Estrogen deficiency leads to apoptosis in dopaminergic neurons in the medial preoptic area and arcuate nucleus of male mice

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The aromatase knockout (ArKO) mouse is unable to synthesize estrogens. Immunohistochemical studies on active caspase-3 and tyrosine hydroxylase (TH) revealed apoptosis of dopaminergic neurons in the medial preoptic area (MPO) and arcuate nucleus (Arc) of the hypothalamus of 1-year-old (1yo) male ArKO mice while no active caspase-3 was detected in wild type (WT). Furthermore, the number of TH-positive cells in the MPO and caudal Arc was significantly decreased in 1yo ArKO compared to WT. RNase protection assays support the presence of apoptosis in 1yo ArKO hypothalamus, revealing an up-regulation of pro-apoptotic genes: FASL, FADD, and caspase-8. Concomitantly, the ratio of bcl-2-related anti-apoptotic genes to pro-apoptotic genes in the hypothalamus of 1yo ArKO mice was significantly down-regulated. Previously, we have reported that no such changes were observed in the hypothalamus of female ArKO mice. Thus, we have provided direct evidence that estrogen is required to maintain the survival and functional integrity of dopaminergic neurons in the MPO and Arc of male, but not female mice.

Introduction

Early reports on the presence of aromatase (the enzyme that converts androgens to estrogens) and estrogen receptors in specific regions of the brain, such as the hypothalamus, amygdala, and hippocampus, suggest a role for estrogen in the brain (McGrire and Lisk, 1969, Naftolin and MacLusky, 1982, Stumpf, 1971, Selmanoff et al., 1977). Since then, several reports have demonstrated neuroprotective effects of estrogen, including the protection of neurons from damage by excitotoxins and free radicals (Emilien et al., 2000, Lee et al., 2001), changes in calcium channel and calcium ion entry (Lee et al., 2001), stabilization of mitochondrial membrane potentials, and prevention of ATP depletion (Mattson et al., 1997, Wang et al., 2001). These neuroprotective effects of estrogen have been implicated in the treatment of brain injury and stroke (Hall ED, 1998, McCullough et al., 2003) and for neurodegenerative diseases such as Alzheimer's disease (Tang et al., 1996, Xu et al., 1998), although this remains controversial. Long-term treatment with 17β-estradiol has been shown to protect against induced apoptosis in neuronal cell cultures (Harms et al., 2001). In addition, several reports have found that estrogen is involved in up-regulation of bcl-2-related anti-apoptotic genes, particularly in the hypothalamus (Dubal et al., 1998, Garcia-Segura et al., 1998, Pike, 1999, Harms et al., 2001).

Previously, estradiol has been shown to localize in tyrosine hydroxylase containing neurons of the rat brain in the arcuate nucleus, periventricular nucleus, and the zona incerta (Grant and Stumpf, 1975, Sar, 1984). In addition, aromatase-immunoreactive (AROM-ir) neurons have been reported in the medial preoptic nucleus (MPO), receiving catecholaminergic inputs identified by the presence of tyrosine hydroxylase staining (Balthazart and Absil, 1997). Estrogen receptors have also been reported to be co-localized with tyrosine hydroxylase in the MPO, arcuate and periventricular nuclei in female rats (Yuri and Kawata, 1994). This indicates that estrogen may play a role in the regulation and integrity of dopaminergic neurons, possibly via its anti-apoptotic effects. Indeed, estrogen and its precursor testosterone have previously been shown to have direct effects on the expansion of dendritic elements of the preoptic area culture, and in addition greatly increase dopamine content of culture medium bathing hypothalamic explant (Kawashima and Takagi, 1994).

Besides providing a protective mechanism, dietary phytoestrogens administered at a young age have been found to change the structure of the sexually dimorphic brain region (anteroventral periventricular nucleus, AVPV) in male but not in female rats (Lephart, 1996). This may have an effect on sexual behavior, as perinatal exposure to aromatase inhibitors during critical periods of male sexual brain differentiation has previously been shown to have a long-term effect of inhibiting sexual behavior of rats (Gerardin and Pereira, 2002). In addition, the medial preoptic area, a key area in the control of male sexual behavior, has previously been shown to have strong inputs to the telencephalon that possesses high densities of ER mRNA (Simerly et al., 1990).

