Molecular Human Reproduction, Vol. 5, No. 11, 995-1002,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular endocrinology |
Regulation of sulphotransferase expression in the endometrium during the menstrual cycle, by oral contraceptives and during early pregnancy
1 Departments of Molecular & Cellular Pathology and 2 Obstetrics & Gynaecology, University of Dundee, Dundee DD1 9SY, UK and 3 Department of Pharmacology & Toxicology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
Abstract
The endometrium plays a key role in reproduction, and this function is tightly regulated by endogenous and xenobiotic steroids. Sulphation, catalysed by members of the sulphotransferase (SULT) enzyme family, is a major deactivating mechanism for steroid hormones and we have investigated the expression and regulation in vivo of SULT in the human endometrium. In the normal cycling endometrium, expression of the phenol sulphotransferases SULT1A1 and SULT1A3 and the oestrogen sulphotransferase SULT1E1 were observed, with SULT1A1 and SULT1E1 expression being higher in the luteal phase than in the follicular phase. No expression of the hydroxysteroid sulphotransferase SULT2A1 was detected at any time in the endometrium. In endometrium from women taking the combined oral contraceptive pill (OCP), SULT1E1 expression was virtually absent, and SULT1A1 expression was substantially reduced. Similarly, in early pregnancy (i.e. first trimester) endometrium, SULT1E1 expression was absent, although SULT1A1 and SULT1A3 expression were unaffected. Our results with normal endometrium support in-vitro data showing that SULT1E1 expression is regulated by progesterone. However, the data obtained from OCP and early pregnancy endometrium suggest that factors other than the concentration of circulating progesterone are involved in the regulation of the expression of this important enzyme in the endometrium.
endometrium/oestrogens/sulphation/sulphotransferase
Introduction
The monthly cyclical changes in endometrial morphology, physiology and biochemistry are strongly influenced by ovarian steroids (principally 17ß-oestradiol and progesterone), and serve to prepare an environment able to accept and implant a fertilized embryo. In most mammalian species progesterone appears to be essential for implantation, whereas oestrogen is permissive but not essential (Ghosh and Sengupta, 1995
). In humans the exact (steroid) hormonal requirement within the intrauterine environment during the peri-implantation period is unknown, although the endometrium is capable of oestrogen synthesis (Tseng et al., 1982; Tseng, 1984) and the peri-implantation conceptus secretes oestrogen (Edgar et al., 1993).
The mid luteal phase production of oestrogen by the ovary and conceptus is complemented by progesterone-dependent increases in the oestrogen-metabolizing enzymes 17ß-hydroxysteroid dehydrogenase type 2 (Tseng and Gurpide, 1975, 1979; Casey et al., 1994) and oestrogen sulphotransferase (Buirchell and Hähnel, 1975; Tseng and Liu, 1981; Clarke et al., 1982) and a decrease in oestrogen receptor levels (Hsueh et al., 1976; Lessey et al., 1988; Snijders et al., 1992; Noe et al., 1999) in the endometrium. Unlike oestrogen sulphotransferase, there appears to be little variation in the expression of endometrial steroid sulphatase (ARSC: the enzyme which hydrolyses the sulphates of various steroid hormones, including oestrogens, and therefore contributes to `sulphation' overall) throughout the menstrual cycle (Prost and Adessi, 1983). Also, the kM for ARSC is considerably higher than that for oestrogen sulphotransferase (Prost and Adessi, 1983; Zhang et al., 1998), suggesting that sulphation may predominate over sulphate conjugate hydrolysis in the presence of low concentrations of oestrogen. These oestrogen-metabolizing enzymes probably serve to protect the endometrium from excess oestrogenic stimulation at this critical period of the reproductive cycle, since overstimulation by oestrogen prior to implantation has been implicated as a risk factor in failure to establish pregnancy (Olson et al., 1983; Sterzik et al., 1988). Thus it is likely that a delicate balance exists between progesterone/oestrogen-mediated preparation of the endometrium for implantation and the enzymatic mechanisms for protecting the endometrium against excessive oestrogenic stimulation. Factors that disrupt this hormonal regulation of the process of implantation are likely to affect reproductive potential and may therefore contribute to infertility (Fauser and Hsueh, 1995). Sulphation plays a key role in steroid hormone function (Roy, 1992; Strott, 1996) since steroid sulphates are unable to influence regulation of gene expression mediated through classical steroid hormone receptors. Sulphation is catalysed by members of the sulphotransferase (SULT) enzyme family (Coughtrie et al., 1998), which comprises at least ten members. In humans there is a single (as far as is known) oestrogen SULT (called SULT1E1) which displays high affinity for endogenous and xenobiotic oestrogens (Aksoy et al., 1994; Forbes-Bamforth and Coughtrie, 1994; Falany et al., 1995).
