Wild-type OVX Research Articles

2-Methoxyestradiol protects from angiotensin II-induced renal CD3+/FOXP3+ T cell-dysregulation via 12/15 lipoxygenase inhibition

We previously showed that 2-methoxyestradiol (2-ME), an active metabolite of 17β-estradiol (E2) and lox (12/15-lipoxygenase) gene disruption, protects against angiotensin (Ang) II-induced hypertension. Moreover, 2-ME reduces Ang II-induced increases in plasma levels of 12(S)-hydroxyeicosatetraenoic acid (HETE), a major arachidonic acid-LOX-generated metabolite in ovariectomized (OVX, endogenous E2 depleted) wild-type (WT) mice. 2-ME also exerts anti-inflammatory effects, and regulatory T cells (Tregs) have been reported to impact Ang II-induced hypertension in rodent models, with premenopausal females being protected relative to males. This study tested the hypothesis that 2-ME protects against the inflammatory effects of Ang II by upregulating Tregs in the kidney via LOX inhibition in female mice. Intact (ovaries present) or OVX WT and OVX-LOX knock-out (KO) mice (8-10 weeks, n=3-4/group) were infused with Ang II (700 ng/kg/min) or saline (vehicle) via osmotic minipumps (S.C.) with or without 2-ME (15 μg/g, I.P., every third day) treatment for 2 weeks. Renal protein expressions of CD3+ T cells and FOXP3+ Tregs were evaluated by immunofluorescence staining and mRNA expressions of tnf-α and il-10 by RT-PCR using TaqMan assay (relative to saline-infused intact WT) in the whole kidney. Results represented as mean ± SEM were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test. The CD3+, tnf-α and il-10 expressions were comparable between saline-infused intact WT, OVX-WT, and OVX-KO (CD3+: 8.3±0.3, 7.0±0.5, 6.3±0.3; tnf-α: 1.0±0.0, 0.9±0.0, 0.8±0.2; il-10: 1.1±0.1, 0.9±0.1, 1.0±0.0). However, saline-infused intact WT and OVX-KO expressed higher Tregs (p<0.05) as compared to OVX-WT (7.6±0.3, 7.3±0.3 vs 4.0±0.5). Ang II increased CD3+, Tregs, and il-10 (p<0.05) but not tnf-α expressions as compared to its vehicle in intact WT (12.6±0.3, 15.3±0.3, 3.7±0.3,0.8±0.1). Ang II also increased CD3+ (11.0±0.6) and Tregs (11.0±0.6) in OVX-KO (p<0.05), without altering mRNA expressions of tnf-α (0.9±0.3) and il-10 (1.5±0.1) . In contrast, Ang II resulted in a greater increase in CD3+ (25.0±1.1) in OVX-WT than OVX-KO and intact WT, besides increasing tnf-α expression (2.5±0.4) (p<0.05). 2-ME reduced CD3+ (16.7±0.9) and tnf-α (1.4±0.4) expressions, and increased Tregs (10.0±1.0) and il-10 (2.0±0.0) expressions in Ang II-infused OVX-WT mice (p<0.05). 2-ME also reduced CD3+ (6.7±0.3, p<0.05) and showed a trend to increase Tregs (14.0±1.1, P=0.056), and il-10 (2.3±0.2, P=0.058) expressions without altering tnf-α (0.7±0.1) expression in Ang II-infused OVX-KO mice. In conclusion, Ang II produces a specific effect on the renal T cell profile, greater increases in proinflammatory T cells in OVX mice, and anti-inflammatory Tregs in intact female mice, most likely mediated by E2 through metabolite 2-ME via inhibition of LOX. R01HL19134-47, AHA Award#940538. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GPR109A gene deletion ameliorates gonadectomy-induced bone loss in mice.

