Brain Research 880 2000 92–101 www.elsevier.com locate bres
Research report
Estrogen receptors and aromatase activity in the hypothalamus of the female frog, Rana esculenta. Fluctuations throughout the reproductive
cycle
a , b
c a
a
Giulia Guerriero , Charles E. Roselli , Marina Paolucci , Virgilio Botte , Gaetano Ciarcia
a
Department of Zoology , Federico II University, Via Mezzocannone, 8, 80134 Naples, Italy
b
Department of Physiology and Pharmacology , O.H.S.U., Portland, OR 97201, USA
c
Faculty of Science , University of Sannio, Benevento, Italy
Accepted 2 August 2000
Abstract
It is well known that certain actions of androgen are mediated through in situ aromatization to estrogen in neural target tissues. This study was undertaken to investigate androgen utilization in the hypothalamus of the female frog, Rana esculenta, through a quantification
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of estrogen receptors and aromatase activity during the reproductive cycle. H-estradiol-binding molecules were present in both the
3 210
cytosol and the nuclear extract of the hypothalamus. These molecules bound specifically H-estradiol with high affinity Kd 10 M and
low capacity cytosol: 1.260.4 fmol mg protein; nuclear extract: 7.960.6 fmol mg protein. Aromatase activity was detected in the microsomal fraction of the hypothalamus using a sensitive in vitro radiometric assay. Both aromatase activity and nuclear estrogen
receptor binding fluctuated in synchrony throughout the reproductive cycle. Western blot analysis of aromatase protein revealed one immunoreactive band with a molecular weight of approximately 56 kDa. In contrast to aromatase enzyme activity, the relative levels of
aromatase protein changed little during the reproductive cycle suggesting that post-translational mechanisms may be involved in regulating estrogen synthesis in the frog brain. A possible role for estrogens in the modulation of the reproductive behavior in this species
is suggested.
2000 Elsevier Science B.V. All rights reserved.
Theme : Endocrine and antonomic regulation
Topic : Hypothalamic-pituitary-gonadal regulation
Keywords : Estrogen receptors; Aromatase; Hypothalamus; Frog
1. Introduction that estrogens, through in situ aromatization of circulating
androgens, mediate certain androgen actions [6,10,39,51]. Sex steroids have potent effects on the central nervous
In male mammals the preoptic area regulates copulatory system. In the adult vertebrate, androgens and estrogens
behavior, concentrates estrogens, contains estrogen recep- control gonadotropin release and evoke the expression of
tors, and synthesizes estrogens from circulating androgens sexual behavior [19,59]. Autoradiographic techniques
[4,9,36,52,58]. show that nuclei in the preoptic area of the hypothalamus,
In contrast to several vertebrate species of males, the limbic structures and mesencephalic nuclei close to the
musk shrew Sunus murinus is the only well-studied optic tectum are responsible for the uptake of androgens
species in which brain aromatization has been shown to be and estrogens in a variety of vertebrates. The critical role
required for the expression of the female sexual behavior. of androgens in neuronal development and maintenance in
Similar to males, aromatase is high in the preoptic area of males is supported by numerous studies. It is well known
female musk shrews and implants of testosterone in this region reinstate sexual receptivity after ovariectomy
[48,57]. Currently, little is known about the function of
Corresponding author. Tel.: 139-081-790-7069; fax: 139-081-790-
aromatization in females of lower vertebrate species,
3326. E-mail address
: guerrierunina.it G. Guerriero.
although circulating androgens are often higher in females
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 7 9 8 - 0
G . Guerriero et al. Brain Research 880 2000 92 –101
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than in males [59]. Aromatase activity AA has been the anterior commissure. Hypothalamic regions were
detected in the preoptic area, hypothalamus and amygdala pooled into three groups of about 20 animals each and
of Rana catesbeiana [11], and aromatization seems to be stored in liquid nitrogen until use. Plasma was obtained
involved in the regulation of male sexual behavior in Rana after centrifugation of the blood at 8003g for 10 min, at
pipiens [7]. Autoradiography and immunocytochemical 48C and stored at 2208C until use. Ovaries were dissected
studies demonstrated, respectively, widespread distribution out and weighed.
of androgen and estrogen-concentrating nerve cells in hypothalamic and limbic structures of Xenopus laevis
2.2. Preparation of microsomal pellet, cytosol and [30,38] and in the urodele Taricha granulosa [14]. How-
nuclear extract ever, a biochemical approach to characterize and quantify
sex steroid receptors was not utilized in any of these All operations were carried out at 48C. Analytical grade
vertebrate studies. chemicals were used. Minced tissues were weighed and
Schlinger and Callard [56] demonstrated in quail that homogenized with 1:4 w v of TEMG 10 mM Tris–HCl,
hypothalamic aromatase regulates testosterone-induced 1 mM EDTA, 1 mM 2-mercaptoethanol and 10 glycerol,
aggressiveness by regulating the quantity of estradiol pH 7.8 containing 0.05 M NaCl homogenization buffer.
available for receptor binding. This was the first demon- The suspension was centrifuged at 8003g for 10 min. The
stration of a quantitative relationship between aromatiza- recovered supernatant was centrifuged at 105,0003g for
tion and estrogen receptor occupancy. It is not currently 60 min. The pellet of this centrifugation was employed for
known whether this relationship exists in the brain of AA detection and is indicated as microsomal pellet, the
amphibians. In Rana esculenta, a distinctive feature of supernatant constituted the cytosol. The 8003g pellet
female reproductive endocrinology is the relatively high crude nuclear pellet was suspended and washed three
level of circulating androgens during the reproductive times with homogenization buffer. Smears of the last pellet
cycle [42,44]. Although some hypotheses have been were inspected by a phase-contrast microscope to assess
advanced to explain the role of androgens in the female the presence and integrity of nuclei. Thereafter, the pellet
frog [16], the role of aromatization has not been addressed. was suspended in 1:4 w v TEMG containing 0.7 M KCl
Thus, the present study was undertaken in order to test the extraction buffer. The suspension was left 1 h in an ice
hypothesis that testosterone acts through conversion to bath, with continuous stirring. It was then centrifuged at
estradiol and subsequent binding to nuclear estrogen 105,0003g for 60 min. The supernatant constituted the
receptors. For this reason, we investigated both the pres- nuclear extract.
ence and amount of both AA and estradiol binding
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molecules in the hypothalamus of the female frog, Rana 2.3.
H-estradiol binding assays esculenta, during the reproductive cycle.
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[2,4,6,7] H-estradiol 90 110 Ci mmol was purchased from Amersham Buckinghamshire, UK. For Scatchard
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2. Materials and methods analysis [55] increasing amounts 0.3–5.0 nM of
