Customised Surface Markers for the Separation of X and Y Bearing Mammalian Sperm

The pre-selection of offspring sex would be hugely beneficial 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, males are preferred. For example, adult male boar meat can have an "off" flavour known as boar taint, thus making it 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.

This project seeks to discover ways to separate sperm bearing an X chromosome (which will give females) and those bearing a Y chromosome (which will give males) before fertilisation occurs. 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 (a) slow and cumbersome and not commercially viable for some species such as pig where large doses of semen are required for artificial insemination and (b) involves staining the DNA and shining an ultraviolet laser on it, both of which can potentially damage the DNA. There is thus a strong demand for more efficient and safer ways of separating X and Y sperm.

This project aims to approach this issue by using modern molecular genetic transgenic techniques to create animals that produce sperm expressing an artificial surface marker. This will be done in such a way that either only X or Y bearing sperm have this marker on the surface. The marker can then be used to separate X or Y bearing sperm from each other. By depleting the semen of sperm that express the surface marker, this will leave an ejaculate population enriched in the sperm of the opposite sex that do not contain the artificial surface marker gene to be used to breed the next generation. In livestock, this would enable sperm selection while preventing genetically modified organisms entering the food chain.

This project seeks to apply transgenic strategies to express surface markers specifically on the surface of X or Y bearing sperm. Transgenes will be targeted to the Y chromosome such that they will only be expressed within Y bearing sperm. This requires (a) that the transgene products are not shared across the cytoplasmic bridges that form the spermatogenic syncytium linking differentiating germ cells, (b) that the transgene is under the control of a post-meiotic spermatid specific promoter active after the X and Y chromosomes have segregated into individual nuclei and (c) that the transgene is placed into active post-meiotic chromatin. We have demonstrated the creation of transgenic constructs that are expressed in post-meiotic germ cells and evade sharing which will form the basis for new transgenes containing domains that direct expression of selectable markers to the surface of mature sperm. This will be achieved through the use of (a) a combination of the acrosome signal sequence and GPI anchoring domain and (b) an alternative strategy using the signal sequence and transmembrane domain of the CD4 type 1 receptor.

Following verification of functionality in cell lines, constructs will be used to create transgenic animals. Evasion of sharing between germ cells and marker expression on the cell surface will be assessed by RNA in situ hybridization to testis sections and immunohistochemical staining for the construct protein product. Transgenes will be targeted to Y chromosome sequences using genome editing technologies (TALENs or CRISPRs). Functionality of sperm with (Y-bearing) or without (X-bearing) the transgene will be assessed by In Vitro Fertilization protocols to determine (a) the extent of sex ratio skew and (b) successful propagation of the transgene to male offspring. These experiments will initially be modelled in mouse as proof of principle and will provide the platform for application to economically important animals such as pigs and cattle.

This project aims to develop a precise technology to express customised cell surface molecules that can be used to facilitate the separation of X and Y-bearing sperm and pre-selection of offspring sex. Specifically, we propose to express avidin or streptavidin on the surface of mature sperm and effect separation of X and Y bearing sperm with the aid of biotin coated magnetic beads. The combination of surface expressed avidin/streptavidin and magnetic biotin coated beads provides a rapid, low technology and scalable means of separating X and Y bearing sperm. These goals, if realised, would have very substantial commercial and animal welfare impacts.

Pre-selection of offspring sex would reduce the number of "undesirable" offspring generated. This will mean less culling of unwanted males/females (and hence improved animal welfare), more efficient meat production requiring smaller breeding populations, and reduced waste generation / environmental impact. This would be a huge benefit to the pig and cattle breeding industries. There is an increased use of biotechnology solutions to animal welfare and environmental sustainability issues in the UK and world-wide, and we believe offspring sex selection via selective tagging of X- and Y-bearing sperm represents a significant commercial opportunity both as an improved method for cattle and as the first commercially viable method for pigs. For example, the UK industry harvests 7.9 million pigs per annum and boar taint is major source of economic loss. Fifty percent are males and if 3% exhibit boar taint 118K pigs are affected. Loss in value to the producer for carcasses downgraded due to boar taint represents an approximately £2.7 million annual loss for the industry. Currently the only alternative is to castrate males for markets with heavy slaughter weights, although legislation to ban castration without the use of anaesthetics is in process in the EU. Chemical castration (Pfizer's Improvac or Improvest), is approved in several countries in the world, but has not been licensed in the UK due to consumer concerns. Markets in the EU, US and the rest of the world would be highly significant. Thus the ability to specifically select females in the pig industry would have very significant economic, welfare and environmental impacts. This impact can be realised in subsequent work through our established partnership with Genus PLC both for cattle and pigs. This would provide the conduit to move from successful proof of principle in the mouse model to a commercial process that can improve the efficiency of meat production.

Successful application of this technology also has impact in terms of government policy on best practice and regulation of animal husbandry. In addition, there will be impacts on public opinion with respect to the use of transgenic approaches in food production. These areas will be explored during the course of the grant.