10/08 - Sex Determination Flashcards
Origin of Germ Cells
- Drosophila and C. elegans egg polarity determines origin of germ cells (posterior end of the fertilized egg)
- Egg polarity not yet demonstrable in mammals
- In mammals, germ cells arise from proximal epiblast (embryonic ectoderm)
What proteins appear essential in inducing formation of primordial germ cells
Bmp4 & Bmp8b
What is the earliest known marker in the origin of germ cells?
TNAP - non-specific tissue alkaline phosphatase
What effect does TNAP KnockOut have on germ cell formation
No effect
Folliculogenesis
Folliculogenesis describes the progression of a number of small primordial follicles into large preovulatory follicles that enter the menstrual cycle.
Once the oocytes are rested in meiosis I; at stage called primordial follicle. You will recruit many oocytes of which only one will be ovulated (until menopause)
What processes begin in folliculogenesis?
Embryogenesis and fertilization
What is the default pathway? And how do we know this?
- Female pathway is the default
- We know this because of Alfred Jost’s experiments with removing the ovaries and testes from rabbit fetuses. Both became female adult.
- In male fetus, the wolfian duct regressed and uterus, oviduct, and upper vagina formed
What actually causes sex determination?
- Y chromosome is a very gene poor region and the majority of genes involving sex determination are on autosomes; not the Y.
Human WT1 gene
- Wilms tumor suppressor/activator gene - regulates transcription
- Causes Frasier syndrome, Denys-Drash symdrome (Wilms tumor + pseudohermaphroditism
- Denys-Drash greater renal tumors risk
- Gonadal dysgenesis
- Gonadoblastoma
- Autosomal dominant
- 46, XY sex reversal
What happens when you knock out the WT1 gene in mice?
- Die between E13.5 and birth
- Fail to develop kidneys and gonads
- Heart, lung, and spleen also affected
- Mouse replicates human phenotype
What happens when you knockout SF1 in mice?
- Die before postnatal day 8
- male and female had internal FEMALE genitalia
What happens when you knockout SF1 in humans?
- phenotypic female
- karyotype XY
- high ACTH
- low cortisone
- low aldosterone
- laparoscopy showed streak gonads, normal mullerian (female) structures otherwise
- Estrogen and progesterone induced periods
- she lacks gonads and has adrenal hypoplasia
Describe the role of ACTH
ACTH: adrenal glands produce cortisol which provides negative feedback on pituitary gland which produces ACTH. If pituitary gland isn’t getting enough cortisol, the pituitary glad will jack up ACTH trying to get all the cortisol possible out of adrenal glands.
Why are streak gonads streaks?
- Because there are no germ cells inside
Describe the phenotype and etiology of 46,XX males
Phenotype: male external genitalia and testes, often well virilized, sterile
Etiology: X-Y interchange during paternal meiosis
What happens if you have an X and a Y chromosome with faulty recombination?
If you have X and Y chromosome (TDF = SRY gene) If there is a faulty recombination and SRY ends up on X chromosome; If this happens; the karyotype would be XY, but phenotypically female. This accounts for 46% of 46,XX males
SRY is TDF
- Expressed from E10.5 to E12.5 (immediately prior to seminiferous tubule appearance)
- Expressed in pre-Sertoli Cells
- Y linked inheritance
- Conserved in mammals
What happens when you add SRY to females?
Will have male genitalia
46, XY females
Sry negative:
Due to Y chromosome deletion from X-Y interchange
Y-autosome translocations possible but rare
Sry mutations:
Mostly in the HMG box
Only 10-20% XY females have Sry mutations
Is SRY the only sex determining gene?
No; but it is the only one on the Y chromosome
Human SOX9
- Mutations in SOX9 cause Campomelic Dysplasia (bone abnormalities)
- 46, XY sex reversal with ambiguous or female genitalia
- Death in neonatal period due to respiratory insufficiency
- SRY-related gene
- Autosomal dominant
BOWING OF THE BONES IS STRIKING IN THESE CASES
What happens when you add SOX9 to female
- will lead to male phenotype
Amh
- Also known as MIS, is a member of the TGF beta family.
- Secreted by Sertoli Cells/Granulosa cells
- Cryptoorchidism
- Causes regression of Mullerian derivatives
Amh/Amhr human mutations
Persistent Mullerian duct syndrome
Typically: male with uterus and fallopian tubes
Amh and its receptor have same phenotype
Mice Amh/Amhr KOs show similar phenotype
These men can be infertile, can also have problem with descent of their testes.
