Supplementary Methods
Fish maintenance. Fish stocks were maintained according to the procedures described in the
Zebrafish Book1
Morpholinos and analysis of genomic structures. The genomic structure of hsd3b was
compiled by scanning the NCBI database using the BLAST program with the hsd3b cDNA
sequence. The morpholino sequences are as follows:
scc mo1: GCCATCACACTCTCTCTCTCTACTT
scc 5-base mismatched mo1: GCGATGACACTGTCTCTCTGTAGTT
scc mo2: TAACATTGCTTACTTTAGCAGGACG
hsd3b mo: CTGCGTTGAAAGCCCGTGTCGAGTC
cyp17 mo: AGCCATTGTCAGCTATATTGAACTG
cyp17 sense mo: CAGTTGAATATAGCTGACAATGGCT
control mo: CCT CTT ACC TCA GTT ACA ATT TAT A
dlx3 mo: ATGTCGGTCCACTCATCCTTAATAA
fluorescein conjugated mo: CCT CTT ACC TCA GTT ACA ATT TAT
A-carboxyfluorescein.
Plasmids
The description of the cyp11a1 genomic BAC clone was previously reported2. Scc
genomic clone was digested with ACCI then subcloned into the Bluescript vector. 12 kb of
scc fragment containing the promoter and about 451 bp of the coding region was linked to a
GFP reporter in phGZK-2 vector (given by Dr. Jen-Leih Wu), Inst. of Zoology, Academia
1
Sinica, Taipei, Taiwan, R.O.C) to form scc-GFP.
The 21 bases of scc mo1 recognition sequence (25 bases) located at 5’UTR adjacent to
ATG of cyp11a1 full length cDNA was deleted by PCR method and sub-cloned into PCS2
expression vector. cyp11a1* RNA was synthesised by MEGAscript SP6 kit (ambion, Inc.
Austin, TX, USA).
200 bp PCR fragment containing cyp17 mo recognition site amplified from
Bluscript –zebrafish cyp17 (given by Dr. Wei Ge, Department of Biology, The Chinese
University of Hong Kong, Shatin, NT, Hong Kong, China.) by primers (T3 primer and
zcyp17 primer: 5’-gggaccggTGATGGGCAGCGAAGGGAGACTTG-3’) was subcloned into
pEGFP-N1 vector (Clontech, Palo Alto, CA, USA ). The cyp17-EGFP plasmid was
constructed by linking the 5’ region of the cyp17 gene containing the binding sequence for
cyp17 mo and the coding sequence of the EGFP gene.
Microinjection and detection of gene expression
Sense or anti-sense RNA was synthesized by in vitro transcription. 1-cell stage embryos
were co-injected with 25 pg scc-GFP with or without 9 ng scc mo1. Sometimes 9ng scc mo1
plus 200 pg cyp11a1 mRNA were injected at the 1-cell stage. One-cell stage embryos were
co-injected with 100 pg cyp17-EGFP with 9 ng cyp17 sense mo or cyp17 antisense mo.
Embryo extracts were collected 16 hours post fertilization and western blot was
performed using anti-GFP antibody (1:1000) (Zymed Laboratories, Inc. South San Francisco,
U.S.A).
Fluorescent images were observed and captured at 70% epiboly stage by Leica MZFIII
fluorescent microscope (Leica Microscopy system Ltd, Heebrugg, Switzerland) with a CCD
camera (Diagnostic Instrument Inc, USA).
For Hsd3b colorimetric assay, embryos were fixed for 1 hour at RT in 4%
paraformaldehyde and wash with phosphate-buffered saline containing 0.1% Tween 20
2
(PBST). Embryos were then incubated in the solution containing 0.1 mg/ml of the Hsd3b
substrate, etiochalan-3β-ol-17-one, 1.5 mg/ml nicotinamide adenine dinucleotide (NAD),
1mg/ml BSA, 1% N,N dimethyl formamide, 8.75 mM EDTA, and 1 mg/ml NBT in PBS for
overnight at room temp. Staining was stopped by washing in PBST and followed by fixing in
4% paraformaldehyde for at least 1 h. All the chemicals used here were purchased from
Sigma (MO, USA).
