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Mechanisms of Hormone Action |
National Institute for Research in Reproductive Health, Indian Council of Medical Research, Parel, Mumbai 400012, India
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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bonnet monkeys, antiprogestin, endometrium, morphology, ultrastructure, ZK 98.299 (onapristone)
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Over the years, several structurally similar synthetic ligands, RU 486, ZK 98.734, and ZK 98.299 have been developed to block progesterone function during the pre- and peri-implantation periods and hence may help regulate fertility [3, 4]. It also has been proposed that the antiprogestins may be useful in the treatment of endometriosis, meningiomas, leiomyomatas [5, 6], and breast cancer [5]. However, the risks of an increased unopposed estrogen action leading to malignancy and other long-term effects as the result of prolonged use of these antiprogestins need to be evaluated in a suitable animal model prior to their therapeutic use.
The antiprogestin onapristone (ZK 98.299) is a 11ß-aryl substituted steroidal progesterone antagonist with high affinity to progesterone receptors (PR) and glucocorticoid receptors [7, 8]. Daily administration of high doses of this antiprogestin (25 mg) blocked the anticipated rise in the midcycle bioactive luteinizing hormone surge, serum estradiol levels, and ovulation [9]. In women, long-term administration of high doses of RU 486 led to the disruption of ovarian function, resulting in amenorrhea [10]. On the other hand, weekly administration of onapristone at lower doses (5.0 or 10.0 mg) had no effect on gonadal hormonal concentrations, ovulation, and menstrual cycle length. However, endometrial development was still found to be impaired in treated bonnet monkeys [11]. Similar observations were recorded in women administered with a daily dose of 1.0 mg RU 486 for 5 mo [12]. This led to the conclusion that the endometrium is more sensitive to antiprogestin action than other target tissues such as the hypothalamus, pituitary, and ovary. However, it still remains to be ascertained whether long-term antiprogestin treatment at low dosages causes endometrial hyperplasia because of an unopposed estrogenic action.
The present study evaluated long-term effects of low-dose onapristone treatment on endometrial development at the subcellular level and set out to determine whether the long-term treatment would induce any hyperplastic changes in the endometrium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female bonnet monkeys (Macaca radiata) weighing between 3.5 and 4.5 kg and showing at least 2 consecutive normal ovulatory menstrual cycles were selected for the study. The monkeys were individually caged under controlled photoperiod and temperature [13]. The study was carried out between the months of September and April. The menstrual cycle length and the duration of menses of each animal was recorded by daily vaginal swab examination. The study was approved by the Institutional Ethics Committee on the Use and Care of Animals in Biomedical Research.
Estimation of Hormonal Levels
Estradiol and progesterone levels were estimated in serum samples by specific radioimmunoassays as described previously [13]. The inter- and intra-assay coefficients of variation were 8% and 7%, respectively, for estradiol and 11% and 13%, respectively, for progesterone. The reagents were provided by the World Health Organization under the Programme for the Provision of Matched Assay Reagents for the Radioimmunoassay of Hormones in Reproductive Physiology [14].
Treatment with Onapristone
Ten adult female bonnet monkeys were randomly divided into three groups. Animals in group 1 (n = 3) were administered subcutaneously (on hind limbs) with vehicle (benzyl benzoate: castor oil, 9:1), and animals in groups 2 (n = 4) and 3 (n = 3) were similarly treated with 2.5 and 5.0 mg onapristone, respectively. Treatment was initiated on Day 5 of the first menstrual cycle, and thereafter it was administered every third day for four to seven consecutive cycles.
Endometrial Biopsies
Endometrial biopsies were collected from control- and onapristone-treated animals during the final treatment cycles. In animals with ovulatory cycles, the biopsy was collected on Day 8 after the midcycle estradiol peak, whereas in animals with anovulatory cycles, biopsy was taken on Day 20 of the cycle.
