Molecular Human Reproduction, Vol. 5, No. 10, 955-960,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular events in the endometrium |
Changes in expression of adenyl cyclase activity in human endometrium during hormone replacement therapy and ovarian stimulation
1 Department of Obstetrics and Gynaecology, University of Genoa, Genoa, Italy 2 Hitachi Chemical Research Center and 3 Worldwide Medical Corporation, Irvine, CA, USA and 4 Clinica Las Condes, Santiago, Chile
Abstract
We have investigated membrane fractions prepared from human endometrium for activity of the signalling adenyl cyclase (AC). We characterized the AC guanine nucleotide-binding proteins (G proteins) and examined the changes in AC activity during evaluation cycles of oestrogen and progesterone replacement therapy as well during ovarian stimulation cycles. AC activity was determined by the conversion of substrate ATP into cyclic AMP under basal conditions and in the presence of guanine nucleotide or forskolin. G proteins were determined by Western Blot using specific polyclonal antibodies against Gs
, Gi1,2 and Gi3. Our results indicate that endometrial AC was highly responsive to activation by both guanine nucleotide and forskolin and its rate of cyclic AMP production was highly pronounced. Mean activity reached 920 pmol/l/min/mg membrane protein in the presence of forskolin, a value ~5-fold higher than those detected in corpus luteum. Hormonal induction of AC activities increased Gs protein, which couples with and stimulates the catalytic component of AC. We conclude that human endometrium is rich in AC and that enzyme activity is induced by oestrogen and progesterone treatment. These data strongly support the concept that the transmembrane signalling AC system and its messenger cyclic AMP are major regulators of endometrial function in the human.
cAMP/human endometrium/implantation/signal transduction/uterine receptivity
Introduction
Adenyl cyclase (AC) is a complex membrane-bound enzymatic system responsible for the synthesis of 3',5' cyclic adenosine monophosphate (cAMP) which is one of the major intracellular signal transduction pathways operating in eukaryotic cells (de Groot and Sassone-Corsi, 1993
). Transmembrane signalling via AC involves the interaction of three main subunits and proceeds as follows: the hormone binds to its receptor (first subunit), and induces the coupling of the receptor to the transmembrane guanine nucleotide (GTP)-binding proteins (G proteins) (second subunit) which either stimulate (via Gs) or inhibit (via Gi) the catalytic component (third subunit) (Spiegel et al., 1992). This last subunit is the effector system, responsible for the intracellular production of cAMP from the adenosine triphosphate (ATP) substrate. Generally, the activity of this enzymatic system is studied on membrane fractions under basal conditions and after hormonal and non-hormonal stimulation of G protein and catalytic component subunits (Rojas et al., 1989). Specifically, Gs protein is directly stimulated by either cholera toxin or guanyl nucleotide, whereas the catalytic component is stimulated by the diterpene forskolin (Rojas et al., 1991).
Cyclic AMP is frequently implicated in several uterine functions both in animals and the human. Accordingly, cAMP has been suggested to be directly involved in decidual reaction and implantation processes (Rankin et al., 1977; Vilar-Rojas et al., 1982). Also, experimental studies have shown that the administration of cholera toxin into ovariectomized hamsters primed with oestrogen intensifies the proliferative endometrial growth and the expression of the progesterone receptors (Alleva et al., 1983). In-vitro exposure to cAMP also resulted in elevated progesterone receptor concentrations in rat uterine cells (Aronica and Katzenellenbogen, 1991). Transfection studies have shown that cAMP is able to induce chicken progesterone receptor expression even in absence of its natural ligand (Denner et al., 1990). Notably, in the human, cAMP has been shown to mediate the prostaglandin-induced decidualization of endometrial stromal cells (Yee and Kennedy, 1991) and to induce decidual prolactin production along with the differentiation of endometrial stromal cells in decidual cells (Tang et al., 1993). However, the physiological source of cAMP supporting these functions in the human is not clear. Also, there is limited information on the capacity of the human endometrium to generate cAMP. In this study we investigated the activity of the transmembrane system AC in human endometrium. We measured its capacity to produce intracellular cAMP and characterized the presence of its associated G proteins. Also, we investigated whether hormones modulate the expression of this enzyme system by assessing AC activity and G proteins during the sequential exposure of endometrium to oestrogen and progesterone therapy. Moreover, an attempt was made to correlate the biochemical findings with the histological examination of endometrial maturation.
