Reduction In Uterine Weight Research Articles

Abstract P232: Estradiol Supplementation Prevents Ovariectomy-Induced Arterial Stiffness and Vascular Fibrosis

Background: Arterial stiffness is an independent predictor of cardiovascular events and mortality. Clinical data shows arterial stiffness increases rapidly after menopause, but mechanisms that mediate this change are not well known. The effect of ovariectomy (ovx) in rodent models is inconsistent in the literature. We hypothesize that the loss of estradiol (E2) contributes to ovx-induced arterial stiffening in a mouse model via fibrotic mechanisms, and that restoration of E2 will prevent these changes. Methods: Eight-week-old female C57/Bl6J mice received bilateral ovx or sham operation. Subcutaneous placebo or E2 (0.36 mg/day) pellets were implanted 2 weeks later to achieve three groups (sham+placebo, ovx+placebo, and ovx+E2). Aortic pulse wave velocity (PWV) was measured as an index of arterial stiffness. Blood pressure (BP) was measured by radiotelemetry. Aortic fibrosis was assessed by histological quantification of Masson’s trichrome staining and expressed as fold change in %fibrosis from sham. Pro-fibrotic genes were assessed in aortic tissue via quantitative PCR. Data presented are mean ± SEM. Results: Successful ovx was confirmed by reduction in uterine weight in the ovx group compared with other groups ( p <0.0001). At 6-weeks-post-surgery (N=16-18/group), PWV was increased in ovx (5.2±0.2 mm/ms) versus sham (3.4±0.1 mm/ms, p <0.0001) and ovx+E2 (3.5±0.1 mm/ms, p <0.0001) groups. In biweekly measures (N=4-5/group), PWV was greater in the ovx group at 4- and 6-weeks-post-surgery compared with sham ( p <0.0001) and ovx+E2 ( p <0.0001) groups, while PWV in the ovx+E2 group did not differ from sham at any time ( p ≥0.6). At 6-weeks post-surgery (N=5/group), there were no observed differences between groups for dark or light cycle systolic or diastolic BP, mean arterial pressure, or pulse pressure. Aortic fibrosis was increased in the ovx group (1.6-fold) compared with sham ( p =0.03) and ovx+E2 (0.9-fold, p =0.02) groups (N=10-12/group). Aortic fibrosis correlated with PWV at 6-weeks-post-surgery (R 2 =0.25, p <0.01). Aortic mRNA expression of collagen1a1 was increased in ovx versus ovx+E2 mice ( p =0.04, N=4-6/group). Conclusion: In conclusion, the restoration of E2 prevents ovx-driven arterial stiffening in female mice via a reduction in fibrotic remodeling of the aorta. The results also support that ovx is an appropriate model to study the mechanisms of menopause-induced arterial stiffening and that anti-fibrotic therapeutics may be beneficial in aging women.

Effects of smokeless tobacco on the uterine wall of adult non pregnant female swiss albino rats

This study was aimed to evaluated effects of smokeless tobacco on the uterine wall of female rats of Swiss albino species. This was a controlled experimental study with three groups of adult female Swiss albino rats. The study was conducted over a period of 31 days, at Animal House, Husbandry, and Veterinary Sciences at Sindh Agricultural University, Tandojam, and the Department of Anatomy, Isra University, Faculty of Medicine & Allied Medical Sciences, Hyderabad, Sindh, Pakistan. Thirty adult female Swiss albino rats were randomly assigned to three groups: Group A (control, no treatment), Group B (treated with 5% smokeless tobacco), and Group C (treated with 10% smokeless tobacco). Blood samples were collected on the 31st day to analyze Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) levels in the serum. After euthanization, uteruses were removed and weighed, followed by histological examination using Hematoxylin & Eosin and Mason's trichrome stains. The experimental groups (B and C) exhibited a significant reduction in uterine weight and decreased serum levels of FSH & LH. Histological examination showed cystically dilated submucosal glands and pronounced atrophy in the uteri of the experimental groups. Additionally, the myometrial wall thickness was also decreased. The use of smokeless tobacco negatively affects the uterine wall of female Swiss albino rats, resulting in histological changes and decreased serum levels of FSH and LH.

Vanillin-Based Indolin-2-one Derivative Bearing a Pyridyl Moiety as a Promising Anti-Breast Cancer Agent via Anti-Estrogenic Activity.

