Activity In Ovariectomized Rats Research Articles

Co-encapsulation of combinatorial flavonoids in biodegradable polymeric nanoparticles for improved anti-osteoporotic activity in ovariectomized rats

In our study a novel nanocombination of flavonoids was synthesized for anti-osteoporotic therapeutics. We chose 2 flavonoids, quercetin and curcumin to be encapsulated in polylactic-co-glycolic acid (PLGA) for anti-osteoporotic activity in induced ovariectomized rats. The synthesized flavonoids co-encapsulated PLGA nanoparticles were analysedfor morphological changes with electron micrography and its zeta potential along with PDI was determined. The encapsulation and loading efficiency studies were performed along with in vitro release studies. Cytotoxicity of the synthesized nanoparticles was evaluated with primary bone cells. The administration of the flavonoid co-encapsulated nanoparticles was done intragastrically after which the physical parameters of the femur, compression test of lumbar vertebra were studied. Weight of ash, % of ash and ash calcium of femoral bone were calculated along with biochemical characterization of serum. The histopathological examination of femur was done too. The synthesized flavonoids co-encapsulated PLGA nanoparticles were small enough (110–135 nm) and exhibited homogeneous distribution. The high encapsulation and loading efficiencies demonstrated a favourable nanocombination. The cytotoxicity results exhibited the cytocompatibility of the nanoparticles. From the in-vivo studies, it was seen that the flavonoids co-encapsulated PLGA nanoparticles inhibited trabecular bone loss and significantly improved bone mineral density and bone microarchitecture. The flavonoids loaded PLGA nanoparticles augmented the protective effect to prevent bone loss after ovariectomy. This study may be furthered for novel therapeutic management of osteoporosis. • Quercetin and curcumin encapsulated in PLGA nanoparticles. • Encapsulation and loading efficiency studies with in-vitro release. • in-vivo studies show inhibited trabecular bone loss and improved bone density. • The protective effect on bone loss got augmented after ovariectomy.

Estrogenic Effect of Asparagus racemosus, Cissus quadrangularis, Punica granatum and Pueraria tuberosa in Post-menopausal Syndrome

Pharmacognosy Research,2021,13,4,238-245.DOI:10.5530/pres.13.4.17Published:October 2021Type:Original ArticleAuthors:Seema Singh, Santosh Kumar, Sunita Singh, Chetna Mishra, Dinesh Tripathi, and S K Verma Author(s) affiliations:Seema Singh1,*, Santosh Kumar1, Sunita Singh2, Chetna Mishra3, Dinesh Tripathi3, SK Verma1 1Department of Respiratory Medicine, King Georges Medical University, Lucknow, Uttar Pradesh, INDIA. 2Department of Microbiology, King Georges Medical University, Lucknow, Uttar Pradesh, INDIA. 3Department of Physiology, King Georges Medical University, Lucknow, Uttar Pradesh, INDIA. Abstract:Background: Asparagus racemosus, Cissus quadrangularis, Punica granatum and Pueraria tuberosa are employed for their supposed properties in treatment in alleviating symptoms of menopause traditionally from ages. The aim of the present study is to evaluate the estrogenic effect of Asparagus racemosus, Cissus quadrangularis, Punica granatum and Pueraria tuberosa in ovariectomized (OVX) rats. Materials and Methods: Female wistar rats were divided into sham control and ovariectomized (OVX), OVX rats receiving standard drug, raloxifene (5.4 mg/kg) and groups treated with 500 mg/kg and 700 mg/kg of test drug (containing equal quantity of Asparagus racemosus, Pueraria tuberose, Cissus quadrangularis and Punica granatum extracts) daily for 90 days. The vaginal cornification, uterine weight, bone loss, biomechanical, biochemical and histopathological observation were carried out to ascertain the effect of test drug in post-menopausal syndrome. Results: The experimental animals treated with mixture of extract of four drugs showed dose dependent activity. The significant increase in uterine weight, femur BMD, femur hardness, was observed. In addition, increased levels of calcium and phosphorus in serum and significant decreased in urine were observed as compared to control OVX group. The histopathological results also confirm the protective effect of extracts. Conclusion: The present findings strongly suggest that Asparagus racemosus, Cissus quadrangularis, Punica granatum and Pueraria tuberosa possess the potent estrogenic activity in ovariectomized rats and substantiate the ethnic use in treatment of postmenopausal osteoporosis. Keywords:Biochemical, Estrogenic, Histopathological, Menopause, Ovariectomized (OVX) rats, Post menopausal syndromeView:PDF (994.49 KB)