Tissue Preparation
Part of the endometrial tissue was fixed immediately in modified Karnovsky fluid [15] prepared in cacodylate buffer for ultrastructural analysis, and the other portion was fixed in Bouin fluid for immunohistochemical analysis. The tissue postfixed in 1% osmium tetroxide for ultrastructural analysis was dehydrated in ascending grades of acetone and embedded in araldite. Semithin (0.5 µ) sections were cut and stained with toluidine blue for morphometric analysis.
Morphologic Features of the Glands
The gland cell height and diameter were measured using an ocular micrometer. Only those gland cells where the cells extended from the basement membrane to cilia in the section were counted. Similarly, the gland diameters were recorded only when the epithelial cells were in the plane from the basement membrane to the ciliated luminal surface. This systematic sampling method was adopted to avoid overestimation arising by the way of a longitudinal or tangential cut through the tubular structures. For each sample, a minimum of 20 fields were examined from different blocks in four to six sections. The number of glands seen under x400 magnification was counted in at least 20 fields, four from each of the four to six blocks, and means were calculated. To calculate the gland mitosis, at least 1000 cells were examined under oil immersion, and only definite mitotic figures with the absence of the nuclear membrane were included in the count. The amount of pseudostratification and secretion in the glandular lumen was also recorded on a scale of 0–3 (0 = absent, 1 = mild, 2 = moderate, 3 = marked).
In the stroma, morphological features recorded were predecidual reaction, amount of leukocytic infiltration, amount of extravasation, and stromal edema (same scoring as used for glands).
Electron Microscopy
The ultrathin sections of endometrial tissue showing interference colors ranging from gold to grey were mounted on uncoated copper grids (200 mesh) and stained with uranyl acetate and lead citrate [16]. These sections were observed under a Philips 400T transmission electron microscope (Philips, Eindhoven, the Netherlands) at 80 kV.
A modified scoring system was adapted to record ultrastructural features of endometrium [17]. The modifications were made mainly for the presence of the nucleolar channel system and the largest mitochondrial diameter. The mean score was calculated for the control and two treatment groups. To calculate the mean score, the number of observations for each score was multiplied by the score and the sum total was divided by the number of observations made.
Immunohistochemical Localization of Endometrial Estrogen Receptor and Progesterone Receptor
In brief, the sections were deparaffinized in xylene and rehydrated through various grades of alcohol in distilled water. Immunoperoxidase staining to localize the estrogen receptor (ER) and PR was performed using Vectastain ABC kits (Burlingame, CA) on sections of endometria from control and treated animals. Sections were blocked with 1% BSA in PBS (120 mM NaCl, 2.7 mM KCl, and 10 mM phosphate buffer, pH 7.4) followed by incubation with the primary antibody for PR (MA1-410; Affinity Bioreagents, Golden, CO) at 1:50 dilution or ER (MA1-310, Affinity Bioreagents) at 1:100 dilution for 1 h at room temperature. After washing with PBS, the sections were incubated with biotinylated goat antimouse secondary antibody (DAKO, Glostrup, Denmark) at 1:100 dilution for 30 min. After a PBS rinse, the endogenous peroxidase was inactivated by a 30-min incubation with 0.3% H2O2 in methanol. This was followed by rehydration in PBS for 30 min. Sections were then incubated with avidin:biotinylated horse radish peroxidase macromolecular complex (ABC) for 30 min followed by an addition of diaminobenzidine in PBS for 5 min to complete the reaction. The sections were dehydrated, cleared in xylene, mounted, and viewed under the microscope.
The resulting staining for ER and PR was evaluated on a Zeiss microscope (Carl Zeiss, Oberkochen, Germany) at low (x10) and high (x40) magnifications. Intensity of the staining was judged as absent (-), weak (+), moderate (++), and strong (+++). Photomicrographs were taken using Kodak Gold-MAX 100 ASA film (Eastman Kodak, Rochester, NY) at x120 magnification.
