Guide to Electroporation and Electrofusion || Production of Genetically Identical Embryos by Electrofusion

23 Production of Genetically Identical

Embryos by Electrofusion

L a w r e n c e Char les S m i t h

Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, Saint Hyacinthe, Québec J2S 7C6, Canada

I. Introduction

II. The Amphibian Cloning Model

A. Methodology

Β. Development Potential

III. The Mammalian Cloning Model

A. Methodology

B. Development Potential

IV. Nuclear-Cytoplasmic Interactions

A. Cell Cycle Stage Effects

B. Cytoplasmic Effects

V. Applications and Conclusions

References

I. Introduction

Recent improvements in micromanipulation techniques have opened new possibil-

ities for experimental research in mammalian embryology. The combined use of the

techniques of nuclear transplantation and cell electrofusion have enabled further

investigations into the genetic and epigenetic mechanisms by which early embryonic

development is controlled. These studies have clarified our understanding of deter-

mination and differentiation in mammalian embryonic cells during the preimplan-

tation stages and also of the abilities of oocyte cytoplasm to reprogram these de-

velopmental events. Live offspring have been derived from nuclear transplanted

Guide to Electroporation and Electrofusion

Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. 371

372 Lawrence Charles Smith

embryos in laboratory and domestic species with the purpose of producing genetically

identical animals ("clones").

This review aims to provide readers with some background knowledge on factors

influencing the development of nuclear transplanted embryos, hoping that this will

stimulate further research on nucleocytoplasmic interactions in early embryos. The

first section is devoted to the cloning model used for amphibia eggs, since this has

been studied in more detail and for a longer t ime than the mammalian model.

Although nuclear transplantation techniques in mammals have been around for at

least one decade, cloning procedures have only become viable in the last 5 years

when they were first successfully applied to farm animals (Willadsen, 1986). The

mammalian section begins by describing the steps leading to the current method-

ology and then outlines the effects the donor nucleus and the recipient cytoplasm

have on the ability of nuclear transplanted embryos to develop normally. The section

on the nuclear-cytoplasmic interactions discusses more fully the events taking place

at fusion, such as the effects of cell cycle stage, cytoskeleton interactions, and others.

The final section concludes by detailing some practical uses for nuclear transplan-

tation and the production of genetically identical mammals .

II. The Amphibian Cloning Model

A. Methodo logy

Current procedures for cloning mammalian embryos originated from nuclear trans-

plantation experiments in amphibians performed initially by Briggs and King (1952).

The transplantation operation in amphibia is carried out in two main steps. First,

the recipient frog eggs are activated by pricking with a glass needle; this causes

rotation and brings the animal pole to lie uppermost . The chromosomes are visualized

as a "black dot" ( i .e . , p igment granules clustered about the second metaphase meiotic

spindle) that can be extirpated surgically by lifting the spindle away from the surface

of the egg with a glass needle (Briggs and King , 1953). Effective enucleation can

also be attained by ablating the chromosomes with either ultraviolet (Gurdon, I960)

or microbeam ruby laser irradiation (Ellinger et al., 1975). The second step involves

the isolation of the donor cells and the nuclear transplantation itself. Donor cells

are isolated, either mechanically with a fine-diameter pipette or chemically with

enzymatic treatment. A given cell is then drawn into the t ip of a sharp micropipette,

the inner diameter of which is somewhat smaller than that of the cell. This causes

rupture of the plasma membrane, but the cytoplasm contents are not dispersed and

remain surrounding the nucleus. The pipet te is then inserted through the membrane

of the recipient enucleated egg and the contents of the broken cell are ejected,

liberating the nucleus into the egg cytoplasm.

Chapter 23 Production of Genetically Identical Embryos 373

B . D e v e l o p m e n t Po t en t i a l

Amphibian nuclear transplantation experiments using several embryonic cell types

have revealed that , as donor nuclei are tested from progressively older stages of

embryogenesis, there is a decrease in the number of individuals that develop normally

(reviews by King , 1966; DiBerardino and Hoffner, 1970; Gurdon, 1974; Briggs,

1977; McKinell , 1978). Al though already containing approximately ten thousand

cells with some degree of morphological differentiation, most , if not all, blastula

nuclei are thought to be able to support normal development when fused to enu-

cleated eggs. However, only a few early gastrula-stage nuclei have been shown to

be totipotent , and a very low percentage of nuclei taken at any later embryonic

stage are fully able to support development (Briggs and King, I960) . Studies carried

out using nuclei derived from regions of larvae already commited to a specific

developmental pathway have shown that only around 0 . 2 % develop to adults and

another 4 % arrest at the larva stage (pluripotent nuclei). Together, these results

suggest that the developmental capacity of nuclei becomes progressively restricted

during the process of cell type determination and differentiation.