An isoflavone enriched diet increases skeletal muscle adaptation in response to physical activity in ovariectomized rats.

This study was to investigate anabolic adaptation of skeletal muscle in response to an isoflavone (ISO) enriched diet, training and their combinations in ovariectomized (OVX) rats. Female Wistar rats were sedentary, performed treadmill uphill running, received ISOs, or a combination of ISOs and running after ovariectomy. Body weight was increased by OVX. Both ISO and training treatment antagonized this increase. The weights of soleus and gastrocnemius muscles were increased only when training and ISOs were combined. In soleus muscle insulin-like growth factor (IGF)-1R, MyoD and Myogenin expressions were only up-regulated by training in Sham groups. However, a stimulation of IGF-1R and MyoD expression could be observed when ISOs and training were combined. In gastrocnemius muscle MyoD and Myogenin expressions were stimulated by either training or ISOs. Additive effects were detected when combining the two interventions. Our results indicate that the combination of ISOs and exercise is more efficient in increasing relative skeletal muscle mass and the expression of molecular markers related to anabolic adaptation in the skeletal muscle of female rats.

Anti-osteoclastogenic, estrogenic, and antioxidant activities of cell suspension cultures and tuber root extracts from Pueraria mirifica

The flavonoid contents of intact tubers and cell culture media were determined and physiological activities of Pueraria mirifica extracts were investigated. The total flavonoid contents from cell culture media (PMC) were higher than from tubers (PMT). Results from in vivo estrogenic activity assays indicated that PMT had a strong estrogenic activity in ovariectomized rats. The same amount of PMC exhibited a weak activity. In vitro osteoclast suppression investigations indicated that both PMT and PMC extracts exhibited anti-osteoclastogenic activities with low toxicities in a standard test cell line. Determination of the antioxidant potential using the DPPH assay revealed that the IC50 value for PMT was lower than for PMC. P. mirifica cell cultures produce more flavonoids and exhibit a mild estrogenic and more antioxidant activities than tubers.

Estrogenic activity of Punica granatum L. peel extract

Abstract Objective To evaluate the estrogenic effect of alcoholic extract of Punica granatum peel (ALPG) in ovariectomized (OVX) rats. Methods Female wistar rats were divided into sham control and ovariectomized (OVX), OVX rats receiving standard drug, raloxifene (5.4 mg/kg) and groups treated with 500 mg/kg and 750 mg/kg of ALPG daily for 90 days. The vaginal cornification, uterine weight, bone loss, biomechanical, biochemical and histopathological observation were carried out to ascertain the effect of test drug in post menopausal syndrome. Results The experimental animals treated with the ALPG showed dose dependent activity. The significant increase in uterine weight, femur BMD, femur hardness was observed. In addition, increased levels of calcium and phosphorus in serum and significant decrease in urine, were observed as compared to control OVX group. The histopathological results also confirm the protective effect of ALPG. Conclusions The present findings strongly suggest that the peel of P. granatum possess potent estrogenic activity in ovariectomized rats and substantiate the ethnic use in treatment of postmenopausal osteoporosis.

Bilateral Ovariectomy in Young Rats: What Happens in Their Livers during Cecal Ligation and Puncture Induced Sepsis?

