Customised Surface Markers for the Separation of X and Y Bearing Mammalian Sperm
Lead Research Organisation:
University of Kent
Department Name: Sch of Biosciences
Abstract
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Technical Summary
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.
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.
Planned Impact
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.
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.
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.
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.
Organisations
Publications
Bergero R
(2021)
Meiosis and beyond - understanding the mechanistic and evolutionary processes shaping the germline genome
in Biological Reviews
Skinner BM
(2019)
A high-throughput method for unbiased quantitation and categorization of nuclear morphology†.
in Biology of reproduction
Rathje CC
(2019)
Differential Sperm Motility Mediates the Sex Ratio Drive Shaping Mouse Sex Chromosome Evolution.
in Current biology : CB
Rathje CC
(2019)
A Conserved Requirement for Fbxo7 During Male Germ Cell Cytoplasmic Remodeling.
in Frontiers in physiology
Ellis PJI
(2020)
Form from Function, Order from Chaos in Male Germline Chromatin.
in Genes
Kopania EEK
(2022)
The contribution of sex chromosome conflict to disrupted spermatogenesis in hybrid house mice.
in Genetics
Vernet N
(2016)
Zfy genes are required for efficient meiotic sex chromosome inactivation (MSCI) in spermatocytes.
in Human molecular genetics
Skinner B
(2022)
Nuclear Morphology Analysis 2.0.0: Improved image analysis software for measuring nuclear shape
in Journal of Open Source Software
Douglas C
(2021)
CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes.
in Nature communications
Description | The goal of this project was to place molecular "flags" on the sperm surface that can be used to separate X- and Y-bearing sperm to allow pre-specification of offspring sex. We developed and tested the molecular "flag" genes in cell culture, and showed that these "flags" can be displayed on the surface of cells, and used to separate different cell types. This was a key early milestone for our project, and indicates that we were initially progressing as expected. Using these "flag" genes , we generated male mice that express these "flag" genes in developing sperm cells, and showed that the mRNA transcripts for the genes were present in only half of the developing sperm cells. This was a second key milestone and showed that our "flag" genes were successfully able to label cells at the mRNA level. Unexpectedly, however, we were not to use the "flags" to successfully sort the sperm from these males. We found that this was because in the developing sperm cells the mRNA is expressed, but not translated into protein. Although unfortunately unsuccessful from an applied point of view, this work tells us something fundamental about the biology of sperm cell production. Subsequent to this setback, based in part on the work performed in this grant (but after the grant period) I was able to develop a novel transgenic method for controlling offspring sex, in collaboration with another group at the Francis Crick Institute. Using this method, embryos of the unwanted sex die shortly prior to implantation, and live-born offspring are all the same sex. We now have a proof-of-principle showing that this system works in mouse and anticipate that it should also work in other livestock species. This work has the potential for dramatic animal welfare gains in the livestock industry, where millions of unwanted male dairy calves and billions of unwanted male layer chickens are culled every year. |
Exploitation Route | In regard to semen sorting, we have shown that this strategy is unlikely to be viable in the near future. We have secured further funding to investigate the mechanisms of transcript sharing in more detail and will use this to inform our work in this area moving forwards. In regard to the transgenic method for offspring sex selection, we are exploring ways to apply this in the livestock industry and seeking funding (from funding bodies and industry) to take this forward ourselves. |
Sectors | Agriculture, Food and Drink |
Description | Genetics Society Summer Studentships 2017 |
Amount | £2,350 (GBP) |
Organisation | The Genetics Society |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 06/2017 |
End | 08/2017 |
Description | KTN/BSAS Summer 2016 Vacation Scholarship Awards |
Amount | £2,500 (GBP) |
Organisation | Biosciences Knowledge Transfer Network |
Sector | Public |
Country | United Kingdom |
Start | 07/2016 |
End | 08/2016 |
Description | Mechanisms of meiotic drive: how do genes break Mendel's laws? |
Amount | £282,284 (GBP) |
Funding ID | RPG-2019-194 |
Organisation | The Leverhulme Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2019 |
End | 08/2023 |
Title | Transgenic mouse lines to allow offspring sex ratio selection |
