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The larynx of adult male Xenopus laevis differs markedly from that of the female; masculinization of the larynx requires androgen secretion during postmetamorphic development. Early in postmetamorphic development, androgen stimulates laryngeal cell proliferation and androgen binding activity is high. Later, androgen induces laryngeal cell differentiation and binding levels decrease. Here we explore the relation between laryngeal differentiation and androgen receptor expression. In untreated females, the larynx expresses high levels of androgen receptor mRNA early in postmetamorphic life; levels decline as females mature. The highest level of androgen receptor message is found in the undifferentiated laryngeal elastic precartilage of both sexes. When juveniles are exposed to androgen, laryngeal cell proliferation is stimulated within 48 hr in both sexes. Short exposures to androgen result in a biphasic response of AR mRNA levels; a marked down-regulation (4 hr) is followed by recovery at 8 hr (males) or 48 hr (females). Following longer periods of androgen treatment (3 weeks), AR mRNA expression is down-regulated and male-typical differentiation of elastic cartilage is induced in both sexes. Thus laryngeal growth responses to androgen are closely related to expression levels of androgen receptor mRNA.
FIG. 1. Northern blot of androgen receptor mRNA expression in normal
developing female larynges. PM2 frogs are 6 months and PM3
frogs 9 months past the end or metamorphosis, respectively. At PM6
(2 years PM) animals are sexually mature adults. Ten micrograms of
total RNA were loaded into each lane. To verify equal loading of RNA,
duplicate gels were run and stained with ethidium bromide.
FIG. 2. Elastic cartilage differentiation and androgen receptor expression in female larynges. Transverse sections through the larynx: ventral
is up and medial is to the right. (A) Bright-field photomicrograph of the EPZ in a PM2 female. At PM2, differentiated elastic cartilage is induced
after 3 weeks of androgen exposure (C). Notice that some undifferentiated EPZ remains at the medial margin. In adult females (PM6), the EPZ
persists (E). Panels on the right show AR mRNA expression in PM2 (B), androgen-treated PM2 (D), and PM6 animals (F). AR mRNA expression
in the hyaline cartilageperichondrium can be seen in D. EPZ, elastic precartilage zone; E. elastic cartilage; H, hyaline cartilage; P, perichondrium.
Scale bar, 10 J.tm.
FIG. 8. Elastic cartilage differentiation and androgen receptor expression in male larynges. Order of presentation, magnification, and abbreviations as in Fig. 2. At PM2, differentiated elastic cartilage is precoeially induced by 3 weeks of androgen exposure (C).
Fw. 4. Northern analysis of AR mRNA expression in 3 week DHTtreated
frogs. (A) AR mRNA expression in laryngeal muscle and cartilage
of control and DHT-treated PM2 males and females. (B) AR
mRNA expression in control and DHT-treated P.M6 male laryngeal
muscle and kidney. Gels were run as in Fig.l except 20 ~goftotalRNA
for kidney was used.
FIG. 5. (Top) Northern blot of PM2laryngeal muscle AR mRNA expression
in response to short-term exposure to DHT. Length of hormone
exposure .is indicated at the top. (Middle) blot was stripped and
reprobed with EF-1(1:. (Bottom) blot was repro bed with a cardiac actin.
Gels were nm as in Fig. 1.
FIG. 6. Androgen-induced proliferation visualized by tritiated thymidine autoradiography in short-term DHT-treated PM2 female and male
larynges. All panels are dark-field photomicrographs taken from the muscle-EPZ border of horizontal sections of the larynx, shown with the
muscle on top. Females are shown on the left and males on the right. In control animals, the labeling index was <1 %. No increase in labeling
was observed 4 (A and B) or 8 (not shown) hr after the initiation of DHT treatment. Significant proliferation is detected in both sexes after 48
hr (C and D) of DHT exposure although more labeled cells are seen in males than in females. Note that in females at this time labeled ce11s are
not as evenly distributed as in males but are more concentrated near the EPZ-musle border. After 96 hr of androgen exposure, DHT-induced
proliferation is still evident in both sexes (E and F) but the response is greater in females (E). M, muscle; HC, hyaline cartilage. Bar, 100 um.
