Molecular and Cellular Endocrinology 171 (2001) 179 – 185 www.elsevier.com/locate/mce
Mechanisms of estradiol inactivation in primate endometrium Bettina Husen a,*, Jerzy Adamski b, Gabriele M. Rune c, Almuth Einspanier a a Department of Reproducti6e Biology, German Primate Center, Kellnerweg 4, D-37077 Goettingen, Germany GSF National Research Center for Health and En6ironment, Institute for Experimental Genetics, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany c Institute of Anatomy, Ernst-Moritz-Arndt-Uni6ersity Greifswald, Friedrich-Loeffler-Str. 23c, D-17487 Greifswald, Germany b
Abstract In uterine endometrium, the level of estradiol is controlled by oxidative 17b-hydroxysteroid dehydrogenase (17HSD) activity which converts the bioactive hormone to the less active compound estrone. At least three different types of 17HSD (types 2, 4 and 8) use estradiol as their preferred substrate and may contribute to the overall rate of estradiol-inactivation in the uterus. In this study the marmoset monkey (Callithrix jacchus) was used for the investigation of the particular contribution of each type of 17HSD. Northern Blots revealed essentially the same tissue distribution as in the human. Likewise, uterine 17HSD enzyme activity increases in the secretory phase of the reproductive cycle, in parallel to the rise in circulating progesterone levels. Northern analysis of uteri from defined time points of the reproductive cycle showed that only the level of 17HSD2 expression is strongly upregulated in the secretory phase, whereas 17HSD4 and 17HSD8 seem to be expressed constitutively. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: 17b-Hydroxysteroid dehydrogenases; Estradiol-inactivation; Endometrium; Marmoset monkey (Callithrix jacchus)
1. Introduction Inactivation of 17b-estradiol by its oxidation to estrone is essential for induction and maintenance of secretory endometrium during the luteal phase of the ovarian cycle. The inactivation is achieved by oxidative 17HSD enzyme activity. This enzyme activity prevails in the secretory phase of the menstrual cycle (Tseng and Gurpide, 1974; Scublinsky et al., 1976) and is stimulated by progesterone (Tseng and Gurpide, 1975, 1979). The first enzyme shown to be involved in this process in the human was 17HSD2 (Casey et al., 1994). Later further candidate enzymes were characterized which may participate in endometrial inactivation of estradiol. So far, many tissues have been shown to express a specific panel of different 17HSDs (for example ovary: Zhang et al. 1996; prostatic carcinoma: Castagnetta et al., 1997; adipose tissue: Corbould et al., 1998; brain: Stoffel-Wagner et al., 1999). This allows a very exact
* Corresponding author. Tel.: +49-551-3851132; fax: + 49-5513851288. E-mail address: [email protected]
control of active hormone levels. To date eight different 17HSD enzymes have been identified in the human (types 1–5 and 8 reviewed by Peltoketo et al., 1999; type 7: Krazeisen et al., 1999; type 10: Yan et al., 1997). With the exception of the aldoketoreductase 17HSD5 all are members of the short chain alcohol dehydrogenase gene family. Conversion of estradiol can theoretically be performed by all oxidative 17HSDs which prefer estradiol as a substrate and these are 17HSD2, 4 and 8. Specific mRNAs for 17HSD2 and 4 have already been shown to be present in human uterus (Casey et al., 1994; Husen et al., 2000). 17HSD4 protein has been shown to play a role in the reproductive cycle of the pig (Husen et al., 1994) and its mRNA is expressed simultaneously with 17HSD2 in human glandular endometrial epithelial cells (Husen et al., 2000). 17HSD8 (Ke6) has been identified only recently and is characterized by a substrate specificity for estradiol which is in the same range as that of 17HSD2 and 17HSD4 (Formitcheva et al., 1998). In contrast to the reductive HSD)s the three enzymes have a rather broad tissue distribution in all species examined. Apart from these common features they differ in many respects.
