Aromatase Knockout Mice Research Articles

Diminished Contracture and Improved Post-Ischemic Functional Recovery in the Aromatase Knockout Mouse Heart

Diminished Contracture and Improved Post-Ischemic Functional Recovery in the Aromatase Knockout Mouse Heart

The importance of ERβ signalling in the ovary

This review examines the evidence for a central role of oestrogen receptor beta (ERbeta or ESR2 as listed in the MGI Database) in folliculogenesis and hence reproductive biology. Knockout mouse models have been a valuable resource in this respect. The ERbeta-null mouse exhibits a granulosa cell phenotype associated with the partial arrest of folliculogenesis and ovulatory dysfunction. Phyto-oestrogens such as genistein, which preferentially activate ERbeta, have been shown to alleviate the ovarian phenotype of the oestrogen-depleted aromatase knockout mouse. In normal adult mice, genistein has been shown to cause reproductive defectives following neonatal administration. Studies of ovarian cancer have also informed the literature. A decline in ERbeta levels in epithelial ovarian cancers has been hypothesised to be associated with severity of disease and prognosis. Whereas the abundant expression of ERbeta in granulosa cell tumours (GCT) of the ovary and evidence that ERbeta signalling is transrepressed by the nuclear factor-kappaB pathway in GCT cell lines suggest a pathogenetic role for ERbeta in GCT. In recent years, studies into the impact of environmental oestrogens (either in the form of pesticides or plastics) on reproductive function have shown that ERbeta-selective toxins cause reproductive dysfunction and impair fertility. It remains to be established as to what genes are regulated by ERbeta in the ovary. Finally, ERbeta has been shown to be regulated by gonadotrophins, the pituitary hormones mediating ovarian function.

Effects of genistein and estrogen receptor subtype-specific agonists in ArKO mice following different administration routes

We have scrutinized the effects of the phytoestrogen genistein and three synthetic estrogen receptor agonists, 17α-ethynylestradiol (EE), propylpyrazole-triol (PPT) and diarylpropionitrile (DPN) in the completely estrogen-free background of aromatase knockout (ArKO) mice by means of two routes of substance administration: oral via diet ( per os; po) or subcutaneous injection (sc) with the intention to evaluate the ArKO mice as sensitive model organism for uterotrophic assays. Additionally, we were aiming to qualitatively analyze effects resulting from oral administration path, in particular for PPT and DPN. Therefore, we analyzed the resulting uterine wet weights (UWW) and epithelial heights as physiological endpoints of function as well as the gonadotropin levels. Moreover, the gene expression profiles of estrogen receptors as well as important uterine and ovarian estrogen-response genes were investigated by real-time PCR. The uterus of ArKO mice responded very sensitive upon the substitution with EE (sc 5 μg/kg BW; po 50 μg/kg BW) in a proliferative manner. This was evaluated inter alia by increased UWW and by up-regulation of the expression of proliferation-associated and estrogen-response genes. It is important to note, that ERα and ERβ-agonist, PPT and DPN respectively (po 5 mg/kg BW and sc 0.5 mg/kg BW), have only been used for sc applications so far. Here, effects resulting from oral application were qualitatively described and evaluated for their applicability. The UWW and expression of proliferation-associated genes were increased following both po and sc treatment with PPT. In contrast, DPN did not exert an increase of the UWW, but a significant decrease of proliferation-associated gene and protein expression. Additionally, a substantial hypoplasia was detectable in the uterine cross-sections of DPN-treated mice. On the other hand, the phytoestrogen genistein (sc 10 mg/kg BW; po 70 mg/kg BW) did not cause detectable uterotrophic responses or large changes of uterine and ovarian gene expression profiles under the applied experimental conditions, but significantly reduced the elevated gonadotropin levels of ArKO mice. In summary, we showed the utility of ArKO mice to detect ER-specific effects, in particular those of PPT and DPN also when applied orally.

