Gonadectomized Mice Research Articles

Effect of estrogen upon methamphetamine-induced neurotoxicity within the impaired nigrostriatal dopaminergic system

In the present study, we investigated whether estrogen remains effective as a neuroprotectant within an impaired nigrostriatal dopaminergic (NSDA) system of gonadectomized female and male mice. In Experiment 1, mice were treated with four different regimens of methamphetamine (MA) to establish a protocol for an impaired NSDA system to be used in subsequent experiments. Based upon the results of Experiment 1, in Experiment 2 gonadectomized female mice received a treatment with either control (saline), low- or high-dose of MA to produce an initial NSDA impairment. At one week post-MA, mice received either estradiol benzoate (10 microg) or vehicle followed 24 h later with low-MA or saline. Estrogen altered the toxic effects of the second invasion of MA as indicated by a significant decrease in striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations. In addition, DA and DOPAC depletion was greater in high- vs. low-dose MA. In gonadectomized male mice (Experiment 3), striatal DA and DOPAC concentrations showed greater decreases following high-, vs. low-doses of MA; however, estrogen did not alter these responses. These results demonstrate that the capacity for estrogen to protect or worsen MA-induced neurotoxicity of dopaminergic neurons is limited to female mice and depends on the condition of the NSDA system.

Biological Responses of Antiprogestins in Mammary Gland, Uterus and Seminal Vesicles of Prepubertal Intact and Adult Gonadectomized Mice

The present study tested and compared the antiproliferative and proliferative activities of three antiprogestins on four separate mouse model systems: prepubertal intact and adult ovariectomized (OVX) females, prepubertal intact and adult castrated males. In prepubertal intact males and females and adult castrated males, norethindrone acetate (NA; a synthetic steroid exhibiting progestational and estrogenic activities)- stimulated mammary growth was decreased by antiprogestins: more by RU 46556 (RU) than RU 38486 (MI) and onapristone (ON). In adult OVX females the inhibitory effect of RU and MI was lower than that of ON. Uterine weights were not significantly decreased by MI, were significantly decreased by RU at a lower daily dose (50 μg) but not affected by a high (500 μg) dose. Seminal vesicle weights were increased by RU but not affected by MI in both NA-treated prepubertal and adult castrated males. In adult castrated but not in prepubertal males ON decreased seminal vesicle weights. In 17β-estradiol (E) plus progesterone (Prog)-treated animals of all four model systems, RU (100 μg/d) acted additively with a submaximal daily dose (10 μg) of antiestrogen ICI 182, 780 (ICI) to produce a lower mammary gland growth rate than ICI alone. In uterus, however, no significant effect of a combination of ICI plus RU was noted when compared with ICI alone. In general, our assay could serve as an in vivo tool for the detection of steroid hormone agonist and antagonist activities of newly synthesized analogs of steroid hormones, and natural and man-made chemicals and extracts of environmental samples.

Sexually dimorphic expression of Usp9x is related to sex chromosome complement in adult mouse brain

We found the expression of Usp9x, an X-linked gene which encodes a ubiquitin protease implicated in synaptic development, to be significantly higher in the adult female mouse brains than in male brains. The sex difference in expression of Usp9x was localized to specific brain regions such as neocortex. Furthermore, in gonadally intact and gonadectomized mice, XX mice expressed Usp9x mRNA and protein more highly than XY mice irrespective of their gonadal type. No sex difference was found in the neonatal brain or peripheral tissues such as the adult kidney. This finding implies that the difference in sex chromosome complement between XY males and XX females could potentially contribute to sexual differentiation of brain structure and function. The relation of genomic dose and Usp9x expression could help explain the neural and behavioural phenotype of women with XO Turner syndrome.

