Estrogen In Mice Research Articles

Targeted estrogen delivery reverses the metabolic syndrome

We report the development of a new combinatorial approach that allows for peptide-mediated selective tissue targeting of nuclear hormone pharmacology while eliminating adverse effects in other tissues. Specifically, we report the development of a glucagon-like peptide-1 (GLP-1)-estrogen conjugate that has superior sex-independent efficacy over either of the individual hormones alone to correct obesity, hyperglycemia and dyslipidemia in mice. The therapeutic benefits are driven by pleiotropic dual hormone action to improve energy, glucose and lipid metabolism, as shown by loss-of-function models and genetic action profiling. Notably, the peptide-based targeting strategy also prevents hallmark side effects of estrogen in male and female mice, such as reproductive endocrine toxicity and oncogenicity. Collectively, selective activation of estrogen receptors in GLP-1-targeted tissues produces unprecedented efficacy to enhance the metabolic benefits of GLP-1 agonism. This example of targeting the metabolic syndrome represents the discovery of a new class of therapeutics that enables synergistic co-agonism through peptide-based selective delivery of small molecules. Although our observations with the GLP-1-estrogen conjugate justify translational studies for diabetes and obesity, the multitude of other possible combinations of peptides and small molecules may offer equal promise for other diseases.

Abstract 10: Disparate Temporal Regulation of Blood Pressure by Estrogens During Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that predominantly affects young women. Due to this partiality towards women, estrogen is commonly implicated in disease development. The potential for estrogens to promote SLE disease progression stems from data showing that removal of estrogens in young female mice with SLE (6-8 week old NZBWF1 mice) delays the development of renal injury and mortality. The primary cause of mortality in women with SLE is cardiovascular disease, and hypertension, a major cardiovascular risk factor, is highly prevalent in this patient population. Based on the presumed role of estrogen to promote SLE, we hypothesized that estrogen promotes hypertension during SLE. To test this, 30 week old SLE mice (NZBWF1) and control mice (NZW/LacJ) were subjected to either ovariectomy (OVX) or a sham operation and a subset of these mice were replete with 17β-estradiol (E2, 5μg/mouse, s.c., 2x/week). At 34 weeks of age, mean arterial pressure (MAP in mmHg) was measured by carotid catheter in conscious freely moving mice. MAP was higher in SLE sham mice compared to control shams (133±3, n=17 vs. 120±3, n=13, p<0.05). Contrary to our hypothesis, OVX at 30 weeks exacerbated the hypertension (154±3, n=9, p<0.05 vs. SLE sham) and prevalence of albuminuria in mice with SLE (83%, 10 of 12 vs 36%, 5 of 14 measured by dipstick > 100 mg/dL). OVX did not alter MAP (115±3, n=9) or albuminuria in control mice. The hypertensive response to OVX was prevented in OVX SLE mice replete with E2 (133±4, n=3) suggesting a protective role for E2 against SLE-associated hypertension. Because previous studies showed that estrogen removal in young mice delayed SLE disease progression, we tested whether OVX starting at 8 weeks of age delayed the development of hypertension. OVX in young SLE mice did not significantly alter the progression of SLE-associated hypertension (141±4, n=4 measured at 34 weeks of age). Consistent with previous work of others, OVX in young SLE mice reduced the prevalence of albuminuria (20%, 1 of 5 vs 33%, 1 of 3). These data suggest that estrogen has a complex temporal role in the pathogenesis of SLE with the potential to promote disease early in life, but protect against the progression of SLE associated hypertension later in the course of the disease.

Bone Marrow Oxytocin Mediates the Anabolic Action of Estrogen on the Skeleton

Estrogen uses two mechanisms to exert its effect on the skeleton: it inhibits bone resorption by osteoclasts and, at higher doses, can stimulate bone formation. Although the antiresorptive action of estrogen arises from the inhibition of the MAPK JNK, the mechanism of its effect on the osteoblast remains unclear. Here, we report that the anabolic action of estrogen in mice occurs, at least in part, through oxytocin (OT) produced by osteoblasts in bone marrow. We show that the absence of OT receptors (OTRs) in OTR(-/-) osteoblasts or attenuation of OTR expression in silenced cells inhibits estrogen-induced osteoblast differentiation, transcription factor up-regulation, and/or OT production in vitro. In vivo, OTR(-/-) mice, known to have a bone formation defect, fail to display increases in trabecular bone volume, cortical thickness, and bone formation in response to estrogen. Furthermore, osteoblast-specific Col2.3-Cre(+)/OTR(fl/fl) mice, but not TRAP-Cre(+)/OTR(fl/fl) mice, mimic the OTR(-/-) phenotype and also fail to respond to estrogen. These data attribute the phenotype of OTR deficiency to an osteoblastic rather than an osteoclastic defect. Physiologically, feed-forward OT release in bone marrow by a rising estrogen concentration may facilitate rapid skeletal recovery during the latter phases of lactation.

