WO1995003398A1 - Enucleation of oocytes - Google Patents

Abstract

A process for enucleating oocytes is described. The process includes: providing a source of oocytes; exposing the oocytes to a density gradient to form an oocyte gradient mix; subjecting the oocyte gradient mix to centrifugation to separate an oocyte fraction including enucleated oocytes; and identifying and isolating the enucleated oocytes. Enucleated oocytes produced according to the process have a reduced lipid content compared to enucleated oocytes prepared by conventional methods. They are also capable of electrofusion at amplitudes of 2 kV/cm or less. Enucleated oocytes produced according to the process may be used to prepare nuclear transplantation embryos, which may in turn be used to prepare multiple genetically similar animals.

Description

ENUCLEATION OF OOCYTES

The present invention relates to a process for the enucleation of oocytes and to the use of such oocytes in a process of nuclear transplantation for the production of nuclear transplantation embryos and multiple offspring of genetic similarity.

The benefits of nuclear transplantation are that it enables the rapid increase in numbers of genetically identical animals or progeny having superior production traits. By removal of genetic diversity from animals, and replacing this with common genetic material, characteristics of the animal may be more accurately quantified.

The process of nuclear transplantation is described as the transfer of an intact nucleus from one cell to another which has had its nuclear DNA removed or destroyed. More particularly, the process involves the introduction of a foreign nucleus into the cytoplasm of an enucleated recipient oocyte. Hence the process may be applied to a recipient oocyte which will receive genetic material from a donor nucleus. Foreign genetic material is generally introduced via fusion which reconstitutes the genome of the oocyte. Fusion with the oocytes then results in a reconstituted, transplanted oocyte of known genetic constitution which has the potential of developing into an embryo whose cells may be used in time for nuclear transplantation or the production of embryonic stem cells thereby increasing the potential number of genetically identical embryos. Resulting embryos can be transferred to recipient females to enable the production of primordial germ cells or embryo development to term. This process is illustrated in Figure 1. Current production of nuclear transplantation embryos, for example, employs micromanipulation to enucleate (remove the chromosomes of) metaphase II (Mil) oocytes. This technique is a highly skilled, technically demanding, expensive and time consuming technique. Other techniques used to enucleate oocytes include the use of UV light or chemicals which may be potentially destructive to the oocyte. Hence, there is no means of enucleation which provides for large numbers of oocytes which remain sufficiently intact so that fusion of foreign genetic material into the oocyte can be carried out successfully. Accordingly, it is an object of the present invention to overcome or at least alleviate one or more of the difficulties and deficiencies of the prior art.

Accordingly in a first aspect the present invention provides a process for enucleating oocytes which process includes: providing a source of oocytes; exposing the oocytes to a density gradient to form an oocyte gradient mix; subjecting the oocyte gradient mix to centrifugation to separate an oocyte fraction including enucleated oocytes; and identifying and isolating the enucleated oocytes.

The present invention thus involves the use of a density gradient in which oocytes are centrifuged to remove the chromosomes. Centrifugation rapidly enucleates large numbers of oocytes. The technique relies on the separation of oocytes into fragments in such a way that the Mil chromosomes are eliminated from the oocyte as it is stretched apart. Foreign genetic material (including DNA, chromosomes or a nucleus) may then be introduced via fusion to reconstitute the genome.

Oocytes may be obtained from any source. For example, they may be of bovine, ovine, porcine, murine, amphibian, equine or wild animal origin. Oocytes may be selected at a particular stage of maturation. The state of maturation may be achieved by in vitro culture as described below or the animal may be monitored to provide oocytes at a particular stage of maturation during an ovulation cycle. This may be effected by administration of fertility hormones and drugs which can synchronise the animal so that the stage of maturation can be determined.

Oocytes selected for enucleation are preferably selected at the stage when the oocytes have extruded the first polar body. Preferably, the oocytes are selected for enucleation at approximately 20 to 30 hours post maturation, more preferably at approximately 24 hours after initiation of maturation. The process of the present invention may include the preliminary step of subjecting the oocytes to in vitro maturation. Maturation may be controlled by protein synthesis and phosphorylation inhibitors. The preliminary step of subjecting the oocytes to in vitro maturation may alter the structure of the oocyte or it may progress the oocyte to a stage where it is more susceptible to enucleation.

The oocytes may be matured in vitro by exposure to suitable culture conditions, hormones and/or growth factors. Preferably the oocytes are matured in the presence of a cell culture medium and serum. Preferably the serum includes growth factors and/or gonadotrophic and/or ovarian hormones. The culture medium may be any culture medium capable of sustaining oocytes in culture. Preferably the cell culture medium is TCM199 and the serum is foetal calf serum (FCS). Where oocytes have been cultured in vitro cumulus cells may be removed to provide oocytes at a suitable stage of maturation for enucleation. Cumulus cells may be removed by pipetting or vortexing for example in the presence of 0.5% hyaluronidase.

Prior to centrifugation, the oocytes are exposed to a density gradient so that after centrifugation, the enucleated oocytes may be separated from other cell fractions such as the zona, lipids, membrane bound vesicles, smooth ER, large organelles including the Mil plate and mitochondria.

The density gradient may be of any suitable type having a suitable density range. The density gradient may be a step gradient. The density gradient may be a Percoll gradient, Ficoll or Sucrose gradient. Preferably the gradient is Percoll, most preferably iso-osmotic Percoll. The Percoll gradient may be made by diluting Percoll to obtain a suitable range of densities. Preferably the gradient includes from approximately 7.0% - 50% Percoll, more preferably approximately 7.5% - 45% Percoll, most preferably the density gradient includes a step gradient of approximately 7.5, 30 and 45% iso osmotic Percoll.

In a preferred aspect, oocytes may be treated prior to centrifugation so as to prepare the oocyte for enucleation. This treatment may make the oocytes more susceptible to enucleation. Preferably they are treated with an agent which make the membranes of oocytes more malleable. Cytochalasins are a preferred group of compounds which inhibit actin polymerisation into microfilaments. Most preferably cytochalasin B is used. Preferably, cytochalasin B is added to the density gradient prior to the centrifugation step. Preferably, the gradient is centrifuged at approximately 1 , 000-10.OOOg for approximately 1-10 seconds, more preferably approximately 5,000g for approximately 4-8 seconds.

In a further preferred aspect the oocytes may be modified by removal of the zona. Accordingly the process according to the present invention may include the step of treating the oocytes to remove the zona. The zona removal may be achieved by treating the oocytes with a proteolytic enzyme or acidified medium, preferably pronase.

Accordingly, in a preferred aspect of the present invention the process for enucleation of oocytes includes: providing a source of oocytes; treating the oocytes to remove zona; exposing the treated oocytes to a density gradient to form an oocyte gradient mix; subjecting the gradient oocyte mix to centrifugation to separate an oocyte fraction including enucleated oocytes; identifying and isolating the enucleated oocytes. In a preferred aspect, the process may further include a first centrifugation step prior to removal of the zona. The oocytes may be centrifuged in the first centrifugation step under high g forces, for example approximately 10,000 g to 200,000 g for approximately 1 to 5 minutes. By centrifuging the zona intact oocyte, then removing the zona and centrifuging to remove chromosomes, the enucleated oocytes isolated may be consistently larger in size and less fragile. The gradient oocyte mix may be centrifuged under lower g forces, for example 5000g for approximately 4-8 seconds.