The endometrium has the capacity to sulphate oestrogens, and enzyme activity is substantially higher in the luteal (secretory) phase of the menstrual cycle (Buirchell and Hähnel, 1975; Pack et al., 1979). Experiments in vitro with endometrium organ culture and endometrial adenocarcinoma cells suggest that oestrogen sulphotransferase is regulated by progesterone in glandular cells (Tseng and Liu, 1981; Clarke et al., 1982; Falany and Falany, 1996), and cyclical changes in glandular and stromal progesterone receptor expression support this (Wang et al., 1998a; Noe et al., 1999). Because of its role in oestrogen inactivation, SULT1E1 may be an important enzyme in reproductive biology, and aberrant sulphation of potent oestrogens such as 17ß-oestradiol may contribute to abnormal endometrial function, such as in infertility. To date there is little detailed information regarding sulphation and SULT in the human endometrium and little is known about the factors regulating sulphation in vivo in humans. We have therefore conducted a thorough examination of SULT isoenzyme expression in the human endometrium, and assessed the effect of the oral contraceptive pill and early pregnancy on SULT expression in vivo. Our results suggest that the level of circulatory progesterone is not the only factor regulating SULT expression in the human endometrium.
Materials and methods
Subjects
Three separate groups of subjects were included in the study. The control group comprised 102 fertile women aged between 19 and 51 years (mean ± SD = 34.3 ± 6.5 years), with a known history of normal conception and childbirth who were undergoing laparoscopic sterilization, hysterectomy or diagnostic laparoscopy. A second group comprised 32 women routinely taking the combined oral contraceptive pill (OCP, oestrogen plus progestin) with mean age 29.8 ± 5.3 years (range 19–42 years), and the third group comprised 28 women undergoing therapeutic termination of pregnancy (by Gemeprost induction and suction termination) during the first 6–13 weeks post conception (mean age 25.1 ± 8.6 years, range 15–42 years). Ethical approval for the study was obtained from the Tayside Committee on Medical Research Ethics, and informed consent was obtained from each participant.
Materials
Pipelle cannulae for endometrial biopsy were obtained from Eurosurgical Ltd, Cranleigh, UK. 3'-phosphoadenosine 5'-phosphosulphate (PAPS), 17ß-oestradiol, 17
-ethinyloestradiol, dopamine, dehydroepiandrosterone (DHEA), pregnenolone, vanillin and goat anti-rabbit IgG–peroxidase conjugate adsorbed with human serum proteins were obtained from Sigma/Aldrich, Poole, UK. [35S]3'-Phosphoadenosine 5'-phosphosulphate (PAP35S, 1.2–2.4 Ci/mmol), 17-[6,7,-3H(N)]ethinyloestradiol (41.2 Ci/mmol), [1,2,6,7–3H(N)]dehydroepiandrosterone (92.0 Ci/mmol) and [7-3H(N)]-pregnenolone (25.0 Ci/mmol) were purchased from Du Pont/NEN, Stevenage, UK. 4-Nitrophenol was purchased from Fluka Chemicals Ltd, Derbyshire, UK. DNA modifying enzymes were obtained from Promega, Southampton, UK, except Bio-X-Act DNA polymerase (Bioline, London, UK). All other reagents were of analytical grade and purchased from commonly used local suppliers.
