Like a Virgin (35 page)

‘Less evolved’ species, such as flies, birds, and fish, can and do generate new eggs throughout their adult life, which means that the ability to produce new eggs was lost somewhere
during evolution, before the emergence of mammals. Yet the ability of males to produce sperm throughout adulthood was conserved in species from flies through to man. Why would the process of
evolutionary selection deem it an advantage to endow women with only a fixed number of eggs that sit stagnant and are subjected to years, if not decades, of ageing before they erupt from a follicle
during ovulation? The more logical, more robust approach would be to keep generating fresh eggs and fresh sperm. At least in theory. But theory aside, new evidence points to signs that mammals also
have the potential to generate new eggs.

Stem cells have received a great deal of attention in the press for their ability to renew on their own, unlike all of our other cells, which only age and die off. Every type of cell, tissue,
and organ in our bodies must be created from the genetic material
contained in the egg and sperm, the precursors of the embryo. This power to generate the totality of
components required to build a human is called
totipotency
. Stem cells derived from embryos are better suited to fulfil this role than those taken from an adult body, because in early
embryos every cell has the potential to generate into any number of different cell types. As development proceeds, and cells become more fixed and decided as to their ultimate fate – that is
to say, after a certain point in development, one cell will only be able to become a brain cell, another will only become a muscle cell, and so on – the stem cells lose their totipotent
potential and become
pluripotent
, able to generate several different cell types but not all. But because extracting embryonic stem cells currently requires destroying an embryo, the
technique is besieged with controversy, particularly in the US. Whether it will become possible to coerce adult stem cells into acting in a similar manner has still not been proven, though
researchers are trying to revert skin stem cells to an embryonic state.

Regardless, egg-making stem cells have been found in the ovaries of adult mice, monkeys, and humans that retain stem cells with the capacity to renew the egg pool. At the moment, there’s
still no evidence that these cells form new eggs naturally inside a woman’s body, but experiments are being conducted to see if they could be coaxed in a dish to make eggs. And if they could
be coaxed to do the same inside a woman’s body, these stem cells could provide an unlimited supply of eggs, and also be used to postpone age-related ovarian failure and perhaps the menopause.
Instead of receiving donor eggs or undergoing a tricky ovary transplant, a woman could receive a transplant of stem cells and let nature take its course.

For now, research into the precursors of eggs has yielded more auspicious results, including the closest anyone has come to
creating
a virgin birth in mammals. In the 1980s, scientists
had made the first attempts to create mice with two fathers or two mothers and, as we have seen, these experiments failed because of the way in which genes are imprinted
– turned on or off by chemicals within the DNA. Trying to create a baby from two sets of DNA regardless of their origin went nowhere; instead of getting one ‘dose’ of a gene, the
offspring usually ended up with double the amount from one parent and none from the other. Then, in 2004, came the breakthrough: Kaguya. Created by a group of Japanese scientists, Kaguya the mouse
was named after a mythical princess whose true parentage was unknown – she was found inside a bamboo reed. In contrast, Kaguya the mouse’s heritage could not have been better recorded.
She was the first mammal to be born without a father and, what is more, the first animal in history to be born to two mothers.

The team, led by Tomohiro Kono at the Tokyo University of Agriculture, suspected that certain portions of the genome were posing the critical stumbling blocks when it came to imprinting. To
circumvent these two problem regions, they realized they could turn to the biology of egg development. Remember that the genes silenced by imprinting are only silenced as the egg grows to maturity.
By using DNA from an egg at an early stage of development, the scientists could gain access to these genes before they were locked. Kaguya was created from constructing an egg out of material from
one mature egg and one immature egg, the equivalent of synthetic fertilization. Admittedly, Kaguya, like Dolly the Sheep before it, won a bit of a reproductive lottery. Out of the 371 reconstructed
eggs that were implanted, only ten live embryos reached maturity, and only two survived outside the womb; Kaguya’s sister was killed so that the genes involved could be studied in more
detail. But within just three years, the scientists had honed the technology to produce fatherless mice that develop at a high success rate – equivalent, in fact, to the rate obtained with in
vitro fertilization
of normal embryos. Like Kaguya, these new generations of mice – all female, of course, since they only have sex chromosomes from eggs – have
proved able to reproduce with males and produce fertile offspring.

To achieve this, Kono’s team deleted two bits of DNA, called
H19
and
Dlk1-Dio3
, which are imprinted in the mother but also serve as key controllers of imprinting across the
genome. The first imprinted gene to be identified,
IGF2
, is imprinted in the mother, and so is expressed in the child from the father’s copy. What was particularly striking is that a
substantial number of genes that have subsequently been discovered to be imprinted act as part of a pathway in which insulin-like growth factor-2 is crucial – the very thing that
IGF2
codes for. And one of these genes is
H19
.

The other critical imprinted region,
Dlk1-Dio3
, contains genes that encode proteins expressed only when they come from the father. The genes in the
Dlk1-Dio3
area are found
throughout the embryo, but after birth, they are predominantly located in the brain, where their instructions for constructing the tiny pieces of machinery that regulate the workings of other genes
do their work. These instructions are expressed only from the chromosome inherited from the mother. Some switch had to be turned, in order for the father’s gene to stop influencing the
offspring.

It was
H19
and
Dlk1-Dio3
that Kono and his team deleted to make Kaguya the mouse. Tampering with these sections effectively allowed them to use the egg’s chromosomes as
though they had come from a sperm.