The structure-based design introduced indoles as an essential motif in designing new selective estrogen receptor modulators employed for treating breast cancer. Therefore, here, a series of synthesized vanillin-substituted indolin-2-ones were screened against the NCI-60 cancer cell panel followed by in vivo, in vitro, and in silico studies. Physicochemical parameters were evaluated with HPLC and SwissADME tools. The compounds demonstrated promising anti-cancer activity for the MCF-7 breast cancer cell line (GI = 6-63%). The compound with the highest activity (6j) was selective for the MCF-7 breast cancer cell line (IC50 = 17.01 μM) with no effect on the MCF-12A normal breast cell line supported by real-time cell analysis. A morphological examination of the used cell lines confirmed a cytostatic effect of compound 6j. It inhibited both in vivo and in vitro estrogenic activity, triggering a 38% reduction in uterine weight induced by estrogen in an immature rat model and hindering 62% of ER-α receptors in in vitro settings. In silico molecular docking and molecular dynamics simulation studies supported the stability of the ER-α and compound 6j protein-ligand complex. Herein, we report that indolin-2-one derivative 6j is a promising lead compound for further pharmaceutical formulations as a potential anti-breast cancer drug.

PMON210 Ovariectomy of Middle-Aged Female Mice Leads to Decreased Fat Oxidation and Energy Expenditure Accompanied by Weight Gain and Dysregulated Blood Glucose

Abstract Introduction In humans, menopause brings upon total body weight gain with increased visceral adiposity and a resulting increase in cardiometabolic risk profile. Although well described, the specific sequence and underlying mechanisms for this remain elusive. Rodent ovariectomy (OVX) is routinely used as an animal model for the study of human menopause but is often performed in young reproducing specimen, failing to replicate the complex interaction of concomitant chronological aging with the loss of sex hormones observed in humans. In this study we used middle-aged ovariectomized mice to explore the effect of concomitant estrogen deficiency with chronological aging on key aspects of cardiometabolic health and energy expenditure. Methods 10-month-old C57BLj/6 female mice, were subjected to a SHAM or OVX procedure and followed for 6 weeks post-surgery (n=5 per group). Non-fasting blood glucose (BG) and body weight were measured weekly. Prior to sacrifice mice were observed in metabolic cages using the Promethion Metabolic Phenotyping System (Sable Systems International). Body composition including total body fat and lean mass were evaluated by EchoMRI. A Reduction in uterine weight was used to confirm successful OVX. Results Relative to baseline weight, OVX mice weighed 13% more than SHAM mice 6-weeks after surgery (p= 0.04; t-test) with no significant change in body composition. Non-fasting BG was consistently higher throughout the study period in OVX vs SHAM mice (p<0.01; ANOVA). OVX mice exhibited an increased respiratory quotient (0.84 vs 0.81; p<0.01; t-test) driven by a reduction in fat oxidation (24 vs 34 g/d/kgeff. mass p<0.000; t-test). OVX mice showed a 17% percent decrease in total energy expenditure in both the light and dark phases (p<0.01), driven by a 38% percent decrease in ambulatory activity (p=0.04) and close to a 3-fold decrease in wheel running (p<0.001). No significant change was observed in overall food or water intake between the groups. Discussion It is difficult to detangle the complex web of causes and effects relating to the deranged metabolic phenotype induced by menopause. Here we show that even without additional insults such as a high-fat-diet or genetic manipulation, the combination of chronological aging at middle-age with the sex hormone deficiency resulting from OVX, led to an increase in total body weight, a dysregulated glucose profile and a deranged energy expenditure pattern. Additional studies are warranted using similar animal models to tease out cause and effect as well explore novel therapeutics and biomarkers for the rapidly increasing population of post-menopausal women at risk for cardiometabolic disease. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Abstract 4379: Not all selective estrogen receptor degraders are equal - Preclinical comparison of AZD9833, AZD9496 and fulvestrant

