A bioassay for the evaluation of antiproliferative potencies of progesterone antagonists.

A bioassay which allows quantification of the antiproliferative potency of progesterone antagonists on the mammary gland was developed. For this purpose, ovariectomized rats were substituted with oestrone and progesterone and a further group simultaneously treated with the progesterone antagonists Mifepristone (= RU 38.468), Onapristone (= ZK 98.299), or ZK 112.993 (Schering AG, Berlin). A morphometric analysis of the tubulo-alveolar buds in the inguinal mammary glands revealed a dramatic antiproliferative effect of the progesterone antagonists after as little as 3 days of treatment. Several less specific mammary gland growth parameters (weight, DNA- and RNA-content) proved to be less sensitive. This bioassay measures the potency of progesterone antagonists to competitively antagonize the specific effects of progesterone on the target organ mammary gland. Further advantages of this bioassay are the use of a hormonally standardized biological system, the quantitative results, the small amount of test compound necessary, as well as the substitution with progesterone and oestrone since the antiproliferative potency of progesterone antagonists on experimental hormone dependent mammary carcinomas is most potently displayed in ovariectomized animals substituted with both sex hormones.     

The antitumor potency of progesterone antagonists is due to their differentiation potential.

A new therapy for the progesterone receptor positive mammary carcinoma may be the treatment with progesterone antagonists. This new class of antihormones causes a strong inhibition of tumor growth comparable to the potency of ovariectomy in a panel of experimental mammary carcinomas. The mechanisms of the strong tumor-inhibiting action of progesterone antagonists on experimental mammary carcinomas mainly depends on a progesterone receptor mediated process leading to induction of terminal differentiation and a blockade of the cell cycle. To further characterize the antitumor mechanism of progesterone antagonists we analyzed the effects of Onapristone and ZK 112.993 on DMBA- and MNU-mammary tumors of the rat and MXT-tumors of the mouse after different therapy intervals. These hormone-dependent mammary tumors normally display intraductal growth in papillary, cribiform or solid formation, whereas after treatment periods of 2-6 weeks with progesterone antagonists they displayed dysplastic ductal and acinous formations, usually filled with secretory material. Whereas tumor size, mitotic index, and the grade of tumor malignancy decreased distinctly, the volume fraction of glandular structures in the tumors as well as the appearance of apoptosis increased 3-fold compared to the controls. In addition, the mammary glands of progesterone antagonist treated animals showed the morphological features of differentiation with the appearance of secretory activity. Interestingly, the staining pattern of some of the lectins used, especially UEA 1 binding pattern, fits to the concept of differentiation since recent studies revealed a higher degree of fucosylation only in benign lesions of human breast cancers. Therefore, these data underline the concept of a differentiation potential of progesterone antagonists on progesterone receptor positive mammary carcinomas.     

Progesterone antagonists: Tumor-inhibiting potential and mechanism of action

A new approach for the treatment of breast cancer could be the use of progesterone antagonists. These compounds were originally developed for the inhibition of progesterone-dependent processes and have been shown to be effective in inhibition of nidation and interruption of pregnancy. Although the roles of progesterone and the progesterone receptor in control of cell growth remain unclear, it was found in progesterone receptor positive mammary carcinoma cell lines that the antiprogestin, Mifepristone, had an inhibitory effect on cell growth-and a growth-inhibiting action on the DMBA-induced mammary carcinoma of the rat. We have shown that the progesterone antagonists, Onapristone and ZK 112993, which possess a reduced antiglucocorticoid activity compared to Mifepristone, exert a strong tumor-inhibiting effect in a panel of hormone-dependent mammary tumor models. The effects of these compounds were in some systems superior to those of tamoxifen or high dose progestins and comparable to ovariectomy. Although prerequisites for their antiproliferative potency are an affinity to the progesterone receptor as well as a sufficient number of available receptors in the tumors, the strong tumor inhibiting potential of the antiprogestins cannot be explained by a classical antihormonal mechanism. Surprisingly, the antitumor activity is evident in spite of elevated serum levels of ovarian and pituitary hormones. It was established by morphometric procedures that treatment with Onapristone triggers differentiation of the mitotically active polygonal tumor epithelial cell towards secretory active glandular structures and acini. All our quantitative light and electron microscopic data indicate that the antitumor action of antiprogestins is accompanied by the initiation of terminal differentiation leading to (apoptotic) cell death. Finally, our flow cytometry studies revealed an accumulation of the tumor cells in the G₀G₁ phase of the cell cycle, which may result from induction of differentiation since a differentiation-specific G₁ arrest has already been proposed for other stem cell systems. It can be concluded from these data that the progesterone receptor antagonists differ in their mode of action from compounds used in established endocrine treatment strategies for mammary carcinoma. The ability of progesterone antagonists like Onapristone to reduce the number of cells in S-phase may offer a significant clinical advantage, since it is established that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors.     

