Molecular tools in reproductive research

1. M olecular tools in reproductive
research
Jerome. A, A.K. Balhara & Inderjeet Singh
(c) Jerome A

2.  All aspects of life are engineered at the
molecular level, without understanding
molecules we can only have a very sketchy
understanding of life itself.
Francis
Crick
 The beginnings of life are closely associated with
the interactions of proteins and nucleic acids.
Florence O.
Bell
 Biotechnology has the potential to bring
tremendous
benefits
John Prescott
(c) Jerome A

3. Fertility
Fertility - measure of reproductive success.
Profitability (Milk / Meat yield), Procreation next generation,
Breeding progress & sustainability of animal production
Royal et al . (2000)
Female
• Behavioural expression of oestrus Van Eerdenburg
(2006)
• proper ovarian cyclicity Opsomer et
al . (2002)
• Success rate of AI Royal et
al . (2000)
• Embryo survival Sheldon et
al . (2006)
Male
• Proper libido
• Production of gametes
Complex feature - Influence of Jerome A
(c) numerous genes, working together to
produce functional gametes, early embryonic and

4. Reproductive loss
Male Con. techniques Female
Low sperm Anoestrus
count Intrinsic Repeat breeding
Abnormal
Extrinsic Endometritis
sperms
COD
Poor libido
Silent oestrus
Genital
diseases MOLECULAR Genetic diseases
TOOLS
Genetic
diseases
(c) Jerome A

5. Molecular Tools ????
(c) Jerome A

6. Molecular tools
 The study of biochemistry at a molecular level.
 Overlaps with biology, chemistry, and genetics.
 Understanding & interactions between the cell’s DNA,
RNA and protein with their regulation mechanism.
Why MT in reproduction?
 Understanding of the underlying mechanisms of
fertility/infertilty
 Improved understanding of genetic variation
 Provide tools to enhance fertility
 Improve diagnosis of fertilityA disorders
(c) Jerome

7. 1869 - DNA discovered (Meischer)
1944 - DNA genetic material (Avery, Mcleod & Mc carrty) H
1953 - DNA structure (Watson& Crick, wilkins)
1959 - Nanotechnogy (Feymann)
I
1961 - RNA discovery s
(Brenner, Jacob & Meseleson )
1972 -
1974 -
Recombinant DNA made in vitro (P. Berg)
I Generation ml. markers
t
1980 - Word nanotechnology (Drexler) o
1986 -
1990 -
Creation of PCR (K. Mullis)
II Generation. mol. markers
r
1995 - Proteomics (Wilkins) y
1997 - Dna microarray (Augenlicht)
2000 - Transcriptomics (ABI sysyems)
2006 - RNAi (Fire and Mello)
(c) Jerome A

8.  Genetic tools
 Molecular biology tools
•Genomics, transcriptomics &
others
•RNA interference (RNAi)
 Proteomics
(c) Jerome A

9. Genetic tools
(c) Jerome A

10. Genetic tools involve the deciphering genetic markers
Genetic marker
A stable and inherited variation at the morpholog
chromosomal,
biochemical or DNA level measured or detected by a suitable
method
Used subsequently to detect the presence of a specific genotyp
or phenotype. High polymorphism, Random distribu
throughout the
genome
Reproductive traits Markers affect oocyte growth, viability, em
growth, semen fertility etc.
DNA isolated from any tissue, at any stage even during
foetal life, longer shelf-life readily exchangeable b/w
labs, at early age/ even at the embryonic and
irrespective of sex .
(c) Jerome A

11. Molecular markers two groups:
I) Method (Southern-blot hybridization-based & PCR-based DNA
II) Utility (Single or multi loci)
Southern-blot hybridization-based markers:
Labeling the genes with probes
Restriction Fragment Length Polymorphisms (RFLP).
Variable number of tandem repeats (VNTR),
PCR-based DNA markers:
Highest resolution of DNA variation can be obtained using
sequence analysis.
Random Amplified Polymorphic DNA (RAPD),
Simple Sequence Repeats (SSR),
Amplified Fragment Length Polymorphisms (AFLP),
Single Nucleotide Polymorphism (SNP)
(c) Jerome A

12. Molecular Markers
Single locus marker
Single locus marker Multi-locus marker
Multi-locus marker
RFLP
RFLP AFLP
AFLP
Microsatellite RAPD
RAPD
Microsatellite
(c) Jerome A

13. Randomly Amplified Polymorphic DNA (RAPD)
PCR based marker with 10-12 base pairs poor
repeatability
Restriction Fragment Length Polymorphism (RFLP):
Genomic DNA digested with Restriction Enzymes
Amplified Fragment Length Polymorphism (AFLP):
Restriction endonuclease digestion of DNA & Ligation
of adaptors
Simple Sequence Repeat or Microsatellite
PCR based markers with 18-25 base pair primers
SNP ( Single Nucleotide Polymorphisms)
Occur much more frequently throughout the genome
(c) Jerome A
DFP : DNA finger printing

14. Application
Determination of twin zygosity & freemartins
Sexing of pre-implanted embryos
Identification of disease carries
– ( Robertsonian translocation between chromosomes 1 and
29)
Various SNPs have been detected in genes of hormones
FSH, LH, GnRH, oxytocin, estrogen, progesterone &
hormone receptors.
( Yang – 2010, Marín – 2007, Szreder – 2004,
Zalata, 2008)
SNP - Disrupt synthesis, mode and place of action of
hormones on the reproductive system
(c) Jerome A