Enucleation may be monitored by any means including staining biopsied karyoplasts or samples of fragments of oocytes. The enucleated oocytes may be identified by identification of fragments without an extrusion cone, containing the oocyte chromosomes.

In a further aspect of the present invention there is provided enucleated oocytes prepared by the processes described above. Such oocytes may have a reduced lipid content compared to enucleated oocytes prepared by conventional methods. Whilst applicants do not wish to be restricted by theory, it is postulated that centrifugation of oocytes partitions the lipid component of oocytes.

Accordingly, the present invention also provides an enucleated oocyte having a reduced lipid content compared to enucleated oocytes prepared by conventional methods.

Enucleated oocytes may have their genomes reconstituted with any foreign genetic material. Whilst applicants do not wish to be restricted by theory, it is postulated that the use of lipid-reduced enucleated oocytes for reconstitution with foreign genetic material produced lipid-reduced embryos which are less susceptible to damage during cryopreservation.

Accordingly, in a further aspect of the present invention there is provided a method for preparing a nuclear transplantation embryo which method includes providing an enucleated oocyte as described above; and a foreign gene sequence; and introducing the foreign gene sequence into the enucleated oocyte to produce a reconstituted, transplanted oocyte; and culturing the transplanted oocyte to produce a nuclear transplantation embryo.

The foreign genetic material may be introduced via fusion which reconstitutes the genome. Enucleated oocytes may for example be reconstituted with blastomeres from embryos via micromanipulation or aggregation. Electrofusion techniques may then be utilised to reconstitute the genome. Whilst the enucleated oocytes may be of unknown genetic potential, this is not critical since the chromosomes have been removed.

Surprisingly, it has been found that it is possible to get adequate electrofusion of the enucleated oocytes according to the present invention using amplitudes of 2kV/cm or less. As a result there is less potential of damage to the cells. Whilst applicants do not wish to be restricted by theory it is postulated that electrofusion is possible at lower amplitudes than would usually be used because of the absence of the zona. Accordingly, the present invention also provides an enucleated oocyte capable of electrofusion at amplitudes of 2 kV/cm or less.

The foreign gene sequence may be provided from any suitable source. The foreign gene sequence may include donor chromosomes. The supply of donor chromosomes foreign to the oocyte may come from an embryo containing up to 100 cells or more or from an embryonic stem (ES) cell or primordial germ cell line of hundreds to millions of cells. The foreign gene sequence may be provided from blastomeres from in vitro or in vivo produced fresh or frozen/thawed embryos. The foreign gene sequence may be from a nuclear transplantation embryo as hereinafter described. It will be understood that each cell has the potential to be fused with the enucleated oocyte and develop into a new embryo.

Preferably, the transplanted oocytes are cultured in a medium including synthetic oviduct fluid ("SOF"), albumin and amino acids. Ovine embryos cultured in SOF supplemented with human serum can produce lambs with high mean birth weights. This can in turn result in a high post-natal mortality rate. Applicants have found high levels of development in vitro and reduced embryo size when serum is replaced with amino acids and albumin. Accordingly, the high mortality problem appears to have been eliminated through supplementation of SOF with albumin and amino acids. Whilst applicants do not wish to be restricted by theory, it is postulated that the culture of embryos in serum-free media, containing albumin and amino acid reduced the lipid component of embryos. This will advantage embryo survival after cryopreservation (freezing and thawing).

Accordingly, in a further aspect of the present invention there is provided a method for producing normal birthweight offspring which method includes providing an embryo derived from oocytes enucleated by centrifugation, and an embryo culture medium including synthetic oviduct fluid, albumin and amino acids; culturing the embryo in the culture medium; transferring the embryo to a recipient female; and allowing the embryo to develop to term. The present invention also privides a method for increasing the viability of an embryo following cryopreservation which method includes providing an embryo derived from oocytes enucleated by centrifugation, and an embryo culture medium including synthetic oviduct fluid, albumin and amino acids; culturing the embryo in the culture medium; and subjecting the embryo to cryopreservation.

Preferably the albumin is bovine serum albumin ("BSA"). Preferably the albumin is present in the medium at a concentration of approximately 10 to 50 mg/ml, more preferably approximately 20 to 40 mg/ml, most preferably approximately 32 mg/ml.

Preferably the amino acids are present in the medium at approximately the concentration present in Eagles Minimal Essential Medium ("MEM"), with the exception of glutamine which is preferably present at a concentration of approximately 1 mM.

Preferably the pH of the medium is approximately 7.4. The medium optionally contains vitamins. These may be present at approximately the concentrations present in MEM. Preferably the osmolarity of the medium is maintained at approximately

270 mOsmol.

Preferably the medium is equilibrated in a gas phase of approximately 5% CO₂, approximately 7% oxygen and approximately 88% N₂.

Preferably the embryos are cultured for approximately 3-8 days, more preferably approximately 5 days.

Preferably the medium is replaced approximately every 48 hours.

In further aspect of the present invention there is provided a nuclear transplantation embryo produced by the method described above. The present invention also provides a nuclear transplantation embryo having a reduced lipid content compared to nuclear transplantation embryos prepared by conventional methods.

The nuclear transplantation embryos may be utilised in the production of genetically identical or similar animals. The nuclear transplantation embryo may be transferred into a recipient female, preferably a synchronised recipient female, utilizing known techniques. The recipient female may be synchronised by administering fertility drugs, steroids or prostaglandins.

Accordingly, in a further aspect of the present invention there is provided a method for preparing a multiple offspring of genetic similarity which method includes providing nuclear transplantation embryos as described above, and recipient females; transferring the nuclear transplantation embryos to the recipient females; and allowing the nuclear transplantation embryo to develop to term. The nuclear transplantation embryos may also be used as donor cells or nuclei for further cycles of nuclear transplantation. The nuclear transplantation embryos may also be used for production of embryonic stem cells or primordial germ cells. The present invention will now be more fully described with reference to the following examples. It should be understood however that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above. Figure 1 illustrates the process of nuclear transplantation.

Figure 2 shows a bovine oocyte before in vitro maturation. Figure 3 shows a bovine karyoplast enucleated by micromanipulation. The metaphase and polar body DNA is visible via epifluorescence.

Figure 4 shows reconstitution by micromanipulation of a blastomere into an enucleated oocyte.

Figure 5 shows an example of oocyte stretching in a Percoll gradient. Figure 6 shows group 2 cytoplasts produced by one centrifugation treatment.

Figure 7 shows group 3 cytoplasts produced by two centrifugation treatments.

Figure 8 shows extrusion cones produced by group 3 cytoplasts 6 hours post centrifugation. Figure 9 shows cytoplast produced by centrifugation with extrusion cone containing metaphase II plate DNA stained.

Figure 10 shows an enucleated cytoplast aggregated with bovine blastomere. Figure 11 shows a two cell bovine NT embryo produced from group 3 cytoplast. The embryo is encased in sodium alginate false zona.

Figure 12 shows a bovine NT blastocyst (6 day old) stage produced by centrifugation.

Figure 13 shows the effect of amplitude and pulse length on electrofusion of zona intact 2 cell mouse embryos.