Estradiol benzoate given 0 or 24 h after the end of a progestagen treatment in postpartum suckled beef cows

The objective of Experiment 1 was to compare the effects of estradiol benzoate (EB) given 0 or 24h after the end of a progestagen treatment on ovulation and CL formation in anestrous cows. Twenty cows were treated with an intravaginal sponge containing 250 mg of medroxiprogesterone acetate (MPA). At sponge insertion, each cow received 3 mg EB and 10 mg MPA im. At device removal, cows received 0.7 mg EB either at that time (EB0) or 24h later (EB24). Ultrasound examinations and blood sampling to determine plasma progesterone concentrations were performed to detect ovulation and CL formation. Ovulation occurred in 77.8 and 81.8% cows in the EB0 and EB24 groups, respectively. Diameter of the ovulatory follicle (EB0 = 10.9 +/- 0.5mm; EB24 = 12.1 +/- 0.8 mm; P = 0.26) and the interval from sponge removal to ovulation (median = 3 days; P = 0.64) did not differ between treatments. Among the cows that ovulated (n = 16), short-lived CL were present in 2/7 and 2/9 cows in the EB0 and EB24 groups, respectively. Plasma progesterone concentrations and CL area did not differ between treatments (P > 0.05). In Experiment 2, cows were treated with the same protocol as in Experiment 1, but at sponge withdrawal all cows received 250 microg cloprostenol and timed artificial insemination (TAI) was performed 48 h after sponge removal. In Replicate 1 (n = 204 multiparous cows), pregnancy rates were 45.0 and 47.5% for EB0 and EB24, respectively (P > 0.05). In Replicate 2 (n = 69 primiparous cows) pregnancy rate did not differ between EB0 and EB24 (51.4% versus 52.9%). In conclusion, EB given 0 or 24h after the end of a progestagen treatment had the same effect on ovulation rate, time to ovulation, diameter of the ovulatory follicle, incidence of short-lived CL, luteal tissue area, and plasma progesterone concentrations of normal lifespan CL, and pregnancy rate after TAI in suckled beef cows.     

Antitumor activity and mechanism of action of different antiprogestins in experimental breast cancer models

Onapristone and other antiprogestins proved to possess a potent antitumor activity in several hormone-dependent experimental breast cancer models. This activity is as strong or even better than that of tamoxifen or ovariectomy in the MXT-mammary tumor of the mouse and the DMBA- and MNU-induced mammary tumor of the rat. The antitumor activity is evident in these models in spite of elevated serum levels of ovarian and pituitary hormones. The detailed analysis of all our data including the morphological (ultrastructure) studies of the mammary tumors of treated animals and the effects on growth and cell cycle kinetics using DNA flow cytometry indicates that the antitumor action of antiprogestins is mediated via the progesterone receptor and related to the induction of terminal cell differentiation leading to increased cell death. The strong antitumor activity of antiprogestins in our experimental breast cancer models does not primarily depend on a classical anti-hormonal mechanism. The antiprogestin-related reduction of the number of mammary tumor cells in the S-phase in our experimental tumor models (G₀G₁ arrest) emphasizes the unique innovative mechanism of action of these new agents in the treatment of human breast cancer.     

Contraceptive potential of an antiprogestin ZK 98.734: reversal of ZK 98.734--induced blockade of folliculogenesis with FSH and LH and their differential effects in bonnet monkeys.