Retention of the Radiotracers 64Cu-ATSM and 64Cu-PTSM in Human and Murine Tumors Is Influenced by MDR1 Protein Expression

Tumor hypoxia is often associated with resistance to chemotherapy. Multidrug resistance type 1 (MDR1) protein is a member of the adenosine triphosphate binding cassette (ABC) proteins, some of which are involved in the multidrug resistance (MDR) phenotype in tumors. Many studies have focused on the role of these proteins in modulating drug resistance, but their effect on retention of imaging agents is less well studied. To study the role of MDR1 expression on the accumulation of (64)Cu-diacetyl-bis(N4-methylthiosemicarbazone) ((64)Cu-ATSM) and (64)Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ((64)Cu-PTSM) in human tumors in vitro and in vivo, we used a model system composed of a low MDR1-expressing parent uterine sarcoma cell line and a daughter cell line selected for overexpression of MDR1. Aromatase knockout (ArKO) mice that spontaneously developed liver tumors were used as an additional in vivo model to study the effect of MDR expression on (64)Cu-ATSM and -PTSM retention. Biodistribution experiments after injection of (64)Cu-ATSM or -PTSM were performed in wild-type mice, ArKO mice, and ArKO mice bearing liver tumors (n = 3-5/group), and in nude mice bearing human tumor xenografts for in vivo PET/CT. Liver expression of Abcb1a and Abcb1b, the MDR1 proteins in mouse liver, was determined by real-time polymerase chain reaction. (64)Cu-ATSM and -PTSM accumulation and efflux studies were conducted in tumor cell lines. The uptake experiments were repeated after knockdown of MDR1 protein expression using MDR1-specific small interfering RNAs. In vivo, the hepatic tumors had a lower percentage injected dose per gram of (64)Cu-ATSM or -PTSM and more highly expressed Abcb1b than did wild-type liver or nontumor-bearing ArKO liver. High MDR1-expressing tumors showed lower tracer activity on PET/CT images. In vitro, cells highly expressing MDR1 had significantly decreased (64)Cu-ATSM and -PTSM retention and enhanced efflux. Knockdown of MDR1 expression significantly enhanced the (64)Cu-ATSM and -PTSM retention and decreased the efflux in MDR1-positive cells. The expression of MDR1 glycoprotein (or its equivalents in mice) affects the retention of (64)Cu-ATSM and -PTSM in the human and murine tumors tested. These results may have implications for clinical hypoxia imaging in tumors and the therapeutic efficacy of (64)Cu-ATSM.

Psychotropic drug-induced locomotor hyperactivity and prepulse inhibition regulation in male and female aromatase knockout (ArKO) mice: role of dopamine D1 and D2 receptors and dopamine transporters

The aim of the present study was to investigate the possible role of oestrogen in schizophrenia by comparing aromatase knockout (ArKO) mice, which are unable to produce oestrogen, with wild-type controls using two behavioural animal models with relevance to the illness, psychotropic drug-induced locomotor hyperactivity and prepulse inhibition (PPI). Baseline PPI was not different between ArKO and controls. Treatment with apomorphine, MK-801 and amphetamine caused disruption of PPI in all groups. However, in female but not male ArKO mice, the effect of both apomorphine and amphetamine was reduced. In female ArKO mice, amphetamine-induced hyperlocomotion was markedly reduced, but in male mice, the genotype difference was far smaller. Female but not male ArKO mice also showed a reduction of phencyclidine-induced locomotor hyperactivity. The density of dopamine transporters, but not D1 and D2 receptors, was significantly increased in the caudate putamen of male but not female ArKO mice compared to wild-type mice. This could represent a compensatory dopaminergic upregulation in male ArKO mice. Because of their lack of oestrogen production, it was anticipated that ArKO mice would display enhanced effects of amphetamine on locomotor activity and PPI. Instead, in these animals, aromatase knockout appeared to be 'protective'. This may represent limitations in the ability to model a complex illness such as schizophrenia in a constitutive knockout model, such as ArKO mice. Moreover, the current results may point at the involvement of other sex steroids, which are also altered in ArKO mice, in dopaminergic control of behaviour.

