The Multiple Roles of Estrogens and the Enzyme Aromatase

L. Martini (Eds.) Progress in Brain Research, Vol. 181 ISSN: 0079-6123 Copyright
2010 Elsevier B.V.

CHAPTER 12

The multiple roles of estrogens and the enzyme aromatase Wah Chin Boon1,2,3,4,
, Jenny D. Y. Chow3,4 and Evan R Simpson3,5 1 Florey Neuroscience Institutes, Parkville, Victoria, Australia Centre of Neuroscience, Melbourne University, Parkville, Victoria, Australia 3 Prince Henry’s Institute, Clayton, Victoria, Australia 4 Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia 5 Department of Biochemistry, Monash University, Clayton, Victoria, Australia 2

Abstract: Aromatase is the enzyme that catalyzes the last step of estrogen biosynthesis. It is expressed in many tissues such as the gonads, brain and adipose tissue. The regulation of the level and activity of aromatase determines the levels of estrogens that have endocrine, paracrine and autocrine effects on tissues. Estrogens play many roles in the body, regulating reproduction, metabolism and behavior. In the brain, cell survival and the activity of neurons are affected by estrogens and hence aromatase. Keywords: aromatase; CYP19; estrogens; brain; neuroprotection

(Allen and Doisy, 1983), it was not until the 1980s that the human aromatase cytochrome P450 protein was extracted from placental microsomes, which demonstrated that indeed a single enzyme is responsible for the multiple reaction steps in the aromatization process (reviewed by Santen et al., 2009). Even then, the structure of aromatase remained unknown for another two decades. In 2009, the aromatase crystal structure was finally revealed (Ghosh et al., 2009), and was the first natural mammalian full-length cytochrome P450 protein to be crystallized.

Aromatase Aromatase cytochrome P450 is the enzyme that catalyzes the last step of estrogen biosynthesis (Fig. 1), that is, the rate-limiting irreversible aromatization of androgens to estrogens. Although actions of estrogens (uterine and vaginal tissue changes during menstrual cycles in guinea pigs) were first described in 1917 (Stockard and Papanicolaou, 1917) and the responsible steroids estrone and 17b-estradiol were purified from urine of pregnant women in the next decades




Corresponding author. Tel.: þ61-3-8344-1888; Fax: þ61-3-9348-1707; E-mail: [email protected]

DOI: 10.1016/S0079-6123(08)81012-6

209

210

CH3 CH3

Cholesterol

HO

Side-chain cleavage (CYP11A) CH3 O CH3

HO

Pregnenolone

Progesterone

3β-HSD2

CYP17

CYP17

17α-OH-pregnenolone

17α-OH-progesterone

3β-HSD2

CYP17

Dehydroepiandrosterone (DHEA)

CYP17

16α-hydroxylase 3β-HSD2

CH3 CH3

CH3

O

O

CH3

OH

CH3 17β-HSD3

16α-OH-DHEA

O Androstenedione

Aromatase (CYP19)

Testosterone

Aromatase (CYP19) CH3

CH3

17β-HSD4

O

HO

Aromatase (CYP19)

CH3

OH OH

OH

CH3

O

OH

17β-HSD1

?

17β-HSD2

HO

HO

Estriol

HO

Estrone

17β-estradiol

Fig. 1. The biosynthesis of estrogens and participating steroidogenic enzymes. CYP11A, cholesterol side-chain cleavage; CYP17, 17a-hydroxylase; 3 -HSD2, 3b-hydroxysteroid dehydrogenase type 2; 17 -HSD1, 17b-hydroxysteroid dehydrogenase type 1; 17 -HSD2, 17b-hydroxysteroid dehydrogenase type 2; 17 -HSD3, 17b-hydroxysteroid dehydrogenase type 3; 17 -HSD4, 17b-hydroxysteroid dehydrogenase type 4; CYP19, aromatase.

211

Aromatization Aromatase belongs to the cytochrome P450 superfamily and thus it is a haem-binding protein (see review by Conley and Hinshelwood, 2001). It is localized at microsomal organelles of estrogen-producing cells. It has a high substrate (androgens) specificity that is conferred by the hydrophobic and polar residues lining the androstenedione cleft, which complements the steroid backbone. To catalyze the conversion of androgens to estrogens, aromatase forms a complex with NADPH-cytochrome P450 reductase (a ubiquitous flavoprotein). Aromatization occurs when the aromatase complex converts C19 androgen substrates to C18 estrogens in three consecutive reactions: (1) hydroxylation (2) oxidation and (3) demethylation. This results in the C19 A-ring being converted into a phenolic ring characteristic of estrogens. Demethylation removes the C19 angular methyl group releasing it as formic acid. These reactions require NADPH cytochrome P450 reductase, which transfers reducing equivalents from NADPH to P450arom during oxidation of the C19 angular methyl group to formic acid. There are three main C19 steroids: dehydroepiandrosterone testosterone and androstenedione. The production of certain estrogens appears to be tissue-specific due to substrate availability: the placenta converts 16a-hydroxydehydroepiandrosterone to estriol; the ovary aromatizes testosterone to estradiol and in adipose tissue androstenedione is aromatized to estrone (Osawa et al., 1987).

Aromatase expression Peripheral tissues Aromatase expression in pre-menopausal women is found in granulosa cells (the major site of expression) and the corpus luteum of the ovary (McNatty et al., 1976). It is also detected in human testicular Leydig and Sertoli cells (Brodie and Inkster, 1993), the epididymis (Carpino et al., 2004), germ cells (Lambard et al., 2004),

syncytotrophoblasts of the placenta (Kilgore et al., 1992) and numerous foetal tissues (Price et al., 1992; Toda et al., 1994). Extragonadal tissues expressing aromatase include adipose mesenchymal tissue (Mahendroo et al., 1993), skin fibroblasts (Harada, 1992), bone osteoblasts and osteoclasts (Nawata et al., 1995), skeletal and smooth muscle (Larionov et al., 2003) and vascular endothelium (Sasano et al., 1999). Estrogen receptors are expressed in the same tissues that express aromatase (Table 1).

Central nervous system Aromatase transcript expression has been detected in rodent and avian brains (as reviewed by Lephart et al., 2001b; Naftolin et al., 2001), markedly in the limbic systems, hypothalamus, preoptic nucleus, sexually dimorphic nucleus, bed nucleus of the striata terminalis, hippocampus (Hojo et al., 2004) and cerebellum (Sakamoto et al., 2003). It has been demonstrated that the male rat foetal hypothalamus expresses higher levels of aromatase than that of female rat (Hutchison et al., 1997). Male porcine hypothalamus has aromatase transcript levels four times that of female counterparts (Corbin et al., 2009) assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). Aromatase expression was also detected in rat pituitary (Galmiche et al., 2006) by RT-PCR. High levels of aromatase activity were detected in the male rat periventricular preoptic nucleus and medial preoptic nucleus; intermediate levels in the suprachiasmatic preoptic nucleus, anterior hypothalamus, periventricular anterior hypothalamus and ventromedial nucleus; and low levels in the arcuate nucleusmedian eminence, lateral preoptic nucleus, supraoptic nucleus (SON), dorsomedial nucleus and lateral hypothalamus (Roselli et al., 1985). Using polyclonal antibody raised against peptide corresponding to rat aromatase sequence (Sanghera et al., 1991), intense immunostaining was observed in neurons of adult rat amygdaloid structures and SON as well as reticular

Table 1. The expression of estrogen receptors and aromatase in normal human tissues

Human tissue

ERa

ERb

Brain

Hypothalamus and forebrain (Osterlund et al., 2000)

Cortex and hippocampus (Gonzalez et al., 2007)

Skin

Fibroblasts (Haczynski et al., 2002)

Fibroblasts (Haczynski et al., 2002)

Cardiovascular

Cardiomyocytes (Mahmoodzadeh et al., 2009); vascular endothelium (Cruz et al., 2008)

Arteries and vascular stroma (Savolainen et al., 2001); smooth muscle cells (Hodges et al., 2000)

Bone Adipose

Osteoblasts, osteoclasts and osteocytes (Hoyland et al., 1997) Adipocytes and stromal cells (Price & O’Brien, 1993)

Osteoblasts, osteoclasts and osteocytes (Vidal et al., 1999) (Pedersen et al., 2001)

Liver

(Grandien, 1996)

Not reported

Placenta

Proliferating trophoblasts (Bukovsky et al., 2003)

Differentiating trophoblasts (Bukovsky et al., 2003)

Granulosa, theca and epithelium (Saunders et al., 2000)

Testis

Granulosa, theca and epithelium (Saunders et al., 2000) Not detected (Makinen et al., 2001)

Urogenital tract Adrenal glands

Not detected (Baquedano et al., 2007)

Zona reticularis (Baquedano et al., 2007)

Uterus

Proliferative glandular and stromal cells (major) (Mylonas et al., 2004)

Prostate

Stromal nuclei only (Leav et al., 2001)

Proliferative glandular and stromal cells (minor), vascular endothelium (Lecce et al., 2001; Mylonas et al., 2004) Multiple cell types (Pasquali et al., 2001)

Foetal tissues

Uterine mesenchyme (Glatstein & Yeh, 1995); Leydig cells (Shapiro et al., 2005b); prostate (Shapiro et al., 2005a); neurons (Gonzalez et al., 2007)

Gonadal cells (Shapiro et al., 2005b); umbilical vein endothelial cells (Toth et al., 2008); prostate (Shapiro et al., 2005a); neurons (Fried et al., 2004)

Gonads Ovary

Sertoli, Leydig and germ cells (Makinen et al., 2001, Moore et al., 1998)

Aromatase gene promoter Promoter 1f (Honda et al., 1994) Promoter 1.4 (Harada, 1992) Promoter 1.7 (endothelial cells) (Sebastian et al., 2002) Promoter 1.6 (Shozu et al., 1998) Promoter 1.4/1.3/II (Mahendroo et al., 1991) N/A (adult) Promoter 1.4 (foetal hepatocytes) (Zhao et al., 1995) Promoter 1a/2a/1.2 Promoter 1.8 (Demura et al., 2008) Promoter II (Means et al., 1991) Promoter II (Bulun et al., 1993) Promoter 1.3/II (Baquedano et al., 2007) Not expressed (Bulun et al., 2005) Promoter II (Ellem et al., 2004) Promoter 1.5 (Toda et al., 1994)

213

thalamic nucleus, olfactory tract and piriform cortex, whereas moderate to light immunoreactivity was observed in the paraventricular and arcuate nuclei and hippocampus. However, contradicting to previous reports, neurons in the bed nucleus stria terminalis, medial basal hypothalamic, and preoptic areas displayed little aromatase immunoreactivity. In contrast, robust aromatase immunoreactivity was detected in the medial preoptic area and hypothalamus of postnatal day 5 rat (Horvath et al., 1997), including periventricular regions, ventromedial and arcuate nuclei, as well as the limbic structures (the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis, lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the cingulate cortex). Despite the fact that there are various studies that have determined aromatase activity and expression in brain regions using animal models (Naftolin and MacLusky, 1982, Naftolin et al., 2001), studies in human postnatal brain tissue are limited due to the difficulty in obtaining fresh human brain tissue samples. Sasano et al., (1998) examined aromatase expression in various postmortem human brain regions using RT-PCR and demonstrated that aromatase is expressed widely in human brain regions such as pons, thalamus, hypothalamus and hippocampus. The expression of aromatase mRNA in human temporal lobe tissues was investigated by Stoffel-Wagner et al. (1998) and reported that aromatase expression levels did not differ significantly between men and women, but aromatase mRNA levels were significantly higher in adults than in children, accounting for differences of expression levels between children and adults. As an extension to the previous findings, Stoffel-Wagner et al., (1999) examined the aromatase expression in biopsy samples from 45 women and 54 men with epilepsy using nested competitive RT-PCR. They detected aromatase expression in hippocampus, temporal and frontal neocortex, with the temporal expressing significantly higher levels than frontal neocortex. No expression differences between sexes were observed in any of the brain regions

investigated. Recent studies have determined that in the human temporal cortex, aromatase is expressed in a large population of pyramidal neurons and in certain interneurons and astrocytes, suggesting that aromatase serves a significant role in human cerebral cortex (Yague et al., 2006). Aromatase expression was detected in normal human pituitary obtained from autopsy (13 males, 6 females, median age: 30 years, interquartile ranges 23–63) via quantitative RT-PCR and aromatase protein with immunohistochemical staining (Kadioglu et al., 2008). Although median relative expression level of aromatase mRNA of men (median DeltaCt = 42.6; interquartile ranges: 7.6–93.9) was higher than women (median DeltaCt = 3.9, interquartile ranges: 0–44.8), the difference is not statistically significant (p = 0.2) due to small sample size and large variations within groups. The aromatase levels were also not correlated with the age of the study subjects (p = 0.42; r = –0.21). As both estrogen receptor a and b are expressed in numerous sites of the brain (Azcoitia et al., 1999; Gonzalez et al., 2007; Mitra et al., 2003), estrogens produced locally in the brain could act in paracrine, intracrine or autocrine manner.

