Exploiting cis-limited antigens in livestock
Lead Research Organisation:
University of Cambridge
Department Name: Pathology
Abstract
The ability to pre-select the sex of offspring would be hugely important in animal husbandry. In many farm species such as pigs or dairy cattle, female offspring are much more economically important than males, while in other species such as racehorses or beef cattle, the opposite situation holds. In particular, meat from adult male boars can have an "off" flavour known as boar taint, meaning it is uneconomical to raise intact male pigs to high weights. Currently, boar taint is avoided by early slaughter or castration of male pigs, which is ethically undesirable as well as economically wasteful.
We are trying to discover ways to sort sperm before fertilisation occurs, i.e. to distinguish between sperm bearing an X chromosome (which will give female offspring) and those bearing a Y chromosome (which will give males). Currently the only way to do this is by using a dye to stain the DNA in the sperm head, and separating the sperm that have more DNA from those that have less - this works because the X chromosome is slightly larger than the Y. This "flow-sorting" process is slow and cumbersome and not commercially viable for some species such as pig. Also, the flow-sorting process involves staining the DNA and shining an ultraviolet laser on it, both of which can potentially damage the DNA. There is thus a strong need for more efficient and safer ways of separating X and Y sperm. We are using two approaches to tackle the problem.
Firstly, we are looking to see whether X and Y sperm naturally carry markers on their surface allowing them to be distinguished. We will do this by using flow-sorting to separate developing X- and Y-bearing sperm and looking to see which genes are active in each cell type. If we find genes which are only switched on in one of the two cell types, these are likely to be be useful in developing ways to separate the two. We will carry out this search in normal mice, in a strain of mice which naturally produce 60% female offspring, and in a transgenic strain which has altered levels of a gene we believe to be involved. From previous work, we know that in these strains with a skewed sex ratio, equal numbers of X- and Y-bearing sperm are produced, but that the Y sperm are less effective at fertilising the egg. This means that these strains will help us to home in on genes of interest - i.e. the ones which make X and Y sperm different from each other.
Secondly, we want to test whether we can separate sperm using an artificial surface marker. To do this, we will breed transgenic mice which express a marker protein in their sperm cells. Ideally, sperm cells containg the transgene will show this "tag" on the cell surface, while those which do not contain the transgene will remain untagged. This will then allow us to separate the two types of sperm and only use untagged sperm to breed the next generation. In livestock, this would enable sperm selection while preventing genetically modified organisms entering the food chain.
We are trying to discover ways to sort sperm before fertilisation occurs, i.e. to distinguish between sperm bearing an X chromosome (which will give female offspring) and those bearing a Y chromosome (which will give males). Currently the only way to do this is by using a dye to stain the DNA in the sperm head, and separating the sperm that have more DNA from those that have less - this works because the X chromosome is slightly larger than the Y. This "flow-sorting" process is slow and cumbersome and not commercially viable for some species such as pig. Also, the flow-sorting process involves staining the DNA and shining an ultraviolet laser on it, both of which can potentially damage the DNA. There is thus a strong need for more efficient and safer ways of separating X and Y sperm. We are using two approaches to tackle the problem.
Firstly, we are looking to see whether X and Y sperm naturally carry markers on their surface allowing them to be distinguished. We will do this by using flow-sorting to separate developing X- and Y-bearing sperm and looking to see which genes are active in each cell type. If we find genes which are only switched on in one of the two cell types, these are likely to be be useful in developing ways to separate the two. We will carry out this search in normal mice, in a strain of mice which naturally produce 60% female offspring, and in a transgenic strain which has altered levels of a gene we believe to be involved. From previous work, we know that in these strains with a skewed sex ratio, equal numbers of X- and Y-bearing sperm are produced, but that the Y sperm are less effective at fertilising the egg. This means that these strains will help us to home in on genes of interest - i.e. the ones which make X and Y sperm different from each other.
Secondly, we want to test whether we can separate sperm using an artificial surface marker. To do this, we will breed transgenic mice which express a marker protein in their sperm cells. Ideally, sperm cells containg the transgene will show this "tag" on the cell surface, while those which do not contain the transgene will remain untagged. This will then allow us to separate the two types of sperm and only use untagged sperm to breed the next generation. In livestock, this would enable sperm selection while preventing genetically modified organisms entering the food chain.
Technical Summary
This project applies two complementary approaches to investigate the molecular control of offspring gender balance: (1) a search for endogenous sex specific antigens in a mouse model system using transcriptomic technology; (2) development of a sperm "tagging" system allowing for antigenic separation of X- and Y-bearing sperm.
