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expert reaction to study looking at method of separating X and Y sperm in mice

Research, published in PLOS Biology, reports on a new technique to separate X-bearing sperm from Y-bearing sperm, allowing alteration of the normal 50/50 male/female offspring ratio.

 

Dr James Turner, who leads the Sex Chromosome Biology lab at the Francis Crick Institute:

“X and Y-sperm carry different sex chromosomes, but they must share the proteins made by these chromosomes in order to develop normally.  This sharing means that no markers specific to X- or Y-sperm have been found, which in turn has hampered attempts to sex sperm.

“The authors report that the X-chromosome TLR proteins escape the rule of sharing, being found only in X-sperm, and they experimentally exploit the biology of TLR to isolate X- from Y-sperm and generate litters of mice that are predominantly of one sex.

“The discovery of a protein that marks only X-sperm is really surprising, so the top priority will be to reproduce this finding, and to understand why this protein proves the exception to the rule.  It is unclear whether the TLR protein escapes the rule of sharing in other animals.  Resolving this point would be essential before the technique could be applied in other settings, for example in the dairy cow industry, where an excess of females is required.”

 

Prof Allan Pacey, Professor of Andrology, University of Sheffield, said:

“There have been many attempts over the years to develop techniques to separate X from Y chromosome bearing sperm and thereby influence the sex of farm animals (for commercial purposes) or humans (for social reasons) born.  We also know that nature is pretty ingenious in how it can influence the sex of offspring born in response to specific environmental or social conditions.  However, to date, the mechanism by which it does this is poorly understood.

“Therefore, this paper is very interesting because it highlights inherent physiological differences between X and Y chromosome bearing sperm in the laboratory.  The experiments reported are very elegant and show that by manipulating just two molecules on the sperm surface it is possible to drastically change the speed at which X or Y bearing sperm can swim.  The authors propose that this works by altering the amount of energy the two types of sperm produce.  This in turn allows enriched populations of X or Y bearing sperm to be collected which when used in IVF are able to skew the proportion of male and female mouse offspring born.

“However, to date, the experiments have only been performed on the sperm from laboratory mice and we don’t know if this effect would be seen in the sperm from other animals, such as cattle (where producing more females is important for dairy herds) or in humans where would be parents may desire a child of a specific sex (although that is currently unlawful in the UK).  However, the data does suggest that sperm are far more complex cells than we’ve previously been aware of and that we still have a lot more to learn about them.”

 

Dr Peter Ellis, Lecturer in Molecular Biology and Reproduction, University of Kent School of Biosciences, said:

Is this study significant?  What does it tell us that is new?

“This study makes the startling claim that there are cell surface markers on X- and Y-bearing sperm cells that ‘label’ these and selectively affect their function.  This type of marker has been sought for many years in many different species, but thus far without success.  If this study were to be replicated – and in particular if it holds true in species other than mice – then the implications could be colossal for both animal and human artificial insemination and assisted reproduction, BUT we are certainly not at that stage yet.

What surprised you the most about the findings?

“The direction of the sex ratio effect runs counter to anything one would predict from evolutionary biology.  While there is some evidence in a range of species that the genes on the sex chromosomes can skew offspring sex ratio, the general expectation is that genes on the X chromosome should favour X-bearing sperm function (producing more female offspring), and genes on the Y chromosome should favour Y-bearing sperm function (producing more male offspring).  In this case, there appears to be a gene on the X chromosome which selectively impairs the motility of X-bearing sperm.  It is very challenging to see how such a system could have arisen and why it would be retained in the genome.

What are the limitations?

“The largest limitation is that so far they have only looked at mice, and so there is as yet no basis to say whether this technique would work in livestock species or in humans.  While the gene sequences themselves are conserved, gene regulation can evolve rapidly and may be species-specific.  Within the mouse system, they have carried out a reasonably thorough investigation, and the IVF experiments in particular are impressive.

“However, given the magnitude of this claim there are some additional simple experiments I would have expected to see.  For example, they could have used flow cytometry to sort sperm cells that have higher or lower levels of TLR7, and then stained these with X and Y chromosome paints to count the number of X- and Y-bearing sperm.  Instead, they relied on immunofluorescence microscopy using only an X paint, which is a less quantitative technique and more susceptible to mis-counting.  Similarly, it would have been relatively simple to carry out mRNA hybridisation on testis slices to show that the mRNAs for TLR7 and TLR8 are only expressed in X-bearing cells.

“Finally, checking the expression of these genes in other species is such an obvious experiment – and the necessary reagents are available – that I’m surprised this is not reported.  I doubt it will be long before someone has a look though!

Do farmers already have a method of producing, say, more female cattle than males?

“Cattle sexing is currently achieved by using a special dye to stain DNA, visualising this with a high-power laser to measure the DNA content and sort the X sperm (with slightly more DNA) from the Y sperm.  This is expensive and laborious, runs the risk of damaging the sperm DNA, and is not practical in many species where the X/Y size difference is less pronounced and more sperm are needed per insemination.