Regulation of aromatase expression Cyp19 (aromatase) gene expression regulation Aromatase is encoded by the Cyp19 gene. More than one copy of the Cyp19 gene has been isolated in fish (gonadal cyp19a1 and brain cyp19a2; Kazeto et al., 2001) and boar (cyp19a1, cyp19a2, cyp19a3; Corbin et al., 2009). It had been hypothesized that multiple isoforms of aromatase may exist in the human body (Osawa et al., 1987). However, from restriction mapping and genomic Southern analyses, Means et al. (1989) demonstrated that there is no evidence for more than one isoform of aromatase existing in the human genome. This was supported by surveying the human genome database after its complete elucidation (Bulun et al., 2003).

214

pathways and transcription factors that exist in different tissues, to which different promoters are responsive (Simpson et al., 1993). Activation of tissue-specific promoters triggers the transcriptional splicing of their associated exons I to the coding exons at a common splice junction (CSJ) 38 bp upstream of the transcription start site, resulting in alternative aromatase transcripts with unique 50 -ends. However, the mechanisms regulating the use of tissue-specific promoters and the resulting tissue-specific alternative splicing are not completely understood. Since 50 -ends are not translated, the resulting aromatase coding sequence and protein sequence are identical regardless of the promoter being used. Although alternative exons I are tissue-specific, they are by no means the only transcript variant present in one particular tissue. For instance, exons I.1, 2a and I.2 are all expressed in the placenta (although the latter two at very low levels), and adipose tissue contains transcript variants of exon I.4, I.3 and PII (Mahendroo et al., 1993). The 50 -UTR of human CYP19A1 and that of some other species have been studied extensively. Of the aromatase species sequenced so

The human CYP19A1 gene is located at chromosome 15q21.1, which consists of nine coding exons (II to X) and a 50 -untranslated region (50 UTR), altogether spanning approximately 123 kb in length (Bulun et al., 2003) (Fig. 2). The coding exons occupy 30 kb and the remaining 93 kb contains alternative promoter and untranslated first exons. To date, 11 tissue-specific alternative promoters/first exons have been characterized: promoter/exon I.1 (placenta major), 2a (placenta minor), I.4 (skin), I.5 (foetal tissues), I.7 (endothelium), 1f (brain), I.2 (placenta minor), I.6 (bone), I.3 (adipose and breast cancer), promoter II/exon PII (gonads) and the newly discovered I.8 (Demura et al., 2008). These promoters allow the regulation of CYP19A1 expression in a tissue-specific manner. For example, promoter II drives aromatase expression in the ovary (Adams et al., 2001; Michael et al., 1995) while promoter 1f is used to direct aromatase expression mainly in the brain (Honda et al., 1994), promoter I.4 to direct expression in adipose tissues (Mahendroo et al., 1993) and promoter I.1 to direct expression in the placenta (Kilgore et al., 1992). This is due to the presence of unique cell signalling

Promoters and untranslated Exons I (∼93 kb)

Coding exons II-X (–30 kb)

3’

5’

Simpson Harada

I.1 1a

2a

I.8 I.4 Ib

Placenta Placenta Minor 2 Major

I.5

Adipose Fetal stomal; tissues skin fibroblast; Foetal tissues Breast cancer Placenta; Multiple tissue s

I.7 If Vascular Brain endothelium Breast cancer

I.2 1e Placenta Minor 1

I.6

I.3 1c

PII II 1d 2

X 10

Bone Ovary; Ovary; Breast Prostate; cancer Testis; Breast cancer

Fig. 2. Human aromatase (CYP19A1) gene. A schematic representation of the CYP19A1 tissue-specific promoters and 50 -untranslated exons 1. Note that exons I.1, 1.4, 1.3, PII and 1.2 from the Simpson system of nomenclature are also known as 1a, 1b, 1c, 1d and 1e, respectively, in the Harada system of nomenclature according to chronological order of discovery.

215

far, that of mouse Cyp19A1 (chromosome 9) has the highest homology of 81% with human CYP19A1, followed by the equine, rat and chicken at 78, 77 and 73%, respectively (Seralini et al., 2003; Simpson et al., 1993). There is also only one copy of Cyp19a1 in the murine genome. So far, five tissue-specific promoters/ exons I have been described in the mouse gene, namely, the brain, ovary- (Honda et al., 1996), testis-specific exon I (Golovine et al., 2003) and the more recently described gonadal adiposespecific exon I (Chow et al., 2009), spanning approximately 75 kb upstream of the mouse Cyp19A1 transcription start site in the order: exon I.4 (gonadal fat; –75 kb), 1f (brain; – 36 kb), Etes (testis; –36 kb), exon I.3 (ovary and testis; –200 bp) and proximal exon PII (ovary). Thus, the regulation of aromatase in the human or rodent body is quite complex with the regulators acting at tissue-specific promoters of one CYP19A1 gene. Regulators of peripheral aromatase expression include hormones (e.g. androgens, estrogens and follicle-stimulating hormones), cytokines and growth factors (e.g. insulin-like growth factor-I) (refer to review by Simpson, 2004). Although no estrogen response elements have been reported in the promoter regions, expression of aromatase can be regulated by estrogens, for example, in the human placenta, estrogens induced estrogen receptor a (ERa) recruitment to the –255- to –155-bp region leading to histone modifications resulting in increased aromatase transcription (Kumar et al., 2009). Expression of aromatase in the avian (Steimer and Hutchison, 1981) and mammalian (Abdelgadir et al., 1994; Lephart, 1996) brain regions such as the hypothalamus can be enhanced by testosterone and estradiol (Balthazart et al., 2001b). However, the steroidal control of aromatase expression is neuroanatomically specific. It had been reported that castration of male rats had no effects on the aromatase activity in the amygdala but significantly reduced aromatase activity levels in the preoptic area to that of female rats (Roselli et al., 1985), and testosterone administration restored the aromatase activity.

Neurotransmitters acting through protein kinase C or G (Abe-Dohmae et al., 1996) at promoter 1f could also control the expression of aromatase. By contrast, expression of aromatase in the fish gonads and brain is regulated by the substantially different 50 -flanking promoters of cyp19a1 and cyp19a2 genes, through the involvement of different regulators/transcription factors. Gonadal-specific aromatase gene cyp19a1 contains three cAMP-responsive elements (CREs), an aryl hydrocarbon-responsive element (AhR/ Arnt), a steroidogenic factor 1 (SF-1) site and a TATA box whereas the brain-specific aromatase gene contains a single CRE, an estrogen-responsive element (ERE), a peroxisome proliferator-activated receptor a/retinoid X receptor a heterodimer-responsive element (PPARa/RXRa) and a TATA box. The predominant transcription initiation sites for cyp19a1 and cyp19a2 transcripts were 28 and 91 bp upstream from the putative translation initiation codon, respectively (Kazeto et al., 2001).

Regulation of aromatase activity The activity of aromatase activity has been shown to be regulated by phosphorylation. The first evidence came from the quail preoptic–hypothalamic homogenate – 15 minute preincubation with 1 mM ATP and 5 mM MgCl2 (a limiting factor for kinases) reduced the aromatase activity significantly down to 16.9% of that of controls (Balthazart, Baillien, & Ball, 2001c). Incidently, the inhibition by ATP/Mg2þ could be blocked by 10 mM staurosporine (a general serine/threonine kinase inhibitor) or 10 mM bisindolymaleimide (a Protein Kinase C (PKC) inhibitor) or 50 mM genistein (a general tyrosine kinase inhibtor) without affecting the basal aromatase activity. Although the exact sites of phosphorylation were not determined emperically in these studies, the quail aromatase sequence has consensus phoshorylation sites for Protein Kinase A (PKA) (T389), PKC (S363) and tyrosine kinase (Y354), which are also conserved in the human aromatase sequence

216

(Balthazart et al., 2001a). Protein kinase A (PKA) and PKC phosphorylate serine and/or threonine amino residues. The above studies are supported by the independent report that phosphorylation of murine aromatase inhibited its activity in COS7 cells (Miller et al., 2008). The mutation S118A blocked phosphorylation of the aromatase and showed increased specific enzymatic activity as compared to the wild-type controls, whereas the mutation S118D (mimics phosphorylation effects) exhibited decreased activity versus wild-type control. The structural importance of the S118 residue is also illustrated by the decreased protein stability of the mutants. In contrast, it has been demonstrated that phosphorylation of human aromatase at Y361 resulted in increased activity in MCF-7 (breast cancer cell line) after estradiol incubation through activation of tyrosine kinase c-Src by ligand-bound ERa (Catalano et al., 2009). This rapid regulation of aromatase would have application in the rapid effects of estrogens on neuronal functions, which will be discussed in the section below.

Multiple roles of aromatase The multiple roles of aromatase were uncovered by studying models of aromatase deficiency – both human and mice.

Human aromatase deficiency Natural mutations of the aromatase gene causing aromatase and estrogen deficiency are very rare in humans and often result from parents of consanguineous marriages. To date, there are only 15 known cases of patients (eight men and seven women) diagnosed with aromatase deficiency (Jones et al., 2007; Lanfranco et al., 2008). Reproductive abnormalities are the primary observations in patients diagnosed with aromatase deficiency. Female patients of aromatase deficiency are usually diagnosed and treated early in

life due to signs of pseudohermaphroditism at birth, pubertal abnormalities (mild virilization, cystic ovaries, hypergonadotrophins, elevated testosterone, low estrogen, enlarged clitoris), amenorrhea at the time of puberty and delayed maturation of bone development. Estrogen replacement therapy resulted in a growth spurt, breast development, menarche, suppression of gonadotropin levels, and resolution of the cysts (Harada et al., 1992; Jones et al., 2007). Aromatase deficiency causes abnormalities that are sexually dimorphic, and hence shedding light on the importance of estrogen unique to each of the genders. Male patients are usually diagnosed and treated much later in life, as pubertal development is normal, but signs of infertility and persistent bone growth become apparent (Jones et al., 2007; Lanfranco et al., 2008). The first male patient with aromatase deficiency was described in 1995 (Morishima et al., 1995). Aromatase deficiency in males does not affect the reproductive development to the same degree as that in females. Aromatase-deficient men develop progressive infertility in adulthood, with decreased sperm motility and/or low sperm counts as the common causes (Carani et al., 1997; Morishima et al., 1995), but sexual activity may also be affected (Carani et al., 1999). Metabolic abnormalities [such as slight truncal obesity, hyperinsulinemia, elevated serum triglyceride and low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol] as well as some liver dysfunction are present in male aromatase-deficient patients (reviewed by Jones et al., 2007). It is still uncertain whether untreated female aromatase-deficient patients will also develop fatty liver disease as observed in male patients. Neither cognitive function nor psychological profiles of these patients were reported.

Aromatase knockout mice The generation of the aromatase knockout (ArKO) mice (Fisher et al., 1998) revealed very similar phenotypes as those observed in aromatase-deficient humans, and has since

217

enabled scientists to study the physiological importance of estrogen in the laboratory. The ArKO mouse model was generated by deleting exon IX of the Cyp19A1 (replaced by a neomycin cassette) resulting in the expression of a nonfunctional aromatase enzyme, thus becoming estrogen-deficient and hyperandrogenic. Both genders presented with disrupted reproductive functions (Fisher et al., 1998; McPherson et al., 2001; Robertson et al., 1999), bone undermineralization (Oz et al., 2001), reduced blood pressure and baroreflex sensitivity (Head et al., 2004), autoimmunity (Shim et al., 2004) as well as an array of metabolic phenotypes such as increased adiposity (Jones et al., 2000; Misso et al., 2003) and hepatic steatosis (Hewitt et al., 2004) (Table 2). These metabolic phenotypes were also reported in the ArKO generated by Toda et al. (2001b). Interestingly, the metabolic phenotypes of the ArKO mice are sexually dimorphic. Female ArKO mice do not develop hepatic steatosis like the male ArKO; however, serum triglyceride was significantly elevated, which was likewise not observed in male ArKO mice. Female ArKO mice also have increased serum cholesterol and HDL levels, and both were reversible upon estrogen replacement. The pituitary of the female ArKO mice is smaller and the plasma growth hormone (GH) levels were decreased (Yan et al., 2005). This is accompanied by decreased transcript levels of GH-secretagogue receptor, GH-releasing hormone receptor in the pituitary that could be corrected by estrogen treatment.