Gonadotropin-Induced Adrenocortical Neoplasia in NU/J Nude Mice

In response to prepubertal gonadectomy certain inbred mouse strains, including DBA/2J, develop sex steroid-producing adrenocortical neoplasms. This phenomenon has been attributed to a lack of gonadal hormones or a compensatory increase in gonadotropins. To assess the relative importance of these mechanisms, we created a new inbred model of adrenocortical neoplasia using female NU/J nude mice. These mice developed adrenocortical neoplasms in response to either gonadectomy or gonadotropin elevation from xenografts of human chorionic gonadotropin (hCG)-secreting Chinese hamster ovary cells. In each instance the adrenal tumors resembled the neoplasms found in gonadectomized DBA/2J mice and were composed of spindle-shaped A cells and lipid-laden B cells. Both cell populations were defined by ectopic expression of GATA-4 and an absence of the adrenocortical markers melanocortin-2-receptor and steroid 21-hydroxylase, but only B cells expressed the gonadal steroidogenic markers inhibin-alpha, LH receptor, P450c17, and P450c19. Expression of sex steroidogenic markers was attenuated in the neoplastic adrenal cortex of hCG-treated vs. gonadectomized mice. Whereas neoplastic adrenals were an obvious source of estradiol in gonadectomized mice, ovaries appeared to be the major source of this hormone in hCG-treated mice. Gonadectomy and hCG treatment elicited comparable increases in serum estradiol, but testosterone levels increased significantly only in hCG-treated mice. We conclude that chronic gonadotropin elevation, caused by either gonadectomy or hCG administration, signals a population of cells in the adrenal subcapsular region of permissive mice to undergo differentiation along a gonadal rather than an adrenal lineage. Thus, NU/J nude mice can be used as a model to study both neoplasia and adrenogonadal lineage specification.

Can Pre-Corneal Elimination Rate Provide Evidence of Polymeric Characteristics?

As certain strains of mice age, hyperplastic lesions resembling gonadal tissue accumulate beneath the adrenal capsule. Gonadectomy (GDX) accelerates this heterotopic differentiation, resulting in the formation of wedge-shaped adrenocortical neoplasms that produce sex steroids. Stem/progenitor cells that reside in the adrenal capsule and retain properties of the adrenogonadal primordium are thought to be the source of this heterotopic tissue. Here, we demonstrate that GLI1+ progenitors in the adrenal capsule give rise to gonadal-like cells that accumulate in the subcapsular region. A tamoxifen-inducible Cre driver (Gli1-creERT2) and two reporters (R26R-lacZ, R26R-confetti) were used to track the fate of GLI1+ cells in the adrenal glands of B6D2F2 mice, a strain that develops both GDX-induced adrenocortical neoplasms and age-dependent subcapsular cell hyperplasia. In gonadectomized B6D2F2 mice GLI1+ progenitors contributed to long-lived adrenal capsule cells and to adrenocortical neoplasms that expressed Gata4 and Foxl2, two prototypical gonadal markers. Pdgfra, a gene expressed in adrenocortical stromal cells, was upregulated in the GDX-induced neoplasms. In aged non-gonadectomized B6D2F2 mice GLI1+ progenitors gave rise to patches of subcapsular cell hyperplasia. Treatment with GANT61, a small-molecule GLI antagonist, attenuated the upregulation of gonadal-like markers (Gata4, Amhr2, Foxl2) in response to GDX. These findings support the premise that GLI1+ progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic tissue.

Androgen, Estrogen and Thyroid Hormone Repress Tear Peroxidase Gene in Lacrimal Gland of Hamster

As certain strains of mice age, hyperplastic lesions resembling gonadal tissue accumulate beneath the adrenal capsule. Gonadectomy (GDX) accelerates this heterotopic differentiation, resulting in the formation of wedge-shaped adrenocortical neoplasms that produce sex steroids. Stem/progenitor cells that reside in the adrenal capsule and retain properties of the adrenogonadal primordium are thought to be the source of this heterotopic tissue. Here, we demonstrate that GLI1+ progenitors in the adrenal capsule give rise to gonadal-like cells that accumulate in the subcapsular region. A tamoxifen-inducible Cre driver (Gli1-creERT2) and two reporters (R26R-lacZ, R26R-confetti) were used to track the fate of GLI1+ cells in the adrenal glands of B6D2F2 mice, a strain that develops both GDX-induced adrenocortical neoplasms and age-dependent subcapsular cell hyperplasia. In gonadectomized B6D2F2 mice GLI1+ progenitors contributed to long-lived adrenal capsule cells and to adrenocortical neoplasms that expressed Gata4 and Foxl2, two prototypical gonadal markers. Pdgfra, a gene expressed in adrenocortical stromal cells, was upregulated in the GDX-induced neoplasms. In aged non-gonadectomized B6D2F2 mice GLI1+ progenitors gave rise to patches of subcapsular cell hyperplasia. Treatment with GANT61, a small-molecule GLI antagonist, attenuated the upregulation of gonadal-like markers (Gata4, Amhr2, Foxl2) in response to GDX. These findings support the premise that GLI1+ progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic tissue.