Studies were undertaken in adult bonnet monkeys to investigate whether treatment with an antiprogestin ZK 98.734 at weekly intervals, starting from day one of menstrual cycle, could arrest ovulation and also to determine if ZK 98.734 induced blockade of ovulation could be reversed with gonadotropins. Adult animals have ovulatory menstrual cycles of normal duration were treated at weekly intervals with ZK 98.734 (25 mg/dose, sc, oil base) for 10 consecutive weeks and its effects on serum levels of estradiol, bioactive LH and progesterone, and endometrial histology were investigated. Following treatment with the antiprogestin they were treated with hMG or hFSH alone. Ovulation was blocked during treatment period in all the animals (n = 14). Typical follicular phase rise in estradiol levels was inhibited, mid cycle surge in the levels of bioactive LH was abolished and serum progesterone levels remained below 1 ng/ml throughout the treatment period. However, prolonged treatment had no significant effect on the basal levels of estradiol which were around 50 pg/ml. ZK 98.734 also had no significant effect on cortisol levels. In animals (n = 4) followed for recovery after the last dose, the treatment cycle length was increased to 117.8 + 6.8 days. In three animals the treatment cycles were anovulatory, whereas in one delayed ovulation with luteal insufficiency was observed. The endometrium had become atrophic. Treatment with hMG (Pergonal: 35 I.U. hLH and 35 I.U. hFSH) or hFSH (Metrodin, 35 I.U.) for 7 consecutive days initiated folliculogenesis and the animals ovulated either spontaneously or after a single im injection of hCG (100 I.U.) on day 8 in ZK 98.734 treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicular growth, estrus and pregnancy after fixed-time insemination in beef cows treated with intravaginal progesterone inserts and estradiol benzoate

An experiment was performed to compare the effects of 3 short-term treatments with progesterone and estradiol benzoate (EB) on follicular growth, synchrony of estrus and pregnancy rate after fixed-time insemination in lactating postpartum beef cows. In Treatment 1 (n = 46), each cow received a progesterone-containing intravaginal insert for 7 d with injection of EB (2 mg, i.m.) at the time of device insertion. In Treatment 2 (n = 46), the insert was used for only 5 d with injection of EB (2 mg, i.m.) at the time of insertion. Cows in Treatment 3 (n = 47) received an insert for 5 d with no EB at the time of insertion. Each cow in the 3 groups received PGF2 alpha (25 mg, i.m.) at the time of insert removal, followed by EB (1 mg, i.m.) 30 h later. The cows were then inseminated 28 to 30 h after treatment with EB (58 to 60 h after insert removal). Treatment with 2 mg EB terminated the growth of the largest ovarian follicle (> 5 mm in diameter) at device insertion in 16/16 and 14/15 cows in Treatments 1 and 2, respectively. Estrus was detected within an 8-h target period (48 to 56 h after insert removal) in 93, 87 and 81% of cows in Treatments 1, 2 and 3, respectively (P > 0.05). Pregnancy rates at 39 d post insemination were 60, 50 and 51% for Treatments 1, 2 and 3, respectively (P > 0.05). The pregnancy rates did not differ between cows that were anovulatory or those that had ovulated before the initiation of treatments (54%), or among cows that were 28 to 40, 41 to 60 or > 60 days post partum at insemination (43, 59 and 54%, respectively). Treatment with progesterone inserts for 5 or 7 d, PGF2 alpha at the time of insert removal and 1 mg EB 30 h later induced the high degree of synchrony of estrus and ovulation necessary for fixed-time insemination.     

Hormonal regulation of protein kinase C in the mouse mammary gland

The hormonal regulation of protein kinase C (PKC) induction over 3 to 14 days was investigated in the mouse mammary gland in vitro and in vivo. In intact mice, estradiol (1 microgram/mouse injected daily for 2 weeks) stimulated PKC activity 70%, while progesterone (1 mg/mouse injected daily) inhibited it by 30%. Prolactin, whose levels were elevated for 2 weeks by two pituitary isografts, had no effect. When mammary gland explants were cultured in insulin and cortisol, the further addition of estradiol (1 ng/ml), progesterone (1 microgram/ml), or prolactin (1 microgram/ml) did not alter PKC activity after 3 days. These data suggest the following conclusions: although previous studies have implicated prolactin in the transient, calcium-phospholipid activation of PKC, it does not appear to elevate total levels of this kinase over prolonged periods. In contrast, the sex steroids do appear to affect long-term levels of this kinase; furthermore, this latter effect may be indirect.     

