10: Sex Determination

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Sex Determination

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The only case in which an organ can have two different fates.

Both the male and female develop an INDIFFERENT GONAD, with the capacity to develop into ovary or testis. Becoming an ovary or testis then goes on to develop/determine the phenotype of the animal.

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Indifferent Gonad

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Capable of developing into (a) ovary, or (b) testis, which goes on to determine phenotype

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Abnormal Reproductive System Development

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If reproductive system develops abnormally, animal usually can LIVE with non-functional reproductive system, (whereas couldn't live w/out functional cardiovascular or respiratory system for example).

(1) Sex Reversed; where XY genotype is phenotypically female
(2) Intersex; mixed male & female phenotype
(3) Hermaphrodites; both male and female sexual tracts

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Control of Sex Determination

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For birds, may want to find a genetic way to control/manipulate sex of offspring. For example, can sort sperm (X & Y) because want female birds (for egg-laying operations).

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Mammals, Sex Determination

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Female: XX
Male: XY
--> Y is what makes an individual "male"

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Sex Chromosome Abnormalities

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Aneuploidy = abnormal number of chromosomes

Examples; (1) Kleinfelter's Syndrome; XXY, a TRISOMY = presence of three copies of a chromosome. One X is enough to make male.
(2) Turner's Syndrome; X, a MONOSOMY = lack a chromosome of normal complement. Even w/ one X, still female.

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Indifferent Gonad, Differentiation

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DEFAULT TRACT: Indifferent gonad, w/out Y present, is a active tract that becomes ovary, and leads to female phenotype.

Y Chromosome present: indifferent gonad becomes testis, and develop male phenotype.

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Male, Sex-determining Gene

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Male has a big X chromosome, with 1100 genes. Male also has small Y chromosome w/ 45 genes, all related to male reproduction. Gene on Y Chromosome determines male sex.

(1) SRY gene, found on sex-determining region of Y chromosome. On short arm of Y chromosome. Only found in mammals. --> A female (phenotype) with XY genotype must have a SRY gene not working/not-functional.

(2) Also, Sox 9 = autosomal gene (not on sex chromosomes) = sex determining gene that acts on indifferent gonad to turn to testicle.

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Evidence Y-Chromosome Determines Male...

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(1) SRY gene is expressed at sex differentiation
(2) Mutated SRY gene in XY is phenotypically female
(3) Transgenic mice; XX mice with SRY gene transgenically inserted = became male
(4) XY male = has normal SRY gene/expression

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SRY on X Chromosome...

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Occasionally during meiosis, get crossing over, where SRY gene can be passed over to X chromosome (resulting in extra material on end of X chromosome, and XY genotype with still a male phenotype).

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SRY Protein, influencing DNA & Transcription...

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SRY protein inserts into minor groove of DNA and bends it The enhancer DNA regions are brought closer together, which can affect transcription

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Sox 9

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An autosomal gene (not found on sex chromosomes), became identified as a major sex determining gene. it acts on the indifferent gonad to turn to testicle.

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Inhibition of Ovarian Pathway

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Increased expression of Sox 9 leads to inhibition of ovarian pathway, and specifically the conversion of the indifferent gonad to testicles.

Sox 9 is in turn regulated by; (1) SRY, and another gene, (2) Steroidogenic Factor 1 (SF1)

Sox 9 needs to be expressed all the time during sex differentiation, so there is an enhancer to increase Sox 9 expression with genes (1) FGF 9, (2) PSD, and (3) DMRT1.

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Ovarian Development Pathway

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In the female, there are a number of genes which are expressed to induce ovarian development. These genes inhibit Sox 9, preventing testis formation.

It is not known what influences the expression of these genes. Such genes include; (1) FOXL2, & (2) WNT4.

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Birds, sex determination

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Female: Heterogametic, ZW
Male: Homogametic, ZZ

Important mechanism that makes an individual male = two Z's --> GENE DOSAGE. (ZZ = male, Z = female)

Genotype ZZW = INTERSEX; shared both male & female characteristics

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Birds; Mechanisms of sex determination

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(1) DMRT 1 gene (same gene found in mammals); stimulates SOX 9, which influences indifferent gonad to become testis. With two copies, becomes male. One copy, becomes female.

(2) W Chromosome: contains genes important for ovary formation.

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Reptiles & Amphibians; Sex determination

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In many cases there are NO sex chromosomes, it's often ENVIRONMENT that determines if male/female.

For example; TEMPERATURE can influence male or female development. Temperature sensitive genes = (1) FOXL2; leads to ovarian development --> female
(2) DMRT1; stimulates Sox 9 --> male

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Snakes, Sex Chromosomes

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Female = ZW
Male = ZZ

May be similar to birds.

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Fish, Sex Chromosomes

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Female = XX
Male = XY

Environment, temperature = influences sex differentiation. This mechanism is not completely understood.

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Ovarian Development, 'By Default?'

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Bipotential gonad, able to develop into testis or ovary. SRY = increases Sox 9 expression --> leads to testis development.

XY genotype, SRY present --> Sox 9 expression --> Testis develop

XX genotype, NO SRY --> Ovaries develop

BUT... There are situations gone wrong; (1) XY, SRY expressed, but Female. (2) XX, No SRY expressed, but still Male.
--> Ovarian development is not just by default. It is more complicated. There are extra-regulatory factors.