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B. Husen et al. / Molecular and Cellular Endocrinology 171 (2001) 179–185
Human 17HSD2 shares B 20% amino acid sequence identity with 17HSD4 and 17HSD8, respectively, whereas 17HSD4 and 8 share 35% identity which is the highest among all 17HSDs known to date (Formitcheva et al., 1998). Unlike 17HSD4 which is localized in peroxisomes (Markus et al., 1995), 17HSD2 and 8 are microsomal proteins. Two of the three enzymes are known to have different additional activities. 17HSD2 can act also as a 20a-hydroxysteroid dehydrogenase (20HSD), converting 20a-dihydroprogesterone into progesterone (Wu et al., 1993). 17HSD4 is a multifunctional enzyme consisting of three functional domains. It is able to perform two steps of peroxisomal b-oxidation and bile acid synthesis using D-3-hydroxy-acyl-CoA derivatives of certain fatty acids as substrates (reviewed by de Launoit and Adamski, 1999). The gene for 17HSD8 (Ke6) was originally identified because it seemed to be essential for normal kidney development (Aziz et al., 1993, 1994, 1996). Especially in primates, 17HSDs exert a key role in controlling the intracellular concentration of bioactive hormone in peripheral target tissues (Labrie et al., 1997, 1998). The knowledge of the particular expression profiles of estradiol-inactivating 17HSDs throughout the reproductive cycle in the uterus may provide clues for a possible pharmacological control of this process and its implications for endometrial dysfunctions in the human. Specific inhibitors for 17HSD1 and 2 are currently tested in several laboratories (Penning, 1996; Tremblay and Poirier, 1998; Tremblay et al., 1999). The aim of this study was to elucidate the contribution of the particular enzymes to the overall conversion rate of estradiol in primate endometrium during the course of the ovarian cycle and to look for a differential regulation. For this purpose the common marmoset monkey (Callithrix jacchus) was used as an experimental model. This species shows similarities to the human and to Old World Monkeys in terms of steroid hormone profiles during the reproductive cycle (Hearn, 1983) and has been extensively used in reproductive and biomedical research (Hillier et al., 1988; Fraser and Lunn, 1999). In this work we show for the first time a simultaneous analysis of mRNA expression and activity of 17HSD2, 4 and 8 in primate uterus.
2. Materials and methods
2.1. Animals A total of 15 female and two male adult marmoset monkeys (Callithrix jacchus) were used in this study. Animals were maintained in pairs at the German Primate Center, Goettingen, under standardized condi-
tions. Stage of the ovarian cycle was determined by assessment of plasma progesterone levels twice a week and at the day of tissue collection (Hodges et al., 1988). Females were monitored during at least three regular cycles which last about 28 days, ovulation occurring around day 10 (Summers et al., 1985). Ovarian cycles were controlled by application of a luteolytic dose of the prostaglandin F-2a analogue Cloprostenol according to Summers et al. (1985). Animals were anaesthesized with isoflurane before tissue collection and euthanasia. Placental tissue was obtained from caesarean sections. All tissues were immediately frozen in liquid nitrogen. Animal experiments reported here were approved by the local Animal Care Committee.
2.2. HPLC analysis of enzyme acti6ity Frozen tissue was pulverized in a dismembrator (Braun, Melsungen, Germany) and the resulting powder was resuspended in 7 vol. of 50 mM KH2PO4, pH 7.8. Conversion of estradiol to estrone was measured by HPLC analysis as described by Adamski et al. (1987). Determination of protein content was performed according to Markwell et al. (1981).
2.3. Preparation of riboprobes For the preparation of marmoset-specific RNAprobes RT-PCR products were obtained as described below and cloned into the vector PCR-Script (Stratagene Europe, Amsterdam, The Netherlands). This template was used according to the instructions of the manufacturer for in vitro-transcription with T3- and T7-polymerase yielding digoxigenin labelled riboprobes (DIG RNA labelling kit, Roche Biochemicals, Mannheim, Germany). The identity of the probes was proven by automated sequencing of the plasmids (Biotechnikum, Greifswald, Germany). The sequences of marmoset 17HSD2, 4 and 8 were in all cases \95% similar to those of the human.
2.4. Northern blotting For Northern blots total RNA was isolated from marmoset tissues using the RNA-Midi Kit (QIAGEN, Hilden, Germany). Total RNA was run on 1% formaldehyde-agarose gels and transferred to positively charged nylon membranes (according to the DIGUser’s Manual, Roche Molecular Biochemicals, Mannheim, Germany). Prehybidization and hybridization were conducted at 68°C. The membranes were subjected to stringent washing and chemiluminescent detection with the substrate CDP-Star™ (Roche Molecular Biochemicals).
B. Husen et al. / Molecular and Cellular Endocrinology 171 (2001) 179–185
2.5. Relati6e quantitati6e RT-PCR Total RNA was isolated from marmoset uteri or placenta as described for Northern Blots, but additionally, residual genomic DNA was removed by treatment with RQ I RNAse-free DNAse (Promega, Mannheim, Germany). Synthesis of cDNA was performed with SuperscriptII-reverse transcriptase (GIBCO BRL, Karlsruhe, Germany) using a mixture of oligo-dT-primers and random primers (Maas-Szabowski, 1997). Specific sequences from 17HSD2 (bp 112 – 408, Wu et al., 1993), 17HSD4 (bp 1–323, Adamski et al., 1995), and 17HSD8 (bp 344–789, Ando et al., 1996) were amplified from this template-cDNA by RT-PCR with Pfu-Polymerase (Stratagene) and with primers designed according to the known human nucleotide sequences. Relative quantitative RT-PCR was performed at an annealing temperature of 56°C. As external control the cDNA of the constitutively expressed 26S ribosomal protein was amplified in a number of 25 cycles (Krazeisen et al., 1999). For 17HSD2, 4 and 8 a number of 35 cycles was performed. Cycle numbers had been tested before to be within the linear dynamic range of amplification.
