assessed. Statistical analysis was performed using the Chi-square test (Table 1) .

Table 1. Cleavage and development of ovine oocytes treated with caffeine.

C = lOmM caffeine. CHXM = cycloheximide. hpm = hours post onset of maturation. Different superscripts indicate significant differences.

Treatment of ovine oocytes for 6 hours (24-30hpm) had no effects on the developmental potential of activated oocytes. The table shows that at 24hrs A23187 alone is insufficient to cause activation of the oocytes, but with cycloheximide (CHXM) activation occurs. At 30hpm activation of oocytes occurs with A23187 alone, however if the oocytes have been treated with caffeine CHXM is required. It can therefore be concluded that caffeine prevents the aquisition of activation competency and therefore prevents aging, this is reflected in the low % of development at 30hpm + C (caffeine) without CHXM.

In conclusion, maintaining the levels of MPF and MAP kinases by caffeine treatment prevented the age related alteration in activation characteristics. As oocytes age they are more susceptible to activation stimuli. Young oocytes require both a calcium releasing stimulus and subsequent inhibition of serine protein kinase activity or inhibition of protein synthesis. In contrast aged oocytes require only a calcium releasing stimulus. Oocytes maintained with elevated kinase levels following caffeine treatment required both stimuli to initiate development. Thus elevated kinase levels prevented the susceptibility to activation associated with oocyte ageing. Example 8: Caffeine treated oocytes as cytoplast recipients for nuclear transfer.

Ovine oocytes aspirated from 2-4 mm follicles were matured in TCM 199 supplemented with 10% FBS, 5 μg/ml FSH, 5 μg/ml LH, 1 μg/ml estradiol, 0.3mM sodium pyruvate and lOOμM cysteamine. Enucleation was carried out at (Anaphase/Telophase I (A/TI) in HSOF containing 5μg/ml Hoechst 33342, 4mg/ml BSA and 7.5μg/ml cytochalasin B. Embryos were reconstructed using serum starved ovine primary foetal fibroblasts. Cell fusion was induced with two DC pulses of 25V/cm for 80μs, delivered by an Eppendorf Multiporator. lOmM caffeine was added to the culture medium as indicated in the tables (2a and 2b) . Oocytes were activated in HSOF containing 5 μg/ml calcium ionophore (A23187), cultured in SOF with 10 μg/ml of cycloheximide (CHXM) and 7.5 μg/ml cytochalasin B for 5h and then transferred to mSOFaaBSA medium, all at 5% CO₂ and 5% O₂ and 90% N₂ at 39^ΩC. On day 2, cleavage was assessed and 5% FBS was added to the culture medium. On day 7, development to blastocyst was assessed. Statistical analysis was performed using the Chi-square test. Table2a. Effects of caffeine treatment of oocytes 18-24hpm on development of ovine embryos .

No. Fused No.

Cytoplast No. NT No. No. NT Cleaved

Recipients blastocysts Cells/blastocyst Oocytes embryos NT embryos

Group (%) (mean + SEM) b) (%)

A 191 163 (85.3) * 149 (91.4) 33 (20.2) 59.0 + 8.7* 139

B 189 124 (89.2) 35 (25.2) 66.0 ± 8.5*^b (73.5) ^b 130

C 184 115 (88.5) 39 (30.0) 88.8 ± 7.1 (70.7) ^b

A: Fusion 20hpm, Activation at 21hpm, B: Fusion 24hpm, Activation at 25hpm, C: lOmM Caffeine 18-24hpm, Fusion 24hpm, Activation at 25hpm

Table2b. Effects of prolonged exposure of the donor chromatin to oocytes cytoplasm in the presence of caffeine.

Cytoplast No. NT No. No. Fused No. Cleaved

Recipients blastocysts Cells/blastocyst NT embryos NT embryos (%)

Group (%) (mean ± SEM)

A 82 68 (82.9) 20 (24.4) 110.4 ± 19.6* B 80 64 (80.0) 14 (17.5) 116.4 ± 35.8* C 79 65 (82.3) 19 (24.1) 142.7 ± 16. l^b

A: Fusion 24hpm, Activation at 25hpm, B: Fusion 24hpm, Activation at 30hpm, C: lOmM Caffeine 24-30hpm, Fusion 24hpm, Activation at 30hpm The use of oocytes treated with caffeine as cytoplast recipients increased the cell numbers of embryos produced by nuclear transfer.