Materials and methods
Tissue collection
Endometrial biopsies used in this study for AC assay and immunoblotting were collected from patients undergoing mock evaluation cycles of hormone replacement therapy (HRT) (mean patients age ±SD: 41.4 ± 1.6) and from oocyte donors undergoing ovarian stimulation (mean patients age ±SD: 26.6 ± 0.5) at Saddleback Women's Hospital, Laguna Hills, CA, USA during the years 1992–1994. Informed consent was obtained from all the patients. For the assessment of AC assay two successive endometrial biopsies were obtained in subsequent dates of the same cycle. This was done on the last day of oestrogen administration (day 0) (n = 7) and on day plus 3 (n = 2) or 6 (n = 2) or 9 (n = 3) of progesterone supplementation during HRT cycles and on the day of transvaginal oocyte aspiration (day 2) (n = 4) and 3 (n = 1) or 5 (n = 1) or 8 (n = 2) days later during ovarian stimulation cycles (days 5, 7 and 10 respectively). Aliquots of tissue samples used for AC assay were utilized also for immunoblotting in three out of seven cases undergoing HRT cycles. Immunoblotting of endometrial tissue proteins was performed in a separate set of six additional women undergoing ovarian stimulation. In any case, part of each tissue sample was reserved for histological examination and dated (Noyes et al., 1950). HRT was achieved by sequential and combined administration of oestradiol and progesterone. 17ß-oestradiol was given per os at increasing dosage (2–6 mg) for a period of 14.3 ± 0.5 days. At this time, concomitant administration of natural progesterone (100 mg i.m., daily) was started. The oestradiol and progesterone serum concentrations measured on day 6 of progesterone hormone supplementation (367 ± 124 and 22 ± 5 ng/ml, respectively) were similar to those observed in natural cycles. Ovarian stimulation was achieved by a combination protocol of luteinizing hormone-releasing hormone (LHRH) analogue plus follicle stimulating hormone (FSH)–human menopausal gonadotrophin as previously published (Bernardini et al., 1993). Collection of rat corpora lutea was performed after ovulation induction with pregnant mare serum gonadotrophin (50 IU, s.c.) and 56 h later with human chorionic gonadotrophin (HCG) (50 IU, s.c.) injections (Birnbaumer et al., 1976). The stimulatory procedure used to induce ovulation induction resulted in a rather homogeneous population of corpora lutea within the ovary, such that the whole ovary could be used to study luteal activity. Human corpora lutea were obtained from ovaries of four women undergoing exploratory laparotomies for benign gynaecological conditions after provision of an adequate consent form. We had previously established the experimental conditions to measure optimal expression of luteal AC and defined its properties and regulation by several hormones and non-hormone activators (Rojas et al., 1991).
Membrane particles preparation
Endometrial tissue was flushed with phosphate-buffered saline (PBS) and weighed. Then, the tissue was minced and homogenized by means of a small Dounce homogenizer (Wheaton Industries, Millville, NJ, USA) (10 strokes with the loose pestle) in 20 volumes of 10 mmol/l Tris–HCl (pH 7.5), 1 mmol/l EDTA, proteinase inhibitors [0.2 mmol/l 4-(2-aminoethylbenzene sulphonylfluoride, hydrochloride: AEBSF), 5 mmol/l N-ethylmaleimide]. The homogenate was then centrifuged at 10 000 g for 45 min, at 4°C, and the resulting pellet resuspended in 5 parts of 27% (vol/wt) sucrose in 1 mmol/l EDTA and 10 mmol/l Tris–HCl, pH 7.5. Similar methodology has been previously used to prepare luteal membrane particles (Rojas et al., 1989).