ArKO brain and behavioral phenotypes Interestingly, the ArKO mouse model presents brain and behavioral phenotypes that were not previously observed in gonadectomized animals (refer to review by Hill and Boon, 2009). We have reported that neuronal apoptosis occurred in the frontal cortex of aged female ArKO mice (Hill et al., 2009) and in the hypothalamus of aged male ArKO mice (Hill et al., 2004) in the absence of external assault. This could be a consequence of decreased levels of anti-apoptotic

gene expressions and increased levels of proapoptotic gene expressions (Hill et al., 2007a, 2009). The ArKO mouse model developed in Harada’s laboratory (Honda et al., 1998), by targeted disruption of exon 1 and 2, had been reported to be more susceptible to neuronal toxins domoic acid (Azcoitia et al., 2001) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Morale et al., 2006). These male ArKO mice showed significant hilar neuronal loss in the hippocampus after injection of a low dose of domoic acid, which had no effect on the control littermates, indicating that aromatase deficiency increases the vulnerability of neurons to neurotoxic degeneration. These neonatal ArKO animals have decreased Purkinje dendritic growth, spinogenesis and synaptogenesis (Sasahara et al., 2007) as compared to the wild type (WT) animals during the same developmental period demonstrating that estrogens are important in promoting synapse formation. Our laboratories have demonstrated that both male and female ArKO mice had impaired spatial reference memory (Martin et al., 2003) although the female ArKO mice performed as well as the WT in the watermaze test (Boon et al., 2005). Our male ArKO mice presented loss of sexual behavior (Robertson et al., 2001) but developed compulsive behaviors such as excessive grooming and wheel-running activities (Hill et al., 2007b). The levels of catechol-Omethyltransferase (COMT) enzyme that metabolize dopamine were lowered in the hypothalamus of the male ArKO mice when compared to the WT mice. The compulsive behavior were ameliorated after estradiol administration (Hill et al., 2007b) with concomitant increase of COMT levels to WT levels. Depressive-like behavior was observed in the female ArKO model developed in Harada’s laboratory (Dalla et al., 2004) whereas the male ArKO did not display such behavior. The sexual and aggressive behaviors were disrupted in the male ArKO mice and they developed infanticide behavior (reviewed by Harada et al., 2009). Toda et al., (2001a) also reported the loss of agressive behavior in their male ArKO mice (Nemoto et al., 2000). Estrogen

Table 2. Key phenotypes of aromatase knockout (ArKO) mice ArKO (Fisher et al., 1998) Cyp19A1 exon 9 deletion

ArKO (Honda et al., 1998) Cyp19A1 exon 1 deletion

ArKO (Nemoto et al., 2000) Cyp19A1 exon 9 deletion

Phenotypes

Male

Female

Male

Female

Male

Female

Reproductive

Age-progressive infertility; impaired spermatogenesis (Robertson et al., 1999); prostate gland hyperplasia (McPherson et al., 2001) " adiposity " liver cholesterol, " serum leptin and insulin Hepatic steatosis

Infertile; disrupted folliculogenesis; haemorrhagic cystic follicles (Britt et al., 2001); underdeveloped uteri

Infertile; no significant external abnormalities

Infertile; underdeveloped uteri; No other significant external abnormalities

Infertile

" adiposity " serum leptin and insulin " serum TG, cholesterol and HDL (Misso et al., 2003)

No info

No info

Hepatic steatosis; impaired gene expression and hepatic enzyme activities of fatty acid b-oxidation (Nemoto et al., 2000) " HDL-cholesterol (Toda et al., 2001c) Insulin resistance (Takeda et al., 2003)

Infertile; anovulation, depletion of follicles; disorganized interstitial cells; haemorrhages in the ovaries (Toda et al., 2001d) Normal hepatic fatty acid b-oxidation (Toda et al. 2001c)

Decreased bone length and density; increased bone formation rate; increase B-cell lymphopioesis (Oz et al., 2001)

No info

No info

" bone resorption similar to females, but at 32 weeks bone loss is less compared to females (Miyaura et al., 2001)

Metabolic

# body lean mass, physical activity, calorie intake (Jones et al., 2000; Hewitt et al., 2003) Bone

Decreased bone formation rate (Oz et al., 2001)

Loss of cancellous bone, " bone resorption from 9 weeks old; more severe at 32 weeks. (Miyaura et al., 2001)

CNS/ behavioral

apoptosis in AN and MPO (Hill et al., 2004, 2007a); obsessivecompulsive behavior (Hill et al., 2007b); # sexual activity (Robertson et al., 2001), reduced spatial reference memory (Martin et al., 2003)

apoptosis in frontal cortex (Hill et al., 2009); reversible middle cerebral artery occlusion resulted in greater total and regional damage in female ArKO mice than ovarectomized WT controls (McCullough et al., 2003). Reduced spatial reference memory (Martin et al., 2003)

Others

Severe autoimmune exocrinopathy (Shim et al., 2004); reduced proliferation and enhances apoptosisrelated death in VSMCs (Ling et al., 2004); reduced acetylcholineinduced release of nitric oxide in aorta (Kimura et al., 2003)

Reduced blood pressure and baroreflex sensitivity (Head et al., 2004); severe autoimmune exocrinopathy (Shim et al., 2004); reduced proliferation and enhances apoptosisrelated death in VSMCs (Ling et al., 2004)

Increase vulnerability to neurotoxin (Azcoitia et al., 2001; Morale et al., 2006); prolonged latencies to mount and decreased numbers of mounts in response to receptive stimulus females; deficits in olfactory and visual cues for sexual partner preference (Bakker et al., 2002); severe deficits in social recognition (Pierman et al., 2008); anxiety and depressive-like symptoms (Dalla et al., 2005) –

Enhanced response to odour cues (Wesson et al., 2006)

Lack of aggressive behavior (Toda et al., 2001b); impairment in mounting behavior (Toda et al., 2001a)

No info

–

Thymic regression and reduced cellularity

–

(Li et al., 2002)

220

replacement could restore the sexual behavior deficit in male (Bakker et al., 2004) but not female ArKO mice (Bakker et al., 2002). In summary, the ArKO mouse models illustrated that aromatase plays important roles in regulating neuronal survival and functions as well as behaviors.

Physiological effects of estrogen Estrogen is considered to be the female steroid also because of its traditional role in the female reproductive system. But today, estrogen is implicated in a far wider range of actions including metabolic regulation, and neurological and behavioral effects in both male and female.

Reproductive effects

and the pattern of fat deposition, and the appearance of axillary and pubic hair also depend on estrogens.

Male Testosterone is the principal hormone for the development of male sexual characteristics. However, current research has shed light on the effects of estrogen in the male reproductive system and some of the effects of testosterone are mediated through its conversion to estrogen. Estrogen is believed to be critical in the normal development of male gonadal functions and spermatogenesis by controlling stem cell number and spermatid maturation in the seminiferous tubules (reviewed by Hess, 2003). The male ArKO testicular phenotype illustrated this role of estrogens (Robertson et al., 1999).

Female The ovary is the principal source of estrogens in the female circulation. Estrogens are responsible for the development of primary reproductive characteristics including growth and maturation of reproductive organs and breasts at puberty and maintaining their adult size and function through the reproductive age. The data gathered from studying the female reproductive phenotype of the ArKO mice confirmed that estrogens are responsible for the estrous cycle, folliculogenesis and ovulation. Female ArKO mice had atrophic uterus (Fisher et al., 1998) and reduced numbers of primordial and primary follicles (Britt et al., 2004) compared with WT mice and did not ovulate. However, oocytes retrieved from the ArKO antral follicles could be fertilized in vitro and developed to the blastocyst stage at the same rate as wild-type and heterozygote littermates (Huynh et al., 2004). Studies on female aromatase-deficient patient also supported these animal data (MacGillivray et al., 1998) and also indicated that secondary characteristics such as feminization of skeleton

Non-reproductive effects in peripheral tissues In peripheral tissues, estrogen generally promotes cellular division, regrowth and anabolism. Estrogen in the cardiovascular system promotes angiogenesis, vasodilation and vasoprotection against atherosclerosis and endothelial cells from apoptosis (reviewed by Mendelsohn, 2009). Nevertheless, estrogen is also known to promote thrombogenesis and therefore blood clotting. Estrogen replacement therapy and/or estrogen-based oral contraceptives are associated with increased risk of venous thrombosis (Canonico et al., 2008). Relevantly, estrogen reduces serum LDLs and hence increases HDLs in the circulation (reviewed by Knopp et al., 2006). In bone, estrogen has pro-formation effects and inhibits resorption to maintain normal bone mass, as well as stimulating epiphyseal closure (Lanfranco et al., 2008). Youthful skin possesses turgor and is free of wrinkles due to the estrogenic effect of hydration and collagen synthesis (Youn et al., 2003).

221

Central nervous system In brief, estrogen in the central nervous system (CNS) exhibits neurotrophic properties (Carrer et al., 2003), promotes synaptogenesis (Frankfurt et al., 1990) and differentiation from as early as foetal development through adulthood (Lephart et al., 2001a; Wang et al., 2003). It plays an important role in neuroprotection. Estrogens also act on hypothalamus and pituitary to inhibit gonadotropin release in men (Raven et al., 2006). Estrogen replacement to aromatase-deficient men decreased basal and gonadotropin-releasing hormone (GnRH)-stimulated secretion of luteinizing hormone (LH), LH-pulsed amplitude as well as lowered frequency of LH pulses (Rochira et al., 2006). Thus, this demonstrated that circulating estrogens exerted effects both at the pituitary level and at the hypothalamic level. However, the discrepancy among testosterone levels, the arrest of spermatogenesis and a slightly inappropriate respective increase of serum follicle-stimulating hormone (FSH) (lower than expected) suggest a possible role of estrogens in the priming and the maturation of hypothalamus–pituitary– gonadal axis in men, an event that has never occurred in these two subjects as a consequence of chronic estrogen deprivation. Estrogens affect all of the hypothalamic nuclei (such as the paraventricular nucleus or arcuate/ventromedial nucleus) that control energy homeostasis and appetite. The activities of hypothalamic neurones are influenced by estrogens through gene regulation and neuronal excitability (reviewed by Roepke, 2009), which are mediated by the classic estrogen receptors present in these nuclei as well as membrane-associated estrogen receptors.

Effects of aromatase in the brain In the 1970s, it was first demonstrated that estrogens could be produced in the brain by the local aromatization of testosterone (Naftolin et al., 1975). Research in this area has been gaining momentum and evidence is mounting to show that locally synthesized estrogens from

pregnenolone (Hojo et al., 2004) could modulate neuronal functions in addition to their neuroprotective effects.