Steroid detection. Embryos were collected at the end of epiboly stage for steroid extraction
and radioimmunoassay as previously described3. Wildtype embryos or embryos injected with
hsd3b morpholino were incubated with 1 M tritium labelled pregnenolone from the early
cleavage stage to the 50% epiboly. Adult ovaries were dissected out and incubated with 1 M
tritium labelled pregnenolone for two hours. Steroids were extracted3 and separated by TLC
plates as previously described4.
RT-PCR. Wildtype and scc mo2 morphants with epibolic delay were collected at the end of
the epiboly stage and homogenized in TRIzol reagent (Invitrogen life technologies, Carlsbad,
CA, USA). Total RNA was extracted several times with phenol/chloroform. 3 μg of total
RNA was reverse transcribed, and its cDNA was analysed by PCR reactions. The sequences
of PCR primers used for analysis are listed as follows:
scc 340 (forward): 5’- GGACGATGGAGGAACAGCCT-3’
scc 695 (reversed): 5’-ACGAGCCACAAAGTCCTGCC-3’
Synthesis of fluorescein conjugated pregnenolone (F-P5)
Fluorescein conjugated pregnenolone, denoted as F-P5, is composed of fluorescein and
pregnenolone bridged by a six-carbon linker via ethereal and thioureido functionalities
(supplementary scheme and experimental procedures).5 It was prepared in six steps with 14%
3
overall yield. Starting from commercially available pregnenolone, the 3-hydroxyl group was
converted to a better leaving group by reacting with p-toluenesulfonyl chloride (TsCl) at
room temperature for 12h to generate pregnenolonyl-p-toluenesulfonate (1) in 81% yield.
This activated compound was treated with 1,6-hexanediol at 80 ℃ in 1,4-dioxane for 12 h to
give monopregnenolonylated ethereal derivative (2) in 41% yield.6 To provide the desired
amino functionality for further reacting with fluorescein isothiocyanate isomer I via thiourea
linkage, the remaining hydroxyl group of 2 was first activated with tosyl chloride in a manner
similar to that for 1, and then subsequently converted to the corresponding azide (4) with
sodium azide in 80% yield. Reduction of 4 to amino group was achieved by reacting with
triphenylphosphine (Ph3P) in tetrahydrofuran (THF) followed by hydrolysis. Subsequent
treatment of the resulting amine with fluorescein isothiocyanate in the presence of
triethylamine (TEA) furnished the fluorescein conjugated pregnenolone (F-P5) in 75% yield.
Spectroscopic data for F-P5 :1H NMR (400MHz, 10% CD3OD/CDCl3) 8.13 (s, 1H),
7.80-7.76 (m, 1H), 7.12 (d, J = 8.4 Hz, 1H), 6.70-6.60 (m, 4H), 6.60-6.50 (m, 2H), 5.35-5.30
(m, 1H), 3.60 (bs, 2H), 3.48 (t, J = 6.4 Hz, 2H), 3.20-3.15 (m, 1H), 2.58 (t, J = 8.8 Hz, 1H),
2.40-2.30 (m, 1H), 2.20-2.15 (m, 2H), 2.11 (s, 3H), 2.12-1.80 (m, 4H), 1.80-1.31 (m, 16H),
1.30-0.80 (m, 4H), 0.98 (s, 3H), 0.60 (s, 3H); 13C (100MHz, 10% CD3OD/CDCl3)  211.2,
181.4, 170.0, 163.6, 160.3, 153.2, 141.4, 140.9, 129.5, 128.1, 124.9, 121.8, 113.1, 110.8,
103.2, 79.9, 68.8, 64.4, 57.6, 51.0, 45.0, 40.0, 39.6, 38.2, 37.8, 37.3, 33.0, 32.8, 32.0, 30.9,
4
29.8, 29.4, 27.8, 26.9, 25.5, 23.8, 22.2, 20.2, 14.0; IR(KBr) 3354, 2933, 1734, 1609, 1541,
1505, 1450, 1331, 1255, 1174, 1111, 851, 730 cm-1; FAB-HRMS calcd for C48H56N2O7S
(M++1) 805.3886; found 805.3881.
Supplementary reaction scheme and experimental procedures
The synthetic scheme for fluorescein conjugated pregnenolone (F-P5) is delineated as
follows.