Figure 14 shows the effect of amplitude and pulse duration on electrofusion of zona absent 2 cell mouse embryos.

Figure 15 shows the effect of amino acids on the development of individual sheep zygotes in culture, a) Effect of amino acids on the percentage of embryos arrested at the 8-16-cell stage, b) Effect of amino acids on blastocyst formation, n = 147 embryos, SOF, control; SOF + 20aa, SOF with all Eagle's amino acids; SOF + non + gin, SOF with nonessential amino acids and glutamine; SOF + ess, SOF with essential amino acids without glutamine. Open bars represent embryos cultured in the same drop of medium for 6 days; closed bars represent embryos transferred to fresh medium every 48 h. Significantly different from control: *p < 0.05; *p < 0.01 ; "like pair significantly different, p < 0.01.

Figure 16 shows bright-field photomicrographs of blastocysts developed after 6 days of culture, a) Blastocyst after culture in medium SOF supplemented with Eagle's amino acids, with the medium renewed every 48 h (x500). b) Blastocyst after culture in medium SOF supplemented with human serum (20%), with the medium renewed every 48 h (x500). Note the vesicles apparent in the trophectoderm.

Figure 17 shows the effect of amino acids, vitamins, an serum on glucose uptake and lactate production, expressed on a per-cell basis, by blastocysts developed after 6 days of culture, SOF, control; SOF + 20aa, SOF medium with all Eagle's amino acids; SOF + 20aa + vit, SOF medium with all

Eagle's amino acids and vitamins. Open bars are glucose uptake (pmol/cell/h). Closed bars are lactate production (pmol/cell/h). *Significantly different from control; p < 0.05.

Figure 18 shows the correlation of glucose uptake and lactate production by in w^'fro-developed blastocysts. a) SOF medium; b) SOF medium with all Eagle's amino acids; c) SOF medium with all Eagle's amino acids and vitamins; d) SOF medium with 20% human serum. Significant correlations between glucose uptake and lactate production in treatments a, b, c; p < 0.01.

Figure 19 shows insofusion controus of bovine embryonic cell types.

EXAMPLE 1 Bovine oocytes aspirated from abattoir collected ovaries (see Fig. 2) were matured in TCM 199 plus FCS and hormones for 22 hours. Cumulus cells were removed by pipetting in 0.5% hyaluronidase and oocytes which had extruded the first polar body selected (176/278; 63%). Enucleation occurred at

24 hours post maturation (hpm). Group 1 oocytes were enucleated via micromanipulation (see Fig. 3). Group 2 oocytes had the zona removed in 0.5%

Pronase and 20 oocytes were centrifuged in 0.4 ml tubes in 100 ml each of 7.5,

30 and 45% iso osmotic Percoll containing 10 μg/ml cytochalasin B at 5000xg for

4 seconds (see Fig. 6). Group 3 oocytes were centrifuged at 15000xg for 2 minutes before having the zona removed and centrifuged as in group 2 (see Fig. 7).

Enucleation of micromanipulated and centrifuged oocytes was checked by staining the biopsied karyoplast or a sample of the fragments with Hoechst 33342 and viewing via epifluorescent microscopy at 32 hpm. Enucleated oocytes were reconstituted with blastomeres from in vivo produced frozen/thawed bovine embryos via micromanipulation (group 1) (see Fig. 4) or by aggregation to a fragment using 100 mg/ml phytohemagglutinin (groups 2 and 3) (see Fig. 10).

Electrofusion was achieved using a BTX ECM 200 and Optimizor with Zimmerman's fusion media. After manual alignment an AC pulse of 8V for 5 seconds was followed by fusion via single DC pulse of 77V(1.75 kV/cm) for 40 μs, fusion was assessed 15 minutes later. Fused oocytes from groups 2 and 3 were placed in a false alginate zona (see Fig. 11 ) and all nuclear transplantation (NT) embryos were cultured in SOF plus amino acids under mineral oil with 4 embryos per 30 ml drop changed every 48 hours. Cleavage was assessed 40 hours post fusion and development to morula day 6 post fusion (see Fig. 12 and Table I).

Table 1

Group Enucleation (%) Fusion (%) Cleavage (%) Morula (%)

1 24/50(48) 15/24(63) 8/15(53) 1/8(13)

2 68/76(90) 11/16(69) 3/11(27) 0 (0)

3 69/78(89) 18/21(86) 6/18(33) 1/11(9)

Transmission electron microscopy (TEM) studies of centrifuged oocytes show the stratification of cytoplasm into 5 phases according to density of its components. ^• Lipid being least dense is found at the upper pole, followed by membrane bound vesicles, smooth ER, a large organelle free region containing the Mil plate and at the lower pole mitochondria are concentrated (see Fig. 5). Centrifugation results in a higher enucleation rate than micromanipulation (89% vs 48%). This is partially due to the emission of identifiable nucleate fragments which possess an extrusion cone containing the Mil plate (see Figs. 8 and 9). Electrofusion rates were similar between groups (63% to 86%). Development of NT embryos produced in groups 1 and 3 was similar (13% vs 9%). Group 2 fragments were darker than the opaque group 3 fragments which contain less lipid and organelles, suggesting that successful NT may rely on a cytoplasmic factor. Full development capacity may be tested via transfer of NT embryos into recipient cows. Enucleation by centrifugation enables the rapid production of large numbers of enucleated oocyte fragments which may be used for the successful production of bovine nuclear transplantation embryos.

EXAMPLE 2 Aim

To increase the efficiency of producing embryos by nuclear transplantation by replacing the micromanipulation steps with high capacity centrifugation and aggregation techniques. Methods

Enucleation by micromanipulation (Group 1 ) by centrifugation remove zona pellucida with 0.5% Pronase centrifuge in 7.5, 30 and 45% isotonic Percoll gradient at

5000 g for 4 seconds (Group 2) preliminary centrifugation with zona intact at 15000 g for 2 minutes (Group 3) enrich enucleated population by selecting for cytoplasts without extrusion cone,

Aggregation micromanipulation or phytohemagglutinin Electrofusion alignment 8 Volts AC for 5 seconds stimulation 1.75 kV/cm DC, 1 pulse for 40 msec,

Culture sodium alginate false zona

SOF + amino acids, 30 ml drops under oil, changed every 48 hours

Table II

Enrichment of Centrifuαed Enucleated Cvtoplasts

Cytoplasts No Cone Enucleated Mean Efficiency

Group* (%) (%) A (%) B (%) (AxB)/100

2 184/325 41/90 143/161 46x89 (57) (46) (89) (41 ) 3 249/423 97/161 84/94 60x89 (59) (60) (89) (53)

See text. Table III

Micromanipulation vs Centrifugation for Nuclear Transplantation

Enucleation Electrofusion Cleavage Morula

Group* (%) (%) (%) (%)

1 270/720 113/180 24/113 5/24

(38) (63) (21) (21 )

2 46x0.89 41/55 7/31 0/7

(41 ) (75) (17) (0)

3 60x0.89 82/118 24/82 5/24

(53) (70) (29) (21 )

* See text.

Discussion

Two types of enucleated cytoplasts are produced by centrifugation. Enucleated cytoplasts are enriched by selection for absence of an extrusion cone. Development of nuclear transplantation embryos produced after centrifugation is equal to those produced by micromanipulation. Conclusions

Enucleation by centrifugation increases the efficiency of nuclear transplantation by rapidly producing large numbers of cytoplasts capable of development after nuclear transplantation.