Secretion of estradiol-17beta by porcine mammary gland of ovariectomized steroid-treated sows

Although the mammary gland of many species secretes estradiol (E(2)), nothing is known of E(2) secretion in the porcine gland. The present study was designed to investigate whether porcine mammary gland was a source of E(2), and to test the influence of individual and combined effects of exogenous progesterone and estradiol benzoate (EB) on the secretion of E(2). Immature crossbred gilts were ovariectomized at 7 months of age followed by 4 weeks later by steroid hormone replacement therapy to produce estradiol and progesterone (P(4)) blood concentrations similar to those observed during a normal estrous cycle. Arterial and venous blood plasma (from carotid artery and anterior mammary vein, respectively) were sampled for 2h at 10 min intervals. Plasma concentrations of progesterone, androstenedione (A(4)), testosterone (T), estrone (E(1)) and estradiol were determined by RIA. In all gilts treated with progesterone alone or in combination with EB, concentrations of P(4), A(4) and E(1) in blood collected from venous outflow were lower compared to concentrations in arterial blood, whereas concentrations of E(2) were higher in blood plasma from the anterior mammary vein compared to plasma from the carotid artery. The results indicated that the porcine mammary gland secreted E(2). Increased concentrations of plasma E(2) collected only from P(4)-treated animals suggested that progesterone activated enzymes involved in steroidogenesis in porcine mammary gland, or those utilized in its metabolism.     

An alteration in the hypothalamic action of estradiol due to lack of progesterone exposure can cause follicular cysts in cattle

Many mammals, including cattle, can develop ovarian follicular cysts, but the physiological mechanisms leading to this condition remain undefined. We hypothesized that follicular cysts can develop because estradiol will induce a GnRH/LH surge on one occasion but progesterone exposure is required before another GnRH/LH surge can be induced by estradiol. In experiment 1, 14 cows were synchronized with an intravaginal progesterone insert (IPI) for 7 days, and prostaglandin F(2alpha) was given on the day of IPI removal. Estradiol benzoate (EB; 5 mg i.m.) was given 3 days before IPI removal to induce atresia of follicles. Cows were given a second EB treatment 1 day after IPI removal to induce a GnRH/LH surge in the absence of an ovulatory follicle. All cows had an LH surge following the second EB treatment, and 10 of 14 cows developed a large-follicle anovulatory condition (LFAC) that resembled follicular cysts. These LFAC cows were given a third EB treatment 15 days later, and none of the cows had an LH surge or ovulation. Cows were then either not treated (control, n = 5) or treated for 7 days with an IPI (n = 5) starting 7 days after the third EB injection. Cows were treated for a fourth time with 5 mg of EB 12 h after IPI removal. All IPI-treated, but no control, cows had an LH surge and ovulated in response to the estradiol challenge. In experiment 2, cows were induced to LFAC as in experiment 1 and were then randomly assigned to one of four treatments 1) IPI + EB, 2) IPI + GnRH (100 microg), 3) control + EB, and 4) control + GnRH. Control and IPI-treated cows had a similar LH surge and ovulation when treated with GnRH. In contrast, only IPI-treated cows had an LH surge following EB treatment. Thus, an initial GnRH/LH surge can be induced with high estradiol, but estradiol induction of a subsequent GnRH/LH surge requires exposure to progesterone. This effect is mediated by the hypothalamus, as evidenced by similar LH release in response to exogenous GnRH. This may represent the physiological condition that underlies ovarian follicular cysts.     

Persistent estrogen responsiveness of ras oncogene-transformed mouse mammary epithelial cells.

Ovarian steroids are associated with the proliferation of normal as well as tumorigenically transformed mammary epithelial cells. The experiments performed in this study were designed to establish that (1) tumorigenic transformation induced by the ras oncogene is associated with alterations in estradiol biotransformation, (2) altered endocrine responsiveness persists in the fully transformed tumor cell phenotype and (3) specific perturbations induced by the ras oncogene can be experimentally downregulated. The ras transfectant pH06T and the tumor-derived T1/Pr1 cells exhibited 3- and 43-fold increases, respectively, in C-16 alpha hydroxylation of estradiol relative to the parental mouse mammary epithelial cells (P less than 0.0001). At the cellular level, this alteration corresponded with approximately 90-fold increase in the anchorage-independent growth of T1/Pr1 cells (P less than 0.0001). Estrogen responsiveness of T1/Pr1 cells was demonstrated by their suppression of growth in phenol red-free and/or tamoxifen-supplemented medium and by the reversal of antiproliferative effect of tamoxifen by phenol red and estradiol. Indole-3-carbinol, a naturally occurring tumor suppressive agent, was able to upregulate C-2 hydroxylation at the expense of C-16 alpha hydroxylation of estradiol. Treatment of T1/Pr1 cells with indole-3-carbinol resulted in a substantial decrease in anchorage-independent growth.     

