Determination of sex Dr habil Kőhidai László

Determination of sex ? Dr. habil. Kőhidai László Dept. Genetics, Cell- & Immunobiology, Semmelweis University 2016.

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 haploide genome House-keeping and specialized genes conserved sequences it does NOT code sex determining factor one of X chrs gets inactive in female (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

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

? § § § 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 § WT 1 § SOX 9 § MIS § DSS § DAX-1 § SRY ‘Steroidogenic’ factor, nuclear receptor which regulates expression of steroid hydroxylases Wilm’s tumor locus determined chrs 11 p 13 ‘SRY-releated HMG-box’, chrs 17 q 24. 3 -25. 1 about 14 genes product of Sertoli-cells; chrs. 19 p 13. 3 regulatory domaine which binds F-1, its promoter binds SRY ‘Dosage Sensitive Sex reversal’, Duplication on chrs Xp 21. 2 -p 22. 2 nuklear hormone receptor, expressed: testis and adrenal gland ‘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)

DAX 1 Ovary WNT 4 SF 1 Bipotencial WT 1 gonade LHX 9 SRY SOX 9 Testis Follicular cells Follicle Theca cells Estrogen Sertoli- SF 1 AMH cells Leydig cells SF 1 Testosteron

Genotype inactive DAX 1 SRY DAX 1 2 copies DAX 1 Gonades Phenotype Testis Male Ovary Female Gonade disgenesis Female Ref. : Genetics Review Group (1995)

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

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

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

X Chromosome DAX 1 Y Chromosome SRY SF 1 WNT 1 Ref. : Aberger F. OVARY SOX 9 TESTIS

Sox 9 Promoter Amh Wt-1 Sox 9 SF 1 Gata Ref. : Arango et al. (1999) Amh

Evolution of huma sex chromosomes (1) Sex chrs. Developes SRY (NRY) blocks Recombination 290 -350 Mill. year Autosome birds recomb. Az NRY expansion (RBMY, RPS 4 Y) NRY expansion (SMCY, UBE 1 Y) 230 -300 Mill year 130 -170 Mill year XY egg layer mammals X-chrs. spec. XY Marsupials Non-recomb.

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

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

Enhancer Promoter Inactive gene Start Hormone-Rec complex Promoter Active gene Activated enhancer m. RNS synthesis REASON: Xq 11 -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 46 XX – SRY translocation or 46 XXY – loss of Y chrs 46 XY – point mutation of Y chrs 46 XX/46 XY 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 (45 X/46 XY – gonadal streak/testis) Testicular feminisation (46 XY – 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 n. Barr = n. X – 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

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

X kromoszóma inaktiválódása (2) Transcriptional „silencing” Timing of asynchron replication Increased macro. H 2 A Hypoacethylated: H 3; H 4

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

XY XX > Mary F. Lyon X XY =

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 Xq 13 (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 § PAR 1 – 2. 6 Mb; PAR 2 – 320 kb § Provide choice for partial meiotic pairing of X-Y chrs § „Obligatory crossing over” in PAR 1 (e. g. Xg blood group, IL-3 receptor)

X chrs PAR 1, PAR 2 Y chrs AR: Androgen receptor CSF 2 RA: Colony-stimulating factor 2 receptor alpha AZF: azoospermia factor ATRX: Alpha-thalassemia/mental retardation syndrome X-linked IL 3 RA: Interleukin 3 receptor alpha DAZ: Deleted in azoospermia DAX 1: DSS-AHC critical region X chromosome gene 1 SHOX: Short stature homeo box HY: Histocompatibility antigen Y FRA-X: Fragile X syndrome IL 9 R: Interleukin 9 receptor RBMY: RNA-binding motif protein Y chromosome DMD: Duchenne muscular dystrophy SRY: Sex-determining region Y chromosome GK: Glycerol kinase USP 9 Y: Ubiquitin-specific protease 9 Y chromosome Kal 1: Kallmann syndrome 1 ZFY: Zinc finger protein Y-linked POLA: DNA polymerase alpha XIST: X inactivation-specific transcript ZFX: Zinc finger protein X-linked

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

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

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