Adenyl cyclase assay
Adenyl cyclase activity was determined by the conversion of ATP into cAMP under basal conditions and in the presence of 100 µmol/l of either GMP-P[NH]P (a stable form of GTP) or forskolin (Birnbaumer et al., 1976). Aliquots (10 µl) of membrane particle preparation (10 µg protein) were assayed in duplicate for AC activity. The final volume of 50 µl contained: [-32]ATP (150 c.p.m./pmol), 0.5 mmol/l ATP, 5.0 mmol/l MgCl2, 1.0 mM EDTA, 1.0 mmol/l [3H]cAMP (15 000 c.p.m.), 25 mmol/l Tris–HCl, pH 7.5, and a nucleoside triphosphate-regenerating system consisting of 20 mmol/l creatine phosphate, 0.2 mg/ml creatine kinase and 0.02 mg/ml myokinase. Incubations were performed at 32°C for 10 min. Termination of AC activity was accomplished by addition of 100 µl of a `stop' solution that contained 40 mmol/l ATP, 10 mmol/l CAMP and 1% sodium dodecyl sulphate. Formed [-32P]cAMP and [3H]cAMP (used as recovery marker) were isolated and separated by [-32P]ATP by double chromatography using Dowex 50 columns and alumina columns according to a procedure previously described (Rojas et al., 1989) and were collected in scintillation fluid for beta counting. The results were expressed in pmol of cAMP generated/mg protein/min and were compared to AC activities found in human and rat corpora lutea (internal controls).
Detection of G proteins
GTP-binding proteins (Gs, Gi1,2, Gi3) were determined as previously reported (Bernardini et al., 1995). Proteins were extracted from the original membrane fraction (centrifugation at 10 000 g) by resuspension of the preparation in 10 parts of PBS (vol/vol) (with proteinase inhibitors) and centrifugation at 100 000 g for 30 min at 4°C. The resulting pellet was resuspended in 5 volumes of original tissue weight in 1% SDS and 10% glycerol and further homogenized. The suspension was boiled for 10 min and then the debris was removed by microcentrifugation for 10 min. Protein content in the homogenate was determined by the BCA protein assay (Pierce Chemical Co., Rockford, IL, USA). Western Blots were performed according to the method of Laemmli (1970). Briefly, proteins were loaded on the gel (12%) under reducing conditions and transferred to Immobilon-P (PVDF) membranes (Millipore Corp., Bedford, MA, USA). After blocking with 2% non-fat dry milk in TBS, the blots were incubated overnight at 4°C with 1:5000 dilution of polyclonal antibodies to fragments of rat Gs, Gi1/2, and Gi3 (DuPont, Boston, MA, USA). The blots were washed and treated with anti-rabbit immunoglobulin G (IgG) horseradish peroxidase conjugate. The molecular sizes of proteins were determined by running standard marker proteins in an adjacent lane. Bands were detected by a system of enhanced chemiluminescence (ECL kit; Amersham, Arlington Heights, IL, USA). Autoradiographies were then processed by scanning densitometry in order to estimate protein concentrations.
Other procedures
To minimize contamination with guanine nucleotide-like material, the components of the nucleoside triphosphate-regenerating system were subjected to purification as previously reported (Rojas et al., 1989). The results are presented as the mean ± SD of two experiments using separated batches of membranes, each carried out in triplicate. In order to guarantee a higher degree of internal control, the immunoblot-membranes deriving from the single experiment were subjected successively to repeated stripping processes and reprobed with different primary antibodies. Statistical significance between experimental groups was evaluated using Student's t-test.