Sexually Dimorphic Expression of Co-Repressor Sin3A in Mouse Kidneys

Using Western blot analysis we found transcriptional co-repressor Sin3A to be expressed at ahigher level in male mouse kidney than in females. HDAC1 (histone deacetylase 1) protein, anotherco-repressor forming complexes with Sin3A, was not higher in males. No sex differences in Sin3Aexpression were found after gonadectomy, suggesting that gonadal secretions in adulthood cause thesex difference in kidney expression of Sin3A. In contrast, HDAC1 levels were higher in castratedgonadal males than in females, which presumably reflects a long-lasting differentiating effect oftesticular secretions in early development on this protein in kidneys. In gonadectomized mice inwhich sex chromosome complement (XX vs. XY) is independent of gonadal type (testes vs. ovaries),there was no difference in the level of Sin3A or HDAC1 expression in kidney in XX or XY mice ofthe same gonadal sex.

Sex differences in the development of angiotensin II-induced hypertension in conscious mice

Sex has an important influence on blood pressure (BP) regulation. There is increasing evidence that sex hormones interfere with the renin-angiotensin system. Thus the purpose of this study was to determine whether there are sex differences in the development of ANG II-induced hypertension in conscious male and female mice. We used telemetry implants to measure aortic BP and heart rate (HR) in conscious, freely moving animals. ANG II (800 ng.kg(-1).min(-1)) was delivered via an osmotic pump implanted subcutaneously. Our results showed baseline BP in male and female mice to be similar. Chronic systemic infusion of ANG II induced a greater increase in BP in male (35.1 +/- 5.7 mmHg) than in female mice (7.2 +/- 2.0 mmHg). Gonadectomy attenuated ANG II-induced hypertension in male mice (15.2 +/- 2.4 mmHg) and augmented it in female mice (23.1 +/- 1.0 mmHg). Baseline HR was significantly higher in females relative to males (630.1 +/- 7.9 vs. 544.8 +/- 16.2 beats/min). In females, ANG II infusion significantly decreased HR. However, the increase in BP with ANG II did not result in the expected decrease in HR in either intact male or gonadectomized mice. Moreover, the slope of the baroreflex bradycardia to phenylephrine was blunted in males (-5.6 +/- 0.3 to -2.9 +/- 0.5) but not in females (-6.5 +/- 0.5 to -5.6 +/- 0.3) during infusion of ANG II, suggesting that, in male mice, infusion of ANG II results in a resetting of the baroreflex control of HR. Ganglionic blockade resulted in greater reduction in BP on day 7 after ANG II infusion in males compared with females (-61.0 +/- 8.9 vs. -36.6 +/- 6.6 mmHg), suggesting an increased contribution of sympathetic nerve activity in arterial BP maintenance in male mice. Together, these data indicate that there are sex differences in the development of chronic ANG II-induced hypertension in conscious mice and that females may be protected from the increases in BP induced by ANG II.

Effects of dihydrotestosterone on adipose tissue measured by serial analysis of gene expression

Intra-abdominal fat accumulation is related to several diseases, especially diabetes and heart disease. Molecular mechanisms associated with this independent risk factor are not well established. Through the serial analysis of gene expression (SAGE) strategy, we have studied the transcriptomic effects of castration and dihydrotestosterone (DHT) in retroperitoneal adipose tissue of C57BL6 male mice. Approximately 50,000 SAGE tags were isolated in intact and gonadectomized mice, as well as 3 and 24 h after DHT administration. Transcripts involved in energy metabolism, such as glyceraldehyde-3-phosphate dehydrogenase, malic enzyme supernatant, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase and monoglyceride lipase, were upregulated by DHT. Transcripts involved in adipogenesis, and cell cycle and cell shape organization, such as DDX5, C/EBPalpha, cyclin I, procollagen types I, III, IV, V and VI, SPARC and matrix metalloproteinase 2, were upregulated by DHT. Cell defense, division and signaling, protein expression and many novel transcripts were regulated by castration and DHT. The present results provide global genomic evidence for a stimulation of glycolysis, fatty acids and triacylglycerol production, lipolysis and cell shape reorganization, as well as cell proliferation and differentiation, by DHT. The novel transcripts regulated by DHT may contribute to identify new mechanisms involved in the action of sex hormones and their potential role in obesity.