Abstract Anti-hormonal therapy has been the mainstay treatment for estrogen receptor (ER) +ve breast cancer for >40 years. The selective ER degrader (SERD) fulvestrant has demonstrated clinical benefit over aromatase inhibitors and the selective ER modulator tamoxifen. However, fulvestrant's efficacy may be limited by the intramuscular route of administration. To overcome this, AstraZeneca has developed orally bioavailable SERDs, the first generation compound AZD9496 and the next generation oral SERD AZD9833. Here we report the preclinical comparison of fulvestrant, AZD9496 and AZD9833. All three compounds induced equal ERα degradation in the ER+ breast cancer cell line MCF7. Importantly, analysis of other ER+ cell lines revealed AZD9833 phenocopied ERα degradation induced by fulvestrant, while AZD9496 only reduced ERα levels to 54% of that achieved by fulvestrant. Activity in the endometrial Ishikawa cell line demonstrated induction of progesterone receptor, an ER target gene, by AZD9496 but not AZD9833. RNAseq analysis of ER+ breast cancer cell lines treated with the three agents with and without estradiol did not reveal differences between the compounds; all estradiol-induced gene expression changes were completely reversed by the SERDs and no ER agonism was detected. By contrast, AZD9833 and fulvestrant produced an equivalent maximal anti-proliferative effect in MCF-7 and CAMA-1 cells, while AZD9496 induced a significantly inferior anti-proliferative effect. The pure anti-estrogen nature of AZD9833 and the partial agonist potential of AZD9496 was confirmed in vivo in a juvenile rat uterine assay. Like fulvestrant, AZD9833 caused a significant reduction in uterine weight and endometrial thickness, while AZD9496 produced a significant increase in both parameters. In MCF7 xenografts, both oral SERDs caused equivalent anti-tumour and pharmacodynamic effects to a supraclinical exposure of fulvestrant. However, in the patient derived xenograft CTC174 harbouring an ESR1 D538G mutation, AZD9833 caused equivalent anti-tumour and ERα degradation effects to supraclinical fulvestrant. By contrast, AZD9496 produced significantly poorer maximal anti-tumour and pharmacodynamic effects in this model compared to fulvestrant. These data demonstrate that AZD9833 phenocopied fulvestrant preclinically, as a SERD and pure anti-estrogen. It is, therefore, a suitable candidate to test the hypothesis that increasing the exposure of a SERD above what is achieved with fulvestrant can provide additional patient benefit. AZD9496, on the other hand, demonstrated inferior ERα degradation in several ER+ breast cancer cell lines and partial agonism. This may explain the result of the clinical window of opportunity study, comparing AZD9496 to fulvestrant, in which AZD9496 failed to demonstrate superior degradation of ERα, downregulation of PR expression and reduction in Ki67 positivity. Citation Format: Mandy Lawson, Natalie Cureton, Michelle DuPont, Oona Delpuech, Dawn Trueman, Pei Zhang, Azadeh Cheraghchi-Bashi-Astaneh, Sladjana Gagrica, Gareth Maglennon, Daniel Sutton, Bairu Zhang, Jonathan Cairns, Jamie Scott, Teresa Klinowska, Christopher J. Morrow. Not all selective estrogen receptor degraders are equal - Preclinical comparison of AZD9833, AZD9496 and fulvestrant [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4379.

MATERNAL AND FETAL TOXICITY-INDUCED BY NICKEL OXIDE NANOPARTICLES ADMINISTRATION IN ALBINO RATS DURING GESTATION

Objective: Despite the widespread of nickel oxide nanoparticles (NiO NPs) and their benefits in all fields, they have many negative effects on human life, especially expectant mothers and their fetus. The purpose of this study was to investigate the possible maternal and developmental toxicity-induced by NiO NPs administration during gestation.
 Methods: Three groups of pregnant rats were administered orally during days 5–19 of gestation, the pregnant rats were haphazardly designed into three groups (six rat/group), as follows: Control group and NiO NPs administered groups, low (4 mg/kg), and high (8 mg/kg) doses.
 Results: NiO NPs administration resulted in severe maternal and developmental toxicity which included reduction in uterine weight, mother weight gain, the average weight of placenta, the number corpora lutea, implantation sites, and the number of live fetuses. Furthermore, high pre/ postimplantation, fetal growth retardation, and morphological and skeletal anomalies, an elevation in liver and brain DNA damage in both mother and fetus, and histopathological alterations in different tissues (placenta, liver, kidney, and brain) of pregnant rats and fetuses. Lipid peroxidation showed a significant elevation in maternal, fetal liver, and brain tissues of NiO NPs ‐administered rats. Furthermore, glutathione content and catalase activity were decreased in both tissues of NiO NPs‐administered rats.
 Conclusion: Finally, the detrimental impacts of NiO NPs in dams and fetuses probably through its potential generation of reactive oxygen species.

Efficacy of a novel orally active SERD AZD9496 against hormone dependent post-menopausal breast cancer depends on inhibition of cellular aromatase activity

