Division of Endocrinology, Central Drug Research Institute, Lucknow 226001, India
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: anti-oestrogen/decidualization/endometrium/receptors
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals, treatment and tissue collection
Adult female rhesus monkeys (Macaca mulatta; body weight 46 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 04°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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
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
).
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allan, G.F, Leng, K. and Tsai, S.Y., et al. (1992) Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation. J. Biol. Chem., 267, 1951319520.
Batra, S., Grundsell, H. and Sjoberg, N.O. (1977) Oestradiol 17ß and progesterone concentration in human endometrium during the menstrual cycle. Contraception, 16, 217224.[ISI]
Baulieu, E.E. (1991) On the mechanism of action of RU 486 In Seppala, M. and Hamberger, L. (eds), Frontiers in Human Reproduction. Ann. NY Acad Sci., 626, 545560.
Beck, C A. and Garner, C W, (1989) Characterization and estrogen regulation of growth factor activity from uterus. Mol. Cell. Endocrinol., 63, 93101.[ISI][Medline]
Boehm, K.D., Diomon, N., Gorodeski, I.G. et al. (1990) Expression of insulin like and platelet derived growth factor genes in human uterine tissues. Mol. Reprod. Dev., 29, 98101.
Burton, K. (1956) A study of the condition and mechanism of diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J., 62, 315322.[ISI]
Chwalisz, K., Hegele-Hartung, C., Fritzimeier, K.H. et al. (1991) Inhibition of the oestradiol mediated endometrial gland formation by the antigestagen onapristone in rabbits: relationship to uterine estrogen receptor. Endocrinology, 129, 312322.[Abstract]
Corden-Cardo, C., Voldavsky, I., Haimovitz-Friedman, A. et al. (1990) Expression of basic fibroblast growth factor in normal human tissue. Lab. Invest., 63, 832840.[ISI][Medline]
Dhar, J.D., Setty, B.S., Durani, S. et al. (1991) Biological profile of 2-[4-(2-N-piperidinoethoxy) phenyl]-3 phenyl (2H) benzo (b) pyran a potent antiimplantation agent in rat. Contraception, 44, 461472.[ISI][Medline]
Dhar, J.D., Dwivedi, A., Srivastava, A. et al. (1994) Structureactitivty relationship of some 2,3-diaryl-2H-1-benzopyrans to their antiimplantation, oestrogenic and antioestrogenic actitivity in rat. Contraception, 49, 609616.[ISI][Medline]
Finn, C.A. (1977) The implantation reaction In Wynn, R. (ed.), Biology of Uterus. Plenum Press, New York, pp. 245308.
Ghosh, D. and Sengupta, J. (1988) Patterns of oestrogen and progesterone receptor in rhesus monkey endometrium during secretory phase of normal menstrual cycle and preimplantation stages of gestation. J. Steroid Biochem., 31, 223229.[ISI][Medline]
Ghosh, D. and Sengupta, J. (1989) Endometrial responses to a deciduogenic stimulus in ovariectomized rhesus monkey treated with oestrogen and progesterone. J. Endocrinol., 120, 5158.[Abstract]
Glasser, S.R. (1990) Biochemical and structural changes in uterine endometrial cell types following natural or artificial deciduogenic stimuli. Trophoblast Res., 4, 377416.
Glasser, S.R. and McCormack, A. (1982) Cellular and molecular aspects of decidualization and implantation In Beier, H.M. and Karlson, P. (eds), Proteins and Steroids in Early Pregnancy. Springer-Verlag, Berlin, pp. 245310.
Greb, R.R., Heikinheimo, O., Williams, R.F. et al. (1997) Vascular endothelial growth factor in primate endometrium is regulated by oestrogen receptor and progesterone receptor ligands in vivo. Hum. Reprod., 12, 12801292.[ISI][Medline]
Ishihara, S., Taketani, Y. and Mizuno, M. (1990) Epidermal growth factor like immunoreactivity in human endometrium. AsiaOceania J. Obstet. Gynaecol., 16, 165168.
Kassis, J.A., Sakai, D. and Walent, J.H. et al. (1984) Primary cultures of oestrogens responsive cell from rat uterus: induction of progesterone receptor and a secretory protein. Endocrinology, 114, 15581566.[Abstract]
Kreitmann-Gimbal, B., Goodmann, A.L., Bayard, F. et al. (1979) Characterization of oestrogen and progesterone receptors in monkey endometrium: methodology and effects of oestradiol and/or progesterone on endometrium of castrate monkeys. Steroids, 34, 749770.[ISI][Medline]
Kreitmann-Gimbal, B., Bayard, F., Nixon, W.E. et al. (1980) Patterns of oestrogen and progesterone receptor in endometrium during the normal menstrual cycle. Steroids, 35, 471479.[ISI][Medline]
Lessey, B.A., Metzger, D.A. and Heney, A.F. et al. (1989) Immunological analysis of oestrogen and progesterone receptor in endometriosis: comparison with normal endometrium during the menstrual cycle and the effect of medical therapy. Fertil. Steril., 51, 409415.[ISI][Medline]
Lowry, O.H., Rosebrough, N.J., Farr., A.L. et al. (1951) Protein measurement with the Folin-phenol reagent. J. Biol. Chem., 193, 265271.
