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Endometrial steroid receptors during decidualization in rhesus monkey (Macaca mulatta); their modulation by anti-oestrogen CDRI-85/287

Endometrial steroid receptors during decidualization in rhesus monkey (Macaca mulatta); their... Abstract With a view to elucidating the hormonal control of decidualization in rhesus monkey, we studied the effects of CDRI-85/287, a potent anti-oestrogen, on endometrial steroid receptors in vivo and in vitro. Compound 85/287 was administered (i.m.) on days 8, 9 and 10 of steroid treatment cycle at a dose of 15 mg/monkey. Deciduoma was induced on day 16. Histological examination of endometrial tissue on days 24 and 30 of the cycle showed an apparent inhibition in uterine epithelial and subepithelial decidual cell plaque formation and a decrease in leukocytic infiltration into the stroma in anti-oestrogen-treated animals. As observed on day 24, a significant decrease in progesterone receptors (PR) (nuclear + cytosolic) was observed in the 85/287-treated group, whereas oestrogen receptor (ER) content remained unaltered. On day 30 total ER as well as total PR content was markedly reduced in treated animals. In-vitro results clearly demonstrated a competitive antagonism of 85/287 at the ER level only. The results are discussed in relation to the histological changes and modulation of steroid receptors, thereby suggesting the decidualization inhibitory activity of anti-oestrogen molecule 85/287 in primate species. anti-oestrogen, decidualization, endometrium, receptors Introduction The non-steroidal oestrogen antagonist CDRI-85/287 2-[4-(2-N-piperidinoethoxy)phenyl]-3-phenyl(2H)benzo(b)pyran has shown potential as an anti-oestrogen and anti-implantation agent in rats (Sharma et al., 1990; Dhar et al., 1991). Like other non-steroidal oestrogen antagonists, e.g. nafoxidine, tamoxifen, ormeloxifene and EM-800, it appears to exert its biological effects via competitive antagonism at the oestrogen receptor (ER) level (Srinivasulu et al., 1992a,b; Dhar et al., 1994; Martel et al., 1998). However, no information is available in primates regarding the biological/pharmacological potential of this molecule. The hormonal control of uterine proliferation and differentiation events during the preimplantation period and early pregnancy have been investigated in detail at the morphological, biochemical and molecular levels in mammals including rodents and humans (Psychoyos, 1973; Finn, 1977; Glasser, 1990; Abrahamson and Zorn, 1993; Tang et al., 1994). Knowledge of these processes suggests that decidualization initiated by an embryonic or artificial stimulus depends upon previous sensitization by a sequence of oestrogen and progesterone secretion and their action. The experimental model established by Ghosh and Sengupta (1989), for the study of plaque and decidual cells in response to artificial traumatization of the uterus in ovariectomized hormone-treated rhesus monkeys, provides an understanding of the hormonal control of endometrial sensitivity for cellular events which normally occur around implantation. Ghosh and Sengupta (1988) have also reported the involvement of sex steroid hormones during preimplantation stages of gestation in terms of changes in quantity of nuclear and cytoplasmic receptors for oestrogen (ER) and progesterone (PR) in the endometrium. Moreover, the enhancing effects of oestradiol on ER and PR are well documented in rhesus monkeys by other groups of investigators (Kreitmann-Gimbal et al., 1979, 1980). The present study was designed to examine the in-vivo effects of a novel anti-oestrogen molecule on ER and PR in endometrium of ovariectomized monkeys receiving hormonal treatments and an artificial deciduogenic stimulus. In addition, we investigated the binding activity of CDRI-85/287 to endometrial steroid receptors in vitro with a view to elucidating the hormonal control of the deciduogenic mechanism in this species. Materials and methods Chemicals [3H]Promegestone (R 5020, sp. act. 87 Ci/mmol), unlabelled R 5020 and [3H]oestradiol (sp. act. 93.3 Ci/mmol) were purchased from Dupont NEN Research Products, USA. Unlabelled diethylstilboestrol (DES) and tamoxifen were purchased from Sigma Chemical Company, St Louis, MO, USA. All other chemicals and reagents were of Analytical grade. Compound CDRI-85/287 synthesized at the Central Drug Research Institute (Sharma et al., 1990) was used throughout the study. Animals, treatment and tissue collection Adult female rhesus monkeys (Macaca mulatta; body weight 4–6 kg) of proven fertility maintained under uniform husbandry conditions at the Institute's Primate House were used in this study. Monkeys were bilaterally ovariectomized under ketamine anaesthesia. After a rest period of 45 days, monkeys were randomly divided into various groups. A total of 12 monkeys were divided into four groups composed of three monkeys each and given oestradiol dipropionate (EDP) followed by progesterone. The treatment schedule and the doses were followed essentially as described by Ghosh and Sengupta (Ghosh and Sengupta 1989). On days 8, 9 and 10, the compound 85/287 was given i.m. in olive oil at a dose of 15 mg/monkey in groups I and II. Animals of groups III and IV received vehicle only and served as control. On day 16 of hormone treatment, monkeys were laparotomized and a sterile nylon thread was inserted through the uterine lumen to induce decidualization. On days 24 and 30 of the treatment cycle, animals were given ketamine anaesthesia, endometrial tissues collected, washed in ice-cold saline to remove blood contamination and stored in liquid nitrogen until assayed. For histology, tissue slices were fixed in Bouin's fluid, embedded in wax at 58°C, sections were cut on a microtome at 5 μm, dehydrated in ethanol, stained with haematoxylin/eosin and examined under microscope. Preparation of nuclear and cytosolic fractions All subsequent steps were carried out at 0–4°C unless otherwise stated. The tissue