Chronic nitric oxide deficiency is associated with altered leutinizing hormone and follicle-stimulating hormone release in ovariectomized rats

Nitric oxide (NO) synthase (NOS) has been found in the gonadotrophs and folliculo-stellate cells of the anterior pituitary. Previous observations from our laboratory suggest that NO may play a role in regulating gonadotropin secretion. Because estrogen secretion by the ovary can influence gonadotropin secretion, we investigated the hypothesis that chronic in vivo NO deficiency has a direct estrogen-independent effect on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Chronic NO deficiency was induced by adding an NOS inhibitor, N-nitro-L-arginine (L-NNA, 0.6 g/l) to the drinking water of ovariectomized (OVX) rats. The control OVX rats were untreated. After 6-8 weeks, the animals were sacrificed, and the pituitaries were removed and perfused continuously for 4 hr in the presence of pulsatile gonadotropin-releasing hormone (GnRH, 500 ng/pulse) every 30 min. S-Nitroso-L-acetyl penicillamine (SNAP, an NO donor, 0.1 mM) or L-nitro-arginine methyl ester (L-NAME, an NOS inhibitor, 0.1 mM) was added to the media and perfusate samples were collected at 10-min intervals. GnRH-stimulated LH and FSH levels were significantly lower in pituitaries from OVX/NO-deficient pituitaries compared with pituitaries from the OVX control group. The addition of SNAP significantly decreased LH and FSH secretion by pituitaries from OVX control animals, but significantly increased their secretion by pituitaries from the OVX/NO-deficient animals. L-NAME also suppressed LH and FSH secretion by pituitaries from the OVX control animals and stimulated their release by pituitaries from the NO-deficient/OVX animals. Immunohistochemistry of frontal sections through the hypothalamus demonstrated that OVX/NO deficiency is associated with increased GnRH in the median eminence. We conclude that NO has a chronic stimulatory effect on LH and FSH release and the subsequent altered secretory responsiveness to NO agonist or antagonist is the result of chronic NO suppression.     

Effects of dehydroepiandrosterone, Premarin and Acolbifene on histomorphology and sex steroid receptors in the rat vagina

To assess the specific estrogenic and/or androgenic effects of a potential novel hormone replacement therapy, we have examined the morphology of the rat vagina 9 months after ovariectomy (OVX) and treatment of OVX animals with dehydroepiandrosterone (DHEA), conjugated estrogens Premarin and the selective estrogen receptor modulator Acolbifene. OVX led to atrophy and inflammatory changes while Acolbifene reduced the inflammation incidence and induced mucification of the vaginal epithelium. Premarin induced a typical keratinized stratified squamous epithelium while DHEA induced stimulation of the vaginal epithelium, with mucous cells typical of an androgenic effect, combined with increased collagen fiber compactness of the lamina propria. On the other hand, after OVX, the vaginal muscle layer decreased by 46%, an effect which was 41 and 100% reversed by DHEA and Premarin, respectively. The present data show particularly interesting effects of DHEA on the three layers of the vaginal wall, namely a highly mucified epithelium, an increased muscularis thickness and increased collagen fiber compactness in the lamina propria. DHEA exerts both androgenic and estrogenic effects on the vaginal mucosa, thus providing a more physiological replacement therapy.     

Relationship between timing of ovariectomy and maintenance of pregnancy in the guinea-pig

The effect of ovariectomy (OVX) and OVX + progesterone (P) treatment on the regulatory profile and uterine function of the guinea-pig are described. Before day 23 of gestation in the intact pregnant guinea-pig, the placental contribution to P-content is small in comparison with the increasing ovarian contribution. After day 30, the ovarian P-content starts to decrease and the placental P-content exceeds the ovarian contribution, indicating the “luteo-placental shift” (LPS) in P-biosynthesis. Thus, when 14 guinea-pigs were ovariectomized around day 21 of gestation all started aborting within 53 hours of OVX. A gradual increase in intrauterine pressure (IUP), a decrease in P-levels in heart and uterine vein plasma and in the uterine and placental tissues and an increase in the levels of PGF in uterine vein plasma and uterine tissue were observed in these animals. However, if the OVX was delayed until after day 30 of gestation, to examine the biological consequences of advanced LPS in P-biosynthesis, there was no increase in IUP and the animals did not abort in the next 5 days. Furthermore, P-therapy following OVX in the day 21 pregnant guinea-pigs prevented the increase in IUP and the animals did not abort. These observations establish for the guinea-pig a correlation between the success in pregnancy maintenance and the degree of the LPS in P-biosynthesis. These studies therefore emphasize the indispensable role of progesterone in pregnancy maintenance.     