EXAMPLE 3 Properties of the zona pellucida during electrofusion of 2 cell mouse embryos

Electrofusion is a valuable technique used to fuse embryonic cells for nuclear transfer. The modification of the nuclear transfer procedure so that the requirement for micromanipulation is alleviated, has changed the mode of electrofusion because the zona pellucida does not surround the embryo. This provides an opportunity for the investigation of the electrical properties of the zona and the effects of 2 cell mouse embryos after they were electrofused and then cultured in vitro to assess development.

Female C57/MIJxCBA/Wehi mice which were 6 to 8 weeks old were superovulated and mated before being killed, then ampullae were dissected and embryos at the 2 cell stage were flushed out. Embryos were electrofused with or without the zona which was removed using 0.5% Pronase. Embryos were washed 4 times for 2.5 minutes in fusion media which consisted of 0.25 M sucrose buffered to pH 7.4 using Trisma base. Blastomeres were aligned for 5 to 10 seconds with an amplitude of 8 volts AC. Electrofusion was achieved with amplitudes varying from 0.4 to 4.8 kV/cm DC using a single pulse of 10, 50 or 100 μs duration. Electrofusion was scored 1 hour after electrical treatment and fused and control embryos were cultured in medium M16 until the blastocyst stage. Statistical analysis was achieved using the SAS general linear model procedure. The results in Figures 13 and 14 show that the electrofusion of embryos with and without the zona is not significantly different (p<0.05) for amplitudes of 2.0 kV/cm or less. However, at amplitudes greater than 2.0 kV/cm the presence of the zona significantly increased electrofusion (P<0.05) than when using either a 50 or 100 μs pulse (47.49; 47.94%). These effects were due to the increased lysis of zona free embryos as the amplitude increased above 2.0 kV/cm and the pulse duration was increased to 50 or 100 μs. The development to the blastocyst stage of embryos electrofused without the zona (36.24%) was significantly lower (P<0.05) than control embryos (95.90%) and embryos electrofused with the zona intact (88.68%). During electrofusion using amplitudes greater than 2.0 kV/cm the zona pellucida provided blastomeres protection against electrically induced lysis. However, at amplitudes of 2.0 kV/cm or less, the zona had no influence or electrofusion. This suggests that the zona was able to protect the embryo from extreme electrical treatment. This protection was not due to a constant resistance of the zona glycoprotein because the effect was not observed at lower amplitudes. Development of zona intact embryos to the blastocyst stage is not effected by electrofusion at any amplitude or pulse duration, however development is decreased with the absence of the zona. This can be explained by the blastomeres of embryos fused without the zona aggregating during culture to form blastocysts which originated from more than 1 embryo.

This study has shown that the zona plays a protective role against cell lysis at high electrofusion amplitudes and that electrofusion does not inhibit development to the blastocyst stage, although embryos cultured without a zona resulted in large blastocysts originating from more than one embryo. This information gives some understanding of the electrical properties of the zona pellucida and the factors involved during electrofusion of embryonic cells.

EXAMPLE 4 Development of a Cell-free defined sheep embryo culture system

Effective culture systems are needed to study embryo metabolic and developmental processes and to support during culture embryos derived from oocytes enucleated by centrifugation. Domestic animal embryo culture systems are largely undefined. Since cell-free, defined media are advantageous in allowing direct examination of media components on development and reducing variability in culture conditions, we have conducted considerable research to develop such a medium. We review here trials leading to an effective cell-free culture system.

The first important observation was that sheep embryos develop to the morula/blastocyst stage in a simple medium, based on the ionic and energy substrate composition of sheep oviduct fluid (Synthetic Oviduct Fluid, SOF), supplemented with bovine serum albumin (BSA), if the oxygen concentration of the atmosphere was lowered to between 0-10%. Further research established that the optimum oxygen concentration is approximately 7%. Further research confirmed that human serum (HS) appears to contain mitogenic factors which are more active than in either sheep serum or HS albumin (HSA). HS was used by us for a number of years as the protein supplement of choice as it supported 80-90% of 1- to 2- cell embryos to blastocysts during 5 d culture with high viability in recipient used (100% recipients pregnant and 70-80% embryos surviving to term). However, in keeping with the recent observation by others, a high post-natal mortality rate was observed (37%) which was mostly attributable to a relatively high birth weight of dead lambs.. This high mortality is a problem which, fortunately, appears has been eliminated through supplementation of SOF with amino acids and BSA. Initial results showed that embryo development was similar in HS and HSA + non- essential amino acids (NEAA). The HSA/NEAA embryos had, however, reduced cell numbers. An explanation why amino acids have failed to stimulate development is that they produce embryo-toxic ammonia. Under the appropriate conditions (SOF + Eagle's amino acid + BSA [SOF aa BSA], changing media every 48 h) 95% of embryos develop to blastocysts with cell numbers similar to in vivo controls and greater than in HS. Comparison of the development and viability of embryos cultured for 5 d in HS or aa BSA showed that a similar percentage of embryos developed to transferable compact morulae/blastocysts in both media (59 v 53%, respectively). Embryos incubated in HS appeared morphologically different from those in aaBSA, having abundant lipid inclusions in the cytoplasm. Recipient pregnancy rate (65%) or embryo survival (48%) did not differ between media or embryo source but lambs from HS were significantly heavier (4.2 kg) than controls (3.4 kg) or aa BS (3.5 kg). Thus, we now routinely culture sheep embryos in a defined medium of SOF aa BSA under 5% C0₂/7% O₂/88% N₂ as it gives high development rates and birth of normal weight lambs.

EXAMPLE 5 The experiments in this study were designed to investigate the role of amino acids, ammonium, vitamins, and culture of embryos in groups on the development of sheep zygotes in culture. Morpholoy, cell number, and metabolism were used to assess the effects of culture systems on embryo development in vitro.

Materials and Methods Superovulation and Collection of Embryos

Embryos were collected for finewool Merino ewes maintained under field conditions at Monash University Research Farm, Tooradin, Victoria, Australia, during the breeding season of 1992-93. Ovulation was synchronised by use of intravaginal progestagen-impregnated sponges (Repromap, Upjohn, Sydney, Australia) containing 60 mg medroxy progesterone acetate. After 14 days, sponges were withdrawn, and 50 μg GnRH (Intervet, Lyppard, Victoria, Australia) was administered intravenously 24 h later. Ovulation in sheep occurs 24 h after GnRH injection. Multiple ovulation was achieved by a single injection of 400 IU eCG (Herior AgVet, Victoria, Australia) and 5.5 ml ovine FSH (Horizon Animal Reproduction, New South Wales, Australia) 48 h before sponge withdrawal. This treatment results in the recovery of 8 embryos on average per ewe. Ewes were run with raddled rams for superovulation hormone treatment to cessation of standing heat. At 20 h post ovulation, zygotes were flushed from the oviduct with a HEPES-buffered modification of Synthetic Oviduct Fluid (SOF) medium. Media Preparation

Embryos were cultures in SOF supplements with 32 mg/ml BSA. The pH of the medium was subsequently adjusted to 7.4 with sodium hydroxide. Amino acids used to supplement this medium were added at the concentration present in Eagle's Minimal Essential Medium (MEM; Eagle H Amino acid metabolism in mammalian cell cultures. Science 1959; 130-432-437, the entire disclosure of which is incorporated herein by reference) with the exception of glutamine, which was present at 1 mM. Vitamins used in this study were also at the concentrations found in MEM. When amino acids and vitamins were used to supplement SOF medium, osmolarity was maintained at 270 mOsmol by reducing the sodium chloride concentration accordingly. For embryo collection and handling, a HEPES-buffered modification of SOF (H-SOF) was used. In this medium, 20 mM NaHC0₃ was replaced with 20 mM HEPES< pH 7.4, and was supplemented with Eagles MEM amino acids and 4 mg/ml BSA.

