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Reproductive Biology andCurrent Status in
Reproductive Biotechnology
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The Science of Reproduction
AnatomyAnatomy
PhysiologyPhysiology
EndocrinologyEndocrinology
EmbryologyEmbryology
HistologyHistology
CytologyCytology
MicrobiologyMicrobiology
NutritionNutrition
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Reproduction
Development of the reproductiveDevelopment of the reproductivesystem in the embryosystem in the embryo
PubertyPuberty ––to produce fertile gametesto produce fertile gametes
FertilizationFertilization ––the sperm and egg meetthe sperm and egg meet
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The Aims of Reproduction in
Perpetuation of the speciesPerpetuation of the species
• Food providing
• Genetic improvement
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Oogenesisin mammals
After the germ cellsthat form oocytesenter the embryonicovary, they dividemitotically a fewtimes and then enterthe prophase of thefirst meiotic division.No further cellmultiplication occurs,
but the oocyteincreases 100-foldmass.
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Spermato-genesis inmammals
Germ cells thatdevelop into spermenter the embryonictestis and becomearrested at the G1
stage of the cellcycle. After birth,they begin to dividemitotically again,forming a populationof stem cell(spermatogonia).
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In the final stage ofmigration, the cells movefrom the gut tube into thegenital ridge, via thedorsal mesentery.
Pathway of primordial germ cellPathway of primordial germ cellmigration in the mouse embryomigration in the mouse embryo
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Development of thegonads and related
structures in mammals
Top panel:Top panel: early in development,there is no difference betweenmales and females in the structures.The future gonads lie adjacent tothe mesonephros.
Bottom panels:Bottom panels: after testes developin the male, their secretion ofMüllerian-inhibitory substance resultin degeneration of the Müllerianduct by programmed cell deathwhereas the Wolffian duct becomesthe vas deferens.
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HH Hypothalamus
PP Pituitary gland
GG Gonad
Development of reproductive tissuesDevelopment of reproductive tissuesand sexual characteristicsand sexual characteristics
GnRHGnRH at appropriate frequencies
GTHGTH
GametogenesisGametogenesis steriodogenesissteriodogenesisAxisAxis
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Endocrine
Autocrine
Paracrine
Affecting the cells away from theAffecting the cells away from theglands which produce hormonesglands which produce hormones
Affecting the cells that secrete themAffecting the cells that secrete them
Affecting other cells in the same organAffecting other cells in the same organ
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Animal breeding and biotechnology
DomesticationDomestication
PropagationPropagation
Selection: breedingSelection: breedingPopulation genetics and statisticsPopulation genetics and statistics
Biotechnology: AI, ETBiotechnology: AI, ETArtificial insemination (AI), embryo transfer (ET),Artificial insemination (AI), embryo transfer (ET),Freezing and thawing techniqueFreezing and thawing technique
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Biotechnology in animal agriculture
Milk production:Milk production: 33--fold between 1945fold between 1945--19951995
Egg production:Egg production: from 134 to 254 betweenfrom 134 to 254 between19401940--19941994
Broilers:Broilers: in 1950, took 84 days to marketin 1950, took 84 days to marketweight of 1.8 kg;weight of 1.8 kg;now, takes 43 days to 1.8 kg andnow, takes 43 days to 1.8 kg andhalf of thehalf of the feedreedingfeedreeding
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BiotechnologyBiotechnology
ReproductiveReproductive--biological procedurebiological procedure
MolecularMolecular--biological procedurebiological procedure
Theriogenology 2001, 56: 1291-1304
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ReproductiveReproductive--biological procedurebiological procedureAIAI
Estrous synchronizationEstrous synchronization
Regulation of parturitionRegulation of parturition
ETET
CryoperservationCryoperservation of gametes and embryosof gametes and embryos
Sexing of sperm and embryosSexing of sperm and embryos
In vitroIn vitro production of embryos (IVP)production of embryos (IVP)
Embryo bisectionEmbryo bisection
Nuclear transfer (NT)Nuclear transfer (NT)
Microinjection technology (Sperm, DNA, RNA...)Microinjection technology (Sperm, DNA, RNA...)
