HUMAN FERTILISATION AND EMBRYOLOGY AUTHORITY
SCIENTIFIC AND CLINICAL ADVANCES GROUP
Scientific and Clinical Advances Group
21st April 2004
Committee:
Meeting Date
9
Agenda Item
SCAG (04/04) 04
Paper Number
New Technologies
Paper Title
Chris O’Toole
Author
Information
For Information or Decision?
Resource Implications
Recommendation to the Committee
The Committee is asked to review this
paper and advice the Executive on those
matters that require further investigation.
NEW TECHNOLOGIES
1.
Members are asked to review the paper on new technologies and
advice the executive on whether any of the techniques require further
consideration.
HUMAN FERTILISATION AND EMBRYOLOGY AUTHORITY
NEW TECHNOLOGIES USED TO CREATE ARTIFICAL GAMETES /
EMBRYOS
1.
1.1.
Purpose
The aim of this paper is to briefly explain the technologies used to
artificially create gametes and embryos. As gametes / embryos
generated from these techniques could be used in both assisted
conception and as a source to derive embryonic stem cells, a brief
introduction is given to explain what happens in normal fertilisation as
well as an introduction to what stem cells are and why researchers are
interested in these cells.
2.
2.1.
Introduction
Gametogenesis
Figure 1. Spermatogenesis
a)
Spermatogenesis
Mammalian sperm are produced in a process
called spermatogenesis (Fig.1). This process
goes on continuously in the epithelical lining of
very long, tightly coiled tubes called
seminiferous tubules which occur in the testes.
Immature germ cells, called spermatogonia,
are located along the outer edge of these tubes
next to the basal lamina, where they divide
continuously by mitosis1. Some of the daughter
cells stop proliferating and differentiate into
primary spermatocytes. These cells then divide
by meiosis2 to produce two secondary
spermatocytes each containing 22 duplicated
chromosomes and either a duplicated X
chromosome or a duplicated Y chromosome.
These secondary spermatocytes then undergo
a second meiotic division to produce four
spermatids each containing a haploid number
of chromosomes (i.e. 22 chromosomes + either
an X or Y chromosome). These four spermatids
then undergo morphological differentiation into
spermatozoa (sperm), which escape into the
1
Mitosis – the usual process by which a nucleus divides into two. Each chromosome
duplicates before beginning mitosis, and mitosis involves separation of the resulting duplicate
chromosomes so that one set of chromosomes goes into each daughter nucleus. As a result
the 2 daughter nuclei have an identical complement of chromosomes and hence genes.
2
Meiosis (reduction division) – Meiosis is a form of cell division in which a cell with 46
chromosomes (referred to as diploid) gives rise to 2 cells with 23 chromosomes (referred to
as haploid). Meiosis takes place only in the formation of the gametes – sperm and egg
(oocyte)
lumen of the seminiferous tubules. They subsequently pass into the
epididymis, a coiled tube overlying the testes, where they are stored until
ejaculation.
b)
Oogenesis
Unlike the continuous sperm production of the
male, the maturation and release of the female
germ cell, the ovum (egg or oocyte), is cyclic
and intermittent (Fig. 2). At birth, normal
human ovaries contain an estimated one
million eggs and no new ones appear after
birth. Thus in marked contrast to the male, the
newborn female already has all the germ cells
she will ever have. The eggs present at birth
are the result of numerous mitotic divisions of
the primitive egg (the oogonia) which occurred
during early foetal development. At some point
the oogonia cease dividing in the foetus (at this
stage the eggs are called primary occytes).
These primary oocytes all begin the first
division of meiosis but do not complete it,
accordingly all the eggs present at birth are
primary oocytes containing 46 chromosomes.
Figure 2. Oogeneis
During each menstrual cycle, just before ovulation,
one or occasionally more than one egg completes the first meiotic division.
However, in this division one of the two daughter cells, the secondary
oocytes, retains nearly all the cytoplasm and the other daughter cell (the
first polar body) is very small and is extruded from the cell. The second
meiotic division occurs after ovulation, indeed it only occurs if the egg is
penetrated by sperm.
2.2.