Tissue collection and processing
Patients with irregular menstrual cycles or who were on regular medication were excluded from the study. Endometrial biopsies were obtained using Pipelle cannula sampling, and tissue was collected into sterile vials containing 250 mmol/l sucrose, 5 mmol/l HEPES, 2 mmol/l 2-mercaptoethanol, pH 7.4 (Buffer A) and stored at –70°C until use. Phase of the menstrual cycle was calculated from the start of the last menstrual period (LMP) and confirmed by standard histological analysis. For data analysis and display the menstrual cycle was divided into five phases: early follicular (EF), days 1–7; late follicular (LF), days 8–13; early luteal (EL), days 14–18; mid luteal (ML), days 19–24; late luteal (LL), greater than 24 days. For preparation of the cytosolic fraction, tissue (~1 g) was thawed and homogenized in 2 volumes Buffer A and homogenates were centrifuged at 11 000 g for 2 min. The resulting supernatants were centrifuged for 30 min at 105 000 g, and the cytosolic fractions (supernatants) were stored in small aliquots at –70°C until use (within 3 months).
Estimation of protein
Protein content of endometrium cytosols was estimated (Lowry et al., 1951) with bovine serum albumin as standard.
Assay for SULT enzyme activity
Depending upon the substrate employed, SULT enzyme activity was measured either with PAP35S (Foldes and Meek et al., 1973) using a modified technique (Jones et al., 1993), or with isotopically labelled substrate (Sharp et al., 1993). All enzyme assays were optimized for substrate and PAPS concentration, buffer composition, cytosolic protein and incubation time.
PAP35S was used for the assessment of SULT activity towards the substrates: 17ß-oestradiol (1.3 µmol/l), phenol (20 µmol/l), 4-nitrophenol (2 µmol/l), vanillin (20 µmol/l) and dopamine (10 µmol/l). The incubation mixture (150 µl) consisted of buffer (60 mmol/l potassium phosphate, pH 6.0 for 17ß-oestradiol; 60 mmol/l Tris/HCl, pH 7.5 for 4-nitrophenol; 6 mmol/l potassium phosphate, pH 7.0 for phenol and dopamine and 60 mmol/l potassium phosphate pH 6.5 for vanillin), 150 µg of endometrium cytosol protein (100 µg for 17ß-oestradiol) substrate dissolved in propylene glycol (steroids) or water (other substrates), 1 mm MgCl2, PAPS (0.7 µmol/l for 17ß-oestradiol, 10 µmol/l for 4-nitrophenol, 5 µmol/l for phenol, 1.1 µmol/l for dopamine and 1.4 µmol/l for vanillin) and 0.02 µmol/l [35S]PAPS. Cytosols were assayed in duplicate with a control incubation containing substrate vehicle only. Reactions progressed at 37°C for either 60 min (17ß-oestradiol), 45 min (4-nitrophenol, vanillin) or 30 min (phenol, dopamine) and were terminated by addition of 200 µl 100 mmol/l barium acetate. Unreacted PAPS was removed by precipitation with 200 µl 100 mmol/l barium hydroxide and 200 µl 100 mmol/l zinc sulphate and following centrifugation for 2 min at 11 000 g. 500 µl of the supernatant were mixed with 4 ml of scintillation fluid (Emuslifier Safe; Canberra Packard, Pangbourne, UK) and radioactivity quantified by liquid scintillation spectrometry.
3H-labelled substrates were used to assay SULT activity towards: 17-ethinyloestradiol (0.5 µmol/l), DHEA (0.4 µmol/l) and pregnenolone (0.67 µmol/l). The incubation mixture (250 µl) comprised: 200 µg endometrium cytosolic protein (300 µg for 17-oethinylestradiol), 0.1 µCi 3H-labelled substrate, unlabelled substrate, 1 mmol/l MgCl2, PAPS (15 µmol/l for 17-oethinylestradiol; 60 µmol/l for DHEA and pregnenolone) and buffer (60 mmol/l Tris/HCl pH 7.5 for DHEA, and pregnenolone; 90 mmol/l potassium phosphate, pH 6.0 for 17-oethinylestradiol). Assays were performed in duplicate and included a control incubation containing no PAPS. After the appropriate incubation time (60 min for DHEA and pregnenolone; 45 min for 17-oethinylestradiol), 3 ml chloroform and 250 µl 250 mm Tris/HCl, pH 8.7 were added and the tubes shaken vigorously before centrifugation at 3 000 g for 3 min. 200 µl of the supernatant were mixed with 4 ml scintillation fluid and radioactivity quantified by liquid scintillation spectrometry.