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MolecularMolecular--biological procedurebiological procedure
Genome analysis (sequencing, mappingGenome analysis (sequencing, mapping……))
Molecular diagnosis (genetic defects, geneticMolecular diagnosis (genetic defects, geneticdescent, genetic diversity)descent, genetic diversity)
Functional genomics (expression patterns,Functional genomics (expression patterns,interactions of genes)interactions of genes)
TransgenicsTransgenics (additive gene transfer, knockout)(additive gene transfer, knockout)
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Factors affecting reproductive efficiency
** Early development of theEarly development of the conceptusconceptus
* Genetic and environmental factors* Genetic and environmental factors
* Nutrition* Nutrition
* Breeds* Breeds
* Fertility and temperature* Fertility and temperature
* Culling strategies* Culling strategies
* Stress* Stress
* Effect of antibiotics and hormones* Effect of antibiotics and hormones
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Advantages of AI
•Genetic improvement e.g., widespread use ofoutstanding sires; improving accuracy of selection throughprogeny test; permitting crossbreeding; introduction of newgenetics
•Control of venereal diseases
•Availability of accurate breeding records
•Economic service
•Safety through elimination of dangerous male
•Use of deep-frozen semen after a donor is dead
•Gender control
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18th centuryovarian transplantation
1948 –glycerolthe discovery of the first cryoprotectant
Long-term organ storage techniques
CryoperservationCryoperservation of gametes and embryosof gametes and embryos
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Freezing injury
* destroy cell function irreversibly* intracellular ice crystal formation andsalt deposits
* changes in the composition of surroundingmilieu at thawing phase
* cytotoxicity of cryoprotectants
Cryopreservation
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Cryoprotective agents* high water solubility; high permeability;
low toxicity* alcohols, amines, sugars and proteins* dimethyl sulfoxide (DMSO); 1,2-propanediol(PROH); ethylene glycol (EG); glycerol
Antifreeze proteins* many animals and plants in nature usecryoprotective agents
* production of antifreeze proteins, mainlyglyopeptides
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1990ssuccessful cryopreservation and transplantation
of ovarian tissue inmouse, sheep and marmoset monkey
2000ovulation occurred in autografted human ovarian tissue
after gonadotropin stimulation
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Histological section of the ovary ofa newborn mouse.
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Appearance of a grafted ovary from a newbornmouse transplanted under the kidney capsule for14 days.
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Histological section of the ovary froma 14-day old mouse.
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The preantral follicle MII oocytesat Day 1 of culture at Day 12 of culture
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Cryopreservation of ovarian tissue
*Clinical Applications*Fertility conservation
Freezing immature primodial follicles in situin the ovarian cortex
Immature oocytes are:* Quiescent (arrested in prophase I)
less liable to cytogenetic errors* Smaller, fewer organelles* Lack of zona pellucida and cortical granules* Repair sublethal damage during
prolonged growth phase
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Options for development of immature oocytes in cryopreservedovarian tissues include autotransplantation and xenoplantation
heterotopic autotransplantation: oocyte retrieval and IVF requiredxenotransplantation: immunological rejection; oocyte retrieval and
IVF required
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Autotransplantation* short life span of ovarian grafts* potential risk of transmission of microscopic metastatic
disease* develop screening methods to detect minimal residual
disease (MRD) –nested PCR; flow cytometry; FISH; cytogenetics
Xenotransplantation* eliminate the possibility of cancer transmission and
relapse* bypass the difficulities of in vitro growth and maturation
of primodial follicles* apply to patients at high risk for hyperstimulation
syndrome or whom hormonal replacement therapy iscontraindicated
* animal pathogens transmitted to human tissue
