Embryonic mouse tissues were obtained from Parkes outbred mice (maintained at
NIMR). For embryo staging, midday on the day o f appearance of the vaginal plug is
taken as 0.5 àpc.
I l l . I I . 2. Plasmid constructs
Mouse S fl and Sox9 cDNA were subcloned into the EcoRV restriction site on the pcDNA3 expression vector (Promega). One copy o f each of the oligonucleotides
SOX/SFl, M UT/SFl, S0X9/MUT and MUT/MUT (Fig.III.l.) was cloned into the
EcoRV restriction site of the pGL3 basic vector (Promega). The reporter plasmids were named SOX/SFl lue, MUT/SFl lue, SFl/MUTluc and MUT/MUTluc.
I l l . I I . 3. Electrophoretic Mobility Shift Assay(EMSA)
Embryonic gonads were dissected at 11.5 dpc and homogenised. Tissue extracts were prepared by three cycles of freeze-thaw lysis in Buffer A (20mM Hepes, 450mM
NaCl, 25% Glycerol, 250mM EDTA, 0.5mM PMSF, 0.5 pg/ml Leupeptin, 0.5pg/ml
Aprotinin, 40 pg/ml Pestatin, lOmM P-Glicerophosphate). Tissue extracts were
prepared by centrifugation at lO.OOOrpm for 15min at 4°C, and supernatants were
stored at -70°C (Scholer et a l, 1989). Protein concentrations were determined using the Bio-Rad protein assay. Synthetic oligonucleotides, probe SRY/SOX, probe SOX/SFl,
probe M UT/SFl, probe SOX/MUT, MUT/MUT (Fig.III.l.) were labelled with
[^^PJdCTP (Amersham) by using T4 polynucleotide kinase (Promega). Tissue extracts
(lOpg) were incubated with 10-20 fmol labelled oligonucleotides for 30 min at room
temperature in 10 pi of retardation mix 2X (20mM TrisHCl, pH 8, 0.2mM EDTA,
20mM MgCl2, 4mM Dithiothreitol, 35% glycerol, 4mg/ml BSA, 50 mM NaCl, 0.5mM
PMSF) containing 1 pg salmon sperm DNA. For supershifts, incubations of VAH2
antibody (Morais da Silva et a l, 1996) with tissue extracts was performed for 45 min prior to the addition of the labelled oligonucleotides. For competition analysis, 100-fold
excess of end-filled unlabelled oligonucleotides were incubated 15min prior to addition
o f labelled oligonucleotides. The DNA-protein complexes were resolved on 5%
I l l . I I . 4 . Cell culture and transient expression assays
COS cells were grown in DMEM supplemented with 10% PCS, L-glutamine and
streptomycine/penicilin in a 5% CO2 atmosphere at 37°C. The cells were transfected
with the lipofectamine method (Gibco). Cells were harvested 48 hours after
transfection. Luciferase assays were performed with the Luciferase kit (Promega),
according to the manufacturers protocol. The data shown are from one experiment and
each value represents the mean of three separate wells, with similar results obtained in
three independent experiments. Bars represent the standard error.
m . m . Results
I I I . I l l . 1. SOX9 binds a conserved CCTTTGA motif in Amh promoter
The alignment of the Amh sequences reveal two conserved elements within the Amh
promoter: the first element (CCTTTGA) is similar to sites recognised by HMG box
type proteins. The second element, designated MIS-RE-1 (AGGTCA) matches the
Electrophoretic mobility shift assays (EMSA) were performed, using protein extracts
from 11.5 àpc testis with the probe SRY/SOX which contains a SOX binding site (Fig.III.l.). These reveal a pattern o f bands that are effectively competed by addition of
an excess of unlabelled probe (Fig.III.2.a, lanes 1,2). In order to address if this shift is
caused by S0X9 binding to the probe, an antibody raised against S0X 9 (VAH2) was
added (Morais da Silva et a l, 1996). The disappearance of the bands with the addition of S0X 9 antibody indicates that the shift is caused by S0X9 protein contained in the
extracts (Fig.III.2.a, lanes 3,4). No change was observed with addition o f preincubated
VAH2 with S0X9 peptide 486 (Morais da Silva et a l, 1996; data not shown) The same experiment was performed with protein extracts from lA.Sàpc testis with similar results (data not shown).
I l l . I l l . 2. SFl and SOX9 bind to the Amh promoter region
SFI protein has been shown to regulate transcription (Shen et a l, 1994). 11.5 àpc
protein extracts were analysed with probe SOX/SFl by EMSA. Two major bands were
detected, which are dependent on each of the binding sites SRY/SOX and MIS-RE-I.
When the experiment is performed with a probe in which the SOX site is mutated
(MUT/SFl) only one band is detected. Conversely when the SFI site is mutated
Fig III.2 S0X 9 binds to the SRY/SOX binding site in the Amh region. EMSA was performed with labelled SRY/SOX probe and 11.5 dpc testis extracts as described in the materials and methods. Lane 1. 100-fold excess probe was used as a competitor. L ane 2. Labelled SOX/SRY probe with 11.5 dpc testis extracts. Addition of 1 mg (Lane 3) and 1.5 mg (Lane 4) of VAH2 antibody to the labelled SOX/SRŸ probe and 11.5 àpc testis extracts. (Unshifted probe not shown).
Fig III.3. Two major bands are detected which are dependent on each of the binding sites SRY/SOX and MIS-RE-1. Lane 1. EMSA performed with 11.5 dpc testis extracts and labelled probe SOX9/SF1. Lane 2. 11.5 dpc testis extracts with the MUT/SFl labelled probe. Lane 3. 11.5 dpc testis extracts with the SOX/MUT labelled probe. Lane 4. 11.5 dpc testis extracts with the MUT/MUT labelled probe.