Sex steroid deficiency plays critical roles in the pathophysiology of bone as the result of uncertain bone remodeling, i.e., increased bone resorption with equivocal bone formation. We have previously shown that GPR109A, a G protein coupled receptor, controls osteoclastogenesis and bone resorption, where global GPR109A deletion decreased osteoclast bone resorption and increased bone mass. Here, we used global GPR109A gene deletion, ovariectomized (OVX) and orchidectomized (ORX) mouse models to probe the role of GPR109A in gonadectomy-induced bone loss in female and male mice. Six months old GPR109A-/- mice and their wild type littermates were allocated to Sham or gonadectomized groups for six weeks. Using densitometric micro-CT confirmed by peripheral quantitative CT (pQCT) scans on tibia and spine, and three-point bending test on femur ex vivo, we found the bone volume, trabecular number, as well as bone mineral density and content in both trabecular and cortical sites were significantly decreased in wild type OVX and ORX compared with respective Sham groups. While bone mass in both male and female GPR109A-/- Sham groups were significantly higher compared with their respective wild type Sham groups, global GPR109A gene deletion ameliorated gonadectomy-induced bone loss. In GPR109A-/- females, most of bone mass and strength parameters measured by micro-CT, pQCT and three-point bending test were not different between Sham and OVX groups. In wild type but not in GPR109-/- mice, bone remodeling marker measurements indicated that both bone resorption (Cathepsin K) and bone formation (osteocalcin) markers were increased in gonadectomized mice compared to sham, with the exception of bone specific ALP, which was decreased in gonadectomized mice. Expression of bone resorption markers (Cathepsin K) were significantly lower, but β-catenin expression was higher in GPR109A-/- mice compared with their wild type littermates. Collectively, these data indicate that global GPR109A deletion ameliorates gonadectomy-induced bone loss through suppression of bone resorption.

Leucine rich amelogenin peptide prevents ovariectomy-induced bone loss in mice.

Amelogenins, major extra cellular matrix proteins of developing tooth enamel, are predominantly expressed by ameloblasts and play significant roles in the formation of enamel. Recently, amelogenin has been detected in various epithelial and mesenchymal tissues, implicating that it might have distinct functions in various tissues. We have previously reported that leucine rich amelogenin peptide (LRAP), one of the alternate splice forms of amelogenin, regulates receptor activator of NF-kappa B ligand (RANKL) expression in cementoblast/periodontal ligament cells, suggesting that the amelogenins, especially LRAP, might function as a signaling molecule in bone metabolism. The objective of this study was to identify and define LRAP functions in bone turnover. We engineered transgenic (TgLRAP) mice using a murine 2.3kb α1(I)-collagen promoter to drive expression of a transgene consisting of LRAP, an internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) to study functions of LRAP in bone formation and resorption. Calvarial cell cultures from the TgLRAP mice showed increased alkaline phosphatase (ALP) activity and increased formation of mineralized nodules compared to the cells derived from wild-type (WT) mice. The TgLRAP calvarial cells also showed an inhibitory effect on osteoclastogenesis in vitro. Gene expression comparison by quantitative polymerase chain reaction (Q-PCR) in calvarial cells indicated that bone formation makers such as Runx2, Alp, and osteocalcin were increased in TgLRAP compared to the WT cells. Meanwhile, Rankl expression was decreased in the TgLRAP cells in vitro. The ovariectomized (OVX) TgLRAP mice resisted bone loss induced by ovariectomy resulting in higher bone mineral density in comparison to OVX WT mice. The quantitative analysis of calcein intakes indicated that the ovariectomy resulted in increased bone formation in both WT and TgLRAP mice; OVX TgLRAP appeared to show the most remarkably increased bone formation. The parameters for bone resorption in tissue sections showed increased number of osteoclasts in OVX WT, but not in OVX TgLRAP over that of sham operated WT or TgLRAP mice, supporting the observed bone phenotypes in OVX mice. This is the first report identifying that LRAP, one of the amelogenin splice variants, affects bone turnover in vivo.

Loss of p27kip1 suppresses the myocardial senescence caused by estrogen deficiency.

Estrogen deficiency accelerates the aging process and increases the risk of developing cardiovascular disease (CVD). Apoptosis is one of the important mechanisms of aging. p27kip1 is a cyclin-dependent kinase inhibitor that can regulate cell cycle, apoptosis, and cell motility. p27kip1 overexpression can inhibit cell cycle and increase apoptosis so it has been considered as a marker of aging. In the present study, bilateral ovariectomy (OVX) was performed as a model for menopause in wild-type (WT) and p27kip1 knockout (KO) mice to assess the effects of p27kip1 loss in myocardial aging caused by estrogen deficiency. We found that myocardial fibrosis and heart weight/body weight ratio of mice in the OVX group and p27kip1 KO group were significantly increased. Echocardiography showed that the left ventricular diameter and volume of the WT OVX group increased significantly and the cardiac function decreased. However, there was no significant difference in the results of echocardiography between the two p27kip1 KO groups. The aging and apoptosis indexes in OVX group were increased significantly, However, the indexes in p27kip1 KO mice were decreased. The expression of antioxidant indexes in OVX group was decreased significantly and p27kip1 KO can improve the antioxidant ability. These results provided that estrogen deficiency increased oxidative stress and apoptosis, accelerated aging of heart. p27kip1 KO can partly delay the aging and apoptosis of heart through upregulated antioxidant enzymes.