Normal Fertilisation
Figure 3. Normal Fertilisation
2.1.1. Upon penetration by the sperm, the egg completes its division and
again one daughter cell with practically no cytoplasm, the second polar
body, is extruded. Approximately 18 hours after the sperm has entered
the egg two spherical objects can be seen within the egg. These are
called the pronuclei, one contains the genetic material from the egg
and the other contains the genetic material from the sperm3. Soon after
this stage the pronuclei fuse, become visible and cell division
(cleavage) begins.
Figure 4. Fertilisation and Embryo Development (source Guy’s and St Thomas’ Hospital
web site)
Fertilisation of the egg by the sperm normally occurs in the fallopian
tube. The fertilised egg then begins to divide first into 2, then 4, then
8 cells (the preimplantation embryo), and continues its journey
towards the uterus. After about 5 days the embryo hatches out of the
outer coating (zona pellucida) and buries itself in the thick lining of
the uterus and begins to grow (the process of implantation).
2.3
In vitro Fertilisation
In Vitro Fertilisation (IVF) treatment involves placing sperm and eggs
together in a culture dish so that fertilisation occurs in the laboratory.
IVF treatment consists of the following stages:
•
Stimulation of the ovaries with hormones to produce eggs.
•
The collection of mature eggs from the ovary of the
female partner.
•
The preparation of motile sperm from the male
partner.
•
Putting together the egg and the sperm in the
laboratory to enable fertilisation and early embryo
growth to occur.
•
Replacing the embryos into the patient’s uterus
(when the fertilised egg has divided twice and
usually consists of between 2 and 8 cells)
Figure 5 Embryo Development
(source Guy’s and St Thomas’
Hospital web site)
2.4. Stem Cells
2.4.1. The various tissues and organs of the human body are made
up of specialised cells, which are adapted to perform certain
functions. In contrast stem cells are cells that have the ability
to give rise to almost all other types of cells and tissues.
2.4.2. Stem cells have the ability to replace damaged cells in the body. This
property has led scientists to investigate the possible use of stem cells
in regenerative medicine. Under certain conditions stem cells can be
induced to become other types of cell, such as blood cells and muscle
3
The female pronuclei contain 22 chromosomes plus the X chromosome. The male pronuclei
contain 22 chromosomes plus either an X or Y chromosome (but not both).
cells, nerve cells, heart cells, or insulin-producing pancreatic cells.
Therefore, stem cells may, in future, be used to replace cells in patients
suffering from a wide range of diseases e.g. diabetes.
2.5. Sources of Stem Cells
2.5.1. Embryonic Stem Cells
After fertilisation embryos keep on dividing to generate the many
different cell types that comprise the human body. After five days a
hollow ball of cells called the blastocyst forms. The outer blastocyst
layer forms the placenta while an inner group of around 50 stem cells
(inner cell mass) will form the developing embryo’s tissues. If isolated
and cultured under the right conditions these stem cells can form any
cell type of the human body, and may in future be used for
transplantation.
Figure 6. Derivation of embryonic stem cells
2.5.2. Foetal Stem Cells
Stem cells have been obtained from foetal tissue, technically called
embryonic germ cells, these stem cells are derived from the region of
the foetus destined to develop into the testes or ovaries.
2.5.3. Adult Stem Cells
Stem cells can be derived from various tissues in adults e.g. stem cells
can be found in bone marrow, blood, skin brain, pancreas and the eye.
The primary role of these stem cells is to maintain, and in some cases,
repair, the tissue in which they are found e.g. stem cells that are found
in the skin will give rise to new skin cells, ensuring that old / damaged
skin cells are replenished. Stem cells can also be obtained from
umbilical cord blood and the placenta.
2.6.
Advantages and Limitations of Embryonic Stem Cells and Adult
Stem Cells
2.6.1. Embryonic Stem Cells
Embryonic stem (ES) cells are very specific class of stem cells which
can be derived from the blastocyst. Due to their ability to reproduce
themselves, and to differentiate into other cell types, stem cells offer
the prospect of developing cell based treatments both to repair or
replace tissues damaged by fractures, burns and other injuries and to
treat a wide range of degenerative diseases e.g. Alzheimer’s disease,
cardiac failure, diabetes and Parkinson’s disease.
a)
Advantages of Embryonic Stem Cells
•
At present, only ES cells can be readily isolated and grown
in culture in significant quantity to be useful.
•
Experiments, in mice, have shown that the culturing of ES
cells does not alter them i.e. cultured ES cells from mice
have been routinely re-implanted into a blastocyst and then
into a mother and have given rise to normal offspring. Thus
cultured ES cells are potential safe for therapeutic use.