Uterine Chromatin Immunoprecipitation-Sequencing Profile of Estrogen Receptor Alpha DNA Binding Mutant Reveals Novel Interactions Between Estrogen Receptor Alpha and Progesterone Receptor Signaling.

Mutation of the first zinc finger of the mouse estrogen receptor a (ERa) disrupts its interaction with estrogen responsive element (ERE) sequences in target gene DNA and leads to altered uterine transcriptional responses. ERa binding sites were profiled using chromatin immunoprecipitation followed by comprehensive sequencing (ChIP-seq) with uteri from the zinc finger mutant ERa “KIKO” mice. Mice were ovariectomized and injected with vehicle (V) or estradiol (E2) for one hour. As was previously seen with WT ERa ChIP-seq, numerous binding sites were found with V treatment (3510 peaks) and E2 increased ERa binding (13773 peaks). Interestingly, only 30% of KIKO and WT ERa binding peaks overlapped. KIKO ERa ChiP-seq DNA sequences were divided into those with ERE motifs (6781 sequences) or without ERE motifs (6992 sequences) and evaluated for enrichment of other transcription factor motifs. Surprisingly, the most significant computed motifs were similar to androgen receptor (AR) or progesterone receptor (PR) binding motifs with 5685 of the KIKO ERa ChIP-seq sites showing one of the AR/PR like motifs. Possible mechanisms accounting for this finding include: 1) Interaction between ERa and PR signaling mediated by PRE/ARE sites. 2) Loss of ERE binding but acquisition of ARE/PRE affinity by the KIKO ERa mutant. The first mechanism was considered by scanning WT and KIKO ERa ChIP-seq peak sequences to predict PREs. 1364 of 17241 WT sites (7.9%) and 2922 of 13773 KIKO sites (21.2%) contained PREs, confirming the presence of PRE motifs in the WT ERa ChIP-seq binding sequences. However, only 449 of the predicted PREs were common between WT and KIKO, indicating that although some interactions are consistent with the first mechanism, not all KIKO ERa interaction with ARE/PRE are described by the scheme. To address the second mechanism, overlap between WT ERa, KIKO ERa and WT PR ChIP-seq peaks were evaluated. Interestingly, 20% of KIKO 1h E2 ERa peaks overlapped with PR 1h progesterone (P) peaks. Transcripts associated with the PRE-motif containing ERa binding peaks were examined in our microarray datasets. Many of these uterine transcripts exhibited ERa mediated E2 regulation in WT and KIKO, including some responses unique to KIKO. ER and/or PR regulated transcripts were selected for further study. Cdkn1a and Errfi1 transcripts are increased by E2 in both WT and KIKO. E2 dependent WT or KIKO ERa binding and P dependent PR recruitment was seen by ChIP-seq at common sites. Fkbp5 and Ihh transcripts are both increased by P and, interestingly, both have increased basal levels and enhanced E2 mediated increases in KIKO compared to WT. PR or ERa ChIP-seq indicates moderate E2 dependent WT ERa binding near both of these genes, but greater E2 dependent KIKO ERa binding and P dependent PR binding at the same sites. Hdc transcript is increased by P, but not by E2, in WT; however, either P or E2 increases Hdc in KIKO. WT ERa ChIP-seq peaks are not seen in the Hdc locus, but KIKO ERa and PR exhibit hormone-dependent recruitment to a common site. In conclusion, we show that the KIKO ERa exhibits novel responses, which suggest this receptor interacts with ARE/PRE like motifs. Together, our observations suggest that WT ERa and PR share sites of interaction in uterine chromatin, which are revealed by use of the ERa DNA binding mutant, consistent with a combination of both of the mechanisms suggested above.