The use of caffeine treated oocytes allows the precise timing of embryo reconstruction in a controlled cytoplasmic environment and improves embryo quality. The use of caffeine allows the level of protein kinase activity to be raised or stabilised for a longer period of time, therefore allowing improved oocytes to be used for longer periods for embryo reconstruction - extending the window in which the oocytes can be used in embryo reconstruction.

Example 9: The Fate of Donor Cell Nucleus after Fusion to Enucleated Oocytes Treated with Caffeine - increase in nuclear envelope breakdown and premature chromatin condensation in donor cell DNA

To examine the fate of a donor cell nucleus transferred to an enucleated nucleus treated with caffeine, primary foetal fibroblast cells (the G0/G1 phase of the cell cycle) were fused to AT-TI enucleated oocytes at 24hpm after incubation with or without lO.OmM caffeine. Caffeine treated and untreated enucleated oocytes used as cytoplast recipient were fixed and examined two hours post fusion. In caffeine treated oocytes the majority of the transferred nuclei underwent nuclear envelope breakdown (NEBD) as compared to the control oocytes (86.2% vs 11.1%) . 36.2% of the nuclei formed a premature metaphase as compared to 3.2% in the control. 1.7% formed unorganised chromosomes vs 0.0% in the control. 48.3% formed a single mass of condensed chromatin vs 7.9% in the control (see Table 3) . The treatment of enucleated oocytes for 6h with lOmM caffeine (18-24hpm) prior to cell fusion significantly increased the occurrence of nuclear envelope breakdown and chromatin condensation in the reconstructed embryos. Table 3

Cumulus cells were removed at 15hpm. Oocytes were enucleated at 16-18hpm and cultured in maturation medium with and without lOmM caffeine for 6 hours. Unenucleated oocytes with the first polar body were used. Nuclear donor cells (G0/G1 phase of the cell cycle) were fused to the cytoplasts with 2 DC pulses of 1.25KV/cm for 60μsec in 0.3M mannitol without calcium ions using an Eppendorf Multiporator. Fused couplets were cultured in mSOF containing 4mg/ml BSA. The fused embryos were fixed 2h post fusion and stained with 1.0% aceto-orcein. Stained NT embryos were examined by phase contrast microscope (Leica, Germany) at x 100 under immersion oil. The results in Table 3 illustrate that by increasing MPF and MAP kinase levels in an oocyte the donor nuclei will undergo nuclear breakdown and chromatin condensation which is favourable for nuclear reprogramming and successful embryo development.

Table 3 also compares the nuclear configuration in constructed embryos using unenucleated and enucleated oocytes (which were not caffeine treated) as cytoplast recipients. From this data it is clear that unenucleated Mil oocytes do not cause nuclear envelope breakdown.

Example 10: Effects of caffeine on the developmental potential of in vitro matured, aged and denuded ovine oocytes.

Oocyte aging is characterised by a decrease in the activities of MPF and MAP kinase, a decrease in development and an increase in polyspermy. Caffeine, a protein phosphatase inhibitor, prevents the decline and in fact increases oocyte kinase activities.

Cumulus oocyte complexes were matured in TCM-199 supplemented with 10% FCS, Sμg.mL^*1 FSH, Sμg.mL ¹ LH, lμg.mL^*1 estradiol, 0.3mM sodium pyruvate and lOOμm cysteamine. At 24hr, oocytes were either fertilised (A) or stripped of their cumulus cells by vortexing, and incubated for a further 6h in the presence (B) or absence (C) of lOmM caffeine in maturation medium. All oocytes were fertilised as previously described (Sinclair KD et al. 1999 J. Reprod^'. Fertil. 116, 177-86) . Briefly, frozen thawed ram semen pellets were used at a final concentration of 2.5-3.5 x 10⁶ sperm. mL^*1. 24hr after fertilisation presumptive zygotes were washed and cultured in SOF media supplemented with 3mg/ml BSA for 2 days, then cultured for another 5 days in SOF supplemented with 10% FCS, all at 5%CO₂, 5%O₂ and 90%N₂ at 392C. Cleavage rates were not different between groups (69%, 74% and 73%) . Development to blastocyst decreased on oocyte aging, caffeine treatment prevented this decline (32%, 30% and 13% respectively (P < 0.05)) . The total cell numbers in blastocysts were not statistically different (92.4 + /- 5.2, 84.7 + /-3.7 and 80.4 + 1-5.8) . Polyspermy rates increased on oocyte aging, caffeine did not significantly reduce polyspermy (2%\ 10%^b and 20%^b)^*. In conclusion, caffeine treatment can prevent the decline in developmental potential of ovine oocytes matured and aged in vitro. The results in brackets are given in the order A, B, C. ^*Different superscripts significant (P < 0.05)

