sex hormones

Question 1

how does the luciferase assay work

Answer 1

used to identify ligands for particular receptors
regulatory region of gene of interest cloned upstream of luciferase gene
candidate ligand can bind to receptor and cause gene expression -> detection of light to determine ligand binding

Question 2

how to determine target gene of a nuclear receptor

Answer 2

clone potential target gene promoters upstream of luciferase gene
test for activation/repression in response to ligand

Question 3

FXR: ligand, target gene and actions

Answer 3

bile acids bind to FXR -> expression of FGF15/19 -> glycogenesis, decreased gluconeogenesis; potential diabetic drug (insulin-independent)

Question 4

FGF21:

1. transcription stimulated by?
2. signals via:
3. effects (3)

Answer 4

1. PPARalpha
2. beta-klotho
3. FGF21 decreases SCN vasopressin expression and AVPV Kiss1 expression -> suppression of female reproduction (as part of adaptive starvation response)
   weight loss and insulin sensitivity in diabetic and obese rodents and humans
   induces CRH -> increased sympathetic nerve activity

Question 5

how are floxed genes used to produce tissue-specific KOs

Answer 5

e. g. delete βklotho seletively in SCN to see if rescues fertility:
1. Generate ‘floxed’ βKlotho mice: βKlotho gene sandwiched between 2 lox P sites (in every cell of mouse)
2. Generate second mouse w cre recombinase under SCN specific promoter ∴ enzyme only made in SCN
3. Cross 2 mice → Cre recombinase causes DNA to fold and excises gene between lox P sites (only in SCN)

Question 6

actions of progesterone (10 targets)

Answer 6

endometrium: implantation, decidualisation
myometrium: suppresses uterine contractility
ovary: ovulation, luteinisation
bone: prevention of bone loss
breast: proliferation, lobular alveolar development
brain: increased sexual behaviour
sperm: acrosome reaction
oocyte: maturation
vascular: decreased platelet aggregation, vasodilatation
immune: decreased T cells

Question 7

progesterone receptor isoform functions in humans

Answer 7

PR-A: high levels antagonise effects of P4

PR-B: high levels mediate effects of P4

Question 8

progesterone intracellular signalling (5 pathways)

Answer 8

receptor signalling mechanisms poorly understood
1. classical: progesterone binds to intracellular PR, dimerises and binds to progesterone response element (promoter), recruits coactivators/corepressors and RNA pol -> gene expression
2. alternative: PR binding to receptor -> c-Src cascade -> MAPK -> recruitment of different transcription factors e.g. CREB -> binds to CRE (cAMP response element)
3. transmembrane receptor: progesterone receptor membrane component (PGRMC-1) -> enters cell in vesicle -> calcium influx -> downstream cascades
PGRMC-1 also forms complex w plasminogen activator inhibitor RNA binding protein-1 (PAIRBP-1); function unknown
4. inhibitory: progesterone binds to GPCR -> decreased cAMP -> decreased CREB

Question 9

regulation of decidualisation

Answer 9

convergence of P4 and cAMP pathways

Question 10

indications for androgen replacement therapy in females

Answer 10

decreased sexual desire, arousal and orgasm

decreased testosterone can also affect mood, bone and muscle mass

Question 11

nuclear receptor: AF1 and AF2 functions

Answer 11

contribute to transcriptional activity of nuclear receptor
AF1: independent of ligand-binding
AF2: part of LBD, dependent on ligand binding

Question 12

nuclear receptor: DNA binding domain structure

Answer 12

2 zinc finger motifs -> can bind to palindromic sequence

Question 13

nuclear receptors: pioneer factors

1. function
2. examples

Answer 13

transcription factors that can directly bind condensed chromatin
e.g. FOXA1, GATA3

Question 14

benefits of HRT (4)

Answer 14

1. reduced vasomotor symptoms: hot flushes, night sweats
2. reduced : mood swings, anxiety
3. reduced risk of osteoporosis
4. CV effects: decreased BP, cholesterol, centripetal fat

Question 15

risks of HRT (3)

Answer 15

breast cancer, stroke, thrombo-embolism

Question 16

androgen receptor signalling

Answer 16

1. Testosterone bound by SHBG (sex hormone binding globulin) in circulation
2. Testosterone: lipophilic, diffuses across membrane
3. 5α-reducatse converts to DHT (more potent androgen)
4. AR sits in cytoplasm complexed w heat shock proteins (act as chaperone proteins: change conformation so that ligand can bind easily)
5. Binding of DHT to AR → release of heat shock proteins from receptor, conformational change, dimerisation
6. Dimerisation exposes nuclear localisation sequence → signals to be transported to nucleus
7. Complex binds to DNA ARE (androgen response element)
8. Recruit transcriptional machinery and coactivators/repressors

Question 17

oestrogen regulated genes

Answer 17

Progesterone Receptor (PR)
Cyclin D1 (cell cycle regulation)
c-myc (cell survival)
TGF-α (growth factor)

Question 18

oestrogen receptor signalling

Answer 18

1. oestrogen diffuses across cell membrane and binds to ER
2. release of hsps and dimerisation
3. binding to palindromic ORE
4. transcription and translation

Question 19

nuclear receptor structure

Answer 19

N-terminal domain, DBD, hinge region, LBD, C- terminus

1. N-terminal domain: recruits transcription machinery
   a. AF-1: constitutively active w/o ligand binding, cofactor recruitment
2. DBD: DNA binding domain, 2 zinc fingers, direct binding to promoters
3. Hinge region: contains nuclear localisation signal (NLS) → nuclear translocation
4. LBD: important for ligand binding and receptor dimerization
   a. AF-2: ligand dependent activity, cofactor binding, stabilisation of dimer