Current research into the single cells of human reproduction, oocytes and spermatogonia, is turning conventional wisdom upside down. Although we were taught in school that a woman is born with all the eggs she’ll ever have, it turns out that’s not so. And as for the belief that older men can make healthy children, but older women risk delivering a child with birth defects or retardation? Well, it’s not that simple. These recent contributions to our understanding of how we procreate would be impossible were it not for advances in genomics, microfluidics, and single-cell analysis techniques.
In the lab of Stephen Quake, professor of bioengineering and Howard Hughes Medical Institute investigator at Stanford University, researchers are studying the potential consequences of changes to the human genome that occur by meiotic recombination or de novo mutation. “Our sperm sequencing study focused on a classical genetics question,” says Quake’s graduate student Jianbin Wang. “How is our genetic information changed during its transmission to the next generation?” In a recent paper published in Cell, Wang and colleagues explore this issue by reporting the first genomic analysis of single human sperm cells (1).
In previous work, the group realized that combining single-cell and whole-genome tools might open new doors experimentally.“In [a study of human haploid genomes], we had detected the shuffle of parental genomes before their transmission to the child, in other words, meiotic recombination,” says Wang. “We realized that the combination of our single-cell and whole-genome haplotyping techniques could be a good way to study such genome changes in the primary sex cells.”
For greater throughput and accuracy, they performed their experiments on a microfluidic chip. Quake is a co-founder of the company Fluidigm, which originated the first microfluidic-based chip for performing biological experiments in tiny volumes of fluid. Movements of the fluids and cells within and through the maze of channels built into the chip are accomplished by tiny, computer-controlled valves that open and close at precise times. The size and architecture of the chip’s chambers and channels also let them isolate and study single cells.
Using microfluidics, the group performed high-throughput, whole-genome amplification of single sperm cells, followed by whole-genome genotyping and single nucleotide polymorphism (SNP) analysis. With these results, they constructed a personal recombination map for the individual sperm donor.
While their results were generally consistent with the two other population-based recombination maps previously published by deCODE Genetics (2) and the International HapMap Consortium (3), Wang and colleagues found previously unidentified areas of high recombination activity, suggesting that the population maps average out individual differences. Indeed, even though the population maps note generalized hotspots of recombination activity, Quake’s group observed hotspots on the individual map that were outside of those areas. They also observed sperm cells with genetic abnormalities, such as missing sex chromosomes and aneuploidy. The group suggests that genomic instability of sperm cells is one possible cause of failed conception events since it is known to halt cell division.
While Quake’s group measured the genomic diversity in one person’s sperm, others were looking at diversity through time. Does the genomic diversity of gametes change as we age? What are the reproductive implications of greater diversity, whether by mutation or otherwise? A recent paper in Nature, from deCODE Genetics headed by CEO Kari Stefansson, addressed mutation rates of men who conceived children at varying ages (4).
By sequencing the entire genomes of 78 parent-child trios in Iceland, they found that de novo mutation rates of SNPs are strongly influenced by the father’s age at the conception of the child, with two mutations added for each year of the father’s age. While not every mutation will have biological consequences, this study runs counter to the conventional belief that the mother’s age at conception is key, while the father’s age can be disregarded. Indeed, Stefansson and colleagues suggest that the father’s age at conception influences the child’s risk of developing autism or schizophrenia, among other diseases.
Eggs that Come and Go
The lab of Jonathan Tilly, director of the Vincent Center for Reproductive Biology at Massachusetts General Hospital, and professor of obstetrics, gynecology, and reproductive biology at Harvard Medical School, has been investigating the opposite sex, namely the secrets of the human egg cell—where they come from and where they go at menopause. Tilly’s group has been riding a turbulent wave of controversy over their discovery in the late 1990s of oogonial stem cells—stem cells in the ovaries that either propagate themselves mitotically, or divide by meiosis to produce eggs. Some still do not believe that such cells exist, despite years of evidence. In a recent Nature Medicine paper, Tilly’s group demonstrated the existence of functional oogonial stem cells in humans (5).