Analyzing the influence of BDNF heterozygosity on spatial memory response to 17β-estradiol

The recent use of estrogen-based therapies as adjunctive treatments for the cognitive impairments of schizophrenia has produced promising results; however the mechanism behind estrogen-based cognitive enhancement is relatively unknown. Brain-derived neurotrophic factor (BDNF) regulates learning and memory and its expression is highly responsive to estradiol. We recently found that estradiol modulates the expression of hippocampal parvalbumin-positive GABAergic interneurons, known to regulate neuronal synchrony and cognitive function. What is unknown is whether disruptions to the aforementioned estradiol–parvalbumin pathway alter learning and memory, and whether BDNF may mediate these events. Wild-type (WT) and BDNF heterozygous (+/−) mice were ovariectomized (OVX) at 5 weeks of age and simultaneously received empty, estradiol- or progesterone-filled implants for 7 weeks. At young adulthood, mice were tested for spatial and recognition memory in the Y-maze and novel-object recognition test, respectively. Hippocampal protein expression of BDNF and GABAergic interneuron markers, including parvalbumin, were assessed. WT OVX mice show impaired performance on Y-maze and novel-object recognition test. Estradiol replacement in OVX mice prevented the Y-maze impairment, a Behavioral abnormality of dorsal hippocampal origin. BDNF and parvalbumin protein expression in the dorsal hippocampus and parvalbumin-positive cell number in the dorsal CA1 were significantly reduced by OVX in WT mice, while E2 replacement prevented these deficits. In contrast, BDNF+/− mice showed either no response or an opposite response to hormone manipulation in both behavioral and molecular indices. Our data suggest that BDNF status is an important biomarker for predicting responsiveness to estrogenic compounds which have emerged as promising adjunctive therapeutics for schizophrenia patients.

Loss of the PGE2 receptor EP1 enhances bone acquisition, which protects against age and ovariectomy-induced impairments in bone strength

PGE2 exerts anabolic and catabolic effects on bone through the discrete actions of four prostanoid receptors (EP1–4). We have previously demonstrated that loss EP1 accelerates fracture repair by enhancing bone formation. In the present study we defined the role of EP1 in bone maintenance and homeostasis during aging and in response to ovariectomy. The femur and L4 vertebrae of wild type (WT) and EP1−/− mice were examined at 2-months, 6-months, and 1-year of age, and in WT and EP1−/− mice following ovariectomy (OVX) or sham surgery. Bone volume fraction, trabecular architecture and mechanical properties were maintained during aging in EP1−/− mice to a greater degree than age-matched WT mice. Moreover, significant increases in bone formation rate (BFR) (+60%) and mineral apposition rate (MAR) (+50%) were observed in EP1−/−, relative to WT, while no change in osteoclast number and osteoclast surface were observed. Following OVX, loss of EP1 was protective against bone loss in both femur and L4 vertebrae, with increased bone volume/total volume (BV/TV) (+32% in femur) and max load at failure (+10% in femur) relative to WT OVX, likely resulting from the increased bone formation rate that was observed in these mice. Taken together these studies identify inhibition of EP1 as a potential therapeutic approach to suppress bone loss in aged or post-menopausal patients.

Disruption of claudin-18 diminishes ovariectomy-induced bone loss in mice

Claudin-18 (Cldn-18), a member of the tight junction family of proteins, is a negative regulator of RANKL-induced osteoclast differentiation and bone resorption (BR) in vivo. Since estrogen deficiency decreases bone mass in part by a RANKL-mediated increase in BR, we evaluated whether estrogen regulates Cldn-18 expression in bone. We found that Cldn-18 expression was reduced in the bones of estrogen deficient mice, whereas it was increased by estrogen treatment in osteoblasts and osteoclasts in vitro. We next evaluated the role of Cldn-18 in mediating estrogen-induced bone loss. Cldn-18 knockout (KO) and littermate wild-type (WT) mice were ovariectomized (OVX) or sham operated at 6 wk of age, and the skeletal phenotype was evaluated at 14 wk of age. PIXImus revealed that total body, femur, and lumbar BMD were reduced 8-13% (P < 0.05) after 8 wk of OVX compared with sham in WT mice. As expected, total body, femur, and lumbar BMD were reduced 14-21% (P < 0.05) in Cldn-18 KO sham mice compared with sham WT mice. However, ovariectomy failed to induce significant changes in BMD of total body, femur, or vertebra in the Cldn-18 KO mice. μCT analysis of the distal femur revealed that trabecular (Tb) bone volume was decreased 50% in the OVX WT mice compared with sham that was caused by a 26% decrease in Tb number and a 30% increase in Tb separation (all P < 0.05). By contrast, none of the Tb parameters were significantly different in OVX Cldn-18 KO mice compared with sham KO mice. Histomorphometric analyses at the Tb site revealed that neither osteoclast surface nor osteoclast perimeter was increased significantly as a consequence of OVX in either genotype at the time point examined. Based on our findings, we conclude that the estrogen effects on osteoclasts may in part be mediated via regulation of Cldn-18 signaling.