A comparison of ovariectomy models for estrogen studies

Many estrogen-replacement studies use ovariectomized animals as controls. However, ovariectomy greatly increases body weight and can enhance the peripheral synthesis of estrogen. Tamoxifen is commonly used as an antiestrogen, but it may elicit mixed agonist or antagonist actions. The aim of our study was to compare vascular function in mesenteric arteries among groups of rats with low estradiol levels. The groups (n = 5, each) of Sprague-Dawley rats were cycling (diestrus), ovariectomized (OVX), OVX + tamoxifen (OVX-T), OVX + 4-hydroxyandrostene-3,17-dione, an aromatase inhibitor (OVX-A) to prevent peripheral synthesis of estrogen, and control-fed OVX to prevent excess weight gain. Body weight was significantly elevated in only the non-control-fed OVX group. Estrogen levels were significantly greater in the cycling rats compared with the other groups, whereas uterine weights were significantly reduced in only the OVX-A and control-fed OVX groups. Methacholine relaxation was blunted only in the OVX-A and control-fed OVX groups, suggesting a possible estrogenic influence in the non-control-fed OVX and OVX-T groups. These data indicate the potential for confounding factors to decrease the efficacy of OVX controls.     

Oxytocin secretion induced by osmotic stimulation in rats during the estrous cycle and after ovariectomy and hormone replacement therapy

Hyperosmolality is a potent stimulus for the secretion of oxytocin. Oxytocinergic neurons are modulated by estrogen and oxytocin secretion in rats varies according to the phase of the estrous cycle, with higher activity during proestrus. We investigated the oxytocin secretion induced by an osmotic stimulus (0.5 M NaCl) in female rats. Plasma oxytocin and the oxytocin contents in the neurohypophysis and the paraventricular and supraoptic nuclei were determined during the morning (8-9 h) and afternoon (17-18 h) of the estrous cycle and after ovariectomy followed or not by hormone replacement. Plasma oxytocin peaked in control animals during proestrus. Oxytocin content decreased in the paraventricular and supraoptic nuclei during proestrus and estrus compared to diestrus and increased in the neurohypophysis during proestrus morning. No significant difference was observed in the oxytocin content of the neurohypophysis, nuclei or plasma between ovariectomized animals and ovariectomized animals treated with estrogen or estrogen plus progesterone. Therefore, any ovarian factor other than estrogen or progesterone seems to play a direct or indirect role in the increase in oxytocin secretion. The osmotic stimulus caused an increase in plasma oxytocin throughout the estrous cycle. A reduction in oxytocin content during diestrus and an increase during proestrus were observed in the paraventricular nuclei. In ovariectomized animals, the treatment with estrogen potentiated the response of oxytocin to the osmotic stimulus, with the response being even stronger in the case of estrogen plus progesterone. In conclusion, the ovarian steroids estrogen plus progesterone could modulate the osmoreceptor mechanisms related to oxytocin secretion.     

Transitory increase in plasma FSH and LH levels induces early vaginal opening and corpus luteum formation in ovarian transplants of rats ovariectomized at 20 days of age

The effect of a transitory increase in plasma FSH and LH levels on puberty has been investigated. Hormonal changes were induced by bilateral ovariectomy (OVX) at 20 days of age followed by implantation of two ovaries (donor animals 20-day-old) beneath the kidney capsule 1, 2 or 3 days later. In an other group of animals the two interventions were made in reversed order. Rats in which OVX was made first showed early vaginal opening. If ovarian implantation preceded OVX, these two interventions did not affect the time of vaginal opening. More than 1/3 of the ovarian implanted animals exhibiting early opening of the vagina contained corpora lutea three days after vaginal opening. FSH and LH levels of OVX + ovary implanted rats were from the third day on following implantations markedly different from the OVX controls and were in the range of intact or sham-operated rats. The present findings indicate that a transitory increase in plasma gonadotropin levels around twenty days of age may lead to precocious puberty.     