Salts and glucose were of Analar grade (BDH, Poole, Dorset, UK). Pyruvate, lactate, glutamine, and phenol red dye were of tissue culture grade (Sigma Chemical Company, St. Louis, MO). Antibiotics and amino acid solutions were obtained from CSL (Parkville, Victoria, Australia). Vitamin solutions were purchased from ICN Biomedicals (Seven Hills, NSW, Australia). HEPES was supplied by Calbiochem (Alexandra, NSW, Australia); and BSA, Miles Pentex Crystalline lot 92, was purchased from Bayer Diagnostics (Kankakee, IL.). Human serum was collected from a female donor, heat-treated at 56°C for 30 min, and stored frozen at -20°C. Embryo Culture

After collection, zygotes were washed twice in H-SOF supplemented with amino acids, and once in the appropriate bicarbonate-buffered medium before being placed in culture. Embryos were cultured in 20-μl droplets of medium under a layer of paraffin oil (Labchem, Ajax Chemicals, Auburn, NSW, Australia) in 35 mm Primaria Petri dishes (Falcon, Becton Dickinson, Victoria, Australia) in a sealed chamber gassed with 5% CO₂, 7% 0₂, and 88% N₂ for 6 days at 39°C. An incubation volume of 20 μl was chosen as this is the minimum volume of culture medium (SOF) that can supply sufficient energy substrates to the sheep embryo over a 6-day period. These calculations were based on the nutrient uptakes of individual sheep embryos. Paraffin oil was pretested for embryo toxicity with mouse zygotes. All media were equilibrated in the culture dishes in a gas phase of 5% CO₂, 7% O₂, and 88% N₂ at 39°C overnight before use.

Culture Experiments

Experiment 1: Effect of specific amino acids and ammonium on zvαote development.

One hundred and forty-seven zygotes were cultured individually in 20-μl drops of medium under paraffin oil, in one of four media: 1) SOF (control); 2) SOF + 20 amino acids; 3) SOF + nonessential amino acids and glutamine; 4) SOF + essential amino acids without glutamine; for each medium, zygotes were cultured in either the same drop of medium for 6 days or in a fresh drop of medium every 48 h. Embryos from each female were randomly distributed evenly among the 8 treatment groups. When females had less than 8 embryos selection of culture treatment was by random numbers. After 144 h of culture, embryo morphology was assessed. Nonessential amino acids and glutamine have been shown to stimulate mouse embryo development in culture, whereas the essential group of amino acids without glutamine inhibit mouse embryo development in culture.

Experiment 2: Effect of embryo grouping and vitamins on zvαote development.

One hundred and eighty-seven zygotes were cultured either singly or in groups of 2 or 4 in one of three media: 1 ) SOF (control); 2) SOF + 20 amino acids; 3) SOF + 20 amino acids + B-group vitamins of Eagle's medium (MEM). Groups of 4 were also cultured in SOF + 20% human serum (for comparison with published studies). All embryos in all media were transferred to fresh medium every 48 h. Zygotes from females were pooled and allocated to one of ten treatment groups by random numbers. After 144 h of culture, embryo morphology was assessed, and blastocyst cell number was determined. The metabolism of at least six embryos per treatment was analysed before cell numbers were determined.

Assessment of Morphology Embryo morphology was determined after 144 h of culture, by use of phase contrast microscopy (200x). The following classifications were employed: <16-cell, less than or equal to 16 identifiable individual cells;' morula, compacted; blastocyst, blastocoel cavity greater than 2.3 volume of the embryo, hatching blastocyst, blastocoel fully expanded and trophectoderm hemiating the zone pellucida.

Assessment of Metabolism and Ammonium Production

Glucose uptake and lactate production by individual sheep blastocysts on Day 6 of culture was determined by means of a noninvasive microfluorometric technique. Briefly, embryos were placed individually in 1 μl of serum-free H-SOF medium containing 0.5 mM glucose as the sole energy substrate. The drops of medium were housed on siliconized microscope slides overlayed with mineral oil, and were incubated at 39°C for up to 3h. Serial 3-nl samples of medium were then taken every 15-20 min and analysed for glucose and lactate. Taking serial samples of medium for analysis ensured that linear rates of nutrient uptakes and metabolite production were recorded. Biochemical assays took place in 30-nl droplets of reagent, also housed on siliconized microscope slides overlayed with mineral oil. The assays themselves were based on conventional methods of enzymatic analysis, employing the puridine nucleotides NADPH, which were generated or consumed in coupled reactions. The fluorescence of the reagent drop (30 nl) was initially quantified by use of a fluorescence microscope with photometer attachment, and subsequently remeasured after the addition of sample (3 nl). The increase in fluorescence microscope with photometer attachment, and subsequently remeasured after the addition of sample (3 nl). The increase in fluorescence was then calibrated by use of standards run with each assay. The mean R² for the standards was 0.999. By means of this noninvasive technique, linear rates of glucose uptake and lactate production could be determined prior to cell number determination. Appearance of ammonium in the culture medium and production by blastocysts after six days of culture from the zygote was also determined fluorometrically. All enzymes and co-factors were obtained from Boehringer-Mannheim (Sydney, Australia). Cell Number Determination

Blastocyst cell number was determined after 144 h of culture, by use of an air-drying technique. Embryos were placed in a solution of 0.2% trisodium citrate for 3 min and then transferred to a microscope slide in a minimal amount of fluid. A drop of fixative, glacial acetic acid (1): ethanol (3), was dropped directly on the embryos and then spread by gentle blowing. Blastomere nuclei were stained with 10% Giesma stain in Gurr's buffer at pH 6.8 and counted. Blastocysts developed in vivo for 7 days were used as the in vivo comparison for cell number analysis. The metabolism of these blastocysts was not assessed for the purpose of this study. Statistical Analysis.

Blastocyst formation was analysed using Chi square. Differences between treatments were determined after arc sine transformation of original data expressed as a proportion, followed by analysis of variance and the bonferroni Multiple Comparison Procedure. Differences in cell numbers were determined by analysis of variance. Differences between treatments were determined by the Bonferroni Multiple Comparison Procedure. There were no significant differences between replicates.