Neuroprotective effects Estrogens have neuroprotective functions in the brain, especially in the elderly (Mulnard et al., 2000). As the plasma levels of estrogens in men are low, local production of estrogens in the brain will have major contributions to the neuroprotection. This holds true for post-menopausal women since their circulating levels of estrogens have plummeted after menopause. This highlights the importance of brain aromatase. Indeed, an increase in aromatase immunoreactivity (aromir) was found in the nucleus basalis of Meynert (NBM) and SON during normal ageing but a decreased arom-ir was found in the SON, infundibular nucleus (INF) and the medial mammillary nucleus (MMN) of Alzheimer’s disease (AD) patients as compared to non-sufferers (Ishunina et al., 2005). The significance of this localized production of estrogens is highlighted by the presence of estrogen receptor a in these brain regions and the expression levels are elevated in AD patients (Ishunina and Swaab, 2003). Another independent study (Yue et al., 2005) reported that female AD brains contained greatly reduced estrogen levels compared with those from age- and gender-matched normal control subjects although both AD and control subjects had comparably low levels of serum estrogen, again confirming that locally synthesized estrogens have neuroprotective effects. Further evidence that local estrogen production is neuroprotective comes from ArKO mouse. Reversible middle cerebral artery occlusion (90 minute; 22 hour reperfusion) resulted in greater total and regional damage in female ArKO mice than ovarectomized WT controls (McCullough et al., 2003). This infers that extragonadal estrogens play a critical role in neuroprotection. In addition, studies using amyloid precursor protein transgenic mice have reported no effect of ovariectomy or estrogen replacement on

222

b-amyloid deposition in the hippocampus and neocortex (Green et al., 2005). However, the estrogen-deficient APP23 mice [generated by cross-breeding the APP23 transgenic mouse (AD model) with the ArKO mouse] presented amyloid plaque formation at a younger age, accompanied by an increased b-amyloid peptide deposition and increased b-amyloid production (Yue et al., 2005). It is noteworthy that increased risk for AD is associated with polymorphisms of the aromatase (CYP19) gene (Huang and Poduslo, 2006; Iivonen et al., 2004). However, the mechanism(s) underlying the neuroprotective effects of brain estrogens has yet to be elucidated completely. One clue would be that AD brains are reportedly under severe oxidative stress, as a result of either amyloid b-mediated generated oxyradicals or perturbed ionic calcium balances within neurons and their mitochondria (Emilien et al., 2000; Xu et al., 2006). Estrogen has strong antioxidant actions (Bhavnani, 2003), and modifies inflammatory responses and may directly reduce amyloid b generation (van Groen and Kadish, 2005; Xu et al., 1998). Estradiol has also been reported to influence the expression of many genes in the brain that are relevant to estradiol’s ability to protect. These include genes involved in the balance of apoptosis and cell survival (reviewed by Wise et al., 2001; Bhavnani, 2003). In the ArKO mice, we have reported neuronal loss in the frontal cortex of aged female ArKO mice (Hill et al., 2009) and also dopaminergic cell loss in the hypothalamus of aged male ArKO mice (Hill et al., 2004, 2007a) in the absence of external assault such as neurotoxin or pathological agents. Increased levels of pro-apoptotic gene expression and decreased levels of antiapoptotic gene expression were detected in the brain regions affected. Hence, aromatase is essential for the survival of neurons. Another suggestion on the neuroprotective mechanism of estrogens is that estrogens upregulate cerebral apolipoprotein E (apoE), which is believed to be involved in neuronal protection and repair (Levin-Allerhand et al., 2001). During this study, ovariectomized mice were treated with

pharmacological levels of 17b-estradiol or placebo for five days. Results indicated an upregulation of apoE but not glial fibrillary acidic protein (GFAP) in the cortex and diencephalons of estradiol-treated mice brains, while both apoE and GFAP were equally upregulated in the hippocampus (LevinAllerhand et al., 2001). It was concluded that estradiol upregulates apoE, a neuroprotective and repair mechanism, in the cortex and diencephalons, while no upregulation of GFAP, a neuronal destructive mechanism, was seen in these areas of the brain. Therefore, estradiol may have neuroprotective effects on the brain by upregulation of apoE in the cortex and diencephalon. Ovariectomized rats with entorhinal cortex lesions showed an inhibition in the increase in GFAP and enhanced neurite outgrowth after estradiol replacement as compared to placebo-treated animals (Rozovsky et al., 2002). Estradiol also seemed to reorganize astrocytic laminin into extracellular fibrillar arrays, which have shown to support neurite outgrowth. Therefore, not only did estradiol inhibit increases of GFAP, but it also actually aids in the growth of neurites (Rozovsky et al., 2002). Similar estradiol repression of astrocyte GFAP neuroinflammatory response was observed in mesencephalic dopaminergic neurons after MPTP lesion to mouse substantia nigra pars compacta (Morale et al., 2006). Brain aromatase is normally expressed in the neurons but after injury such as neurotoxin kainic acid lesion, novel aromatase in astrocytes was detected by immunostaining and was co-localized with GFAP (Garcia-Segura et al., 1999). No aromatase immunostaining was detectable in astrocytes of control animals.

Sustaining brain glucose metabolism Estrogens may play an important role of sustaining glucose as the primary fuel source in the brain. The evidence presented so far includes the observation that estrogens increased the expression of neuronal glucose transporter subunits, for example, Glut1, Glut3 and Glut4 and glucose transport across the blood–brain barrier endothelium (Cheng et al., 2001). Simultaneously,

223

estrogens increased glycolytic enzyme activity of hexokinase, phosphofructokinase and pyruvate kinase (Kostanyan and Nazaryan, 1992). In addition, estrogens increase the levels of pyruvate dehydrogenase (Nilsen et al., 2007), which converts pyruvate, the product of glycolytic metabolism, to acetyl-CoA, which feeds into the tricarboxylic acid cycle for production of ATP. It may not be a coincidence that estrogens also increase the levels of ATP synthase and proteins involved in mitochondrial oxidative phosphorylation electron transfer (Nilsen et al., 2007). Thus, estrogen treatment increases glucose metabolism of the brain and this may be an important mechanism by which estrogens protect the brain from ageassociated metabolic decline. Significant decrease in the metabolism of the posterior cingulate cortex was observed during two-year follow-up examination in post-menopausal women not on estrogen replacement therapy whereas age-matched estrogen-treated women did not present such a decline (Rasgon et al., 2005).

Modulating effects on neurons

neurons substantia nigra pars compacta and dopamine transporter innervation of the caudate-putamen in adulthood (Morale et al., 2008).

Levels of neurotransmitter–receptor subunits Estrogens may influence the expression pattern and/or levels of neurotransmitter–receptor subunits mediated via the classic estrogen receptor pathway. For example, in the hippocampus, both ERa and ERb are present. Using the estrogen receptor-specific agonists, ERa selective agonist propylpyrazole triol (PPT) [1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazoletriol] and the ERb selective agonist DPN [2,3-bis (4-hydroxyphenyl) propionitrile] it was demonstrated that only diarylpropiolnitrile (DPN) administration had effects on the expression of alpha-amino-3-hydroxyl-5methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR2 and GluR3, increasing and decreasing levels respectively, whereas estradiol, DPN and PPT increased AMPA-type glutamate receptor subunit GluR1 in the female rat stratum radiatum of dorsal hippocampus (Waters et al., 2009a).

Levels of neurotransmitters Physiological levels of estradiol have acute stimulatory effects on the dopaminergic activity in the ovarectomized rat striatum (Pasqualini et al., 1995). Subcutaneous injection of 17b- (but not 17a-) estradiol stimulated in situ tyrosine hydroxylase (TH) activity in a rapid dose-dependent manner, releasing newly synthesized dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). This stimulation of DA synthesis was a consequence of an increase in TH levels. Incubation of striatal slices in the presence of 10(-9) M 17b- (but not 17a-) estradiol indeed evoked an approximate twofold increase in the Ki(DA) of one form of the enzyme. We have reported that less TH-positive neurons are present in the aged male hypothalamus of the ArKO mouse (Hill et al., 2004). Others have reported that aromatase deficiency from early embryonic life in the ArKO mice also significantly impaired the functional integrity of TH-positive

Dendritic spine formation Aromatase has been detected in axons and axon terminals in rat brains by immunostaining (Horvath et al., 1997). Previously, it has been shown that estrogens increase dendritic spine density and synaptogenesis in female (Frankfurt et al., 1990) and male rat (de Castilhos et al., 2008). Furthermore, ERa has also been detected by immunostaining in several extranuclear sites including dendrites, spines, terminals and axons in the hippocampus (McEwen et al., 2001; Romeo et al., 2005) with pro-estrous female rats having significantly more ERa in the dendrites than diestrous or male rats (Romeo et al., 2005). Estradiol and the ERa selective agonist PPT could induce significant increase in dendritic spines in vitro (Jelks et al., 2007); estrogen receptors have been localized along dendrites of cultured hippocampal neurons expressing N-Methyl-D-Aspartate

224

(NMDA) receptor subunit 1 through immunocytochemistry. The presence of aromatase in the axon terminals may infer that locally produced estrogens are acting directly on the localized estrogen receptors at synapses. One of the proposed mechanisms is that estrogens increase scaffolding proteins at the synpase such as post-synaptic density-95 (PSD-95) protein. Strong evidence has implicated that estrogens could increase the protein levels by upregulating the translation rate without increasing the transcription. It has been demonstrated in the NG108-15 neuroblastoma culture that estrogens rapidly stimulate an increase in PSD-95 protein levels without a concomittant rapid increase in PSD-95 mRNA levels. This stimulation could be inhibited by ERa antagonist ICI 182,780 or the PI3K inhibitor LY294002 (Akama and McEwen, 2003). The mechanism is mediated by Akt (protein kinase B) and 4E-BP1 (eukaryotic initiation factor-4E binding protein 1) phosphorylation. The translation of the PDS-95 transcripts at the dendrites is arrested by the binding of a protein complex containing 4E-BP1. Estrogens activate the phosphorylation of Akt (mediated through action of ERa), which in turn phosphorylates 4E-BP1 and leads to the dissociation of the inhibiting complex resulting in the translation of new PSD-95 immediately at the spine to increase spine maturation and synaptic formation. All this occurs in the dendrite, without involving the genomic action of estrogens (Waters et al., 2009b). Later, it is demonstrated in vitro that estrogen-induced phosphorylation of Akt is first apparent at 10 minute and maximal at 30 minute and could be blocked by an inhibitor of phosphatidylinositol-3kinase (PI3K) (Mannella and Brinton, 2006). The same study also showed that there is protein–protein interaction between ER and the PI3K regulatory subunit p85 in cultured cortical neurons. These in vitro data are supported by studies using cortical synaptoneurosome preparations (Dominguez et al., 2007), further illustrating that estrogens act at the synapses to modify dendritic spine formation and plasticity. Further evidence supporting this mechanism is the localization of phosphorylated Akt by immunostaining at the dendritic spines of rat

hippocampus. With the use of electron microscopy (Znamensky et al., 2003), the phosphorylated Akt could be detected at (i) dendritic spines (both cytoplasm and plasmalemma); (ii) spine apparati located within 0.1 micron of dendritic spine bases; (iii) endoplasmic reticula and polyribosomes in the cytoplasm of dendritic shafts and (iv) the plasmalemma of dendritic shafts. The localization density is correlated to the natural fluctuations of estrogen levels across the estrous cycle (Znamensky et al., 2003), which have previously been shown to cause cyclic changes in dendritic spine density and synaptogenesis in the rat hippocampus (Woolley and McEwen, 1992) levels. Estrogens also induce PSD-95 to form a ternary complex with calcium-activated neuronal NO synthase (nNOS) and glutamate NMDA receptor channels harbouring NR2B subunits as demonstrated in primary rat preoptic neuronal culture. Coupling of nNOS to NMDA receptor is accompanied by nitric oxide (NO) production. Again, this is an ERa-mediated event as the protein– protein interaction as well as NO production can be abolished by an ERa antagonist, ICI 182,780 (d’Anglemont de Tassigny et al., 2009). Dendritic spines are composed from actin microfilaments and recently, there are reports that estrogens have non-genomic effects on the re-modelling of actin cytoskeleton (reviewed by Sanchez and Simoncini, 2009). 17b-Estradiol addition to primary rat cortical cultures leads to phosphorylation and activation of WAVE1 (Wiskott-Aldrich syndrome protein (WASP)family verprolin homologous), which controls actin polymerization through the actin-related protein (Arp)-2/3 complex (Sanchez et al., 2009) in the neurons. This is mediated by ERa through the G protein/c-Src signalling cascade. Another non-genomic estrogen mechanism proposed is the activation of PI3 kinase that results in enhanced glutamate release from pre-;psynaptic neurons and leads to the activation of ionotropic glutamate receptors, which then activate mitogen-activated protein (MAP) kinases, thereby inducing dendritic spine formation during estradiol-induced organization of the hypothalamic synaptic patterning (Schwarz et al., 2008).

