genetics of sex determination and develo

Credit seminar : 

Credit seminar O On GENETICS OF SEX DETERMINATION AND DEVELOPMENT Speaker: Saurav Kumar Panigrahi Ph.D. (AGB).

Evolution of sex : 

Evolution of sex Asexual reproduction. Sexual reproduction. Production of haploid gametes. Fusion – diploid zygote. Parts of evolution: Origin of sexual reproduction. Evolution and maintenance.

Sex chromosome homology : 

Sex chromosome homology Evolved - autosomal pair. Homology - many X-Y shared sequences in differentiated regions of chromosomes. eg. YCR and YAR genes homologues on X. X born - mutated and inactive genes on Y . Y genes - structural and functional change. SRY and RBMY have X homologue - SOX3 and RBMX . (Delbridge et al. 1999)

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FIG.1

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FIG. 2

Degradation of genes -Y : 

Degradation of genes -Y Acquisition of sex determining allele (mammals – SRY). Degradation - male specific gene package together to prevent recombination. Dosage inequality b/w sexes. Inactivation of one of X chromosome – dosage compensation or Lyons hypothesis.

Cont… : 

Cont… Comparative genome mapping ( Mammals , marsupials , monotremes) 200 years Addition to prevent homology – X and degradation – Y Y has been saved from extinction Mortality of Y chromosome – No SRY New sex determining allele. (Just et al. 1995)

SRY (humans ) and SRY (mouse) : 

SRY (humans ) and SRY (mouse) Short arm of Y chromosome SRY - Equated with TDF in man and mouse Complex with WT1 (Wilms tumor 1) transcription in testis development. complete organogenesis. Zinc finger Y(ZFY) - candidate for TDF sertoli cells and AMH (Page et al.1987)

SRY : 

SRY SOX9 Mammals , birds and reptiles. Activates AMH or Mullerian inhibiting substance expression with orphan receptor (SF – 1). (Graves et al. 1998)

Importance of sexual reproduction : 

Importance of sexual reproduction Genetic variation among siblings - Weismann(1889) , Fisher and Burt (2000) - Tangled bank hypothesis – Michael Ghiselin or Graham Bell ,1982 Creates novel genotype rapidly - Fisher and George Williams. Resistance to parasites – Red queen hypothesis. Van Valen 1973 Removal of deleterious genes Reconstruction of mutation free individuals - Muller Deterministic mutation hypothesis – kondrashov 1988.

Sex determination : 

Sex determination Theories : Aristotle (384-322 BC) Females – Building material Males – Life giving power dynamics Theory of evolution – Darwin 1859 Neo Darwinism – August Weisman(1834-1914) Modern view (1960) Paradox of recombination. Paradox of meiosis. Paradox of variability.

Mechanisms of sex determination. : 

Mechanisms of sex determination. Environmental Association with females. Egg size. Incubation temperature. Chromosomal Molecular basis Genic balance Y linked genes

Environmental : 

Environmental Association with females: eg. Sea worm - Bonelia Free living – females Attached to probosis - males Egg size : eg. Sea worm - Dinophilus large - females small - males

Incubation temperature : 

Incubation temperature eg. Turtles , alligators ,crocodiles. 1. M M&F F 230c 280c 300c 350c 2. F M&F F < 300c > 330c 3. F M F < 250c > 300c

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FIG.3

Chromosomal sex determination : 

Chromosomal sex determination XX – F and XO – M. (Orthopteran insects , grass hoppers) XO - F and XX – M ( Fumeans ) . XX - F and XY - M (humans, mice, fishes, amphibians, drosophila) XY –F and XX – M ( birds , reptiles). 2n – F and n – M ( honey bees, ants and termites).

Genic balance : 

Genic balance Drosophila Bridges, 1921 Correlation of sex of individual with chromatin constitution. Balance b/w genes for maleness (autosomes) and those for femaleness (X chromosomes). Sex index = No. of X chromosomes = X No. of autosomal sets A

Contd… : 

Contd… Index ratio ? 1 = Super female 1 = Female 0.5 – 1 = Intersex 0.5 = Male ? 0.5 = Super male Intersexes are neither male nor female and are completely sterile.

Mechanism of X/ A action : 

Mechanism of X/ A action

Sex determination with Y linked genes(Molecular basis) : 

Sex determination with Y linked genes(Molecular basis) ( mammals ,amphibians and humans ) . HY antigen. TDF through SRY. Dosage compensation – man, mouse and drosophila. Regulates expression of X linked genes.

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FIG. 5

Sex related disorders : 

Sex related disorders Hermaphrodites. Gynandromorphs. Androgen insensitivity syndrome . Intersex . Chromosomal abnormalities Mammals: - 47XXY - 45XO - 47XY –Downs, Edwards, Pataus syndrome. Drosophila –XO,XXY.

Hermaphroditism or Intersexuality. : 

Hermaphroditism or Intersexuality. Ovarian follicles and testicular tubules in same or different gonads. True Pseudo Freemartinism Behavioural – Nymphomania and Adrenal virilism. Animals : Sequential : ( Protoandry or Protogyny ) Simultaneous or Synchronous Gonadal dysgenesis

Gynandromorphs : 

Gynandromorphs Part female (Gyn) and part male (Andro). Regional disharmony in distribution of sex chromosomes. Male and female parts run longitudinally or transversely. Sex mosaics. Arise from XX zygotes. XO – male phenotype XX – female phenotype. Impossible in vertebrates.

Androgen insensitivity syndrome : 

Androgen insensitivity syndrome Androgen resistance syndrome. Mutation of Tfm gene. Not responding to testosterone. Various degrees Complete - Female - 46 XY and testis Incomplete - Male features – puberty. Testicular Feminization. .