O
O
O
(a)
(b)
O
S O
O
HO
HO
O
(c)
O
S O
O
(e), (f)
O
2
1
N3
O
O
(d)
O
4
3
O
O
CO2H
O
H
N
H
N
O
S
F-P5
HO
Reagents and reaction conditions: (a) TsCl, pyridine, r.t., 12h, 81%; (b) 1,6-hexandiol,
1,4-dioxane, 80 ℃, 12h, 41%; (c) TsCl, pyridine, r.t., 4h, 70%; (d) NaN3, DMF, 80 ℃, 12h,
80%; (e) PPh3, H2O, THF, r.t., 5h; (f) FITC, TEA, DMF, r.t., 12h, 75%.
General Method: All reagents and starting materials were obtained from commercial
suppliers (Acros, Aldrich, Sigma and Merck) and were used without further purification. IR
spectra were recorded on a Nicolet 550 series II spectrometer. 1H and 13C NMR spectra were
5
recorded using a Varian Mercury Plus 400 spectrometer. The proton and carbon chemical
shifts are given in ppm using CDCl3 (δH 7.24 and 77.0) as internal standard. Low resolution
mass spectra were recorded with a JEOL-102A mass spectrometer. Melting points were
determined with a Meltemp apparatus and are uncorrected. N,N-dimethylformamide (DMF)
was dried with CaH and distilled under reduced pressure prior to use. Thin-layer
chromatography (TLC) was performed on silica gel 60F-54 (Merck) plates, and the spots
were visualized under UV light and/or phosphomolybdic acid-ethanol. Flash column
chromatography was conducted on Kiesegel 60 (230-400 mesh Merck).
Experimental procedures:
O
O
S O
O
1
3β-(Toluene-4-sulfonyloxy)-pregn-5-en-20-one (1)
A pyridine solution (15 mL) of pregnenolone (1.58 g, 5 mmol) was cooled to 0 oC with an
ice bath, and then treated with TsCl (1.42g, 7.5 mmol). The reaction mixture was
subsequently warmed up to room temperature and stirred at this temperature for 20 h before
poured into 20 mL water with stirring. White precipitates were formed and 10% HCl was
added until the solution is neutral. The precipitate was filtered, and the solid was washed
with deionized water several times and dried in vacuo to give the crude product, which was
further recrystallized in EtOAc to yield compound 1 as colorless solids, mp 136-138 ℃.
6
Yield: 1.9 g (0.134 mmol, 81%). Rf= 0.45 (EtOAc/Hexane, 1:3); 1H NMR (400MHz, CDCl3)
 7.71 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 5.25-5.22 (m, 1H), 4.30-4.20 (m, 1H),
2.45 (t, J = 8.8 Hz, 1H), 2.37 (s, 3H), 2.22-2.18 (m, 1H), 2.17-2.04 (m, 2H), 2.04 (s, 3H),
1.97-1.70 (m, 4H), 1.70-1.25 (m, 8H), 1.20-0.87 (m, 4H), 0.90 (s, 3H), 0.54 (s, 3H); 13C
NMR (100MHz, CDCl3) 208.8, 144.1, 138.5, 134.3, 129.5, 127.3, 122.9, 81.9, 63.4, 56.6,
49.6, 43.8, 38.7, 38.6, 36.8, 36.2, 31.6, 31.4, 28.5, 24.4, 22.7, 21.6, 20.9, 19.1, 13.2; IR
(KBr) 2965, 2939, 2893, 2874, 1698, 1360 cm-1; FAB-MS m/z (rel intensity) 471.2 (M+ +
H, 16%).
O
HO
O
2
Pregn-5-en-20-one-3-yl 6-hydroxyhexyl ether (2)
A 1,4-dioxane (5 mL) solution of compound 1 (427 mg, 1 mmol) was treated
1,6-hexanediol (590 mg, 5 mmol). The resulting mixture was heated to 80 ℃ and
maintained at this temperature for 12h. After cooling to room temperature, the volatiles
were removed under reduced pressure, and the residue was purified by chromatography on
a silica gel column (EtOAc/Hexane, 1:4) to yield compound 2 as white solids.