Several investigators have tested the developmental capacity of amphibian nuclei

from adult cells. The most advanced stage of development reported from using

nuclei of adult cells has been a feeding larva obtained from spermatogonia cells of

Rana pipens (DiBerardino and Hoffner, 1971). Among adult somatic nuclei tested

in Xenopus, there are 33 cases of early larvae obtained in support of nuclear pluri-

potency (Laskey and Gurdon, 1970; Gurdon et al., 1975; McAvoy et al., 1975;

Wabl et al., 1975; Brun, 1978). One of these larvae was reported to be normal and

originated from a crest cell nucleus of the intestine, but this apparent normal larva

died during an early larval stage (McAvoy et al., 1975). The remaining nuclear

transplant larvae were morphologically abnormal and the percent success ranged

from 0.3 to 6 . 0 % . N o adult frog has yet developed from transplanted adult nucleus.

In order to determine the nature of the developmental restrictions displayed by

nuclei from advanced cell types, an extensive series of studies was performed on

abnormal embryos and larvae of Rana nuclear transplants (reviewed by DiBerardino,

1979)· Most abnormal nuclear transplants examined exhibited abnormalities in

chromosome number and/or structure that , in most cases, arose during the first cell

cycle of the egg (DiBerardino and Hoffner, 1970). These abnormalities are now

known to be the cause of developmental arrest in this species. The most severe

chromosomal alterations cause developmental arrest at the blastula stage, whereas

relatively minor karyotypic alterations permit development to early larval stages.

Evidence has been presented that most of these numerical and structural changes in

the chromosomes (1) involve chromosomal loss, (2) are a reflection of chromosomal

differentiation acquired progressively though embryogenesis, and (3) are not a result,

for the most part , of technical damage (DiBerardino, 1979, 1980; DiBerardino and


Hoffner, 1980). It appears that the cytoplasmic cell cycle of the amphibian egg,

which is much faster than the nuclear cycle of advanced cell types, induces premature

changes in transplanted nuclei resulting in incomplete D N A replication, chromo-

some breaks and arrangements, and hypoaneuploidy in the most severely affected

embryos.

III. The Mammalian Cloning Model

A. Methodo logy

As the volume of individual mammalian eggs is close to one thousand times smaller

than that of frog eggs, it is not surprising that more refined methods of microsurgery

were required before the development of techniques for nuclear transplantation could

be usefully applied to mammals . The successful microinjection of embryonic nuclei

into the cytoplasm of one-cell rabbit (Bromhall, 1975) and mouse embryos (Mod-

linski, 1978) suggested that nuclei from later embryonic stages were able to par-

ticipate with the egg's genome in supporting preimplantation development. The

first report of mice born from nuclear transplantation came from the work of Illmensee

and Hoppe (1981), showing that nuclei derived from the inner cell mass rather than

trophectoderm of mouse blastocysts were able to support development to the m o r u l a -

blastocyst stage in 3 4 % of the transferred embryos, and that 19% of these would

develop to term. These reports brought many scientists to speculate that this tech-

nique would provide a means of making an infinite number of genetically identical

copies from a single embryo. However, it was later reported that a t tempts of other

laboratories using this technique were unsuccessful (Marx, 1983; McGrath and

Solter, 1984a; McLaren, 1984). Suggestions were made that Illmensee and Hoppe

could not have properly enucleated the recipient zygotes, which would have allowed

for their development to full term (McGrath and Solter, 1984a).

In support of these suggestions were the results from a novel method developed

for nuclear transplantation by McGrath and Solter (1983a). Their technique avoids

the necessity of penetrating the plasma membrane of either the donor or the recipient

egg. This is possible by placing the eggs in cytoskeletal inhibitors for a short period

before microsurgery, followed by zona pellucida penetration with a sharp micro-

pipette and sucking out the two pronuclei surrounded by a piece of plasma mem-

brane—a karyoplast. The donor karyoplast, which can be obtained from any em-

bryonic or somatic cell, is injected under the zona pellucida of the enucleated

recipient egg (Fig. 1). The following step of this noninvasive method for nuclear

transplantation relies on an effective means for fusing the nuclear (karyoplast) and

cytoplasmic (cytoplast) portions enclosed in the zona pellucida. Contrary to the poor

levels of success obtained using Illmensee and Hoppe's invasive technique, McGrath

and Solter's method provided virtually 100% of success, and the yield of live mice

is very high when fertilized eggs are used as nuclear donors.