The aromatase knockout mouse (ArKO) lacks aromatase, the enzyme that converts androgens to estrogens, and therefore lacks any classical endogenous estrogen (Fisher et al., 1998). Disruption of the aromatase gene has been shown to cause significant changes in male sexual behavior, namely a marked deficit in mounting behavior (Robertson et al., 2001). This paper characterizes the consequences of chronic lack of estrogen in the male ArKO rodent brain and in addition correlates these effects with other phenotypes of the ArKO mouse. We provide novel evidence that estrogen is required for the regulation and maintenance of dopaminergic neurons in the hypothalamic region of the male mouse.

Section snippets

TUNEL assay

TUNEL assay was performed on sagittal whole brain sections of five 20-week-old ArKO and WT as well as five 1yo ArKO and WT mice. TUNEL labels DNA strand breaks that occur during apoptosis, and can therefore be a relatively reliable indicator of apoptosis. TUNEL staining of whole sagittal sections revealed fluorescein labeled DNA strand breaks occurring throughout the hypothalamus of 1yo male ArKO mice with most staining concentrating in the medial preoptic area (MPO) and arcuate nucleus (Fig. 1A

Discussion

The present study describes the consequences of estrogen deprivation on the male rodent brain, by examining the brain of the male aromatase knockout (ArKO) mouse. Our data demonstrate that the absence of estrogen promotes apoptosis in the dopaminergic neurons of the arcuate nucleus of the hypothalamus and MPO of 1yo male mice, as characterized by TUNEL, protein expression of active caspase-3, and a significant decrease in the expression ratio of anti:pro-apoptotic bcl-2-related genes. In

Animals

ArKO mice (C57Black/6J/J129) were generated by disruption of the Cyp19 gene (Fisher et al., 1998). Homologous null or wild-type (WT) offspring were bred by crossing heterozygous ArKO, and genotyped by PCR. Animals were housed under SPF conditions and had ad libitum access to water and soy-free mouse chow (Glen Forrest Stockfeeders, Western Australia). Mice were killed by cervical dislocation, the brains removed and placed in Bouin's fixative for histological studies or dissected in RNAlater

Acknowledgment

This work was supported by USPHS grant number R37AG08174.

References (53)

  • A. Pecci et al.

    Progestins prevent apoptosis in a rat endometrial cell line and increase the ratio of bcl-XL to bcl-XS

    J. Biol. Chem.

    (1997)
  • A. Sainsbury et al.

    Hypothalamic regulation of energy homeostasis

    Best Pract. Res. Clin. Endocrinol. Metab.

    (2002)
  • H. Sawada et al.

    Estradiol protects dopaminergic neurons in a MPP+Parkinson's disease model

    Neuropharmacology

    (2002)
  • M.X. Tang et al.

    Effect of oestrogen during menopause on risk and age at onset of Alzheimer's disease

    Lancet

    (1996)
  • B. Wiedenmann et al.

    Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles

    Cell

    (1985)
  • K. Yuri et al.

    Region-specific changes of tyrosine hydroxylase-immunoreactivity by estrogen treatment in female rat hypothalamus

    Brain Res.

    (1994)
  • H. Zha et al.

    Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2

    J. Biol. Chem.

    (1996)
  • H. Zhang et al.

    Combinatorial interaction of human bcl-2 related proteins: mapping of regions important for bcl-2/bcl-x-s interaction

    Biochem. Biophys. Res. Commun.

    (1995)
  • D.M. Armstrong et al.

    Distribution of dopamine-, noradrenaline-, and adrenaline-containing cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes

    J. Comp. Neurol.

    (1982)
  • J. Balthazart et al.

    Identification of catecholaminergic inputs to and outputs from aromatase-containing brain areas of the Japanese quail by tract tracing combined with tyrosine hydroxylase immunocytochemistry

    J. Comp. Neurol.

    (1997)
  • D.B. Dubal et al.

    Estradiol protects against ischemic injury

    J. Cereb. Blood Flow Metab.

    (1998)
  • G. Emilien et al.

    Prospects for pharmacological intervention in Alzheimer disease

    Arch. Neurol.

    (2000)
  • C.R. Fisher et al.

    Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene

    Proc. Natl. Acad. Sci. U. S. A.

    (1998)
  • L.M. Garcia-Segura et al.

    Estradiol upregulates Bcl-2 expression in adult brain neurons

    NeuroReport

    (1998)
  • F. Giuliano et al.

    Dopamine and male sexual function

    Eur. Urol.

    (2001)
  • L. Grant et al.

    Hormone uptake sites in CNS biogenic amines systems

    Anat. Neuroendocrinol., Karger

    (1975)
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