These results demonstrate that caffeine treated aged oocytes remain able to be fertilised and develop as compared to non-caffeine treated oocytes. The results also demonstrate that the development of IVF embryos is improved by using oocytes with an increased or stabilised activity of MPF and/or MAP kinase.

Claims

1. An oocyte with a stabilised or an increased activity of one or more protein kinases.

2. An oocyte according to claim 1 wherein the one or more protein kinases are selected from the group comprising maturation/M-phase promoting factor (MPF) and mitogen-activated protein (MAP) kinase.

3. An oocyte according to claim 1 or claim 2 which has been treated with a protein kinase stabilising or increasing agent to cause the stabilisation or increase in protein kinase activity in the oocyte.

4. An oocyte according to any preceding claim in which the protein kinase activity is increased by at least about 20%.

5. An oocyte according to any preceding claim in which the protein kinase activity is increased by at least about 50%.

^' 6. An oocyte according to any preceding claim with one or more improved qualities selected from the group comprising • the oocyte is more likely to produce a viable embryo; • an embryo produced from the oocyte is more likely to produce a viable offspring; • normal age related changes in the oocyte are reduced; • the oocyte produces a blastocyst with more cells; • an embryo produced from the oocyte hatches earlier; • the oocyte displays nuclear breakdown; • an embryo produced from the oocyte has an increased developmental potential; • the oocyte displays rejuvenated developmental potential; • the oocyte has increased chromatin formation; • the oocyte displays improved nuclear reprogramming; and • the oocyte has an increased window of time in which it is in a condition in which it can be fertilized.

7. An oocyte according to any preceding claim wherein the oocyte is arrested at metaphase of the second meiotic division (Mil) .

8. An oocyte according to any preceding claim wherein the oocyte is enucleated.

9. An oocyte according to any preceding claim wherein the protein kinase content is stabilised or increased by treating the oocytes with a protein phosphatase inhibitor.

10. An oocyte according to claim 9 wherein the protein phosphatase inhibitor is caffeine.

11. An oocyte according to claim 9 wherein the activity of protein kinase is increased or stabilised by any one of the following means: • injection into the oocyte of cell cytoplasm with increased activity; • injection into the oocyte of purified and/or recombination protein; • introduction into the oocyte of a DNA or RNA vector which induces synthesis of a protein kinase, prevents deactivation of a protein kinase and/or causes activation of an inactive protein kinase; and • treatment of the oocyte with compounds which inhibit or activate cellular factors resulting in increased kinase activities.

12. An oocyte according to any preceding claim which is derived from a human or non-human animal.

13. An oocyte according to claim 12 wherein the non-human animal is selected from the group comprising cow, bull, pig, goat, sheep, horse, camel, rodent, cat and dog.

14. The use of an oocyte according to any preceding claim in the generation of a human or non-human animal embryo.

15. The use of claim 14 wherein the embryo is produced using an assisted conception technique.

16. The use of claim 15 wherein the assisted conception technique is selected from the group comprising nuclear transfer, in vitro fertilisation, intracytoplasmic sperm injection and parthogenesis.

17. The use of any one of claims 14 to 16 wherein the embryo is generated by introducing into the oocyte a donor nucleus or a donor intact genome devoid of any nuclear membrane. ,

18. The use of any one of claims 14 to 17 wherein the embryo generated is genetically selected and/or genetically modified.

19. The use of any one of claims 14 to 18 wherein the embryo generated is transpecies.

20. The use of an embryo generated by any one of claims 14 to 19 to provide embryonic stem cells and/or isogenic stem cells.

21. A human or non-human embryo, foetus or animal produced from an oocyte according to any of claims 1 to 12.

22. An embryo, foetus or a human or non-human animal according to claim 21 produced by assisted conception, such as nuclear transfer, in vitro fertilisation, intracytoplasmic sperm injection or parthogenesis.