The estrogenic effects of clomiphene during the neonatal period in the rat

The ability of clomiphene and its isomers to cause estrogenic responses during the neonatal period in the rat was examined. Rats were injected s.c. with clomiphene (CL), zuclomiphene (ZUC) or enclomiphene (ENC) on days 1,3, and 5 of life and the stimulation of the reproductive tract and estrogen receptor binding was observed. Uterine weight and DNA content were increased significantly by day 7 in animals treated with clomiphene or zuclomiphene. Uterine epithelial hypertrophy was present in all groups by day 10 and hyperplasia was present in the animals treated with ZUC and CL. The time of vaginal opening was greatly accelerated in all drug treated groups with the earliest day of opening occurring on day 7. Ovarian hemorrhage and blood in the periovarian sac occurred between days 12-14 and continued to be present through day 25. Drug treatment caused the estrogen receptor to accumulate in the nuclear fraction of the uterus and to be depleted from the cytosol fraction. We conclude that clomiphene administered to neonatal rats causes estrogenic stimulation of the reproductive tract in a fashion similar to other estrogens. This stimulation may account for the reproductive tract abnormalities which develop in rats treated with those drugs during the neonatal period.     

Estrogenic effects of the new opioid antagonist naltrexone-estrone azine on pituitary luteinizing hormone secretion in ovariectomized rats.

The effect of the new opioid antagonist naltrexone-estrone azine (EH-NX) on pituitary luteinizing hormone (LH) secretion in the ovariectomized rat was studied. EH-NX is a hybrid between the steroid component estrone and the opioid antagonist naltrexone (NX). It is a potent and long-acting opioid antagonist in vitro and in vivo, but its effect upon in vivo LH secretion has not been tested before. The aims of the study were to investigate whether, unlike naltrexone, EH-NX can stimulate LH secretion without the need of additional estrogen pretreatment and whether EH-NX has peripheral estrogenic effects upon the uterine weight, when administered chronically to long-term ovariectomized rats. Female rats were injected subcutaneously with EH-NX 21 days after ovariectomy. The effects of EH-NX injections on LH secretion were compared to the effects of NX and estrone hydrazone (EH) alone, or in combination, with or without estradiol-benzoate (EB) pretreatment. Inhibition of LH secretion and uterine proliferation were observed in rats treated chronically with EH-NX in dosages of 0.250 mg/kg bw and higher. These effects were similar to those caused by EH and EB. In short-term OVX rats EH-NX appeared to act faster than EH. In contrast to NX, no stimulatory effect on LH secretion was seen with EH-NX in EB primed OVX rats. These results surprisingly demonstrate that EH-NX behaves like an estrogen and not like an opioid antagonist. The unexpected pharmacological profile of this new drug may open up doors for several medical applications.     

Combinatorial effects of the phytoestrogen genistein and of estradiol in uterus and liver of female Wistar rats