Study the Pathological Effects of the Combination of Estrogen and Progesterone Hormones on Some Organs Experimentally Induced in Mice

The aim of this study was design to investigate the pathological changes for one month after therapeutic and toxic doses of subcutaneous injection of estrogen and progesterone combination hormones in mice, on the target organs testis and epidydimus in males and uterus and ovary in females. As well as the effects on non-target organ of Brain, liver spleen, intestine, stomach, kidney and lung in both sexes. The results showed sever pathological changes in male's testis and epidydimus and in females, uterus and the ovary. It is characterized by some pathological changes in toxic group less severity than in the therapeutic group.Also, in non-target organs brain and spleen of toxic group of males and females showed some pathological changes while therapeutic group almost appear normal. The liver and kidney were affected in both groups (therapeutic and Toxic) in males and females. Other organs like intestine stomach, Lung doesn't showed any change in both groups.

Pro-survival Effects of 17β-Estradiol on Osteocytes Are Mediated by Nitric Oxide/cGMP via Differential Actions of cGMP-dependent Protein Kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.

The citrus flavonone hesperetin inhibits growth of aromatase-expressing MCF-7 tumor in ovariectomized athymic mice

Aromatase is responsible for the rate-determining reaction in estrogen synthesis and is a prime target for treating estrogen-receptor-positive breast cancer. Previous in vitro study has demonstrated that apigenin (APG), naringenin (NGN) and hesperetin (HSP) are three of the most potent natural aromatase inhibitors. Because the enzyme inhibition could potentially block breast cancer development, we employed an established postmenopausal breast cancer model to examine the chemopreventive effect of these flavonoids in vivo. Athymic mice were ovariectomized and transplanted with aromatase-overexpressing MCF-7 cells. Dietary administration of HSP at 1000 ppm and 5000 ppm significantly deterred the xenograft growth, while a null effect was observed in mice treated with APG or NGN. Further study illustrated that plasma estrogen in HSP-treated mice was reduced. Messenger RNA expression of the estrogen-responsive gene pS2 was also decreased in the tumors of mice treated with 1000 and 5000 ppm HSP. On the other hand, western analysis indicated that cyclin D1, CDK4 and Bcl-x(L) were reduced in the tumors. This study suggested that HSP could be a potential chemopreventive agent against breast carcinogenesis through aromatase inhibition.

Comparative Effects of Sesame Seed Lignan and Flaxseed Lignan in Reducing the Growth of Human Breast Tumors (MCF-7) at High Levels of Circulating Estrogen in Athymic Mice

Flaxseed (FS) has a breast tumor-reducing effect, possibly because of its high content of secoisolariciresinol diglucoside (SDG) lignan. Sesame seed (SS) is rich in the lignan sesamin (SES) but is non-protective. Both lignans are metabolized to estrogen-like enterodiol and enterolactone. The objective of this study was to differentiate the effects of SDG and SES on established human estrogen receptor-positive breast tumors (MCF-7) in athymic mice with high serum estrogen to help explain the different effects of FS and SS. Mice were fed for 8 wk the basal diet (BD, control) or BD supplemented with 1 g/kg SDG or SES. SES reduced palpable tumor size by 23% compared to control, whereas SDG did not differ from SES or control. Both treatments reduced tumor cell proliferation, but only SES increased apoptosis. SDG and SES reduced human epidermal growth factor receptor 2 and endothelial growth factor receptor expressions, but only SES reduced downstream pMAPK. Neither treatment affected IGF-1R, vascular endothelial growth factor receptor-2, Akt, pAkt, or MAPK of the growth factor signaling pathway. Thus, at high serum estrogen levels, SDG may not account for the tumor reducing effect of FS. SES was more effective than SDG in reducing breast tumor growth, but its effect may have been lost when consumed as a component of SS.

Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells

Interferon Regulatory Factor-3 (IRF-3) plays a central role in the induction of interferon (IFN) production and succeeding interferon-stimulated genes (ISG) expression en route for restraining hepatitis C virus (HCV) infection. Here, we established a stable Huh7.5-IRF3ER cell line expressing a fusion protein of IRF-3 and mouse estrogen receptor (ER) to examine IFN production and anti-HCV effects of IRF-3 in retinoic acid inducible-gene-I (RIG-I) deficient Huh 7.5 cells. Homodimerization of the IRF-3ER fusion protein was detected by Western blotting after treatment with the estrogen receptor agonist 4-hydrotamoxifen (4-HT) in Huh7.5-IRF3ER cells. Expression of IFN-α, IFN-β, and their inhibitory effects on HCV replication were demonstrated by real-time polymerase chain reaction (PCR). Peak expression of IFN-α and IFN-β was achieved 24-hours post 4-HT treatment, coinciding with the appearance of phosphorylated signal transducer and activator of transcription (STAT) proteins. Additionally, HCV viral replication declined in time-dependent fashion. In previous studies, a novel IFN-mediated pathway regulating expression of 1-8U and heterogeneous nuclear ribonucleoprotein M (hnRNP M) inhibited HCV internal ribosomal entry site (IRES)-dependent translation. When expression of ISGs such as 1-8U and hnRNP M were measured in 4-HT-treated Huh7.5-IRF3ER cells, both genes were positively regulated by activation of the IRF-3ER fusion protein. In conclusion, the anti-HCV effects of IRF-3ER homodimerization inhibited HCV RNA replication as well as HCV IRES-dependent translation in Huh7.5-IRF3ER cells. The results of this study indicate that IRF-3ER homodimerization is a key step to restore IFN expression in Huh7.5-IRF3ER cells and in achieving its anti-HCV effects.

Multimodal In Vivo Imaging and Blood Monitoring of Intrinsic and Extrinsic Apoptosis

Noninvasive detection and in vivo imaging of apoptosis plays a critical role in the development of therapeutics in many different fields including cancer. We have developed an apoptosis biosensor by fusing green fluorescent protein (GFP) to the N-terminus of the naturally secreted Gaussia luciferase separated by a caspase-3 cleavage peptide consisting of aspartic acid (D), glutamic acid (E), valine (V), and aspartic acid (D) or DEVD. We showed that this fusion is retained in the cytoplasm of cells in an inactive form. Upon apoptosis, the DEVD peptide is cleaved in response to caspase-3 activation, freeing ssGluc, which can now enter the secretory pathway where it is folded properly and is released from the cells and can be detected in the conditioned medium in culture or in blood of live animals ex vivo over time. Because Gluc is secreted from cells via conventional pathway through the endoplasmic reticulum (ER), Golgi and vesicles, we showed that the presence of Gluc in these compartments in response to apoptosis can be visualized in vivo using bioluminescence imaging. This reporter provides a valuable tool for imaging and real-time monitoring of apoptosis and is compatible with high-throughput functional screening application in cultured cells and animal models.

Construction of recombinant lentivirus carrying mouse estrogen receptor α and identification in infected neurons

Objective To construct a recombinant lentivirus carrying C57BL/6 mouse estrogen receptor α(Erα) and to infect mouse neurons,so as to pave a way for further studying the relationship of Erα with some nervous system diseases.Methods Erα gene was inserted into the main virus vector LV-GFP-flag to construct recombinant lentiviral vector LV-Erα-flag,which was confirmed by agarose gel electrophoresis(AGE) and DNA sequencing.Recombinant lentivirus V-Erα-flag was produced by 293T cells following the co-transfection of LV-Erα-flag with the packaging plasmids pHelper 1.0 and pHelper 2.0,and the virus titer was examined.The neurons of C57BL/6 mouse were cultured using a serum-free culture medium,and then control lentivirus V-GFP-flag was used to infect the neurons.The infection efficiency and apoptosis rate were examined by flow cytometry to obtain optimal multiplicity of infection(MOI).GFP expression was detected daily under an inverted fluorescent microscope.After that,V-Erα-flag with the optimal MOI was used to infect neurons;the expression of Erα mRNA and protein in the neurons was detected by quantitative real-time PCR and Western blotting analysis.Results AGE and DNA sequencing confirmed that LV-Erα-flag was successfully constructed,and packaged into 293T cells with a titer of 2×10~8 TU/ml.Control lentivirus V-GFP-flag could infect neurons,with the infection efficiency being(89.8±4.03)% and the cell apoptosis rate being only(3.6±0.29)% when MOI=5.Neurons could survive in the culture for at least 8 weeks,during which the GFP was persistently expressed,indicating the lentivirus could efficiently and stably infect the neurons.The expression of Erα mRNA and protein was greatly increased in neurons infected with V-Erα-flag(MOI=5).Conclusion The recombinant lentivirus carrying Erα has been constructed successfully,which can infect neurons and lead to increase the expression of Erα mRNA and protein.

