Molecular tools in reproductive research


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Molecular tools in reproductive research

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Molecular tools in reproductive research

  1. 1. M olecular tools in reproductiveresearch Jerome. A, A.K. Balhara & Inderjeet Singh (c) Jerome A
  2. 2.  All aspects of life are engineered at themolecular level, without understandingmolecules we can only have a very sketchyunderstanding of life itself. FrancisCrick The beginnings of life are closely associated withthe interactions of proteins and nucleic acids. Florence O.Bell Biotechnology has the potential to bringtremendous benefits   John Prescott (c) Jerome A
  3. 3. FertilityFertility - 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 gametesComplex feature - Influence of Jerome A (c) numerous genes, working together toproduce functional gametes, early embryonic and
  4. 4. Reproductive loss Male Con. techniques FemaleLow sperm Anoestrus count Intrinsic Repeat breeding Abnormal Extrinsic Endometritis sperms CODPoor libido Silent oestrus Genital diseases MOLECULAR Genetic diseases TOOLS Genetic diseases (c) Jerome A
  5. 5. Molecular Tools ???? (c) Jerome A
  6. 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 offertility/infertilty Improved understanding of genetic variation Provide tools to enhance fertility Improve diagnosis of fertilityA disorders (c) Jerome
  7. 7. 1869 - DNA discovered (Meischer)1944 - DNA genetic material (Avery, Mcleod & Mc carrty) H1953 - DNA structure (Watson& Crick, wilkins)1959 - Nanotechnogy (Feymann) I1961 - RNA discovery s (Brenner, Jacob & Meseleson )1972 -1974 - Recombinant DNA made in vitro (P. Berg) I Generation ml. markers t1980 - Word nanotechnology (Drexler) o1986 -1990 - Creation of PCR (K. Mullis) II Generation. mol. markers r1995 - Proteomics (Wilkins) y1997 - Dna microarray (Augenlicht)2000 - Transcriptomics (ABI sysyems)2006 - RNAi (Fire and Mello) (c) Jerome A
  8. 8.  Genetic tools Molecular biology tools •Genomics, transcriptomics & others •RNA interference (RNAi) Proteomics (c) Jerome A
  9. 9. Genetic tools (c) Jerome A
  10. 10. Genetic tools involve the deciphering genetic markers Genetic marker A stable and inherited variation at the morphologchromosomal, 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 distributhroughout thegenomeReproductive 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. 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 usingsequence analysis. Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeats (SSR), Amplified Fragment Length Polymorphisms (AFLP), Single Nucleotide Polymorphism (SNP) (c) Jerome A
  12. 12. Molecular MarkersSingle locus markerSingle locus marker Multi-locus marker Multi-locus marker RFLP RFLP AFLP AFLP Microsatellite RAPD RAPD Microsatellite (c) Jerome A
  13. 13. Randomly Amplified Polymorphic DNA (RAPD) PCR based marker with 10-12 base pairs poorrepeatabilityRestriction Fragment Length Polymorphism (RFLP): Genomic DNA digested with Restriction EnzymesAmplified Fragment Length Polymorphism (AFLP): Restriction endonuclease digestion of DNA & Ligationof adaptorsSimple Sequence Repeat or Microsatellite PCR based markers with 18-25 base pair primersSNP ( Single Nucleotide Polymorphisms) Occur much more frequently throughout the genome (c) Jerome A DFP : DNA finger printing
  14. 14. ApplicationDetermination of twin zygosity & freemartinsSexing of pre-implanted embryosIdentification of disease carries – ( Robertsonian translocation between chromosomes 1 and 29)Various SNPs have been detected in genes of hormonesFSH, LH, GnRH, oxytocin, estrogen, progesterone &hormone receptors. ( Yang – 2010, Marín – 2007, Szreder – 2004,Zalata, 2008)SNP - Disrupt synthesis, mode and place of action ofhormones on the reproductive system (c) Jerome A
  15. 15. Various SNPs in cytokines regulating embryonic stage duringpregnancyIFN-tau , GH , PRL , GHR , PRLR , STAT5A , OPN , UTMP Male Khatib,(2009 ) GH- testicular developmentα -Actinins ( ACTN1)   &  γ -actin ( ACTG2 )Fertility collagen type I alpha 2 gene Gorbani(2009) Sperm concentration, motility, semen volume perejaculate, plasma droplets rate, abnormal sperm rateand the fertility traits, integrin (c) Jerome A
  16. 16. Genomics & Proteomics (c) Jerome A
  17. 17. Oogenesis, spermatogenesis, Folliculogenesis & embryogenesis- complex and coordinated biological processesSeries of events - induce morphological & functional changeswithin the follicle, sperm leading to gamete differentiation,development with corresponding behaviour.Expression profiling of transcriptomes / proteome ofreproductive tissues, usually across different stages, conditions.High throughput technologies - Microarray, Proteomics (c) Jerome A
  18. 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 AInferior embryos (Corcoran,
  19. 19. Candidate genes for the developmental competence of bovineOocytes, sperm and embryos.Oocyte, sperm & embryo competence, uterine receptivitymarkers:Competent oocyte- STAT3, AURKA, SMARCC1, CYP19, PlGF genesDegenerated oocyte- IGFBP5, GADD45A, TSP2, Fas, Bcl(chromatin modeling, cytoplasmic development, activator of transcription,apoptosis ) Vallee, 2005 (c) Jerome A
  20. 20. Transcriptome Analysis of Bull Semen.Spermatozoa are terminally differentiated cells producedduring the complex process of spermatogenesis. Lalancette - 2008 ESTRUS BEHAVIOURHigh fertile: Immunoglobulin superfamily, Protein phosphatae,SCO-spondinLow fertile: Gamma actin, Desmin Bauersachs,2005 (c) Jerome A
  21. 21. Uterine receptivity UTMP AGRN, LGALS3BP, LGALS9, USP18, PARP12 andBST2.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. 22.  Epigenomics, Evolutionary genomics, Toxicogenomics, Nutrigenomics Mitochondriomics (Stoughton, 2005; Ness,2007). (c) Jerome A
  23. 23. Proteomics (c) Jerome A
  24. 24. Proteomics  is the large-scale study of proteins, structures & functions.Proteins are vital parts of living organisms, as they are the maincomponents of the physiological metabolic pathways of cells.Identification of new fertility biomarkers, specific to normal/abnormal reproductive health status.Two analysis: Total protein complement, Positionalproteomics. (c) Jerome A
  25. 25. BULL FERTILITY High fertility group : Glyceraldehyde 3-phosphate dehydrogenase, Phosphatidylethanolamine- 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. 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. 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. 28. RNA interference (RNAi) (c) Jerome A
  29. 29. RNA interference (RNAi) is a system within living cells thattakes part in controlling which genes are active and howactive they are.RNAs are the direct products of genes, and these smallRNAs can bind to specific other RNAs and either increaseor decrease their activityControl of gene expression and in functional genomics(post-transcrip -tional gene silencing) (c) Jerome A
  30. 30. Selective degradation of maternal and embryonic transcripts in invitro produced bovine oocytes and embryos using sequence specificdouble-stranded RNAEmbryos with dsRNA specific for some genes the interference effectis specific & can block expression of maternal gene products.To study the effects of genes’ loss of function in developingembryos 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. 31. Conclusion Reproduction Normal / Diseases Female / male CON. M OLECULARTECHNIQUES TOOLS (c) Jerome A
  32. 32. (c) Jerome A