(1) Male mice with deletions on Yq show a non-Mendelian sex ratio among their offspring caused by a functional difference in the fertilising ability of X- and Y-bearing sperm. We reason that this functional difference must be rooted in a difference in the transcript content of the developing X- and Y- cells. Alternative explanations, such as a difference in translation efficiency or post-translation modification, pose a bootstrapping problem: how does that difference itself arise? We will carry out a systematic search for the predicted "responder" gene via microarray analysis of purified, flow sorted spermatid populations in Yq deleted mice, in matched controls, and in mice transgenic for a candidate distorter gene.
(2) We will generate "marker" transgenes that selectively label transgene-bearing sperm. IVF protocols for sorting these sperm will be developed, and finally the tag system will be deployed in a mouse model to allow for sperm selection.
(1) Male mice with deletions on Yq show a non-Mendelian sex ratio among their offspring caused by a functional difference in the fertilising ability of X- and Y-bearing sperm. We reason that this functional difference must be rooted in a difference in the transcript content of the developing X- and Y- cells. Alternative explanations, such as a difference in translation efficiency or post-translation modification, pose a bootstrapping problem: how does that difference itself arise? We will carry out a systematic search for the predicted "responder" gene via microarray analysis of purified, flow sorted spermatid populations in Yq deleted mice, in matched controls, and in mice transgenic for a candidate distorter gene.
(2) We will generate "marker" transgenes that selectively label transgene-bearing sperm. IVF protocols for sorting these sperm will be developed, and finally the tag system will be deployed in a mouse model to allow for sperm selection.
Planned Impact
This project aims to discover specific cell surface antigens allowing the separation of X and Y-bearing sperm and the pre-selection of offspring sex. We also aim to develop synthetic "tags" selectively identifying transgenic sperm. These goals, if realised, would have very substantial commercial impacts. Beneficiaries include, but are not limited to:
1) Farming industry
Pre-selection of offspring sex would reduce the number of "undesirable" offspring generated. This will mean less culling of unwanted males/females, more efficient meat production requiring smaller breeding populations, and reduced waste generation / environmental impact.
2) Rare species conservation
Captive breeding programmes for endangered species rely critically on the generation of breeding females to maintain the population. Offspring sex selection would thus help ensure the stability of such schemes.
3) Research and biotechnology companies producing transgenic animals
In some species, current methods of targeted transgenesis require the creation of chimeric founders, which transmit the transgene to subsequent generations at high or low levels depending on the degree of germ line chimerism. The ability to specifically sort transgenic sperm from non-transgenic sperm would make this latter step more efficient.
In terms of timescale, if we are able to identify suitable cell surface markers on X and Y sperm, then commercial development and testing could begin rapidly, within the duration of this project, subject of course to regulatory approval. The sperm tagging project is currently pre-competitive and will require further rounds of optimisation before a commercial sex selection technology can be developed (5-7 years). However, if the search for endogenous cell surface markers is unsuccessful, then the transgenic tag technology is a plausible route to selection of X and Y sperm, given that flow-sorting is slow, cumbersome and not commercially viable in most farm species.
1) Farming industry
Pre-selection of offspring sex would reduce the number of "undesirable" offspring generated. This will mean less culling of unwanted males/females, more efficient meat production requiring smaller breeding populations, and reduced waste generation / environmental impact.
2) Rare species conservation
Captive breeding programmes for endangered species rely critically on the generation of breeding females to maintain the population. Offspring sex selection would thus help ensure the stability of such schemes.
3) Research and biotechnology companies producing transgenic animals
In some species, current methods of targeted transgenesis require the creation of chimeric founders, which transmit the transgene to subsequent generations at high or low levels depending on the degree of germ line chimerism. The ability to specifically sort transgenic sperm from non-transgenic sperm would make this latter step more efficient.
In terms of timescale, if we are able to identify suitable cell surface markers on X and Y sperm, then commercial development and testing could begin rapidly, within the duration of this project, subject of course to regulatory approval. The sperm tagging project is currently pre-competitive and will require further rounds of optimisation before a commercial sex selection technology can be developed (5-7 years). However, if the search for endogenous cell surface markers is unsuccessful, then the transgenic tag technology is a plausible route to selection of X and Y sperm, given that flow-sorting is slow, cumbersome and not commercially viable in most farm species.