Immunoblot analysis
Endometrial cytosolic proteins were resolved on (SDS)–polyacrylamide gels (11% acrylamide monomer) (Laemmli, 1970) and transferred to nitrocellulose (Towbin et al., 1979). Immunostaining of SULT was performed using the ECL detection system essentially as described by the manufacturer (Amersham, Little Chalfont, UK). Primary antibodies used were IgG fractions of rabbit anti-rat liver oestrogen SULT (Borthwick et al., 1993) and rabbit anti-human liver oestrogen SULT (Falany et al., 1995), and the secondary antibody was a horseradish peroxidase-conjugated anti-rabbit IgG adsorbed against human serum proteins.
Bacterial expression and purification of recombinant human SULT1E1
An EST (expressed sequence tag) which shared high sequence identity with the human liver SULT1E1 cDNA (Aksoy et al., 1994; Falany et al., 1995) was identified by searching the EMBL nucleotide sequence database (accession number AA334071). This clone, which was isolated from a whole human embryo cDNA library prepared in the vector pBluescript SK–, was purchased from The Institute of Genomic Research through the American Type Culture Collection (Rockville, MD, USA) and the insert sequenced using automated sequencing on an ABI373A sequencer (Applied Biosystems, Warrington, UK). Sequencing revealed a clone of 956 bp with an open reading frame of 885 nucleotides coding for an amino acid of 294 residues (subunit molecular weight 35 129 Da) which was 100% identical to the adult human liver SULT1E1 within the coding region. The full sequence of this cDNA has been deposited in the EMBL nucleotide sequence database with the accession number Y11195. For expression in E.coli, the coding region was (PCR)-amplified (using Bio-X-Act DNA polymerase) from the vector using the forward and reverse oligonucleotide primers 5'-GTGTACCCATATGAATTCTGA-3' and 5'-GTAAATCTAGACCTTCTTAGATCT-3' which contain recognition sites for the restriction enzymes NdeI and XbaI, respectively. The PCR product was ligated into the vector pCRII (Invitrogen, Carlsbad, CA, USA) and then digested with NdeI and XbaI. The resulting fragment was ligated into the vector pCW which had previously been digested with NdeI and XbaI. Following sequencing of the pCW/SULT1E1 expression construct, it was transformed into E.coli strain JM109. For protein expression, cultures in 500 ml LB broth containing 100 µg/ml ampicillin were established and expression was induced by the addition of 2 mmol/l isopropylthiogalactoside. Cells were harvested after incubation at 30°C for 18 h. Recombinant protein was purified from lysed E.coli cell pellets by 30–70% ammonium sulphate fractionation followed by ion exchange chromatography on DEAE–Sepharose and affinity chromatography on 3',5'-ADP agarose essentially as described previously (Borthwick et al., 1993). Purification was monitored using 17ß-oestradiol SULT enzyme assay, SDS (PAGE) and immunoblotting.
Results
Sulphotransferase enzyme activities and protein expression in the endometrium during the normal menstrual cycle
To identify which sulphotransferases are expressed in the human endometrium, we first employed substrates generally considered to reflect the activity of four SULT isoforms: 4-nitrophenol and phenol (SULT1A1/P-PST); dopamine and vanillin (SULT1A3/M-PST); 17ß-oestradiol and 17-oethinylestradiol (SULT1E1/EST); DHEA, and pregnenolone (SULT2A1/HST). Enzyme assay conditions were optimized using pooled luteal phase endometrium cytosols, and then used to assess SULT activities in individual cytosols prepared from early and late follicular, and early, mid and late luteal endometrium biopsies from fertile women. Figure 1 shows that enzyme activities for SULT1E1 (A and B), SULT1A1 (C and D) and SULT1A3 (E and F) were readily detectable in endometrium from all stages of the menstrual cycle, whereas SULT2A1 activity (G and H) was not detectable in any of the samples analysed. SULT1E1 activity toward both 17ß-oestradiol and 17-ethinyl oestradiol was low in the follicular phase, rose sharply in the early luteal phase and reached a peak in the mid luteal phase. Activity towards 4-nitrophenol and phenol (SULT1A1) was again lower in the follicular phase than the luteal phase, peaking in the early luteal phase; however, the increase in activity between the follicular phase and early luteal phase was not as marked as with the substrates for SULT1E1. In contrast, SULT1A3 activity towards the substrates dopamine and vanillin did not vary significantly across the cycle. There was substantial inter-individual variation in the SULT1E1 enzyme activity at all stages of the cycle, something also observed with hepatic SULT1E1 expression (Song et al., 1998).