Results Effects of Amino Acids on the Development of Zygotes Cultured Individually The ability of different media to alleviate cleavage arrest during development in culture is shown in Figure 15a. Forty-three percent of zygotes grown in SOF medium without amino acids (control) were arrested during cleavage around the 8-16-cell stage. A similar percentage of embryos (47%) were arrested when placed in fresh medium every 48 h. In the presence of all 20 amino acids, 41% of embryos exhibited developmental arrest, similar to that observed in the absence of amino acids. However, when embryos were cultured with amino acids and placed in fresh medium every 48 h, significantly fewer embryos (6%) blocked at the 8-16-cell stage compared to embryos grown in the same drop of medium for 6 days (p < 0.05) and compared to embryos developed in SOF (p < 0.01)). In the presence of Eagle's nonessential amino acids and glutamine and with the medium replaced every 48 h, significantly fewer embryos underwent cleavage arrest (13%) compared to embryos grown in SOF (p < 0.05). Cessation of embryo development at the 8-16-cell stage in the presence of essential amino acids with glutamine was similar to that observed in SOF whether the embryos were moved every 48 h (24%) or remained in the same drops for the duration of culture (53%).

The ability of different culture conditions to support blastocyst development from the zygote is shown in Fig. 15b. In SOF medium, zygote development to the blastocyst was low (19%). Placing the embryos in fresh medium every 48 h had no significant effect on development. When all amino acids were included in the medium formulation, blastocyst development for embryos grown in the same drop was elevated (45%), and it was significantly increased (62%, p < 0.01 ) when the medium was renewed every 48 h. Neither nonessential amino acids and glutamine, nor the essential amino acids without glutamine had an effect on blastocyst development compared to that in SOF medium. In subsequent experiments, therefore, SOF medium containing all Eagle's amino acids was utilized, the medium being renewed every 48 h. Effect of Embryo Grouping and Vitamins on Zygote Development

Zygotes cultured individually in SOF medium formed significantly fewer blastocysts compared to those cultured in other media (p < 0.01) (Table IV).

Table IV

Effect of amino acids. vitamins and culturing embryos i in groups on sheep zvqote development

Stage of development after 6 days of culture

No. embryos

Media* per drop <16 cell Morula Blastocyst* Percentage of

(n) (%) (%) (%) blastocysts hatching

SOF 1 (15) 53 27 20 0^a

SOF 2 (18) 39 33 28 o^b

)

SOF 4 (12) 37 25 38 34^ab N ) aa 1 (20) 0 25 75 47° aa 2 (40) 12 30 58 31^d aa 4 (20) 0 5 95 ₇gcd aa + vit 1 (18) 12 33 55 60 aa + vit 2 (20) 15 15 70 57 aa + vit 4 (16) 6 6 88 65

HS 4 (12) 8 25 67 75

*Media as per Materials and Methods; aa = amino acids, vit = vitamin, HS = human serum ^a,bLike pairs are significantly different: p < 0.05. ^{c d}Like pairs are significantly different: p < 0.01. "There were siαnificant increases in blasti Dcvst for. mation to < 0.01) at each embrvo densiti when SOF was SUDI Dlemented with amino acids (with or without vitamins).

Culturing embryos in groups had no significant effect on the number of embryos arrested or forming blastocysts in any o the media employed (Table 1 ), although blastocyst formation was elevated as group size increased. However, increasing the number of embryos per drop significantly increased the blastocyst hatching rate in SOF medium with (p < 0.01) or without (p < 0.05) amino acids. In contrast, group size had no effect on hatching when embryos were cultured in the presence of vitamins. There was also a trend of increasing blastocyst cell number when zygotes were cultured in increasing group sized in medium SOF (Table V).

TABLE V Effect of amino acids, vitamins and culturing embryos in groups of blastocyst cell number.#

Number of embryos per drop +

Media * 1 2 4

SOF 52 ± 7^a 65 ± 8^cα 75 ± 7^β'⁹

SOF + aa 105 ± 12^aj 136 ± 12^cjk 173 ± 6^θhk*

SOF + aa + vit 141 ± 21^b 124 ± 13^d 158 ± 13^fi*

SOF + HS nd nd 103 ± 15^ghi

In vivo 160 ± 9 - -

⁺n = 187

#Cell numbers expressed as mean ± SEM.

Media as per Materials and Methods; aa = amino acids, vit = vitamins, HS human serum ^a'^{c,I ,}Like pairs significantly different; p < 0.05 ^b.^d.^β.^f.^g.^h.k _{Uke pajrs S}jg_njfjcant|v different; p < 0.01

^•Equivalent cell numbers as in vivo controls.

The mean blastocyst cell number obtained in SOF, 65, was significantly below numbers obtained for embryos grown in groups of 4 in serum (103, p < 0.01 ) or for the in Vo-developed controls (160, p < 0.01 ).

In contrast, when embryos were grown in increasing group sizes in the presence of amino acids, there were significant increases in cell numbers as group size increased. Blastocyst cell numbers increased from 105 when embryos were grown singly, to 136 (p < 0.05) and 173 (p < 0.01 ) when embryos were grown in groups of 2 and 4, respectively (Table V). Cell numbers in each group size in the presence of amino acids were significantly greater than those obtained in medium SOF (p < 0.01 ). Cell numbers of blastocysts cultured individually in the presence of amino acids were equivalent to those of embryos developed in serum in groups of 4. Embryos cultured in groups of 2 and 4 had a significantly greater number of cells at the blastocyst stage than did embryos developed in serum (p < 0.05 and 0.01 respectively) and equivalent to blastocysts developed in vivo.

The supplementation of medium containing amino acids with vitamins had no significant effect on blastocyst cell number for embryos cultured singly or in groups (Table V).

Figure 16 shows typical blastocysts developed in the presence of either amino acids or 20% serum. Blastocysts cultured in the presence of amino acids exhibited an appearance comparable to that of embryos developed in vivo, which were characterised by a translucent trophectoderm and well-defined inner cell mass. In contrast, blastocysts developed in the presence of serum took on a dark, granular morphology with the apparent inclusion of lipid-like vesicles. Embryo Metabolism.

In SOF medium, growing embryos in groups had no effect on glucose uptake or lactate production (Table VI).

Table VI

Metabolism of sheep blastocvsts after culture

Glucose uptake Lactate production

Media* No. embryos per drop (pmol/embryo/h) (pmol/cell/h)* (pmol/embryo/h)

SOF 1 19.2 ± 7.6 0.35 ± 0.07 47.8 ± 5.4 0.91 ± 0.08

SOF 2 10.5 ± 4.7 0.18 ± 0.07 43.6 ± 12.0 0.75 ± 0.19

SOF 4 28.6 ± 5.8 0.37 ± 0.07 65.5 ± 7.2 0.86 ± 0.12 I to

SOF + aa 1 35.9 ± 6.0^ab 0.43 ± 0.04 72.2 ± 10.0^do 0.84 ± 0.07

I

SOF + aa 2 64.1 ± 14.3^a 0.45 ± 0.06 117.9 ± 29.0^d 0.79 ± 0.01

SOF + aa 4 67.6 ± 10.5^b 0.35 ± 0.05 145.8 ± 20.6^Θ 0.76 ± 0.10

SOF + aa + vit 1 107.7 ± 9.8° 0.83 ± 0.08 236.9 ± 28.9^f 1.76 ± 0.22

SOF + aa + vit 2 71.8 ± 12.9 0.56 ± 0.10 164.5 ± 39.2 1.19 ± 0.31

SOF + aa + vit 4 42.3 ± 7.9° 0.46 ± 0.08 111.0 ± 17.1^f 1.20 ± 0.19

SOF + HS 4 68.6 ± 10.2 0.62 ± 0.09 200.9 ± 24.7 2.05 ± 0.22

⁺n = at least 6 embryos per treatment (except for SOF, where n = 2 for embryos cultured singly and n = 4 embryos cultured in groups of 2 and 4). *Media as per Materials ad Methods; aa = amino acids, vit = vitamins, HS = human serum. ^{c d}Like pairs significantly different; p < 0.05. ^a.^b,e,f.|j_ke p_ajrs significantly different; p < 0.01. *On a per cell basis, the number of embryos per drop had no effect on metabolism in any media.

