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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

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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.