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To confirm whether the SULT activity towards oestrogens in the endometrium reflected the expression of the oestrogen sulphotransferase SULT1E1, we performed Western immunoblot analysis on endometrium cytosol using an antibody prepared against purified recombinant human liver SULT1E1 (Falany et al., 1995
). Figure 2A and B shows that the antibody recognized a number of polypeptides (there is substantial amino acid sequence identity between members of the human SULT1 family), but the SULT1E1 protein could be identified based on its co-migration with authentic purified recombinant human SULT1E1. The level of SULT1E1 enzyme protein expression varied considerably between individuals within each phase of the cycle (as was observed with the enzyme activity). However, the level of enzyme activity (17ß-oestradiol as substrate) correlated strongly with the level of protein expression (e.g. Figure 2C, r2 = 0.59, P < 0.005 by linear regression analysis).
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Effect of the combined oral contraceptive pill on sulphotransferase expression in the endometrium
Sulphotransferase enzyme activity was determined in endometrial biopsies from 39 women using the OCP. These biopsies displayed dramatically reduced sulphotransferase activity towards 17ß-oestradiol and 17
-ethinyl oestradiol, substrates for the oestrogen sulphotransferase SULT1E1 (Figure 3A shows data obtained with 17ß-oestradiol). To determine whether this effect was also observed with other sulphotransferases, SULT enzyme activity was assessed with 4-nitrophenol (for SULT1A1), dopamine (for SULT1A3) and dehydroepiandrosterone (for SULT2A1). Figure 3B, C and D indicates that, in the endometrium from women using the OCP, SULT1A1 activity (4-nitrophenol as substrate) was significantly decreased (but not abolished), whereas dopamine sulphation (representing SULT1A3) was only slightly reduced (not statistically significant) and there was no effect on the SULT2A1 activity, which was still absent from all samples. These data suggest that the SULT enzymes are differentially regulated by hormonal contraceptives in the endometrium. However, it is not possible to rule out the influence of gland atrophy under OCP, since the site of expression of the individual enzymes is not known. The suppression of SULT1E1 enzyme protein expression was confirmed using immunoblot analysis (Figure 4), which showed that none of the endometrium samples from women taking the OCP displayed expression of SULT1E1 enzyme protein.
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Sulphotransferase expression in endometrium during early pregnancy
Another major biological effector of endometrial function is pregnancy, and we therefore determined the effect of pregnancy on oestrogen sulphation in the endometrium. We examined the expression of various sulphotransferases in endometrial biopsies obtained from a group of 28 women undergoing therapeutic terminations during the first 6–13 weeks of pregnancy. As with the OCP, SULT1E1 enzyme activity (as assessed with 17ß-oestradiol as substrate) was absent from all 28 samples, whereas SULT1A1 (4-nitrophenol as substrate) and SULT1A3 (vanillin as substrate) activities were virtually unaffected (Table I
). SULT1E1 enzyme protein was also absent from early pregnancy endometrium as assessed using immunoblot analysis (not shown). Again, SULT2A1 activity (DHEA as substrate) was undetectable in all samples, similar to the control group. This was regardless of the method of termination used. Thus, a specific suppression of oestrogen sulphation occurs in the first trimester of pregnancy.
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Discussion
The endometrium is a major oestrogen target tissue and many factors control and regulate its response to oestrogenic stimulation. Metabolism, and inactivating pathways such as sulphate conjugation in particular, exert an important influence on this response so it is of considerable interest to study sulphation and its regulation in the human endometrium.