ovulation between day 8 and 10. Specific 17HSD enzyme activity (Table 1) measured in uteri from the proliferative phase was below 0.5 mU/mg protein throughout the proliferative phase. During this time period serum progesterone concentration did not exceed 3 ng/ml. In the secretory phase which was characterised by elevated progesterone levels enzyme activity increased more than ten-fold.
3.2. Tissue distribution of 17HSD2, 4 and 8 mRNA
Northern blot analysis of the distribution of 17HSD2, 4 and 8 mRNA in marmoset tissues was performed to facilitate the comparison of this primate model with the human (Fig. 1). Each of the three enzymes was present in many different tissues as already reported for the human (Casey et al., 1994; Adamski et al., 1995; Ando et al. 1996). A hybridization signal for 17HSD2 was detected in placenta, liver, kidney and intestine. 17HSD4 was additionally found in adrenal, testis, lung and pancreas. The broadest tissue distribution was observed for 17HSD8 which was expressed more or less in all tissues examined with the exception of immature ovary. It was evident that the full-length-transcripts detected with marmoset-specific riboprobes have the same length and number as the known human transcripts.
3.1. Enzyme acti6ity
3.3. Expression of 17HSD2 and 17HSD4 -mRNA
Overall enzymatic conversion of estradiol to its less active metabolite estrone in marmoset uterus homogenates was monitored by HPLC analysis. In marmoset monkeys of our colony the reproductive cycle has a duration of 28 days. The proliferative phase of the endometrium begins at day 1, when serum progesterone levels from the preceding cycle have decreased below 10 ng/ml progesterone. The secretory phase starts after
To investigate which of the estradiol-inactivating 17HSDs contribute to the variation of enzyme activity in the uterus during the reproductive cycle, changes in their mRNA expression were monitored by Northern Blot analysis in animals from six different time points of the cycle (Fig. 2). All three 17HSDs investigated were detected in marmoset uterus, including 17HSD8 which has never been
Table 1 Conversion of estradiol to estrone assessed by HPLC analysis in marmoset uterus homogenates and corresponding serum concentrations of progesterone in five animals from the proliferative and four animals from the secretory phase of the reproductive cycle Day of cycle Proliferative phase
Estradiol Estrone (mU/mg protein)
Serum progesterone (ng/ml)
3 7 7 7 8
0.48 0.03 0.05 0.10 0.10 x¯ =0.15 9 0.18
B1.5a B1.5a B1.5a 2.8 B1.5a
19 19 22 25
3.89 8.66 5.67 9.26 x¯ =6.87 92.53
48.3 28.1 49.7 40.0 x¯ =41.5 9 9.9
Detection limit of the progesterone assay.
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17HSD8. The two latter enzymes seemed to be expressed constitutively.
4. Discussion In the marmoset uterus estradiol-inactivating enzyme activity changes during the course of the reproductive cycle in the same way as was shown earlier in the human (Tseng and Gurpide, 1974, 1975). It is low in the proliferative phase and high in the progesteronedominated secretory phase. This finding, together with the similarities observed in the tissue-specific expression of the estradiol-inactivating 17HSDs indicated that the marmoset monkey is a good model to study the physiological functions of these enzymes with respect to the special features of primates and, most importantly, of humans.
Fig. 1. Northern Blot analysis of the tissue distribution of 17HSDs in marmoset monkeys. Total RNA was extracted from different tissues as described in Section 2. Per lane 5 mg of total RNA were loaded. Blots were hybridized with digoxigenin-labelled marmoset-specific probes to 17HSD mRNA as indicated. 17HSD2, 4 and 8 show the same distribution as known in humans.
studied in this tissue before. A prominent regulation was only observed in the expression pattern of 17HSD2. The hybridization signal for this enzyme was virtually absent in the proliferative phase and increased during the secretory phase. The expression level of 17HSD4 was higher than that of 17HSD2 during the whole reproductive cycle, but it remained constant at all time points examined. Also, for 17HSD8 no variations in its expression level could be detected. The result of the Northern Blots was verified in a larger number of animals by a relative quantitative RT-PCR approach which did not require as much RNA as the Northern Blot (Fig. 3). Again, a marked variation in mRNA expression during the cycle is only observed for 17HSD2, but not for 17HSD4 or
Fig. 2. Northern Blot analysis of the expression of 17HSD2, 17HSD4 and 17HSD8 mRNA in marmoset uterus during the reproductive cycle. Total RNA was extracted as described in Section 2. Numbers at the bottom indicate the day of the cycle when the uteri where collected (3, 7, 8 = proliferative phase, days 13, 18, 23 =secretory phase). For the uteri and placenta (PI), 5 mg of total RNA were loaded per lane, for liver (L), 2 mg of total RNA. Liver and placenta were used as positive controls. Blots were hybridized with digoxigenin-labelled marmoset-specific probes to 17HSD mRNA as indicated. A regulated mRNA-expression is observed only for 17HSD2. 17HSD4 and 8 are expressed constitutively.