Treatment of hormone sensitive breast cancer tumors with endocrine therapy such as antiestrogens or aromatase inhibitors has improved the outcome significantly. Studies including our own have shown that downregulation of ERα with pure antiestrogen fulvestrant in combination with aromatase inhibitors may prolong responsiveness of the tumors to endocrine therapy. Fulvestrant has been studied as second line or first line treatment for post-menopausal hormone receptor positive breast cancers as a single agent or in combination with AIs. Studies have also suggested that further escalation of dose may improve benefit. However, dose escalation of fulvestrant, which is administered via intramuscular injection, is difficult due to its poor solubility. To overcome this shortcoming of an injectable drug, a novel orally active antiestrogen, AZD9496 was developed. In addition to being orally active, AZD9496 is designed as a selective ERα downregulator (SERD). In the current study, we compared the effect of AZD9496 and fulvestrant on the growth of MCF-7Ca (human estrogen receptor positive MCF-7 cells stably transfected with human placental aromatase gene) xenografts grown in ovariectomized athymic nude mice. AZD9496 was also compared to fulvestrant in vitro as a single agent or in combination with anastrozole. Our current study shows that AZD9496 is equally effective as fulvestrant at controlling the growth of hormone sensitive human breast cancer tumors. Similar to fulvestrant, AZD9496 inhibits cellular aromatase activity through ERα mediated signaling. However, unlike fulvestrant, combination of AZD9496 with anastrozole did not produce increased tumor inhibition. Our results show that AZD9496 was significantly better at inhibiting cellular aromatase which contributed to its anticancer activity. Next, we measured the effect of AZD9496 on the mouse uterus. Uterine weight of mice treated with AZD9496 was significantly lower than that for mice treated with androstenedione. This reduction in uterine weight was due to AZD9496 mediated inhibition of aromatase activity and not a direct effect on uterine ERα expression. We also observed that anti-cancer efficacy of AZD9496 depended on its ability to inhibit cellular aromatase. These results suggest that AZD9496 may be a better alternative to fulvestrant due to its selectivity for mammary ER and ability to inhibit aromatase in addition of downregulating ERα that can be obtained upon oral administration. As such, AZD9496 may prove to be a better option than fulvestrant for the treatment of hormone sensitive human breast cancer.

Using three-point bending to evaluate tibia bone strength in ovariectomized young mice.

It is well known that estrogen deficiency induces a deterioration of bone strength in aged females. The aim of this study is to determine the effect of estrogen depletion on tibia bone strength in sexually mature mice that are still undergoing skeletal maturation. At 8weeks of age, C57BL/6 female mice underwent an ovariectomy (OVX) or sham (SHAM) surgery. Mice were killed at 2, 4, or 8weeks post-surgery. Tibia length and cross-sectional area continued to increase in both treatment groups until 4weeks post-surgery. Compared to SHAM mice, OVX mice demonstrated a significant reduction in uterine weight and plasma estrogen levels. Three-point bending was used to quantify the mechanical properties (breaking point, stress, stiffness, and elasticity) of the tibia. The tibias from the SHAM mice had a higher breaking point than all the age-matched OVX mice. At 8weeks post-surgery, the tibias from the SHAM mice demonstrated higher elasticity, stress, and stiffness than the younger SHAM mice and the age-matched OVX mice. Compared to the SHAM mice, our study suggests that (1) there is a reduction in the mechanical strength of tibias from young OVX mice, and (2) the greatest decline in tibia strength of the OVX mice was once they reached skeletal maturity.

Abstract 60: Mig-6 suppresses development and progression of endometrial cancer by inhibiting ERK2 phosphorylation

Abstract Endometrial cancer is the most common cancer of the female reproductive system. PTEN is mutated or absent in more than half of human endometrial cancers. The major pathologic phenomenon of endometrial cancer is the loss of ovarian steroid hormone control over uterine epithelial cell proliferation and apoptosis. Mig-6 suppresses estrogen signaling. Here, we show the significance of MIG-6 in human endometrial cancer through sample analysis, where MIG-6 expression is inversely associated with ERK phosphorylation; this relationship is tightly correlated with endometrial cancer progression. To determine the tumor suppressor function of Mig-6 in the development of endometrial cancer, we generated Mig-6 conditional overexpression mice (R26Mig-6LSL). To assess the effects of Mig-6 on the PTEN/PI3K/AKT signaling pathway in uterine tumorigenesis, mice with Pten floxed (Ptenf/f) and R26Mig-6LSL were bred to the PRCre mouse model to generate overexpression of Mig-6 and ablation of Pten in the uterus (PRcre/+ R26Mig-6LSL Ptenf/f). PRcre/+ R26Mig-6LSL Ptenf/f showed significantly increased survival time and uterine weight compared to PRcre/+ Ptenf/f mice. Gross morphology and histological analysis displayed dramatically suppressed development of endometrial cancer in double mutant mice compared to ablation of Pten alone. Immunohistochemical analysis showed significantly increased apoptosis and decreased proliferation in epithelial cells of PRcre/+ R26Mig-6LSL Ptenf/f mice compared to PRcre/+ Ptenf/f mice. Interestingly, the expression of pERK1/2 was significantly decreased in PRcre/+ R26Mig-6LSL Ptenf/f mice compared to PRcre/+ Ptenf/f mice. To examine whether inhibition of ERK phosphorylation suppresses tumor progression in endometrial cancer, PRcre/+Ptenf/f mice were treated with U0126, an effective inhibitor of MAPK/ERK kinase. PRcre/+Ptenf/f mice treated with U0126 exhibited a significant reduction in uterine weight. Histopathological analysis of the entire animal cohort showed that inhibition of ERK phosphorylation suppressed endometrial cancer progression in PRcre/+ Ptenf/f mice, as reflected by the arrest of tumors at the hyperplastic or normal stage, whereas tumors from PRcre/+Ptenf/f mice treated with vehicle advanced to endometrial cancer. These results demonstrate that activation of ERK signaling is critical for endometrial cancer development and progression in Pten mutation. Our findings highlight a crucial tumor suppressor role for MIG-6 in progression of PTEN-null endometrial cancer by inhibiting ERK phosphorylation. As MIG-6 is a mediator of progesterone signaling, the activity of which can suppress unopposed-estrogen signaling, our studies provide a potential new drug target for the intervention of metastatic human endometrial cancer. (This work was supported by NIH, U54 HD007495 to J.P.L, NIH, R01 HD057873 and American Cancer Society Research Grant, RSG-12-084-01-TBG to J.W.J.) Citation Format: Jung-Yoon Yoo, Tae Hoon Kim, Hong Im Kim, Jane Li, Gordon B. Mills, Russell R. Broaddus, John P. Lydon, Ho-Geun Yoon, Jae-Wook Jeong. Mig-6 suppresses development and progression of endometrial cancer by inhibiting ERK2 phosphorylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 60. doi:10.1158/1538-7445.AM2014-60