Effects of two estradiol esters (benzoate and cypionate) on the induction of synchronized ovulations in Bos indicus cows submitted to a timed artificial insemination protocol

The effects of estradiol benzoate (EB) and estradiol cypionate (EC) on induction of ovulation after a synchronized LH surge and on fertility of Bos indicus females submitted to timed AI (TAI) were evaluated. In Experiment 1, ovariectomized Nelore heifers were used to evaluate the effect of EB (n = 5) and EC (n = 5) on the circulating LH profile. The LH surge timing (19.6 and 50.5 h; P = 0.001), magnitude (20.5 and 9.4 ng/mL; P = 0.005), duration (8.6 and 16.5 h; P = 0.001), and area under the LH curve (158.6 and 339.4 ng/mL; P = 0.01) differed between the EB and EC treatments, respectively. In Experiment 2 (follicular responses; n = 60) and 3 (pregnancy per AI; P/AI; n = 953) suckled Bos indicus beef cows submitted to an estradiol/progesterone-based synchronization protocol were assigned to receive one of two treatments to induce synchronized ovulation: 1 mg of EB im 24 h after progesterone (P4) device removal or 1 mg of EC im at P4 device removal. There was no difference (P > 0.05) between EB and EC treatments on follicular responses (maximum diameter of the ovulatory follicle, 13.1 vs. 13.9 mm; interval from progesterone device removal to ovulation, 70.2 vs. 68.5 h; and ovulation rate, 77.8 vs. 82.8%, respectively). In addition, P/AI was similar (P < 0.22) between the cows treated with EB (57.5%; 277/482) and EC (61.8%; 291/471). In conclusion, despite pharmacologic differences, both esters of estradiol administered either at P4 device removal (EC) or 24 h later (EB) were effective in inducing an LH surge which resulted in synchronized ovulations and similar P/AI in suckled Bos indicus beef cows submitted to TAI.     

The use of GnRH or estradiol to facilitate fixed-time insemination in an MGA-based synchronization regimen in beef cattle

Two experiments were conducted to compare pregnancy rates when GnRH or estradiol were given to synchronize ovarian follicular wave emergence and ovulation in an MGA-based estrus synchronization program. Crossbred beef cattle were fed melengestrol acetate (MGA, 0.5 mg per day) for 7 days (designated days 0-6, without regard to stage of the estrous cycle) and given cloprostenol (PGF; 500 microg intramuscular (im)) on day 7. In Experiment 1, lactating beef cows (n=140) and pubertal heifers (n=40) were randomly allocated to three groups to receive 100 microg gonadorelin (GnRH), 5 mg estradiol-17beta and 100 mg progesterone (E+P) in canola oil or no treatment (control) on day 0. All cattle were observed for estrus every 12 h from 36 to 96 h after PGF. Cattle in the GnRH group that were detected in estrus 36 or 48 h after PGF were inseminated 12 h later; the remainder were given 100 microg GnRH im 72 h after PGF and concurrently inseminated. Cattle in the E+P group were randomly assigned to receive either 0.5 or 1.0 mg estradiol benzoate (EB) in 2 ml canola oil im 24 h after PGF and were inseminated 30 h later. Cattle in the control group were inseminated 12 h after the first detection of estrus; if not in estrus by 72 h after PGF, they were given 100 microg GnRH im and concurrently inseminated. In the absence of significant differences, all data for heifers and for cows were combined and the 0.5 and 1.0 mg EB groups were combined into a single estradiol group. Estrus rates were 57.6, 57.4 and 60.0% for the GnRH, E+P and control groups, respectively (P=0.95). The mean (+/-S.D.) interval from PGF treatment to estrus was shorter (P<0.001) and less variable (P<0.001) in the E+P group (49.0+/-6.1 h) than in either the GnRH (64.2+/-15.9 h) or control (66.3+/-13.3 h) groups. Overall pregnancy rates were higher (P<0.005) in the GnRH (57.6%) and E+P (55.7%) groups than in the control group (30.0%) as were pregnancy rates to fixed-time AI (47.5, 55.7 and 28.3%, respectively). In Experiment 2, 122 crossbred beef heifers were given either 100 microg GnRH or 2 mg EB and 50 mg progesterone in oil on day 0 and subsequently received either 100 microg GnRH 36 h after PGF and inseminated 14 h later or 1 mg EB im 24 h after PGF and inseminated 28 h later in a 2 x 2 factorial design. Pregnancy rates were not significantly different among groups (41.9, 32.2, 33.3 and 36.7% in GnRH/GnRH, GnRH/EB, EB/GnRH and EB/EB groups, respectively). In conclusion, GnRH or estradiol given to synchronize ovarian follicular wave emergence and ovulation in an MGA-based synchronization regimen resulted in acceptable pregnancy rates to fixed-time insemination.     