Manganese-Induced NF-κB Activation and Nitrosative Stress Is Decreased by Estrogen in Juvenile Mice

Manganese toxicity can cause a neurodegenerative disorder affecting cortical and basal ganglia structures with a neurological presentation resembling features of Parkinson's disease. Children are more sensitive to Mn-induced neurological dysfunction than adults, and recent studies from our laboratory revealed a marked sensitivity of male juvenile mice to neuroinflammatory injury from Mn, relative to females. To determine the role of estrogen (E2) in mediating sex-dependent vulnerability to Mn-induced neurotoxicity, we exposed transgenic mice expressing an NF-κB-driven enhanced green fluorescent protein (EGFP) reporter construct (NF-κB-EGFP mice) to Mn, postulating that supplementing male mice with E2 during juvenile development would attenuate neuroinflammatory changes associated with glial activation, including expression of inducible nitric oxide synthase (NOS2) and neuronal protein nitration. Juvenile NF-κB-EGFP mice were separated in groups composed of females, males, and males surgically implanted with Silastic capsules containing 25 μg of 17-β-estradiol (E2) or vehicle control. Mice were then treated with 0 or 100 mg/Kg MnCl(2) by intragastric gavage from postnatal days 21-34. Manganese treatment caused alterations in levels of striatal dopamine, as well as increases in NF-κB reporter activity and NOS2 expression in both microglia and astrocytes that were prevented by supplementation with E2. E2 also decreased neuronal protein nitration in Mn-treated mice and inhibited apoptosis in striatal neurons cocultured with Mn-treated astrocytes in vitro. These data indicate that E2 protects against Mn-induced neuroinflammation in developing mice and that NF-κB is an important regulator of neuroinflammatory gene expression in glia associated with nitrosative stress in the basal ganglia during Mn exposure.

Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line

To investigate the functions of signal transducers and activators of transcription 1 (STAT1)-induced anti-hepatitis C viral (HCV) effects, a stable Huh7.5 cell line (Huh7.5-STAT1ER) was established that constitutively expresses a fusion protein (STAT1ER) of STAT1 and the mouse oestrogen receptor (ER), which forms STAT1ER homodimers after 4-hydroxytamoxifen (4-HT) treatment. This inducible and cytokine/receptor-independent STAT1 activation system allowed us to investigate the anti-HCV effects of STAT1ER activation after inducing IFN-stimulated gene (ISG) expression. The anti-HCV effects of dimerized STAT1ER fusion protein were determined by real-time PCR in a time-dependent fashion post-HCV (JFH-1) infection. HCV (JFH-1) RNA decreased 48% at 72 h after 4-HT treatment. To distinguish the inhibitory effects of STAT1ER activation on HCV RNA replication or HCV internal ribosomal entry site (IRES)-mediated translation, a dicistronic pRL-HL construct was used in the studies. Both cellular (Cap-dependent) and HCV IRES-mediated (Cap-independent) translation were decreased by 63% and 57% at 72 h post-STAT1ER activation in the STAT1ER cell line. In our previous studies, interferon-induced transmembrane protein 3 [(IFITM3) (1-8U)] was found to inhibit HCV RNA replication. Subsequently, elevated expression of the 1-8U gene was confirmed by Western blotting in the Huh7.5-STAT1ER cell line. To further investigate the 1-8U function with both in vivo and in vitro studies, the 1-8U gene was found to suppress cellular and HCV IRES-mediated translation.

Complex organization of the 5′-untranslated region of the mouse estrogen receptor α gene: Identification of numerous mRNA transcripts with distinct 5′-ends

The 5′-untranslated region (5′-UTR) of the estrogen receptor α (ERα) gene plays an important role in determining its tissue-specific expression. We examined the 5′-UTRs of the mouse ERα mRNA variants in depth using the Basic Local Alignment Search Tool (BLAST), rapid amplification of 5′-cDNA ends (5′-RACE) and RT-PCR. We demonstrated the presence of multiple variants containing unique 5′-UTRs. We mapped the cDNA sequences onto the mouse genome, and found that both alternative splicing from four different leader exons (A, C, F1, and H) to exon 1, and combinations of 12 internal exons (X1, X2, X3, X4, F2/X5, X6, X7, X8, X9, X10, X11, and B) generate multiple ERα transcripts. Mouse exon B, that has homologies with human exon B and rat exon 0T, was used as an internal exon, not as a leader exon. RT-PCR analysis revealed distinct expression patterns of the variants, suggesting that the alternative promoter usage and alternative splicing are regulated in a tissue-specific manner. Our results indicate that the genomic organization of the mouse ERα gene is complicated as previously shown in the rat ERα gene. In addition, both alternative promoter usage and alternative splicing contribute to the remarkable mRNA diversity of the mouse ERα gene.