•
ES cells have the potential to regenerate all normal cell
types in the body (at present ES cells are the only cell type
to have this potential).
•
ES cells are undifferentiated therefore, it is not necessary
to dedifferentiate ES cells prior to differentiation into a new
cell type.4
b)
Limitations of Embryonic Stem Cells
•
Use of ES cells therapeutically may be problematic in that
transplanted ES cells may be rejected by the recipient’s
immune system. This is because the ES cells will not have
been derived form the patient’s own embryos.
2.6.2. Adult Stem Cells
Experiments have shown that stem cells from blood can be induced to
differentiate into neural cells. Therefore adult stem cells may have a
greater therapeutic potential than had previously been thought.
a)
4
Advantages of Adult Stem Cells
•
In general adult stem cells would be derived from cells
taken from the patient to be treated therefore, the
transplanted adult stem cells would not be rejected by the
recipient’s immune system
In the course of human development, a single cell, the fertilised egg, gives rise to more than
200 cell types. The process whereby less specialised cells turn into more specialised cell
types is called differentiation.
b)
3.
Limitations of Adult stem Cells
•
It will not always be possible to derive stem cells from
patients e.g. in a patient suffers from a genetic disorder or
some types of cancer, adult stem cells derived from these
patients may retain damaging genetic alterations therefore
could not be used therapeutically.
•
It is difficult to grow (culture) adult stem cells and to get them
to differentiate into new cell types.
•
The mechanism of how adult stem cells differentiate into
new cell types is poorly understood i.e. it is unknown
whether adult stem cells give rise to cells of different tissue
types by transdifferentiation or by dedifferentiation to a
pluripotent cell, which then differentiates into new cell types.
•
Experiments have shown that adult stem cells transplanted
into mice do not differentiate into new cell types in sufficient
numbers to cure the disease.
•
Even though adult human bodies contain stem cells they
have limited potential for differentiation into new cell types
as the purpose of these cells is to give rise to specific cell
lineages. In fact if these cells did have the potential to
differentiate into all cell types it would be disastrous as it
could result in the wrong cell types developing into the
wrong tissue e.g. neural cells developing onto heart tissue.
Therefore it remains uncertain whether adult stem cells
could be manipulated into undergoing dedifferentiation and
redifferentiation into other cell types.
Artificial Creation of Embryos
3.1. Parthenogenesis
3.1.1. Parthenogenesis (Greek for virgin birth) – is a technique in which an
egg cell is activated without being fertilised by a sperm cell i.e. a
human egg cell develops into an embryo without the genetic input from
a sperm cell.
Figure 7. Illustration of Parthenogenesis
3.1.2. Parthenotes are, according to current thinking, unlikely to develop into
viable foetuses.
3.1.3. Parthenogentically activated embryos may provide a valuable source of
stem cells and these stem cells would have the advantage that if used
by the women who provided the egg they would be unlikely to be
rejected after transplantation. In theory a patient in need of a particular
cell or tissue type provides the egg, this is then activated using
chemicals or an electric current to form an preimplantation embryo.
The inner cell mass is removed and the resulting stem cells are
differentiated into the type of tissue the patient needs. Note: only a
woman could benefit from this treatment.
3.2.
Cell Nuclear Replacement
Cell nuclear replacement (CNR) is the process of inserting the nucleus
of an adult cell into a donated egg from which the original nucleus has
been removed. Following cell nuclear replacement the recipient egg is
induced to divide by chemicals and an embryo is produced.
Figure 8.Illustrations of Cell Nuclear Replacement
CNR could, potentially, be used to produce cells / tissues for patients that
would not be rejected by their immune system. A somatic (adult) cell would be
taken from the patient and injected into an enucleated donor egg and after
artificial activation the embryo would be cultured to the blastocyst stage.
Embryonic stem cell would be isolated from the inner cell mass of the
blastocyst and differentiated in vitro to produce cells or tissues for
transplantation. Using these cells / tissues in therapy would have advantages
over using embryonic stem cells isolated from embryos created by IVF,
because the genetic material would be derived from the person to be treated
and so would not be rejected by their immune system.