Sex hormones and behaviour : 

Sex hormones and behaviour Androgens : Testosterone Estrogen Hypothalamus differentiation Estrogen availability – male. Absence – female Testicular feminization.

Sex reversal : 

Sex reversal Transformation of sexual characterization. Types : Complete Incomplete. Not possible: Mature individuals Sexual dimorphism In mammals – late embryonic stage- Impossible. In birds: Gonadectomy through disease Development through sex cords.

Sex selection : 

Sex selection 3 stages: Pre conceptional or semen sexing . Pre implantation genetic diagnosis (PGD) or Embryo sexing. Post implantation selection. Pre conceptional: Veterinary medicine. Large scale insemination programmes. Patented by XY Inc. of Fort Collins, USA

Flow cytometer : 

Flow cytometer

2.Sperm selection by gradients : 

2.Sperm selection by gradients Centrifugation through gradient layers of albumin. Y spermatozoa - bright spot containing quinacrine dihydrochloride dye. Alters X :Y spermatozoa ratio. Alternatives for albumin - Percoll , Ficoll or sephadex.

3.Immunological sexing : 

3.Immunological sexing Bradley (1989), Blecher et. al (1999). HY antigen on plasma membrane (post acrosomal region and mid piece ) Treatment with HY antiserum kills Y bearing sperms

4. Based on motility : 

4. Based on motility Density gradient centrifugation + sperm migration over BSA ingredients in laminar flow medium. Swim up:Differential swimming ability of X and Y X – more linear swimming ability.

Advantages of sexed semen : 

Advantages of sexed semen 1.calving ease : -10.9 %difficulty as bull. - 5.3% difficulty as heifer. Advantages of less calving problems : Increased milk production . More live calves / pregnancy. Reduced culling of 1 lactation heifers. Reduced open days and semen costs. Lower vet bills.

2. Genetic progress. : 

2. Genetic progress. In dairy cattle - 15 % increase (Van Vleck, 1981) MOET nucleus scheme - increases by 0.4-1.4% ( Montaldo et. al 1988) MOET+ sexed semen + MAS Farming system - male dairy calves. Avg. lactation length -extended to 18, 20, or 24 Months. Top 50 cows - with X bearing sperms and bottom 50 - with Y bearing beef semen. ( Hohenboken,1999 )

3.Biosecurity: : 

3.Biosecurity: Expansion of herd with own replacements. Harmful diseases Milk quality. 4. Breeders dream

5.In vitro embryo production(IVP) : 

5.In vitro embryo production(IVP) More commercially applicable than in-vivo. Genetically useful cows. In vitro - <1,00,000 sperms /100 ova. ? of ova fertilized : Same for sexed and unsexed semen Blastocyst production - 70% in sexed semen

Limitations of semen sexing : 

Limitations of semen sexing Sorting speed. In vivo conception rate: 35 – 40 % with sexed semen 50 – 60 %with unsexed semen 3. Cost

Pre implantation genetic diagnosis(PIGD) : 

Pre implantation genetic diagnosis(PIGD) 1. Antibody. 2. DNA analysis. 3. RAPD markers - ovine embryos. 4. Loop mediated isothermal amplification (LAMP). 5. PCR - Y specific probes. 6. Follicular fluid steroid levels. 7. Sexing kit.

1.Antibody: : 

1.Antibody: sex specific proteins (SSP) on outer surface of cells. Preparation of antibodies. Elimination of male or female embryo. Selection of embryos. Grown in lab – transfer to animals.

2.DNA analysis : 

2.DNA analysis By using DNA of embryos. Limitations: Invasive procedure . Chances of infection. Embryo growing. Complex procedure.

3.RAPD markers in Ovines : 

3.RAPD markers in Ovines Highly specific to Ovines. PCR primers - ovine specific Y chromosome ZFY/ZFX with randomly amplified polymorphic marker (Ucd043). 100 % accuracy - detects 10 pg of male DNA and 50 ng of female DNA.

4.Loop mediated isothermal amplification : 

4.Loop mediated isothermal amplification DNA amplification method . DNA of single blastomeres - extracted using heat or NaOH or proteinase K . Product detected by turbidity of reaction mixture. Accuracy- 88.9 - 94.4% Pregnancy rate : 57.4 % Same principle in sexing kit.

5. Y specific probes : 

5. Y specific probes Isolation and cloning of specific DNA sequences 3 sets of primers. 1’st pair - bovine specific satellite sequence amplified in both male and females. Other 2 pairs recognise bovine Y specific sequences. Highly specific for bovine embryos.

Follicular fluid steroid levels : 

Follicular fluid steroid levels Based on level of testosterone in follicular fluid. High concentration of testosterone Y - Chromosome. Dominant females- male offspring.

Uses of sex determination in embryos : 

Uses of sex determination in embryos Knowing karyotype. Abnormals aborted. Embryo sexing and cloning. eg. Production of bile salt stimulated lipase in females Control of gender and fertility- acquatic species - Monosex population Temperature dependent sex determination.

Post implantational sex determination : 

Post implantational sex determination By karyotyping. HY antigens. Amniocentesis using DNA probes. Ultrasonography. Involves termination of pregnancy if it is of undesired sex.

Prognosis : 

Prognosis Rate of adaptation –more than AI or milk recording.Young et al.(1988) 1st sexed calf -1990 and 1st sexed foal - 1988. Acquisition by dairy producers. Endangered species. Ethics and legal issues. Transgenic bull - X chromosomal sperms.

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