was washed three times and homogenized in 5 vol. of 10 nmol/l Tris buffer, pH 7.4 containing 1 nmol/l magnesium chloride, 1 nmol/l monothioglycerol and 10% glycerol (TMMG) giving 10 s bursts with 1 min cooling intervals using a polytron PT-10 homogenizer (Brinkmann Instruments, Westbury, NY, USA). Cytoplasmic and nuclear fractions were separated by centrifugation (800 g, 10 min) and the supernatant centrifuged at 130 000 g for 60 min, to yield the clear cytosol fraction. The pellet was then washed three times with TMMG buffer and centrifuged at 800 g for 10 min. The washed nuclear pellet was resuspended in assay buffer to the original volume and was used for ER and PR assays. Oestrogen and progesterone receptor assays Prior to assay, the cytosol was incubated with an equal volume of dextran-coated charcoal (0.05% dextran and 0.5% activated charcoal in Tris buffer pH 7.4) (DCC) for 60 min at 4°C with occasional stirring to remove endogenous steroids. Cytosol was centrifuged at 800 g for 15 min, twice. The clear supernatant was used for binding assay. ER and PR were measured using methods described by Ghosh and Sengupta and Okulicz et al. with some modification (Ghosh and Sangupta, 1988; Okulicz et al., 1990). In brief, for determination of cytosolic receptors, the cytosol (200 μl) was incubated in triplicate with saturating concentration of [3H]oestradiol (25 nmol/l) or [3H]R 5020 (25 nmol/l) in the absence or presence of 1 μmol/l DES (for ER) or 1 μmol/l R 5020 (for PR) for 18 h at 4°C. At the end of the incubation period the bound and free steroids were separated by a brief incubation wth 500 μl of DCC suspension for 10 min. Tubes were then centrifuged at 800 g for 10 min at 4°C. The supernatant obtained (bound fraction) was transferred to the vials containing scintillation fluid. For determination of nuclear ER and PR, the nuclear fraction (200 μl) was incubated in triplicate with [3H]oestradiol for 30 min at 37°C or with [3H]R 5020 for 16 h at 4°C in the absence or presence of unlabelled 1 μmol/l DES or R 5020 for ER and PR respectively. Following incubation, 1 ml chilled buffer was added to each tube and free steroid was removed by washing with buffer and centifuged at 800 g three times at 4°C. Bound radioactivity was then extracted by 1 ml 100% ethanol twice at 30°C. Ethanolic extracts were pooled and transferred to scintillation vials and counted for radioactivity at the counting efficiency of 60%. Cytosolic protein and nuclear DNA were estimated by the methods of Lowry et al. (Lowry et al., 1951) and Burton (Burton, 1956) respectively. Specific binding was computed from the difference between the total binding (i.e. in the absence of unlabelled DES or R 5020) and non-specific binding (i.e. in the presence of unlabelled DES or R 5020). Receptor concentrations were expressed as fmol/100 μg protein (for cytosolic receptors) and fmol/100 μg DNA (for nuclear receptors). Values were expressed as mean ± SD. Competitive binding assay Endometrial cytosol (obtained from the control animals) were incubated with [3H]oestradiol (2.5 nmol/l) or [3H]R 5020 (2.5 nmol/l) at 4°C for 22 h. Parallel incubations were run in the presence of various concentrations of unlabelled steroid or tamoxifen or CDRI-85/287 dissolved in DMF:buffer (1:1) as described elsewhere (Dhar et al., 1994). Following incubation, bound and free steroids were separated by addition of 500 μl of DCC suspension as described above. Bound fractions at each concentration of competitor were assessed for radioactivity. The concentration of competitor required for 50% inhibition of 3H-steroid binding were determined by a graph between percentage bound ligand versus molar concentraion of the competitors. Statistical analsysis Significance of differences between various treatment groups was carried out by using Student's t-test. Results Histology In hormone-treated monkeys, decidualization was characterized by a marked luminal and epithelial plaque acinar cell formation on day 24 of the cycle (Figure 1A,B) which increased on day 30 (Figure 2A). Hypertrophy and hyperplasia was evident in plaque acinar cells with metrial glands in the peripheral endometrial stroma lined with cuboidal epithelial cells. Administration of the compound 85/287 showed an apparent inhibition in uterine epithelial and subepithelial decidual plaque cell formation indicating their degeneration (Figure 1C,D). Clumping and vacuolization was discernible in decidual plaque cells both on days 24 and 30 of the cycle in treated monkeys (Figures 1C,D, 2B,C). Low cuboidal epithelium of metrial glands and proliferation in epithelial cells were noted. The stromal response to decidualization was marked by a higher degree of oedema with leukocytic infiltration in control hormone-primed decidualized uterine endometrium on day 24. However, the degree of response was more on day 30. CDRI-85/287 relatively decreased the leukocytic infiltration into the stroma, whereas mitotic activity showed an increase. ER and PR concentrations in endometrium The results of cytosolic and nuclear ER and PR are illustrated in Figure 3. When expressed as fmol/100 μg protein and per 100 μg DNA, on day 24 of treatment cycle there was a marked decrease (P < 0.001) in cytosolic as well as nuclear progesterone receptor content in the 85/287-treated group of monkeys (PRc 43.7 ± 8.5, PRn 44.6 ± 13.6) as compared to that of control decidualized endometrium (PRc 256.6 ± 34.0, PRn 260.2 ± 54.9; Figure 3B). In addition, no significant change in ER content was observed in the treated group. However, the nuclear ER were increased in concentration (control, ERn 79.8 ± 8.4; treated 208.6 ± 36.7) (P < 0.05) (Figure 3A). On day 30 of the cycle, cytosolic ER and PR concentrations were significantly decreased (P < 0.001) as compared to that on day 24 of the cycle, in untreated decidualized endometrium. Interestingly, administration of 85/287 caused a significant reduction (P < 0.001) in the concentrations of nuclear ER as well as PR in both the