BOVINE SEMEN SEXING:

OPPORTUNITIES FROM
TRANSGENIC TECHNOLOGIES
Dr. Muralidhar METTA
MVSc, PhD (Austria), Postdoc (Belgium)
Why semen sexing?
• Most desired technology in livestock industry
• Can accelerate genetic improvement of livestock
• In cattle production, an efficient and accurate system of
sperm sexing combined with AI would have potential
dramatic effects on the efficiency of milk and meat
production
• Dairy industry –
– Economic production of replacement heifers
• Beef industry –
– Production of steers for a commercial unit
Current methods of semen sexing
• Attempts based on various differences between X- and Y-
bearing spermatozoa –
– Density
– pH sensitivity
– Swimming speed
– Surface charge
– H-Y antigen content
– Motility characters
• Breakthrough invention-
– Separation based DNA content
(X- bearing spermatozoa contains 3-4% more DNA than Y-)
Current methods of semen sexing

Beltsville sperm sexing technology

Current methods of semen sexing
• Sexing of morula or blastocyst stage embryos by genotyping
biopsies with Y-specific probes is also used quite widely

• Problems –
– Reduced fertility
– DNA binding dye may cause mutations
– Expensive (3 times costlier than unsorted sperm)
– Accuracy is about 90%
– Obtaininng sufficient quantities of sexed semen
Alternatives for sperm/embryo sexing?

• Marking of one of the sex-chromosome with

surface expressing molecule

X X X Y
female male
Alternatives for sperm/embryo sexing?

• Marking of one of the sex-chromosome with

surface expressing molecule
• Transgenics?

X Y
Alternatives for sperm/embryo sexing?

Express at right developmental stage (embryo sexing)

Alternatives for sperm/embryo sexing?

Express post-meiotically (sperm sexing)

Alternatives for sperm/embryo sexing?

Feasible to engineer genes on to mammalian sex

chromosome to show expression in only one sex

Pirottin et al., 2005;

PNAS
Mammalian sex determination

The Y chromosome plays main role in sex

determination
Mammalian spermatogenesis
Issues of transgenics or semen sexing
• Gene expression during spermatogenesis
• Male meiotic sex chromosome inactivation
• Haploid gene expression
Issues of transgenics or semen sexing
• Gene expression during spermatogenesis

Transcriptionally
inactive

Relics of
spermatogenesis
A right promotor?
Issues of transgenics or semen sexing
• Male meiotic sex chromosome inactivation

Cloutier and Turner, 2007;

Current Biology
Inactivation due to unsynapsis ? ssty1, ssty2 and sly on the Y chromosome are
expressed at spermatid stage
Issues of transgenics or semen sexing
• Haploid gene expression
Issues of transgenics or semen sexing
• Haploid gene expression

Wild type smok gene and its

regulatory elements are
sufficient to have haploid gene
expression

Véron et al., 2009; Genome Research

Technologies
RMCE
Technologies
Technologies
CRISPR- CAS9
Haploid Gene expression in mouse
• Creation of transgenic mice expressing green
fluorescent protein specific to Y-chromosome
bearing spermatids – can we avoid diffusion of
gene products across spermatids?
Construct

mP1 Promoter

Smok2b 3’UTR

Smok2b 5’UTR
EGFP
Smok2b CDS
 pcDNA3- pcDNA3-EGFP pcDNA3
smok-EGFP (+ve control) (-ve control)
Translation potential of smok-GFP fusion gene
Analysis of the transgenic founders
[43]
Determine sex, ear tagging and
Tail biopsy at 3 weeks of age for DNA
extraction
PCR with eGFP specific primers to detect presence of transgene

founders founders founders

+ve
-ve
-ve
+ve
-ve
-ve
Analysis of F1 offspring
Founder sex F1 male F1 positive female F1 positive
2504 male 23 8 2 15 0
2507 female 27 11 5 16 6
2533 male 54 29 0 25 0
2540 male 37 18 2 19 2
2541 female 42 27 17 15 6
2550 male 28 10 3 18 6
2554 male 16 7 1 9 5
2556 male 37 18 12 19 9
2562 female 13 7 3 6 0
    277 135 45 142 34
Immunohistochemistry on testis cryosections

Testis of F1 offspring from 2507 individual

Immunohistochemistry on testis cryosections

GFP –ve
spermatozoa
Unanswered……
• RNA- FISH – to prove haploid expression
• Generation of homozygous transgenic controls
• Immunocytochemistry on sperm cells
• IHC with α-tubulin for spermatozoa
Issues with transgenic technologies
• Genetic considerations
– Phenotypic
– Long demonstration phase
– Genetic lag – requires elite animals
• IP considerations – protected by patents
• Regulatory requirements, consumer acceptance and
political support
Conclusion
• With transgenic technology, it is feasible to
engineer bovine Y-chromosome with an
application for semen sexing
Acknowledgements
Dr. Michel Georges,
DVM, PhD
Director
GIGA-ULG
Belgium
THANK YOU

Questions?