Figure 1 Technical procedure use for the enucleation of pronuclear zygotes followed by the collection and transplantation of karyoplasts. (a) Positioning of pronuclear zygote for enu-cleation; (b) removal of male and female pronuclei in a karyoplast; (c) nuclear donor four-cell stage embryo positioned for microsurgery; (d) removal of four-cell stage karyoplast; (e) positioning of donor karyoplast into zona pellucida; (f) recipient cytoplast and donor karyoplast ready for fusion.


Several methods are available for fusion, some of which are either unreliable and

toxic, such as polyethylene glycol (PEG) and lysolecithin or, as for inactivated Sendai

virus solutions, laborious to obtain and dangerous to raise due to its highly infectious

nature. The latter also has the inconvenience that it is impossible to ascertain exactly

the moment of fusion, since it occurs between 15 to 60 min after manipulation

according to the amount and activity of the solution injected into the perivitelline

space. However, possibly due to its high levels of success and to the ease with which

manipulations are performed, Sendai-mediated fusion has been the most widely used

approach in mouse nuclear transplantations. Willadsen (1986) reported the use of

electric pulses to fuse the nuclear donor cell to the cytoplasm of the enucleated egg.

Although Sendai virus-mediated fusion was successfully achieved in sheep, electro-

fusion techniques are more effective and less variable when species other than mice

are used.

Electrofusion has been successfully used to fuse blastomeres and for nuclear

transplantation experiments in mammalian embryos derived from species as diverse

as mice (Kubiak and Tarkowski, 1985; Tsunoda et al, 1987a; Kono and Tsunoda,

1988; Barra and Renard, 1988; Clement etal., 1988), rabbit (Ozil and Modlinski,

1986; Stice and Robl, 1988; Clement etal, 1988), sheep (Willadsen, 1986; Smith

and W i l m u t , 1989), cattle (Prather et al, 1987; Bondioli et al, 1990), and pigs

(Clement et al, 1988; Prather et al, 1989). Embryonic cells (karyoplasts and

cytoplasts) to be fused are placed in a chamber with electrofusion medium and

positioned between two electrodes connected to a pulse generator (Fig. 2). Successful

fusion can be attained using a large range of parameters for the direct current (DC)

fusing pulse. Normally, a field intensity of around 1.0 kV/cm with durations between

50 and 100 \ks is used. Two or three consecutive pulses separated by a fraction of

a second may, in some instances, improve the levels of success. Fusion seems to be

caused by the reversible electrical breakdown in the zone of membrane contact

between the cytoplasm and nuclear donor cells. This breakdown is followed im-

mediately by their repair into a single membrane, leading initially to the formation

of small pores between the two cells, which continue growing and joining together

until fusion is complete (Zimmerman and Vienken, 1982). Alternating current (AC)

pulses may be used to align the cells so as to position their membranes perpendicular

to the electrical field where conditions for fusion are most suitable. The preceding

AC pulse is particularly important when fusing enucleated oocytes to cells with

reduced diameters since the polarization caused by the AC field will aid in bringing

their membranes into contact for the DC fusing pulse (Smith and W i l m u t , 1989).

Willadsen (1986) reported the development to term of sheep embryos derived

from the transplantation of embryonic blastomere nuclei to the cytoplasm of sec-

ondary oocytes. The relevance of his findings was not exclusively related to the

ability of a single eight-cell blastomere to support development, since this had

already been indicated in earlier chimeric studies (Willadsen and Fehilly, 1983),

but also that , as for amphibian nuclear transfers, the resulting fused embryo would


Figure 2 Schematic diagram of a chamber used for the electrofusion of mammalian embryos. Two ΙΟΟ-μπι-diameter platinum electrodes are stuck to the bottom of a glass petri dish separated at 200 μπι from each other.

develop and differentiate as if commencing from the t ime of fertilization, indicating

an ability of the oocyte's cytoplasm for reprogramming the developmental "clock"

of the donor nucleus. Al though the technique used by Willadsen is similar to that

used with frogs, in that both techniques utilize the secondary oocytes as the recipient

cytoplasm, it also uses the noninvasive technique for transplanting the nucleus into

the cytoplasm.