cellular compartments (control, ERn 232.2 ± 91.0, treated 34.7 ± 11.4; control PRn 83.4 ± 38.7; treated, 32.4 ± 8.0; control, PRc 264.7 ± 112.5, treated 109.4 ± 3.4) (Figure 3C and D). Our findings suggest in general a decrease in cytosolic ER and cytosolic PR as a result of 85/287 treatment in both groups of animals. The rise in nuclear ER as observed on day 24 appears to be due to translocation of the receptor to the nuclear compartment. In-vitro binding of 85/287 to endometrial ER and PR In order to examine the in-vitro competitive binding to both steroid receptors, i.e. ER and PR, IC50 of oestradiol, R5020, tamoxifen and CDRI-85/287 were determined for [3H]oestradiol and [3H]R 5020 binding in endometrial cytosol (Table I). Tamoxifen and 85/287 showed IC50 ~50- and 350-fold that of oestradiol respectively for [3H]oestradiol binding, whereas R 5020 could not inhibit [3H]oestradiol binding even up to 100 μmol/l concentration. In the case of [3H]R 5020 binding only R 5020 showed IC50 of 16 nmol/l whereas the rest of the competitors including 85/287 showed IC50 >1000-fold that of R 5020. These results clearly demonstrate the competitive antagonism of tamoxifen and 85/287 at ER level only. However, 85/287 appears to be more potent as a competitor. Discussion The obligatory role of progesterone in the elicitation of endometrial responses in decidual cell reactions is well documented, whereas the role of oestrogen appears to be subtle (Glasser and McCormack, 1982). Steroid dynamics in the target organ vary and depend upon the nature of hormone sensitization, treatment and hormonal milieu at the cell level. The steroid receptor values of the present study are comparable to those reported in ovariectomized monkeys following sequential oestradiol and progesterone treatment (Kreitmann-Gimbal et al., 1979; West and Brenner, 1985; West et al., 1986; Ghosh and Sengupta, 1989). It is well known that oestradiol increases the concentration of its own receptor as well as that of PR in normal endometrium (Kassis et al., 1984; Lessey et al., 1989), at the same time progesterone in adequate amounts counteracts these oestrogenic effects. Moreover, unoccupied PR also play a role in the control of PR biosynthesis in primate endometrium as suggested by Chwalisz et al. (Chwalisz et al., 1991). Anti-oestrogens also counteract such oestrogenic effects/action (Lessey et al., 1989). Here too our findings show that treatment with anti-oestrogen CDRI-85/287 decreased the amount of both receptors. The results indicate that ER and PR synthesis is ultimately dependent upon the presence of oestradiol. It has been reported that plasma and endometrial oestradiol and progesterone concentration in primates as well as humans varies with respect to physiological conditions and is regulated by tissue specific variables including distribution, binding, intracellular metabolism and clearance of steroid hormones (Resko et al., 1976; Batra et al., 1977; Poortman et al., 1983). The observation that concentrations of PRn closely parallel those of progesterone in tissue and plasma substantiates the requirement of progesterone for inducing and maintaining the gestational changes in the endometrial tissue; however, despite the apparent decrease in cytoplasmic PR and nuclear ER, the total receptor content for both hormones remained unchanged (Ghosh and Sengupta 1988, 1989). In the present study the ER-mediated anti-oestrogenic action of 85/287 (Table I) may probably be one of the factors leading to the inhibition of decidualization-stimulating events. Endometrial proliferation is well known to be an oestrogen-dependent event (prerequisite for decidualization) as observed in our studies showing inhibition of eosinophilic inflitration, stromal oedema, glandular proliferation and epithelial cell height. It has been demonstrated that oestradiol in a physiological amount has induced the synthesis of peptide growth factors and their specific receptors in order to promote endometrial mitogenesis and tissue growth generally (Beck and Garner, 1989; Boehm et al., 1990; Corden-Cardo et al., 1990; Ishihara et al., 1990; Reynolds et al., 1990). It is pertinent to note here that modulation of growth factors by anti-oestrogens/antiprogestins has also been reported (Greb et al., 1997; MacGregor and Jordon, 1998). The data presented here indicate that the antiprofliferative effects of anti-oestrogen upon endometrium are due to diminution of the ER pool as well as the altered synthesis/concentrations of PR. Interestingly on day 24, we observed an increase in nuclear ER concentrations. Several investigators have reported that injection of progesterone under proliferative, secretory, midcycle or oestradiol-primed conditions results in a change in distribution of PR concentrations with increase in nuclear and a concomitant decrease in cytoplasmic receptor concentrations. Similar effects might be involved after anti-oestrogen treatment under oestrogen-primed conditions leading to the altered distribution of ER in different cellular compartments (Neulen et al., 1996). The mechanisms underlying the antideciduogenic effects of anti-oestrogen in primate endometrium vary. Antiprogestins have been reported to stabilize the inactive so-called `8S' form of PR as well as to limit its transformation towards the activated `4S' form (Renoir et al., 1989). Recent studies suggest that progesterone acts through cycle-specific PR isoforms A and B associated with the fluctuation in their ratios (Mangal et al., 1997). Receptor-bearing RU 486 bound to PRE (progesterone response element), however, cannot promote transcription from respective genomic sites (Baulieu, 1991). Similarly in a few reports anti-oestrogens such as tamoxifen and H 1285 have been suggested to affect the post-transcriptional events involving the above mechanism in breast cancer/uterine tissue (Ruh et al., 1990; Allan et al., 1992). Thus, anti-oestrogenic activities may derive from receptor transformation/ERE (oestrogen response element) mediated mechanisms. In this context it has also been reported that opposite reactivity of the two forms of ER (8S and 4S) with tamoxifen aziridine and ERE support the hypothesis that they may constitute separate entities with a different physiological role (Navarro et al., 1998). Our observations suggest that anti-oestrogens may have decidualization inhibitory activity in primate endometrium and also indicate the anti-oestrogenic potential of this benzopyran derivative (85/287) in higher species. However, further extensive studies on the pharmacological profiles of such novel series of non-steroidal moieties are warranted. Table I. In-vitro competitive binding of 85/287 Competitor  IC50a value    [3H] oestradiol binding  [3H]R 5020 binding  aConcentration of the competitor required for 50% inhibition of radioligand binding. Data are mean of three experiments.  