Neuropeptide Y mediates the short-term hypometabolic effect of estrogen deficiency in mice

Estrogen deficiency increases body weight or total and central adiposity and decreases energy expenditure. Hypothalamic neuropeptide Y (NPY) expression is altered by estrogen deficiency in rodents, but the long-term consequences on energy homeostasis are unknown. To investigate the role of NPY in the changes in energy expenditure and physical activity, as well as the associated changes in body weight and composition in response to short-term and long-term estrogen deficiency. Sham and ovariectomy (OVX) operations were performed at 8 weeks of age in wild-type (WT) and NPY(-/-) mice. Energy expenditure, physical activity, body composition and weight, as well as food intake were measured at 10-18 days (short-term) and 46-54 days (long-term) after OVX. OVX influences energy homeostasis differently at early compared with later time-points. At the early but not the late time point, OVX in WT mice reduced oxygen consumption and energy expenditure and tended to reduce resting metabolic rate. Interestingly, these effects of short-term estrogen deficiency were ablated by NPY deletion, with NPY(-/-) mice exhibiting significant increases in energy expenditure and resting metabolic rate. In addition to these hypermetabolic effects, OVX NPY(-/-) mice exhibited significantly lower body weight and whole-body fat mass relative to OVX WT controls at the short-term but not the long-term time point. Food intake and physical activity were unaltered by OVX, but NPY(-/-) mice exhibited significant reductions in these parameters relative to WT. The effects of estrogen deficiency to reduce energy metabolism are transient, and NPY is critical to this effect as well as the early OVX-induced obesity.

Are estrogen and leptin the sole factors affecting the estrous cycle of ob/ob and ovariectomized mice?

Hormones like leptin and estrogen have been suggested to increase energy expenditure and modulate estrous cycle. The aim of this study was to investigate the stages of estrous cycle in mouse models with contrasting leptin and estrogen levels. Estrous cycle of Wild type (WT), WT ovariectomized (OVX), ob/ob and ob/ob OVX was observed by obtaining vaginal smear and staining with Papanicolaou (PAP) procedure. Quantitative analysis showed cellular morphology and predominance of cellular content across the estrous cycle. Microscopical and quantitative counts showed a 4–5 day regular cycle in WT mouse, WT OVX were acyclic, infertile and hardly showed any cells, ob/ob and ob/ob OVX were both acyclic, infertile and were at persistent estrous or persistent metaestrous phase. Enzyme‐Immunometric Assay results showed that leptin levels were about 3 fold higher in WTOVX than WT mouse (p&lt;0.0001) with beta‐estradiol values being negligible in WTOVX. The beta‐estradiol level for ob/ob and WT were similar. These results suggest that although leptin and estrogen play an important role in the estrous cycle, they are not the sole factor influencing the cycle. It is probable that in the absence of leptin and decrease in sex steroid hormones with increase in acyclicity, the central nervous system probably interprets the circulation as absence of energy stores and shuts down reproduction indicated by the changes in the estrous cycle.

Involvement of dynorphin in anxiogenic effects of estrogen

Methods Dyn KO mice were generated by replacement of the entire coding region of the prodynorphin gene [2] and backcrossed onto C57bl/6N. Age and testing experiencematched female intact and ovariectomized (OVX) Dyn KO and wildtype (WT) mice at 3-8 months age were tested in all experiments. Anxiety (open field test, OF; elevated plus maze test, EPM; light dark test, LDT) and stressrelated behaviour (forced swim test, FST; tail suspension test, TST) was investigated in correlation to the estrous cycle in intact female WT and Dyn KO mice and in OVX WT and Dyn KO mice treated with the general estrogen receptor (ER) agonist 17β-estradiol (E2), and specific agonists for ERα (PPT), ERβ (DPN) or GPER (G1) two hours before testing.