Study ObjectivePediatric ovarian masses comprise a heterogeneous group of benign and malignant lesions. Surgical methods consist of emergency or programmed surgery with tumoral resection and uni/bilateral oophorectomy or salpingo-oophorectomy. We examined whether bilateral ovariectomy (OVX) worsens liver injury during the onset of cecal ligation and puncture (CLP)-induced sepsis in rats. DesignThe rat groups were: sham, bilateral-OVX, sepsis, and OVX-sepsis. SettingsAfter OVX operation, rats were allowed to recover for 12 weeks. At the end of recovery, CLP was applied 16 hours after sepsis induction. Main OutcomeThere was a significant difference in the numerical density of hepatocytes only between the sepsis and the OVX-sepsis groups. Serum ALT and AST were increased significantly in the OVX-sepsis group. NF-κB activation after OVX increased after induction of sepsis. OVX-sepsis group showed marked thrombosis in portal vein branches and the central vein, degeneration in the bile ducts, and widespread ischemic areas in liver sections. Intra-inflammatory cell invasion was observed in both the portal and intrasinusoidal areas. DiscussionThis study indicates that increases in liver NF-κB activity in ovariectomized rats following CLP-induced sepsis correlates with elevated levels of serum ALT and AST and with histopathologic changes in rat liver. Bilateral OVX therefore appears to play a role in the activation of NF-κB or in production of cytokines in liver cells. Thus, we provided novel insight into the effects of OVX on liver injury following CLP-induced sepsis.

Testing of the estrogenic activity and toxicity of Stephania venosa herb in ovariectomized rats

Stephania venosa Spreng is a traditional herb which has been used for cancer treatment as well as an aphrodisiac. The scientific literature strongly supports its in vitro antiproliferative effects on cancer cell lines and has suggested developing this plant as a potential anticancer drug. However, the in vivo steroidogenic activity and toxicity of this plant have never been tested. We analyzed the levels of five key isoflavones in the plant extract by quantitative HPLC and then evaluated the in vivo estrogenic activity and toxicity in ovariectomized rats, in comparison with the phytoestrogen-rich plant, Pueraria mirifica. Twenty rats were first ovariectomized, and then seven days later divided into four groups and gavaged daily with 0, 10 and 100 mg/kg body weight/day of S. venosa, or 100 mg/kg body weight/day of P. mirifica for 28 days. A trace amount of puerarin, daidzin and daidzein with a subtle amount of genistein and genistin were isolated from the S. venosa tuber extract. S. venosa tuber powder, at both doses, did not exhibit any detectable estrogenic activity in ovariectomized rats, as assessed by the vaginal cytology and uterotropic assays, whilst P. mirifica induced a remarkable vaginal and uterine proliferation. S. venosa induced a toxicological effect on the hematological values and histopathological appearance of metabolic organs. Taken together, these results suggest that S. venosa has no discernable estrogenic activity but that it is toxic, at least to ovariectomized rats. Thus, the use of this plant for anticancer treatment needs to be reassessed or used with caution.

Ginsenoside Rb1 inhibits osteoclastogenesis by modulating NF-κB and MAPKs pathways

Ginsenosides (GSS), the main active components of ginseng, have been reported possessing anti-osteoporosis activity in ovariectomized rats. However, the active ingredient and the mechanisms underlying the anti-osteoporosis activity of GSS have not been clearly elucidated. In the present study, we determined the effect of ginsenoside Rb1, a major component of ginsenosides, on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. Ginsenoside Rb1 inhibited RANKL-induced osteoclast differentiation from Raw264.7 cells without cytotoxicity. Ginsenoside Rb1 also inhibited RANKL-induced TNFα mRNA expression in Raw264.7 cells. Pretreatment with ginsenoside Rb1 significantly inhibited RANKL-induced the gene expression of c-Fos and nuclear factor of activated T-cells c1 (NFATc1), which are two essential and crucial transcription factors for osteoclast formation. Rb1 inhibited RANKL-induced nucleus translocation and activation of NF-κB, the upstream factor of c-Fos and NFATc1. Among the three well known mitogen-activated protein kinases (MAPKs), Rb1 inhibited RANKL-induced JNK and p38 phosphorylation, but not ERK1/2. Taken together, our data suggest that ginsenoside Rb1 is one of the effective components of GSS for the anti-osteoporosis activity and can inhibit osteoclastogenesis by suppressing RANKL-induced activation of both JNK and p38 MAPKs and NF-κB pathways, and consequently down-regulating the gene expression of c-Fos and NFATc1 in osteoclast precursors.