B. D e v e l o p m e n t Po ten t i a l

As for amphibian, many factors are involved in the ability of mammalian nuclear

transplanted embryos to develop normally. Because these techniques are still fairly

recent in mammals , our understanding of each factor remains l imited. The following

sections will try to point out some possible reasons why many embryos fail to develop

after nuclear transplantation.

1. Nuclear Donor Cell

All cells in an embryo have a nucleus containing a comparable amount of D N A

{In), which is derived by continuous replication and segregation at mitosis from a

single fusion product of the D N A carried by male (n) and female (n) gametes at

fertilization. Assuming that this concept of nuclear equivalence is correct, and there

are many reasons to believe so, one can presume that because later embryonic stages

possess more cells with genetically identical nuclei, these are more suitable for

cloning. There are some factors that l imit the extent to which embryos should

develop when allocated for use as nuclear donors in the cloning procedure.

The first factor relates to the loss of developmental potential at the expense of

cellular determination or differentiation. As described earlier in amphibia, nuclei

derived from cells that have commit ted themselves to a particular pathway seem to

lose the ability to be readily reprogrammed and are unable to regain the totipotent

status of their ancestral fertilization genome. An early report in mice showed that ,

although 9 5 % of enucleated zygotes fused with zygote pronuclei will develop to

blastocyst, only 13% will develop to blastocyst when 2-cell nuclei are fused and

none when four-, eight-, 16-cell, and inner cell mass (ICM) nuclei are fused to

enucleated zygotes, indicating a rapid loss in potential already by the 2-cell stage

(McGrath and Solter, 1984a). These findings have been confirmed by other labo-

ratories (Robl et al., 1986; Surani et al., 1987; Tsunoda et al., 1987b; Smith et al.,

1988), and suggestions have been made that blastomere nuclei lose their potential

at the t ime of maternal-zygotic transition (MZT) due either to genome differentiation

or to toxicity of stage-specific factors (Solter et al., 1986; Solter, 1987).

More recent findings in several mammalian species have shown that nuclei derived

from stages beyond the M Z T are still able to support development (Table 1).

Comparisons between results from several mammals indicate that embryos derived

from species that experience M Z T within a single or two cells after oocyte activation

tend to perform poorly after nuclear transplantation. In mice, where M Z T occurs

at the early two-cell stage (Flach et al., 1982), it has been reported that a small

proportion of nuclei from the eight-cell (McGrath and Solter, 1986) and from the

inner cell mass (Tsunoda et al., 1988, 1990) will support development to the

blastocyst stage when fused to enucleated secondary oocytes. Again in mice, by

using karyoplasts derived from embryos at different stages within the two-cell cycle,


Table 1

Pre- and Postimplantation Development Potential of Nuclear Transplanted Embryos Derived from the Fusion of Embryonic Cells to the Cytoplasm of "Enucleated" Secondary

Oocytes in Several Mammalian Species*

Morula Embryonic cell Number or Number Established Full-term

Species type fused blastocyst transferred pregnancy development Reference

Mouse 2-Cell 86 35% n.a. n.a. n.a. (1) 8-Cell 36 None n.a. n.a. n.a. (1) ICM 80 3% n.a. n.a. n.a. (1) ICM 83 4% n.a. n.a. n.a. (2) PGC 135 10% 59 10% None (1)

Pig 2-Cell 11 9% 33 n.a. None (3) 4-Cell 83 8% 34 n.a. 3% (3) 8-Cell 57 19% 21 n.a. None (3)

Rabbit 8-Cell 70 68% 85 2% 1% (4) 16-Cell n.a. n.a. 110 n.a. 21% (5) 32-Cell 67 76% n.a. n.a. n.a. (5) 3 2-Cell 67 55% 67 n.a. n.a. (6) ICM 52 37% n.a. n.a. n.a. (6) TE 27 None n.a. n.a. n.a. (6)

Sheep 8-Cell 76% 42% 4 n.a. 75% (7) 16-Cell 29 48% 6 50% n.a. (7) 16-Cell 49 35% 14 21% 14% (8) ICM 16 56% 8 13% 13% (8)

Cattle <8-Cell 111 12% 12 n.a. None (9) < 16-Cell 50 16% 7 n.a. 29% (9) <32-Cell 24 8% n.a. n.a. n.a. (9) Day 5 morula 604 32% n.a. n.a. n.a. (10) Day 5.5 morula 139 40% n.a. n.a. n.a. (10) Day 6 morula 54 53% n.a. n.a. n.a. (10) Morula n.a. n.a. 463 23% 22% (10)