Progesterone receptor involvement in independent tumor growth in MPA-induced murine mammary adenocarcinomas

We have developed a model of hormonal carcinogenesis in BALB/c female mice, in which MPA induced ductal mammary adenocarcinomas, expressing high levels of estrogen and progesterone receptors (ER and PR). A series of tumor lines, retaining both PR and ER expression, were obtained from selected tumors, which are maintained by syngeneic passages. In this model progesterone behaves as the growth-stimulating hormone (progesterone-dependent or PD tumors), whereas estrogens induce tumor regression. Through selective treatments we were able to derive a series of progesterone-independent (PI) variants. These lines do not require progesterone treatment to grow in ovariectomized female BALB/c mice, but retain, however, the expression of ER and PR. The aim of this paper is to investigate a possible regulatory role of the progesterone receptor (PR) on PI tumor growth. ER and PR were detected by immunocytochemistry in all lines studied. They were also characterized using biochemical assays and Scatchard plots. No differences in Kd of PR or ER were detected in PI variants. AR or GR were not detected in tumor samples using biochemical assays. Estradiol (5 mg silastic pellet) induced complete tumor regression in all tumors tested. We also evaluated the effects of different antiprogestins on tumor growth. Onapristone (10 mg/kg/day) and mifepristone (4.5 mg/kg/day) were able to induce complete tumor regression. The antiandrogen flutamide (5 mg silastic pellet) had no effect on tumor growth in agreement with the lack of androgen receptors. We used an in vitro approach to corroborate that the antiprogestin-induced inhibition was not attributable to an intrinsic effect. Cultures of a selected PI line were treated with PR antisense oligodeoxynucleotides (ASPR) to inhibit in vitro cell proliferation. A significant decrease of 3H-thymidine uptake was observed in cells of a PI line growing in the presence of 2.5% charcoalized fetal calf serum and 0.8-20 microg/ml ASPR. It can be concluded that the PR pathway is an essential path in the growth stimulation of PI tumors.     

Review of the pharmacological properties of toremifene

New compounds were synthesized with the aim to develop new anti-estrogenic antitumor drugs. The biological properties of the molecules were screened by (1) estrogen receptor (ER) binding, (2) effect on MCF-7 cells, (3) uterotrophic effect and inhibition of estradiol induced uterotropic effect and (4) antitumor effect in DMBA induced rat mammary cancer. One of the molecules, Fc-1157a = toremifene, exhibited the following characteristics: competitive inhibition of [3H]estradiol binding to ER (IC50 = 0.3 mumol/l), inhibition of MCF-7 cell growth in a concentration-dependent manner and cell-killing effect at higher than 3 mumol/l concentrations. Minimal estrogenic dose of toremifene on rat uterus weight was about 40 times higher than that of tamoxifen. Toremifene had statistically significant effect against DMBA-induced rat mammary cancer. Further screening consisted of antitumor, pharmacokinetic and safety studies. Toremifene inhibited the growth of ER-negative, glucocorticoid sensitive, mouse uterine sarcoma in a dose-dependent manner. Pharmacokinetics and metabolism of toremifene resembled closely those of tamoxifen, but since the chlorine atom of the toremifene molecule was not metabolically cleaved tamoxifen and toremifene did not have chemically similar metabolites. Toremifene was well tolerated in animal toxicity studies. No hyperplastic or neoplastic nodules, which were seen in almost all high-dose (48 mg/kg for 24 weeks) tamoxifen-treated rats, were found in toremifene-treated rats (dose 48 mg/kg). In clinical phase I studies in healthy voluntary postmenopausal women, no side effects were reported, at doses less than or equal to 460 mg, neither after a single dose nor after five daily doses. At the dose of 680 mg two out of five persons experienced vertigo and headache. Toremifene, at the dose of 68 mg daily, had antiestrogenic effect on estradiol-induced human vaginal epithelial cells. Clinical phase II studies have confirmed that toremifene has a promising antitumor effect.     