23. An embryo, foetus or a human or non-human animal according to claim 21 or claim 22 which is genetically selected and/or genetically modified.

24. A method of producing a human or non-human embryo comprising treating an oocyte to increase the activity of one or more protein kinases in the oocyte.

25. A method of producing an oocyte with improved qualities comprising treating the oocyte to increase the activity of one or more protein kinases in the oocyte.

26. A method of increasing or stabilising the activity of one or more protein kinases in an oocyte comprising treating the oocyte with a protein phosphatase inhibitor.

27. A method according to claim 26 wherein the protein phosphatase inhibitor is caffeine.

28. A method of therapeutic cloning comprising the step of stabilising or increasing the activity of one or more protein kinases in an oocyte.

29. A stem cell produced by the method of therapeutic cloning according to claim 28.

30. A method of reproductive cloning comprising the step of stabilising or increasing the activity of one or more protein kinase in an oocyte.

31. A method of claim 28 or 30 in which a genetically modified donor nucleus or a genetically modified intact genome devoid of a nuclear membrane is introduced into the oocyte.

32. A method for the treatment of infertility in a human or non-human animal comprising treating a human or non-human animal oocyte to stabilise or increase activity of one or more protein kinase.

33. A method of assisted conception comprising treating an oocyte to stabilise or increase activity of one or more protein kinase.

34. A method for the production of animal, human or non-human, embryos by intracytoplasmic sperm injection (ICSI) comprising treating an oocyte to stabilise or increase the activity of one or more protein kinase .

35. The use of a protein phosphatase inhibitor to increase the activity of one or more protein kinase in an oocyte.

36. A method for the prevention or reversal of human or non-human oocyte ageing comprising treating an oocyte to stabilise or increase the activity of one or more protein kinase in an oocyte.

37. A method of reconstituting a human or a non-human animal embryo, the method comprising treating an oocyte to increase or stabilise the protein kinase concentration.

38. A method according to claim 37 wherein a donor nucleus is transferred into the oocyte.

39. A method according to claim 38 wherein the nucleus is transferred by cell fusion.

40. A method according to claim 37 wherein an intact genome, which is not bounded by a nuclear envelope, is transferred into the oocyte

41. A method according to any one of claims 38 to 40 wherein the nucleus or intact genome is transferred by injection.

42. A method according to any one of claim 38 or 41 in which the donor nucleus or intact genome is genetically modified.

43. A method according to any one of claims 37 to 42 wherein the oocyte is enucleated.

44. A method according to any one of claim 37 to claim 42 wherein the oocyte and/or the donor nucleus or intact genome is selected from the group comprising cow, bull, pig, goat, sheep, horse, camel, rodent, cat or dog.

45. A method according to any of claims 37 to 43 wherein the oocyte is from a different species to the donor nucleus or intact genome.

46. A method according to claim 45 wherein the donor nucleus is transferred to the oocyte using nuclear transfer techniques.

47. A transpecies human or non-human embryo, foetus or animal produced according to the method of claim 45 or 46.

48. A method of preparing a non-human animal, the method comprising: (a) reconstituting a non-human animal embryo according to any one of claims 37 to 46; (b) causing a non-human embryo to develop to term from the embryo ; (c) optionally, breeding from the non-human animal so formed.

49. A method of producing a cell or cell line, the method comprising reconstituting a non-human animal embryo according to any one of claims 37 to 46 and clonally expanding cells from the embryo to produce a cell or cell line.

50. A method for the production of a chimeric human or non-human animal embryo wherein the recipient embryo is produced according to the method of any one of claims 24 to 27, 34, 35 to 46, 48 or 49 or using an oocyte according to any one of claims 1 to 13, and the embryonic stem cells or early embryo cells are obtained from an embryo produced according to the method of any one of claims 24 to 27, 34, 35 to 46, 48 or 49 or using an oocyte according to any one of claims 1 to 13.

51. A method according to claim 50 in which the genetic material of the recipient embryo and/or the embryonic stem cells/early embryo cells have been genetically modified.

52. A chimeric human or non-human animal embryo produced from an embryo, produced from the method of claim 50 or 51.

53. A chimeric human or non-human animal foetus or offspring produced from an embryo according to claim 52.