'References: (1) Tsunoda et al., 1990; (2) Tsunoda et al., 1988; (3) Prather et al., 1989; (4) Tsunoda et al., 1989; (5) Collas and Robl, 1990; (6) Collas and Robl, 1991; (7) Willadsen, 1986; (8) Smith and Wilmut, 1989; (9) Prather etal., 1987; (10) Bondioli et al., 1990; n.a. = not available; ICM = inner cell mass; PGC = primordial germ cell; TE = trophectoderm cells.

nuclei derived from embryos just before the M Z T are slightly less able to support

blastocyst development than those derived from embryos after the M Z T (Smith et

al., 1988). In pigs, where M Z T occurs at the four-cell stage (Norberg, 1973), no

significant difference could be detected between using nuclei from two- (9%), four-

(8%) or eight-cell (19%) blastomeres in nuclear transplantations to enucleated sec-

ondary oocytes (Prather et al., 1989).


Species that experience M Z T at or after the eight-cell stage (three or more cell

cycles after oocyte activation) tend to perform better in nuclear transplantation trials.

In sheep, where M Z T occurs at the eight-cell stage (Crosby et al., 1988), it has

been reported that eight-cell (42%) and 16-cell (48%) nuclei are able to support

development to blastocyst at similar proportions (Willadsen, 1986) and also that

nuclei from 16-cell (35%) and the ICM (56%) stages of early blastocysts are similarly

competent in supporting development both to blastocyst and to term (Smith and

Wi lmu t , 1989). In cattle, where M Z T occurs around the eight-cell stage (Camous

et al., 1986), reports have indicated similar development potentials are obtained

from nuclei derived from embryos collected at day 5 (32%), day 5.5 (40%), and

day 6 (53%) (Bondioli et al., 1990). Therefore, it seems that , at least up to the

embryonic stages tested in farm animals, there is no detectable loss of developmental

potential of embryonic nuclei dur ing the preimplantat ion stages utilized to this

data. However, reports using rabbit nuclear transplanted embryos have indicated a

slight decrease in potential at later embryonic stages. Al though M Z T occurs at the

eight-cell stage in rabbits (Manes, 1977), the potential of nuclei derived from eight-

cell (68%), 32-cell (55%), or from the inner cell mass cells (37%) seems to decrease

slightly during development (Tsunoda et al., 1989; Collas and Robl, 1990, 1991).

Another important finding made in this species is the striking fall in potential (0%)

when using commit ted blastocyst trophectoderm cells in nuclear transplantations

(Collas and Robl, 1991).

Technical aspects should also be considered when deciding on the best embryonic

stage from which to derive viable cells for nuclear transplantation. At compaction,

cells from the outer layer of the embryo develop t ight junctions making disaggre-

gation into single cells increasingly more difficult. Al though calcium-free medium

and/or trypsinization can be used to aid in separating cells, such treatments may be

detrimental to the viability of their nuclei even after short periods of exposure. The

compact outer layers of late morula and blastocysts can be destroyed through an

immunosurgical technique (Solter and Knowles, 1975), permit t ing an easier dis-

aggregation of their inner cells as reported for the use of cells from the inner cell

mass of blastocysts in sheep nuclear transplantations (Smith and W i l m u t , 1989).

Moreover, cell diameter decreases considerably during the cleavage stages, and their

reduced size causes problems during the fusion procedure.

2. Recipient Cytoplasm

Recipient cytoplasm for mammalian nuclear transplantation was derived initially

from enucleated zygotes at early stages after fertilization (McGrath and Solter,

1983a). As described in the previous section, nuclear transplantation experiments

in mice using cytoplasm from pronuclear zygotes provided very limited support

from nuclei beyond the two-cell stage in mice (McGrath and Solter, 1984a). Nuclear

transplantations to zygotic cytoplasm have also been at tempted in other mammals


with very poor results. In rats, al though 2 2 % of the embryos derived from pronuclear

transfers developed to term, no pregnancies could be attained when using nuclei

derived from two-, four-, or eight-cell blastomeres (Kono et al, 1988). In cattle,

pronuclear zygotes were centrifuged to reveal the pronuclei for removal and later

fused to karyoplasts from early stage blastomeres (Robl et al., 1987). Bovine results

were similarly discouraging, with only 17% of the pronuclear transplantations de-

veloping to the morula-blastocyst stage after 5 days culture in a sheep's oviduct and

not a single normal development after transplantations of two-, four-, and eight-

cell blastomere nuclei.