The knowledge about safety of phytoestrogens on proliferative endpoints in the endometrium is rather limited, particularly when low amounts of estrogens are present like in postmenopausal women. Therefore, we now studied how genistein (GEN) exposure affects proliferative endpoints in the endometrium in estrogenized animals. We investigated the effects of GEN (10 mg/(kg day) BW) on uterine proliferation and on general uterine response markers in intact female rats and ovariectomized (OVX) female rats co-treated with different doses of estradiol (E2; 1 or 4 μg/(kg day) BW). In parallel we investigated generalized hepatic effects of GEN in this co-stimulatory protocol. In agreement to our previous results, GEN treatment of OVX animals for 3 days results in a faint stimulation of the uterine wet weight. In intact animals and in OVX animals co-treated with E2 no effects of GEN on uterine wet weight were detectable. GEN treatment did not affect the uterine epithelial height in intact animals but resulted in a decrease of the protein and mRNA expression of the proliferation marker PCNA. In OVX animals co-treated with E2, GEN antagonized the E2 stimulated increase of the uterine epithelial height and epithelial PCNA expression. Besides PCNA, GEN effects on the uterine mRNA expression of IGF-1, IGF-1R, Complement C3, estrogen receptor- (ER) and -β (ERβ), as well as progesterone receptor were investigated in intact and OVX co-treated animals. Overall there was a tendency in all combinatorial groups that GEN counteracts E2 function in uterine tissue. Surprisingly, while investigating estrogenic response markers in liver, we observed very strong effects of GEN on hepatic marker gene expression. GEN significantly down-regulated CaBP9K and IGFBP1 mRNA levels in intact animals. In OVX animals hepatic CABP9K and IGFBP1 mRNA levels were not affected by E2 treatment. GEN treatment, even in combination with E2, decreased the hepatic CaBP9K expression below the levels observed in untreated animals. Interestingly co-treatment of OVX rats with low dose E2 and GEN resulted in a significant increase of IGFBP1 mRNA expression. Summarising our results we conclude that (1) GEN treatment in the presence of E2 is safe regarding proliferative responses in the endometrium of adult animals; (2) the observation of differences of the GEN activity in intact and OVX/E2 substituted animals can be taken as a hint that GEN may interact mechanistically with progestins which has to be proven in detail in future investigations and (3) the detection of strong effects of the phytoestrogen GEN on hepatic gene expression may point to the need of future investigations to rule out the possibility of adverse responses in this organ.     

Effect of cortisol on estradiol secretion and its reception in the morning and evening hours in 30-day-old female rats

Cortisol administration (250 micrograms) to 5-day-old female rats was studied for its effect on the diurnal (morning-evening) rhythm of the estradiol secretion and reception on the 30th day of life. This stimulus had no influence on the estradiol level in the morning time. In the evening the estrogen concentration in the control group decreased while in the cortisol-treated group there were no changes. Disturbance of the circadian periodicity in the serum estradiol concentration in cortisol-treated animals was not induced by changes in the secretory ability of adrenals and ovaries, which was estimated by measurement of the estradiol production by glands in vitro. This disturbance was not a result of changes in hormone reception in uterine cytosol. The increased estradiol level in cortisol-treated rats in the evening is in good agreement with effects caused by neonatal glucocorticoid treatment in the reproductive system.     

Estrogen status and skeletal muscle recovery from disuse atrophy.

Although estrogen loss can alter skeletal muscle recovery from disuse, the specific components of muscle regrowth that are estrogen sensitive have not been described. The primary purpose of this study was to determine the components of skeletal muscle mass recovery that are biological targets of estrogen. Intact, ovariectomized (OVX), and ovariectomized with 17beta-estradiol replacement (OVX+E2) female rats were subjected to hindlimb suspension for 10 days and then returned to normal cage ambulation for the duration of recovery. Soleus muscle mass returned to control levels by day 7 of recovery in the intact animals, whereas OVX soleus mass did not recover until day 14. Intact rats recovered soleus mean myofiber cross-sectional area (CSA) by day 14 of recovery, whereas the OVX soleus remained decreased (42%) at day 14. OVX mean fiber CSA did return to control levels by day 28 of recovery. The OVX+E2 treatment group recovered mean CSA at day 14, as in the intact animals. Myofibers demonstrating central nuclei were increased at day 14 in the OVX group, but not in intact or OVX+E2 animals. The percent noncontractile tissue was also increased 29% in OVX muscle at day 14, but not in either intact or OVX+E2 groups. In addition, collagen 1a mRNA was increased 45% in OVX muscle at day 14 of recovery. These results suggest that myofiber growth, myofiber regeneration, and extracellular matrix remodeling are estrogen-sensitive components of soleus muscle mass recovery from disuse atrophy.     

Estrogenic activity of B-fluorinated o-carborane-1,2-bisphenol synthesized via S(N)Ar reaction

We previously identified o-carborane bisphenol BE360 (4) as a selective estrogen receptor modulator (SERM), which ameliorated bone loss without inducing estrogenic action in uterus of OVX and ORX mice. Here, we synthesized a fluorinated derivative, B-fluorinated o-carborane bisphenol BE310 (5) by means of S(N)Ar reaction. Compound 5 was a partial ER agonist, like 4, with little change of ERα and ERβ selectivity as compared with 4. However, its agonistic activity was 40 times weaker than that of 4. Thus, 5 is a novel SERM candidate with potential for reduced estrogenic side effects, and in vivo evaluation as an anti-osteoporosis agent seems warranted.     

