BIOLOGY OF REPRODUCTION 74, 931–937 (2006)
Published online before print 1 February 2006.
Effect of Steroid Milieu on Gonadotropin-Releasing Hormone-1 Neuron Firing Pattern
and Luteinizing Hormone Levels in Male Mice1
Justyna Pielecka and Suzanne M. Moenter2
Departments of Internal Medicine and Cell Biology, University of Virginia, Charlottesville, Virginia 22908
GnRH neuronal function is regulated by gonadal hormone
feedback. In males, testosterone can act directly or be converted
to either dihydrotestosterone (DHT) or estradiol (E2). We
examined central steroid feedback by recording firing of green
fluorescent protein (GFP)-identified GnRH neurons in brain
slices from male mice that were intact, castrated, or castrated
and treated with implants containing DHT, E2, or E2þDHT.
Castration increased LH levels. DHT or E2alone partially
suppressed LH, whereas E2þDHT reduced LH to intact levels.
Despite the inhibitory actions on LH, the combination of
E2þDHT increased GnRH neuron activity relative to other
treatments, reflected in mean firing rate, amplitude of peaks in
firing rate, and area under the curve of firing rate vs. time.
Cluster8 was used to identify peaks in firing activity that may be
correlated with hormone release. Castration increased the
frequency of peaks in firing rate. Treatment with DHT failed to
reduce frequency of these peaks. In contrast, treatment with E2
reduced peak frequency to intact levels. The frequency of peaks
in firing rate was intermediate in animals treated with E2þDHT,
perhaps suggesting the activating effects of this combination
partially counteracts the inhibitory actions of E2. These data
indicate that E2mediates central negative feedback in males
primarily by affecting the pattern of GnRH neuron activity, and
that androgens combined with estrogens have a central
activating effect on GnRH neurons. The negative feedback
induced by E2þDHT to restore LH to intact levels may mask an
excitatory central effect of this combination.
estradiol, gonadotropin-releasing hormone, luteinizing hormone,
GnRH neurons form the final common pathway regulating
reproductive function. GnRH is released in pulses [1–3] that
stimulate the synthesis and secretion of the pituitary gonado-
tropins LH and FSH and are required for fertility in both males
and females [4–7]. Changes in GnRH, and resulting LH, pulse
patterns are largely regulated by feedback actions of the
steroids released by the ovaries or testes [8–16]. In males,
testosterone is the predominant gonadal steroid in the
circulation, but it is often converted in target tissues via
aromatization to estradiol (E2) or via 5-alpha reduction to
dihydrotestosterone (DHT) [17–19].
Although GnRH neurons express the beta isoform of the E2
receptor, many studies indicate that these cells lack the alpha
isoform of the E2receptor and androgen receptors [20–24]. This
suggests that feedback effects from either steroid are likely
indirect, mediated by steroid-sensitive neurons either locally
within the hypothalamus or in distant brain regions. In this
regard, both androgen and estrogen receptors are found in the
in steroid feedback regulation [28–32]. GnRH neurons and
immortalizedGnRH neuronalGT1cellshavealso been reported
to express more recently described membrane-associated forms
of both the estrogen and the androgen receptor, suggesting an
alternative possibility for direct steroid feedback [33, 34].
There is evidence for both androgen and estrogen action in
testosterone-mediated negative feedback. Estrogen action is
suggested by suppression of LH by local hypothalamic implants
containing E2but not DHT [35, 36]. In patients lacking estrogen
receptors or aromatase activity, LH release is elevated [37, 38];
mouse aromatase knockouts also exhibit elevated LH .
Androgen action is suggested by reductions in serum LH levels
after systemic treatment with DHT in rodents, sheep, and
primates [10, 13, 40]. Additionally, reduction of androgen
levels [41, 42]. Reports of feedback actions in estrogen receptor
a knock-out mice are mixed, with one study showing an
elevation in LH in gonad-intact males , suggesting a role for
the alpha isoform of the E2receptor, but others showing no
change [35, 44]. In one of the latter studies , castration
increased, and high-dose DHT treatment reduced LH levels,
suggesting androgen receptor-mediated negative feedback. In
men with idiopathic hypogonadotropic hypogonadism, testos-
terone and E2similarly suppressed LH release from the pituitary
in response to a known GnRH signal, suggesting either
conversion to E2or similar action of these two steroids at the
pituitary in humans [45, 46]. In contrast, both E2and
dihydrotestosterone were reported to act centrally and not at
the pituitary to affect negative feedback on the reproductive
neuroendocrine system in sheep .
It is thus evident that a consensus has not yet emerged
regarding the active steroid(s) mediating testosterone feedback.
This may be in part because of species differences, but could
also be because of differences in technical approach.
Specifically, many studies probe feedback by examining LH
levels, which represent the combined central and pituitary
actions making positive identification of the site of feedback
action difficult. In the present studies we investigated how
steroid milieu affects the firing pattern of green fluorescent
protein (GFP)-identified GnRH neurons to examine central
actions, and compared this to LH levels of male mice.
1Supported by the National Institute of Child Health and Human
Development/National Institutes of Health through cooperative agree-
ment U54HD28934 and the Ligand Assay and Analysis Core. A
preliminary report of this work was presented at the 38th annual
meeting of the Society for the Study of Reproduction, 24–27 July 2005,
Quebec City, Quebec, Canada.
2Correspondence: Suzanne M. Moenter, Department of Internal
Medicine, P.O. Box 800578, University of Virginia, Charlottesville,
VA 22908. FAX: 434 9820088; e-mail: firstname.lastname@example.org
Received: 23 November 2005.
First decision: 8 December 2005.
Accepted: 31 January 2006.
? 2006 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
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ANDROGENS CAN ACTIVATE GNRH NEURONS IN MALES