225

Allosteric effects

Conclusion

Aromatase has been detected by immunocytochemistry in axon terminals of vertebrate brains including quail, rat, monkey and human tissues (Naftolin et al., 1996) or at the surface of presynaptic vesicles (Hojo et al., 2004). Locally synthesized estrogens could serve as allosteric modulators of neurotransmitter receptors or ionotropic receptors. It has been reported that 17b-estradiol acts as a non-serotonin-competitive allosteric antagonist for the 5-hydroxytryptamine type 3 (5-HT3) receptor at the membrane surface of HEK-293 cells (Wetzel et al., 1998). 17b-Estradiol has also been reported to directly bind to the b subunit of the maxi-K channels (hSlo) and rapidly activates the channels (Valverde et al., 1999). As a consequence, estrogens can modulate the electrical activity of neurons within seconds. It has been demonstrated by electrophysiological studies that extremely low concentrations of estrogens acutely potentiate L-type voltage-gated Ca(2þ) channels (VGCCs) in hippocampal neurons, hippocampal slices, and HEK-293 cells transfected with neuronal L-type VGCC, via an ERindependent mechanism (Sarkar et al., 2008). Dihydropyridine site-specific L-type VGCC antagonist could displace membrane-bound estrogens, inferring that estrogens are interacting directly with the channels to rapidly induce Ca2þ influx (Sarkar et al., 2008). Remarkably, 17bestradiol pre-treatment of aged hippocampal neurons could normalize age-dysregulated intracellular Ca concentration responses after glutamate stimulation to that of middle-aged neurons in vitro (Brewer et al., 2006). In vitro study using neonatal hypothalamic neurons has also shown that estradiol pre-treatment could extend the duration of depolarization actions of gammaaminobutyric acid (GABA) as well as doubling the amplitude of Ca2þ transient induction (PerrotSinal et al., 2001) but the underlying mechanism remains to be elucidated. In summary, brain estrogens produced by brain aromatase are ‘neuroactive steroids’ that alter neuronal excitability by interacting with specific neurotransmitter receptors at the cell surface (Balthazart and Ball, 2006; Rupprecht and Holsboer, 1999).

Estrogens play multiple roles in regulating physiological functions. Mounting evidence has been gathered to support that estrogens act in paracrine or autocrine manners in the brain to regulate neuronal survival and brain functions. This is possible due to the expression of aromatase in specific brain regions/nuclei and cell populations. Since aromatase expression levels are highly regulated at the genomic levels and the aromatase activity are regulated by phosphorylations, the levels of brain estrogens could be tightly controlled.

References Abdelgadir, S. E., Resko, J. A., Ojeda, S. R., Lephart, E. D., McPhaul, M. J., & Roselli, C. E. (1994). Androgens regulate aromatase cytochrome P450 messenger ribonucleic acid in rat brain. Endocrinology, 135, 395–401. Abe-Dohmae, S., Takagi, Y., & Harada, N. (1996). Neurotransmitter-mediated regulation of brain aromatase: Protein kinase C- and G-dependent induction. Journal of Neurochemistry, 67, 2087–2095. Adams, M. M., Oung, T., Morrison, J. H., & Gore, A. C. (2001). Length of postovariectomy interval and age, but not estrogen replacement, regulate N-methyl-D-aspartate receptor mRNA levels in the hippocampus of female rats. Experimental Neurology, 170, 345–356. Akama, K. T. & McEwen, B. S. (2003). Estrogen stimulates postsynaptic density-95 rapid protein synthesis via the akt/ protein kinase B pathway. Journal of Neuroscience, 23, 2333–2339. Allen, E. & Doisy, E. A. (1983). Landmark article Sept 8, 1923. An ovarian hormone. Preliminary report on its localization, extraction and partial purification, and action in test animals. By Edgar Allen and Edward A. Doisy. JAMA, 250, 2681–2683. Azcoitia, I., Sierra, A., & Garcia-Segura, L. M. (1999). Localization of estrogen receptor beta-immunoreactivity in astrocytes of the adult rat brain. Glia, 26, 260–267. Azcoitia, I., Sierra, A., Veiga, S., Honda, S., Harada, N., & Garcia-Segura, L. M. (2001). Brain aromatase is neuroprotective. Journal of Neurobiology, 47, 318–329. Bakker, J., Honda, S. I., Harada, N., & Balthazart, J. (2002). The aromatase knock-out mouse provides new evidence that estradiol is required during development in the female for the expression of sociosexual behaviors in adulthood. Journal of Neuroscience, 22, 9104–9112. Bakker, J., Honda, S., Harada, N., & Balthazart, J. (2004). Restoration of male sexual behavior by adult exogenous

226 estrogens in male aromatase knockout mice. Hormones and Behavior, 46, 1–10. Balthazart, J., Baillien, M., & Ball, G. F. (2001a). Phosphorylation processes mediate rapid changes of brain aromatase activity. The Journal of Steroid Biochemistry and Molecular Biology, 79, 261–277. Balthazart, J., Baillien, M., & Ball, G. F. (2001b). Phosphorylation processes mediate rapid changes of brain aromatase activity. The Journal of Steroid Biochemistry and Molecular Biology, 79, 261–277. Balthazart, J., Baillien, M., & Ball, G. F. (2001c). Rapid and reversible inhibition of brain aromatase activity. Journal of Neuroendocrinology, 13, 63–73. Balthazart, J. & Ball, G. F. (2006). Is brain estradiol a hormone or a neurotransmitter? Trends in Neurosciences, 29, 241–249. Baquedano, M. S., Saraco, N., et al. (2007). Identification and developmental changes of aromatase and estrogen receptor expression in prepubertal and pubertal human adrenal tissues. Journal of Clinical Endocrinology and Metabolism, 92 (6), 2215–2222. Bhavnani, B. R. (2003). Estrogens and menopause: Pharmacology of conjugated equine estrogens and their potential role in the prevention of neurodegenerative diseases such as Alzheimer’s. The Journal of Steroid Biochemistry and Molecular Biology, 85, 473–482. Boon, W. C., Diepstraten, J., van der Burg, J., Jones, M. E., Simpson, E. R., & van den Buuse, M. (2005). Hippocampal NMDA receptor subunit expression and watermaze learning in estrogen deficient female mice. Brain Research. Molecular Brain Research, 140, 127–132. Brewer, G. J., Reichensperger, J. D., & Brinton, R. D. (2006). Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons. Neurobiology of Aging, 27, 306–317. Britt, K.L., Drummond, A.E., et al. (2001). The ovarian phenotype of the aromatase knockout (ArKO) mouse. The Journal of Steroid Biochemistry and Molecular Biology, 79 (1–5), 181–185. Britt, K. L., Saunders, P. K., McPherson, S. J., Misso, M. L., Simpson, E. R., & Findlay, J. K. (2004). Estrogen actions on follicle formation and early follicle development. Biology of Reproduction, 71, 1712–1723. Brodie, A. & Inkster, S. (1993). Aromatase in the human testis. The Journal of Steroid Biochemistry and Molecular Biology, 44, 549–555. Bukovsky, A., Caudle, M.R., et al. (2003). Placental expression of estrogen receptor beta and its hormone binding variant – comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells. Reproductive Biology and Endocrinology, 1, 36. Bulun, S.E., Imir, G., et al. (2005). Aromatase in endometriosis and uterine leiomyomata. The Journal of Steroid Biochemistry and Molecular Biology, 95(1–5), 57–62. Bulun, S. E., Sebastian, S., Takayama, K., Suzuki, T., Sasano, H., & Shozu, M. (2003). The human CYP19 (aromatase P450) gene: Update on physiologic roles and genomic

organization of promoters. The Journal of Steroid Biochemistry and Molecular Biology, 86, 219–224. Canonico, M., Plu-Bureau, L. G.D., & Scarabin, P. Y. (2008). Hormone replacement therapy and risk of venous thromboembolism in postmenopausal women: Systematic review and meta-analysis. BMJ, 336, 1227–1231. Carani, C., Qin, K., Simoni, M., Faustini-Fustini, M., Serpente, S., Boyd, J., et al. (1997). Effect of testosterone and estradiol in a man with aromatase deficiency. The New England Journal of Medicine, 337, 91–95. Carani, C., Rochira, V., Faustini-Fustini, M., Balestrieri, A., & Granata, A. R. (1999). Role of oestrogen in male sexual behaviour: Insights from the natural model of aromatase deficiency. Clinical Endocrinology (Oxford), 51, 517–524. Carpino, A., Romeo, F., & Rago, V. (2004). Aromatase immunolocalization in human ductuli efferentes and proximal ductus epididymis. Journal of Anatomy, 204, 217–220. Carrer, H. F., Cambiasso, M. J., Brito, V., & Gorosito, S. (2003). Neurotrophic factors and estradiol interact to control axogenic growth in hypothalamic neurons. Annals of the New York Academy of Sciences, 1007, 306–316. Catalano, S., Barone, I., Giordano, C., Rizza, P., Qi, H., Gu, G., et al. (2009). Rapid estradiol/ERalpha signaling enhances aromatase enzymatic activity in breast cancer cells. Molecular Endocrinology, 23, 1634–1645. Cheng, C. M., Cohen, M., Wang, J., & Bondy, C. A. (2001). Estrogen augments glucose transporter and IGF1 expression in primate cerebral cortex. The FASEB Journal, 15, 907–915. Chow, J. D., Simpson, E. R., & Boon, W. C. (2009). Alternative 50 -untranslated first exons of the mouse Cyp19A1 (aromatase) gene. The Journal of Steroid Biochemistry and Molecular Biology, 115, 115–125. Conley, A. & Hinshelwood, M. (2001). Mammalian aromatases. Reproduction, 121, 685–695. Corbin, C. J., Berger, T., Ford, J. J., Roselli, C. E., Sienkiewicz, W., Trainor, B. C., et al. (2009). Porcine hypothalamic aromatase cytochrome P450: Isoform characterization, sexdependent activity, regional expression, and regulation by enzyme inhibition in neonatal boars. Biology of Reproduction, 81, 388–395. Cruz, M.N., Agewall, S., et al. (2008). Acute dilatation to phytoestrogens and estrogen receptor subtypes expression in small arteries from women with coronary heart disease. Atherosclerosis, 196(1), 49–58. Dalla, C., Antoniou, K., Papadopoulou-Daifoti, Z., Balthazart, J., & Bakker, J. (2004). Oestrogen-deficient female aromatase knockout (ArKO) mice exhibit depressive-like symptomatology. European Journal of Neuroscience, 20, 217–228. Dalla, C., Antoniou, K., et al. (2005). Male aromatase-knockout mice exhibit normal levels of activity, anxiety and depressive-like symptomatology. Behavioural Brain Research, 163(2), 186–193. d’Anglemont de Tassigny, X., Campagne, C., Steculorum, S., & Prevot, V. (2009). Estradiol induces physical association of neuronal nitric oxide synthase with NMDA receptor and promotes nitric oxide formation via estrogen receptor activation in primary neuronal cultures. Journal of Neurochemistry, 109, 214–224.

227 de Castilhos, J., Forti, C. D., Achaval, M., & Rasia-Filho, A. A. (2008). Dendritic spine density of posterodorsal medial amygdala neurons can be affected by gonadectomy and sex steroid manipulations in adult rats: A Golgi study. Brain Research, 1240, 73–81. Demura, M., Reierstad, S., Innes, J. E., & Bulun, S. E. (2008). Novel promoter I.8 and promoter usage in the CYP19 (aromatase) gene. Reproduction Science, 15, 1044–1053. Dominguez, R., Liu, R., & Baudry, M. (2007). 17-Beta-estradiol-mediated activation of extracellular-signal regulated kinase, phosphatidylinositol 3-kinase/protein kinase B-Akt and N-methyl-D-aspartate receptor phosphorylation in cortical synaptoneurosomes. Journal of Neurochemistry, 101, 232–240. Ellem, S.J., Schmitt, J.F., et al. (2004). Local aromatase expression in human prostate is altered in malignancy. Journal of Clinical Endocrinology and Metabolism, 89(5), 2434–2441. Emilien, G., Beyreuther, K., Masters, C. L., & Maloteaux, J. M. (2000). Prospects for pharmacological intervention in Alzheimer disease. Archives of Neurology, 57, 454–459. Fisher, C. R., Graves, K. H., Parlow, A. F., & Simpson, E. R. (1998). Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proceedings of the National Academy of Sciences of the United States of America, 95, 6965–6970. Frankfurt, M., Gould, E., Woolley, C. S., & McEwen, B. S. (1990). Gonadal steroids modify dendritic spine density in ventromedial hypothalamic neurons: A Golgi study in the adult rat. Neuroendocrinology, 51, 530–535. Fried, G., Andersson, E., et al. (2004). Estrogen receptor beta is expressed in human embryonic brain cells and is regulated by 17beta-estradiol. European Journal of Neuroscience, 20(9), 2345–2354. Galmiche, G., Corvaisier, S., & Kottler, M. L. (2006). Aromatase gene expression and regulation in the female rat pituitary. Annals of the New York Academy of Sciences, 1070, 286–292. Garcia-Segura, L. M., Wozniak, A., Azcoitia, I., Rodriguez, J. R., Hutchison, R. E., & Hutchison, J. B. (1999). Aromatase expression by astrocytes after brain injury: Implications for local estrogen formation in brain repair. Neuroscience, 89, 567–578. Ghosh, D., Griswold, J., Erman, M., & Pangborn, W. (2009). Structural basis for androgen specificity and oestrogen synthesis in human aromatase. Nature, 457, 219–223. Glatstein, I.Z. & Yeh, J. (1995). Ontogeny of the estrogen receptor in the human fetal uterus. Journal of Clinical Endocrinology and Metabolism, 80(3), 958–964. Golovine, K., Schwerin, M., & Vanselow, J. (2003). Three different promoters control expression of the aromatase cytochrome p450 gene (cyp19) in mouse gonads and brain. Biology of Reproduction, 68, 978–984. Gonzalez, M., Cabrera-Socorro, A., Perez-Garcia, C. G., Fraser, J. D., Lopez, F. J., Alonso, R., et al. (2007). Distribution patterns of estrogen receptor alpha and beta in the human cortex and hippocampus during development and adulthood. The Journal of Comparative Neurology, 503, 790–802. Grandien, K. (1996). Determination of transcription start sites in the human estrogen receptor gene and identification of a