Endogenous n‐3 fatty acids protect ovariectomy induced bone loss by attenuating osteoclastogenesis

AbstractBeneficial effects of n‐3 fatty acids (FA) on bone mineral density (BMD) have been reported in mice, rats and human beings, but the precise mechanisms involved have not been described. This study used the Fat‐1 mouse, a transgenic model that synthesizes n‐3 FA from n‐6 FA to directly determine if outcome of bone health were correlated with n‐3 FA. Ovariectomized (Ovx) and sham operated wild‐type (WT) and Fat‐1 mice were fed an AIN‐93M diet containing 10% corn oil for 24 weeks. BMD was analysed by dual energy x‐ray absorptiometry. Fat‐1 Ovx mice exhibited significantly lower level of osteotropic factors like receptor activator of NF‐κB ligand and tartrate‐resistant acid phosphatase (TRAP)5b in serum and higher BMD in distal femoral metaphysis, proximal tibial metaphysis, femoral diaphysis and lumbar vertebra as compared to WT Ovx mice. LPS‐stimulated bone marrow (BM) cells from Fat‐1 Ovx mice produced significantly lower level of pro‐inflammatory cytokines like tumour necrosis factor‐α, interleukin (IL)‐1‐β, IL‐6 and higher level of anti‐inflammatory cytokines like IL‐10, IFN‐γ and higher level of nitric oxide as compared to BM cells from WT Ovx mice. LPS‐stimulated COX‐II activity as well as NF‐κB activation in BM cells from Fat‐1 Ovx mice was significantly less as compared to BM cells from WT Ovx mice. Furthermore, Fat‐1 BM cells generated significantly less number of TRAP osteoclast‐like cells as compared to WT BM cells. In conclusion, we offer further insight into the mechanisms involved in preventing the BMD loss in Ovx mice by n‐3 FA using a Fat‐1 transgenic mouse model.

Endogenous n-3 fatty acids protect ovariectomy induced bone loss by attenuating osteoclastogenesis

Beneficial effects of n-3 fatty acids (FA) on bone mineral density (BMD) have been reported in mice, rats and human beings, but the precise mechanisms involved have not been described. This study used the Fat-1 mouse, a transgenic model that synthesizes n-3 FA from n-6 FA to directly determine if outcome of bone health were correlated with n-3 FA. Ovariectomized (Ovx) and sham operated wild-type (WT) and Fat-1 mice were fed an AIN-93M diet containing 10% corn oil for 24 weeks. BMD was analysed by dual energy x-ray absorptiometry. Fat-1 Ovx mice exhibited significantly lower level of osteotropic factors like receptor activator of NF-kappaB ligand and tartrate-resistant acid phosphatase (TRAP)5b in serum and higher BMD in distal femoral metaphysis, proximal tibial metaphysis, femoral diaphysis and lumbar vertebra as compared to WT Ovx mice. LPS-stimulated bone marrow (BM) cells from Fat-1 Ovx mice produced significantly lower level of pro-inflammatory cytokines like tumour necrosis factor-alpha, interleukin (IL)-1-beta, IL-6 and higher level of anti-inflammatory cytokines like IL-10, IFN-gamma and higher level of nitric oxide as compared to BM cells from WT Ovx mice. LPS-stimulated COX-II activity as well as NF-kappaB activation in BM cells from Fat-1 Ovx mice was significantly less as compared to BM cells from WT Ovx mice. Furthermore, Fat-1 BM cells generated significantly less number of TRAP osteoclast-like cells as compared to WT BM cells. In conclusion, we offer further insight into the mechanisms involved in preventing the BMD loss in Ovx mice by n-3 FA using a Fat-1 transgenic mouse model.

Signal crosstalk between estrogen and peroxisome proliferator-activated receptor α on adiposity

Peroxisome proliferator-activated receptor alpha and estrogen are believed to be involved in metabolic changes leading to obesity. To test this relationship, we divided female wildtype and PPAR alpha-deficient mice fed on a high fat diet into the following groups: mock-operated, ovariectomized (OVX), and E(2)-treated. The visceral white adipose tissue and plasma cholesterol levels were increased significantly in wild type OVX and decreased in the E(2)-treated group, but interestingly not in PPAR alpha-deficient mice. The mRNA levels of lipoprotein lipase in adipose tissue were also increased in only wild type OVX and decreased significantly in E(2)-treated mice. These novel results suggest the possibility of signaling crosstalk between PPAR alpha and E2, causing obesity in vivo.