B. Husen et al. / Molecular and Cellular Endocrinology 171 (2001) 179–185
Fig. 3. Expression of 17HSD2, 17HSD4 and 17HSD8 mRNA in marmoset uterus during the reproductive cycle analyzed by relative quantitative RT-PCR. Numbers on the top indicate the day of the cycle when the uteri where collected (3,7,8 proliferative phase, days 13, 18, 22, 23=secretory phase). For most time points, cDNA from two different animals was available. M = molecular weight marker, C =control RT-PCR without cDNA template. The expression of 17HSD2 is an upregulated, in the secretory phase of the cycle. No regulation is observed in 17HSD4 and 8.
The occurrence of oxidative 17HSDs in multiple peripheral tissues including liver, lung and tissues of the gastrointestinal tract can be explained by a possible role in the detoxification of environmental estrogens (Corton et al., 1996). In target tissues of estradiol they are thought to exert a protective effect against excessive estrogen action (Elo et al., 1996; Takeyama et al., 1998; reviews: Labrie et al., 1997; Peltoketo et al., 1999). According to their enzymatic characteristics three 17HSDs were expected to be active in the uterus. Early studies of Tseng and Gurpide (1974) showed a moderate increase of 20a-hydroxysteroid dehydrogenase activity in parallel to the increase in 17HSD activity in human endometrium. Consequently, the expression of 17HSD2 which possesses additional 20a-activity was shown to be upregulated in the secretory phase of the reproductive cycle in humans (Casey et al., 1994; Mustonen et al., 1998). This is confirmed by our present results in the marmoset. Interestingly, Zeitoun et al. (1998) reported that, in contrast to eutopic secretory endometrium, 17HSD2 mRNA and protein are absent from endometriotic lesions which are considered as estrogen dependent structures.
The role of 17HSD4 in estradiol-inactivation is under discussion, since it proved to be a multifunctional enzyme which also efficiently catalyzes two steps of peroxisomal b-oxidation (Leenders et al., 1996; Dieuaide-Noubhani et al., 1996; Qin et al., 1997). Nevertheless, in pig, it could be demonstrated by immunofluorescence staining that there is an increase in 17HSD4 protein expression during the secretory phase. Moreover, this is accompanied by a reorganization of peroxisomes, the subcellular compartment where 17HSD4 is localized (Husen et al., 1994; Markus et al., 1995). In the marmoset, we found a strong expression of 17HSD4, but we were not able to detect any changes in the level of mRNA-expression, although we used two different experimental approaches (Northern Blot and relative quantitative RT-PCR). This result may represent either a primate-specific phenomenon or it may be due to post-translational events which cause a regulation of 17HSD4 at the protein level. These aspects need further investigation. This is the first study to show that 17HSD8 can participate in estradiol inactivation in the uterus. In the rat, its mRNA expression was shown in the cumulus cells of ovarian follicles and in testes (Formitcheva et al., 1998. Other reproductive organs were not examined. Like 17HSD4, 17HSD8 mRNA expression level in marmoset uterus remained constant throughout the cycle, indicating that these two enzymes may provide a base level of 17HSD activity, independent of cyclic events. From our present results it may be concluded that the fine-tuning of estradiol action in the endometrium of primates can be achieved by the expression of three different 17HSD enzymes, with different additional enzymatic activities. In healthy cycling endometrium the regulation of receptor occupancy with bioactive estrogen is mainly performed by 17HSD2. Apart from that, a base level of estradiol inactivation seems to be constantly provided by 17HSD4 and 17HSD8. With specific effectors, a differential regulation according to physiological requirements should be possible.
Acknowledgements The authors are grateful to Ingo Schwabe, Kerstin Fuhrmann, Angelika Jurdzinski and Alexandra Marten for their help in sample preparation and monitoring of the animals. We are indebted to Keith Hodges for providing his expertise on the reproductive physiology of Callithrix jacchus. We thank S. Rensing for veterinary care and maintenance of animals and for assistance with sample collection. This work was supported in part by a DFG-grant to J. Adamski and by DFG-grant Ei 333/6-1 to A. Einspanier.
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