Anti-osteoporotic activity of aqueous-methanol extract of Berberis aristata in ovariectomized rats

Traditionally Berberis aristata is employed for its supposed properties in treatment of joint pain and also used in alleviating symptoms of menopause. The aim of the present study is to evaluate the antiosteoporotic effect of Berberis aristata in ovariectomized (OVX) rats. Sprague-Dawley rats were divided into sham and OVX groups. The OVX rats were further divided into four groups, which received standard estrogen (0.0563 mg/kg) and 100, 300, and 500 mg/kg aqueous-methanol extract of Berberis aristata, daily for 42 days. The uterine weight, bone loss, ash content, biomechanical, biochemical and histopathological observation were carried out for antiosteoporotic activity. The experimental animals treated with Berberis aristata aqueous-methanol extract showed dose dependent activity. The significant increase in uterine weight, femur BMD, ash content and lumbar hardness were observed. In addition, increased levels of calcium and phosphorus in serum and significant decreased in urine were observed as compared to control OVX group. The histopathological results also confirm the protective effect of extract. The present findings strongly suggest that Berberis aristata possess the potent antiosteoporosis activity in ovariectomized rats and substantiates the ethnic use in treatment of postmenopausal osteoporosis.

Moderate intensity of regular exercise improves cardiac SR Ca2+ uptake activity in ovariectomized rats

The impact of regular exercise in protecting cardiac deteriorating results of female sex hormone deprivation was evaluated by measuring changes in intracellular Ca2+ removal activity of sarcoplasmic reticulum (SR) in ovariectomized rats following 9-wk treadmill running exercise at moderate intensity. Despite induction of cardiac hypertrophy in exercised groups of both sham-operated and ovariectomized rats, exercise training had no effect on SR Ca2+ uptake and SR Ca(2+)-ATPase (SERCA) in hormone intact rat heart. However, exercise training normalized the suppressed maximum SR Ca2+ uptake and SERCA activity in ovariectomized rat heart. While exercise training normalized the leftward shift in pCa (-log[Ca2+])-SR Ca2+ uptake relation in ovariectomized rats, no effect was detected in exercised sham-operated rats. Similar phenomena were also observed on SERCA and on phospholamban (PLB) phosphorylation levels; exercise training in ovariectomized rats enhanced SERCA expression to reach the level as that in sham-operated rats, in which there were no differences in SERCA and phospho-PLB levels between sedentary and exercised groups. In addition, the reduction in phospho-Thr(17) PLB in myocardium of ovariectomized rats was abolished by exercise training. These results showed that regular exercise maintains the molecular activation of cardiac SR Ca2+ uptake under normal physiological conditions and is able to induce a protective impact on cardiac SR Ca2+ uptake in ovarian sex hormone-deprived status.

