Determination of sex

? Determination of sex Dr. habil. Kőhidai László Dept. Genetics, Cell- & Immunobiology, Semmelweis University 2017.

Determination of sex • Characterization of sex chrs • Main types of sex chrs linked inheritance • Pathological forms

Main determining factors of sex • Environmental factors • size of body (marine worms) • temperature (reptiles) • Genetical • allelic + environment (wasps) • chromosomal • Ratio of X chrs and autosomes • autonomic e.g. Drosophila • not autonomic e.g. human

! General characteristics of X chromosome • 5% of haploidgenome • House-keeping and specializedgenes • conservedsequences • itdoes NOT code sex determiningfactor • one of X chrsgetsinactiveinfemale (1961)

General characteristics of Y chromosome ! • It was a homologue pair of X chrs in a previous phase of phyligeny • Phenotype of somatic cells is determined by the testicular hormones • Y chrs acts on testis developement by TDF • SRY gene product = TDF (1991) • SRY codes a Zn-fingered transcription factor • SRY is expressed ONLY in: - gonadal tissues - at the development of testis - presence of gonocytes is not a prerequisite

Housekeeping Testis Other places X chrs has a homologue NO homologue of X chrs Y chrs.

! Known genes of Y chromosome ? ! • SRY – sex region of X (see below) • ZFY – code of a Zn-fingered protein • AZY – responsible for development of sperm (AZ=azospermia) • H-Y – cell surface antigene, present on each cell of males • MSY – long palindromic sequences on the q arm of Y chrs

! Genes of early sex differentiation • SF-1 ‘Steroidogenic’ factor, nuclear receptor which regulates expression of steroid hydroxylases • WT1 Wilm’s tumor locus determined chrs 11p13 • SOX9 ‘SRY-releated HMG-box’, chrs 17q24.3-25.1 about 14 genes • MIS product of Sertoli-cells; chrs. 19p13.3 regulatory domaine which binds F-1, its promoter binds SRY • DSS ‘Dosage Sensitive Sex reversal’, Duplication on chrs Xp21.2-p22.2 • DAX-1 nuklear hormone receptor, expressed: testis and adrenal gland • SRY ‘Sex-determining region product of Y gene, transcription factor

SRY gene • Yp 11.3 • only one, 850 bp exon • highly conserved • 79 amino acid containing HMG box ( ‘Highly Mobile Group of proteins)

Follicular cells Ovary DAX1 WNT4 Follicle Theca cells Estrogen SF1 WT1 LHX9 Bipotencial gonade SF1 Sertoli- cells AMH SRY SOX9 Testis SF1 Leydig cells Testosteron !

Genotype inactive DAX1 SRY SRY DAX1 DAX1 2 copies DAX1 Gonade disgenesis Testis Ovary Gonades Phenotype Male Female Female Ref.: Genetics Review Group (1995)

Sexually not differentiated Female Male Ref.:Langman (1981)

Week 4 Testis Ovary Week 6 Week 8 Week 8 Ref.:Langman (1981)

Testis Ovary Week 20 Week 16 Ref.:Langman (1981)

! X Chromosome DAX1 Y Chromosome SRY SF1 SOX9 TESTIS WNT1 OVARY Ref.: Aberger F.

Wt-1 Sox9 SF1 Sox9 Amh Wt-1 Promoter Amh SF1 Sox9 Gata Amh ! Ref.: Arango et al. (1999)

recomb. X-chrs. spec. Non-recomb. ! Evolution of huma sex chromosomes (1) NRY expansion (SMCY, UBE1Y) 130-170 Mill year Sex chrs. Developes SRY (NRY) blocks Recombination 290-350 Mill.year Az NRY expansion (RBMY, RPS4Y) 230-300 Mill year XY egg layer mammals XY Marsupials Autosome birds

NRY expansion (AMELY, KALP) 30-50 Myr X-Y translocation PCDHY 3-4 Myr NRY expansion (CASKP, DBY) 80-130 Myr Translocation expansion to PARp 80-130 Myr XY Homo sapiens XY Not human anthropoids XY Non-anthropoid mammals recomb. X-chrs. spec. notrecomb. ! Evolution of huma sex chromosomes (1) Protocadherin X/Y