Our results confirm earlier studies (Buirchell and Hähnal, 1975; Tseng and Liu, 1981; Clarke et al., 1982) suggesting that endometrial oestrogen sulphation is tightly associated with the menstrual cycle, being low in the follicular phase and high in the luteal phase. We also extensively studied other sulphotransferase enzyme activities and their in-vivo regulation in the endometrium, using probe substrates that are generally accepted to reflect specific SULT isoforms. The hydroxysteroid sulphotransferase SULT2A1 (or HST) was not expressed in any of the 169 endometrium cytosol samples studied, suggesting that sulphation is not an important modulator of androgen activity in the endometrium. Other androgen-metabolizing enzymes are, however, present in human endometrium. For example 17ß-hydroxysteroid dehydrogenase type 2 (Wu et al., 1993) is expressed (under progestin control) (Bonney et al., 1985; Casey et al., 1994) and may ensure minimal androgen (testosterone) synthesis in this tissue, thereby obviating sulphation as a means of modulating androgenic activity. We found that additional members of the phenol sulphotransferase family were expressed at significant levels in the endometrium, as was shown in the Ishikawa endometrial adenocarcinoma cell line (Falany and Falany, 1996). SULT1A1 and SULT1A3 enzymes appeared less heavily influenced by the phase of the menstrual cycle than SULT1E1, and dopamine sulphation was remarkably constant across all samples studied. The precise function of these enzymes in the endometrium is unknown; however, they are involved in the metabolism of potent endogenous chemicals such as iodothyronines (SULT1A1 and SULT1A3) and catecholamines (SULT1A3). The human endometrium expresses triiodothyronine (T3) receptors (Kirkland et al., 1983) and may therefore be a thyroid hormone-responsive tissue. Sulphation has a dramatic effect on iodothyronine metabolism, blocking the conversion of the prohormone thyroxine (T4) to the receptor active T3 and accelerating the formation of inactive reverse T3, as well as directly inactivating T3 (Visser, 1994; Visser, 1996). Thus the expression of these phenol sulphotransferases, which are known to metabolize iodothyronines (Young et al., 1988; Wang et al., 1998b; Kester et al., 1999a) may play a role in local regulation of the thyroid hormone responsiveness of the endometrium, as with SULT1E1 and oestrogens. Indeed, SULT1E1 appears to be the major sulphotransferase involved in sulphation of T4 (Kester et al., 1999b). Similarly, the endometrium may also be responsive to catecholamines, including dopamine, as there is evidence from animal studies of the presence of catecholamine biosynthetic enzymes and receptors in reproductive tissues (e.g. Mitchell and Ahmed, 1992; Kim et al., 1997). In humans, sulphation is a major pathway of catecholamine metabolism and inactivation, with >95% of circulating dopamine existing as the sulphate conjugate, which probably originates from extraneuronal tissues including the gastrointestinal tract (Goldstein et al., 1995; Rubin et al., 1996; Eisenhofer et al., 1997). It is of course possible that these phenol sulphotransferases are expressed in the endometrium as part of the body's chemical defence mechanisms directed against toxic or potentially toxic xenobiotics (Coughtrie, 1996). Thus the human endometrium is richly endowed with the capacity to sulphate endogenous compounds and xenobiotics.
