THE FAR WALL OF THE CAVE OF YENGA

How to make female sperm

In other Web pages, you have learned how to make male sperm, fix male sperm, and enhance male sperm, focusing on pre-sperm male germ cells. How does that help us make female sperm? We once again turn to the work of Professors Ralph Brinster and James Zimmermann at the University of Pennsylvania, who invented methods for fixing male sperm. In their 1991 patent application for fixing male sperm, they address the problem of providing alternatives to using germ cells for transplantation (germ cells are less numerous and more sensitive to acquire than related stem cells). On page 7 of their patent application, they write:

The primitive cells used in accordance with the invention can come from other individuals or in vitro culture. Examples of primitive cells that can be used include totipotent stem cells, embryonal carcinoma cells, embryonic stem cells, sperm cells from other males, primordial germ cells, other primitive cells, etc. Primitive sperm cells from seminiferous tubules, embryonic stem cells grown in culture, or primitive cells from body organs are prime candidates.

Now Brinster and Zimmermann are guessing a bit here on the ability to use non-germ stem cells (there are subtle effects like imprinting to take into account), but that's for scientists to argue and research (for example, professor George Daley of the Harvard Medical School is studying how to convert stem cells into sperm). What is crucial of the last sentence of this paragraph:

The use of female (XX) cells is also within the scope of the present invention.

That's right, in 1991, Brinster and Zimmermann proposed the possibility of female sperm. What they are proposing is that you can make female sperm by injecting certain types of female cells into a male's testicles. Prof. Brinster had a five year NIH research grant to continue his studies on sperm cells.

Extremely radical stuff, making it not surprising that they decided not to tell anyone (patent applications and patents are a good place to hide information since few people read them in detail). Controversy aside, there is another reason not to publicize this suggestion - it probably does not work.

For males to make sperm from transplanted germ cells, they need properly imprinted adult diploid germ cells (with one X and one Y chromosome), and with Y chromosome having sperm-making genes to be transplanted into the testes. Adult females interested in making their own female cell have a few problems - adult female germ cells are haploid, are not imprinted male, and don't contain the necessary Y chromosome sperm-making genes. Thus Brinster and Zimmermann's suggestion of using female (XX) cells probably will not work. Solution? Create a properly imprinted adult female germ cell through cloning, and add elements of a Y chromosome. But is this possible? The key to answering these questions is the phenomenon of epigenetics.

Earlier observations about female sperm

Brinster and Zimmermann were not the first to suggest the possibility of female sperm. As far back as 30 years ago, scientists were considering the possibility of male eggs and female sperm. In a June 1977 paper, a group of English scientists, in a paper on male mouse eggs, wrote that "Evidently the sex of a germ cell is not an autonomous property but is determined by the nature of the gonad in which it finds itself.". In the paper they talk of XX/XY chimeric mice, many of which were fertile males, raising the possibility that some XX germ cells from these males could become sperm (XX germ cells similar to female germ cells, but also suggesting the need for the presence of the Y chromosome in some cells). In 1981, another English scientist, Anne McLaren, reported that sex-reversed XX male mice produced prospermatogonia in the embryo, but that such "female" sperm cells died soon after birth.

Assuming male XXY germ cells can split meiotically, to achieve female sperm, we need to create XXY germ cells from an adult female. To do so, you need to combine two techniques: cloning and human artificial chromosomes. On this Web page, we will discuss how to do so as follows:

But first, a few extremely important questions: Will XXY female germ cells be healthy? Will females born of XY female sperm be healthy if their genetic structure is XXY? The interests of women wanting to make female sperm are important, but not as important as the health of children born from such female sperm. The health issue here focuses on having XXY cells in a female, where the Y chromosome only has the sperm making genes and none of the sex organ making genes (especially SRY). Will such females be healthy? This concern is avoided if the use of XXY germ cells can result in the creation of only X or only Y sperm (the same as in males). But if the sperm have both X and Y chromosomes, then a fertilized egg will have an XXY configuration.

At least in one case, the answer is yes. In 2000, German scientists reported the existence of a health mother and daughter who both are SRY-negative 47,XXY females. The mother is fertile enough to have three children, one son and two daughters (one of whom is also SRY-negative 47,XXY). The existence of the mother is quite rare - one in 20 million female births. But that she was able to live long and healthy enough to successfully have three children implies that SRY-negative 47,XXY cells of all types function healthily. Similarly, in 1999 an Israeli group reported that the replication of non-sexual chromosomes (the paired alleles) was normal in embryos with three sex chromosomes

Cloning females to make female germ cells

Note: the use of cloning to produce female germ cells (discussed below) is offered to show that there is at least one biological pathway that leads to female sperm. The more desired pathway would be to make use of retrodifferentiation and transdifferentiation techniques to convert some form of adult stem cell into a germ cell, thereby eliminating the need for cloning and embryonic stem cells.