Recrystallization in EtOAc could provide purer product, mp 145-146 ℃. Yield: 170 mg
(0.408 mmol, 41％). Rf = 0.26 (EtOAc/Hexane, 1:2); 1H NMR (400MHz, CDCl3) 
7
5.34-5.31 (m, 1H), 3.63 (t, J = 6.4 Hz, 2H), 3.44 (t, J = 6.8, 2H), 3.15-3.05 (m, 1H), 2.51 (t,
J = 9.2 Hz, 1H), 2.40-2.30 (m, 1H), 2.20-2.12 (m, 2H), 2.11 (s, 3H), 2.07-1.8 (m, 4H),
1.70-1.31 (m, 16H), 1.30-0.98 (m, 4H), 0.98 (s, 3H), 0.61 (s, 3H); 13C NMR (100MHz,
CDCl3)  209.2, 140.9, 121.0, 78.8, 68.0, 63.7, 62.9, 57.0, 50.1, 44.1, 39.2, 38.9, 37.4, 37.0,
32.8, 31.9(2×), 31.6, 30.2, 28.5, 26.1, 25.7, 24.6, 22.9, 21.2, 19.5, 13.3; IR (KBr) 3410,
2971, 2930, 2897, 2853, 1705, 1464 cm-1; FAB-MS m/z (rel intensity) 417.3 (M+ + H,
11%).
O
O
S O
O
O
3
Pregn-5-en-20-one-3-yl 6-[(p-tolylsulfonyl)oxy]hexyl ether (3)
Compound 3 was prepared in 70% yield by a procedure similar to that for 1 except using
column chromatography to purify the product instead of recrystallization. The crude product
was purified on a silica gel column (EtOAc/hexane 1:6) to give the desired product as white
solids, mp 80-82 ℃. Yield: 440 mg (77.2 mmol, 70%). Rf = 0.20 (EtOAc/Hexane, 1：6); 1H
NMR (400MHz, CDCl3) 7.75 (d, J = 8 Hz, 2H), 7.31 (d, J = 8 Hz, 2H), 5.33-5.30 (m, 1H),
4.00 (t, J = 6 Hz, 2H), 3.38 (t, J = 6.4 Hz, 2H), 3.15-3.05 (m, 1H), 2.50 (t, J = 8 Hz, 1H),
2.42 (s, 3H), 2.35-2.27 (m, 1H), 2.20-2.12 (m, 2H), 2.10 (s, 3H), 2.08-1.75 (m, 4H),
1.75-0.97 (m, 20H), 0.97 (s, 3H), 0.60 (s, 3H); 13C NMR (100MHz, CDCl3)  208.5, 144.1,
8
140.5, 132.7, 129.3, 127.4, 120.7, 78.8, 70.6, 67.8, 63.7, 57.0, 50.2, 44.2, 39.3, 39.0, 37.5,
37.1, 32.1(2×), 31.8, 30.2, 29.1, 28.7, 25.9, 25.5, 24.8, 23.1, 22.0, 21.4, 19.7, 13.6; IR (KBr)
2928, 2856, 2362, 1699, 1352 cm-1; FAB-MS m/z (rel intensity) 571.3 (M+ + H, 20%).
O
N3
O
4
Pregn-5-en-20-one-3-yl 6-azidohexyl ether (4)
To 2 mL DMF was added compound 3 (130 mg, 0.23 mmol) and NaN3 (30 mg, 0.46 mmol).
The resulting solution was heated to 80 ℃ and maintained at this temperature for 12 h. DMF
was removed under reduced pressure, and the resulting white slurry was treated with EtOAc
to yield solid precipitates. The precipitate was filtered, and the filtrate was washed with brine
(x2). The combined organic phases were dried over anhydrous MgSO4, filtered, and
concentrated to yield compound 4, mp 89-90 ℃. Yield: 81 mg (0.184 mmol, 80%). Rf= 0.45
(EtOAc/Hexane, 1：4); 1H NMR (400MHz, CDCl3)  5.34-5.31 (m, 1H), 3.42 (t, J = 6.8 Hz,
2H), 3.23 (t, J = 7.2 Hz, 2H), 3.15-3.05 (m, 1H), 2.49 (t, J = 9.2 Hz, 1H), 2.40-2.30 (m, 1H),
2.20-2.10 (m, 2H), 2.09 (s, 3H), 2.07-1.8 (m, 4H), 1.70-1.31 (m, 16H), 1.30-0.98 (m, 4H),
0.97 (s, 3H), 0.60 (s, 3H); 13C NMR (100MHz, CDCl3)  209.1, 140.9, 121.0, 78.8, 67.8,
63.7, 56.9, 51.4, 50.1, 44.0, 39.2, 38.9, 37.3, 37.0, 31.9(2×), 31.6, 30.1, 28.9, 28.5, 26.6, 25.9,
24.6, 22.9, 21.2, 19.5, 13.3; IR (KBr) 2934, 2853, 2094, 1644, 1474, 1361, 1116 cm-1;
FAB-MS m/z (rel intensity) 442.3 (M+ + H, 14 %).