When using cytoplasm from two-cell enucleated blastomeres, Robl et al. (1986)

were able to obtain morulae and blastocysts after fusion with karyoplasts derived

from eight-cell mouse blastomeres. However, they also reported that these apparently

normal blastocysts were unable to support development beyond mid-gestation after

transfer to synchronized females. W i t h a slight modification of the technique for

using two-cell recipient cytoplasm, Tsunoda et al. (1987) were able to produce a

small number of offspring from nuclei derived from eight-cell embryos but concluded

that this technique would not prove useful in cloning mammalian embryos. Gen-

erally, recipient cytoplasm from secondary oocytes utilized in mouse nuclear trans-

plantations has provided very little improvement when compared to the postfertil-

ization cytoplasm sources (Fig. 3). However, some development to blastocyst was

achieved with eight-cell (5%) and ICM (3%) stages after fusion to enucleated oocytes

(McGrath and Solter, 1986; Tsunoda et al., 1990). Moreover, primordial germ cell

(PGC) nuclei have also been reported to support development to blastocyst (10%)

and to mid-gestation (10%) after fusion to secondary oocyte cytoplasm (Tsunoda et

al, 1990).

Secondary oocytes have been the only cytoplasm recipient stage to support full-

term development after nuclear transplantation in mammals . Liveborn have been

produced in several species including sheep (Willadsen, 1986; Smith and W i l m u t ,

1989), cattle (Prather etal, 1987; Marx, 1988; Bondioli etal, 1990), rabbit (Stice

and Robl, 1988), and pigs (Prather et al, 1989). The optimal stage for util izing

secondary oocytes for nuclear transplantations ( t iming after ovulation or maturation)

has not yet been determined, but there seems to be an increased potential after a

short period of aging (Ware et al, 1989). However, this beneficial effect is likely

to be related to improved levels of success in causing activation by artificial stimuli

after aging. The beneficial effect of aging the oocyte has also been observed in rabbit ,

where further improvement can be obtained by increasing the number of pulses at

or after electrofusion (Collas and Robl , 1990).

In general, secondary oocytes have been obtained directly from the oviduct either

during surgical intervention or at slaughter. W i t h the current efficiency of the

cloning procedure, the collection of in vivo matured oocytes comprises a substantial

proportion of the total cost for obtaining a nuclear transplanted offspring. Al though

Prather et al (1987) reported l imited development using in vitro matured oocytes,


Figure 3 Microsurgical methods used in the preparation of sheep secondary oocytes and embryos for nuclear transplantation by electrofusion. (a) Secondary oocyte positioned for enucleation with the first polar body (1PB) facing the beveled microsurgical pipette, (b) Removal of approximately one-quarter of the cytoplasm surrounding the 1PB in a membrane-bound cell fragment. A single cell derived from either (c) 16- or 32-cell embryos or (d) inner cell mass of early blastocysts has been positioned within the zona pellucida of an enucleated secondary oocyte for the electrofusion procedure.

recent improvements in the methods for in vitro maturat ion (Gordon and Lu, 1990)

are likely to make this the most cost-effective route for obtaining recipient oocytes

for nuclear transplantation. This slaughterhouse source of cytoplasm has been suc-

cessfully used for obtaining live offspring in cattle and sheep nuclear transplantation.

One limitation for the use of secondary oocytes as recipient cytoplasm concerns

the enucleation procedure. W i t h the exception of the rabbit (Stice and Robl, 1988),

metaphase chromosomes cannot be readily visualized in secondary oocytes from other

farm species due to the presence of large lipid vesicles in the cytoplasm. This leads

to the need for using the position of the first polar body as an indicator of the

position of the chromosomal plate followed by the aspiration of cytoplasm from the

adjacent area (Fig. 4). However, possibly due to displacement or degeneration of


1 0 0 T

1 -ce l l 2 -ce l l 4 -ce l l 8 -ce l l I.C.M.