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Priorities for Research
Cryopreservation techniquesoptimal dehydration times, cooling and thawing rateseffective cryoprotectants
Ischemic-reperfusion injuryfacilitation of angiogenesis
Transplantation sitetissue survival and follicle access
Safety of autotransplantationscreening methods for detection of MRD
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Molecular breeders
Nuclear transferMammalian embryogenesisby asexual reproduction
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Cell transplantationMale gametes have potential ofdeveloping in a surrogate environment
Molecular breeders
Nuclear transferMammalian embryogenesisby asexual reproduction
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primodial germ cells gonocytes spermatogonia
Genital ridge birth puberty
quiescent dividing and differentiating
Production of spermatozoa
spermatogoniaStem cell spermatogonia
Proliferative spermatogonia
Differentiating spermatogonia
self-renewal
Stem cell
Apr spermatogonia chain of 4,8,16 Aal spermatogoniaAs spermatogonia
B spermatogonia A2, A3, A4 spermatogonia A1 spermatogonia
spermatocytes
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Transplantation procedure
Inject into the seminiferous tubules
Inject into rete cavity
Inject into efferent ducts
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Colonization of donor testicular cells collected fromday 5 trangenic mice, carrying a bacterial geneencoding -galactosidase
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normal mouse transgenic mouse
Promoter: metallothioninePromoter: metallothionine--11
TransgeneTransgene: rat GH: rat GH
Science 1982, 385:810-813
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The Methods to produce transgenic animals
Microinjection
Retroviral vector infection
Sperm vector
Embryonic stem cell (ES cells):partical gun / transfection / electroporation
Nuclear transfer
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Transgenic offspring / transferred injectedzygotes - 1-4%: inefficientinefficient
Random integrationRandom integration into the host genome
VariableVariable expression (position effects)
TimeTime-consuming
RequirementRequirement of intellectual, financial andmaterial resources
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Retroviral vector infection
Theriogenology 2001, 56: 1345-1369
Embryos orEmbryos or oocytesoocytes are exposed in vitro to concentratedare exposed in vitro to concentratedvirus solutions or incubated over a single layer ofvirus solutions or incubated over a single layer of
virusvirus--producing cells.producing cells.
The virus enters the perivitelline spacethrough a slit in the ZP
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The injection of virus under the ZP ofThe injection of virus under the ZP of oocytesoocytes withwithsubsequent IVM and IVF.subsequent IVM and IVF.
Theriogenology 2001, 56: 1345-1369
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The main advantage of retroviralThe main advantage of retroviral--mediated genemediated genetransfer intotransfer into animlasanimlas
•The technical ease
•the sequence of DNA transferred is limited by size
•the inserted gene is not always expressed in thesecond generation
•many founders are mosaic, with multiple insertionsites (breeding experiments required)
The disadvantage of retroviralThe disadvantage of retroviral--mediated genemediated genetransfer intotransfer into animlasanimlas
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The Aims of producing transgenic animals
Biological, biomedical, veterinary and genetic
research
Agriculture: enhance growth and development
Increase disease resistance
Xenotransplantation
Produce foreign proteins
Gene therapy
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Improvement of agriculture traits
Dietary modifications of animal productsDietary modifications of animal products
Environmentally friendly farm animalsEnvironmentally friendly farm animals
49Nature Biotech. 2001, 19: 741–745
Total phosphorus content (on a dryTotal phosphorus content (on a drymatter basis) of fecal matter frommatter basis) of fecal matter fromnonnon--transgenic pigs ( ) andtransgenic pigs ( ) andtransgenic pigs ( ) of line WAtransgenic pigs ( ) of line WAfed differentfed different levelsoflevelsof soybeansoybeanmeal as the sole source ofmeal as the sole source ofdietary phosphorus.dietary phosphorus.
DM, Dry matter content of feces.