Transcription factor GATA-4 is a marker of anaplasia in adrenocortical neoplasms of the domestic ferret (Mustela putorius furo).

Adrenocortical neoplasms are a common cause of morbidity in neutered ferrets. Recently we showed that gonadectomized DBA/2J mice develop adrenocortical tumors that express transcription factor GATA-4. Therefore, we screened archival specimens of adrenocortical neoplasms from neutered ferrets to determine whether GATA-4 could be used as a tumor marker in this species. Nuclear immunoreactivity for GATA-4 was evident in 19/22 (86%) of ferret adrenocortical carcinomas and was prominent in areas exhibiting myxoid differentiation. Normal adrenocortical cells lacked GATA-4 expression. Two other markers of adrenocortical tumors in gonadectomized mice, inhibin-alpha and luteinizing hormone receptor, were coexpressed with GATA-4 in some of the ferret tumors. No GATA-4 expression was observed in three cases of nodular hyperplasia, but patches of anaplastic cells expressing GATA-4 were evident in 7/14 (50%) of tumors classified as adenomas. We conclude that GATA-4 can function as a marker of anaplasia in ferret adrenocortical tumors.

Adult Gonadal Hormones Selectively Regulate Sexually Dimorphic Quantitative Traits Observed in Experimental Allergic Encephalomyelitis

Experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS) are characterized by strong sexual dimorphisms, many of which may be due to genetically controlled sex hormone effects on the immune system, the central nervous system (CNS), or both. In the present study we used 487 gonadectomized and 376 intact age-matched F(2) mice generated through crosses of B10.S/SgMcdJ and SJL/J mice to assess the role of adult gonadal hormones in regulating clinical and histopathological quantitative traits (QT) associated with EAE in the context of genetic heterogeneity. We found that gonadectomy resulted in different effects, depending on the QT and the sex of the mouse. Ovariectomized mice on average had lower cumulative clinical disease scores, shorter duration of clinical signs, and increased peak disease scores. This trend was accompanied by a significant increase in the incidence of acute, progressive EAE which is more frequently seen in intact and orchiectomized males. Although spinal cord (SC) inflammation was the better predictor of clinical signs of EAE in both sexes, ovariectomized females had considerable reductions in nearly all histopathological QT in both the brain and SC. Orchiectomy resulted in modestly significant increases in disease severity and peak score and earlier onset of clinical signs. With the exception of SC demyelination and lesion scores, orchiectomy had no effect on histopathological QT. Importantly, gonadectomy reduced but did not completely abolish any of the sexually dimorphic clinical QT seen in intact mice. It did however, lead to a significant sexual dimorphism in incidence and severity not seen in intact mice. For histopathological QT, no sexual dimorphism was detected for brain lesions in either intact or gonadectomized mice. In contrast, SC histopathological QT exhibited significant sexual dimorphisms, which were impacted by gonadectomy. The results from this study indicate that within the context of genetic heterogeneity, circulating gonadal hormones influence both clinical and histopathological QT in this model of MS, but they do not solely account for the sexual dimorphisms seen in these traits. Thus, additional mechanisms must play a role in regulating gender differences in autoimmune disease of the CNS.

Transcription Factors GATA‐4 and GATA‐6 in Human Adrenocortical Tumors

Transcription factors GATA‐4 and GATA‐6 are expressed during normal adrenocortical development in mice and humans, and in vitro studies have linked them to adrenal steroidogenesis. GATA‐4 is highly expressed in the adrenocortical tumors of gonadectomized mice, whereas GATA‐6 is down‐regulated in the tumor area. Based on these findings we studied GATA‐4 and GATA‐6 expression in 39 human adrenocortical tumors using RT‐PCR, Northern analysis and immunohistochemistry. 6/18 adenomas and 4/11 carcinomas were positive for GATA‐4 mRNA. GATA‐6 mRNA was expressed in 19/19 adenomas and 9/10 carcinomas, and GATA‐6 immunoreactivity was remarkably lower in adrenocortical carcinomas than in adenomas (p < 0.05). Some of the steroidogenically active human adrenocortical cells (NCI‐H295R) were weakly positive for GATA‐4, whereas steroidogenically inactive cells (ACT‐1) were totally GATA‐4 negative. In contrast, both cell lines expressed GATA‐6. GATA expression patterns similar to the animal models can thus be observed in human adrenocortical tumors, but the pathophysiological significance of these findings remains to be elucidated.