What are the potential uses of this research in the future?  Does it mean that parents could pick the sex of their children in the future?

“If it is indeed the case that these genes act as X-specific sperm markers in species other than mouse, then yes, it potentially allows for routine sex selection in any such species – however, I repeat that this is only conjecture at present and remains to be tested.  Routine sex skewing in livestock animals would be a major boon with dramatic benefits for animal welfare in many species.  Sex skewing in humans would be an ethical minefield with the potential for unpredictable and disruptive social consequences.

How does this study contribute to our wider understanding of this area of research?

“As an evolutionary biologist it certainly raises a very puzzling question – why on Earth would the X chromosome contain genes that, when activated, prevent the X from being inherited!

Anything else you feel is important?

“One thing I notice is that the mouse knockout models for TLR7 and TLR8 have been studied for several years, and neither shows any sex ratio abnormalities.  While the current study looks at receptor activation rather than gene knockout, it seems strange that there would be no effect on sex ratio in the knockout animals.  Similarly, there is a mouse mutant called “Yaa” (Y autoimmune accelerator, named for its effects on autoimmune disease) that has a copy of TLR7 on both the X and Y chromosomes – and again no effects on sex ratio are reported for this mouse.

“They also haven’t given very precise details for the swim-up separation (e.g. the size and shape of container used for the swim up, which affects the path length), so it will be challenging for anyone to replicate this work from the data as presented.”

 

Prof Robin Lovell-Badge FRS, Group Leader, The Francis Crick Institute, said:

“In most mammals, female and male sex is specified at fertilisation with the inheritance of an X or a Y chromosome respectively from the father at fertilisation (although the acquisition of most male and female characteristics does not begin until the development of ovaries or testes much later in embryonic development).  Usually sperm bearing an X or a Y are present in equal numbers and have an equal chance of fertilising an egg (which always carry an X chromosome), hence the sex ratio is about 50:50.  Even though the X chromosome is much larger than the Y and it carries many more genes, there is sharing of X and Y-linked gene products throughout almost all of sperm formation, until the very final stages when the bridges break down between otherwise distinct X and Y-bearing spermatids, the stage before swimming sperm with tails are produced.

“Surprisingly, Umehara and colleagues now show that in mouse sperm this sharing may not be the case for a couple of proteins encoded by X chromosome genes that are made around the time that the bridges disappear.  These related proteins, Toll-like receptor 7 and 8, which are on the cell surface, are consequently present more on X-bearing than Y-bearing sperm.  A drug that mimics natural ligands (which can be thought of as keys) for these receptors (akin to locks) activates them and somehow slows down the swimming of the X-bearing sperm.

“The authors of this very nice paper show that this can be used to separate the X- and Y-bearing sperm such that when each population is used to fertilise mouse eggs they can bias the sex ratio of resulting mouse embryos and pups from approximately 50% males and females to over 80% of either male or female.  This could be very useful if it was shown to work well with a number of farm animals where it is beneficial to have an excess of males (e.g. beef cattle) or females (e.g. dairy cattle).  Perhaps the involvement of Toll-like receptor 7 and 8 can also explain some natural cases where sex ratios vary from 50:50 in some mammals depending on environmental conditions, including perhaps some pathogens as pointed out by the authors.  There is also some evidence that stress in humans may have a subtle influence on the likelihood of having a boy or a girl.  However, there is a long way to go to prove exactly how the activity of these receptors has an effect on sperm motility, whether the human equivalents show the same expression and would have the same effect.  And while the mice born after the sperm sorting apparently appeared normal, it would be essential to verify that there were no long-term effects of activating these receptors prior to fertilisation.  In other words, do not try this at home in attempts to bias the likelihood of having a boy or a girl!”

 

‘Activation of Toll-like receptor 7/8 encoded by the X chromosome alters sperm motility and provides a novel simple technology for sexing sperm’ by Takashi Umehara et al. was published in PLOS Biology at 19:00 UK time on Tuesday 13 August 2019. 

DOI: 10.1371/journal.pbio.3000398

 

Declared interests

Prof Allan Pacey: “Chairman of the advisory committee of the UK National External Quality Assurance Schemes in Andrology, Editor in Chief of Human Fertility and Trustee of the Progress Educational Trust (all unpaid).  Also, recent work for the World Health Organisation, British Broadcasting Corporation, Purple Orchid Pharma (paid consultancy with all monies going to University of Sheffield).  Co-applicant on a research grant from the Medical Research Council (ref: MR/M010473/1).”

Dr Peter Ellis: “Previous BBSRC funding to develop novel methods of sperm separation, current Leverhulme Trust funding to look at evolutionary biology of transmission ratio skewing including sex ratio skewing.”

Prof Robin Lovell-Badge: Robin Lovell-Badge is on PLOS’ Board of Directors but he is not paid by them and board members have no direct role in choosing which papers are published.

None others received.

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