The estrogenic component of tibolone reduces adiposity in female aromatase knockout mice

To explore the effects of tibolone on adiposity in the absence of aromatase and determine which of the hormonal properties of tibolone are exerting these effects. In this study, vehicle; tibolone; estrogenic (ethinyl estradiol [EE]), progestogenic (ORG2058), or androgenic (dihydrotestosterone) compounds; or a combination of ORG2058 + EE was administered to 6-month-old ovariectomized aromatase knockout (ArKO) mice for a period of 6 weeks. In response to tibolone or EE-alone treatments, omental adipose tissue and infrarenal adipose tissue weights were significantly reduced (P = 0.004 and P = 0.01; P = 0.009 and P = 0.014, respectively) compared with those in ovariectomized and vehicle-treated ArKO mice. In contrast, adipose tissue weight tended to increase after ORG2058-alone treatment. Furthermore, EE in the presence of ORG2058 (ORG2058 + EE group) results in little effect on adiposity when compared with that in ovariectomized and vehicle-treated ArKO mice, showing that ORG2058 can negate the effect of EE. Dihydrotestosterone treatment did not have an impact on adipose tissue mass. Adipocyte volume and numbers followed the same treatment trends. In summary, our study in the ArKO mouse has confirmed the efficacy of tibolone as a hormone therapy to reduce adipose tissue accumulation after menopause and also shows that aromatization of tibolone is not required to elicit these estrogenic effects.

Regarding “Prostatic hormonal carcinogenesis is mediated by in situ estrogen production and estrogen receptor alpha signaling”

It was recently demonstrated that antiestrogens prevented prostate cancer (PRCA) in men. The source of estradiol (E2) that contributes to carcinogenesis, as well as the selected estrogen receptor (ER) signaling pathway, is unknown. To evaluate estrogen's effects in carcinogenesis, we developed a new model of PRCA utilizing testosterone and E2 to stimulate PRCA. To determine whether local in situ production of E2 affected incidence of PRCA, aromatase-knockout (ArKO) mice were evaluated. In contrast to the wild-type mice, ArKO mice had reduced incidences of PRCA, which implicates in situ production of E2 as an important determinant of PRCA. To determine whether E2-mediated responses were due to ER alpha or ER beta signaling, ER alpha-knockout (alphaERKO) or ERbeta-knockout (betaERKO) mice were used. Prostates from betaERKO mice underwent biochemical and histological carcinogenesis similar to wild-type mice, whereas prostates from alphaERKO mice remained free of pathology. These data suggest that effective prevention of carcinogenesis will require antagonism of ER alpha but not ER beta. This mouse model provides a means to examine genetic gain and loss of function and determine the efficacy of therapeutics on prostatic carcinogenesis.

Estrogens, Brain, and Behavior: Lessons from Knockout Mouse Models

The use of animal models to effectively replicate problems such as hormone deficiencies, neurologic diseases, and brain injury and stroke has certainly made a vast contribution to understanding the neuroprotective effects of estrogen in the brain. Studies using gonadectomy procedures followed by 17beta-estradiol replacement have effectively demonstrated the positive effects that estrogen provides in cognitive performance and memory performance tasks. A major problem with such studies is that local brain aromatase (the estrogen-synthesizing enzyme) may still convert locally produced androgens to estrogens. Hence, such "estrogen-deficient" models may not be completely void of estrogen. The generation of the aromatase knockout (ArKO) and estrogen receptor knockout (ERKO) mouse models has enabled researchers to characterize the effects of complete estrogen deficiency within the brain and hence behavior. This review aims to compare and contrast the results of these various mouse models.

Functional Analysis of Neurosteroidal Oestrogen Using Gene‐Disrupted and Transgenic Mice