What do we know about female development
- Very little
- Only two genes have been identified
- DAX1 not really involved
- Wnt4 = weak association
AHC (DAX-1, Nr0b1, Chr. Xp21.3)
Nuclear hormone receptor with DNA binding domain
Deletions cause congenital adrenal hypoplasia, and hypogonadism
Expressed in Sertoli cells
Duplication of Xp21 (XY individuals) develop as females, external genitalia, impaired testes by histology (DSS- dosage sensitive)
Female fertility is unaffected
Maybe duplication of gene drives female development? But when you knock out Ahch gene; you find that female development is not affected.
Does knocking out Ahch affect female development in mice?
No
Wnt4 mouse KO
Lacks mullerian structures (uterus, upper vagina, fallopian tubes)
Germ cells are lacking
Testosterone biosynthesis activated in females
WNT4 mutant humans are masculinized
Mayer-Rokitansky-Kuster-Hauser syndrome Primary amenorrhea Short vagina No uterus, no fallopian tubes Ovaries of normal size Aplastic right kidney Elevated levels of androstenedione, testosterone, DHEAS
GENITAL AMBIGUITY
True hermaphroditism (46, XX ovotesticular disorder of sex development)
Female pseudohermaphroditism (46, XX disorders of sex development)
Male pseudohermaphroditism (46, XY disorders of sex develpoment)
Pseudohermaphroditism
The discrepancy between gonadal tissue (histologic diagnosis) and external genitalia appearance is called pseudohermaphroditism.
If gonadal tissue is a testis, term male pseudohermaphrodite.
If ovary is present the term is female pseudohermaphrodite.
Archaic term. Coined prior to X and Y knowledge.
Disorders of sexual development/differentiations is a better term
46,XX ovotesticular disorder of sex development (true hermaphroditism)
Both testicular and ovarian tissue
Oocytes, not just fibrous stroma
Testicular tubules or spermatozoa Male external genitalia (70%) Pubertal feminization (Most) Uterus (90%) (Tubal occlusion associated) Pregnancies (46,XX) Some gonadal neoplasia
46, XX DISORDERS OF SEX DEVELOPMENT AND ADRENAL HYPERPLASIA (female pseudohermaphroditism )
21-Hydroxylase deficiency
11alpha-hydroxylase deficiency
17 alpha-OH PROGESTERONE
Elevation in serum indicates block at 21-hydroxylase site in adrenal biosynthetic pathways
Reliable rapid assay
21-HYDROXYLASE DEFICIENCY (FEMALES)
Phenotype: Ambiguous genitalia Uterus and ovaries normal Fertility with treatment normal Salt wasting Diagnosis: Elevated 17-OH Progesterone
Treatment: Cortisol
Mineralocorticoids
11beta-HYDROXYLASE DEFICIENCY
Phenotype:Ambiguous genitalia (XX)
Hypertension due to salt retention (XX and XY)
Hypertension in 11 differentiates it from 21 hydroxylase deficiency
Lab: Deoxycortisol and deoxycorticosterone levels are elevated.
Treatment:Cortisol
AROMATASE DEFICIENCY (CYP19)
Clitoral hypertrophy Affected XX and (tall) XY sibs, delayed epiphyseal closure Virilization of the pregnant mother Primary amenorrhea Autosomal recessive Therapy: Estrogen!