Publications
Cocquet J
(2012)
A genetic basis for a postmeiotic X versus Y chromosome intragenomic conflict in the mouse.
in PLoS genetics
Riel JM
(2013)
Deficiency of the multi-copy mouse Y gene Sly causes sperm DNA damage and abnormal chromatin packaging.
in Journal of cell science
Vernet N
(2014)
The expression of Y-linked Zfy2 in XY mouse oocytes leads to frequent meiosis 2 defects, a high incidence of subsequent early cleavage stage arrest and infertility.
in Development (Cambridge, England)
Vernet N
(2016)
Zfy genes are required for efficient meiotic sex chromosome inactivation (MSCI) in spermatocytes
in Human Molecular Genetics
| Description | This grant was focussed on identifying regions of certain gene expressed in mouse testis that restrict the mRNA to a single male germ cell. As male germ cells develop, they are connected by bridges that allow male the germ cells to develop as a cohort. This means that genes expressed as mRNA and protein can diffuse between cells and this has the effect of equalising expression of genes on the X and Y chromosomes between X and Y bearing germ cells. Certain genes do not show this sharing and hence contain signals that restrict them to the cell within which they are expressed. This grant has sought to use such sequences to make constructs carrying markers that can be exploited for sex selection which, when introduced in to male germ cells, will not be shared between X and Y bearing cells. We have based these constructs carrying the killer red protein (which when exposed to light can ablate the cell within which it is expressed) on the Smok 2b gene known to avoid sharing in sperm - it is said to be cis-limited. By linking parts of this gene to killer red we have made gene constructs to make transgenic animals; that is, mice carrying the transgene in their germ cells. These founder animals were shown to be able to transmit this transgene to their offspring. Using the approach of RNA in situ hybridization where a probe for the transgene is used to test the expression of the gene in individual germ cells, we were able to show that only 50% of the cells at the round spermatid/elongating spermatid stage of non-dividing and maturing germ cells contained the expressed gene. This indicates that we have been successful in using sequences in the Smok 2 b gene to limit sharing of the transgene. Work is on going to target the killer red transgene to either the X or Y chromosomes and hence (because the product will be limited to either an X or Y bearing cell) be able to selectively ablate X or Y bearing sperm. This would have great significance for the animal breading industries where a particular sex is preferred. The second part of this project was focussed on searching for sex-linked genes that do not show sharing between germ cells. We postulate that such genes exist because of a mouse model we are examining that shows a sex ratio skew in favour of females as a consequence of deletions of the mouse Y chromosome long arm. In a previous study we have shown that this deletion leads to an up-regulation of genes on the X chromosome indicating (a) a genomic conflict between the X and Y and (b) that a gene on the X (or regulates a gene elsewhere that) is responsible for the sex skew in favour of females. We have reasoned that such cis-limited gene products (mRNA or protein may be tightly bound to cellular structures such as the cytoskeleton or cell membranes to prevent their sharing with other cells. As a result we have carried out a cell sub compartment fractionation procedure for germ cells at two stages of development after they have stopped dividing; round spermatids and elongating spermatids. This procedure separates out tightly bound, moderately bound and free messenger RNA molecules. We have conducted detailed micro-array analysis of the these messenger RNA fractions to determine the number of genes in each fraction and which of those are derived from the X chromosome. This has identified about 80-90 genes on the X that are in the tightly/moderately bound fractions that are candidates for cis-limited genes. We are currently investigating these genes for their cis-limitation. |
| Exploitation Route | We have demonstrated that cis-limitation of a transgene is possible by introducing the correct signals. We need to take this work forward to introduce the transgene into the X or Y chromosome to demonstrate selective ablation of X or Y bearing cells with the killer red protein. This technology would be very useful for the animal breeding industry, especially pigs where the preferred sex is female. Transfer of this approach to pigs would therefore be a very useful economic and welfare development as it would avoid culling and castration of male pigs. |
| Sectors | Agriculture, Food and Drink |
| Description | Collaboration with Professor Bruce Whitelaw |
| Organisation | University of Edinburgh |
| Department | The Roslin Institute |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We have established a collaboration with Professor Bruce Whitelaw to develop technologies for semen sexing in farm animal species. We are providing intellectual property and technical know-how in regard to the construction of selectable markers. Details are commercially sensitive. |
| Collaborator Contribution | We have established a collaboration with Professor Bruce Whitelaw to develop technologies for semen sexing in farm animal species. He is providing transgenesis technology and technical assistance in clone preparation. Details are commercially sensitive. |
| Impact | No outputs with impact yet. |
| Start Year | 2014 |