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Mechanisms of ovary development...

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ACTIVE signaling is required to transform indifferent gonad to ovary to (1) repress testis development, and (2) promote ovary development.

SRY gene --> negatively regulates FACTOR Z Gene = which represses male development

Research carried out to determine candidates for anti-testis & pro-ovary genes.

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Anti-testis & Pro-ovary genes

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Research in mouse embryos for candidates of anti-testis & pro-ovary genes.

Looking for genes expressed in indifferent gonad that are downregulated in male, and expressed in female; Discovered two...
(1) Wnt-4
(2) DAX 1

Research in female to male sex reversal situation (XX, no SRY, male) discovered...PIS region, containing;
(3) PISRT1 (polled gene in goats, when homozygous leads to female to male sex reversal), and
(4) FOXL2

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Anti-testis/Pro-ovary Gene Functions

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Genes DAX1, Wnt4, PISRT1 & FOXL2 --> (1) Need to repress male development;
(a) Sox 9; indifferent gonad develop into testis
(b) SF1; increase Sox 9 expression
(c) Testosterone prod'n,
(d) Anti-mullerian hormone (AMH); prevents mullerian development in embryo (--> which forms female repro tract)

Also are involved with; (2) germ cell migration and mitosis, (3) somatic cell development and estrogen synthesis, (4) folliculogenesis and follicular development, and (5) mullerian duct formation and development.

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SRY & Anti-testis/Pro-ovary genes

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SRY has a negative effect on genes (1) DAX1, (2) Wnt4, (3) PISRT1, and (4) FOXL2 --> Which is the FACTOR "Z" that SRY needs to control.

Inhibiting these anti-testis/pro-ovary genes removes the repression/inhibition on factors of male development (Sox9, SF-1, Testosterone, and AMH)

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FGF9 & Wnt4

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A balance of expression of (1) FGF9 and (2) Wnt4 is important in determining Sox9 expression. Both are in equal balance in developing mammalian gonad before sex determination

FGF9 = is activated by Sox9, and forms positive feedback loop with Sox9. FGF9 also blocks Wnt4.

Wnt4 = involved with (a) mullerian duct development, (b) prevents leydig cell differentiation & testis blood vessel formation, and (c) stimulates DAX1; another anti-testis/pro-ovary gene --> inhibits SF-1, preventing testosterone synthesis, wolffian duct development, and preventing mullerian duct regression.

If XY mice lose Fgf9, they develop ovaries, while XX mice that lose Wnt4 develop incomplete testes. This suggests that vertebrate sex determination results from the interplay between these two opposing signals.

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DAX1 & SF-1

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DAX1 = an "anti-testis/pro-ovary" gene expressed in females. Fxn: inhibits SF-1 expression, which prevents testosterone synthesis, wolffian duct development, and prevents mullerian duct regression.

SF-1 = Male development gene. (a) promotes expression of StAR gene, involved w/ testosterone synthesis & wolffian duct development (form part of male repro tract). (b) promotes expression of antimullarian hormone (AMH) gene, resulting in mullerian duct (form part of female repro tract) regression

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Factor Z Candidate

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Factor Z = a factor that represses male development, but is negatively regulated by the SRY gene (found on Y chromosome, involved w/ bipotential indifferent gonad becoming testis).

FOXL2 = a gene (a) involved w/ ovarian development, somatic cell differentiation, & follicle development, and (b) inhibits SOX9 expression. A candidate for Factor Z, it may be negatively regulated by SRY gene.

PISRT1 gene = also inhibits SOX9 and encourages ovarian development. When a homozygous mutation exists for this gene, an individual has SOX9 expression and female to male sex reversal.

How can get: XX genotype, no SRY, but male phenotype = (a) FOXL2 knocked out in female, or (b) homozygous for PIS mutations.

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Germ cells in Developing Ovary

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Germ cells are very important in developing ovary.

--> If ovaries developed w/out germ cells (called a 'streak' gonad), then (a) NO supporting somatic cells develop, (b) male markers may be expressed.

--> With germ cells present; signaling b/w germ cells (c-kit receptor) and somatic cells (by kit-ligand, a cytokine that binds to c-kit). c-Kit is expressed in primordial germ cells of males and females. Kit-ligand is expressed along the pathways that the germ cells use to reach their terminal destination in the body, and in the final destinations for these cells, helping guide the germ cells to their appropriate locations in the body.

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Oocyte-granulosa cell signaling

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Important in subsequent follicular development:
(1) follicular recruitment and steroidogenesis: AMH (granulosa cells)
(2) Primary to Secondary follicle: GDF9 (oocyte)
(3) Growth of preantral follicles: c-kit & kit-ligand

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Sex Determination Timeline: (In Bovine Female Fetus)

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6 weeks: Beginning of morphological differences preceded by differences in steroidogenesis. (Ovaries differentiated, produce estradiol, but no noticeable morphological difference yet.)

Day 60: Development of Mullerian ducts

B/w Day 60 & 70 can do sexing (transrectal ultrasound). Genital tubercle present, moves from mid-position b/w tail & umbilicus to under tail (if no androgens). Genital folds remain separate.

Day 70: Regression of Wolffian ducts

Day 100: Primordial follicles in ovary