The mean glucose uptake for all embryos was 20.4 ± 4.1 pmol/embryo/h, an mean lactate production was 54.3 ± 5.9 pmol/embryo/h. Similarly, when glucos uptake and lactate production were expressed as pmol/cell/h, group size had n effect on either parameter (Table VI), the mean glucose uptake and lactat production being 0.03 ± 0.05 and 0.84 ± 0.08 pmol/cell/h, respectively.

In contrast to results with embryos cultured in SOF medium, both glucose uptake and lactate production increased significantly with embryo grou size when all amino acids were included in the medium (Table VI). Glucose uptake increased from 35.9 pmol/embryo/h by embryos developed individually, to 67.6 pmol/embryo/h by embryos grown in groups of 4 (p < 0.01). Similarly, lactate production increased from 72.2 pmol/embryo/h for embryos grown individually, to 145.8 pmol/embryo/h for embryos grown in groups of 4 (p < 0.01). However, when nutrient uptake and metabolite production were expressed on a per-cell basis, differences between umbers of embryo/drop[ were not apparent, the mean glucose uptake and lactate production being 0.41 ± 0.05 and 0.80 ± 0.10 pmol/cell/h respectively (Table VI).

On a per-embryo basis, glucose uptake and lactate production by blastocysts were significantly higher in the presence of amino acids compared to SXOF medium when embryos were cultured in groups of 2 or 4 (p < 0.05). However, there was no difference between the two media when metabolism was expressed on a per-cell basis; i.e., the increase in glucose uptake and lactate production can be attributed to the increase in cell number.

When vitamins were included in the medium formation, a significant decrease in glucose uptake and lactate production was obtained as the group size increased from 1 to 4 embryos (p < 0.01) (Table VI). Glucose uptake decreased from 107.7 pmol/embryo/h by embryos cultured individually to 42.3 pmol/embryo/h for blastocysts developed in groups of 4 (p < 0.01). Lactate production decreased from 236.9 pmol/embryo/h to 111.0 pmol/embryo/h for embryos cultures individually and in groups of 4 respectively (p < 0.01). Similar results in the other two culture treatment when metabolism of embryos cultured in the presence of vitamins was expressed on a per-cell basis, culture in groups had no effect (p < 0.06). However, on a per-cell basis, glucose uptake and lactate production were significantly greater than in embryos grown in the other two culture groups (p < 0.01 ). The mean glucose uptake and lactate production per cell in the presence of vitamins were 0.63 ± 0.08 and 1.42 ± 0.12 pmol/cell/h respectively.

Because there was no significant effect of growing embryos individually or in groups when metabolism was expressed on a per-cell basis, all 3 groups for each medium were therefore considered as one unit in a further comparison with embryos grown in groups of 4 in serum (Fig. 17). Glucose uptake and lactate production by all blastocysts grown in SOF or in the presence of amino acids was equivalent. However, the addition of vitamins to SOF with amino acids caused a significant increase in both glucose uptake and lactate production per cell. A similar pattern of metabolism was observed for embryos developed in the presence of serum. There was no significant difference between embryo metabolism in the presence of vitamins or serum.

The relationship between glucose uptake and lactate production by each blastocyst is shown in Figure 18. There was a significant correlation between the nutrient uptake and metabolite release in all groups (p < 0.01 ) except serum. Ammonium Production

The level of ammonium in a 20-μl drop of medium containing all of Eagle's amino acids was determined to be 0.191 mM at 48 h, 0.357 mM at 96 h, and 0.419 mM at 144 h (n = 6 replicates for each time point). There was a linear increase in ammonium concentration with time, R² = 0.94. The production of ammonium by individual cultured blastocysts was 7.38 ± 1.46 pmol/embryo/h, 0.07 ± 0.01 pmol/cell/h (n = 17). Note We have subsequently assessed the viability of sheep zygotes cultured to the blastocyst stage in groups of 4 in SXOF medium with amino acids (the medium being renewed every 48 h). The pregnancy rate achieved for such cultured embryos was 62% (n = 26 embryos; 13 recipients). The pregnancy rate for in vivo developed controls transferred on the same day was 78% (n = 16 embryos; 8 recipients). EXAMPLE 6

Lamb birth weight following transfer is affected by culture system used for pre-elonαation development of embryos

Ovine embryos cultured in Synthetic Oviduct Fluid (SOF) supplemented with human serum (SOF+HS) can produce lambs with high mean birth weights. We have found high levels of development in vitro when serum was replaced with amino acids and albumin (SOFaaBSA). Here we compare both in vitro development and post-transfer viability of sheep embryos derived from oocytes enucleated by centrifugation and cultured for 5 days in either SOF+HS or SOFaaBSA. Synchronised recipients (N=63) received 2 embryos ipsilateral to a functional corpus luteum. A further group (N=16) were naturally mated to serve as flock fertility and lambing controls. All were scanned by ultrasonography at 55 d following transfer or mating, then allowed to lamb. Overall, development of cleaved embryos to a transferable quality (ie. good to excellent compact morulae or blastocysts) was not affected by medium (SOFaaBSA = 53+5% vs. SOF+HS = 59+5%). Embryos incubated in SOF+HS appeared morphologically different from those in SOFaaBSA, having abundant lipid inclusions present in the cytoplasm. Pregnancy rate (PR, 65%) or embryo survival (ES, 48%) of recipients at scanning did not differ between media treatments. The recipient PR was significantly lower than the control flock (94%, P<0.05) but ES (63%) was similar. Weight of lambs from SOF+HS (4.2+0.2kg) was significantly heavier than controls (3.4+0.2, PO.01 ) or SOFaaBSA (3.5+0.2, P<0.05). Reasons for this birth weight difference are unclear, but our data demonstrates that an embryo culture system which produced embryos that lack abundant lipid inclusions produces lambs with birth weights similar to controls. Altered metabolic or growth factor mediated responses may be a consequence of exposure to serum.

TABLE 7 Electrofusion of in vitro produced bovine embryonic cells Electrofusion of cells used in nuclear transfer involves the combination of an alternating current for cell alignment and a direct current pulse to induce membrane fusion. This is followed by further membrane contact with the formation of an unstable flat membrane diaphragm which deteriorates to allow cell mixing. The characteristics of cell membranes are obtained by electrofusion of identical cells. Optimal electrofusion results are used to produce isofusion contours for each cell type. The intersection of isofusion contours provides the parameters for electrofusion of different cell types. This study investigated the electrofusion of in vitro matured (IVM) oocytes, oocytes enucleated by centrifugation and blastomeres from day 3 to 6 bovine embryos produced after in vitro fertilisation (IVF).