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Transgenic animals in biomedicine
As models for human diseasesAs models for human diseases
AlzheimerAlzheimer’’s diseases disease
Physiol. Behav. 2001, 73:873-886.
Amyloid precursor protein (APP)Presenilins (PS1 or PS2)
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For pharmaceutical productionFor pharmaceutical production
TRENDS in Biotech. 2004, 22:286-294.
> 2007Blood substituteGoatPhase I / IIVariousAbs
2008Hemophilia ACattlePhase IHAS
> 2008Cystic fibrosissheepExperimentalhFVIII
2007Lung emphysemaGoat / sheepPhase II / III-AT
2006Dissolving coronaryclots
GoatPhase II / IIITPA
2005Genetic heparinresistance
GoatPhase IIIAT III
PotentialPotentialmarket datemarket date
TherapeuticTherapeuticapplicationapplication
ProductionProductionspeciesspecies
DevelopmentalDevelopmentalphasephase
ProteinProtein
Proteins produced in the mammary gland of transgenic farm animals
AT III: antithrombin; TPA: tissue plasminogen; -AT: antitrypsin; hFVIII; human clotting factor;Human serum albumin;
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Adv. Biochem. Engin/Biotechnol. 2004, 91: 171–189
Procedure for generation of transgenic birds
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XenotransplantationXenotransplantation of porcine organs toof porcine organs tohuman patientshuman patients
••Similar size of organs to human organsSimilar size of organs to human organs
••Anatomy and physiology are not too different from thoseAnatomy and physiology are not too different from thosein humanin human
••Short reproduction cycles and large littersShort reproduction cycles and large litters
••Grow rapidlyGrow rapidly
••Maintenance at high hygienic standard at relative low costsMaintenance at high hygienic standard at relative low costs
••Domestic speciesDomestic species
Reasons for pigs as the optimal donor animalsReasons for pigs as the optimal donor animals
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••Overcoming the immunological hurdlesOvercoming the immunological hurdles** hyperacutehyperacute rejection response (HAR)rejection response (HAR)
** acute vascular rejection (AVR)acute vascular rejection (AVR)
** cellular rejection and potentially chronic rejectioncellular rejection and potentially chronic rejection
••Prevention of transmission of pathogens fromPrevention of transmission of pathogens fromthe donor animal to the human recipientthe donor animal to the human recipient
** porcine endogenous retroviruses (porcine endogenous retroviruses (PERVsPERVs))
••Compatibility of the donor organs with the humanCompatibility of the donor organs with the humanorgan in terms of anatomy and physiologyorgan in terms of anatomy and physiology
Prerequisites for successfulPrerequisites for successful xenotransplantationxenotransplantation
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••Overcoming the immunological hurdles** Synthesis of human complement regulatory proteins inSynthesis of human complement regulatory proteins in
transgenic animalstransgenic animals
** Knockout the antigenic structures on the surface of the porcineKnockout the antigenic structures on the surface of the porcineorganorgan
** Induce a permanentInduce a permanent chimerisimchimerisim
Anim. Reprod. Sci. 2003, 79:291-317.
Success rates of RCA-transgenic porcine organs after transplantation to primate recipients
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••Prevention of transmission of pathogens fromPrevention of transmission of pathogens fromthe donor animal to the human recipientthe donor animal to the human recipient
* knock* knock--outout -------------- difficultdifficult
* RNA interference (RNAi)* RNA interference (RNAi)
57Virology 2004, 325:18-23
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100
80
60
40
20
0
gag1
gag2
pol1pol2
pol3pol4
pol5env2
env1
control
Efficacy of siRNAs targeting PERV
58Gene Therapy 13:478-486, 2006
The potential uses of RNAi in ES cells.
59Gene Therapy 13:464-477, 2006
Schematic diagram to illustrate siRNA targets important for tumor-host interaction.siRNA technology can be used to target molecules that are important for tumorangiogenesis, invasion, metastasis and immune evasion.
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