Making human babies using Kaguya-style genetic tinkering
should be possible in the future. But doing so will yield only female offspring, unless we can
get hold of a Y chromosome, even one manufactured in a lab. In 2007, a first step in this direction was taken: in a painstaking process, a synthetic chromosome was assembled using lab-made
chemicals – that is, copies of the chemicals that make up DNA. The artificial chromosome contained 381 genes containing 582,970 base pairs – paired letters of the DNA alphabet. The
pioneering biologist behind this construction was Dr Craig Venter, whose company, Celera Genomics, helped to unravel the sequence of the human genome, in parallel with the government-backed Human
Genome Project, in 2003.

The initial design of Venter’s artificial chromosome was based on a parasitic bacterium called
Mycoplasma genitalium
, which is considered the smallest naturally occurring genome in
cell form. Venter’s team extracted the bacterium’s own DNA and inserted the synthetic reconstruction in its place. When they finally succeeded, they branded the creation as the first
truly new artificial life form on earth. In Venter’s words, the artificial chromosome was ‘a very important philosophical step in the history of our species. We are going from reading
our genetic code to the ability to write it.’ Learning to write genetic code will be more complicated when it comes to creating artificial eggs and sperm, especially on the scale of
Homo
sapiens
’ twenty-three thousand genes, even after taking account of the ‘non-coding’ portions of the genome.

Still, the ability to create artificial eggs and sperm from stem cells is hailed as the technology that will finally bring an end to infertility. And rightly so, as it will also help us to
uncover many of the remaining secrets surrounding how reproduction works. Experiments to make artificial eggs and sperm are likely to yield an increased understanding of genetic imprinting and the
diseases that arise when imprinting goes awry. Since the cells that
become the placenta can also be derived from these stem cells, this research could allow scientists to
investigate how the early placenta develops and how disorders arise in it. And of course, artificial germ cells would allow individuals to bypass donors, avoiding the ethical issues of the egg and
sperm trade. In fact, because the children produced from these cells will not be ‘artificial’ babies, scientists prefer to call them in vitro-derived cells.

In vitro-derived cells should be able to withstand freezing and be stored for future use, just as their ‘natural’ donated counterparts already are. The freezing procedures used today
are much the same as they were in the early 1950s, when the modern technique was established. Scientists had been able to freeze and store sperm by the 1930s, but had not found a way to ensure that
the sperm were not damaged, rendering them useless for reproduction. In 1949, two British scientists, C. Folge and A. D. Smith, were part of a team who finally succeeded in ‘reviving’
sperm after preservation, by using glycerol to maintain the sperm’s structural integrity as it is plunged into temperatures around minus 196 degrees Celsius (minus 320 degrees F), and the
process was improved substantially a few years later by the ‘father of cryobiology’, American zoologist Jerome K. Sherman. UK law currently only allows sperm frozen in this way to be
kept for ten years, but as there is no evidence of any changes in quality over time, in theory, sperm suspended like this can last forever – no matter the source of the sperm.

Things are not as straightforward, however, when it comes to freezing eggs. Unlike sperm, which are quite small, the egg is big – the biggest cell in the body. When an egg is frozen, the
large cell’s greater fluid content often sustains ice-crystal damage. Even with an improved technique, known as vitrification, which flash-freezes the egg to avoid crystallization, there is
only a ten percent success rate. If a young woman has her own eggs removed, chances are that when she is older and decides to use
them, the process may very well have failed.
And seeing that for women, removing eggs is quite invasive and uncomfortable, being able to make eggs in a lab from a woman’s DNA is very attractive: it would mean that most of the process
takes place outside of her body with far less risk.

So, how close are we? Bone marrow stem cells have proved extremely promising in the early experiments to create in vitro-derived germ cells: given the right signals, bone marrow stem cells are
capable of becoming sperm. Further, three types of stem cells exist in bone marrow and it contains the cell-level blueprints for much of the body, including the heart, lungs, liver, kidney, bone,
cartilage, fat, muscle, tendon, skin, and even the brain.

Regardless of the provenance of human embryonic stem cells, the proteins that act as signposts for developing male (sperm) and female (egg) cells can be detected in them. In fact, eggs have been
made in the lab from both female and male embryonic stem cell lines. This is because the male embryonic stem cells have not yet expressed the
SRY
gene, which triggers the development of the
testes and eventually the generation of sperm. Female embryonic stem cells, on the other hand, can only give rise to eggs. So without an artificial Y chromosome, women could only ever make
artificial eggs, while men could make either artificial eggs or sperm. However, by keeping eggs fashioned from embryonic stem cells in a culture (that is, in a nutrient/chemical soup incubated with
a prescribed mixture of gases at an appropriate temperature), scientists have been able to create parthenogenic embryos – embryos that begin to develop despite never having been fertilized by
sperm. (Of course, this isn’t all that peculiar when you consider that parthenogenesis is a relatively normal phenomenon; whenever eggs are kept in culture, they tend to start dividing on
their own.)

In early 2006, two laboratories, one based in Germany and
the other in the UK, reported some remarkable results using embryonic stem cell lines. Earlier, scientists had
successfully developed immature sperm, or spermatogonial stem cells (SSCs), from embryonic stem cells that, when they were injected into mouse eggs, developed into early embryos. The new research
went one better. The teams transplanted SSC artificial sperm into the testes of mice that had no sperm of their own. After four months, the scientists observed sperm in some of these mice,
generated from the transplanted cells. Unfortunately, the sperm did not move, or move very far, unaided; they weren’t ever going make it to an egg. So to help the process along, the sperm
were removed from the mice testes and injected into unfertilized eggs. Out of 210 eggs, 65 embryos were produced and transferred into surrogate mice mothers. Seven of these became baby mice,
fathered by artificial sperm. But the baby mice sired this way were not very healthy, and they died at ages well below the average life expectancy of mice conceived naturally. Much remains to be
worked out before artificial sperm are ready for humans.