Ovarian steroids and hypothalamic norepinephrine release: studies using in vivo brain microdialysis

This study employed microdialysis in urethane-anesthetized female rats to monitor ovarian steroid-dependent changes in KCl-evoked levels of extracellular norepinephrine (NE) in the ventromedial hypothalamus. An initial KCl stimulus (Sl) increased NE from low or undetectable levels in all animals. A second KCl stimulus (S2) given several hr later evoked 40% less NE release than did Sl in ovariectomized (OVX) females or OVX females given only estrogen or progestin. In contrast, the two KCl-evoked NE releases were equivalent in OVX females administered both estrogen and progestin. These results suggest that ovarian steroids may act as presynaptic modulators of NE release in the ventromedial hypothalamus.     

Effects of para-nonylphenol on 92 kDa gelatinase secretion by human peripheral lymphocytes and U937 cells in vitro

Much attention has focused on environmental estrogenic chemicals such as para-nonylphenol which disrupt various tissues via the estrogen receptor. We studied effects of para-nonylphenol on gelatinase secretion by human lymphocytes in vitro. para-Nonylphenol (0.05-50 microM) dose dependently suppressed 92 kDa gelatinase secretion. The suppressive effect of 25 and 50 microM para-nonylphenol was completely blocked by tamoxifen. We also studied the effects of para-nonylphenol (0.05-50 microM) on 92 kDa gelatinase secretion by human leukemia U937 cells. para-Nonylphenol suppressed 92 kDa gelatinase secretion in a dose-dependent manner. The suppressive effect of 50 microM para-nonylphenol was completely blocked by tamoxifen. Estradiol did not significantly suppress 92 kDa gelatinase secretion. Our results suggest that para-nonylphenol suppressed 92 kDa gelatinase secretion via the estrogen receptor, however, para-nonylphenol interacts with the estrogen receptor in a manner distinct from estradiol. As this assay system is simple and rapid, it may prove useful to evaluate toxic effects of para-nonylphenol on human blood cells.     

A potential role for catecholamines in the development and progression of carcinogen-induced mammary tumors: hormonal control of beta-adrenergic receptors and correlation with tumor growth.

In order to gain further knowledge on the beta-adrenergic receptor system in DMBA-induced rat mammary tumors, we have studied the correlation between changes in tumoral beta-adrenergic receptor concentration and distribution, progesterone receptor status and tumor growth after ovariectomy and treatment with various ovarian and adrenal steroids, or induction of hyperprolactinemia. Autoradiographic localization of beta-adrenergic receptors in ovariectomized (OVX) animals shows very weak labeling with [125I]cyanopindolol. In these tumors, the connective tissue is predominant, while the epithelial cell content is very low. Similarly, when direct measurements of [125I]cyanopindolol are performed with membrane preparations, beta-adrenergic receptor concentration is sharply reduced 2-3 weeks following ovariectomy or treatment with LHRH against [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide. This effect on the beta-adrenergic receptor population in the tumor is accompanied by the well known effect of castration on tumor growth and progesterone receptor levels, namely a marked regression of tumor growth and a significant decrease in progesterone receptor concentration. Treatment of OVX rats with 17 beta-estradiol (E2) alone or in combination with progesterone (P) caused a highly significant increase in beta-adrenergic and progesterone receptor levels, as well as tumor growth. A similar sharp increase in the value of the three parameters studied was observed following daily treatment of OVX rats with dehydroepiandrosterone (DHEA) or androst-5-ene-3 beta,17 beta-diol (5-ene-diol). The autoradiographic localization of beta-adrenergic receptors in OVX rats treated with 5-ene-diol showed that the epithelial cells were numerous with a high degree of labeling. On the other hand, treatment of OVX animals with the androgen dihydrotestosterone (DHT) did not produce significant changes in beta-adrenergic receptor levels or tumor growth. Finally, endogenously-induced hyperprolactinemia by implanting three anterior pituitary glands under the kidney capsule of OVX animals resulted in a significant increase in beta-adrenergic and progesterone receptor levels as well as tumor growth. The positive correlation observed between changes in beta-adrenergic receptor concentration, progesterone receptor levels and tumor growth indicates a high sensitivity of the beta-adrenergic receptor population of DMBA-induced rat mammary tumors to the hormonal milieu, and suggests that the beta-adrenergic receptor system may represent a valuable parameter of hormone responsiveness.     