9
O
O
CO2H
O
H
N
H
N
S
O
F-P5
HO
5-{3-[6-(Pregn-5-en-20-one-3-yloxy)-hexyl]-thioureido}-2-(6-hydroxy-3-oxo-3H-xanthen9-yl)-benzoic acid (F-P5)
To a solution of 4 (79 mg, 0.18 mmol) in 5 mL of THF was added PPh3 (51 mg, 0.196 mmol)
and H2O (32 μL, 0.18 mmol). The mixture was stirred at room temperature for 4 h, and then
brought to reflux for 1h. The volatiles were removed under reduced pressure, and the crude
amino compound was obtained and used for further reaction without purification. Fluorescein
isothiocyanate isomer I (140 mg, 0.36 mmol) and the crude amino product were dissolved in
2 mL DMF and to the solution was added triethylamine (20 μL, 0.18 mmol). The resulting
solution was stirred at room temperature for 12 h, and then concentrated under reduced
pressure. The residue was purified by chromatography on a silica gel column (CHCl3/CH3OH
13:1) to give the desired product (Fluorescein-pregnenolone (F-P5)) as orange solids, mp
170-173 oC. Yield: 108 mg (0.134 mmol, 75%). Rf = 0.21 (CHCl3/MeOH, 9:1); 1H NMR
(400MHz, 10% CD3OD/CDCl3) 8.13 (s, 1H), 7.80-7.76 (m, 1H), 7.12 (d, J = 8.4 Hz, 1H),
6.70-6.60 (m, 4H), 6.60-6.50 (m, 2H), 5.35-5.30 (m, 1H), 3.60 (bs, 2H), 3.48 (t, J = 6.4 Hz,
2H), 3.20-3.15 (m, 1H), 2.58 (t, J = 8.8 Hz, 1H), 2.40-2.30 (m, 1H), 2.20-2.15 (m, 2H), 2.11
(s, 3H), 2.12-1.80 (m, 4H), 1.80-1.31 (m, 16H), 1.30-0.80 (m, 4H), 0.98 (s, 3H), 0.60 (s, 3H);
C (100MHz, 10% CD3OD/CDCl3)  211.2, 181.4, 170.0, 163.6, 160.3, 153.2, 141.4, 140.9,
13
10
129.5, 128.1, 124.9, 121.8, 113.1, 110.8, 103.2, 79.9, 68.8, 64.4, 57.6, 51.0, 45.0, 40.0, 39.6,
38.2, 37.8, 37.3, 33.0, 32.8, 32.0, 30.9, 29.8, 29.4, 27.8, 26.9, 25.5, 23.8, 22.2, 20.2, 14.0;
IR(KBr) 3354, 2933, 1734, 1609, 1541, 1505, 1450, 1331, 1255, 1174, 1111, 851, 730 cm-1;
FAB-HRMS calcd for C48H56N2O7S (M++1) 805.3886; found 805.3881.
Reference:
1. Westerfield, M. The ZEBRAFISH BOOK:A guide for the laboratory use of zebrafish
(Danio rerio). (University of Oregon press, 1995).
2. Hsu, H. J., Hsiao, P., Kuo, M. W. & Chung, B. C. Expression of zebrafish cyp11a1 as a
maternal transcript and in yolk syncytial layer. Gene Expr Patterns 2, 219-22 (2002).
3. Takase, M., Ukena, K., Yamazaki, T., Kominami, S. & Tsutsui, K. Pregnenolone,
pregnenolone sulfate, and cytochrome P450 side-chain cleavage enzyme in the amphibian
brain and their seasonal changes. Endocrinology 140, 1936-44 (1999).
4. Bair, S. R. & Mellon, S. H. Deletion of the mouse P450c17 gene causes early embryonic
lethality. Mol Cell Biol 24, 5383-90 (2004).
5. An analogue with a two-carbon linker was synthesized, but it performed poorly compared
with the reported one. Presumably the fluorophore is too close to the recognition site
creating steric interference for binding to the corresponding protein. Compounds 1-4 and
F-P5 were fully characterized by IR, MS, 1H and 13C NMR spectra.
6. Ponpipom, M. M. et al. Glycolipids as host resistance stimulators. J Med Chem 33, 861-7
(1990).
11