The brain aromatase (oestrogen synthase) hypothesis predicts that oestrogen plays important roles in both sexual behaviours and brain sexual differentiation. To elucidate the functions of oestrogen in the brain, we generated aromatase knockout (ArKO) mice, which showed undetectable oestrogen and enhanced androgen levels in blood. These ArKO mice exhibited an enhanced appetite and disorders in sexual motivation, sexual partnership preference, sexual performance, aggressive behaviour, parental behaviour, infanticide behaviour and exploratory (anxiety) behaviour. We characterised the brain-specific promoter of the mouse aromatase gene, and identified several crucial cis-acting elements and the minimal essential promoter region for brain-specific expression. Next, we introduced a transgene of human aromatase, controlled by the minimal promoter region, into the ArKO mouse. The resulting mouse (ArKO/hArom), whose preoptic area, hypothalamus and amygdala were exposed to oestrogens only in the perinatal period, and then to enhanced androgens and no oestrogens in adulthood, showed near recovery from behavioural disorders. These results suggest that local oestrogens acting in specific brain regions are involved in the organisation of sex-specific neural networks during the perinatal period. Finally, we examined effects of oestrogens on gene expression within specific brain regions in mice during the perinatal period using DNA microarray analysis. This assay revealed both up-regulated and down-regulated brain-specific genes, including those related to neuronal function. Specifically, genes involved in energy metabolism, cell proliferation/apoptosis and secretory/transport system were altered in ArKO mice compared to wild mice. These results suggest that brain oestrogens participate in the sexual differentiation of the brain by influencing gene expression.

Postnatal Development of an Estradiol-Kisspeptin Positive Feedback Mechanism Implicated in Puberty Onset

The regulation of GnRH neurons by kisspeptin is critical for normal puberty onset in mammals. In the rodent the kisspeptin neurons innervating GnRH neurons are thought to reside in the rostral periventricular area of the third ventricle (RP3V). Using kisspeptin immunocytochemistry we show that kisspeptin peptide expression in the RP3V of female mice begins around postnatal d 15 (P15) and rapidly increases to achieve adult-like levels by P30, the time of puberty onset. Ovariectomy of female pups at P15 resulted in a 70-90% reduction (P < 0.01) in kisspeptin peptide expression within the RP3V of P30 or P60 mice. Replacement of 17-beta-estradiol (E2) in P15-ovariectomized mice from P15-30 or P22-30 resulted in a complete restoration of kisspeptin peptide expression in the RP3V (P < 0.01). Kisspeptin-immunoreactive fibers throughout the hypothalamus, including the arcuate nucleus, followed the same pattern of estrogen-dependent expression. To test the absolute necessity of estrogen for kisspeptin expression in the RP3V, aromatase knockout mice were examined. Kisspeptin-immunoreactive cells were detected in the arcuate nucleus, but there was a complete absence of kisspeptin peptide in RP3V neurons of aromatase knockout adult females. These results demonstrate that E2 is essential for the prepubertal development of kisspeptin peptide within RP3V neurons and suggest that an E2-kisspeptin positive feedback mechanism exists before puberty. This implies that RP3V kisspeptin neurons are E2-dependent amplifiers of GnRH neuron activity in the prepubertal period.

Regulation of progestin receptors in medial amygdala: Estradiol, phytoestrogens and sex

Exposure to estrogens during critical developmental periods and in adulthood affects sex differences in the brain. We examined the roles of estradiol (E2) and phytoestrogens, and their interactions, on potential sex differences in brain. We used aromatase knockout (ArKO) mice, which cannot produce endogenous estrogens, along with wild type (WT) littermates. Mice were gestated, raised and maintained on a diet either rich in phytoestrogens or a diet virtually void of soy-derived phytoestrogens. Adult males and females were gonadectomized and received implants filled with 17-β-estradiol to induce progestin receptors (PR), while controls received empty implants. Mice were sacrificed five days later and brain sections containing the posterodorsal medial amygdala (MePD) were processed for PR immunoreactivity. Activation of sex differences in PR required adult E2 treatment. A diet high in phytoestrogens was required for expression of sex differences in PR after E2 treatment. Our data underscore the important contribution of dietary phytoestrogens for the development of sex differences in PR-ir in the adult mouse medial amygdala. We hypothesize that both aromatization of androgens to estrogens and dietary sources of additional estrogens are part of the normal requirement for sex differences in the rodent brain.