Intensity of immunohistochemical staining for ER and PR in both endometrial glands and stroma for each animal in the control and treatment groups was evaluated by two independent observers using the semiquantitative HSCORE = Pi(i + 1) method, where i is the intensity of staining (a value of 1, 2, or 3, corresponding to weak, moderate, or strong staining, respectively) and Pi is the percentage of staining cells for each intensity (varying from 0% to 100%) [18]. At least 1000 cells were counted, which corresponded to four to six sections per slide.
Statistical Analyses
Unpaired Student t-test was applied to determine differences in hormonal concentrations. One-way ANOVA with Duncan multiple range test was applied to determine group differences in morphometric parameters. Analysis of semiquantitative HSCORE for significance was carried out using Mann-Whitney rank sum test. The data were considered to be significant when P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animals in the control group displayed follicular and luteal phases of normal length with optimal serum concentrations of estradiol and progesterone. Serum concentrations of estradiol and progesterone during the pretreatment and four treatment cycles in a representative animal (#266) from the 2.5-mg treatment group are shown in Figure 1. All cycles were ovulatory in two animals treated with 2.5 mg onapristone for four cycles. Serum concentrations of estradiol and progesterone on the day of biopsy were 50 pg/ml and 3.28 ng/ml, respectively, in these animals. However, animals that were treated with 2.5 mg for 6 cycles became anovulatory. Thus the duration of the treatment did adversely affect the serum concentrations of gonadal hormones.
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All animals treated with 5.0 mg onapristone also showed anovulation as evident by low serum concentrations of estradiol and progesterone (Fig. 2). However, the anovulatory cycles were of normal duration in these animals. Serum concentrations of estradiol increased gradually during the midfollicular phase, which was followed by a sharp decline. The profile appeared to be similar to that seen in normal cycles. However, serum concentrations of progesterone did not increase above 1 ng/ml (Table 1).
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Morphological Analysis
Endometria from the control animals in the midluteal phase showed well-developed glands with secretory activity, stromal edema (Fig. 3A), and formation of spiral arteries, whereas in the 2.5-mg treatment group endometrial glands showed increased mitotic activity, epithelial pseudostratification, and reduced diameter compared with the control group. Stroma exhibited the early signs of compaction (Fig. 3B). In the higher dose group (5.0 mg), diameter of the glands was further decreased. The stroma showed no signs of vascular and stromal differentiation (Fig. 3C).
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The results of the morphometric evaluation of glandular and stromal features of endometria in control and treatment groups are presented in Tables 2 and 3, respectively. Endometria from the 2.5-mg-treated ovulatory animals showed weak signs of secretion in the glandular cells. Mitotic activity in endometrial glandular cells was also evident in these animals (#186, #266). However, in the animals that became anovulatory (#104, #244) following 2.5 mg onapristone treatment, endometrial glands were inactive and apparently devoid of secretory activity.
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Endometria from the animals in the 5.0-mg treatment group showed a further decrease in the height and diameter of the glands resulting in an increased number of glands per high power field (P < 0.05; Table 2). The glands did not show any secretory activity. Pseudostratification of glandular cells was seen with the absence of subnuclear vacuoles.
Stromal features such as predecidual reaction, edema, and mitosis scored much lower in treatment groups as compared with the control group (Table 3). The mitotic index remained unaffected in endometrial stroma in 2.5-mg-treated ovulatory animals. An increased number of mast cells were seen in the endometrial stromal tissue from the 2.5-mg treatment group as compared with controls. Blood vessels were significantly dilated in the compact spindly stroma in the 5.0-mg treatment group as compared with vehicle-treated animals.
