Mechanisms underlying sexual differentiation

Question 1

Three potential sources of sex differences.

Answer 1

• Y chromosome in males only.
• Two X’s in females vs. one in males (Female-only X inactivation).
• Parental Origin’s of X’s

Question 2

Outline differences between X and Y chromosomes. Why do X chromosomes contrain so many genes relevant to brain functioning? (c.155 million, ~1500 vs. c.60 million, ~350 pseudo, 78 protein-coding).

Answer 2

• The human X and Y chromosome.
  o X chromosome
   Average size: ~155 million bps, containing ~1500 genes. Many involved in brain function and neurodevelopment.
  • Why? One common idea is that at some point in evolution, female mammals decided to select male mammals in mating on the basis of how smart they were.
  o Y chromosome
   Small size: ~60 million bps, contains ~350 (pseudo)genes; 78 protein-coding, most genes involved in sperm production, some expressed in the brain.

Question 3

Male-limited expression on Y What is SrY? How was its function discovered? What does it do and how? (bipotential gonads -> switches on = differentiation –> androgens –. masculinisation.

Answer 3

o SRY (Sex-determining Region on Y) can influence brain development/function indirectly via gonadal hormones.
o Was identified as the testis-determining factor in the early 1990s.
 XY, XXY, and XXXY subjects all male.
 Sry-transgenic XX mice sterile males.
 Translocation of part of Y chromosome containing SRY to X chromosome can cause XX male syndrome.
 Rare XY females with mutations of SRY (Swyer syndrome).
o SRY encodes transcription factors that regulate gene expressions in cells. During early mammalian development, organisms have bipotential gonads (could differentiate into either ovaries or testes), if SRY switches on, causes gonads to differentiate along a sex-differentiated route in testes. Testes begin to secrete a variety of androgens, including testosterone. Androgens bind to androgen receptors in specific tissues. These are suspended in the cytoplasm, but once the androgen binds to it, the complex moves to the nucleus where it can exert effects on DNA. This initiates the process of masculinisation causing differentiation of the brain and male specific tissues.

Question 4

How do androgens affect masculinisation (inc. pre- and perinatal [organisational] and postnatal [activational] puberty effects?

Answer 4

Androgen receptors are highly concentrated in sexually dimorphic brain regions, e.g. amygdala, medial pre-optic area, paraventricular nucleus, anteroventral periventricular nucleus, temporal cortex.
 Androgens can affect masculinisation
• In critical pre- and perinatal periods (organisational) effects):
o Genital virilisation (4-6 weeks gestation)
o Gender determination (2nd trimester)
o Increase <4-6 months postnatally (unknown function).
• In later postnatal life, e.g. during puberty (activational) effects.
 Not affecting gross structure, only having subtle effects.

Question 5

ASD as the “extreme male brain” systematising vs. empathising. in utero testosterone. 2D:4D ration (indirect).

Answer 5

 Males and females differ with regard to “systematising” and “empathising” behaviours (and underlying neural substrates).
 Autism posited to represent an extreme of the male pattern (impaired empathising, enhanced systematising).
 Some evidence that in utero testosterone levels may correlate with alter male behaviours.
 However, indirect measures (2D:4D finger length ratio), or small sample sizes from amniotic fluid samples = increased likelihood of autistic behaviours in children (latter probably biased sample).
 Potentially, increased testosterone = increased risk of autism. The genes underlying the metabolism of these sex-specific steroids and their receptors might be associated with autism.

Question 6

What is the evidence that genes can directly cause sexual differentiation independently (i.e. without the mediation) of hormones? Gynandromorphs. Extra credit: why does this only occur in birds, insects, crustaceans?