Nuclear donor s t age

Figure 4 Comparison between the development potential of different recipient cytoplasm

stages with regard to their ability in supporting the development to the morula or blastocyst

stage after fusion to karyoplasts derived from preimplantation stage embryos. MZT = timing

of the maternal-zygotic transition. O, 1-cell cytoplasm; · , 2-cell cytoplasm; Δ, oocyte

cytoplasm.

the first polar body, an improper removal of chromosomes still occurs in a substantial

proportion of manipulated secondary oocytes. Unsuccessful enucleation has been

reported to occur in approximately one-third of the a t tempts , which, possibly due

to aneuploidy, lead to abnormal cleavage and developmental arrest during the early

preimplantation stage (Willadsen, 1986; Smith and W i l m u t , 1989). Al though the

success of enucleation can be ascertained using D N A vital stains, further research

is required to assess the effect this may have on development due to the possible

damaging of mitochondrial D N A and other cytoplasmic components.

IV. IMuclear-Cytoplasmic Interactions

When fusion is complete, donor nucleus and recipient cytoplasm will initiate in-

teractions, which, when compatible, will lead to the normal development of a nuclear

transplanted embryo. This section discusses the cellular and molecular interactions

that take place at and after fusion.


A. Cell Cycle Stage Effects

Experiments with mouse pronuclear zygotes have indicated that cell cycle stage

synchrony between nucleus and cytoplasm is beneficial for further development in

vitro (Smith et al., 1988). This observation has been further extended to transplan-

tations between two-cell embryos where asynchronous exchanges were highly del-

eterious to further development (unpublished observation). This effect may be ex-

plained either by the disruption of the cell cycle oscillatory mechanisms or by

incompatible nuclear-cytoplasmic interactions in controlling critical developmental

steps (Smith et al., 1990). Possibly resulting from incompatible nuclear-cytoplasmic

interactions, sheep embryos recovered from ligated oviducts 5 days after nuclear

transplantation either do not cleave at all (and may fragment after a period of aging)

or cleave a few times but cannot compact and blastulate as normal embryos (Smith

and W i l m u t , 1989). Because observations have been performed mostly after a long

period of in vivo culture, it is unclear exactly at what stage these embryos arrest

development. Better methods for culturing livestock embryos in vitro ( i .e . , coculture

with oviductal cells) coupled with detailed biochemical studies on their transcrip-

tional and translational activities will certainly enable a clearer understanding of

these nuclear-cytoplasmic incompatibilit ies.

B . Cytop lasmic Effects

Nucleo—cytoplasmic compatibili ty has been shown to improve when nuclear trans-

planted embryos are placed into a medium containing cytochalasin Β (CB) for a

short period immediately after fusion. In sheep, nuclei derived from both 16-cell

stages ( 3 5 % vs. 11%) and ICM cells (56% vs. 0%) of early blastocysts were sig-

nificantly better able to support development to morulae and blastocysts after nuclear

transplantation when placed in medium containing 7.5 p g CB for 1 hr after the

electric stimulus as compared with placing them into medium alone (Smith and

W i l m u t , 1989). A similar t reatment was also effective in improving development

to blastocyst (44% vs. 11%) in rabbit 32-cell nuclear transplantations (Collas and

Robl, 1990). Together, these results indicate that fused nuclei are affected by

cytoskeletal mechanisms operating during the activation of the recipient cytoplasm,

possibly leading to chromosomal anomalies by extrusion into pseudo first polar

bodies. However, an abstract report has shown that the effect of cytochalasin Β is

not apparent in cattle nuclear transplantation, which may indicate species variations

in nucleo-cytoplasmic interactions after fusion (Levanduski and Westhusin , 1990).

Czolowska et al. ( 1984) observed the swelling of thymocyte nuclei in activated

ovum cytoplasm to equal that of pronuclei when nuclear introduction coincided

with activation, but diminished with increasing t ime between activation and nuclear

introduction. Nuclear swelling has also been observed in mouse and rabbit nuclear


transplantations (Robl et al., 1986; Stice and Robl, 1988). Moreover, Szollosi etal.

(1988) showed that nuclear membrane breakdown and further reassembly of a new

nuclear envelop occurred only when fusions were performed immediately before or

up to 30 min after oocyte activation. Possibly, the nuclear membrane functions as

a barrier to a few specific factors in the oocyte cytoplasm required to reprogram the

genome's developmental pathway. Another explanation for the poor performance of

nuclear transplantations to enucleated zygotes as compared with secondary oocytes

is that developmentally important factors are sequestered within or around the

nuclear membrane and removed dur ing pronuclear enucleation.