novel, tissue-specific, estrogen receptor-mRNA isoform. Molecular and Cellular Endocrinology, 116(2), 207–212. Grandien, K. (1996). Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, tissue-specific, estrogen receptor-mRNA isoform. Mol Cell Endocrinol, 116(2): 207–212. Green, P. S., Bales, K., Paul, S., & Bu, G. (2005). Estrogen therapy fails to alter amyloid deposition in the PDAPP model of Alzheimer’s disease. Endocrinology, 146, 2774– 2781. Harada, N. (1992). A unique aromatase (P-450AROM) mRNA formed by alternative use of tissue-specific exons 1 in human skin fibroblasts. Biochemical and Biophysical Research Communications, 189, 1001–1007. Harada, N., Ogawa, H., Shozu, M., & Yamada, K. (1992). Genetic studies to characterize the origin of the mutation in placental aromatase deficiency. The American Journal of Human Genetics, 51, 666–672. Harada, N., Wakatsuki, T., Aste, N., Yoshimura, N., & Honda, S. I. (2009). Functional analysis of neurosteroidal oestrogen using gene-disrupted and transgenic mice. Journal of Neuroendocrinology, 21, 365–369. Haczynski, J., Tarkowski, R., et al. (2002). Human cultured skin fibroblasts express estrogen receptor alpha and beta. International Journal of Molecular Medicine, 10(2), 149–153. Head, G. A., Obeyesekere, V. R., Jones, M. E., Simpson, E. R., & Krozowski, Z. S. (2004). Aromatase-deficient (ArKO) mice have reduced blood pressure and baroreflex sensitivity. Endocrinology, 145, 4286–4291. Hess, R. A. (2003). Estrogen in the adult male reproductive tract: A review. Reproductive Biology and Endocrinology, 1, 52. Hewitt, K. N., Pratis, K., Jones, M. E., & Simpson, E. R. (2004). Estrogen replacement reverses the hepatic steatosis phenotype in the male aromatase knockout mouse. Endocrinology, 145, 1842–1848. Hewitt, K. N., Boon, W. C. et al. (2003). The aromatase knockout mouse presents with a sexually dimorphic disruption to cholesterol homeostasis. Endocrinology, 144 (9), 3895–3903. Hill, R. A. & Boon, W. C. (2009). Estrogens, brain, and behavior: Lessons from knockout mouse models. Seminars in Reproductive Medicine, 27, 218–228. Hill, R. A., Chow, J., Fritzemeier, K., Simpson, E. R., & Boon, W. C. (2007a). Fas/FasL-mediated apoptosis in the arcuate nucleus and medial preoptic area of male ArKO mice is ameliorated by selective estrogen receptor alpha and estrogen receptor beta agonist treatment, respectively. Molecular and Cellular Neuroscience, 36, 146–157. Hill, R. A., Chua, H. K. et al. (2009). Estrogen deficiency results in apoptosis in the frontal cortex of adult female aromatase knockout mice. Mol Cellular Neurosci, 41(1), 1–7. Hill, R. A., McInnes, K. J., Gong, E. C., Jones, M. E., Simpson, E. R., & Boon, W. C. (2007b). Estrogen deficient male mice develop compulsive behavior. Biological Psychiatry, 61, 359–366. Hill, R. A., Pompolo, S., Jones, M. E.E., Simpson, E. R., & Boon, W. C. (2004). Estrogen deficiency leads to apoptosis in dopaminergic neurons in the medial pre-optic area and

228 arcuate nucleus of male mice. Molecular and Cellular Neuroscience, 27, 466–476. Hill, R. A., Pompolo, S., Jones, M. E., Simpson, E. R., & Boon, W. C. (2004). Estrogen deficiency leads to apoptosis in dopaminergic neurons in the medial preoptic area and arcuate nucleus of male mice. Molecular and Cellular Neuroscience, 27, 466–476. Hodges, Y.K., Tung, L., et al. (2000). Estrogen receptors alpha and beta: prevalence of estrogen receptor beta mRNA in human vascular smooth muscle and transcriptional effects. Circulation, 101(15), 1792–1798. Hojo, Y., Hattori, T. A., Enami, T., Furukawa, A., Suzuki, K., Ishii, H. T., et al. (2004). Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017alpha and P450 aromatase localized in neurons. Proceedings of the National Academy of Sciences of the United States of America, 101, 865–870. Honda, S., Harada, N., Ito, S., Takagi, Y., & Maeda, S. (1998). Disruption of sexual behavior in male aromatasedeficient mice lacking exons 1 and 2 of the cyp19 gene. Biochemical and Biophysical Research Communications, 252, 445–449. Honda, S., Harada, N., & Takagi, Y. (1994). Novel exon 1 of the aromatase gene specific for aromatase transcripts in human brain. Biochemical and Biophysical Research Communications, 198, 1153–1160. Honda, S., Harada, N., & Takagi, Y. (1996). The alternative exons 1 of the mouse aromatase cytochrome P-450 gene. Biochimica et Biophysica Acta, 1305, 145–150. Horvath, T. L., Roa-Pena, L., Jakab, R. L., Simpson, E. R., & Naftolin, F. (1997). Aromatase in axonal processes of early postnatal hypothalamic and limbic areas including the cingulate cortex. The Journal of Steroid Biochemistry and Molecular Biology, 61, 349–357. Hoyland, J.A., Mee, A.P., et al. (1997). Demonstration of estrogen receptor mRNA in bone using in situ reverse-transcriptase polymerase chain reaction. Bone, 20(2), 87–92. Huang, R. & Poduslo, S. E. (2006). CYP19 haplotypes increase risk for Alzheimer’s disease. Journal of Medical Genetics, 43, e42. Hutchison, J. B., Beyer, C., Hutchison, R. E., & Wozniak, A. (1997). Sex differences in the regulation of embryonic brain aromatase. The Journal of Steroid Biochemistry and Molecular Biology, 61, 315–322. Huynh, K., Jones, G., Thouas, G., Britt, K. L., Simpson, E. R., & Jones, M. E. (2004). Estrogen is not directly required for oocyte developmental competence. Biology of Reproduction, 70, 1263–1269. Iivonen, S., Corder, E., Lehtovirta, M., Helisalmi, S., Mannermaa, A., Vepsalainen, S., et al. (2004). Polymorphisms in the CYP19 gene confer increased risk for Alzheimer disease. Neurology, 62, 1170–1176. Ishunina, T. A. & Swaab, D. F. (2003). Increased neuronal metabolic activity and estrogen receptors in the vertical limb of the diagonal band of Broca in Alzheimer’s disease: Relation to sex and aging. Experimental Neurology, 183, 159–172.

Ishunina, T. A., van Beurden, D., van der, M. G., Unmehopa, U. A., Hol, E. M., Huitinga, I., et al. (2005). Diminished aromatase immunoreactivity in the hypothalamus, but not in the basal forebrain nuclei in Alzheimer’s disease. Neurobiology of Aging, 26, 173–194. Jelks, K. B., Wylie, R., Floyd, C. L., McAllister, A. K., & Wise, P. (2007). Estradiol targets synaptic proteins to induce glutamatergic synapse formation in cultured hippocampal neurons: Critical role of estrogen receptor-alpha. Journal of Neuroscience, 27, 6903–6913. Jones, M. E., Boon, W. C., McInnes, K., Maffei, L., Carani, C., & Simpson, E. R. (2007). Recognizing rare disorders: Aromatase deficiency. Nature Clinical Practice Endocrinology and Metabolism, 3, 414–421. Jones, M. E., Thorburn, A. W., Britt, K. L., Hewitt, K. N., Wreford, N. G., Proietto, J., et al. (2000). Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity. Proceedings of the National Academy of Sciences of the United States of America, 97, 12735–12740. Kadioglu, P., Oral, G., Sayitoglu, M., Erensoy, N., Senel, B., Gazioglu, N., et al. (2008). Aromatase cytochrome P450 enzyme expression in human pituitary. Pituitary, 11, 29–35. Kazeto, Y., Ijiri, S., Place, A. R., Zohar, Y., & Trant, J. M. (2001). The 50 -flanking regions of CYP19A1 and CYP19A2 in zebrafish. Biochemical and Biophysical Research Communications, 288, 503–508. Kilgore, M. W., Means, G. D., Mendelson, C. R., & Simpson, E. R. (1992). Alternative promotion of aromatase P-450 expression in the human placenta. Molecular and Cellular Endocrinology, 83, R9–R16. Kimura, M., Sudhir, K., et al. (2003). Impaired acetylcholineinduced release of nitric oxide in the aorta of male aromatase-knockout mice: regulation of nitric oxide production by endogenous sex hormones in males. Circulation Research, 93(12), 1267–1271. Knopp, R. H., Paramsothy, P., Retzlaff, B. M., Fish, B., Walden, C., Dowdy, A., et al. (2006). Sex differences in lipoprotein metabolism and dietary response: Basis in hormonal differences and implications for cardiovascular disease. Current Cardiology Reports, 8, 452–459. Kostanyan, A. & Nazaryan, K. (1992). Rat brain glycolysis regulation by estradiol-17 beta. Biochimica et Biophysica Acta, 1133, 301–306. Kumar, P., Kamat, A., & Mendelson, C. R. (2009). Estrogen receptor alpha (ERalpha) mediates stimulatory effects of estrogen on aromatase (CYP19) gene expression in human placenta. Molecular Endocrinology, 23, 784–793. Lambard, S., Galeraud-Denis, I., Saunders, P. T., & Carreau, S. (2004). Human immature germ cells and ejaculated spermatozoa contain aromatase and oestrogen receptors. Journal of Molecular Endocrinology, 32, 279–289. Lanfranco, F., Zirilli, L., Baldi, M., Pignatti, E., Corneli, G., Ghigo, E., et al. (2008). A novel mutation in the human aromatase gene: Insights on the relationship among serum estradiol, longitudinal growth and bone mineral density in an adult man under estrogen replacement treatment. Bone, 43, 628–635.