Oestradiol  ~10 nmol/l  >500 nmol/l  R 5020  >100 μmol/l  ~16 nmol/l  Tamoxifen  ~400 nmol/l  >100 μmol/l  CDRI-85/287  ~3500 nmol/l  >100 μmol/l  Competitor  IC50a value    [3H] oestradiol binding  [3H]R 5020 binding  aConcentration of the competitor required for 50% inhibition of radioligand binding. Data are mean of three experiments.  Oestradiol  ~10 nmol/l  >500 nmol/l  R 5020  >100 μmol/l  ~16 nmol/l  Tamoxifen  ~400 nmol/l  >100 μmol/l  CDRI-85/287  ~3500 nmol/l  >100 μmol/l  View Large Figure 1. View large Download slide View large Download slide View large Download slide View large Download slide (A) Endometrial deciduogenic response in ovariectomized hormone-primed rhesus monkey on day 24 of the cycle shows a characteristic decidual plaque cell formation (arrow head). Note also the uterine glandular epithelial plaques (arrow). (B) A magnified view of A exhibits plaque acinar cells depicting hypertrophy and hyperplasia. (C) Compound CDRI-85/287 treatment showing significant degenerative changes in endometrial decidual plaque cells reaction marked by degeneration of plaques, condensation of acinar cell nuclei and presence of cytoplasmic vacuoles (arrow) after 24 days. Note also a decreased epithelial cell height in metrial glands. (D) A magnified view of C showing degeneration of decidual plaque cells. Haematoxylin–eosin. Scale bars = 188 μm (A, C), 94 μm (B, D). Figure 1. View large Download slide View large Download slide View large Download slide View large Download slide (A) Endometrial deciduogenic response in ovariectomized hormone-primed rhesus monkey on day 24 of the cycle shows a characteristic decidual plaque cell formation (arrow head). Note also the uterine glandular epithelial plaques (arrow). (B) A magnified view of A exhibits plaque acinar cells depicting hypertrophy and hyperplasia. (C) Compound CDRI-85/287 treatment showing significant degenerative changes in endometrial decidual plaque cells reaction marked by degeneration of plaques, condensation of acinar cell nuclei and presence of cytoplasmic vacuoles (arrow) after 24 days. Note also a decreased epithelial cell height in metrial glands. (D) A magnified view of C showing degeneration of decidual plaque cells. Haematoxylin–eosin. Scale bars = 188 μm (A, C), 94 μm (B, D). View large Download slide View large Download slide View large Download slide Figure 2. (A) Showing an increased endometrial decidual plaque cell response in ovariectomized, horomone-primed rhesus monkeys on day 30 of the cycle. Metrial glands show an increase in epithelial cell height (arrow). (B) Compound CDRI-85/287 administration caused a marked inhibition in decidual plaque cell reaction indicating an intensive degeneration in plaque acinar cells (arrow). (C) More highly magnified picture of B showing clearly the clumps of acinar cells. Haematoxylin–eosin. Scale bars = 188 μm (A, B), 94 μm (C). View large Download slide View large Download slide View large Download slide Figure 2. (A) Showing an increased endometrial decidual plaque cell response in ovariectomized, horomone-primed rhesus monkeys on day 30 of the cycle. Metrial glands show an increase in epithelial cell height (arrow). (B) Compound CDRI-85/287 administration caused a marked inhibition in decidual plaque cell reaction indicating an intensive degeneration in plaque acinar cells (arrow). (C) More highly magnified picture of B showing clearly the clumps of acinar cells. Haematoxylin–eosin. Scale bars = 188 μm (A, B), 94 μm (C). Figure 3. View largeDownload slide Oestrogen receptor (ER) and progesterone receptor (PR) in cytosolic (c) and nuclear (n) compartments in monkey endometrium on day 24 (A, B) and day 30 (C, D) of the treatment cycle; data are mean of three observations. □ Control group; ▪ 85/287-treated group; error bars indicate standard deviation. Significance is based on comparisons between control and treated groups. Figure 3. View largeDownload slide Oestrogen receptor (ER) and progesterone receptor (PR) in cytosolic (c) and nuclear (n) compartments in monkey endometrium on day 24 (A, B) and day 30 (C, D) of the treatment cycle; data are mean of three observations. □ Control group; ▪ 85/287-treated group; error bars indicate standard deviation. Significance is based on comparisons between control and treated groups. 1 To whom correspondence should be addressed at: Division of Endocrinology, Central Drug Research Institute, Post Box No. 173, Lucknow 226001, India The authors are grateful to Dr C.M.Gupta, Director and Dr V.P.Kamboj, former Director, for their keen interest in this study. We thank Dr R.S.Kapil for the generous supply of compound 85/287. We acknowledge Ms Kanak Lata for technical assistance in animal experimentation and Mr Balvir Singh for protein and DNA estimations of the samples. This study was supported by a grant from the Ministry of Health and Family Welfare, Government of India. 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( 1986) Differential suppression of progesterone receptor by progesterone in the reproductive tract of female macaques. J. Steroid Biochem. , 25, 497–503. Google Scholar © European Society of Human Reproduction and Embryology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Reproduction Oxford University Press