Answer 6

 Some evidence comes for gynandromorphic organisms. Observations of these suggest that sex-linked genes may act directly on the brain and contribute to sexually dimorphic phenotypes independently of hormones. For example, in a gynandromorphic zebra finch- half of their body exhibited male plumage, the other half had female plumage. In the brain, the hemisphere aligning with the male plumage also contained a male-specific song circuit. Particularly strange since every cell in every part of the body is surrounded by the same hormone levels. Can’t be due to hormones. Looking at the genetics, on the male side it had male sex chromosomes, on the female side, had female sex chromosomes, suggesting that underlying genetics caused the unique morphology.
 This bilateral asymmetry has only been observed in birds, insects, and crustaceans. This is due to events in mitosis when the organism is only a few cells large- where one of the cells does not split its sex chromosomes correctly, leading to one of the two cells having male chromosomes, the other having female. For example, an XY cell duplicates to XXYY, which would usually split into two XY cells, but may very rarely divide into an X and an XYY cell. If this happens early enough, then a large proportion of cells will be X, and the other half will be XYY.

Question 7

Outline the Four-Core Genotypes model and its utility: gonadal vs karyotypical phenotye 40, XX; 40, XY-; 40, XXSry; 40, XY-Sry.

Answer 7

In this model, take a set of common parents producing 4 different types of offspring. Some are both karyotypically and gonadally female (40, XX), some are karyotypically male but gonadally female due to a mutation in the Sry gene leading to development of female phenotype, similar to Swyer syndrome in humans (40, XY-), some have a female karyotype with an Sry region attached to one of the autosomes such that they develop testes (40, XXSry), others effectively normal males with male karyotype (Sry deleted then added back) and male gonad (40, XY-Sry).  Using this model, can test whether a particular phenotype segregates with gonadal type (testes/ovaries), or with sex chromosome complement (XX/XY).
• I.e. can parse out the effects of hormones vs. genetics.

Question 8

In the Four-Core Genotype Model, most sexually dimorphic phenotypes separate with gonadal type, what doesn’t?

Answer 8

Sex-linked gene effects have been described on nociception, habit formation (observation of increased propensity to addiction disorders, see below), aggression, social interaction style, and reinforcer sensitivity.

Question 9

How can SRY directly affect brain function (DA & VTA)

Answer 9

 SRY can influence brain function directly (e.g. dopaminergic activity in the substantia nigra of the VTA), maybe SRY predisposes to some dopaminergic related conditions such as Parkinsons.

Question 10

How may geographically linked Y haplotypes confer either risk (or protection) in males? (R1b, age of haplotype + IQ, alcohol dependence, hypertension, aggression)

Answer 10

 There are many haplotypes (“flavours”) of Y chromosomes in males, some are more prevalent in certain regions than others. For example, R1b is most dominant in Britain.
• In terms of IQ scores, if you have the older version of the Y chromosome and ADHD, you tended to have lower IQ scores than those with the more recent version.
 Some evidence of other Y haplogroup effects in alcohol dependence, hypertension, and aggressive behaviour.

Question 11

Higher expression of X-linked genes in females: Is X-inactivation a complete process?

Answer 11

o Generally, one female X is silenced to ensure equal X-linked activity with males.
o However, ~20% of all X-linked genes are more highly expressed in female brain as they are expressed from both X chromosomes. I.e. X-inactivation is not a complete process.

Question 12

What are some functional consequences of aberrant X-linked gene dosage, i.e. errors in X-inactivation?

Answer 12

Reduced X-linked gene dosage = Turner Syndrome (45, X), Increased = Klinefelter syndrom (47, XXY).

Question 13

What are symptoms of Turner syndrome? Why are these believed to be genetic and not hormonal effects?

Answer 13

Ovarian dysfunction, and is linked to the following:
Impaired attention and social cognition (increased ADHD and autism diagnoses).
Impaired maths/reading.
Impaired visuospatial skills.
Increased anxiety (though causality?)
Genetic, not hormonal because: Many TS cognitive deficits maintained throughout development. Deficits not completely rescued by hormone supplementation. Similar deficits not seen in females with premature ovarian failure.

Question 14

How have candidate genes for TS phenotypes been identified using deletion mapping (Zinn et al., Xp22.3 example, neurocognitive function.