Description | In collaboration with the Francis Crick Institute we have developed mouse lines with integrated Cas9 on either the X or Y chromosomes, and a line with an autosomally integrated gRNA targeting the essential gene Top1. When a male carrying either X-Cas9 or Y-Cas9 is crossed to a female homozygous for the Top1 gRNA, this leads to inactivation of Top1 and embryo arrest in either female or male embryos respectively, allowing 100% efficient specification of offspring sex. This has the potential to reduce culling of live-born animals in many areas of research where only a single sex of offspring is required. |
Type Of Material | Model of mechanisms or symptoms - mammalian in vivo |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | We are monitoring the uptake of this method within the community. The publication of this method generated very substantial press coverage related to the potential for future use in livestock sex selection, including reporting on BBC1 News at Ten, BBC Radio 4, BBC World Service, and multiple national newspapers. |
URL | https://www.nature.com/articles/s41467-021-27227-2 |
Title | Nuclear Morphology Analysis |
Description | We have created an image analysis tool to quantitatively assess nuclear shape in sperm samples. It has been designed to automatically detect objects in fluorescence microscopy images, and from the detected objects identify and align mouse and pig sperm nuclei. The morphological analysis is based on a modified Zahn-Roskies transform, converting the outline of each nucleus into a trace describing the underlying curvature at each point along the periphery. Specific biological features of the sperm - such as the hook of mouse sperm, or the tail attachment region - can be identified from these traces, and used to align and compare nuclei. The software is implemented in Java as a plugin to the open source image analysis program ImageJ. |
Type Of Technology | Software |
Year Produced | 2016 |
Open Source License? | Yes |
Impact | We have used the software to compare sperm shape in a range of mouse strains, and in mice with a Y chromosome deletion affecting sex-ratio skew. This has identified regions of the nucleus that particularly differ under different genetic backgrounds, and points to developmental pathways impacted. The software has allowed rapid analysis of thousands of nuclei, uncovering subtle differences otherwise undetectable by conventional manual analysis. We have also developed collaborations with other groups to use and extend the software for analysis of mouse sperm in lines with particular genetic defects, as well as pig sperm in fertile and sub-fertile boars. |
URL | https://bitbucket.org/bmskinner/nuclear_morphology/wiki/Home |
Description | 2018 Article about sex chromosome evolution in The Conversation |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | This was an article in The Conversation (an online science magazine) discussing aspects of sex chromosome evolution, as a followup to a book chapter I (co-)authored. The article has so far received almost 2 million views, was reported widely in national and international media, and translated into multiple other languages for increased reach. |
Year(s) Of Engagement Activity | 2018 |
URL | https://theconversation.com/the-y-chromosome-is-disappearing-so-what-will-happen-to-men-90125 |
Description | 2019 Article in The Conversation about turtle sex determination |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | I wrote a commentary article for the online magazine The Conversation, which has a worldwide reach in promoting the latest scientific research. As of February 2021 the article has been read over 9,000 times |
Year(s) Of Engagement Activity | 2019 |
URL | https://theconversation.com/turtle-embryos-can-choose-their-own-sex-shows-new-research-but-why-12107... |
Description | 2020 Article in The Conversation about the potential presence of SARS-CoV-2 virus in semen |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | I wrote a commentary article for the online magazine The Conversation, which has a worldwide reach in promoting the latest scientific research. As of February 2021 the article has been read over 300,000 times |
Year(s) Of Engagement Activity | 2020 |
URL | https://theconversation.com/coronavirus-found-in-semen-of-young-men-with-covid-19-138059 |
Description | Class debate - Animal Research Ethics (Canterbury Christchurch University) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Undergraduate students |
Results and Impact | I give a guest lecture and class debate at Canterbury Christchurch University discussing the ethics of animal use in research and teaching them about the surrounding legislative framework. As part of this, I use my own work (lay summary) as one of a range of examples of the type of research done, the purpose for which it is done, and so on. This always sparks questions and debate within the class, from a range of different ethical standpoints. Feedback from the academics at Christchurch indicate that this session is a valued and useful part of the curriculum for their Animal Studies course. |
Year(s) Of Engagement Activity | 2016,2017,2018 |