The road to their present work was not easy. For decades, nearly everyone has been taught that women are born with all the eggs they will use for their lifetime. Yet Tilly’s group had observed, while examining ovarian tissue samples, that many more eggs die than can be accounted for by the rate of ovarian follicle atresia, i.e immature oocyte loss. “We had a mathematical dilemma in terms of oocyte numbers,” says Tilly. “Much to our shock, the net change in healthy oocyte numbers was exceeded by a factor of threefold in terms of the number of dead oocytes that we could count. There were far more oocytes dying than we could account for by simply doing a subtraction. Nobody had actually measured dead oocytes before.”
The simplest explanation, according to Tilly, is that germline stem cells in the ovaries are generating oocytes throughout life. “Our belief is that these cells play a very important role in maintaining the reserve of oocytes that adult females have at their disposal for use,” he says. In fact, in other species such as Drosophila, females make eggs throughout life, just as males do with sperm. Tilly doesn’t see any reason to expect that humans would be an exception. “Why would the survival of humans depend on a population of cells that are 40 years old?” he says. “That makes zero sense.”
One of the necessary steps to convince others of the existence of these stem cells in humans was to purify them from human ovarian tissue—for which he turned to single-cell techniques. In his recent paper, his group established a purification protocol using fluorescence-activated cell sorting (FACS) detection of an antigen exposed on the surface of the stem cells. “FACS gives you the ability to march the cells, single file, down a tube and pick the ones you want, one by one,” says Tilly. “So, from our perspective, having the ability to do this on a single-cell level was the only way to go, if we were really going to tease apart the cells and figure out what their function is.”
The isolated oogonial stem cells can make eggs that can be fertilized in vitro. But to demonstrate further that these cells are functional, Tilly’s group transfected the purified stem cells with green fluorescent protein (GFP), then injected them into human ovarian tissue. The subsequent formation of follicles containing oocytes that were labeled with GFP strongly supports the existence of functional oogonial stem cells in women.
Right now, Tilly is excited about clinical applications that could delay the timing of menopause, when virtually no eggs remain because the stem cells have stopped producing new eggs. His group has generated a transgenic mouse model in which oocyte loss was slowed down, which they termed a “no-menopause” mouse. Even though these mice aged normally, they experienced none of the detrimental health effects that some women suffer after menopause – including alopecia, cataracts, cognitive decline, bone loss, and weight gain, among others. “People talk about fertility because it’s the hot buzz thing,” says Tilly. “But my personal interest is actually much grander. Can we actually delay menopause for quality-of-life reasons?”
Such transgenic technology is a long way off for women, but what if? “What if you could actually sustain an ovarian reserve well into older age through this type of technology?” asks Tilly. “I think it’s a pretty viable way to think about the utility of these cells; a kind of ovarian fountain of youth, so to speak.” Such new beginnings—made possible with new single-cell technologies—are fitting given the long strides researchers are making in our understanding of human sex cells.
1. Wang, J., H. C. Fan, B. Behr, and S. R. Quake. 2012. Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell 150(2):402-412.
2. Kong, A., G. Thorleifsson, H. Stefansson, G. Masson, A. Helgason, D. F. Gudbjartsson, G. M. Jonsdottir, S. A. Gudjonsson, S. Sverrisson, T. Thorlacius, et al. 2008. Sequence variants in the RNF212 gene associate with genome-wide recombination rate. Science 319(5868):1398-1401.
3. Consortium, T. I. H. 2005. A haplotype map of the human genome. Nature 437(7063):1299-1320.
4. Kong, A., M. L. Frigge, G. Masson, S. Besenbacher, P. Sulem, G. Magnusson, S. A. Gudjonsson, A. Sigurdsson, A. Jonasdottir, A. Jonasdottir, et al. 2012. Rate of de novo mutations and the importance of father/'s age to disease risk. Nature 488(7412):471-475.
5. White, Y. A. R., D. C. Woods, Y. Takai, O. Ishihara, H. Seki, and J. L. Tilly. 2012. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. Nat Med 18(3):413-421.