Estrogen receptor-α and -β and aromatase knockout effects on lower limb muscle mass and contractile function in female mice

Estrogen (E2) is reported to regulate skeletal muscle mass and contractile function; whether E2 exerts its effects through estrogen receptor-alpha (ERalpha) or -beta (ERbeta) is unclear. We determined the effect of ERalpha or ERbeta elimination on muscle mass and contractile function in multiple muscles of the lower limb, muscles with different locomotor tasks and proportions of fiber types I and II: soleus (Sol), plantaris (Plan), tibialis anterior (TA), and gastrocnemius (Gast) in mature female mice. To determine E2 elimination effects on muscle, we also used aromatase (Ar) knockout (KO) and wild-type (WT) mice. ERalpha and ArKO body weights were approximately 10 and 20% higher than WT. Although muscle mass tended to show a commensurate increase in both groups, only the TA was significantly larger in ERalpha (P<0.05). Ratios of muscle mass to body mass revealed significantly lower values for Gast and TA in ArKO mice (P<0.05). Tetanic tension (Po) per calculated anatomical cross-sectional area (aCSA) in ERalpha KO was lower in TA and Gast than in WT. Lower Po/aCSA in ERalpha KO Gast and TA was also supported histologically by significantly less Po/fiber areas (P<0.05). ArKO mice also had lower Po/aCSA in Gast and TA compared with WT. ERbeta KO and WT mice were comparable in all measures. Our results support the hypothesis that E2 effects on skeletal muscle are mediated in part via the ERalpha but that E2 effects may be mediated via more than one mechanism or receptor.

Estrogen deficiency results in apoptosis in the frontal cortex of adult female aromatase knockout mice

The aromatase knockout (ArKO) mouse is completely estrogen deficient. We previously detected apoptosis in the hypothalamus of 1 year-old male ArKO mice. This study shows that 12 week-old female ArKO mice display spontaneous apoptosis of pyramidal neurons in the frontal cortex while wild-type (WT) littermates show no signs of apoptosis. Concomitantly, bcl-2 related anti-apoptotic genes are down-regulated whereas the pro-apoptotic gene TRADD is up-regulated in the female ArKO frontal cortex. This phenotype can be rescued by 3-week replacement of 17β-estradiol. Furthermore, the apoptosis phenotype is exacerbated in 12–15 month-old female ArKO mice, which have 30% less neurons in the frontal cortex and lower brain weights than WT counterparts. These data show that estrogens are essential for the survival of female cortical neurons even in the absence of pathological conditions or external assaults. Our observations also demonstrate the sexually dimorphic susceptibility of neurons to estrogen deficiency.

Age-Dependent Effects of Testosterone in Experimental Stroke

Although male sex is a well-recognized risk factor for stroke, the role of androgens in cerebral ischemia remains unclear. Therefore, we evaluated effects of testosterone on infarct size in both young adult and middle-aged rats (Wistar, 3-month versus 14-month old) and mice (C57/BL6, 3-month versus 12-month old) subjected to middle cerebral artery occlusion. In young adult groups, castrates displayed less ischemic damage as compared with intact males and castrates with testosterone replacement (Cortex: 24% in castrates versus 42% in intact versus 40% with testosterone; Striatum: 45% versus 73% versus 70%) at 22 h reperfusion. Surprisingly, supplementing testosterone in middle-aged rats to the physiologic levels ordinarily seen in young males reduced infarction (Cortex: 2% with testosterone versus 31%; Striatum: 38% with testosterone versus 68%). Testosterone effects on infarct size were blocked by the androgen receptor (AR) antagonist flutamide and further confirmed in young versus middle-aged mice. Baseline cerebral aromatase mRNA levels and activity were not different between young and middle-aged rats. Aromatase activity increased in ischemic tissue, but only in young males. Lastly, stroke damage was not different in aging aromatase knockout mice versus wild-type controls. Our findings indicate that testosterone's effects in experimental stroke are age dependent, mediated via AR, but not cerebral aromatase.