In Utero and Lactational Exposure to Fluoxetine in Wistar Rats: Pregnancy Outcomes and Sexual Development

This study evaluated the reproductive effects of fluoxetine exposure in utero and during lactation on pregnancy outcomes and the sexual development of offspring. Pregnant Wistar rats were treated daily with fluoxetine (0.4, 1.7 and 17mg/kg/day) or distilled water by gavage from gestation day (GD) 7 to lactation day (LD) 21. A significant reduction in maternal body weight was observed during pregnancy and lactation in dams exposed to 17mg/kg fluoxetine. Hormone analysis revealed an increase in progestagen and glucocorticoid metabolites on GD 15 and oestrogen and progestagen metabolites on LD 7 in dams treated with 17mg/kg fluoxetine. Oestrogen metabolites also were increased on LD 7 in dams treated with 0.4mg/kg fluoxetine. Besides that, an increase in the weight of the adrenal glands and a reduction in uterine weight in dams exposed to highest dose of fluoxetine were observed. Finally, pup birthweight and the viability and weaning indices also were reduced in animals exposed to 17mg/kg fluoxetine. Overall, maternal hormonal changes were only observed at the highest dose tested, which also induced maternal and foetal toxicity. No significant changes were seen in dams or offspring exposed to therapeutic-like doses.

In vivo loss of estrogen increases voltage‐gated calcium channel function in mesenteric arterial smooth muscle cells

The presence of estrogen in premenopausal women protects against the development of vascular dysfunctions, such as hypertension. Previous studies from our lab demonstrated a slight (p=0.12) increase in voltage‐gated, L‐type Ca2+ channel (CaL) expression in mesenteric arteries from ovariectomized (OVX) mice. Therefore, in this study we hypothesized that the in vivo loss of estrogen will increase Ca2+ influx in mesenteric arterial smooth muscle cells. Mice (C57BL/6) underwent an ovariectomy or sham surgery at 8 weeks of age. At 12 weeks, the mice were sacrificed, mesenteric arteries dissected, and smooth muscle cells isolated. Lack of gonadal estrogen production was verified by a reduction in uterine weights. No significant difference (p=0.496) in weight gain between mice groups was observed. Ratiometric fluorescent imagining was conducted on isolated cells loaded with the Ca2+ fluorescent dye, fura‐2AM. Cells isolated from the mesenteric arteries of OVX mice (n=23) demonstrated an increased (p<0.001) intracellular Ca2+response to the CaL agonist, FPL64176 (1×10−6), compared to the sham mice (n=15). We plan to conduct whole cell Ca2+ currents on these cells in the future. These results suggest that the loss of in vivo estrogen leads to the enhanced influx of Ca2+into arterial cells which may lead to a chronic elevation of vascular tone in postmenopausal women. Support: NIGMM of the NIH, Grant #P20 GM103429–11