Ultrastructural Analysis
The electron microscopic analysis of endometrial glandular and stromal cells in vehicle-treated animals revealed characteristic features of a developed secretory endometrium (Fig. 4, A–D; Fig. 5A). The gland cell nuclei demonstrated an euchromatin pattern with only little chromatin condensation. Nucleoli were well developed. The nuclear envelope and endoplasmic reticulum system were extensively developed to produce a labyrinthine complex throughout the cytoplasm. The glandular cytoplasm also showed some secretory material such as glycoprotein and free glycogen particles (Fig. 4, A and B). A well-developed Golgi system with a number of dilated vacuoles and vesicles were observed (Fig. 4, A and B). A ribosomal endoplasmic reticulum (RER) system, represented by short and dilated cisternae enclosing the electron lucent material, was most prominent in basal cytoplasm surrounded by numerous small and large mitochondria. The mitochondria exhibited a well-organized internal structure with transverse cristae, whereas few were irregularly developed with a loss of their cristae (Fig. 4, C and D). Numerous long microvilli were seen on the cell surface.
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The stromal cells showed lipid droplets, predecidual changes with well-developed RER, and several phagocytic vesicles with cross-sectioned collagen fibers (Fig. 5A).
Endometria from 2.5-mg-treated animals (Fig. 4E) showed a general loss of cell organelles. A delay in maturation of the endometrium was indicated by proliferative changes and small round glands in two animals (#186, #246) that remained ovulatory even after onapristone treatment. Endometria of the two anovulatory animals (#104, #244) showed degenerative changes like vacuolation and formation of myelin bodies in glandular and stromal cells (Figs. 4F and 5B). The desmosomes were absent with characteristic infolding of the plasma membrane (Fig. 4G). The cytoplasmic organelles were fewer, and nuclei with prominent nucleoli were seen. Fibrous stroma was marked by the presence of vacuoles in the cytoplasm (Fig. 5C).
At a higher dose (5.0 mg), total degeneration with an increased appearance of lipofuchsin bodies, loss of other cellular organelles, and marked vacuolation was observed in endometrial glandular cells (Fig. 4H). An increase in the intracellular spaces and pseudostratification of the nuclei could also be seen. There was accumulation of secretions in the spaces between glandular cells. The nuclei were highly indented, giving rise to a segmented appearance, and were without nucleoli (Fig. 4H). There was very little lumen with few microvilli at the luminal surface. The stroma also showed accumulation of secretory material and lipids in the intercellular space in otherwise degenerative cells (Fig. 5D). The mean scores for ultrastructural analysis of endometrial cell organelles are given in Table 4.
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Endometrial ER and PR
No significant change was observed in the localization and expression of ER in the nuclei of endometrial glandular epithelial and stromal cells between control and treated animals (Fig. 6). Similarly, localization or expression of the endometrial PR protein in the glandular as well as in the stromal compartments remained unaltered in 2.5-mg-treated animals (Fig. 7b) as compared with the control animals (Fig. 7A). However, the expression of PR was significantly (P < 0.01) reduced in the endometrial glandular epithelial as well as stromal cells in 5.0-mg-treated animals (Fig. 7C) as compared with controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Morphometric analysis revealed a drastic reduction in the diameter and height of the endometrial glandular cells in treated animals, thereby indicating impaired glandular function following antiprogestin treatment. Two animals (#104, #232) that became anovulatory following a 2.5-mg onapristone treatment for six cycles showed myelin bodies and mast cells in the endometrial stroma. Myelin bodies could have originated because of the degeneration of cellular organelles, especially mitochondria, whereas an increase in the number of mast cells could have occurred because of fibrosis [19]. These mast cells were not seen in the endometria from control and treated ovulatory animals.
Animals (#186, #266) that remained ovulatory following a 2.5-mg onapristone treatment for four cycles showed a concomitant increase in the mitotic index, whereas endometria from the animals that became anovulatory following treatment with 2.5 or 5.0 mg of onapristone showed a negligible mitotic index in glandular and stromal cells. In these anovulatory animals, the serum progesterone concentrations were low and the estradiol levels were within the range usually encountered during the normal follicular phase [13]. This should have led to increased cell proliferation in the endometrial epithelial cells. However, we failed to note any increase in mitotic activity in the glandular epithelium of endometria obtained from the treated animals. In contrast, severely atrophied endometrium was observed in these animals.