Endogenous GABA Release Inhibits the Firing of Adult Gonadotropin-Releasing Hormone Neurons

The effect of endogenous gamma-aminobutyric acid (GABA)(A) receptor-mediated signaling on the excitability of adult male and female GnRH neurons was examined using gramicidin perforated-patch electrophysiology in GnRH-LacZ and GnRH-GFP (green fluorescent protein) transgenic mouse models. In both lines of mice, approximately 80% of GnRH neurons (n = 42) responded to the selective GABA(A) receptor antagonist bicuculline (20 microm) with a rapid and reversible membrane depolarization and/or increase in firing rate. Approximately 16% of GnRH neurons gave no response, and two neurons were inhibited by bicuculline. The same depolarizing responses (78%) were obtained from adult gonadectomized GnRH-GFP mice. The depolarizing response to bicuculline persisted in the presence of tetrodotoxin, demonstrating that even action potential-independent GABA release was acting to reduce GnRH neuron membrane potential. These observations show that endogenous GABA signaling through the GABA(A) receptor exerts a powerful net inhibitory effect upon the excitability of mature GnRH neurons.

Effects of steroid hormones on (Na +, K +)-ATPase activity inhibition-induced amnesia on the step-through passive avoidance task in gonadectomized mice

Inhibition of sodium-potassium adenosine 5′-triphosphatase ((Na +, K +)-ATPase) activity causes edema and cell death in the central nervous system, and impairment of learning and memory. Several sex steroid hormones have a protective effect against neuronal cell damage and the hypofunction of learning and memory. To examine the possible roles and mechanism of action of steroid hormones against amnesia induced by ouabain, an inhibitor of (Na +, K +)-ATPase, gonadectomized male mice were administrated ouabain (0.1 μg per mouse) intracisternally (i.cist.), and the learning and memory abilities of the mice were assessed by a step-through passive avoidance task. Subcutaneous (s.c.) administration of 17β-estradiol (βE2; 10 μg kg −1) or testosterone (TES; 1 mg kg −1) improved the memory impairment induced by ouabain, while administration of dihydrotestosterone (1 mg kg −1) or corticosterone (COR) (1 mg kg −1) did not. Treatment with the estradiol receptor antagonists, tamoxifen (TAM) (10 mg kg −1; s.c. or 0.1 μg; i.cist.) and 4-hydroxytamoxifen (10 mg kg −1; s.c.), or the androgen receptor antagonist, cyproterone (10 mg kg −1; s.c. or 1 μg; i.cist.), did not influence the protective effect of βE2 or TES on ouabain-induced amnesia. Moreover, we studied the effects of several free radical scavengers—17α-estradiol (10 μg kg −1; s.c.), α-tocopherol (VE: 200 mg kg −1; per os (p.o.), ascorbic acid (VC: 200 mg kg −1; p.o.), or VE+VC (200 mg kg −1 each; p.o.)—on ouabain-induced amnesia, and compared those effects with that of βE2. The administration of free radical scavengers had no significant effect on memory impairment. These results indicate that βE2 and TES ameliorate the amnesia induced by inhibition of (Na +, K +)-ATPase activity, and that the protective effect of βE2 is caused by a non-genomic, rather than a genomic effect or a radical scavenging action. Additionally, the ameliorative effect of TES does not appear to involve free radical scavenging, but its aromatization to estrogen could contribute to the non-genomic action of βE2.

Mouse strain susceptibility to gonadectomy-induced adrenocortical tumor formation correlates with the expression of GATA-4 and luteinizing hormone receptor.