The impact of n-hexane on the secretion of mouse estrogen and progesterone

Aim: To study the toxic effect of n-hexane on sex hormone levels, including its effect on ovarian sex hormone secretion levels and the underlying mechanism. 1) In vivo experiment: The mice were exposed to n- hexane by static inhalation of doses of 0, 3.0, 15.1 or 75.8 ml/m3 once a day (4 hours), 7 days a week, for 5 consecutive weeks. After exposure, mouse sera were sampled to measure estradiol (E2) and progesterone (P4) levels by electrochemiluminescence immunoassays (ECLIA). 2) In vitro experiment: mice at different stages of the estrous cycle were sacrificed and their minced tissue pieces were incubated in medium containing hexane-2,5-dione (2,5-HD). Then, the E2 and P4 levels in the media were measured by ECLIA. In vivo assays, the serum P4 levels in each group were lower than the control group, which was statistically significant (F = 6.196, P 2 level of 75.8 mL/m3 dosage group was decreased (96.9522 ± 7.8652)) compared to the control group (119.1600 ± 25.6420), with statistical significance (P 4 levels of 15.1 mL/m3 and 75.8 mL/m3 dosage groups were also decreased (18.5938 ± 12.485pmol/L, 13.5550 ± 5.7789 pmol/L) and were statistically significantly (F = 6.196, P In vitro, the E2 levels at various stages of the estrous cycle were significantly different from the control groups (P In vitro, different concentrations of 2, 5-HD significantly inhibited the P4 level (P 4 levels at various stages of the estrous cycle were also significantly different from controls (P 0.05). The results indicate that n-hexane may inhibit release of ovarian E2 and P4, which may be the main reason for the interference of the reproductive endocrine system by n-hexane.

Increased PTHrP and Decreased Estrogens Alter Bone Turnover but Do Not Reproduce the Full Effects of Lactation on the Skeleton

During lactation, calcium is mobilized from the maternal skeleton to supply the breast for milk production. This results in rapid but fully reversible bone loss. Prior studies have suggested that PTHrP, secreted from the breast, and estrogen deficiency, due to suckling-induced central hypogonadism, combine to trigger bone resorption. To determine whether this combination was sufficient to explain bone loss during lactation, we raised PTHrP levels and decreased levels of estrogens in nulliparous mice. PTHrP was infused via osmotic minipumps and estrogens were decreased either by using leuprolide, a long-acting GnRH agonist, or by surgical ovariectomy (OVX). Bone mineral density declined by 23.2 ± 1.3% in the spine and 16.8 ± 1.9% in the femur over 10 d of lactation. This was accompanied by changes in trabecular architecture and an increase in both osteoblast and osteoclast numbers. OVX and PTHrP infusion both induced a modest decline in bone mineral density over 10 d, but leuprolide treatment did not. The combination of OVX and PTHrP was more effective than either treatment alone, but there was no interaction between PTHrP and leuprolide. None of the treatments reproduced the same degree of bone loss caused by lactation. However, both forms of estrogen deficiency led to an increase in osteoclasts, whereas infusion of PTHrP increased both osteoblasts and osteoclasts. Therefore, although the combination of PTHrP and estrogen deficiency contributes to bone loss, it is insufficient to reproduce the full response of the skeleton to lactation, suggesting that other factors also regulate bone metabolism during this period.