Postnatal uterine development in Inverdale ewe lambs

Postnatal development of the uterus involves, particularly, development of uterine glands. Studies with ovariectomized ewe lambs demonstrated a role for ovaries in uterine growth and endometrial gland development between postnatal days (PNDs) 14 and 56. The uterotrophic ovarian factor(s) is presumably derived from the large numbers of growing follicles in the neonatal ovary present after PND 14. The Inverdale gene mutation (FecXI) results in an increased ovulation rate in heterozygous ewes; however, homozygous ewes (II) are infertile and have 'streak' ovaries that lack normal developing of preantral and antral follicles. Uteri were obtained on PND 56 to determine whether postnatal uterine development differs between wild-type (++) and II Inverdale ewes. When compared with wild-type ewes, uterine weight of II ewes was 52% lower, and uterine horn length tended to be shorter, resulting in a 68% reduction in uterine weight:length ratio in II ewes. Histomorphometrical analyses determined that endometria and myometria of II ewes were thinner and intercaruncular endometrium contained 38% fewer endometrial glands. Concentrations of estradiol in the neonatal ewes were low and not different between ++ and II ewes, but II ewes had lower concentrations of testosterone and inhibin-alpha between PNDs 14 and 56. Receptors for androgen and activin were detected in the neonatal uteri of both ++ and II ewes. These results support the concept that developing preantral and/or antral follicles of the ovary secrete uterotrophic factors, perhaps testosterone or inhibin-alpha, that acts in an endocrine manner to stimulate uterine growth and endometrial gland development in the neonatal ewes.

POSTNATAL UTERINE DEVELOPMENT IN INVERDALE EWE LAMBS

Postnatal development of the uterus involves, particularly, development of uterine glands. Studies with ovariectomized ewe lambs demonstrated a role for ovaries in uterine growth and endometrial gland development between postnatal days (PNDs) 14 and 56. The uterotrophic ovarian factor(s) is presumably derived from the large numbers of growing follicles in the neonatal ovary present after PND 14. The Inverdale gene mutation (FecXI) results in an increased ovulation rate in heterozygous ewes; however, homozygous ewes (II) are infertile and have 'streak' ovaries that lack normal developing of preantral and antral follicles. Uteri were obtained on PND 56 to determine whether postnatal uterine development differs between wild-type (++) and II Inverdale ewes. When compared with wild-type ewes, uterine weight of II ewes was 52% lower, and uterine horn length tended to be shorter, resulting in a 68% reduction in uterine weight:length ratio in II ewes. Histomorphometrical analyses determined that endometria and myometria of II ewes were thinner and intercaruncular endometrium contained 38% fewer endometrial glands. Concentrations of estradiol in the neonatal ewes were low and not different between ++ and II ewes, but II ewes had lower concentrations of testosterone and inhibin-alpha between PNDs 14 and 56. Receptors for androgen and activin were detected in the neonatal uteri of both ++ and II ewes. These results support the concept that developing preantral and/or antral follicles of the ovary secrete uterotrophic factors, perhaps testosterone or inhibin-alpha, that acts in an endocrine manner to stimulate uterine growth and endometrial gland development in the neonatal ewes.

Effect of postnatal exposure to benzo[ a]pyrene on the uterus of immature rats

The objective of this study was to investigate the morphological effects of postnatal exposure to benzo[ a]pyrene (B[ a]P) on the development of the uterus, uterine estrogen receptor (ER α) expression, and the uterine response to estrogen stimulation using the uterotrophic bioassay in rats. Neonates were injected on each postnatal day (PND) 1–14 with B[ a]P (0.1, 1.0 and 10.0 mg/kg), ethynylestradiol (EE; 1.0 μg/kg) or vehicle (control group). All animals were killed on PND 23. Postnatal administration of B[ a]P with doses of 1.0 and 10.0 mg/kg induced significant ( P<0.01) reduction of uterine weight and significantly lowered ( P<0.05) ER α expression in the luminal epithelium. The increase in uterine weight and luminal epithelium heights after EE stimulation (1.0 μg/kg) on PND 20–22 was significantly higher ( P<0.01) in all groups in comparison with corresponding non-stimulated groups. However, the uterotrophic response in rats postnatally exposed to EE and B[ a]P was significantly lower ( P<0.01) than in controls. In the control and EE groups, EE stimulation on PND 20–22 induced a significant ( P<0.01) decrease in ER α immunoreactivity of the luminal epithelium. In contrast, rats postnatally treated with B[ a]P showed no change in the density of ER α immunostaining when detected after estrogenic stimulation. The present study showed that postnatal exposure to B[ a]P caused pathological changes in constitution and maturation of uterine ER α resulting in disturbed morphological development and uterine dysfunction in immature rats.