Certain inbred strains of mice, including DBA/2J, develop adrenocortical tumors in response to gonadectomy. Spindle-shaped cells with limited steroidogenic capacity, termed A cells, appear in the subcapsular region of the adrenal gland, followed by sex steroid-producing cells known as B cells. These changes result from unopposed gonadotropin production by the pituitary, but the adrenocortical factors involved in tumorigenesis have not been characterized. GATA-4, a transcription factor normally expressed in fetal, but not adult, adrenocortical cells, was found in neoplastic cells that proliferate in the adrenal cortex of gonadectomized DBA/2J mice. GATA-4 mRNA was detected in the adrenal glands of female mice 0.5 months after ovariectomy and reached a maximum by 4 months. Castrated male mice developed adrenocortical tumors more slowly than gonadectomized females, and the onset of GATA-4 expression in the adrenal was delayed. In situ hybridization and immunohistochemistry revealed GATA-4 mRNA and protein in A and B cells, but not in normal adrenocortical cells. mRNA encoding another factor associated with adrenocortical tumorigenesis, LH receptor (LHR), was detected in A and B cells. In addition, transcripts for P450 17 alpha-hydroxylase/C17-C20 lyase, an enzyme essential for the production of sex steroids, and inhibin-alpha were found in B cells. Unilateral ovarian regeneration, a phenomenon known to occur in gonadectomized mice, was observed in a subset of DBA/2J mice undergoing complete ovariectomy. In these animals, adrenocortical tumor progression was arrested; A cells and GATA-4 expression were evident, but there was no expression of LHR or P450 17 alpha-hydroxylase/C17-C20 lyase. Strain susceptibility to adrenocortical tumorigenesis (DBA/2J >> FVB/N) correlated with the expression of GATA-4 and LHR, implicating these factors in the process of adrenocortical neoplasia in response to continuous gonadotropin stimulation.

Hepatic expression of PPARα, a molecular target of fibrates, is regulated during inflammation in a gender-specific manner

Dyslipidemia, inflammation and gender are major risk factors in cardiovascular disease. Here we show that hepatic expression of Peroxisome proliferator-activated receptor α (PPARα), a nuclear receptor that regulates lipid metabolism and inflammation, is regulated in a gender-specific manner during lipopolysaccharide (LPS)-induced systemic inflammation. Immediately following LPS-induced systemic inflammation, hepatic PPARα mRNA level decreased dramatically in mice. It was restored to baseline within 24 h in females but remained below baseline for >72 h in male mice. In gonadectomized mice of both sexes, PPARα mRNA level was restored to baseline within 48 h after the initial decrease.

Interactive effects of testosterone and superior cervical ganglionectomy on attraction thresholds to volatile urinary odors in gonadectomized mice

Volatile urinary odors contribute to mate recognition in mice after their detection by the main olfactory epithelium (MOE). We used a habituation/dishabitution task to ask whether the capacity of gonadectomized mice of both sexes to detect and investigate decreasing concentrations of volatile urinary odors from either breeding males or estrous females is modulated by administering androgen or estrogen and if so, whether any effects of these sex steroids are altered by disrupting the sympathetic innervation of the MOE via bilateral superior cervical ganglionectomy (SCGx). In tests given, beginning 51 days after gonadectomy without steroid treatment both male and female subjects detected even the lowest concentrations (1:120 and 1:160 dilutions by volume) of male urinary odors, provided they were SCGx as opposed to sham operated. In subsequent tests given after estradiol benzoate (EB) followed later by 5α-dihydrotestosterone (DHT) treatments, neither male nor female subjects detected low concentrations of male urinary odors regardless of whether or not their SCG’s were intact. Administration of testosterone (T) prior to a final series of tests restored the ability of gonadectomized subjects of both sexes to detect low concentrations of male urinary odors regardless of their SCG status. This suggests that T, but not its neural metabolites estradiol, or DHT, facilitates responsiveness to low concentrations of male odors in mice of both sexes. In tests given 51 days after gonadectomy without steroid treatment most male and female subjects readily detected the three highest concentrations of estrous female urinary odors whereas SCGx males and females failed to detect the lowest concentrations of these odors. After treatment with EB and then with DHT, gonadectomized mice of both sexes generally failed to detect the three lowest concentrations of estrous female urinary odors regardless of their SCG status. After T treatment; however, subjects of both sexes again detected most dilutions of estrous female urine, provided their SCG’s were intact. Again, these results suggest that T, but not estradiol or DHT, facilitates responsiveness to estrous female urinary odors. Provided such an activational effect of T is present, sympathetic, noradrenergic inputs to the MOE may enhance odorant contrast, as previously suggested [Nat. Neurosci. 2 (1999) 106], by reducing the responsiveness of olfactory neurons to low (1:120 and 1:160 dilutions) concentrations of some biologically significant odorants (e.g. male urinary odors) while facilitating their responsiveness to low to moderate (1:80 dilution) concentrations of others (e.g. estrous female urinary odors).