EFFECT OF OVERIECTOMY AND OF ESTROGEN ADMINISTRATION UPON DUODENAL ULCERATION INDUCED BY CYSTEAMINE

Duodenal ulcers were induced in ovariectomized, intact and old mice using cysteamine hydrochloride. Under these experimental conditions ovariectomy and old age strikingly increased sensitivity to ulcer induction while estrogen administration showed a decrease in sensitivity to ulcer induction. Nevertheless, the administration of estrogen in old mice showed no effects in either intact or overiectomized mice. This change in ulcer sensitivity reflected from histological, histochemical and biochemical studies. The histological study was performed by using haematoxylin-eosin and haematoxyline staining technique. The histochemistry of the duodenal region was studied by using periodic acid Schiff reaction (PAS) for glycoproteins. The biochemical study was performed to study various constituents of glycoproteins like hexose, fucose, sialic acid and the protein. The result showed that ulcer severity was more in overiectomized cysteamine treated mice and old mice treated with cysteamine. The histological studies showed that ovariectomy decreased or did not show any change in the ulcer sensitivity considering cryptus Lieburkuhn and Brunner's glands. The same result reflected by differential intensity in the staining property of the Brunner's gland. The biochemical study showed that the glycoprotein contents were reduced many times in overiectomized cysteamine treated mice and their reversal in estrogen administered ovariectomized cysteamine injected mice. These findings prove that estrogen protects the duodenal ulcer from cysteamine administration.

Genetically-Induced Estrogen Receptor Alpha (ESR1) Overexpression Does Not Adversely Affect Fertility or Penile Development in Male Mice.

Previously, we reported that ESR1 overexpression resulting from neonatal exposure to estrogens in rats and wild-type mice was associated with infertility and mal-developed penis characterized by reduced length and weight and abnormal accumulation of fat cells. The objective of this study was to determine if mutant male mice overexpressing ESR1 are naturally infertile or have reduced fertility and/or develop abnormal penis. The fertility parameters, including fertility and fecundity indices, numbers of days from the day of cohabitation to the day of delivery, and numbers of pups per female, were not altered from controls, as a result of ESR1 overexpression. Likewise, penile morphology, including the length, weight, and diameter and os penis development, was not altered from controls. Conversely, weights of the seminal vesicles and bulbospongiosus and levator ani (BS/LA) muscles were significantly (P < 0.05) lower as compared to controls; however, the weight of the testis, the morphology of the testis and epididymis, and the plasma and testicular testosterone concentration were not different from controls. Hence, this study concludes that the genetically-induced ESR1 overexpression in male mice does not adversely affect fertility, penis, testis or epididymis, but significantly reduces weights of the seminal vesicles and BS/LA muscles. (poster)

The Importance of LAT in the Activation, Homeostasis, and Regulatory Function of T Cells

LAT (linker for activation of T cells) is a transmembrane adaptor protein that plays an essential role in TCR-mediated signaling and thymocyte development. Because LAT-deficient mice have an early block in thymocyte development, we utilized an inducible system to delete LAT in primary T cells to study LAT function in T cell activation, homeostasis, and survival. Deletion of LAT caused primary T cells to become unresponsive to stimulation from the TCR and impaired T cell homeostatic proliferation and long term survival. Furthermore, deletion of LAT led to reduced expression of Foxp3, CTLA-4, and CD25 in T(reg) cells and impaired their function. Consequently, mice with LAT deleted developed a lymphoproliferative syndrome similar to that in LATY136F mice, although less severe. Our data implicate that LAT has positive and negative roles in the regulation of mature T cells.

The Reliability of Rabbit Monoclonal Antibodies in the Immunohistochemical Assessment of Estrogen Receptors, Progesterone Receptors, and HER2 in Human Breast Carcinomas

The reliability of the rabbit monoclonal antibodies SP1, SP2, SP3, and 4B5 was immunohistochemically assessed on a range of 96 invasive breast carcinomas and the results compared with those achieved with established antibody markers for estrogen receptors (6F11), progesterone receptors (PgR636), and HER2 (polyclonal A0485 and clone CB11), with HER2 status validated by fluorescence in situ hybridization (FISH) and silver in situ hybridization. Optimal results depended on the duration of microwave antigen-retrieval time and the use of a high pH buffer for rabbit and mouse estrogen receptor antibodies (SP1 and 6F11), although only on antigen-retrieval duration for the progesterone receptors SP2 and PgR636. The highest rate of concordance between HER2 overexpression and HER2 gene amplification was with the rabbit monoclonal antibodies (SP3 and 4B5) and FISH. Rabbit monoclonal antibodies are reliable alternatives to established antibody markers for the immunohistochemical testing of estrogen receptors, progesterone receptors, and HER2 in breast cancer.