229 Larionov, A. A., Vasyliev, D. A., Mason, J. I., Howie, A. F., Berstein, L. M., & Miller, W. R. (2003). Aromatase in skeletal muscle. The Journal of Steroid Biochemistry and Molecular Biology, 84, 485–492. Leav, I., Lau, K.M., et al. (2001). Comparative studies of the estrogen receptors beta and alpha and the androgen receptor in normal human prostate glands, dysplasia, and in primary and metastatic carcinoma. The American Journal of Pathology, 159(1), 79–92. Lecce, G., Meduri, G., et al. (2001). Presence of estrogen receptor beta in the human endometrium through the cycle: expression in glandular, stromal, and vascular cells. Journal of Clinical Endocrinology and Metabolism, 86(3), 1379–1386. Lephart, E. D. (1996). A review of brain aromatase cytochrome P450. Brain Research. Brain Research Reviews, 22, 1–26. Lephart, E. D., Call, S. B., Rhees, R. W., Jacobson, N. A., Weber, K. S., Bledsoe, J., et al. (2001a). Neuroendocrine regulation of sexually dimorphic brain structure and associated sexual behavior in male rats is genetically controlled. Biology of Reproduction, 64, 571–578. Lephart, E. D., Lund, T. D., & Horvath, T. L. (2001b). Brain androgen and progesterone metabolizing enzymes: Biosynthesis, distribution and function. Brain Research. Brain Research Reviews, 37, 25–37. Levin-Allerhand, J., McEwen, B. S., Lominska, C. E., Lubahn, D. B., Korach, K. S., & Smith, J. D. (2001). Brain region-specific up-regulation of mouse apolipoprotein E by pharmacological estrogen treatments. Journal of Neurochemistry, 79, 796–803. Li, C. L., Toda, K., et al. (2002). Estrogen deficiency results in enhanced expression of smoothened of the hedgehog signaling in the thymus and affects thymocyte development. International Immunopharmacology, 2(6), 823–833. Ling, S., Dai, A., et al. (2004). Endogenous estrogen deficiency reduces proliferation and enhances apoptosis-related death in vascular smooth muscle cells: insights from the aromataseknockout mouse. Circulation, 109(4), 537–543. MacGillivray, M. H., Morishima, A., Conte, F., Grumbach, M., & Smith, E. P. (1998). Pediatric endocrinology update: An overview. The essential roles of estrogens in pubertal growth, epiphyseal fusion and bone turnover: Lessons from mutations in the genes for aromatase and the estrogen receptor. Hormone Research, 49(Suppl. 1), 2–8. Mahendroo, M.S., Means, G.D., et al. (1991). Tissue-specific expression of human P-450AROM. The promoter responsible for expression in adipose tissue is different from that utilized in placenta. The Journal of Biological Chemistry, 266(17), 11276–11281. Mahendroo, M. S., Mendelson, C. R., & Simpson, E. R. (1993). Tissue-specific and hormonally controlled alternative promoters regulate aromatase cytochrome P450 gene expression in human adipose tissue. The Journal of Biological Chemistry, 268, 19463–19470. Mahmoodzadeh, S., Fritschka, S., et al. (2009). Nuclear factorkappaB regulates estrogen receptor-alpha transcription in the human heart. The Journal of Biological Chemistry, 284(37), 24705–24714.

Makinen, S., Makela, S., et al. (2001). Localization of oestrogen receptors alpha and beta in human testis. Molecular Reproduction and Development, 7(6), 497–503. Mannella, P. & Brinton, R. D. (2006). Estrogen receptor protein interaction with phosphatidylinositol 3-kinase leads to activation of phosphorylated Akt and extracellular signalregulated kinase 1/2 in the same population of cortical neurons: A unified mechanism of estrogen action. Journal of Neuroscience, 26, 9439–9447. Martin, S., Jones, M., Simpson, E., & van den Buuse, M. (2003). Impaired spatial reference memory in aromatasedeficient (ArKO) mice. NeuroReport, 14, 1979–1982. McCullough, L. D., Blizzard, K., Simpson, E. R., Oz, O. K., & Hurn, P. D. (2003). Aromatase cytochrome P450 and extragonadal estrogen play a role in ischemic neuroprotection. Journal of Neuroscience, 23, 8701–8705. McEwen, B., Akama, K., Alves, S., Brake, W. G., Bulloch, K., Lee, S., et al. (2001). Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation. Proceedings of the National Academy of Sciences of the United States of America, 98, 7093–7100. McNatty, K. P., Baird, D. T., Bolton, A., Chambers, P., Corker, C. S., & McLean, H. (1976). Concentration of oestrogens and androgens in human ovarian venous plasma and follicular fluid throughout the menstrual cycle. Journal of Endocrinology, 71, 77–85. McPherson, S. J., Wang, H., Jones, M. E., Pedersen, J., Iismaa, T. P., Wreford, N., et al. (2001). Elevated androgens and prolactin in aromatase-deficient mice cause enlargement, but not malignancy, of the prostate gland. Endocrinology, 142, 2458–2467. Means, G. D., Mahendroo, M. S., Corbin, C. J., Mathis, J. M., Powell, F. E., Mendelson, C. R., et al. (1989). Structural analysis of the gene encoding human aromatase cytochrome P-450, the enzyme responsible for estrogen biosynthesis. The Journal of Biological Chemistry, 264, 19385–19391. Means, G.D., Kilgore, M.W., et al. (1991). Tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tissues. Molecular Endocrinology, 5(12), 2005–2013. Mendelsohn, M. E. (2009). Estrogen actions in the cardiovascular system. Climacteric, 12(Suppl. 1), 18–21. Miyaura, C., Toda, K., et al. (2001). Sex- and age-related response to aromatase deficiency in bone. Biochemical and Biophysical Research Communications, 280(4), 1062–1068. Michael, M. D., Kilgore, M. W., Morohashi, K., & Simpson, E. R. (1995). Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. The Journal of Biological Chemistry, 270, 13561–13566. Miller, T. W., Shin, I., Kagawa, N., Evans, D. B., Waterman, M. R., & Arteaga, C. L. (2008). Aromatase is phosphorylated in situ at serine-118. The Journal of Steroid Biochemistry and Molecular Biology, 112, 95–101. Misso, M. L., Murata, Y., Boon, W. C., Jones, M. E., Britt, K. L., & Simpson, E. R. (2003). Cellular and molecular

230 characterization of the adipose phenotype of the aromatasedeficient mouse. Endocrinology, 144, 1474–1480. Mitra, S. W., Hoskin, E., Yudkovitz, J., Pear, L., Wilkinson, H. A., Hayashi, S., et al. (2003). Immunolocalization of estrogen receptor beta in the mouse brain: Comparison with estrogen receptor alpha. Endocrinology, 144, 2055–2067. Moore, J.T., McKee, D.D., et al. (1998). Cloning and characterization of human estrogen receptor beta isoforms. Biochemical and Biophysical Research Communications, 247(1), 75–78. Morale, M. C., L’episcopo, F., Tirolo, C., Giaquinta, G., Caniglia, S., Testa, N., et al. (2008). Loss of aromatase cytochrome P450 function as a risk factor for Parkinson’s disease? Brain Research Reviews, 57, 431–443. Morale, M. C., Serra, P. A., L’episcopo, F., Tirolo, C., Caniglia, S., Testa, N., et al. (2006). Estrogen, neuroinflammation and neuroprotection in Parkinson’s disease: Glia dictates resistance versus vulnerability to neurodegeneration. Neuroscience, 138, 869–878. Morishima, A., Grumbach, M. M., Simpson, E. R., Fisher, C., & Qin, K. (1995). Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. Journal of Clinical Endocrinology and Metabolism, 80, 3689–3698. Miyaura, C., K. Toda, et al. (2001). Sex- and age-related response to aromatase deficiency in bone. Biochem Biophys Res Commun, 280(4): 1062–1068. Mulnard, R. A., Cotman, C. W., Kawas, C., van Dyck, C. H., Sano, M., Doody, R., et al. (2000). Estrogen replacement therapy for treatment of mild to moderate alzheimer disease: a randomized controlled trial. Alzheimer’s disease cooperative study. JAMA, 283(8), 1007–1015. Mylonas, I., Jeschke, U., et al. (2004). Immunohistochemical analysis of estrogen receptor alpha, estrogen receptor beta and progesterone receptor in normal human endometrium. Acta Histochemica, 106(3), 245–252. Naftolin, F., Horvath, T. L., & Balthazart, J. (2001). Estrogen synthetase (aromatase) immunohistochemistry reveals concordance between avian and rodent limbic systems and hypothalami. Experimental Biology and Medicine (Maywood), 226, 717–725. Naftolin, F., Horvath, T. L., Jakab, R. L., Leranth, C., Harada, N., & Balthazart, J. (1996). Aromatase immunoreactivity in axon terminals of the vertebrate brain. An immunocytochemical study on quail, rat, monkey and human tissues. Neuroendocrinology, 63, 149–155. Naftolin, F. & MacLusky, N. J. (1982). Aromatase in the central nervous system. Cancer Research, 42, 3274s–3276s. Naftolin, F., Ryan, K. J., Davies, I. J., Reddy, V. V., Flores, F., Petro, Z., et al. (1975). The formation of estrogens by central neuroendocrine tissues. Recent Progress in Hormone Research, 31, 295–319. Nawata, H., Tanaka, S., Tanaka, S., Takayanagi, R., Sakai, Y., Yanase, T., et al. (1995). Aromatase in bone cell: Association with osteoporosis in postmenopausal women. The Journal of Steroid Biochemistry and Molecular Biology, 53, 165–174. Nemoto, Y., Toda, K., Ono, M., Fujikawa-Adachi, K., Saibara, T., Onishi, S., et al. (2000). Altered expression of fatty acid-

metabolizing enzymes in aromatase-deficient mice. The Journal of Clinical Investigation, 105, 1819–1825. Nilsen, J., Irwin, R. W., Gallaher, T. K., & Brinton, R. D. (2007). Estradiol in vivo regulation of brain mitochondrial proteome. Journal of Neuroscience, 27, 14069–14077. Osawa, Y., Higashiyama, T., Fronckowiak, M., Yoshida, N., & Yarborough, C. (1987). Aromatase. The Journal of Steroid Biochemistry, 27, 781–789. Osterlund, M.K., Grandien, K., et al. (2000). The human brain has distinct regional expression patterns of estrogen receptor alpha mRNA isoforms derived from alternative promoters. Journal of Neurochemistry, 75(4), 1390–1397. Oz, O. K., Hirasawa, G., Lawson, J., Nanu, L., Constantinescu, A., Antich, P. P., et al. (2001). Bone phenotype of the aromatase deficient mouse. The Journal of Steroid Biochemistry and Molecular Biology, 79, 49–59. Pasqualini, C., Olivier, V., Guibert, B., Frain, O., & Leviel, V. (1995). Acute stimulatory effect of estradiol on striatal dopamine synthesis. Journal of Neurochemistry, 65, 1651–1657. Pasquali, D., Staibano, S., et al. (2001). Estrogen receptor beta expression in human prostate tissue. Molecular and Cellular Endocrinology, 178(1–2), 47–50. Pedersen, S.B., Bruun, J.M., et al. (2001). Demonstration of estrogen receptor subtypes alpha and beta in human adipose tissue: influences of adipose cell differentiation and fat depot localization. Molecular and Cellular Endocrinology, 182(1), 27–37. Perrot-Sinal, T. S., Davis, A. M., Gregerson, K. A., Kao, J. P., & McCarthy, M. M. (2001). Estradiol enhances excitatory gamma-aminobutyric [corrected] acid-mediated calcium signaling in neonatal hypothalamic neurons. Endocrinology, 142, 2238–2243. Pierman, S., Sica, M., et al. (2008). Activational effects of estradiol and dihydrotestosterone on social recognition and the arginine-vasopressin immunoreactive system in male mice lacking a functional aromatase gene. Hormones and Behavior, 54(1), 98–106. Price, T., Aitken, J., & Simpson, E. R. (1992). Relative expression of aromatase cytochrome P450 in human fetal tissues as determined by competitive polymerase chain reaction amplification. Journal of Clinical Endocrinology and Metabolism, 74, 879–883. Price, T.M. & O‘Brien, S.N. (1993). Determination of estrogen receptor messenger ribonucleic acid (mRNA) and cytochrome P450 aromatase mRNA levels in adipocytes and adipose stromal cells by competitive polymerase chain reaction amplification. Journal of Clinical Endocrinology and Metabolism, 77(4), 1041–1045. Rasgon, N. L., Silverman, D., Siddarth, P., Miller, K., Ercoli, L. M., Elman, S., et al. (2005). Estrogen use and brain metabolic change in postmenopausal women. Neurobiology of Aging, 26, 229–235. Raven, G., de Jong, F. H., Kaufman, J. M., & de Ronde, W. (2006). In men, peripheral estradiol levels directly reflect the action of estrogens at the hypothalamo-pituitary level to inhibit gonadotropin secretion. Journal of Clinical Endocrinology and Metabolism, 91, 3324–3328.

