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Using Northern analysis, in situ hybridization, and nuclease protection assays, the expression and regulation of androgen receptor messenger RNA (AR mRNA) was examined in the CNS of juvenile Xenopus laevis. Only one of the AR mRNA isoforms expressed in X. laevis is transcribed in the CNS as shown by Northern blot analysis. Nuclease protection assays demonstrate that the expression of AR mRNA is higher in the brain stem than in the telencephalon and diencephalon. Although expression of AR mRNA is widespread throughout the CNS, cells of cranial nervenucleus IX-X (N.IX-X) and spinal cord display the highest in situ hybridization signals in their cytoplasm. Double labeling using horseradish peroxidase and digoxigenin labeled AR probes reveals that laryngeal and anterior spinal cord motor neurons express AR mRNA. More cells express AR mRNA in N.IX-X of males than of females. The number of AR expressing cells in N. IX-X decreases following gonadectomy in both sexes, and dihydrotestosterone (DHT) treatment for 1 month reverses this effect. Increased expression of AR mRNA in the brain of DHT treated animals is also apparent in nuclease protection assays. Sex differences in number of AR expressing cells and hormone regulation of AR mRNA expression in motor nuclei may influence neuromuscular systems devoted to sexually differentiated behaviors.
Figure 1 (A) AR cDNA and probes used for Northern
blots, in situ hybridization, and nuclease protection assays.
Probes are either complementary to 843 nucleotides
(nt) of the DNA and ligand binding domains
(Northern) or to the first 495 nt of the ligand binding
domain of the AR (nuclease and in situ, assays). The
probe for nuclease protection assays contained, in addition,
19 nt of the Sp6 promoter and 43 nt of the pCR-I1
vector. (B) Northern blot analysis demonstrates that
only a single transcript of approximately 9.6 kb is expressed
in the CNS of PM 1 X . luevis males and females.
Figure 2 (A) Diagram of Xenopus luevis brain. Two regions were surveyed for AR mRNA
expression by nuclease protection assays: the anteriorbrain (AB) and brain stem region containing
N. IX-X (IX-X). (B) Sensitivity of the nuclease protection assay. Signals due to the
protected fragment increase linearly with increasing amounts of total RNA (2.5, 5, and 10 pg)
in the assay. (C) Nuclease protection assay comparing expression of AR mRNA in IX-X and
AB. Optical density analysis reveals that expression in the brain stem is -58% higher than in
the anteriorbrain. Insert: Total RNA from the brain stem and anteriorbrain analyzed in 1%
agarose gel. (B, C) MW lane: RNA molecular weight markers; arrowheads, 562 and 363 nt. P
lane: Undigested AR probe. tRNA lane: As an internal control, the AR probe was hybridized
to tRNA. Occasionally, a band of undigested probe was seen in the tRNA lane and in the 2.5
and 10 wg lanes.
Figure 3 Nonisotopic hybridization signals in N. IX-X in control experiments. The tissue
was hybridized with (A) 10 pg/pL, (B) 2 pg/pL, and (C) 0.1 pg/pL of probe in the hybridization
solution. No hybridization signals were found in sections hybridized with (D) sense
probe or pretreated with RNAse A. Scale bar (E) = 50 pm.
Figure 4 Hybridization signals in X . luevis CNS. Horizontal sections of the brain are shown
from (A) rostra1 to (F) caudal. In each figure, up is anterior and down is posterior. Cells labeled
with the AR probe display a blue cytoplasmic signal. Brain stem nuclei were identified according
to Simpson et al. ( 1986). (A) Weak hybridization signals were found in the stnatum (St),
preoptic area (Pa), and thalamus (Th). (B) Hybridization signals were observed in the vestibular
(Ve) and lateral line (NLL) nuclei; the AR probe did not hybridize to granule cells of the
cerebellum (Cer). (C) hybridization signals in cells of the nucleusisthmus (Is) and dorsal
tegmental nucleus ofthe medulla (Dt). No hybridization signals with the AR probe were found
in the central grey (Cg). (D) Hybridization signals in the trigeminal (V) and facial (VII) motor
nuclei. (E) In situ hybridization signals in the motor nucleus of cranial nerves IX-X (IX-X)
and reticular formation (Re). (F) Motor neuron columns in the anterior spinal cord display
high AR hybridization signals. (G) Cells in the postotic or acoustic ganglion were strongly
labeled with AR probe. (H) Section at the level of the spinal cord: no hybridization signals
were apparent in sections hybridized with sense probe. Original magnifications in (A-F) and
(H)areasin(A).Scalebar= (A) 100pm,(G)25pm.
Figure 5 (A) Motor neurons in N. IX-X and (B) motor neurons that project to the arm were
labeled with HRP (brown reaction) and AR probe (blue reaction). Some N. IX-X motor
neurons express AR mRNA (arrowheads) whereas others do not (arrows). Scale bar = (A) 30
pm, (B) 50 pm.
Figure 6 AR mRNA expressing cells of N. IX-X in (A, C) gonadectomized and (B, D)
gonadectomized/DHT treated (A, B) males and (C, D) females. IX-X, nucleus IX-X. An
increase in hybridization signal was observed in cells of gonadectomized/ DHT treated groups
as compared to untreated/gonadectomized animals. Scale bar = 100 pm.
Figure 7 Androgen receptor mRNA expressing cells in
N. IX-X of males (striped bars) and females (white
bars). Mean values +_ S.E.M. are presented; number of
animals analyzed are given with each bar. Intact females
have significantly fewer AR mRNA expressing cells than
males ( Newmann-Keuls, p < 0.05 ). Gonadectomized
(GX) males have significantly fewer AR mRNA expressing
cells than intact males ( Newman-Keuls, p < 0.05).
Gonadectomized and DHT treated (GX-DHT) males
and females have significantly more AR mRNA expressing
cells than untreated gonadectomized animals
(Newman-Keuls, p < 0.01 ). Gonadectomized females
treated with estradiol (GX-E2) have significantly fewer
AR mRNA expressing cells than DHT treated females
( Newman-Keuls, p < 0.05).
Figure 8 Nuclease protection analysis of gonadectomized
(GX) and GX/DHT treated (DHT) males and
females. Total RNA ( 5 p g ) from the anteriorbrain ofthe
experimental groups was hybridized to the AR probe and
digested with RNAse. GX/DHT treated animals have
relatively more AR mRNA than gonadectomized animals.
MW lane: RNA molecular weight markers are indicated
with arrowheads, 562 and 363 nt (below). Plane:
Undigested AR probe. tRNA lane: No protected fragment
was found after hybridization of the probe to
tRNA. Insert: Total RNA from the respective experimental
groups was compared in a 1 % agarose gel.