In-vitro experiments have demonstrated that oestrogen sulphotransferase activity and expression are induced by progesterone (Tseng and Liu, 1981; Clarke et al., 1982; Falany and Falany, 1996), and this is consistent with the cyclical nature of their expression in endometrium biopsy material. However, additional information regarding the regulation of SULT expression in vivo was not available. We therefore studied endometrium SULT expression in women taking the OCP and during the early stages of pregnancy (6–13 weeks post conception). Oestrogen sulphation by cytosols prepared from endometrial biopsies was abolished (early pregnancy) or dramatically reduced (OCP). A reduction in endometrial cytosol SULT1A1 enzyme activity was also seen in women taking the OCP, but this was not found with SULT1A3 activity. Similarly, early pregnancy did not seem to affect SULT1A1 and SULT1A3 expression. It is possible that changes in endometrium structure (i.e. altered ratio of stromal to glandular epithelial cells) under the hormonal influences of the OCP and pregnancy influence the measurement of SULT expression and activity. However, the differences we observed were specific for SULT1E1, and SULT1A3 expression in particular was not affected. The results obtained with the OCP are consistent with the role of progesterone in the induction of SULT1E1 expression in the endometrium. The OCP suppresses ovulation through inhibition of pituitary follicle stimulating hormone and blocking the oestrogen-controlled positive feedback which triggers the ovulatory luteinizing hormone (LH) surge. Thus, circulating progesterone and oestrogen are reduced and a reduction in SULT1E1 expression would be anticipated. This also suggests that these endocrine effects predominate over any direct effect of progestin on SULT1E1 expression (Falany and Falany, 1996). However, our observations on endometrium from early pregnancy, where SULT1E1 activity was completely abolished, suggest that the regulation of SULT1E1 expression is more complex. During the first trimester of pregnancy, maternal progesterone concentrations continue to rise from those seen at ovulation, and therefore it might be expected that SULT1E1 expression would be the same or greater than that present in the normal mid luteal endometrium. Since this was not the case, additional factor(s) are presumably involved in the suppression of SULT1E1 expression in early pregnancy. It is possible that the elevated concentrations of oestrogen also seen during early pregnancy could exert this effect; however, ß-oestradiol at a concentration of up to 10 µmol/l had no effect on oestrone sulphation in the Ishikawa endometrial adenocarcinoma cell line (Falany and Falany, 1996). Another potential candidate is human chorionic gonadotrophin, which dramatically rises in the maternal circulation during the first trimester, peaking at about 10 weeks gestation (Carr, 1995)and which may suppress the expression of SULT1E1. Evidence suggests that SULT1E1 activity is expressed at basal levels in both glandular and stromal cells, but that it is only responsive to progestin in the glandular cells (Tseng and Liu, 1981). Recent data demonstrating the differential expression of progesterone receptors A and B (PRA, PRB) in stromal and glandular cells (Wang et al., 1998a) indicate a potential mechanism whereby this could occur. It is particularly interesting that these investigators found very low levels of PRA and PRB in glandular epithelium in early pregnancy (although stromal PRA appears to remain high), where SULT1E1 activity was absent. Investigation of these and other possibilities in in-vitro systems such as endometrial explant culture (e.g. Tseng and Gurpide, 1975) and endometrium-derived cell lines such as Ishikawa cells (e.g. Falany and Falany, 1996) may shed more light on this obviously complex regulatory pathway. Overall, this reduced oestrogen catabolism in early pregnancy and during OCP use may actually have a beneficial effect, resulting in enhanced endometrial maintenance.
The precise contributions of sulphation (by SULT1E1) and sulphate conjugate hydrolysis (by ARSC) in modulating levels of active oestrogen in tissues such as endometrium remain unclear. It is possible to demonstrate substrate cycling between DHEA and its sulphate in in-vitro reconstituted cytosol/microsomal systems (Kauffman et al., 1998), and by inference this may occur in vivo. Given that SULT1E1 expression varies considerably throughout the menstrual cycle whereas ARSC activity is similar in follicular and luteal phases, the potential for such substrate cycling may be greatest at the peak of SULT1E1 expression in the mid-luteal phase. The recent development of highly selective and potent inhibitors of ARSC may provide one mechanism for further investigating the role of this enzyme in determining the outcome of sulphoconjugation (Purohit et al., 1995, 1998)
We have carried out a detailed investigation of sulphotransferase expression in the human endometrium and our results are consistent with a major role for sulphation of oestrogens in the normal endometrium. However, this function is presumably no longer required during pregnancy. We have provided further evidence for the influence of progesterone on SULT1E1 expression but have also demonstrated that additional factors are likely to regulate the expression of this protein in the endometrium. The role of the other sulphotransferases expressed in the endometrium remains as yet unclear.
Acknowledgments
This work was generously supported by a grant from the Scottish Hospital Endowments Research Trust (to M.W.H.C. and J.A.M.), and in part by equipment grants from the Wellcome Trust (to M.W.H.C.) and Tenovus Scotland (to M.W.H.C. and J.A.M.). We thank Drs Ron Gilissen and Rana Dajani for helpful discussions, and Dr David Edgar for his input at the early stages of this work.
Notes
4 Present Address: Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia 
5 To whom correspondence should be addressed at: Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee DD1 9SY, UK
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Submitted on December 16, 1998; accepted on July 8, 1999.
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