Neuroactive steroids inhibit spinal reflex potentiation by selectively enhancing specific spinal GABA A receptor subtypes

Recently, we demonstrated a spinal GABA A receptor (GABA AR)-dependent inhibition on the induction of repetitive stimulation-induced spinal reflex potentiation. However, it remains unclear whether steroid hormones modulate such an inhibition. Here, we show that progesterone is capable of producing GABA ARs-dependent inhibition of the induction of spinal reflex potentiation by actions through neurosteroid metabolites. Progesterone (5 mg/kg, twice daily for 4 days) up-regulates the expression of GABA AR α2, α3, α4 and δ subunits, and is associated with attenuated repetitive stimulation-induced spinal reflex activity in ovariectomized rats. These changes were blocked by finasteride (50 mg/kg, twice daily), an antagonist of neurosteroid synthesis from progesterone, but not by the progesterone receptor antagonist, RU486 (100 mg/kg, twice daily). The induction of spinal reflex potentiation was attenuated after a short (30 min) intrathecal treatment with the neurosteroids, allopregnanolone (ALLOP, 10 μM, 10 μL) and 3α,5α-tetrahydrodeoxycorticosterone (THDOC, 10 μM, 10 μL). Acute intrathecal administration of the GABA AR antagonist, bicuculline (10 μM, 10 μL) reversed the inhibition produced by progesterone, THDOC and allopregnanolone. These results imply that progesterone-mediated effects on GABA AR expression and neural inhibition are regulated by neurosteroids synthesis rather than progesterone receptor activation.

Effect of OS-0544, a selective estrogen receptor modulator, on endothelial function and increased sympathetic activity in ovariectomized rats

Estrogens are known to contribute to endothelial function and sympathetic activity, both of which are strongly associated with the pathogenesis of ischemic heart disease. In addition, estrogens improve impaired lipid profile, a risk factor of endothelial dysfunction. In this study, we investigated the effects of OS-0544, a structurally new selective estrogen receptor modulator (SERM), on endothelial function, sympathetic activity, and plasma cholesterol level in ovariectomized (OVX) rats. Female Sprague–Dawley rats were ovariectomized and orally treated with OS-0544 (or OS-0689, the ( R)-enantiomer of OS-0544), or 17β-estradiol (E 2) for 4 weeks, starting the next days after ovariectomy or for 1 week, starting 6 weeks after ovariectomy. Ovariectomy significantly increased vasopressin-induced mean blood pressure (AVP-MBP) (57 ± 3.3 mm Hg vs. 46 ± 3.5 mm Hg, P < 0.05) and decreased acetylcholine (Ach)-induced maximum vasorelaxation response (69 ± 5.6% vs. 81 ± 4.0%, P < 0.05). OS-0544 significantly inhibited AVP-MBP elevation (46 ± 3.5 mm Hg vs. 57 ± 3.3 mm Hg, P < 0.05) and decreased Ach-induced maximum vasorelaxation response (90 ± 3.3% vs. 69 ± 5.6%, P < 0.05) in OVX rats. In addition, OS-0689 as well as E 2 significantly reduced (up to 67%) the increase in sympathetic activity in OVX rats. Moreover, like E 2, OS-0544 significantly decreased plasma cholesterol level in OVX rats. These results demonstrate that OS-0544 has vascular protective effect on vascular function after ovariectomy. It is therefore believed that OS-0544 has vascular protective effect in postmenopausal woman.

Sex steroids modulate rat anterior pituitary and liver iodothyronine deiodinase activities.

In this study, we investigated the sex hormone regulation of 5'-iodothyronine deiodinase activity, which is responsible for enzymatic conversion of thyroxine into the bioactive form, triiodothyronine. Pituitary homogenates and liver microsomes from: 1) ovariectomized rats injected with 17-beta-estradiol benzoate and/or progesterone (0.7 and 250 microg/100 g body weight, respectively, subcutaneously, over 10 days); 2) male castrated rats treated or not with 0.4 mg/100 g body weight testosterone propionate, intramuscular, over 7 days, were assayed for type 1 and type 2 deiodinase activity in the pituitary. Enzyme activities were measured by release of (125)I from deiodination of (125)I reverse triiodothyronine under varying assay conditions. Estrogen stimulated anterior pituitary and liver type 1 deiodinase activity in ovariectomized rats (45 and 30 %, p < 0.05). Progesterone inhibited the liver enzyme (40 %, p < 0.05), and had no effect on the pituitary, but in both tissues, blocked estrogen stimulatory effect on type 1 deiodinase. In males, testosterone normalized the reduced liver type 1 deiodinase of castrated rats. However, in the pituitary, castration increased (50 %) type 1 deiodinase independent of testosterone treatment, suggesting the existence of a inhibitory testicular regulator of pituitary type 1 enzyme. Treatments did not alter pituitary type 2 deiodinase activity. In conclusion, gonads and sex steroids differentially modulate type 1 deiodinase activity in rat pituitary and liver.

