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

Lead Research Organisation: University of Cambridge
Department Name: Pathology

Abstract

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.

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.

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. 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.
 
Title Additional file 10: Figure S1. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Tissue sampling. Sampling porcine ear tissues using an ear notcher (A). B and C represent the procedure, the tube in D contains tissue in preservative. For each animal additional husbandry and morphological information (e.g. weight, height, length: D) was recorded on farm. (TIF 400Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_10_Figure_S1_of_Origin_and_phylo...
 
Title Additional file 10: Figure S1. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Tissue sampling. Sampling porcine ear tissues using an ear notcher (A). B and C represent the procedure, the tube in D contains tissue in preservative. For each animal additional husbandry and morphological information (e.g. weight, height, length: D) was recorded on farm. (TIF 400Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_10_Figure_S1_of_Origin_and_phylo...
 
Title Additional file 2: Figure S3. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Variation in coat colours of local Ashanti Dwarf pigs (ADP) of Ghana. Image A and B show spotting and patchy coat colours appearing in the litters of selected â purebredâ ADPs, whilst C and D show spotted and belted patterns in local livestock of known mixed ancestry. (TIF 535Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_2_Figure_S3_of_Origin_and_phylog...
 
Title Additional file 2: Figure S3. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Variation in coat colours of local Ashanti Dwarf pigs (ADP) of Ghana. Image A and B show spotting and patchy coat colours appearing in the litters of selected â purebredâ ADPs, whilst C and D show spotted and belted patterns in local livestock of known mixed ancestry. (TIF 535Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_2_Figure_S3_of_Origin_and_phylog...
 
Title Additional file 3: Figure S4. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Distribution of coat colour in local Ghanaian pigs. Panel 4A shows the distribution of coat colour in local ADP compared with local crossbred animals. Panel 4B shows the distribution of coat colour phenotypes by agro-ecological zones: Guinea Savannah (GSZ: UWR and NR); Forest (FZ: ER and AR); Coastal (CZ: CR and GAR); and by local pig classification into ADP or crossbred. In each panel, nâ =â total number of animals sampled. (TIF 98Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_3_Figure_S4_of_Origin_and_phylog...
 
Title Additional file 3: Figure S4. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Distribution of coat colour in local Ghanaian pigs. Panel 4A shows the distribution of coat colour in local ADP compared with local crossbred animals. Panel 4B shows the distribution of coat colour phenotypes by agro-ecological zones: Guinea Savannah (GSZ: UWR and NR); Forest (FZ: ER and AR); Coastal (CZ: CR and GAR); and by local pig classification into ADP or crossbred. In each panel, nâ =â total number of animals sampled. (TIF 98Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_3_Figure_S4_of_Origin_and_phylog...
 
Title Additional file 4: Figure S5. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Distribution of Asian and European MC1R alleles in local pigs. Panels A and B show the distribution based on whether the alleles defined are of European or Asian origin by local classification (ADP or crossbred) and by agro-ecological zone (GSZ = Guinea Savannah zone; FZ = Forest Zone; CZ = Coastal Zone). In panels C and D the haplotypes are defined in greater detail. In each panel, n = total number of animals sampled (TIF 158 kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_4_Figure_S5_of_Origin_and_phylog...
 
Title Additional file 4: Figure S5. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Distribution of Asian and European MC1R alleles in local pigs. Panels A and B show the distribution based on whether the alleles defined are of European or Asian origin by local classification (ADP or crossbred) and by agro-ecological zone (GSZ = Guinea Savannah zone; FZ = Forest Zone; CZ = Coastal Zone). In panels C and D the haplotypes are defined in greater detail. In each panel, n = total number of animals sampled (TIF 158 kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_4_Figure_S5_of_Origin_and_phylog...
 
Title Additional file 6: Figure S6. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description A: Principal Component Analysis of local pigs of Ghana based on SNP genotyping. (AR = Ashanti region; CR = Central region; ER = Eastern region; GAR = Greater Accra region; NR = Northern region; UWR = Upper West region). B: PCA analysis of Ghanaian pigs and European and Asian populations. (TIF 244 kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_6_Figure_S6_of_Origin_and_phylog...
 
Title Additional file 6: Figure S6. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description A: Principal Component Analysis of local pigs of Ghana based on SNP genotyping. (AR = Ashanti region; CR = Central region; ER = Eastern region; GAR = Greater Accra region; NR = Northern region; UWR = Upper West region). B: PCA analysis of Ghanaian pigs and European and Asian populations. (TIF 244 kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_6_Figure_S6_of_Origin_and_phylog...
 
Title Additional file 7: Figure S8. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description FST values along chromosome 7, comparing ADP subgroup 1 and Duroc. The horizontal lines show the 99% and 95% genome-wide FST threshold values. Distinct regions of interest can be seen at ~30Â Mb and 55Â Mb with high FST. (TIF 139Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_7_Figure_S8_of_Origin_and_phylog...
 
Title Additional file 7: Figure S8. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description FST values along chromosome 7, comparing ADP subgroup 1 and Duroc. The horizontal lines show the 99% and 95% genome-wide FST threshold values. Distinct regions of interest can be seen at ~30Â Mb and 55Â Mb with high FST. (TIF 139Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_7_Figure_S8_of_Origin_and_phylog...
 