Answer 14

• Find individuals with varying mutations of the X chromosome (missing various bits of the X)
o E.g., TS syndrome with terminal deletion with particular phenotype (e.g. impaired attention), get another individual with TS and impaired attention but larger terminal deletion, another individual with TS with same phenotype but has interstitial deletion. Look for common region of deletion- within that interval there must be a gene responsible for that particular phenotype.
o Has been done in TS by Zinn et al., implication of Xp22.3 involvement in neurocognitive function.

Question 15

Deletion mapping in TS: Thus, increased expression of certain X-linked genes in females relative to males confer protection for psychiatric disorders. How has this been tested in 39, XO mice against their 40, XX counterparts (attention endophenotype; Xp22.3 homologue, STS as Y-linked pseudogene escapes X-inactivation; protective effect of X-linked genes (STS is X-linked in humans)

Answer 15

o 39, XO mouse model is attentionally impaired like in TS; deficits can be rescued by addition of small Y*X chromosome containing STS (steroid sulfatase) gene, to the extent that their attention does not differ from their 40, XX counterparts (Davies et al., 2009).
o X-linked gene STS (Xp22.3) influences ADHD risk.
o STS escapes X-inactivation; non-expressed Y-linked pseudogene.
o Does higher expression in females protect against developing ADHD (thus explaining the sex-specific aetiological nature of ADHD), or more specifically, STS expression protects against deficits in attention?

Question 16

Why are inactive allosomes “heterochromatin sinks”, and how may this impact the autosome?

Answer 16

• Maintenance of heterochromatinization of inactive X chromosome and Y chromosome depends on enzymes that modify the epigenome.
• Possible gender-specific reductions in the availability of these enzymes to modify autosomal epigenome (and therefore regulate gene expression). Because you get less heterochromatin produced in male cells due to smaller area of the Y genes, maybe less capacity to regulate autosomal genes (perhaps explanation of sex differences in gene expressions not related to allosomes).

Question 17

How does male hemizygosity make them more vulnerable to X-linked mutations.

Answer 17

 As males only have one X chromosome, effects of a mutation will be manifest.
 As females have a second X, effects of a mutation on one X will be “masked” by the other.
 Most famous example is colour-blindness.

Question 18

Outline the X-linked explanation for IQ variability (10% X ch. mutation in LDs [cf. other disorders], “superhaplotypes” vs. “poor” haplotypes.

Answer 18

 We know that mutations of the X chromosome can potentially have consequences for learning disability.
 We know that in the X chromosome, about 10% are mutated in LDs, much higher than in other disorders.
 Females have two X’s with many genes on each. Each gene has two alleles (e.g., an average version and a “super” version, the latter may lead to improved cognition/intelligence). When the mother produces eggs, the two X chromosomes can recombine and then get parcelled into one of two eggs. Rarely, all the “super” alleles end up on one X chromosome, leading to a “superhaplotype”. That egg could be fertilised by sperm containing a Y chromosome, thus offspring will be male. Thus, every cell in his body will contain the X with the superhaplotype- thus he will be very intelligent.
 Alternatively, the sperm could contain the X chromosome, chances are that this sperm will be more average. If this happens, the fertilised egg will develop into a female with the super X plus the average X. This means that the average X is going to pull the phenotype towards the mean- i.e. regression towards the mean. Thus, explains the disproportionately high number of males at the higher end of the spectrum.
 Thus, also explains the other end of the spectrum, e.g. all “poor” alleles collected on the X chromosome of the female.

Question 19

What does X inactivation predict of sex differences between twin pairs?

Answer 19

 Identical female twins differ more than identical male twins.  This is because, although they share the same genes, female twins may have different patterns of X-inactivation (might be altered in utero or environmental factors).

Question 20

Parental origin of X chromosome: Turner syndrome Xp vs. Xm, same-opposite word task (Skuse et al., 1997).