雌激素和孕激素对雌性小鼠甲状腺C细胞的影响

探讨降钙素在老年女性骨质疏松症发病中的作用 ,观察了雌、孕激素对雌性小鼠甲状腺 C细胞的影响。去卵巢小鼠 (OVX)分别肌肉注射苯甲酸雌二醇 (FB)、己烯孕酮 (HPC)和苯甲酸雌二醇加乙烯孕酮 (EB+HPC)两个月。用药剂量根据小鼠口龄和体重的不同而不同。采用免疫组织化学方法显示降钙素 (CT)阳性细胞并对其进行细胞形态计量学分析。结果显示 :(1)去卵巢小鼠与正常对照组相比 ,甲状腺 C细胞数目剧增 ,给予 EB、HPC、EB+HPC治疗的各组小鼠甲状腺C细胞数目与正常对照组水平相近。 (2 )各组小鼠甲状腺 C细胞的大小无明显改变 ,平均直径间无显著性差异 (P>0 .0 5 )。雌、孕激素缺乏可引起雌性小鼠甲状腺 C细胞增生。 C细胞增生也可能是雌激素缺乏引起的骨质疏松的后果     

Fertility in beef cattle given a new or previously used CIDR insert and estradiol, with or without progesterone

The objective was to compare pregnancy rates following fixed-time AI (FTAI) in beef cattle given a new or previously used CIDR insert and injections of estradiol, with or without progesterone, to synchronize follicular wave emergence. In Experiment 1, heifers (n=616) received a new or once-used CIDR insert for 9 days and were given 1mg estradiol cypionate (ECP), with or without 100 mg of a commercial progesterone preparation (CP4), at CIDR insertion. Heifers were treated with PGF at CIDR removal and 0.5 mg ECP i.m. 24h later, with FTAI 55 to 60 h after CIDR removal. Pregnancy rate was not affected by either the number of CIDR uses (P=0.59; 48.3% versus 46.2% for new versus once-used CIDRs, respectively) or the addition of progesterone (P=0.42; 45.6% versus 48.8% for ECP+CP4 and ECP, respectively). In Experiment 2 (replicated at two locations), heifers (n=56) and lactating beef cows (n=307) received a once- or twice-used CIDR and an i.m. injection of 1mg estradiol benzoate (EB), with or without 100 mg progesterone, at CIDR insertion. Cattle received PGF in the ischiorectal fossa at CIDR removal (Day 7) and 1mg EB i.m. 24h later, with FTAI 52 to 56 h after CIDR removal. Pregnancy rate was affected by location (P<0.002; 46.0% versus 61.1% for Locations A and B, respectively), parity (P<0.04; 67.9% versus 53.1% in heifers and cows, respectively), and numbers of times the CIDR had been used (P<0.03; 62.4% versus 48.4% for once- and twice-used CIDRs, respectively). However, the addition of progesterone to the injection of EB at CIDR insertion did not affect pregnancy rate (P=0.6). In Experiment 3, heifers (n=187) received one new, one once-used, one twice-used or two twice-used CIDRs for 7 days and 2 mg EB plus 50 mg of CP4 at the time of CIDR insertion. Heifers were treated with PGF at CIDR removal and 1mg EB i.m. 24 h later, with FTAI 52-56 h after CIDR removal. Pregnancy rate was not affected by treatments (P=0.28, 57.5, 63.8, 47.9, 47.9% for one new, one once-used, one twice-used, or two twice-used CIDRs, respectively). In summary, pregnancy rate to FTAI did not differ between cattle synchronized with a new or once-used CIDR, but pregnancy rate was lower in cattle synchronized with a twice-used CIDR; however, the insertion of two twice-used CIDRs did not affect pregnancy rates. The addition of an injection of progesterone to the estradiol treatment at CIDR insertion did not enhance pregnancy rate to FTAI.     