15. Various SNPs in cytokines regulating embryonic stage during
pregnancy
IFN-tau , GH , PRL , GHR , PRLR , STAT5A , OPN , UTMP
Male
Khatib,
(2009 )
GH- testicular development
α -Actinins ( ACTN1) & γ -actin ( ACTG2 )
Fertility collagen type I alpha 2 gene Gorbani
(2009)
Sperm concentration, motility, semen volume per
ejaculate, plasma droplets rate, abnormal sperm rate
and the fertility traits, integrin
(c) Jerome A

16. Genomics &
Proteomics
(c) Jerome A

17. Oogenesis, spermatogenesis, Folliculogenesis & embryogenesis
- complex and coordinated biological processes
Series of events - induce morphological & functional changes
within the follicle, sperm leading to gamete differentiation,
development with corresponding behaviour.
Expression profiling of transcriptomes / proteome of
reproductive tissues, usually across different stages, conditions.
High throughput technologies - Microarray,
Proteomics
(c) Jerome A

18. Oocyte maturation (Vallee,2005)
Non–regressed/regressed Corpus luteum (Casey,
2005)
Oviductal cell function
(Bauersachs, 2003)
Endometrium during estrus (Bauersachs,
2005)
Pregnancy (Hashizume,
2007)
Implantation , embryonic development
(Ushizawa, 2004)
Early fetal development (Mamo,
2006)
Uterine Transcriptomic changes during pregnancy (Ishiwata,
2003)
(c) Jerome A
Inferior embryos (Corcoran,

19. Candidate genes for the developmental competence of bovine
Oocytes, sperm and embryos.
Oocyte, sperm & embryo competence, uterine receptivity
markers:
Competent oocyte- STAT3, AURKA, SMARCC1, CYP19, PlGF genes
Degenerated oocyte- IGFBP5, GADD45A, TSP2, Fas, Bcl
(chromatin modeling, cytoplasmic development, activator of transcription,
apoptosis )
Vallee, 2005
(c) Jerome A

20. Transcriptome Analysis of Bull Semen.
Spermatozoa are terminally differentiated cells produced
during the complex process of spermatogenesis.
Lalancette - 2008
ESTRUS BEHAVIOUR
High fertile: Immunoglobulin superfamily, Protein phosphatae,
SCO-spondin
Low fertile: Gamma actin, Desmin
Bauersachs,
2005
(c) Jerome A

21. Uterine receptivity
UTMP AGRN, LGALS3BP, LGALS9, USP18, PARP12 and
BST2.
Embryos
Normal embryo :
GLUT1 , G6PD, GAPDH , tubulin, DNMT , GATA6, IFITM3,
Glut3 (Unregulated)
Degenerated embryo :
CDX2 , ALOX15, BMP15, PLAU, PLAC8, FAS, caspase-3,
HSP (Upregulated)
Ishiwata, 2003
(c) Jerome A

22.  Epigenomics,
 Evolutionary genomics,
 Toxicogenomics,
 Nutrigenomics
 Mitochondriomics
(Stoughton, 2005; Ness,
2007). (c) Jerome A

23. Proteomics
(c) Jerome A

24. Proteomics is the large-scale study of proteins, structures &
functions.
Proteins are vital parts of living organisms, as they are the main
components of the physiological metabolic pathways of cells.
Identification of new fertility biomarkers, specific to normal/
abnormal reproductive health status.
Two analysis: Total protein complement, Positional
proteomics.
(c) Jerome A

25. BULL FERTILITY
High fertility group :
Glyceraldehyde 3-phosphate dehydrogenase, Phosphatidyl
ethanolamine- binding protein 1 , bovine mitochondrial F1-ATPase, actin-
related proteinT2 , glutathione peroxidase, ropporin & enolase 1
Peddinti, 2009
EMBRYO PROTEOMIC
Proteomic analysis - unique protein profiles of embryos
embryonic development
Protein profiles of embryonic samples 32 potential
proteins/biomarkers
Abnormal embryoes - down-regulation of 10 biomarkers
(c) Jerome A Katz-Jaffe,
2009

26. Proteomic analysis of bovine conceptus fluids during early pregnancy
2-DE coupled with MALDI-TOF-MS/MS
>200 2-DE generated protein spots
(74 individual protein species identified)
MS/MS peptide identification of 105 LC-ESI-MS/MS
generated protein identities.
179 individual protein species specific for pregnancy (PAG,
PRL)
Kim,2009
(c) Jerome A

27. Applying high-throughput genomic ,proteomic technologies,
with mathematical modeling-
 Sub-fertility, infertility, loss of pregnancy & even
offspring with poor health status.
 Temporal sequence of events –interaction of gametes,
maternal communication with gametes & embryos in
health and disease.
 Oestrus mechanism and behaviour
 Fertility molecular markers for both in males and females
(c) Jerome A

28. RNA interference
(RNAi)
(c) Jerome A

29. RNA interference (RNAi) is a system within living cells that
takes part in controlling which genes are active and how
active they are.
RNAs are the direct products of genes, and these small
RNAs can bind to specific other RNAs and either increase
or decrease their activity
Control of gene expression and in functional genomics
(post-transcrip -tional gene silencing)
(c) Jerome A

30. Selective degradation of maternal and embryonic transcripts in in
vitro produced bovine oocytes and embryos using sequence specific
double-stranded RNA
Embryos with dsRNA specific for some genes the interference effect
is specific & can block expression of maternal gene products.
To study the effects of genes’ loss of function in developing
embryos without the complications of gene knockout method.
Korakot, 2006
Selective blocking / down regulation of C-mos, Cyclin B1,
Connexin, E-cadherin & Oct-4
Lethal genes disrupting reproduction can be silenced
(c) Jerome A

31. Conclusion
Reproduction
Normal / Diseases
Female / male
CON. M OLECULAR
TECHNIQUES TOOLS
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32. (c) Jerome A