Answer 20

o Turner syndrome: 45, Xp (X of paternal origin) or 45,Xm (X of maternal origin)
 Skuse et al. (1997): from social-cognitive questionnaire, girls who inherited single X chromosome from mum suffered more from behavioural inhibition.
• Explicitly tested with a neuropsychological paradigm: the Same-Opposite Word Task. Time difference between the Same and Opposite tasks gives a “Behavioural Flexibility Index”. XM girls were impaired compared to XP. Suggests the latter is protective. Potentially females may benefit of social cognition improving gene from Dad, suggesting that may be why they have decreased incidence of disorders such as ASD.

Question 21

Why and how parental origin of X matters, need to identify gene variants (Davies et al., 2005; serial reversal learning paradigm: Xlr3b gene.

Answer 21

 Possible using 39, XPO mice, and 39, XMO mice. Compared in terms of behaviour in a serial reversal learning paradigm.
• A test of behavioural flexibility requiring elements of the same psychology as the Same-Opposite Word Task.
• Acquisition: Mice given a binary choice (dark or light arm) on a Y-maze (only one arm rewarded).
• Reversal (x3): Upon acquisition (>85% correct responding), contingencies changed such that previously rewarded arm is unrewarded and vice versa.
• The less behaviourally flexible the animal is, the more errors it will make in learning the new S-R association.
• 39, XMO mice acquired at the same rate as 39, XPO mice, but the former is less behaviourally flexible than the latter (and 40, XX counterparts), i.e. they tended to perseverate in going down the same arm (Davies et al., 2005).
 Then wanted to look at gene expression in the brains of the two karyotypes in order to identify candidate genes.
• Identified one differentially expressed X-linked gene (Xlr3b): expression difference between male and female mouse brain.
• So, can Xlr3b explain TS and sex difference data? Humans do not have an analogous gene. The search goes on.

Question 22

How can these sex-linked mechanisms have downstream effects on the autosomes (methylation, Spiers et al., 2015

Answer 22

o These sex-linked genetic mechanisms can have downstream effects on gene expression on the autosomes as mentioned before (e.g. epigenetics; Spiers et al., 2015).
 Examined methylation in “healthy” 100 males, 79 female human foetal brain samples.
 ~2% of probes assessed suggested sex-specific differential methylation of genome
 ~93.5 of these located on X chromosome; remaining ~6.5% on the autosome
 ~50% of autosomal DMRs hypermethylated in females.

Question 23

How may male risk for neurodevelopmental disorder be enhanced?

Answer 23

 Presence of a Y chromosome (higher circulating testosterone: predicts increased risk in females exposed to high testosterone).
 Reduced expression of X-inactivation escaping genes.
 Expression of deleterious X-linked mutations.
 Possession of a maternally inherited X chromosome only.

Question 24

How may female protection to neurodevelopmental disorder be conferred?

Answer 24

 Lack of a Y chromosome
 Increased expression of X-inactivation escaping GENES.
 Protection against X-linked mutations by possession of a second X.
 Possession of a paternally inherited X chromosome

Question 25

What is the evidence of a female protective model (Jacquemont et al., 2014, 15,585; female probands inc. deleterious mutations;Chang et al., 2015, truncating mutations impacting upon more highly brain-expressed genes than in male. ADHD (Martin et al., 2014)

Answer 25

 Jacquemont et al. (2014) screened a large sample (15,585) of male and female individuals diagnosed with a neurodevelopmental disorder. Female probands had a significant excess of deleterious autosomal mutations (CNVs, SNVs) compared to males when matched for phenotype severity. Truncating mutations in females tend to impact upon more highly brain-expressed genes than in males (Chang et al., 2015). Females buffered against adverse effects of genetic mutations (sex-linked/hormonal mechanisms?)
• Cardiff work has shown that healthy girls have higher polygenic scores for ADHD genetic risk variants than healthy boys when matched for ADHD-related traits (Martin et al., 2014)