Ultrastructural analysis of endometria from treated animals revealed the presence of large vacuoles, membranous inclusions, and myelin-like bodies. These features were suggestive of the disintegration of cytoplasmic proteins. Vacuolation is one of the structural indicators of energy deficit and permeability disorder of membranes in the endometria from treated animals.
The presence of abundant ribosomes and a rough endoplasmic reticulum indicated that the machineries for protein synthesis and transport were present but probably dysfunctional in endometria from the treated animals. Marked shrinkage in mitochondrial size and electron density also indicated some changes in the lipoprotein complex of the membrane, resulting in the formation of myelin bodies. This could have caused impaired endometrial growth in the treated animals.
Our data also suggest that the histomorphological measurements by quantitative morphometry are useful tools for the evaluation of steroidal effects on endometrial stroma and glandular epithelium, as suggested previously [20, 21]. Progesterone-induced secretory activity was profoundly reduced in the endometria from onapristone-treated groups. This is in agreement with the reports of Gemzell-Danielsson et al. [22] and Dockery et al. [23].
Formation of new blood vessels (angiogenesis) in a menstrual cycle-dependent pattern is a distinct process during endometrial development [24, 25]. Progesterone is known to exert mitogenic effects on spiral arteries in endometrium. Decline in the expression of specific angiogenesis and vasopermeability factors (i.e., vascular endometrial growth factor/vascular permeability factor [VEGF/VPF]) has been observed following treatment with mifepristone [26, 27]. In the present study, an intense dilatation of stromal vessels was observed following treatment with onapristone. These results are in agreement with the reports by Reinsch et al. [28] and Slayden et al. [29], where a decrease in blood flow in the endometrium was observed following long-term treatment with antiprogestins. It is likely that the reduced vascular support deprives the endometrium of nutrition and oxygen and causes atrophy. The pronounced atrophy of the endometrium in the animals subjected to long-term onapristone treatment at low doses may also arise because of the derangement in the expressions of progesterone-dependent growth factors such as leukemia inhibitory factor, transforming growth factor [30], keratinocyte growth factor [31], and insulin-like growth factor II [32].
Our data support earlier observations that demonstrated antimitogenic effects of mifepristone administered at 2 mg/day for 30 day [33]. The inhibitory effects of mifepristone on endometrial growth were also observed in humans [34] and monkeys [35]. In the present study an attempt has been made to determine whether the antiprogestin-induced antiestrogenic effects are reflected as change in the concentration or localization of endometrial ER and PR, as both of these receptor proteins are estrogen-dependent. However, we did not find any significant change in the levels of endometrial ER protein in 2.5-mg- and 5.0-mg-treated groups, whereas a decrease in the PR localization was observed following 5.0-mg treatment. This is in contrast to a report demonstrating elevation in endometrial ER and PR after chronic administration of antiprogestin [35]. Our data suggest that the antiestrogenic effect of ZK 98.299 are mediated through a mechanism that does not operate via changes in ER and PR levels. The precise mechanism of antiprogestin action on endometrium still needs to be elucidated. It is likely that the effects of antiprogestins are elicited through some perturbation in postreceptor events. This study also suggests that long-term treatment with antiprogestins does not lead to endometrial hyperplasia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 31 May 2002.
First decision: 17 June 2002.
Accepted: 20 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) between two gland cells, giant M, and disorganized RER were observed in glandular cell cytoplasm (original magnification x8500). E) Degeneration and loss of most of the cellular organelles was observed in endometrial glandular cells. Apical end of gland cells showed fewer microvilli (original magnification x1400). F) N of the glandular cells with prominent NC and myelin bodies (MY) in the cytoplasm (original magnification x1400). G) The glandular cell cytoplasm with accumulation of L with interdigitation of plasma membrane of cells (