Peri- and postimplantational studies on nuclear transplanted embryos are limited

in mammals (Table 1). It has been reported that al though apparently normal in

morphology, many of the morula—blastocyst stage embryos derived from nuclear

transplantation fail to produce pregnancy after transplantation to the uteri of syn-

chronized recipients (Prather et al., 1987; Smith and W i l m u t , 1989; Bondioli et

al., 1990). Al though many seem to die soon after implantat ion, causing an extended

estrous cycle, others may develop much further, leading to abortions at later stages

of gestation. It has also been noted that fetuses derived from nuclear transplanted

embryos are likely to be larger than usual and/or to extend slightly beyond the

normal gestation t ime (Smith and W i l m u t , 1989; Dr. K. Bondioli, personal com-

munication).

V. Applications and Conclusions

The technique of nuclear transplantation in mammals is the most rigorous test to

ascertain whether embryonic or somatic nuclei retain all of the genetic information

found in a zygote nucleus. However, other biological applications of this technique

have included (1) studies on the role of paternally and maternally derived genes in

development ("imprinting") (McGrath and Solter, 1984b; Surani et al., 1984), (2)

studies to test whether events that take place dur ing embryonic development are

inherited through the nucleus or cytoplasm (McGrath and Solter, 1983b; McGrath

and Solter, 1984c; Mann, 1986), and (3) the control of cleavage in early embryos

(Smith etal., 1990).

In practice, the technique of nuclear transplantation already provides a means

for cloning embryos in many mammalian species (Fig. 5). Therefore, it is likely

that this technique will soon become more widely available for producing genetically

identical animals either for research or for commercial application. In research, clones

could be used to eliminate the variation caused by the genotype allowing for better

assessment of nongenetic factors such as environment, disease, experimental treat-

ments , etc. For animal breeders, cloning will bring the advantage of speeding the

annual genetic gain of selection programs and enable a much shorter interval to

disseminate the gain to the commercial herds. Commercial organizations using these


CYTOPLASM DONOR NUCLEAR DONOR CLONES

in vivo culture

Figure 5 Schematic representation of the techniques used for cloning mammalian embryos. Pathways used for obtaining donor nuclei and recipient cytoplasm are presented, followed by the cyclic procedure for deriving cloned embryos. The cloned embryos can be either recycled, frozen to be used at a later stage, or transferred to recipients to provide cloned offspring.

techniques have reported the successful use of nuclear transplanted embryos as nuclear

donors for further transplantations (serial transplantations). Considering that at the

present level of success one can derive approximately five embryos from every initial

nuclear donor embryo and that the same process could be repeated for each of the

resulting nuclear transplanted embryos, after four generations of asexual reproduction

(or 4 weeks), a total of 625 genetically identical cloned embryos would be available

for transplantation into synchronized recipients (Wi lmut and Smith, 1989). It is

likely that as many clones would be required for any reason, but this example serves

to illustrate the potential of the nuclear transplantation procedure.


Recent reports have indicated progress in deriving embryonic stem (ES) cells

from farm animals, which may become a valuable source of genetically identical

nuclei for cloning (Evans et al., 1990). The ease of selecting for integration and

expression of transfected ES cells in vitro will also allow for a more efficient route

for producing transgenic livestock than the current methodologies of pronuclear

injection and chimeras produced from transfected ES cells. At this point , it is also

important to note some of the possible disadvantages in the cloning procedure. One

aspect concerns the decrease in the genetic variability by inbreeding. Large foun-

dation populations should be obtained when using cloning in conjunction with

M O E T (multiple ovulation and embryo transfer) selection schemes. It is also im-

portant to verify the degree to which cytoplasmic inheritance can influence animal

production, since clones will be exposed not only to different uterine and neonatal

environments but also to a different ooplasm. The maternal inheritances of mi to-

chondrial genes are examples of differences that may arise between nuclear trans-

plantation-derived clones. Other potential factors of variation to be considered are

the random inactivation of X chromosomes in females and the possibility of differ-

ential expression or mutat ion after nuclear transplantation in cloned embryos. These

final comments help to strengthen the notion that many aspects of the interactions

between nucleus and cytoplasm after transplantation remain unknown and that it

is only by both fundamental and applied research that further improvements may

be achieved with the cloning of mammalian species.

Acknowledgments

The author thanks Dr. A. K. Goff for helpful comments and NSERC, CAAB, and FCAR for their financial support during the preparation of this manuscript.

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Guide to Electroporation and Electrofusion || Production of Genetically Identical Embryos by Electrofusion