Dietary fish oil is antihypertrophic but does not enhance postischemic myocardial function in female mice

Clinically and experimentally, a case for omega-3 polyunsaturated fatty acid (PUFA) cardioprotection in females has not been clearly established. The goal of this study was to investigate whether dietary omega-3 PUFA supplementation could provide ischemic protection in female mice with an underlying genetic predisposition to cardiac hypertrophy. Mature female transgenic mice (TG) with cardiac-specific overexpression of angiotensinogen that develop normotensive cardiac hypertrophy and littermate wild-type (WT) mice were fed a fish oil-derived diet (FO) or PUFA-matched control diet (CTR) for 4 wk. Myocardial membrane lipids, ex vivo cardiac performance (intraventricular balloon) after global no-flow ischemia and reperfusion (15/30 min), and reperfusion arrhythmia incidence were assessed. FO diet suppressed cardiac growth by 5% and 10% in WT and TG, respectively (P < 0.001). The extent of mechanical recovery [rate-pressure product (RPP) = beats/min x mmHg] of FO-fed WT and TG hearts was similar (50 +/- 7% vs. 45 +/- 12%, 30 min reperfusion), and this was not significantly different from CTR-fed WT or TG. To evaluate whether systemic estrogen was masking a protective effect of the FO diet, the responses of ovariectomized (OVX) WT and TG mice to FO dietary intervention were assessed. The extent of mechanical recovery of FO-fed OVX WT and TG (RPP, 50 +/- 4% vs. 64 +/- 8%) was not enhanced compared with CTR-fed mice (RPP, 60 +/- 11% vs. 80 +/- 8%, P = 0.335). Dietary FO did not suppress the incidence of reperfusion arrhythmias in WT or TG hearts (ovary-intact mice or OVX). Our findings indicate a lack of cardioprotective effect of dietary FO in females, determined by assessment of mechanical and arrhythmic activity postischemia in a murine ex vivo heart model.

Estradiol or diarylpropionitrile administration to wild type, but not estrogen receptor beta knockout, mice enhances performance in the object recognition and object placement tasks

Cognitive processes mediated by the hippocampus and cortex are influenced by estradiol (E 2); however, the mechanisms by which E 2 has these effects are not entirely clear. As such, studies were conducted to begin to address the role of actions at the β form of the intracellular estrogen receptor (ERβ) for E 2’s cognitive effects in adult female mice. We investigated whether E 2 improved performance of wild type (WT) and ERβ knockout (βERKO) mice in tasks considered to be mediated by the cortex and hippocampus, the object recognition and object placement tasks. WT and βERKO mice were ovariectomized (ovx) and E 2 (0.1 mg/kg), an ERβ selective ER modulator (SERM), diarylpropionitrile (DPN; 0.1 mg/kg), or oil vehicle was administered to mice following training in these tasks. We hypothesized that if E 2 has mnemonic effects, in part, due to its actions at ERβ, then WT mice administered E 2 or DPN would have improved performance compared to vehicle WT controls, which would not be different from βERKO mice administered vehicle, E 2 or DPN. Alternatively, activation of ERα (with E 2, which is a ligand for both ERα and ERβ) may produce opposing effects on cognition and/or the activation of ERα and ERβ vs. either receptor isoform alone may produce a different pattern of effects. Results obtained supported the hypothesis that ERβ activation is important for mnemonic effects. Ovx WT, but not βERKO, mice administered E 2 or DPN had a greater percentage of time exploring a novel object in the object recognition task and a displaced object in the object placement task. Thus, actions at ERβ may be important for E 2 or SERMs to enhance cognitive performance of female mice in the object recognition and placement tasks.

Estrogen receptor-α mediates estrogen protection from angiotensin II-induced hypertension in conscious female mice

It has been shown that the female sex hormones have a protective role in the development of angiotensin II (ANG II)-induced hypertension. The present study tested the hypotheses that 1) the estrogen receptor-alpha (ERalpha) is involved in the protective effects of estrogen against ANG II-induced hypertension and 2) central ERs are involved. Blood pressure (BP) was measured in female mice with the use of telemetry implants. ANG II (800 ng.kg(-1).min(-1)) was administered subcutaneously via an osmotic pump. Baseline BP in the intact, ovariectomized (OVX) wild-type (WT) and ERalpha knockout (ERalphaKO) mice was similar; however, the increase in BP induced by ANG II was greater in OVX WT (23.0 +/- 1.0 mmHg) and ERalphaKO mice (23.8 +/- 2.5 mmHg) than in intact WT mice (10.1 +/- 4.5 mmHg). In OVX WT mice, central infusion of 17beta-estradiol (E(2); 30 microg.kg(-1).day(-1)) attenuated the pressor effect of ANG II (7.0 +/- 0.4 mmHg), and this protective effect of E(2) was prevented by coadministration of ICI-182,780 (ICI; 1.5 microg.kg(-1).day(-1), 18.8 +/- 1.5 mmHg), a nonselective ER antagonist. Furthermore, central, but not peripheral, infusions of ICI augmented the pressor effects of ANG II in intact WT mice (17.8 +/- 4.2 mmHg). In contrast, the pressor effect of ANG II was unchanged in either central E(2)-treated OVX ERalphaKO mice (19.0 +/- 1.1 mmHg) or central ICI-treated intact ERalphaKO mice (19.6 +/- 1.6 mmHg). Lastly, ganglionic blockade on day 7 after ANG II infusions resulted in a greater reduction in BP in OVX WT, central ER antagonist-treated intact WT, central E(2) + ICI-treated OVX WT, ERalphaKO, and central E(2)- or ICI-treated ERalphaKO mice compared with that in intact WT mice given just ANG II. Together, these data indicate that ERalpha, especially central expression of the ER, mediates the protective effects of estrogen against ANG II-induced hypertension.