3.3. Ooplasmic Transfer
Normally, ooplasmic (or cytoplasmic) transfer is used to correct for deficient
factors in oocytes in assisted reproductive technologies. In this technique
small amounts (5-15%) of cytoplasm of high quality donor oocytes are
injected into recipient oocytes that are assumed to be deficient in factors
important for further embryonic development. In theory ooplasmic transfer
could be used to correct for those mitochondrial diseases which involve
mitochondrial DNA. As these diseases may be inherited through the maternal
line, women at risk of passing a disease to their offspring may have diseased
mitochondria in the cytoplasm of their oocytes. Therefore, in these cases
ooplasmic transfer could be useful, in that some of the cytoplasm of the
oocytes containing diseased mitochondria could be replaced with the
cytoplasm taken from a woman who did not have diseased mitochondria in
their oocytes.
However, it may be possible to transfer the cytoplasm of an egg into an adult
somatic cell (the reverse of cell nuclear replacement). The resulting cell would
become an embryo like structure from which embryonic stem cells could be
derived.
Figure 9. Illustration of Ooplasmic Transfer
In ooplasmic transfer a small amount of cytoplasm is removed from an oocyte
and inserted into an adult somatic cell. The cytoplasm, contained in an egg,
as well as having properties that influence the activation of the egg it is also
necessary for the development of the embryo. Theoretically, ooplasmic
transfer the diploid nucleus of the new cells could be induced to divide and the
resulting structure would have embryonic properties from which stem cells
could be derived.
4.
Artificial Creation of Gametes
4.1. Nuclear Transplantation to Create New Oocytes
4.1.1. In nuclear transplantation the nucleus plus a small amount of
cytoplasm (karyoplast) is removed from the donor oocyte and
transferred into a recipient oocyte that has its nucleus removed.
(source: Tsai et al., 2000 Human Reproduction 15: 988-998)
Figure 10. Nuclear Transplantation (a) Enucleation of immature oocytes – the top picture illustrates
the isolation of the karyoplast (the nucleus and same proportion of cytoplasm) from aged oocytes and
the lower picture illustrates the removal of the nucleus from the younger oocyte. (b) Grafting,
electrofusion and reconstruction – this illustrates the insertion of the karyoplast into the younger
enucleated oocyte. The grafted oocyte is exposed to an electrical pulse to induce nucleus-cytoplasm
fusion and reconstitute the oocyte.
4.1.2. This method could be used to reconstruct oocytes of older patients and
if successful these oocytes would be capable of fertilisation using
husband / partner / donor sperm. Studies on human oocytes have
shown that following the transfer of karyoplasts from immature donor
eggs (Germinal Vesicle) into immature recipient cytoplasts (enucleated
oocytes) 60% of the reconstructed oocytes went on to mature normally
(the relatively low success rate was probably due to the media used to
mature the oocytes in vitro) (Takeuchi et al., 1998, 1999).
4.2.
a)
Haploidisation
Creating new Oocytes
A process known as haploidisation could be used to create new
oocytes (fig. 11). The technique is similar to cell nuclear replacement in
that a nucleus from an adult diploid somatic cell (containing 46
chromosomes) is inserted into an oocyte which has had its nucleus
removed. However, unlike cell nuclear replacement where chemicals
are used to trigger the oocyte to divide as though it had been fertilised
in the normal way, in haploidisation the genetic properties of the
oocytes initiates the diploid nucleus to undergo a meiosis-like reduction
division so that half the number of chromosomes are excluded in a
polar body (this would be similar to what occurs in nature when an
oocyte undergoes the first meiotic division), and the resulting oocyte
would be haploid (i.e. contain 23 chromosomes). Furthermore, unlike
cell nuclear replacement, these haploid oocytes would still need to be
fertilised with the paternal gamete in order to develop into an embryo
and produce offspring.
(source: Tsai et al., 2000: Human Reproduction 15: 988-998)
Figure 11. Manufacturing oocytes (a) a somatic cell containing the patient’s genome can be used to
manufacture oocytes when transferred into an enucleated immature oocyte. (b) Enucleation of an
immature oocyte –the nucleus of an immature oocyte is removed to produce a cytoplast. (c) Grafting,
electrofusion, reconstruction and haploidisation – the somatic cell nucleus (extracted as the karyoplast)
is inserted into the enucleated oocyte. The grafted oocyte is exposed to an electrical impulse to induce
nucleus-cytoplasm fusion and reconstitute an immature oocyte that undergoes haploidisation and
extrudes the first polar body.