Sensitivity and specificity of bioassay of estrogenicity on mammary gland and uterus of female mice

Young intact (18 days of age) and adult ovariectomized (OV-X, ovariectomized between 21 to 24 days of age) C3H/Di mice were used to measure the estrogenicity on the basis of the growth response of mammary epithelial structures and weight of the uterus. The percentage area of the mammary fat pad occupied by mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.001 microg.d(-1). The maximum effective dose of estradiol was 0.01 microg.d(-1) and the dose 10 microg.d(-1) of estradiol decreased mammary size to control levels (inverted-U-shaped dose-response curve). Progesterone alone progressively stimulated mammary growth in young intact females from dose 125 microg.d(-1), in adult OV-X animals from dose 1000 microg.d(-1). Both in young intact and adult OV-X animals, uterine weight progressively increased during estradiol treatment. Progesterone alone had no effect on uterine weight in young intact animals; in adult OV-X animals, uterine weight was increased starting from dose 250 microg.d(-1). Progesterone acted synergistically with estradiol to produce higher mammary growth than that in females treated with estradiol alone. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate and inhibited by cortisol in both young intact and adult OV-X animals. Testosterone inhibited estradiol plus progesterone stimulated growth of mammary gland only in OV-X animals, but stimulated uterine weights in both young intact and adult OV-X animals. Spleen weight and size of mammary lymph nodes were not affected by estradiol, progesterone, norethindrone acetate or testosterone, but were decreased by cortisol. Cortisol also decreased the percent area of the mammary fat pad occupied by mammary epithelial structures, but had no effect on weight of the uterus. These results show that bioassay of estrogenicity in females is not specific. Mammary and uterine growth is stimulated not only by estrogens but also by progesterone and testosterone, respectively.     

Studies on extrahypophyseal sites of estrogen action in the induction of ovulation in rats.

To discover possible extrahypophyseal sites of estrogen action in the induction of ovulation, the influence of a s.c. injection of estradiol benzoate (EB) on cell nuclear sizes in the limbic-medial preoptic continuum of progesterone-pretreated cyclic rats was evaluated. The ovulatory dose of 5 mug EB caused a significant increase of nuclear volumes in the medial preoptic nucleus and the anterior and posterior parts of the medial amygdaloid nucleus. Precocious ovulation was induced in prepuberal female rats by unilateral implantation of a molten EB: cholesterol mixture into the posterior part of the mediocortical amygdala (PMCA), but not by implantation into the anterior part of this region (AMCA) or the medial preoptic area (MPA). In adult females injected s.c. with 2.0 mg progesterone on the day post estrus, bilateral implantation of 0.1 or 0.2 mug crystalline EB on the following day did not abolish the delaying effect of progesterone on the preovulatory LH increase and ovulation, when the implants were located in the MPA, lateral septum (LS), bed nucleus of the stria terminalis (BST), AMCA, PMCA or dorsal hippocampus (DHPC), whereas intrapituitary implants were highly effective. However, the bilateral introduction of large tallow pellets containing 0.1 mug EB each, into the LS, BST, AMCA or PMCA advanced ovulation in rats with progesterone-induced 5-day cycles. Equal pellets did neither induced ovulation nor an LH increase after implantation into the MPA or the DHPC. The results suggest that the anterior pituitary, mediocortical amygdala, BST and LS, but not the MPA or DHPC, are sites of the stimulatory feedback of estrogen on gonadotropin secretion in female rats, and that the amygdaloid response to estrogen differs between prepuberal and cyclic females.     

Physiologic role of relaxin on mammary gland growth in rats

The role of relaxin in stimulating growth of the mammary gland was assessed in ovariectomized and intact male rats for a period of 20 days. In addition to relaxin alone, the ovarian mammogenic hormones estradiol and progesterone were used in combination with relaxin and with each other to evaluate responses of mammae. Indices for mammary growth included wet weight, dry fat-free tissue, DNA, RNA, total protein, and collagen. Quantitative estimates of DNA and collagen represented the best indicators of parenchymal and stromal growth, respectively. Because changes in body weights were significantly different among hormonally administered groups, these were included as well. In Ovariectomized young rats, relaxin alone and in combination with estradiol and progesterone increased all indices significantly (P less than 0.01). The collagenous portion of total protein was high for the group receiving relaxin alone (62%) compared with the control group (46%). Relaxin administered along with estradiol and progesterone increased collagen accumulation to 73%, compared with 54% in the estradiol + progesterone group. Relaxin did not significantly increase growth indices when administered to male rats at 10 and 20 micrograms/day, while 30 micrograms stimulated a significant increase in total protein (P less than 0.05), suggesting that 30 micrograms of relaxin/day may be considered the basal concentration needed to induce a physiologic response in males. Relaxin induced a growth effect on mammae by synergizing with progesterone and estradiol in order to stimulate parenchymal proliferation, as noted by a DNA increase, and to increase stromal distensibility of the mammary pad by invoking accumulation of collagen and total protein in substituting for mammary adipose tissue.