Breaking news. In 2004, scientists at Boston's Massachusetts General Hospital published data suggesting that adult human females retain a small number of diploid germ cells. If so, such cells could be extracted and used to make female sperm, eliminating the need for cloning.

Germline stem cells and follicular renewal in the postnatal mammalian ovary
Jonathan Tilly et.al.
Nature, v428 2004, 145-149
[TIL04]

Adult females have no diploid germ cells. During the embryonic stage, female eggs go through one round of meiotic division into haploid cells, before they enter into a rest state until puberty.

TIMING   MALE GERM CELLS FEMALE GERM CELLS
3 weeks post-fertilization germ cells on wall of yolk sac germ cells on wall of yolk sac
4 weeks post-fertilization germ cells start migrating germ cells start migrating
5 weeks post-fertilization germ cells arrive at primitive gonads germ cells arrive at primitive gonads
6 weeks post-fertilization germ cells in genital ridges germ cells in genital ridges
7 weeks post-fertilization male sex organs start forming female sex organs start forming
12 weeks post-fertilization sperm are imprinted ??? egg meiosis starts
20 weeks post-fertilization testes develop egg meiosis pauses
BIRTH    
11-13 years - puberty sperm meiosis starts egg meiosis resumes - eggs are imprinted ???

Adding sperm-making genes to the female germ cells

Adding spermatogenesis genes

As mentioned earlier, adult female germ cells are different from adult male germs cells in at least two ways - they are not diploid and they do not have a Y chromosome, lacking in particular the sperm making genes, especially the DAZ gene. Cloning helps us achieve obtaining female diploid cells, but how about the sperm making genes? Now, female cells already have many genes used for making sperm (many located on the female's X chromosome).

However, studies show that genes on the male's Y chromosome are necessary to promote spermatogenesis. There are three types of genes on the Y chromosome: pseudoautosomal genes that are present as well on the X chromosome and for the most part are expressed outside the testis; homologous genes located within the X-Y homologous regions, where the X and Y versions express highly similar proteins that act in many cells in the body; and Y-specific genes located on the long arm (Yq) that are expressed only in the testis.

The male Y chromosome has some genes (DAZ, RBM, TSPY) that when missing or defective render men unable to make sperm. Indeed the acronym DAZ stands for Deleted-in-AZoospermia, i.e. this gene is known to be absent in men unable to make sperm. One researcher, Page, studies these sperm making genes in detail, and has even patented the genes.

The Y chromosome is much smaller than the X chromosome, and has been shrinking for tens of millions of years. The Howard Hughes Medical Institutes have a nice animation of the evolution of the Y chromosome.

So, by taking those Y chromosome genes that are needed for making sperm, inserting them into a human artificial chromosome, and then inserting the human artificial chromosomes into the female germ cells (similar to adding genes to male germ cells), we have created female germ cells ready to be transformed into sperm.

More roughly, one could clone the female and then inject DNA fragments of the sperm making genes directly into fertilized eggs. Then as the fertilized egg matures and eventually produces germ cells, many of the germ cells could have copies of the DNA fragments. This process is very risky, but is a possibility. Ironically, the idea of injecting DNA fragments into embryos dates back to the early 1980s in work done, once again, by Ralph Brinster and his colleages at the University of Pennsylvania.

Having bits of Y chromosomes in female cells impacts the female little, since the genes only express in the testicular environment. For example, in 2002 it was reported that a woman with an extra Y chromosome in all of her cells, where the Y chromosome was missing the sex determining SRY gene, was not only healthy but fertile enough to have children. Suppresion of the expression of Y sperm making germs in non-gonadal cells can be achieved by adding transactivators to the artificial chromosome so that the genes are activated only when an external drug is administered.

X-inactivation

Normal males have one active X chromosome. To match this activity level, females with two X chromosomes have one of the X chromosomes inactivated (the inactivated X is called a Barr body). In fact, no matter how many X chromosomes a cell has, all but one are inactivated. Scientists have found a region of the X chromosome that initiates this inactivation, the X-inactivation center (Xic). The most important part of the Xic is a gene, the Xi-specific transcript (Xist for mice, XIST for humans). The inactivation starts from the Xic, and spreads out across the entire X chromosome. The Xist gene is one of the few genes that doesn't code for a protein, and for males is only expressed in the XY body. In females, the two X chromosomes are randomly inactivated (some cells inactivating the maternal X chromosome and some the paternal X chromosome). This makes women somewhat of a mosaic (think of Calico cats).

At the beginning of spermatogenesis for males, the X chromosome is activated. Most likely, both X chromosomes in a female diploid germ cell should be activated as well (which will occur if the germ cells are extracted as above). For sperm-making alone, the Xist contributes little (sperm are produced in mice with no Xist gene).