Nestorone®: a progestin with a unique pharmacological profile

Nestorone® (Nestorone 16-methylene-17α-acetoxy-19-norpregn-4-ene-3,20-dione), formerly referred to as ST 1435, is a potent progestin when given parenterally via sustained release formulations. The pharmacological profile of Nestorone was compared with that of levonorgestrel and 3-keto-desogestrel by steroid receptor binding studies and by in vivo bioassays in rats and rabbits. 3-Keto-desogestrel showed the highest binding affinity to progesterone receptors (PR) followed by Nestorone, levonorgestrel, and progesterone. The binding affinity of Nestorone to androgen receptors (AR) was 500- to 600-fold less than that of testosterone. However, both levonorgestrel and 3-keto-desogestrel showed significant binding (40 to 70% of testosterone) to AR. None of the progestins bound to estrogen receptors (ER). The progestational activity of Nestorone, levonorgestrel, and progesterone was compared using McPhail index in immature rabbits and pregnancy maintenance and ovulation inhibition tests in rats after subcutaneous (s.c.) administration. In all three tests, Nestorone was the most potent progestin. The progestational activity of Nestorone was also compared after oral and s.c. administration in rabbits. The potency of Nestorone was over 100-fold higher upon s.c. administration than via the oral route. The androgenic activity of progestins, based on the stimulation of ventral prostate (androgenic target) and levator ani (anabolic target) growth in castrated immature rats, showed good correlation with their binding affinity to AR. Nestorone showed no androgenic or anabolic activity. Nestorone did not bind to sex hormone binding globulin (SHBG), whereas both levonorgestrel and 3-keto-desogestrel showed significant binding to SHBG. The estrogenic/antiestrogenic activity of Nestorone was investigated in immature ovariectomized rats. In contrast to estradiol and levonorgestrel, Nestorone showed no uterotropic activity in ovariectomized rats. Despite significant binding to glucocorticoid receptors (GR), Nestorone showed no glucocorticoid activity in vivo. It is concluded that a strong progestational activity, combined with lack of androgenic, estrogenic, and glucocorticoid-like activities, confer special advantages to Nestorone for use in contraception and hormone replacement therapy.

Raloxifene and estradiol benzoate both fully restore hippocampal choline acetyltransferase activity in ovariectomized rats