Increased osteoblastogenesis and decreased bone resorption protect against ovariectomy-induced bone loss in thrombospondin-2-null mice

Although bone is composed primarily of extracellular matrix (ECM), the dynamic role that the ECM plays in regulating bone remodeling secondary to estrogen loss is relatively unexplored. Previous studies have shown that mice deficient in the matricellular protein thrombospondin-2 (TSP2-null) form excess endocortical bone; thus, we postulated that enhanced bone formation in TSP2-null mice could protect against ovariectomy (OVX)-induced bone loss. Wild-type (WT) OVX mice showed a significant loss of both midfemoral endocortical and proximal tibial trabecular bone, but OVX did not significantly alter TSP2-null bone. TSP2-null mice showed an increase in bone formation, as indicated by a 70% increase in serum osteocalcin two weeks post OVX and a two-fold increase in bone formation rate (BFR) five weeks post OVX as measured by dynamic histomorphometry. WT animals showed only a 20% increase in serum osteocalcin at two weeks and no change in BFR at five weeks. This increase in bone formation in TSP2-null OVX mice was accompanied by a three-fold increase in osteoprogenitor number. Although these results provide a partial explanation for the maintenance of bone geometry post-OVX, TSP2-null mice five weeks post-OVX also showed a significantly lower level of bone resorption than OVX WT mice, as determined by serum levels of the amino-terminal telopeptide of type I collagen (NTx). We conclude that the absence of TSP2 protects against OVX-induced bone loss by two complementary processes: increased formation and decreased resorption.

Mice lacking the integrin beta5 subunit have accelerated osteoclast maturation and increased activity in the estrogen-deficient state.

Integrin alphavbeta5 is expressed on osteoclast precursors and is capable of recognizing the same amino acid motif as alphavbeta3. Three-month-old beta5(-/-) female OVX mice had increased osteoclastogenesis ex vivo, and microCT assessment of trabecular bone volume was 53% lower than WT-OVX animals. These preliminary data suggest alphavbeta5 integrin's presence on osteoclast precursors may inhibit of osteoclast formation. Osteoclasts are unique resorptive skeletal cells, capable of degrading bone on contact to the juxtaposed matrix. Integrin alphavbeta5 is expressed on osteoclast precursors, structurally similar to alphavbeta3, and capable of recognizing the same amino acid motif. Given the structural relationship and reciprocal regulation of alphavbeta3 and alphavbeta5, the purpose of this study was to evaluate how alphavbeta5 might contribute to osteoclast maturation and activity. Three-month-old wildtype (WT) and beta5(-/-) female mice had ovariectomy (OVX) or sham operations. The osteoclastogenic capacity of marrow-derived precursors, the kinetic, the circulating, and structural parameters of bone remodeling, was determined after 6 weeks of paired feeding. OVX increased osteoclastogenesis ex vivo and in vivo. Osteoclast formation and prolonged pre-osteoclast survival were substantially enhanced in cultures containing beta5(-/-) cells whether obtained from sham-operated or OVX mice. Expression of cathepsin K, beta3 integrin subunit, and calcitonin receptor were accelerated in cultured beta5(-/-)osteoclasts. beta5(-/-) osteoclasts from OVX animals showed a 3-fold enhancement of net resorptive activity, with quantitative muCT showing trabecular bone volume loss after OVX 53% greater in beta5(-/-) OVX compared with similarly treated WT OVX mice (p < 0.05). alpha5beta3 seems to be an inhibitor of osteoclast formation, in contrast to alphavbeta3. In addition, loss of alphavbeta5 seems to accelerate osteoclast formation in the OVX model. Further examination of alphavbeta5 signaling pathways may enhance our understanding of the activation of bone resorption.