The Novel Estrogen Receptor, G Protein-Coupled Receptor 30, Mediates the Proliferative Effects Induced by 17β-Estradiol on Mouse Spermatogonial GC-1 Cell Line

Many studies have indicated that estrogens could have a role in the regulation of testicular function. However, it remains uncertain whether estrogens are able to directly activate signaling pathways in male germ cells. Estrogens are synthesized by the enzyme aromatase and classically act by binding to estrogen receptors (ERs)-alpha and ERbeta. Knockout mice for both receptor isoforms exhibit a testicular phenotype that is less severe than aromatase knockout mice, suggesting the existence of an estrogen-binding receptor that may compensate for the lack of ERs. Recently studies using estrogen-sensitive tumor cell lines have demonstrated that the G-protein-coupled receptor (GPR)-30 binds and mediates estrogen action through the activation of the epidermal growth factor receptor (EGFR)/ERK/fos transduction pathway. The present study investigated the ability of 17beta-estradiol (E2) to activate this pathway in the mouse spermatogonial cell line (GC-1). Using the GC-1 cell line as a model system, we demonstrated that GC-1 cells express GPR30 and ERalpha but not ERbeta. E2, the selective GPR30 agonist G1, and the selective ERalpha agonist 4,4',4''-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol activated the rapid ERK1/2-fos signaling cascade. This response was abrogated by the EGFR inhibitor AG1478, ERK inhibitor PD98059 and ER inhibitor ICI 182780, or by silencing GPR30 expression. Moreover, E2 and G1 up-regulated cyclin D1 expression and GC-1 cell proliferation. Our results indicate for the first time that estrogens, through a cross talk between GPR30 and ERalpha, activate the rapid EGFR/ERK/fos pathway, which in turn stimulate mouse GC-1 cell proliferation. Further studies to elucidate the involvement of rapid estrogen signaling pathways in the regulation of male fertility are warranted.

A novel method for assessing sex-specific and genotype-specific response to injury in astrocyte culture

Female astrocytes sustain less cell death from oxygen–glucose deprivation (OGD) than male astrocytes. Arimidex, an aromatase inhibitor, abolishes these sex differences. To verify sex-dependent differences in P450 aromatase function in astrocyte cell death following OGD, we developed a novel method that uses sex-specific and genotype-specific single pup primary astrocyte cultures from wild-type (WT) and aromatase-knockout (ArKO) mice. After determining sex by external and internal examination as well as PCR and genotype by PCR amplification of tail cDNA, we established cultures from 1–3-day-old male and female WT and ArKO mice pups and grew them to confluence in estrogen-free media. Cell death was measured by lactate dehydrogenase (LDH) assay. Our study shows that, while WT female astrocytes are more resistant to OGD than WT male cells, sex differences disappear in ArKO cells. Cell death is significantly increased in ArKO compared to WT in female astrocytes but not male cells. Therefore, P450 aromatase appears to be essential in endogenous neuroprotection in females, and this finding may have clinical implications. This innovative technique may also be applied to other in vitro studies of sex-related functional differences.

Estrogen receptor (ER) α and β and Aromatase knock‐out (ArKO) effects on muscle development in male mice

ERs have been identified in male muscle but whether estrogen (E2) exerts its effects through ERα or ERβ is unclear. Direct E2 effects on developing muscle were also determined in ArKO. All comparisons were made to corresponding wild type (WT) controls. Muscle mass, particularly in fast‐fibered gastrocnemius (Gast) and Tibialis Anterior (TA) (not soleus or plantaris) was diminished in ERα but not ERβ. Most absolute mass measures, and 3 of 4 muscles normalized to body weight were significantly lower in ArKO mice compared with WT. Normalized muscle mass/body weight was still lower in ERAα Gast and TA but not in the ERβ group. There was no difference between WT and KO mice in peak force, force/area, force/velocity, time to peak tension or ½ relaxation time. Total protein and contractile protein myosin in Gast and TA were similar in WT and KO, further indication that contractile function was not affected by KO status. Results suggest ERαKO and ArKO may directly regulate muscle growth without affecting contractile function. The presence or absence of ERβ does not alter skeletal muscle function in male mice. Estrogen may be required for complete muscle size during development.Support: Spinal Injuries Board, Research Board

Activational effects of estradiol and dihydrotestosterone on social recognition and the arginine-vasopressin immunoreactive system in male mice lacking a functional aromatase gene