During spermatogenesis for males, the X chromosome (and Y chromosome) becomes inactivated. This occurs during meiosis, when the X and Y chromosomes separate a bit from the other chromosomes and form an XY body. The Y chromosome gets inactivated by having the X chromosome's inactivation spread over to the Y chromosome. The X chromosome remains inactivated in meiotic spermatocytes, postmeiotic spermatids and sperm.

For females, the 2 X and 1 Y chromosomes would have to form an XXY body. The coupling of the chromosomes is different from that in an XY body (one X and Y strongly coupled with the other X loosely coupled, or with the two X chromosomes strongly coupled with the Y loosely coupled, the loosely coupled chromosome eventually discarded). The formation of an XXY-body and proper inactivation for altered female germ cells is dependent on the existence of pure non-mosaic 47,XXY germ cells undergoing meoisis (discussed above).

The Howard Hughes Medical Institutes have a nice animation of the X-inactivation process.

XY body

Transplanting the enhanced female germ cells into the testes

Much like enhancing male sperm, once the female germs cells are prepared, they are injected into the testicles of a man host, volunteer or paid, where the female germ cells proceed to be transformed into sperm. Click here for the discussion of the male version of injecting germ cells back into the testicles. As mentioned, the testis is an immune privileged organ, meaning that the male's immune system won't automatically reject the transplanted female cells. And given the common origin of the male and female sex organs, the female cells should be able to grow in the male's sex organs.

Filtering/cleaning out defective sperm

As mentioned, female germ cells with an artificial chromosome are similar to a male condition known as Klinefelter syndrome, males with two X chromosomes and one Y chromosomes - such males are known as 47,XXY males. Such males are known to be able to produce sperm, but many of the sperm are defective, much more than the defective sperm produced by normal males. For all of these males, the assisted reproduction industry has produced a variety of equipment to filter/clean defective sperm, equipment which can be used for female sperm. For example, such equipment can be used to select female sperm that only have an X chromosome (those without the artificial Y chromosome).

Forget the males - using artificial testicles

To partially having to rely on men to help make female sperm, it should be possible to grow human female sperm using mice. To do this, first, testicular tissue is taking from a man and implanted under the skin of a mouse with a "knocked-out" immune system. Then altered female germ cells can be injected into the testicular tissue to create female sperm. Sounds bizarre, but scientsts have made this process work for rhesus macque monkey, a fellow primate to man.

Now there are some women who might want to avoid anything from men - to make this as much an all female process as possible. For such women, there is an alternative to male testis, for this is America - artificial testicles, that is, an in-vitro environment for growing germ cells and sperm cells, preferably using an environment that uses only human cells and chemicals . One approach to artificial testis will be the use of one area of nanotechnology research - nanofibers coated with chemicals that help germ cells differentiate into sperm.

Imprinting, sperm, cloning, cures

The key determining factor in cloning humans for therapeutic purposes and/or making sperm, is controlling the gene imprinting process and related epigenetic phenomenon. Such control will takes decades to develop. The PNAS paper from the UPenn lead group, "Reprogramming of promordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning", has an interesting conclusion:

In conclusion, although germ cells by their very nature are often thought to be totipotent and therefore hypothetically ideal nuclear donors for cloning experiments, they are in fact not competent to act as nuclear donors to produce viable cloned offspring that can complete development. PGCs have typically erased or are in the process of establishing epigenetic marks. Thus germ cells should perhaps be viewed as "hemipotent" donors for nuclear transfer.

The very nature of have their imprinting erased, which makes them less useful for therapeutic cloning purposes, makes such germ cells more useful creating female sperm.

The University of Calgary's Virtual Embryo project has a nice introduction to imprinting.

In 1999, singer Natalie Merchant released a song titled "Wonder". I would like to think she was subconsciously inspired to write the song due to a future premonition of the first woman born from female sperm, who will truly be an almost unexplainable wonder. The lyrics: 


Doctors have come
From distant cities
Just to see me
Stand over my bed
Disbelieving what they're seeing

They say I must be one of the wonders
Of god's own creation
And as far as the can see they can offer
No explanation

Newspapers ask
Intimate questions
What confessions
They reach into my head
To steal the glory
Of my story

They say I must be one of the wonders
Of god's own creation
And as far as the can see they can offer
No explanation

I believe
Fate smiled and destiny
Laughed as she came to my cradle
"know this child will be able"
Laughed as my body she lifted
"know this child will be gifted
With love, with patience
And with faith
She'll make her way"

People see me
I'm a challenge
To your balance
I'm over your heads
How I confound you
And astound you
To know I must be one of the wonders
Of god's own creation
And as far as you can see you can offer me
No explanation

I believe
Fate smiled and destiny
Laughed as she came to my cradle
"know this child will be able"
Laughed as she came to my mother
"know this child will not suffer"
Laughed as my body she lifted
"know this child will be gifted
With love, with patience
And with faith
She'll make her way"