Effects of thyroid hormones on the receptor level in estrogen target organs

The influence of thyroid hormones on the turnover of cytoplasmic estrogen receptors in the liver, kidney and uterus of intact and ovariectomized female rats was studied under in vivo conditions. Thyroidectomy had no significant effect on the receptor level in the uterus but caused a substantial reduction of the receptor content in the liver and kidney. In livers of intact and ovariectomized animals receptor values were reduced with 70 and 80%, respectively, 30 days after thyroidectomy. Substitution with triiodothyronine (T3) restored the hepatic estrogen receptor concentration in thyroidectomized rats to the preoperative level. If rats that had been both ovariectomized and thyroidectomized were substituted with thyroid hormone for the same time period, the receptor level was increased but did not reach the level seen in animals that had been ovariectomized only. The effects of thyroid hormone substitution was found to be dose dependent and paradoxical. Thus, a high dose of 50 micrograms/day of triiodothyronine given to intact animals for nine days caused a 30% reduction in the hepatic receptor content. The same level of reduction was seen in the ovariectomized rat given a hormone dose of only 1 micrograms/day. When this type of rats was treated with the higher dose of triiodothyronine the reduction in hepatic estrogen receptors was 50%. These results are discussed in relation to existing information concerning the multihormonal regulation of estrogen receptor concentration in the rat liver.     

Chronic Activation of the G Protein-Coupled Receptor 30 with Agonist G-1 Attenuates Heart Failure

G protein-coupled receptor (GPR) 30 is a novel estrogen receptor. Recent studies suggest that activation of the GPR30 confers rapid cardioprotection in isolated rat heart. It is unknown whether chronic activation of GPR30 is beneficial or not for heart failure. In this study we investigated the cardiac effect of sustained activation or inhibition of GPR30. Female Sprague–Dawley rats were divided into 7 groups #2Q1: sham surgery (Sham), bilateral ovariectomy (OVX), OVX+estrogen (E₂), OVX+isoproterenol (ISO), OVX+ISO+G-1, OVX+ISO+E₂+G15, OVX+ISO+E₂. ISO (85 mg/kg×17 day, sc) was given to make the heart failure models. G-1(120 µg/kg·d×14 day) was used to activate GPR30 and G15 (190 µg/kg·d×14 day) was used to inhibit GPR30. Concentration of brain natriuretic peptide in serum, masson staining in isolated heart, contractile function and the expression of β₁ and β₂- adrenergic receptor (AR) of ventricular myocytes were also determined. Our data showed that ISO treatment led to heart failure in OVX rats. G-1 or E₂ treatment decreased concentration of brain natriuretic peptide, reduced cardiac fibrosis, and enhanced contraction of the heart. Combined treatment with β₁ (CGP20712A) and β₂-AR (ICI118551) antagonist abolished the improvement of myocardial function induced by G-1. We also found that chronic treatment with G-1 normalized the expression of β₁-AR and increased the expression of β₂-AR. Our results indicate that chronic activation of the GPR30 with its agonist G-1 attenuates heart failure by normalizing the expression of β₁-AR and increasing the expression of β₂-AR.     

Dynamics of folliculogenesis of sexually mature and neonatal ovarian tissue under conditions of long-term heterotopical transplantation

In this work, a comparative analysis of dynamics of morphological development and endocrine function of transplants of sexually mature and neonatal ovarian tissue was performed under conditions of bilateral ovariectomy of animals-recipients. It has been established that at transplantation of the sexually mature ovarian tissue there were observed all stages of folliculogenesis and preservation of endocrine function at long-term observation (up to 100 days). At transplantation of neonatal ovarian tissue there was revealed a pathological picture of its development: hyperplasia of stromal structure and formation of cysts and cystomae. Follicles of different degree of maturity were revealed in 30 % of animals at the 30th day of observation. A significant increase of concentration of sex hormones as compared with ovariectomied animals has been shown only at early stages after implantation (30 days).