On group of researchers (Tesarik et al. 2001) have reported the ability
to fertilise reconstructed oocytes. In this study the researchers
removed the nuclei from the cumulous oophorus cells that surround
oocytes in the follicular fluid, which had been obtained from a woman
who had failed to produce viable oocytes following ovarian stimulation.
These nuclei were inserted into six enucleated oocytes that had been
donated by another woman undergoing IVF treatment. Within an hour
the nuclei had become invisible indicating that they had undergone a
meiotic division. The researchers then injected these reconstructed
oocytes with partner sperm and following 5 hours in culture a structure
similar to the second polar body was extruded from three of the
oocytes. By 10 hours after sperm injection two normal looking pronuclei
appeared in two of the oocytes and both of these oocytes underwent
the first cleavage to produce 2-cell embryos. Both these embryos were
cryopreserved for eventual future transfer to the patient’s uterus.
Therefore this study has shown that human oocytes can be used to
reprogram adult cell nuclei, allowing the reconstruction of genetically
own oocytes for patients who have ovarian function failure.
b)
Eggs fertilised without Sperm
Researchers have found a way to fertilise eggs using genetic material
from adult cells. To do this the researchers mimicked the process that
takes place during normal fertilisation when two sets of chromosomes
in an egg are separated and one set is ejected, leaving the remaining
set to combine with the single set from the sperm.
Scientists from the Monash Institute of Reproduction and Development
in Australia have demonstrated the fertilisation of mouse oocytes using
somatic cells as male germ cells. These researchers injected nuclei
taken from adult male somatic cells (i.e. these nuclei are diploid thus
contain 46 chromosomes) into non-enucleated oocytes (these oocytes
were at metaphase II – the first meiotic division stage of development).
Following activation, using chemicals, of the injected oocytes, two
second polar bodies were extruded and two pronuclei were formed,
each containing a haploid set of chromosomes, one derived from
oocytes chromosomes and the other derived from the somatic cell’s
chromosomes. The fertilisation rate following injection of nuclei taken
from adult male fibroblast cells was 29%, out of which 80 – 90% went
on to cleave to the two cell stage and ~ 50% reached the morula stage.
However, very few developed to the blastocyst stage and no live
offspring were born. This study demonstrates that nuclei taken from
adult somatic cells undergo haploidisation when injected into
metaphase II oocytes and therefore have the ability to fertilise oocytes
thus acting like diploid male germ cells.
If this technique proves to be successful it could be used to help men
who are unable to have children because they have no sperm, or germ
cells with the potential to become sperm. In these circumstances an
adult cell e.g. a skin cell from the male could be used to fertilise
oocytes of his wife / partner, thus ensuring that the resulting embryo
would have the chromosomes of both partners. Theoretically, this
technique could also be used to enable lesbian couples to have their
own biological children. However, this could prove problematic as
aspects of development are controlled by a parental gene.
4.3
Derivation of Gametes from Embryonic Stem Cells
Embryonic stem cells derived in vitro are generally considered to be
pluipotent rather than totipotent because of their inability to differentiate
into germ line cells [the gametes (sperm and eggs) and precursor cells
from which the gametes are derived from].
4.3.1 Derivation of oocytes – Researchers from the University of
Pennsylvania have reported the ability of in vitro cultured mouse
embryonic stem cells to develop into female germ line cells (Hϋbner et
al., 2003 Derivation of oocytes from mouse embryonic stem cells.
Science 300: 1251-1256). This group demonstrated that embryonic
stem cells, under appropriate conditions, differentiated into early
female germ line cells (oogonia) which had the ability to undergo
meiosis and to recruit adjacent cells. These follicle-like structures went
on to develop into blastocysts.
4.3.2 Derivation of sperm – A group of researchers in Japan have reported
the ability to derive germ cells from embryonic stem cells in vitro
(Toyooka et al., 2003 Embryonic stem cells can form germ cells in vitro
Proc Natl Acad Sci USA 100: 11457-11462). They demonstrated that
embryonic stem cells can differentiate, in vitro, to from germ line cells
and that these cells undergo spermatogenesis when transplanted into
reconstituted testicular tubules. Therefore embryonic stem cells can
differentiate into germ line cells that have the ability to undergo meiosis
and produce sperm.