Endometrial steroid receptors during decidualization in rhesus monkey (Macaca mulatta); their modulation by anti-oestrogen CDRI-85/287

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Abstract

Abstract With a view to elucidating the hormonal control of decidualization in rhesus monkey, we studied the effects of CDRI-85/287, a potent anti-oestrogen, on endometrial steroid receptors in vivo and in vitro. Compound 85/287 was administered (i.m.) on days 8, 9 and 10 of steroid treatment cycle at a dose of 15 mg/monkey. Deciduoma was induced on day 16. Histological examination of endometrial tissue on days 24 and 30 of the cycle showed an apparent inhibition in uterine epithelial and subepithelial decidual cell plaque formation and a decrease in leukocytic infiltration into the stroma in anti-oestrogen-treated animals. As observed on day 24, a significant decrease in progesterone receptors (PR) (nuclear + cytosolic) was observed in the 85/287-treated group, whereas oestrogen receptor (ER) content remained unaltered. On day 30 total ER as well as total PR content was markedly reduced in treated animals. In-vitro results clearly demonstrated a competitive antagonism of 85/287 at the ER level only. The results are discussed in relation to the histological changes and modulation of steroid receptors, thereby suggesting the decidualization inhibitory activity of anti-oestrogen molecule 85/287 in primate species. anti-oestrogen, decidualization, endometrium, receptors Introduction The non-steroidal oestrogen antagonist CDRI-85/287 2-[4-(2-N-piperidinoethoxy)phenyl]-3-phenyl(2H)benzo(b)pyran has shown potential as an anti-oestrogen and anti-implantation agent in rats (Sharma et al., 1990; Dhar et al., 1991). Like other non-steroidal oestrogen antagonists, e.g. nafoxidine, tamoxifen, ormeloxifene and EM-800, it appears to exert its biological effects via competitive antagonism at the oestrogen receptor (ER) level (Srinivasulu et al., 1992a,b; Dhar et al., 1994; Martel et al., 1998). However, no information is available in primates regarding the biological/pharmacological potential of this molecule. The hormonal control of uterine proliferation and differentiation events during the preimplantation period and early pregnancy have been investigated in detail at the morphological, biochemical and molecular levels in mammals including rodents and humans (Psychoyos, 1973; Finn, 1977; Glasser, 1990; Abrahamson and Zorn, 1993; Tang et al., 1994). Knowledge of these processes suggests that decidualization initiated by an embryonic or artificial stimulus depends upon previous sensitization by a sequence of oestrogen and progesterone secretion and their action. The experimental model established by Ghosh and Sengupta (1989), for the study of plaque and decidual cells in response to artificial traumatization of the uterus in ovariectomized hormone-treated rhesus monkeys, provides an understanding of the hormonal control of endometrial sensitivity for cellular events which normally occur around implantation. Ghosh and Sengupta (1988) have also reported the involvement of sex steroid hormones during preimplantation stages of gestation in terms of changes in quantity of nuclear and cytoplasmic receptors for oestrogen (ER) and progesterone (PR) in the endometrium. Moreover, the enhancing effects of oestradiol on ER and PR are well documented in rhesus monkeys by other groups of investigators (Kreitmann-Gimbal et al., 1979, 1980). The present study was designed to examine the in-vivo effects of a novel anti-oestrogen molecule on ER and PR in endometrium of ovariectomized monkeys receiving hormonal treatments and an artificial deciduogenic stimulus. In addition, we investigated the binding activity of CDRI-85/287 to endometrial steroid receptors in vitro with a view to elucidating the hormonal control of the deciduogenic mechanism in this species. Materials and methods Chemicals [3H]Promegestone (R 5020, sp. act. 87 Ci/mmol), unlabelled R 5020 and [3H]oestradiol (sp. act. 93.3 Ci/mmol) were purchased from Dupont NEN Research Products, USA. Unlabelled diethylstilboestrol (DES) and tamoxifen were purchased from Sigma Chemical Company, St Louis, MO, USA. All other chemicals and reagents were of Analytical grade. Compound CDRI-85/287 synthesized at the Central Drug Research Institute (Sharma et al., 1990) was used throughout the study. Animals, treatment and tissue collection Adult female rhesus monkeys (Macaca mulatta; body weight 4–6 kg) of proven fertility maintained under uniform husbandry conditions at the Institute's Primate House were used in this study. Monkeys were bilaterally ovariectomized under ketamine anaesthesia. After a rest period of 45 days, monkeys were randomly divided into various groups. A total of 12 monkeys were divided into four groups composed of three monkeys each and given oestradiol dipropionate (EDP) followed by progesterone. The treatment schedule and the doses were followed essentially as described by Ghosh and Sengupta (Ghosh and Sengupta 1989). On days 8, 9 and 10, the compound 85/287 was given i.m. in olive oil at a dose of 15 mg/monkey in groups I and II. Animals of groups III and IV received vehicle only and served as control. On day 16 of hormone treatment, monkeys were laparotomized and a sterile nylon thread was inserted through the uterine lumen to induce decidualization. On days 24 and 30 of the treatment cycle, animals were given ketamine anaesthesia, endometrial tissues collected, washed in ice-cold saline to remove blood contamination and stored in liquid nitrogen until assayed. For histology, tissue slices were fixed in Bouin's fluid, embedded in wax at 58°C, sections were cut on a microtome at 5 μm, dehydrated in ethanol, stained with haematoxylin/eosin and examined under microscope. Preparation of nuclear and cytosolic fractions All subsequent steps were carried