Germinal vesicle stage oocytes were aspirated from ovaries collected at an abattoir, then matured in TCM 199 with FCS and hormones for 24 hours. Oocytes which extruded the first polar body were selected. Entire oocytes were then electrofused together after the zona was removed. Enucleated oocytes were prepared with a 1 or 2 step centrifugation process and cytoplasts greater than 50 μm were considered enucleated if an extrusion cone was not observed. Embryos were produced after IVF with frozen/thawed semen then cultured until required in SOF with amino acids and BSA. Prior to electrofusion oocytes and blastomeres were equilibrated for 10 minutes with 4 washes in fusion medium (0.25 M sucrose, pH 7.4). Alignment of two identical cell types was achieved with an AC pulse of 8 volts amplitude for 5 to 10 seconds. Electrofusion was achieved with a single DC pulse of 10, 50 or 100 μs with amplitudes between 0.2 and 2.0 kV/cm in an electrofusion chamber which consisted of parallel wires with a diameter of 0.5 mm and a separation of 0.5 mm.

A total of 2100 electrofusion attempts involved changes in cell type, pulse duration and amplitude. For all cell types a pulse duration of 10 μs resulted in significantly greater electrofusion (52%, p < 0.05) than either a 50 or 100 μs pulse (41 %, 36%). Electrofusion was significantly greater with a DC pulse above an amplitude of 1.0 kV/cm (55%, p < 0.05) than below 1.0 kV/cm (33%) when a 50 μs pulse was used.

Optimal electrofusion of identical cells was achieved with a 10 μs pulse and amplitudes greater than 1.0 kV/cm. When the isofusion contours for each cell type are compared (figure 19) the parameters for electrofusion of different cell types can be determined. The isofusion contours intersect at various pulse durations and amplitudes. This shows that selection of optimal parameters for electrofusion of different cell types depends on the isofusion contour for the cell type. This study has provided some understanding of the electrofusion behaviour of bovine embryonic cell types in electric fields. It also allows the prediction of parameters for electrofusion of any combination of bovine embryonic cell type used in nuclear transfer. Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims

THE CLAIMS

1. A process for enucleating oocytes which process includes: providing a source of oocytes; exposing the oocytes to a density gradient to form an oocyte gradient mix; subjecting the oocyte gradient mix to centrifugation to separate an oocyte fraction including enucleated oocytes; and identifying and isolating the enucleated oocytes.

2. A process according to claim 1 wherein prior to exposing the oocytes to the density gradient the oocytes are treated to remove zona.

3. A process according to claim 2 wherein the zona are removed by exposing the oocytes to a proteolytic enzyme or acidified medium.

4. A process according to claim 3 wherein the zona are removed by exposing the oocytes to pronase.

5. A process according to claim 2 further including a first centrifugation step prior to removal of the zona.

6. A process according to claim 5 wherein the first centrifugation step is at approximately 10,000 to 200,000g for approximately 1 to 5 minutes.

7. A process according to claim 1 wherein the density gradient is a step gradient.

8. A process according to claim 7 wherein the density gradient includes from approximately 7 to 50% Percoll.

9. A process according to claim 1 wherein the oocyte gradient mix is centrifuged at approximately 1 ,000 to 10,000g for approximately 1 to 10 seconds.

10. A process according to claim 9 wherein the oocyte gradient mix is centrifuged at approximately 5000g for approximately 4 to 8 seconds.

11. A process according to claim 1 further including a preliminary step of selecting oocytes at approximately 20 to 30 hours post maturation when the oocytes have extruded a first polar body.

12. A process according to claim 11 wherein the oocytes are selected from oocytes that have been subjected to in-vitro maturation.

13. A process according to claim 12 wherein the in-vitro maturation includes exposing the oocytes to a cell culture medium including:

(a) serum and/or

(b) growth factors and/or (c) gonadotrophic hormones and/or

(d) ovarian hormones.

14. A process according to claim 11 wherein the oocytes are selected from an animal in which ovulation has been synchronised.

15. A process according to claim 11 wherein the oocytes are treated with an agent to increase malleability of the oocyte membrane.

16. A process according to claim 15 wherein the agent is Cytochalasin B.

17. A process according to claim 15 wherein the agent is included in the density gradient.

18. An enucleated oocyte prepared by the process of claim 1.

19. An enucleated oocyte having a reduced lipid content compared to enucleated oocytes prepared by conventional methods.

20. An enucleated oocyte capable of electrofusion at amplitudes of 2 kV/cm or less.

21. A method for preparing a nuclear transplantation embryo which method includes providing an enucleated oocyte according to claim 18; 19 or 20; and a foreign gene sequence; and introducing the foreign gene sequence into the enucleated oocyte to produce a reconstituted, transplanted oocyte; and culturing the transplanted oocyte to produce a nuclear transplantation embryo.

22. A method according to claim 21 wherein the foreign gene sequence is introduced into the enucleated oocyte by electrofusion.

23. A method according to claim 22 wherein the electrofusion is performed at an amplitude of 2 kV/cm or less.

24. A method according to claim 21 wherein the transplanted oocyte is cultured in a culture medium including synthetic oocyte fluid, albumin and amino acids.

25. A nuclear transplantation embryo produced by the method of claim 21.

26. A nuclear transplantation embryo having a reduced lipid content compared to nuclear transplantation embryos prepared by conventional methods.

27. A method according to claim 21 wherein the foreign gene sequence is from a nuclear transplantation embryo according to claim 25 or 26.

28. A method for preparing multiple genetically similar animals which method includes providing nuclear transplantation embryos according to claim 25 or 26; and recipient females; transferring the nuclear transplantation embryos to the recipient females; and allowing the nuclear transplantation embryos to develop into live young.

29. Animals produced by the method of claim 27.

30. A method for producing normal birthweight offspring which method includes providing an embryo derived from oocytes enucleated by centrifugation, and an embryo culture medium including synthetic oviduct fluid, albumin and amino acids; culturing the embryo in the culture medium; transferring the embryo to a recipient female; and allowing the embryo to develop to term.

31. A method for increasing the viability of an embryo following cryopreservation which method includes providing an embryo derived from oocytes enucleated by centrifugation, and an embryo culture medium including synthetic oviduct fluid, albumin and amino acids; culturing the embryo in the culture medium; and subjecting the embryo to cryopreservation.

32. A method according to claim 30 or 31 wherein the albumin is bovine serum albumin.

33. A method according to claim 30 or 31 wherein the albumin is at a concentration of approximately 20 to 40 mg/ml culture medium.

34. A method according to claim 33 wherein the albumin is at a concentration of approximately 32 mg/ml culture medium.

35. A method according to claim 30 or 31 wherein the amino acids are at approximately the concentration present in Eagle's Minimal Essential Medium, with the exception of glutamine which is at a concentration of approximately 1 mM.

36. Use of an enucleated oocyte according to claim 18, 19 or 20 in cryopreservation.

37. Use of a nuclear transplantation embryo according to claim 25 or 26 in cryopreservation.

38. Use of a nuclear transplantation embryo according to claim 25 or 26 as donor cells or nuclei for further cycles of nuclear transplantation.

39. Use of a nuclear transplantation embryo according to claim 25 or 26 for production of embryonic stem cells or primordial germ cells.