In rodents, parts of the arginine-vasopressin (AVP) neuronal system are sexually dimorphic with males having more AVP-immunoreactive cells/fibers than females. This neuropeptide neuronal system is highly sensitive to steroids and has been proposed to play an important role in the processing of olfactory cues critical to the establishment of a social memory. We demonstrate here that gonadally intact male aromatase knockout (ArKO) mice, which cannot aromatize androgens into estrogens due to a targeted mutation in the aromatase gene, showed severe deficits in social recognition as well as a reduced AVP-immunoreactivity in several brain regions. To determine whether this reduction is due to a lack of organizational or activational effects of estrogens, we assessed social recognition abilities and AVP-immunoreactivity in male ArKO and wild-type (WT) mice when treated with estradiol benzoate (EB) in association with dihydrotestosterone propionate (DHTP) in adulthood. Adult treatment with EB and DHTP restored social recognition abilities in castrated ArKO males since they showed normal female-oriented ultrasonic vocalizations and were able to recognize an unfamiliar female using a habituation–dishabituation paradigm. Furthermore, adult treatment also restored AVP-immunoreactivity in the lateral septum of ArKO males to levels observed in intact WT males. These results suggest that social recognition in adulthood and stimulation of AVP expression in the adult mouse forebrain depend predominantly on the estrogenic metabolite of testosterone. Furthermore, our results are in line with the idea that the organization of the AVP system may depend on androgen or sex chromosomes rather than estrogens.

Evidence for the existence of an estrogen-responsive sexually dimorphic group of cells in the medial preoptic area of the 129SvEv mouse strain

The current study describes the presence of sexually dimorphic cell groups within the MPO of the 129SvEv, but not the C57BL/6J strain of mice. We detected galanin-positive clusters of cells located in the MPO of 129SvEv and C57BL/6J mice, with sex differences found only in the 129SvEv strain. Aromatase-positive and dense Nissl-counterstained clusters of cells were identified only in the MPO of 129SvEv mice, but not in the C57BL/6J strain. Furthermore, this study has demonstrated that the volume of these sexually dimorphic cell groups is regulated by estrogen, but not testosterone, as male aromatase knockout mice (which have high levels of testosterone, but no estrogen) show reduced cell group volumes. These structural changes, which occur in an estrogen-deficient state may influence male sexual behaviors.

255. Hormonal manipulation on the phenotype of ArKO female mice

Gonadotrophins and steroid hormones are vital in controlling the cyclical pattern of ovarian follicular development essential for fertility. Previous studies have shown that ArKO (aromatase knockout) female mice are infertile due to the absence of oestrogen, elevated levels of circulating gonadotrophins and testosterone and folliculogenic disruption. Therefore, the aim of this study was to determine the effects of E2 (oestradiol-17β) replacement, Acyline (GnRH antagonist) and Flutamide (anti-androgen) treatment on ArKO female mice. WT and ArKO female mice (C57B6/J129; 16 weeks old; n = 6–8/grp) were assigned into three main groups: group 1 - received either E2 (0.05 mg) pellet or placebo, group 2 - received either a single s.c. injection of acyline (1.5 mg/kg/week) or placebo and group 3 – received either flutamide (25 mg) pellet or placebo for 3 weeks. Mice were subjected to daily vaginal smears. The ovaries and uterine horns were collected and weighed. One ovary and the uterine horns were fixed in formalin for histological assessment, while the other ovary was snap frozen in Ultraspec solution for RNA isolation and gene expression studies. Serum was collected for hormone measurements. All female ArKO mice exhibited an abnormal cycle that alternated between diestrus and early oestrus. E2 replacement restored the oestrus cycle in ArKO female mice but acyline and flutamide treatment did not. Histologically, hemorrhagic cystic follicles were present in all placebo, acyline and flutamide treated ArKO ovaries, however, E2 replacement improved the ovarian and uterine phenotypes. E2 replacement and acyline treatment also led to a decrease in serum gonadotropin levels in ArKO mice. In summary, E2 replacement could reverse the abnormal reproductive phenotype of the ArKO female mice. This study suggests that the reproductive phenotype of the ArKO female mouse is due to the direct effect of oestrogen and not due to the elevated circulating levels of gonadotrophins and testosterone. Supported by NH&amp;MRC (Regkeys 241000, 338510 and 198705)