out at 0–4°C unless otherwise stated. The tissue was washed three times and homogenized in 5 vol. of 10 nmol/l Tris buffer, pH 7.4 containing 1 nmol/l magnesium chloride, 1 nmol/l monothioglycerol and 10% glycerol (TMMG) giving 10 s bursts with 1 min cooling intervals using a polytron PT-10 homogenizer (Brinkmann Instruments, Westbury, NY, USA). Cytoplasmic and nuclear fractions were separated by centrifugation (800 g, 10 min) and the supernatant centrifuged at 130 000 g for 60 min, to yield the clear cytosol fraction. The pellet was then washed three times with TMMG buffer and centrifuged at 800 g for 10 min. The washed nuclear pellet was resuspended in assay buffer to the original volume and was used for ER and PR assays. Oestrogen and progesterone receptor assays Prior to assay, the cytosol was incubated with an equal volume of dextran-coated charcoal (0.05% dextran and 0.5% activated charcoal in Tris buffer pH 7.4) (DCC) for 60 min at 4°C with occasional stirring to remove endogenous steroids. Cytosol was centrifuged at 800 g for 15 min, twice. The clear supernatant was used for binding assay. ER and PR were measured using methods described by Ghosh and Sengupta and Okulicz et al. with some modification (Ghosh and Sangupta, 1988; Okulicz et al., 1990). In brief, for determination of cytosolic receptors, the cytosol (200 μl) was incubated in triplicate with saturating concentration of [3H]oestradiol (25 nmol/l) or [3H]R 5020 (25 nmol/l) in the absence or presence of 1 μmol/l DES (for ER) or 1 μmol/l R 5020 (for PR) for 18 h at 4°C. At the end of the incubation period the bound and free steroids were separated by a brief incubation wth 500 μl of DCC suspension for 10 min. Tubes were then centrifuged at 800 g for 10 min at 4°C. The supernatant obtained (bound fraction) was transferred to the vials containing scintillation fluid. For determination of nuclear ER and PR, the nuclear fraction (200 μl) was incubated in triplicate with [3H]oestradiol for 30 min at 37°C or with [3H]R 5020 for 16 h at 4°C in the absence or presence of unlabelled 1 μmol/l DES or R 5020 for ER and PR respectively. Following incubation, 1 ml chilled buffer was added to each tube and free steroid was removed by washing with buffer and centifuged at 800 g three times at 4°C. Bound radioactivity was then extracted by 1 ml 100% ethanol twice at 30°C. Ethanolic extracts were pooled and transferred to scintillation vials and counted for radioactivity at the counting efficiency of 60%. Cytosolic protein and nuclear DNA were estimated by the methods of Lowry et al. (Lowry et al., 1951) and Burton (Burton, 1956) respectively. Specific binding was computed from the difference between the total binding (i.e. in the absence of unlabelled DES or R 5020) and non-specific binding (i.e. in the presence of unlabelled DES or R 5020). Receptor concentrations were expressed as fmol/100 μg protein (for cytosolic receptors) and fmol/100 μg DNA (for nuclear receptors). Values were expressed as mean ± SD. Competitive binding assay Endometrial cytosol (obtained from the control animals) were incubated with [3H]oestradiol (2.5 nmol/l) or [3H]R 5020 (2.5 nmol/l) at 4°C for 22 h. Parallel incubations were run in the presence of various concentrations of unlabelled steroid or tamoxifen or CDRI-85/287 dissolved in DMF:buffer (1:1) as described elsewhere (Dhar et al., 1994). Following incubation, bound and free steroids were separated by addition of 500 μl of DCC suspension as described above. Bound fractions at each concentration of competitor were assessed for radioactivity. The concentration of competitor required for 50% inhibition of 3H-steroid binding were determined by a graph between percentage bound ligand versus molar concentraion of the competitors. Statistical analsysis Significance of differences between various treatment groups was carried out by using Student's t-test. Results Histology In hormone-treated monkeys, decidualization was characterized by a marked luminal and epithelial plaque acinar cell formation on day 24 of the cycle (Figure 1A,B) which increased on day 30 (Figure 2A). Hypertrophy and hyperplasia was evident in plaque acinar cells with metrial glands in the peripheral endometrial stroma lined with cuboidal epithelial cells. Administration of the compound 85/287 showed an apparent inhibition in uterine epithelial and subepithelial decidual plaque cell formation indicating their degeneration (Figure 1C,D). Clumping and vacuolization was discernible in decidual plaque cells both on days 24 and 30 of the cycle in treated monkeys (Figures 1C,D, 2B,C). Low cuboidal epithelium of metrial glands and proliferation in epithelial cells were noted. The stromal response to decidualization was marked by a higher degree of oedema with leukocytic infiltration in control hormone-primed decidualized uterine endometrium on day 24. However, the degree of response was more on day 30. CDRI-85/287 relatively decreased the leukocytic infiltration into the stroma, whereas mitotic activity showed an increase. ER and PR concentrations in endometrium The results of cytosolic and nuclear ER and PR are illustrated in Figure 3. When expressed as fmol/100 μg protein and per 100 μg DNA, on day 24 of treatment cycle there was a marked decrease (P < 0.001) in cytosolic as well as nuclear progesterone receptor content in the 85/287-treated group of monkeys (PRc 43.7 ± 8.5, PRn 44.6 ± 13.6) as compared to that of control decidualized endometrium (PRc 256.6 ± 34.0, PRn 260.2 ± 54.9; Figure 3B). In addition, no significant change in ER content was observed in the treated group. However, the nuclear ER were increased in concentration (control, ERn 79.8 ± 8.4; treated 208.6 ± 36.7) (P < 0.05) (Figure 3A). On day 30 of the cycle, cytosolic ER and PR concentrations were significantly decreased (P < 0.001) as compared to that on day 24 of the cycle, in untreated decidualized endometrium. Interestingly, administration of 85/287 caused a significant reduction (P < 0.001) in the