X chromosome Human Cat Conserved loci in mammalian X and Y chrs Y chromosome Human Cat

! Testicular feminisation Genotype: XY Testosteron in sera is normal Testis in the abdominal cavity Feminine statue • Reasons: • error of differentiation • after testosteron action? • testosteron can influence development of Wolff-tubule at differentiation? Reason: MUTATION OF TESTOSTERON RECEPTOR

Promoter Enhancer Start Inactive gene Hormone-Rec complex Promoter Active gene mRNS synthesis Activated enhancer REASON: Xq11-12 mutation – androgenic receptor

Hermaphroditism ! • True hermaphroditism • Both gonads (ovary and testis) or its tissue is present in the body • Male OR female external sexual organs are dominant • Forms • 46XX – SRY translocation or 46XXY – loss of Y chrs • 46XY – point mutation of Y chrs • 46XX/46XY mosaicism • (Hermaphroditism is frequent in invertebrates and plants)

Hermaphroditism ! Pseudo hermaphroditism • Gonad of one sex and the opposite external sex organs • Ph masculinus • Mixed gonad dysgenesis (45X/46XY – gonadal streak/testis) • Testicular feminisation (46XY – pointmutation of Y chrs – testis releases estrogen; ligand is not bind by receptors) • Ph femininus • Adrenogenital syndrome (androgen threatments can induce it)

! Inactive X chromosome Form of „gene dose compensation” Only 1 X chrs is active when there are X chrs’ in excess number nBarr = nX – 1 X recessive mutations: some cells in female are „functional hemizygotes” – this may result disease An other form of gene dose compensation: differences in activity of X chrs in the two sex

XY XX ‘Drumstick’ Barr-bodies

Xist expression - inhibitor factor Inhibitor factor LINE helps in sreading the effect Xist RNA coats the chromosome ! Inactivation of X chromosome (1)

Transcriptional „silencing” Timing of asynchron replication Increased macroH2A Hypoacethylated: H3; H4 ! Inactivation of X chromosome (2)

Xist is active on both X chrs’ Inactive X chrs is covered by RNA Only the inactive, „RNA-coated” Xchrs is detectable Xist transcription in embryonic stem cells

X XY = XY XX > Mary F. Lyon

Inactivation of X chromosome ! • Takes place randomly in the early phase of development in healthy female • The same X chrs gets inactive in the offspring generations of cells • A product of Xq13 (Xist) is significant in the process • Virtually all genes of X chr turn into inactive phase (except genes responsible for inactivation) • Female are mosaic for inactive X chrs as maternal and paternal X chrs get inactive, too • Male: constitutional hemizygotes • Nők: functional hemizygotes

! PAR regions PAR = pseudo autoszomal region • Never gets inactive • Telomeric position on the two sex chromosomes • PAR1 – 2.6 Mb; PAR2 – 320 kb • Provide choice for partial meiotic pairing of X-Y chrs • „Obligatory crossing over” in PAR1 (e.g. Xg blood group, IL-3 receptor)

! Frequent problems resulting disfunctions in sexual differentiation • mutations of SRY • disturbed biosynthesis of androgens • mutations of androgen receptor • errors of AMH • XY/XO mosaicism • Wnt and WT-1 mutations (differentiation of gononephrotom)

Sex limited inheritance The trait is present in the genotype of both sex, however it is expressed only in one sex E.g. hair, menstruation, pelvic parameters

X X X X X X X Y Y Y X X • Incomplet sex restriction Crossing over between pseudoautosomal regions of X and Y chrs.

Sex controlled inheritanceThe trait is expressed in both sex, however its degree is different Normal features: Deepness of sound Baldness BB+ B+B+ Diseases: gout 80% M Cleft lip/palate Anencephaly - F Spina bifida Male-baldness (androgenes) Female– normal Male and Female - baldness

Determination of sex