231 Robertson, K. M., O’Donnell, L., Jones, M. E., Meachem, S. J., Boon, W. C., Fisher, C. R., et al. (1999). Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene. Proceedings of the National Academy of Sciences of the United States of America, 96, 7986–7991. Robertson, K. M., Simpson, E. R., Lacham-Kaplan, O., & Jones, M. E. (2001). Characterization of the fertility of male aromatase knockout mice. Journal of Andrology, 22, 825–830. Rochira, V., Zirilli, L., Genazzani, A. D., Balestrieri, A., Aranda, C., Fabre, B., et al. (2006). Hypothalamic-pituitary-gonadal axis in two men with aromatase deficiency: Evidence that circulating estrogens are required at the hypothalamic level for the integrity of gonadotropin negative feedback. European Journal of Endocrinology, 155, 513–522. Roepke, T. A. (2009). Oestrogen modulates hypothalamic control of energy homeostasis through multiple mechanisms. Journal of Neuroendocrinology, 21, 141–150. Romeo, R. D., McCarthy, J. B., Wang, A., Milner, T. A., & McEwen, B. S. (2005). Sex differences in hippocampal estradiol-induced N-methyl-D-aspartic acid binding and ultrastructural localization of estrogen receptor-alpha. Neuroendocrinology, 81, 391–399. Roselli, C. E., Horton, L. E., & Resko, J. A. (1985). Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system. Endocrinology, 117, 2471–2477. Rozovsky, I., Wei, M., Stone, D. J., Zanjani, H., Anderson, C. P., Morgan, T. E., et al. (2002). Estradiol (E2) enhances neurite outgrowth by repressing glial fibrillary acidic protein expression and reorganizing laminin. Endocrinology, 143, 636–646. Rupprecht, R. & Holsboer, F. (1999). Neuroactive steroids: Mechanisms of action and neuropsychopharmacological perspectives. Trends in Neurosciences, 22, 410–416. Sakamoto, H., Mezaki, Y., Shikimi, H., Ukena, K., & Tsutsui, K. (2003). Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell. Endocrinology, 144, 4466–4477. Sanchez, A. M., Flamini, M. I., Fu, X. D., Mannella, P., Giretti, M. S., Goglia, L., et al. (2009). Rapid signaling of estrogen to WAVE1 and moesin controls neuronal spine formation via the actin cytoskeleton. Molecular Endocrinology, 23, 1193– 1202. Sanchez, A. M. & Simoncini, T. (2009). Extra-nuclear signaling of ERalpha to the actin cytoskeleton in the central nervous system. Steroids. (in press). Sanghera, M. K., Simpson, E. R., McPhaul, M. J., Kozlowski, G., Conley, A. J., & Lephart, E. D. (1991). Immunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal antibodies. Endocrinology, 129, 2834–2844. Santen, R. J., Brodie, H., Simpson, E. R., Siiteri, P. K., & Brodie, A. (2009). History of aromatase: Saga of an important biological mediator and therapeutic target. Endocrine Reviews, 30, 343–375. Sarkar, S. N., Huang, R. Q., Logan, S. M., Yi, K. D., Dillon, G. H., & Simpkins, J. W. (2008). Estrogens directly potentiate neuronal L-type Ca2þ channels. Proceedings of the National Academy of Sciences of the United States of America, 105, 15148–15153.

Sasahara, K., Shikimi, H., Haraguchi, S., Sakamoto, H., Honda, S., Harada, N., et al. (2007). Mode of action and functional significance of estrogen-inducing dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell. Journal of Neuroscience, 27, 7408–7417. Sasano, H., Murakami, H., Shizawa, S., Satomi, S., Nagura, H., & Harada, N. (1999). Aromatase and sex steroid receptors in human vena cava. Endocrine Journal, 46, 233–242. Sasano, H., Takashashi, K., Satoh, F., Nagura, H., & Harada, N. (1998). Aromatase in the human central nervous system. Clinical Endocrinology (Oxford), 48, 325–329. Saunders, P.T., Millar, M.R., et al. (2000). Differential expression of estrogen receptor-alpha and -beta and androgen receptor in the ovaries of marmosets and humans. Biology of Reproduction, 63(4), 1098–1105. Savolainen, H., Frosen, J., et al. (2001). Expression of the vasculoprotective estrogen receptor subtype beta in rat and human cardiac allograft vasculopathy. Transplantation Proceedings, 33(1–2), 1605. Schwarz, J. M., Liang, S. L., Thompson, S. M., & McCarthy, M. M. (2008). Estradiol induces hypothalamic dendritic spines by enhancing glutamate release: A mechanism for organizational sex differences. Neuron, 58, 584–598. Sebastian, S., Takayama, K., et al. (2002). Cloning and characterization of a novel endothelial promoter of the human CYP19 (aromatase P450) gene that is up-regulated in breast cancer tissue. Molecular Endocrinology, 16(10), 2243–2254. Seralini, G. E., Tomilin, A., Auvray, P., Nativelle-Serpentini, C., Sourdaine, P., & Moslemi, S. (2003). Molecular characterization and expression of equine testicular cytochrome P450 aromatase. Biochimica et Biophysica Acta, 1625, 229–238. Shapiro, E., Huang, H., et al. (2005a). Immunolocalization of estrogen receptor alpha and beta in human fetal prostate. The Journal of Urology, 174(5), 2051–2053. Shapiro, E., Huang, H., et al. (2005b). Immunolocalization of androgen receptor and estrogen receptors alpha and beta in human fetal testis and epididymis. The Journal of Urology, 174(4 Pt 2), 1695–1698, discussion 1698. Shim, G. J., Warner, M., Kim, H. J., Andersson, S., Liu, L., Ekman, J., et al. (2004). Aromatase-deficient mice spontaneously develop a lymphoproliferative autoimmune disease resembling Sjogren’s syndrome. Proceedings of the National Academy of Sciences of the United States of America, 101, 12628–12633. Shozu, M., Zhao, Y., et al. (1998). Multiple splicing events involved in regulation of human aromatase expression by a novel promoter, I.6. Endocrinology, 139(4), 1610–1617. Simpson, E. R. (2004). Aromatase: Biologic relevance of tissuespecific expression. Seminars in Reproductive Medicine, 22, 11–23. Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Corbin, C. J., & Mendelson, C. R. (1993). Tissuespecific promoters regulate aromatase cytochrome P450 expression. The Journal of Steroid Biochemistry and Molecular Biology, 44, 321–330. Steimer, T. & Hutchison, J. B. (1981). Androgen increases formation of behaviourally effective oestrogen in dove brain. Nature, 292, 345–347.

232 Stockard, C. R. & Papanicolaou, G. N. (1917). A rhythmical “heat period” in the guinea-pig. Science, 46, 42–44. Stoffel-Wagner, B., Watzka, M., Schramm, J., Bidlingmaier, F., & Klingmuller, D. (1999). Expression of CYP19 (aromatase) mRNA in different areas of the human brain. The Journal of Steroid Biochemistry and Molecular Biology, 70, 237–241. Stoffel-Wagner, B., Watzka, M., Steckelbroeck, S., Schwaab, R., Schramm, J., Bidlingmaier, F., et al. (1998). Expression of CYP19 (aromatase) mRNA in the human temporal lobe. Biochemical and Biophysical Research Communications, 244, 768–771. Takeda, K., Toda, K., et al. (2003). Progressive development of insulin resistance phenotype in male mice with complete aromatase (CYP19) deficiency. Journal of Endocrinology, 176(2), 237–246. Toda, K., Saibara, T., Okada, T., Onishi, S., & Shizuta, Y. (2001a). A loss of aggressive behaviour and its reinstatement by oestrogen in mice lacking the aromatase gene (Cyp19). Journal of Endocrinology, 168, 217–220. Toda, K., Simpson, E. R., Mendelson, C. R., Shizuta, Y., & Kilgore, M. W. (1994). Expression of the gene encoding aromatase cytochrome P450 (CYP19) in fetal tissues. Molecular Endocrinology, 8, 210–217. Toda, K., Takeda, K., Akira, S., Saibara, T., Okada, T., Onishi, S., et al. (2001b). Alternations in hepatic expression of fattyacid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator. The Journal of Steroid Biochemistry and Molecular Biology, 79, 11–17. Toda, K., Takeda, K., et al. (2001c). Alternations in hepatic expression of fatty-acid metabolizing enzymes in ArKO mice and their reversal by the treatment with 17beta-estradiol or a peroxisome proliferator. The Journal of Steroid Biochemistry and Molecular Biology, 79(1–5), 11–17. Toda, K., Takeda, K. et al. (2001). Targeted disruption of the aromatase P450 gene (Cyp19) in mice and their ovarian and uterine responses to 17beta-oestradiol. J Endocrinol, 170 (1), 99–111. Toth, B., Saadat, G., et al. (2008). Human umbilical vascular endothelial cells express estrogen receptor beta (ERbeta) and progesterone receptor a (PR-A), but not ERalpha and PR-B. Histochemistry and Cell Biology, 130(2), 399–405. Valverde, M. A., Rojas, P., Amigo, J., Cosmelli, D., Orio, P., Bahamonde, M. I., et al. (1999). Acute activation of Maxi-K channels (hSlo) by estradiol binding to the beta subunit. Science, 285, 1929–1931. van Groen, T. & Kadish, I. (2005). Transgenic AD model mice, effects of potential anti-AD treatments on inflammation and pathology. Brain Research. Brain Research Reviews, 48, 370–378. Vidal, O., Kindblom, L.G., et al. (1999). Expression and localization of estrogen receptor-beta in murine and human bone. Journal of Bone and Mineral Research, 14(6), 923–929. Wang, L., Andersson, S., & Gustafsson, J. A. (2003). Estrogen receptor (ER)beta knockout mice reveal a role for ERbeta in migration of cortical neurons in the developing brain. Proceedings of the National Academy of Sciences of the United States of America, 100, 703–708.

Waters, E. M., Mitterling, K., Spencer, J. L., Mazid, S., McEwen, B. S., & Milner, T. A. (2009a). Estrogen receptor alpha and beta specific agonists regulate expression of synaptic proteins in rat hippocampus. Brain Research, 1290, 1–11. Waters, E. M., Mitterling, K., Spencer, J. L., Mazid, S., McEwen, B. S., & Milner, T. A. (2009b). Estrogen receptor alpha and beta specific agonists regulate expression of synaptic proteins in rat hippocampus. Brain Research, 1290, 1–11. Wetzel, C. H., Hermann, B., Behl, C., Pestel, E., Rammes, G., Zieglgansberger, W., et al. (1998). Functional antagonism of gonadal steroids at the 5-hydroxytryptamine type 3 receptor. Molecular Endocrinology, 12, 1441–1451. Wise, P. M., Dubal, D. B., Wilson, M. E., Rau, S. W., Bottner, M., & Rosewell, K. L. (2001). Estradiol is a protective factor in the adult and aging brain: Understanding of mechanisms derived from in vivo and in vitro studies. Brain Research. Brain Research Reviews, 37, 313–319. Wesson, D.W., Keller, M., et al. (2006). Enhanced urinary odor discrimination in female aromatase knockout (ArKO) mice. Hormones and Behavior, 49(5), 580–586. Woolley, C. S. & McEwen, B. S. (1992). Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat. Journal of Neuroscience, 12, 2549–2554. Xu, H., Gouras, G. K., Greenfield, J. P., Vincent, B., Naslund, J., Mazzarelli, L., et al. (1998). Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides. Nature Medicine, 4, 447–451. Xu, H., Wang, R., Zhang, Y. W., & Zhang, X. (2006). Estrogen, beta-amyloid metabolism/trafficking, and Alzheimer’s disease. Annals of the New York Academy of Sciences, 1089, 324–342. Yague, J. G., Munoz, A., Monasterio-Schrader, P., Defelipe, J., Garcia-Segura, L. M., & Azcoitia, I. (2006). Aromatase expression in the human temporal cortex. Neuroscience, 138, 389–401. Yan, M., Jones, M. E., Hernandez, M., Liu, D., Simpson, E. R., & Chen, C. (2005). Oestrogen replacement in vivo rescues the dysfunction of pituitary somatotropes in ovariectomised aromatase knockout mice. Neuroendocrinology, 81, 158–166. Youn, C. S., Kwon, O. S., Won, C. H., Hwang, E. J., Park, B. J., Eun, H. C., et al. (2003). Effect of pregnancy and menopause on facial wrinkling in women. Acta Dermato-Venereologica, 83, 419–424. Yue, X., Lu, M., Lancaster, T., Cao, P., Honda, S., Staufenbiel, M., et al. (2005). Brain estrogen deficiency accelerates Ab plaque formation in an Alzheimer’s disease animal model. Proceedings of the National Academy of Sciences of the United States of America, 102, 19198–19203. Znamensky, V., Akama, K. T., McEwen, B. S., & Milner, T. A. (2003). Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites. Journal of Neuroscience, 23, 2340–2347. Zhao, Y., Mendelson, C. R. et al. (1995). Characterization of the sequences of the human CYP19 (aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes. Molecular Endocrinology, 9(3), 340–349.