MacGregor, J.I. and Jordon, V.C. (1998). Basic guide to the mechanism of antioestrogen action. Pharmacol. Rev., 50, 151196.
Mangal, R.K., Wiehle, R.D., Poindextery III, A.N. et al. (1997) Differential expression of uterine progesterone receptor forms A and B during the menstrual cycle. J. Steroid Biochem. Mol. Biol., 63, 195202.[ISI][Medline]
Martel, C., Provencher, L., Li, X. et al. (1998). Binding characteristics of novel nonsteroidal antioestrogens to the rat uterine oestrogen receptors. J. Steroid Biochem. Mol. Biol., 64, 199204.[ISI][Medline]
Navarro, D., Leon, L., Chirino, R. et al. (1998) The two nature oestrogen receptor forms of 8S and 4S present in cytosol from human uterine tissues display opposite reactivities with the antioestrogen tamoxifen aziridine and oestrogen responsive element. J. Steroid Biochem. Mol. Biol., 64, 4958.[ISI][Medline]
Neulen, J., Willians, R.F., Breckwoldt, M. et al. (1996) Noncompetitive antioestrogenic action of progesterone antagonist in primate endometrium: Enhancement of oestrogen and progesterone receptors with blockade of post-receptor proliferative mechanism. Hum. Reprod., 11, 15331537.
Okulicz, W. C., Sarater A. M. and Hoberg, L. M. et al. (1990) Biochemical and immunohistochemical analysis of oestrogen and progesterone receptors in rhesus monkey uterus during the proliferative and secretory phases of artificial menstrual cycle. Fertil. Steril., 53, 913920.[ISI][Medline]
Poortman, J., Thijssen, J.H.H, Landighem, A.A. Jr et al. (1983) Subcellular distribution of androgens and oestrogen in target tissue. J. Steroid Biochem., 19, 939945.[ISI][Medline]
Psychoyos, A. (1973) Endocrine control of egg implantation In Greep, R.O. and Astwood, A.G. (eds), Handbook of Physiology. American Physiological Society, Washington, DC, pp. 187215.
Renoir, J.M., Radanyi, C. and Baulieu, E.E. (1989) The antiprogesterone RU-486 stabilizes the hetro oligomeric, non-DNA binding, 8S form of the rabbit uterus cytosol progesterone receptors. Steroids, 53, 120.[Medline]
Resko, J.A., Boling, J.L., Brenner, R.M. et al. (1976) Sex steroids in reproductive tract tissue, regulation of oestradiol concentrations by progesterone. Biol. Reprod., 15, 153157.[ISI][Medline]
Reynolds, R.K., Talavera, F., Roberts, J.A. et al. (1990) Regulation of epidermal growth factor and insulin like growth factor receptors by oestradiol and progesterone in normal and neoplastic endometrial cell cultures. Gynecol. Oncol., 38, 396406.[ISI][Medline]
Ruh, M.F., Turner, J.W., Paulson, C.M. et al. (1990) Differences in the form of the salt transformed oestrogen receptor when bound by oestrogen versus antiestrogen. J. Steroid Biochem., 36, 509516.[ISI][Medline]
Sharma, A.P., Saeed, A., Durani, S. et al. (1990) Structureactivity relationship of antioestrogens. Effect of the side chain and its position on the activity of 2,3-diaryl-2H-1-benzopyrans. J. Med. Chem., 33, 32163222.[ISI][Medline]
Srinivasulu, S., Dwivedi, A., Singh, M.M. et al. (1992a) CDRI-85/287: Studies on competition to oestrogen binding sites in the immature rat uterus. Ind. J. Exp. Biol., 30, 11151117.[Medline]
Srinivasulu, S., Singh, M.M., Dwivedi, A. et al. (1992b) Duration of antioestrogenecity of compound CDRI- 85/287: A new antiimplantation agent. Ind. J. Exp. Biol., 30, 968971.[Medline]
Tang, B., Guller, S. and Gurpide, E. (1994) Mechanism of human endometrial stromal cell decidualization. Ann. NY Acad. Sci., 734, 1925.[ISI][Medline]
West, N.B. and Brenner, R.M. (1985) Progesterone mediated suppression of estradiol receptor in cynomologus macaque cervix endometrium and oviduct during sequential oestradiol progesterone treatment. J. Steroid Biochem., 22, 2937.[ISI][Medline]
West, N.B., Hess, D.L. and Brenner, R.M. (1986) Differential suppression of progesterone receptor by progesterone in the reproductive tract of female macaques. J. Steroid Biochem., 25, 497503.[ISI][Medline]
Submitted on August 24, 1998; accepted on January 14, 1999.