TERATOGENIC 46, XX disorders of sex development
Virilization of female fetuses possible if androgens or androgenic progestens given pregnant mother in first trimester
Once (1950’s, 1960’s) relatively frequent but now rare: - Doses of inadvertent oral contraceptives low
- Hormonal support now rare
Can occur with danazol
46, XY disorders of sex development (male pseudohermaphroditism )
45,X/46,XY mosaicism Testicular biosynthetic errors 5-reductase deficiency Complete and partial androgen insensitivity Agonadia Leydig cell agenesis
45,X/46,XY
Variable phenotype: female or ambiguous external genitalia
Variable frequency of somatic anomalies (Turner stigmata)
Uterus usually present
Increased gonadal neoplasia
SELECTION BIAS IN 45,X/46,XY
Most cases detected in neonates show female or ambiguous external genitalia
Approximately 85% of cases detected in utero (amniocentesis, CVS) show male external genitalia
5 alpha-REDUCTASE DEFICIENCY
Enzyme Block: T DHT
Normal male levels of T but DHT low
T/DHT ratio elevated
Males only, autosomal recessive condition Normal male external genitalia, ambiguous genitalia, or Normal female genitalia. Male gonads with testicles and Wolffian structures Often raised as girls, male gender identity, and virilization with puberty (deepening voice, clitoral enlargement, testes descent) Jeffrey Eugenides won a Pulitzer Prize for his 2002 novel Middlesex
HORMONE RECEPTOR DEFECTS
Estrogen
Androgen
Anti-mullerian hormone
PARTIAL ANDROGEN INSENSITIVITY
Variable extent of genital virilization (Labial fusion and clitoral hypertrophy)
Hypospadias
Feminization (breast development at puberty) despite testosterone being higher than in unaffected XY individuals
Complete Androgen Insensitivity
46XY individuals have bilateral testes Female external genitalia Blind ending vagina No mullerian derivatives Cells unable to respond to testosterone Gonadal neoplasia in 5% Well developed breasts
Evaluation of infant with ambiguous genitalia
Three generation family history
Prenatal history, prenatal ultrasound, testing done
Physical examination:
Chromosomes (microarray)
Ultrasound to assess internal anatomy.
Adrenal and gonadal steroid secretion
GONADAL FAILURE IN MONOSOMY X
Germ cells present in 45,X abortuses and neonates.
Pathogenesis must therefore involve increased rate of atresia.
Such a pathogenesis consistent with occasional 45,X pregnancy.
OVARIAN MAINTENANCE DETERMINANTS
Located in several regions of Xp and Xq
Loci in regions Xp11 and Xq13 most important
Elucidating function of these genes would be relevant for normal ovarian development
X-chromosome genes
BMP15- bone morphogenetic protein 15
FMR1- fragile X mental retardation
POF1B- premature ovarian failure region 1
PGMRC1-progesterone membrane receptor
Inverdale and Hanna sheep
Missense and Nonsense mutations in Bmp15 gene, X chromosome
Heterozygous sheep have increased fertility
Homozygous sheep are infertile
Humans with Bmp15 mutations described and found to be infertile
FRAGILE X SYNDROME
X-linked recessive gene localized to
Xq27.3 protein FMR1
Initially detected through chromosomal
fragility (thus “Fragile X”)
1:4000 Males: Mental retardation (most
common genetic cause in males)
1: 4000-8000 Females (Borderline IQ)
CGG REPEATS IN FRAGILE X SYNDROME
Affected: >200
Premutation: 54-200
Normal: 5-54
FMR1 AND POF
10-15% of premutation carriers manifest POF.
Premutation in 1-5% sporadic POF and 10-15% familial POF.
Prevalence in premutation carriers increases as (CGG)n increases up to 80-99, but plateaus thereafter or decreases.
Not increased in full mutation.
XX GONADAL DYSGENESIS
IN FINLAND
Frequency: 1/8300 liveborn females
Of 75 cases, 57 sporadic
Aggregated in north central Finland where consanguinity rate 12%
Segregation ratio 0.23 for female sibs
FSHR MOUSE MODEL
Knockout shows primordial, primary and secondary follicles
Females are sterile
Males are fertile (reduced sperm count)
Closely mimics human phenotype
FSHB MUTATIONS
Three subjects reported to date
Clinical: Primary amenorrhea, Infertility, Normal adrenarche and absent thelarche
Small ovaries and uteri
Mutations necessary in both copies of FSH to have a phenotype
Autosomal recessive inheritance
FSH BETA KNOCKOUT IN MICE
Phenotype closely mimicks what is observed in humans
Females are sterile
Males are fertile
BLEPHAROPHIMOSIS-PTOSIS EPICANTHUS (FOXL2)
- Autosomal dominant: 3q2224
- Type II: Premature ovarian failure
- Gene: Winged-helix/forkhead transcription factor (FOXL2)
- Expressed in mesenchyme of mouse ovaries and eyelids
FOXL2 BLOCKS ADULT OVARY TO TESTES TRANSDIFFERENTIATION
- Conditional excision of Foxl2 caused follicular structures of the ovary to look like seminiferous tubules of the testes
- Up-regulation testes determining genes: Sox9 and Dmrt1.
- Adult lineage reprogramming
- Active repression of the Sertoli cell-promoting gene Sox9, maintains mammalian ovarian phenotype through adulthood.
Future of Sex disorders
Molecular studies to delineate anti-male and pro-female molecules.
Further genetic classification of DSDs
Effects of environment on germline quality and transgenerational effects