Selective estrogen receptor modulators (SERMs) demonstrate tissue-specific estrogen receptor (ER) agonist or antagonist properties. Raloxifene, a prototypical SERM, has ER agonist properties in bone and on cholesterol metabolism but full antagonist properties in the uterus and breast. To characterize the ER agonist/antagonist profile of raloxifene in the brain, we have examined its effect on the activity of a known estrogen-responsive gene product, choline acetyltransferase (ChAT), in the hippocampus and other brain regions of 6-month-old ovariectomized (OVX) Sprague–Dawley rats. Three weeks post-ovariectomy, animals received estradiol benzoate (EB, 0.03 mg or 0.3 mg kg -1 day -1 for 3 or 10 days); raloxifene HCl (3.0 mg kg -1 day -1 for 3 or 10 days), tamoxifen (3.0 mg kg -1 day -1 for 10 days) or vehicle (20% CDX). As previously reported, ChAT activity decreased by approximately 20%–50% in the hippocampus of OVX compared with SHAM-operated control rats with no change in ChAT activity observed in the hypothalamus. Raloxifene or EB reversed the OVX-induced decrease in ChAT activity in the hippocampus but did not change ChAT activity in the hypothalamus. Animals that received combined EB (0.03 mg/kg) plus raloxifene (1 mg/kg) or tamoxifen alone (3.0 or 10 mg/kg) also showed increased hippocampal ChAT activity. Raloxifene failed to increase uterine weight and blocked the estrogen-induced increase in uterine weight, while another SERM, tamoxifen, increased uterine weight. These data demonstrate that raloxifene has estrogen-like properties on hippocampal ChAT activity in vivo, and suggest that benzothiophene SERMs may exert estrogen-like beneficial effects on cholinergic neurotransmission in brain without producing peripheral stimulation of breast or uterine tissue.

Estrogenic and progestational activity of 7 α-methyl-19-nortestosterone, a synthetic androgen

Synthetic androgens exhibit estrogenic/antiestrogenic and progestational activities in addition to their androgenic effects. To investigate the pharmacological action of the synthetic androgen, 7 α-methyl-19-nortestosterone (MENT), we examined its action in female rodents. The criteria employed for estrogenic/antiestrogenic effects were, uterine weight increase, vaginal cornification, induction of progesterone receptors (PR) synthesis and stimulation of peroxidase activity in the uteri of ovariectomized rats and mice. MENT increased uterine weight in a dose dependent manner, but did not cause vaginal cornification or stimulate PR synthesis in the uterus. The uterotropic activity of MENT was 200-fold lower than that of estradiol. Estrogen receptor (ER) bound [ 3H]-E 2 was displaced by E 2 and MENT with ED 50 values of 70 pg and 250 ng, respectively, a 3,500 fold difference in their binding affinity. The low binding of MENT to ER, in contrast to its relatively high uterotropic action, suggested that receptors other than ER may be involved in its action on the uterus. The progestational activity of MENT in immature rabbits using the McPhail index assay was comparable to that of progesterone. Binding affinities of MENT and progesterone to PR were also comparable. However, the action of MENT on the uterus does not seem to be a progestational effect since mifepristone, an antiprogestin, had no effect on MENT-induced uterine growth. Specific androgen receptors (AR) in uterine cytosol were demonstrated. The involvement of AR in MENT action was confirmed by using an antiandrogen (flutamide) and an antiestrogen (ICI-182) in ovariectomized mice. Although MENT did not block the uterotropic effect of E 2, it inhibited the E 2-induced cornification of vaginal epithelium, induction of uterine PR synthesis and increase in uterine peroxidase activity in ovariectomized rats. The antiestrogenic effect of MENT was also blocked by flutamide. These results suggest that the uterotropic and antiestrogenic effects of androgens are mediated via AR. It is concluded that the increase in uterine weight caused by MENT is attributable to its anabolic effects.

Dissection of the molecular mechanism of action of GW5638, a novel estrogen receptor ligand, provides insights into the role of estrogen receptor in bone.

The estrogen receptor (ER) mixed agonists tamoxifen and raloxifene have been shown to protect against bone loss in ovariectomized rats. However, the mechanism by which these compounds manifest their activity in bone is unknown. We have used a series of in vitro screens to select for compounds that are mechanistically distinct from tamoxifen and raloxifene in an effort to define the properties of an ER modulator required for bone protection. Using this approach, we identified a novel high affinity ER antagonist, GW5638, which when assayed in vitro functions as an ER antagonist, inhibiting the agonist activity of estrogen, tamoxifen, and raloxifene and reversing the "inverse agonist" activity of the pure antiestrogen ICI182,780. Thus, GW5638 appears to function as an antagonist in these in vitro systems, although in a manner distinct from other known ER modulators. Predictably, therefore, GW5638 alone displays minimal uterotropic activity in ovariectomized rats, but will inhibit the agonist activity of estradiol in this environment. Unexpectedly, however, this compound functions as a full ER agonist in bone and the cardiovascular system. These data suggest that the mechanism by which ER operates in different cells is not identical, and that classical agonist activity is not required for the bone protective activity of ER modulators.

