Supplemental Figure Legends
Supplemental Figure 1. Effects of maternal injection with vehicle (CTL), flutamide (FLU) or
DHT propionate from GD 60 to 90 on oSDN development. A, treatment differences in thionin
volume; B, treatment differences in oSDN length. Data (mean ± SEM) were analyzed by 2-way
ANOVA followed by LSD test. Bars with different superscripts differ significantly (P < 0.05).
Supplemental Figure 2. Effects of maternal injections with vehicle (CTL), flutamide (FLU) or
DHT propionate from GD 60 to 84 on preoptic area (POA) GnRH and Kiss1 gene expression in
GD 85 ovine fetuses. Data (means ± SEM) were analyzed by 2-way ANOVA.
Supplemental Table 1. Ovine primer sequences used in qtRT-PCR
Gene
Sense
Antisense
LHβ
TGTCATCCTGCCGCCCATGC
GGAGCCGAACGGAGGCGAAG
FSHβ
TGCCCAGGATGAAGTCCGTCCAGT ACCAAGTCCCGGGTGTAGCAAT
α-subunit CTCCAGCGAGGTCTAAGAAG
CACTGTGGCCTTGGTAAATG
KISS
TTCCAGGACCGGCATCTTC
GAGCCTGTGGTTCTAGGATTC
GnRH
TGCTCTGGTCAACACTGGTC
GAGGAGAATGGGACTGGTGA
GAPDH TCATCCATGACCACTTTG
AGTAGAAGCAGGGATGATGT
Length
74
175
84
133
154
145
Accession #
NM001009380
NM001009798
NM001009464
JQ716394
U02517
NM001190390
Supplemental Table 2. Effects of maternal injection with flutamide (Flu) and DHT propionate
(DHTP) from GD 60 to 90 on physical measures and serum testosterone concentrations of GD
135 ovine fetuses.
Measure
Treatment
CTL
CTL
Flu
Flu
DHTP
DHTP
male
female
male
female
male
female
Testosterone†
225±55
147±16*
153±26
122±26*
191±35
109±14*
(pg/ml)
Brain wt (g)
52.9±1.0
49.0±0.7
51.4±1.1
50.3±1.0
52.8±1.2
51.3±1.6
A.Pit wt (mg)
46.8±9.3
37.2±7.6
43.3±4.8
33.2±5.4
40.0±7.6
35.0±3.6
Body wt (kg)
4.1±0.2
3.3±0.1
3.7±0.3
3.2±0.3
4.1±0.4
3.9±0.4
CRL (cm)
53.0±0.7
48.9±0.5
50.3±1.3
49.4±1.3
52.0±1.8
51.4±1.6
AGD/AUD
0.8±.01
0.1±.01*
0.9±.01
0.1±.01*
0.9±.01
0.1±.01*
n
6-7
4-7
6-7
5-7
5-6
7
†, Serum testosterone was measured in umbilical artery blood. Data (means±SEM) were
analyzed by two-way ANOVA. The mean concentration of serum testosterone was significantly
greater in males than in females [F(1,34) = 4.6; P <0.05], but was not significantly affected by
treatment [F(2.34) = 1.5; P = 0.2]. There were no significant sex or treatment effects on brain
weight (wt), anterior pituitary (A.Pit.) wt, body wt or crown-rump length (CRL). The expected
larger ratio of anogential distance to anoumbilical distance (AGD/AUD) found in males
compared to females [F(1,35) = 10,963: P < 0.0001] was not affected by treatment [F(2,35) =
1.0; P = 0.4]. *, Indicates significant main effect of sex, P < 0.05.
Supplemental Table 3. Effects of maternal injections with flutamide (Flu) or DHT propionate
from GD 60 to 84 on physical measures in GD 85 ovine fetuses.
Treatment
CTL
CTL female
Flu
Flu
DHTP
DHTP
Measure
male
male
female
male
female
Body wt (g)
477±12
474±8
525±18
505±17
514±16
500±23
Brain wt (g)
13±0.5
12±0.2
13±0.3
13±0.5
13±0.4
13±0.5
CRL (cm)
26±0.4
26±.4
27±0.4
26±0.6
26±.3
26±.4
AUG/AUD
0.9±0.01 0.09±0.01*
0.9±0.003 0.1±0.02* 0.9±0.004
0.1±0.003*
n
6-7
5
5-6
4-9
14
8
Data (means±SEM) were analyzed by two-way ANOVA. There were no significant sex or
treatment effects on body weight (wt), brain wt or crown-rump length (CRL). The expected
larger ratio of anogential distance to anoumbilical distance (AGD/AUD) found in males
compared to females [F(1,43) = 10,228: P < 0.0001] was not affected by treatment [F(2,43) =
0.6; P = 0.5]. *, Indicates significant main effect of sex, P < 0.05. *, Indicates significant main
effect of sex, P < 0.05; no treatment effects were observed.