Title Additional file 8: Figure S7. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description FST values around the LCORL region of porcine chromosome 8. The upper panel shows population 1 against other European breeds, and the lower panel the population 2 ADPs against the same European breeds. The yellow bar represents the region 5â to the LCORL locus, which harbours the peak SNP adjacent to this gene (*). (TIF 67Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_8_Figure_S7_of_Origin_and_phylog...
 
Title Additional file 8: Figure S7. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description FST values around the LCORL region of porcine chromosome 8. The upper panel shows population 1 against other European breeds, and the lower panel the population 2 ADPs against the same European breeds. The yellow bar represents the region 5â to the LCORL locus, which harbours the peak SNP adjacent to this gene (*). (TIF 67Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_8_Figure_S7_of_Origin_and_phylog...
 
Title Additional file 9: Figure S2. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Distribution of mitochondrial sequences across agro-ecological zones. Panel 2A shows the distribution of sequences according to their clustering into Asian or European clades by region: Guinea Savannah Zone (GSZ: UWR and NR); Forest Zone (FZ: ER and AR); Coastal Zone (CZ: CR and GAR). Panel 2B further subdivides the data according to local pig classification into ADP or crossbred. Panels 2C and 2D provide the same breakdown by sequence haplotype. In each panel, nâ =â total number of animals sampled. (TIF 150Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_9_Figure_S2_of_Origin_and_phylog...
 
Title Additional file 9: Figure S2. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Distribution of mitochondrial sequences across agro-ecological zones. Panel 2A shows the distribution of sequences according to their clustering into Asian or European clades by region: Guinea Savannah Zone (GSZ: UWR and NR); Forest Zone (FZ: ER and AR); Coastal Zone (CZ: CR and GAR). Panel 2B further subdivides the data according to local pig classification into ADP or crossbred. Panels 2C and 2D provide the same breakdown by sequence haplotype. In each panel, nâ =â total number of animals sampled. (TIF 150Â kb) 
Type Of Art Film/Video/Animation 
Year Produced 2017 
URL https://springernature.figshare.com/articles/figure/Additional_file_9_Figure_S2_of_Origin_and_phylog...
 
Description We have designed a series of constructs that permit insertion of the protein product into cell membranes and allow expression of markers on the cell surface in cell lines that can be detected with specific antibodies. We have made transgenic animals with the same constructs where gene expression is under the control of a testis spermatid specific promoter. The constructs are expressed as RNA in mice testis but we have been unable thus far to detect protein expression in vivo. We are currently addressing this issue.
Exploitation Route Not at this stage.
Sectors Agriculture

Food and Drink

 
Title Additional file 1: Table S1. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Number of animals sampled per agro-ecological region, based on farms and locations. Table S2. A summary of mitochondrial haplotypes. Table S3. Results from Y chromosome derived polymorphisms. Table S4. Summary of coat colour information, sequence polymorphisms, and predicted haplotypes in the MC1R gene from animals collected in Ghana. Table S5. A summary of the intervals defined by FST analysis, with genes, flanking genes for each intergenic region, and genes at peaks commonly seen between between ADP subset 1 and ADP subset 2 comparisons with other breeds identified in bold. Table S6. DAVID functional annotation results showing clustering of genes (pâ 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/dataset/Additional_file_1_Table_S1_of_Origin_and_phylog...
 
Title Additional file 1: Table S1. of Origin and phylogenetic status of the local Ashanti Dwarf pig (ADP) of Ghana based on genetic analysis 
Description Number of animals sampled per agro-ecological region, based on farms and locations. Table S2. A summary of mitochondrial haplotypes. Table S3. Results from Y chromosome derived polymorphisms. Table S4. Summary of coat colour information, sequence polymorphisms, and predicted haplotypes in the MC1R gene from animals collected in Ghana. Table S5. A summary of the intervals defined by FST analysis, with genes, flanking genes for each intergenic region, and genes at peaks commonly seen between between ADP subset 1 and ADP subset 2 comparisons with other breeds identified in bold. Table S6. DAVID functional annotation results showing clustering of genes (pâ 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/dataset/Additional_file_1_Table_S1_of_Origin_and_phylog...
 
Title Nuclear Morphology Analysis 
Description The software allows the detection of stained nuclei from microscope images, and automatically orients them and detects key landmarks. This allows for quantitative morphological analysis of large numbers of nuclei. It has been developed initially for analysis of cells with asymmetric nuclei such as mouse and pig sperm. The software is written in Java and implemented as a plugin to the open source image analysis program ImageJ. 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact It has allowed us to establish collaborations leading to the application of this software in number of different biological systems. These are: (1) Comparison of nuclear shape of sperm from a number of different mouse line; (2) Nuclear shape in neutrophils in the disease process ARDS; (3) Comparison of morphology of sperm in mice where intrauterine exposures (endocrine insults) are correlated with intergenerational changes in non-coding RNA expression; (4) Comparison of sperm morphology in fertile versus sub-fertile boars. 
URL https://bitbucket.org/bmskinner/nuclear_morphology/wiki/Home