An endogenous cholinergic rhythm may be involved in the circadian changes of tuberoinfundibular dopaminergic neuron activity in ovariectomized rats treated with or without estrogen.

We recently reported that a circadian change in the activities of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons exists in ovariectomized (OVX) rats treated with or without estrogen. The involvement of an endogenous cholinergic control mechanism was the focus of this study. Adult female Sprague-Dawley rats OVX for 2 weeks and treated with or without a long-acting estrogen (poly-estradiol phosphate, 0.1 mg/rat, sc) were used in the study. An intracerebroventricular cannula for drug injection was implanted into the lateral cerebroventricle of each rat 6 days before experiment. TIDA neuron activity was determined by measuring the concentrations of 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylalanine in the median eminence by HPLC plus electrochemical detection. Serum PRL levels were determined by RIA. Neither atropine nor mecamylamine, two cholinergic receptor antagonists, had any effect on TIDA neuron activity in the morning before 1200 h, when endogenous TIDA neuron activity is high. Both drugs, however, exhibited a dose-related stimulating effect on the TIDA neuron activity in the afternoon, when endogenous activity is low. The estrogen-induced afternoon PRL surge was also blocked by a single injection of atropine or mecamylamine at 1300 h. The rhythmic changes in endogenous TIDA neuron activity and their responses to atropine were also observed in OVX rats with no estrogen replacement. In conclusion, a daily change in endogenous cholinergic neuron activity may be responsible for the change in TIDA neuron activity in female rats, which is a prerequisite for the PRL surge.

Differential effects of sex steroids on uterine and renal ODC activity in ovariectomized rats

Many hormones are known to induce the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme for polyamine biosynthesis, in their target tissues. Using ovariectomized rats, we have compared the effects of sex hormones on ODC activity in the uterus and the kidney which contain estrogen and androgen receptors. The results show that 1) both estrogen and androgen stimulate renal ODC activity, 2) estrogen but not androgen effectively increases ODC activity in the uterus, 3) estrogen at higher dosage can stimulate renal ODC activity to an extent similar to that in the uterus, 4) daily treatment with estradiol for 5 days results in the desensitization of uterine ODC activity, but not that of renal ODC activity to the hormonal stimuli. Although both uterus and kidney are targets of sex hormones, our results indicate that estrogen and androgen have differential influences on the ODC activity in these two organs.

Disruption of Estrogen-induced Facilitation of Spontaneous Activity in Female Rats by Preoptic Transection

We examined the effects of preoptic knife cuts on estrogen-induced facilitation of locomotor activity in ovariectomized rats to study the possible involvement of the preoptic area in the facilitation of activity. Animals housed in a cage with a running wheel received ovariectomy and one of the following brain surgeries : anterior cut (AC), posterior cut (PC), or dorsal cut (DC) of the preoptic area. Running activity was significantly suppressed following the ovariectomy. The activity of animals with DC was significantly lower than that of the ovariectomized controls. One week after the surgery, they were treated with estradiol-17β using silastic capsules. Recovery of activity in the running wheel was found in animals with AC and in the controls, but the activity was still lower in both PC and DC animals. Open field tests were also carried out on 3 consecutive days starting on the 5th day following the treatment of estrogen. Only DC animals showed significantly lower activity in the open field tests than control animals. These results indicate that facilitation of wheel-running and open field activities by estrogen may be related to different neural mechanisms in the rat forebrain, and the preoptic area may be actively involed in estrogen-induced facilitation of wheel-running but not of open field activity.