Mice lacking the plasminogen activator inhibitor 1 are protected from trabecular bone loss induced by estrogen deficiency.

Bone turnover requires the interaction of several proteases during the resorption phase. Indirect evidence suggests that the plasminogen activator/plasmin pathway is involved in bone resorption and turnover, and recently we have shown that this cascade plays a role in the degradation of nonmineralized bone matrix in vitro. To elucidate the role of the plasminogen activator inhibitor 1 (PAI-1) in bone turnover in vivo, bone metabolism was analyzed in mice deficient in the expression of PAI-1 gene (PAI-1-/-) at baseline (8-week-old mice) and 4 weeks after ovariectomy (OVX) or sham operation (Sham) and compared with wild-type (WT) mice. PAI-1 inactivation was without any effect on bone metabolism at baseline or in Sham mice. However, significant differences were observed in the response of WT and PAI-1-/- mice to ovariectomy. The OVX WT mice showed, as expected, decreased trabecular bone volume (BV/TV) and increased osteoid surface (OS/BS) and bone formation rate (BFR), as assessed by histomorphometric analysis of the proximal tibial metaphysis. In contrast, no significant change in any of the histomorphometric variables studied was detected in PAI-1-/- mice after ovariectomy. As a result, the OVX PAI-1-/- had a significantly higher BV/TV, lower OS/BS, lower mineral apposition rate (MAR) and BFR when compared with the OVX WT mice. However, a comparable decrease in the cortical thickness was observed in OVX PAI-1-/- and WT mice. In addition, the cortical mineral content and density assessed in the distal femoral metaphysis by peripheral quantitative computed tomography (pQCT), decreased significantly after ovariectomy, without difference between PAI-1-/- mice and WT mice. In conclusion, basal bone turnover and bone mass are only minimally affected by PAI-1 inactivation. In conditions of estrogen deficiency, PAI-1 inactivation protects against trabecular bone loss but does not affect cortical bone loss, suggesting a site-specific role for PAI-1 in bone turnover.

Absence of gonadotropin surges and gonadotropin-releasing hormone self-priming in ovariectomized (OVX), estrogen (E2)-treated, progesterone receptor knockout (PRKO) mice.

It is well known that estrogen (E2) stimulates expression of progesterone receptors (PRs), thereby inducing responsiveness of several tissues to the actions of progesterone (P). Recent studies have also suggested, however, that biological actions previously ascribed to E2 alone may also be mediated by activation of E2-induced PRs, even independently of signal changes in P concentrations. In the present experiments, the progesterone receptor knockout (PRKO) mice were used to assess the role of PR activation in the positive feedback actions of E2 on gonadotropin release. Ovariectomized (OVX) PRKO mice were tested for their capacity to mount primary gonadotropin surges in response to exogenous E2, and to exhibit a GnRH self-priming effect in response to sequential injections of the decapeptide. Wild-type (WT) and PRKO mice were OVX, treated with both 17beta-estradiol and estradiol benzoate (EB), and then killed at 1900 h on day 7 postOVX. Plasma LH RIA revealed that WT mice exhibited surges in response to the E2 treatment; the PRKO mice, however, showed no elevation in plasma LH above untreated controls. Instead, plasma LH levels in E2-treated, OVX PRKO mice decreased significantly in comparison to untreated OVX PRKO mice, suggesting that E2 can exert a negative feedback influence on LH release in PRKO mice, despite the absence of positive feedback effects. A slight but significant rise in plasma FSH was observed in E2-treated OVX WT mice in comparison to untreated controls: an effect not seen in E2-treated OVX PRKO mice, reinforcing the observation that estrogen's positive feedback effects are compromised in PRKO mice. In a second experiment, E2-treated OVX WT and PRKO mice were given either one or two pulses of GnRH 60 min apart, and killed 10 min later. The WT mice were found to exhibit a robust GnRH self-priming effect, as WT mice receiving two GnRH pulses displayed LH responses approximately 2-fold greater than those receiving only one pulse. By contrast, PRKO mice receiving two GnRH pulses exhibited no additional increase in plasma LH levels. We conclude that PR activation is obligatory for expression of the GnRH self-priming effect as well as for generation of E2-induced LH and FSH surges. The extent to which failure of LH surge secretion in PRKO mice is due to the absence of GnRH self-priming, lack of hypothalamic GnRH surges, and/or defects in other processes remains to be determined. These observations clearly demonstrate, however, that the presence of PR is an absolute requirement for the transmission of E2-induced signals leading to gonadotropin surges.