Effect of estrogen on calcium-handling proteins, β-adrenergic receptors, and function in rat heart

Regulation of cellular Ca 2+ cycling is central to myocardial contractile function. Loss of Ca 2+ regulation is associated with cardiac dysfunction and pathology. Estrogen has been shown to modify contractile function and to confer cardioprotection. Therefore, we investigated the effect of estrogen on expression of rat heart myocardial Ca 2+-handling proteins and beta-adrenergic receptor (β 1-AR) and examined functional correlates. Female rats were sham-operated (SHAM) or ovariectomized. Two weeks after ovariectomy rats were injected (i.p.) daily with estradiol benozoate (OVX + EB) or sesame oil (OVX) for 2 weeks. Protein abundance was measured by immunoblotting and mRNA was quantified by real-time RT-PCR. OVX significantly decreased estrogen and progesterone levels and EB replacement returned both estrogen and progesterone to physiological levels. OVX induced a 75% reduction of uterine weight and a gain in body weight. Replacement restored weights to SHAM level. OVX increased and estrogen-replacement normalized abundance of β 1-AR and L-type Ca 2+ channel (Cav1.2) protein. OVX decreased sodium–Ca 2+ exchange protein (NCX) and estrogen restored protein abundance to SHAM levels. Sarcoplasmic reticular ATPase (SERCA), phospholamban (PLB), and ryanodine receptor (RyR) abundance was not altered by hormone status. Levels of mRNA encoding for β 1-AR, Cav1.2, and NCX were not influenced by OVX or estrogen replacement. OVX had no effect on SERCA and PLB mRNA level but estrogen replacement elicited a significant increase compared to OVX and SHAM. Estrogen-dependent changes in Ca 2+-handling proteins and β 1-AR are theoretically consistent reduced myocellular Ca 2+ load. However, hormone-dependent alterations in protein were not associated with changes in contractile function.

Effect of isoflavone administration on age-related hepatocyte changes in old ovariectomized femal Wistar rats

Aging seems to be due to the accumulation of oxidative damage in cells and molecules. On the other hand, menopause and ovariectomy induce deleterious effects on different organs and systems that have been shown to be counteracted by estrogens and in a not so evident form also with phytoestrogens. The present study has investigated whether the administration of a commercial soy extract that contains approximately 10% isoflavones was able to modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female Wistar rats. Eighteen 22-month-old animals that had been previously ovariectomized at 12 months of age were divided into four groups: ovariectomized control rats, estradiol-treated ovariectomized females and ovariectomized rats treated with isoflavones. Six intact female rats of 2 months of age were used as reference group. Hepatocytes were isolated and cultured, and carbon monoxide (CO) and nitric oxide (NO) release, as well as adenosyl triphosphate (ATP), cyclic guanosyl monophosphate (cGMP), phosphatidylcholine (PC) and lipid peroxide (LPO) content of cells were evaluated. Uterus was also removed and weighed. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content as compared to young animals. Age also induced an increase in LPO cell content. NO, CO and cGMP were augmented with age, and PC synthesis showed a dramatic reduction. Treatment with either estradiol or isoflavones were able to improve all the mentioned parameters altered in hepatocytes isolated from old ovariectomized rats, and the magnitude of the improvement was similar for both treatments. Ovariectomy induced a significant reduction in uterine weight, which was significantly counteracted by estradiol treatment but not by isoflavone administration. In conclusion, the administration of a soy extract containing isoflavones seems to prevent oxidative changes in hepatocytes isolated from old ovariectomized female rats, without modifying uterus weight.

In vivo estrogen bioactivities and in vitro estrogen receptor binding and transcriptional activities of anticoagulant synthetic 17β-aminoestrogens

Estrogenic activities of the two 17β-aminoestrogen (AE) derivatives, prolame and butolame, were studied upon coagulation, serum luteinizing hormone (LH) and uterine weight, including endometrial morphology in castrated female rats. We have also investigated the ability of these two compounds, as well as another AE pentolame, to activate transcription through the estrogen receptor α (ERα) and the estrogen receptor β (ERβ). Administration of prolame and butolame to castrated animals increased significantly ( P < 0.01) the mean clotting time when compared with that obtained in the group of control animals. Butolame was a more potent anticoagulant than prolame ( P < 0.01), as judged by their corresponding IC 50 (5.4 ± 0.65 and 66.6 ± 2.57 μg/animal, respectively). In contrast, estradiol significantly shortened blood clotting times ( P < 0.005). Both prolame and butolame caused a significant inhibition of serum LH levels (EC 50 8.10 ± 0.79 and 17 ± 64 μg/animal, respectively), and restored castration-induced reduction in uterine weight of ovariectomized rats (EC 50 4.14 ± 1.57 and 17.0 ± 1.78 μg/animal, respectively). In terms of the effects of prolame, butolame and pentolame in transient transfection assays, all the three AE activated ER dependent reporter gene expression, however, only at high concentrations. Prolame had the highest activity followed by butolame and pentolame. Induction of transcription by these compounds was preferentially mediated through the ERα, especially in the case of pentolame where little, if any, activation occurred through the ERβ. None of the compounds showed antagonistic activities through either ER subtype. The overall data suggest that modifications in the structure and length of the amino-alcohol side-chain at C-17 might have an impact on the affinity and estrogenic intrinsic properties of AE at the level of diverse target tissues.

Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption.

Osteopontin is one of the major noncollagenous bone matrix proteins produced by osteoblasts and osteoclasts, bone cells that are uniquely responsible for the remodeling of mineralized tissues. Osteoclasts express the alphavbeta3 integrin, which is one of the receptors for osteopontin. Recent knockout studies revealed that noncollagenous bone matrix proteins are functionally important in regulation of bone metabolism. However, the significance of the presence of osteopontin in in vivo has not been known. We report here that osteopontin knockout mice are resistant to ovariectomy-induced bone resorption compared with wild-type mice. Microcomputed tomography analysis indicated about 60% reduction in bone volume by ovariectomy in wild-type mice, whereas the osteopontin-deficient mice exhibited only about 10% reduction in trabecular bone volume after ovariectomy. Reduction in uterine weight was observed similarly in both wild-type and osteopontin-deficient mice, indicating the specificity of the effect of osteopontin deficiency on bone metabolism. We propose that osteopontin is essential for postmenopausal osteoporosis in women. Strategies to counteract osteopontin's action may prove effective in suppressing osteoporosis.

Increase in calcium responsiveness of cardiac myofilament activation in ovariectomized rats

To evaluate a possible role of ovarian sex hormones in the Ca 2+ responsiveness of cardiac myofilament activation, the relations of pCa (−log Ca 2+ molar concentration) to actomyosin adenosine triphosphatase (ATPase) activity of isolated myofibrillar preparations from 8–10 week ovariectomized (Ovx) rat hearts were compared with those from sham-operated hearts. Deficiency of ovarian sex hormones in plasma of ovariectomized rats was indirectly verified by a significant reduction in uterine weights. Body weights of the ovariectomized rats were significantly greater than those of sham-operated controls. Despite a significant increase in heart weight of 10 week ovariectomized animals, the percent of heart weight-to-body weight ratio was not different from control group. The maximum myofibrillar ATPase activity at pH 7.0 was significantly suppressed after ovariectomy in both eight and ten week groups. However, the maximum ATPase activity at pH 6.5 was significantly suppressed only in 10 week ovariectomized hearts. Surprisingly, in every condition with depressed maximum myofibrillar ATPase activity, the pCa-actomyosin ATPase relationships of ovariectomized cardiac myofilaments demonstrated a significant leftward shift in pCa 50 (−log half-maximally Ca 2+ activation) from those of sham-operated controls. There was, however, no change in the Hill-coefficient of these cardiac myofilaments after ovariectomy. Analysis of myofilament proteins using gel electrophoresis demonstrated neither change nor loss of any thin filament proteins. These results indicate a possible modulating effect of ovarian sex hormone deficiency on the Ca 2+ responsiveness of cardiac myofilament activation by induction of myofilament Ca 2+ hypersensitivity but suppression of maximum myofibrillar ATPase activity.

Effects of melatonin on estrogen receptor expression in the forebrain of outbred (Lak.LVG) golden hamsters

Studies have shown that the pineal gland via its hormone, melatonin, induces the involution of male and female reproductive systems in seasonally reproducing animals. Melatonin has direct inhibitory effects on both hypothalamic and pituitary functions, which are also exquisitely sensitive to the feedback effects of estradiol. Since melatonin can modulate estrogen receptor (ER) expression in other tissues, immunocytochemical and ribonuclease protection analyses were used to examine the effects of 12 weeks of daily late afternoon injections of melatonin on ER protein and mRNA levels in the hypothalamus of Lak.LVG golden hamsters. Significant decreases in ER-immunoreactivity were noted in the medial preoptic area (MPOA) and bed nucleus of the stria terminalis (BNST) in response to melatonin, while other hypothalamic areas which express ER, e.g. the anterior hypothalamus, showed less dramatic changes. Hypothalamic ER mRNA was decreased in response to melatonin in both intact and ovariectomized animals by 25%. In intact, cycling female hamsters, there was a significant reduction in uterine weight after melatonin treatment. These results suggest that melatonin exerts its anti-reproductive effects in hamsters by modulating ER levels in neurons of the MPOA and BNST, thereby influencing steroid feedback mechanisms.