concentrations of nuclear ER as well as PR in both the cellular compartments (control, ERn 232.2 ± 91.0, treated 34.7 ± 11.4; control PRn 83.4 ± 38.7; treated, 32.4 ± 8.0; control, PRc 264.7 ± 112.5, treated 109.4 ± 3.4) (Figure 3C and D). Our findings suggest in general a decrease in cytosolic ER and cytosolic PR as a result of 85/287 treatment in both groups of animals. The rise in nuclear ER as observed on day 24 appears to be due to translocation of the receptor to the nuclear compartment. In-vitro binding of 85/287 to endometrial ER and PR In order to examine the in-vitro competitive binding to both steroid receptors, i.e. ER and PR, IC50 of oestradiol, R5020, tamoxifen and CDRI-85/287 were determined for [3H]oestradiol and [3H]R 5020 binding in endometrial cytosol (Table I). Tamoxifen and 85/287 showed IC50 ~50- and 350-fold that of oestradiol respectively for [3H]oestradiol binding, whereas R 5020 could not inhibit [3H]oestradiol binding even up to 100 μmol/l concentration. In the case of [3H]R 5020 binding only R 5020 showed IC50 of 16 nmol/l whereas the rest of the competitors including 85/287 showed IC50 >1000-fold that of R 5020. These results clearly demonstrate the competitive antagonism of tamoxifen and 85/287 at ER level only. However, 85/287 appears to be more potent as a competitor. Discussion The obligatory role of progesterone in the elicitation of endometrial responses in decidual cell reactions is well documented, whereas the role of oestrogen appears to be subtle (Glasser and McCormack, 1982). Steroid dynamics in the target organ vary and depend upon the nature of hormone sensitization, treatment and hormonal milieu at the cell level. The steroid receptor values of the present study are comparable to those reported in ovariectomized monkeys following sequential oestradiol and progesterone treatment (Kreitmann-Gimbal et al., 1979; West and Brenner, 1985; West et al., 1986; Ghosh and Sengupta, 1989). It is well known that oestradiol increases the concentration of its own receptor as well as that of PR in normal endometrium (Kassis et al., 1984; Lessey et al., 1989), at the same time progesterone in adequate amounts counteracts these oestrogenic effects. Moreover, unoccupied PR also play a role in the control of PR biosynthesis in primate endometrium as suggested by Chwalisz et al. (Chwalisz et al., 1991). Anti-oestrogens also counteract such oestrogenic effects/action (Lessey et al., 1989). Here too our findings show that treatment with anti-oestrogen CDRI-85/287 decreased the amount of both receptors. The results indicate that ER and PR synthesis is ultimately dependent upon the presence of oestradiol. It has been reported that plasma and endometrial oestradiol and progesterone concentration in primates as well as humans varies with respect to physiological conditions and is regulated by tissue specific variables including distribution, binding, intracellular metabolism and clearance of steroid hormones (Resko et al., 1976; Batra et al., 1977; Poortman et al., 1983). The observation that concentrations of PRn closely parallel those of progesterone in tissue and plasma substantiates the requirement of progesterone for inducing and maintaining the gestational changes in the endometrial tissue; however, despite the apparent decrease in cytoplasmic PR and nuclear ER, the total receptor content for both hormones remained unchanged (Ghosh and Sengupta 1988, 1989). In the present study the ER-mediated anti-oestrogenic action of 85/287 (Table I) may probably be one of the factors leading to the inhibition of decidualization-stimulating events. Endometrial proliferation is well known to be an oestrogen-dependent event (prerequisite for decidualization) as observed in our studies showing inhibition of eosinophilic inflitration, stromal oedema, glandular proliferation and epithelial cell height. It has been demonstrated that oestradiol in a physiological amount has induced the synthesis of peptide growth factors and their specific receptors in order to promote endometrial mitogenesis and tissue growth generally (Beck and Garner, 1989; Boehm et al., 1990; Corden-Cardo et al., 1990; Ishihara et al., 1990; Reynolds et al., 1990). It is pertinent to note here that modulation of growth factors by anti-oestrogens/antiprogestins has also been reported (Greb et al., 1997; MacGregor and Jordon, 1998). The data presented here indicate that the antiprofliferative effects of anti-oestrogen upon endometrium are due to diminution of the ER pool as well as the altered synthesis/concentrations of PR. Interestingly on day 24, we observed an increase in nuclear ER concentrations. Several investigators have reported that injection of progesterone under proliferative, secretory, midcycle or oestradiol-primed conditions results in a change in distribution of PR concentrations with increase in nuclear and a concomitant decrease in cytoplasmic receptor concentrations. Similar effects might be involved after anti-oestrogen treatment under oestrogen-primed conditions leading to the altered distribution of ER in different cellular compartments (Neulen et al., 1996). The mechanisms underlying the antideciduogenic effects of anti-oestrogen in primate endometrium vary. Antiprogestins have been reported to stabilize the inactive so-called `8S' form of PR as well as to limit its transformation towards the activated `4S' form (Renoir et al., 1989). Recent studies suggest that progesterone acts through cycle-specific PR isoforms A and B associated with the fluctuation in their ratios (Mangal et al., 1997). Receptor-bearing RU 486 bound to PRE (progesterone response element), however, cannot promote transcription from respective genomic sites (Baulieu, 1991). Similarly in a few reports anti-oestrogens such as tamoxifen and H 1285 have been suggested to affect the post-transcriptional events involving the above mechanism in breast cancer/uterine tissue (Ruh et al., 1990; Allan et al., 1992). Thus, anti-oestrogenic activities may derive from