Reversal of Bone Loss in Mice by Nongenotropic Signaling of Sex Steroids

Many of the actions of estrogens and androgens are not explained by their receptors interacting with DNA, but there is no firm evidence that these nongenotropic actions are biologically important. The authors recently demonstrated strong anti-apoptotic effects of sex steroids on mouse osteoblasts and osteocytes. They are mediated by alterations in signaling cascades that modulate the activity of transcription factors. These sex steroid effects are not gender specific; they require only the ligand-binding domain of the receptor. The effects can be consistently reproduced by estren (4-estren-3α,17β-diol), a synthetic compound lacking classic transcriptional activity. Studies in adult mice showed that 17β-estradiol (E2), dihydrotestosterone (DHT), and estren lessened osteoblastic apoptosis and stimulated osteoclastic apoptosis. The osteoblastic apoptosis induced by gonadectomy in the lumbar spine was prevented by a replacement dose of E2, DHT, or estren in both male and female mice. Estren, unlike E2, failed to stimulate in vitro transcription of the C3 gene in the uterus, which is mediated by estrogen response elements. Estren was at least as effective as E2 for preserving spinal and global bone mineral density in female mice. After ovariectomy, estren proved better than E2 in augmenting bone mineral density in the hindlimb. In addition, estren was at least as effective as DHT replacement in mice having orchidectomy, as reflected by greater compression strength. Closure of the bone marrow cavity was noted with a pharmacologic dose of E2 but not with estren. Histomorphometric analysis of the lumbar spine confirmed significantly greater cortical and trabecular width in ovariectomized mice treated with estren compared with others given E2. Estren-treated animals had more osteoblasts on the trabeculae, and these cells produced much more unmineralized matrix. Both estren and E2 decreased the rate of bone formation and numbers of osteoclasts compared with ovariectomized mice. In contrast to E2 or DHT, estren did not alter uterine or seminal vesicle weight in gonadectomized mice. These findings mean that it is possible to separate the skeletal effects of sex steroids from their reproductive effects. Mechanism-specific ligands such as estren may be preferable to estrogens for hormone replacement therapy (HRT), especially in view of increasing concern about the safety of current HRT regimens.

Gonadal effects on plasma ACE activity in mice

Angiotensin-converting enzyme (ACE) regulates blood pressure and is an important target in the management of hypertension. Hypertension is a gender biased disease. Plasma ACE activity is significantly higher in male mice (309 U/l) than female mice (237 U/l) and is reduced significantly upon gonadectomy to 224 and 209 U/l, respectively. Although, the gonads influence plasma ACE activity in both male and female mice, the effect is more pronounced in male mice. Plasma ACE is derived from the cleavage of tissue ACE and lung has the highest concentration of tissue ACE. However, lung ACE activity is not gender dimorphic but increases significantly upon gonadectomy in both male and female. ACE mRNA level in the lung is not influenced by gender or gondaectomy. Therefore, the gonads affect plasma ACE activity by influencing cleavage of tissue ACE to plasma ACE and/or decrease stability of plasma ACE in gonadectomized mice is mediated.

Immediate-early Fos Protein Levels in Brainstem Neurons of Male and Female Gonadectomized Mice Subjected to Cold Exposure

The expression of the immediate early gene c-fos has been used extensively as a marker for neural activation in response to acute and chronic stressful stimuli in brain and spinal cord. The present study examined the expression of Fos protein in the brainstem nuclei of male and female gonadectomized mice in response to cold stress. Free-floating sections were processed immunohistochemically for Fos protein using standard avidin-biotin complex methods. The number of Fos-positive neurons in each nucleus was determined. Although the experiment was designed to look for gender differences, results were equivalent between females and males. After mice were exposed to a cold ambient temperature (4°C) for 2 h, elevated numbers of Fos-positive neurons were counted in the medullary gigantocellular reticular nucleus, medullary raphe nucleus, nucleus of the solitary tract and in locus coeruleus. However, no elevated expression was found in nucleus ambiguus, nor in neurons of the A1 group, nor the C2 or C3 group. Similar to rats, these results with mice reveal a widespread arousal system in the lower brainstem activated by cold stress in both genders. These findings in gonadectomized mice have set the stage for investigations following hormonal and genetic manipulations.