receptor transformation/ERE (oestrogen response element) mediated mechanisms. In this context it has also been reported that opposite reactivity of the two forms of ER (8S and 4S) with tamoxifen aziridine and ERE support the hypothesis that they may constitute separate entities with a different physiological role (Navarro et al., 1998). Our observations suggest that anti-oestrogens may have decidualization inhibitory activity in primate endometrium and also indicate the anti-oestrogenic potential of this benzopyran derivative (85/287) in higher species. However, further extensive studies on the pharmacological profiles of such novel series of non-steroidal moieties are warranted. Table I. In-vitro competitive binding of 85/287 Competitor  IC50a value    [3H] oestradiol binding  [3H]R 5020 binding  aConcentration of the competitor required for 50% inhibition of radioligand binding. Data are mean of three experiments.  Oestradiol  ~10 nmol/l  >500 nmol/l  R 5020  >100 μmol/l  ~16 nmol/l  Tamoxifen  ~400 nmol/l  >100 μmol/l  CDRI-85/287  ~3500 nmol/l  >100 μmol/l  Competitor  IC50a value    [3H] oestradiol binding  [3H]R 5020 binding  aConcentration of the competitor required for 50% inhibition of radioligand binding. Data are mean of three experiments.  Oestradiol  ~10 nmol/l  >500 nmol/l  R 5020  >100 μmol/l  ~16 nmol/l  Tamoxifen  ~400 nmol/l  >100 μmol/l  CDRI-85/287  ~3500 nmol/l  >100 μmol/l  View Large Figure 1. View large Download slide View large Download slide View large Download slide View large Download slide (A) Endometrial deciduogenic response in ovariectomized hormone-primed rhesus monkey on day 24 of the cycle shows a characteristic decidual plaque cell formation (arrow head). Note also the uterine glandular epithelial plaques (arrow). (B) A magnified view of A exhibits plaque acinar cells depicting hypertrophy and hyperplasia. (C) Compound CDRI-85/287 treatment showing significant degenerative changes in endometrial decidual plaque cells reaction marked by degeneration of plaques, condensation of acinar cell nuclei and presence of cytoplasmic vacuoles (arrow) after 24 days. Note also a decreased epithelial cell height in metrial glands. (D) A magnified view of C showing degeneration of decidual plaque cells. Haematoxylin–eosin. Scale bars = 188 μm (A, C), 94 μm (B, D). Figure 1. View large Download slide View large Download slide View large Download slide View large Download slide (A) Endometrial deciduogenic response in ovariectomized hormone-primed rhesus monkey on day 24 of the cycle shows a characteristic decidual plaque cell formation (arrow head). Note also the uterine glandular epithelial plaques (arrow). (B) A magnified view of A exhibits plaque acinar cells depicting hypertrophy and hyperplasia. (C) Compound CDRI-85/287 treatment showing significant degenerative changes in endometrial decidual plaque cells reaction marked by degeneration of plaques, condensation of acinar cell nuclei and presence of cytoplasmic vacuoles (arrow) after 24 days. Note also a decreased epithelial cell height in metrial glands. (D) A magnified view of C showing degeneration of decidual plaque cells. Haematoxylin–eosin. Scale bars = 188 μm (A, C), 94 μm (B, D). View large Download slide View large Download slide View large Download slide Figure 2. (A) Showing an increased endometrial decidual plaque cell response in ovariectomized, horomone-primed rhesus monkeys on day 30 of the cycle. Metrial glands show an increase in epithelial cell height (arrow). (B) Compound CDRI-85/287 administration caused a marked inhibition in decidual plaque cell reaction indicating an intensive degeneration in plaque acinar cells (arrow). (C) More highly magnified picture of B showing clearly the clumps of acinar cells. Haematoxylin–eosin. Scale bars = 188 μm (A, B), 94 μm (C). View large Download slide View large Download slide View large Download slide Figure 2. (A) Showing an increased endometrial decidual plaque cell response in ovariectomized, horomone-primed rhesus monkeys on day 30 of the cycle. Metrial glands show an increase in epithelial cell height (arrow). (B) Compound CDRI-85/287 administration caused a marked inhibition in decidual plaque cell reaction indicating an intensive degeneration in plaque acinar cells (arrow). (C) More highly magnified picture of B showing clearly the clumps of acinar cells. Haematoxylin–eosin. Scale bars = 188 μm (A, B), 94 μm (C). Figure 3. View largeDownload slide Oestrogen receptor (ER) and progesterone receptor (PR) in cytosolic (c) and nuclear (n) compartments in monkey endometrium on day 24 (A, B) and day 30 (C, D) of the treatment cycle; data are mean of three observations. □ Control group; ▪ 85/287-treated group; error bars indicate standard deviation. Significance is based on comparisons between control and treated groups. Figure 3. View largeDownload slide Oestrogen receptor (ER) and progesterone receptor (PR) in cytosolic (c) and nuclear (n) compartments in monkey endometrium on day 24 (A, B) and day 30 (C, D) of the treatment cycle; data are mean of three observations. □ Control group; ▪ 85/287-treated group; error bars indicate standard deviation. Significance is based on comparisons between control and treated groups. 1 To whom correspondence should be addressed at: Division of Endocrinology, Central Drug Research Institute, Post Box No. 173, Lucknow 226001, India The authors are grateful to Dr C.M.Gupta, Director and Dr V.P.Kamboj, former Director, for their keen interest in this study. We thank Dr R.S.Kapil for the generous supply of compound 85/287. We acknowledge Ms Kanak Lata for technical assistance in animal experimentation and Mr Balvir Singh for protein and DNA estimations of the samples. This study was supported by a grant from the Ministry of Health and Family Welfare, Government of India. 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Journal

Human Reproduction – Oxford University Press

Published: Apr 1, 1999

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