Results
Presence of adenyl cyclase activity and G proteins in human endometrium
Adenyl cyclase activity in membrane preparations of human endometrium obtained during oestrogen replacement therapy (proliferative phase) were studied under basal conditions (no additions) and in the presence of 100 µmol/l GMP-P(NH)P (G) and 100 µmol/l forskolin. The addition in separate experiments of GMP-P(NH)P and forskolin demonstrated a dramatic increase of the AC activity levels above those observed during basal conditions (no additions) (363 ± 118 and 920 ± 257 versus 47 ± 19) (Figure 1). Adenyl cyclase activity in membrane fractions of human endometrium were significantly higher (P < 0.001) than AC activity reported traditionally for maximally stimulated human and rat luteal AC (5–10-fold higher) (Table I).
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These data demonstrate that human endometrium has the potential to produce an extremely large amount of cAMP. Endometrial AC was very sensitive to activation by GMP-P[NH]P (stable form of GTP) and forskolin indicating the presence of Gs
and catalytic component respectively in human endometrium. The presence of different members of the family of the GTP-binding proteins in human endometrium is shown in Figure 2. Gs protein expression resulted in two separate bands migrating at 48 and 42 kDa and the 42 kDa band was significantly increased by progesterone supplementation (Figure 2a). Gi 1,2 and 3 proteins migrated at 40 kDa and their expression did not show any significant modification during the different phases of the cycle (Figure 2b). No significant changes were noted for the Gi 3 proteins (data not shown).
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Modulation by hormone replacement therapy of AC activity and G proteins expression
Adenylyl cyclase activity in membrane preparations from human endometrium obtained during progesterone supplementation (secretory changes) was studied under basal conditions and in the presence of 100 µmol/l guanyl nucleotide analogue and 100 µmol/l forskolin. On day 3 of progesterone therapy, maximal AC activity reached 660 ± 17 pmol/mg/min as determined by GMP-P(NH)P and 1830 ± 17 pmol/mg/min as determined by forskolin treatment (Figure 3
). Regardless of the experimental protocol, 3–6 days of progesterone therapy significantly increased the level of AC activity compared to that observed when progesterone was not added to oestradiol (proliferative phase) (P < 0.001). On day 9 of progesterone supplementation AC activity returned to levels similar to that observed during the proliferative phase of the cycle (day 0). Densitometer analysis of immunoblots showed that the intensity for 42 kDa band representing Gs protein was low on day 0 (proliferative phase), but increased significantly after progesterone supplementation (on days 3, 6 and 9) (P < 0.001). On the contrary, the inhibitory GTP-binding proteins expression remained constant throughout the different phases of the cycle (Figure 2
). Taken together our data provide evidence for a temporal correlation between Gs protein expression and increase of AC activity, both being upregulated by steroid hormone therapy. Although the pattern of results was similar in both types of cycles (HRT and ovarian stimulation), AC activities of endometrial membranes prepared from HRT were significantly higher than those of ovarian stimulation cycles (Table II). This could not be ascribed to changes in the Gs protein expression since the protein was found to be normally expressed in both types of endometria (data not shown). When the AC results were plotted according to the local degree of endometrial maturation (evaluated by conventional histological examination and regardless of the number of progesterone injections), the highest AC activities were detected during the early luminal and glandular epithelial changes (days 16–20 of the cycle). Conversely, at the time of initial decidualization process (days 23–27 of the cycle), AC activity decreased significantly (P < 0.001) (Figure 4).
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Discussion
In this study we have demonstrated the presence of a complete and functional signal transduction system in human endometrium. Our data indicate that human endometrium is particularly rich in AC, the enzyme responsible for the intracellular synthesis of cAMP. The presence of this enzyme in membrane fractions of human decidua of pregnant women was originally reported in 1979 (Whitsett and Johnson, 1979). Since then, only one study has reported the presence of AC in membrane preparations of human endometrium during normal menstrual cycles (Tanaka et al., 1993). However, in both studies the AC activities detected in human endometrium (40–110 pmol/mg/min) were markedly lower than that reported here. It is very likely that this discrepancy is due to methodological differences. In fact, previous studies used different cellular fractions as a source of AC and assessed only cAMP accumulation as determined by binding proteins. In contrast, we used a particulate fraction rich in adenylate cyclase and quantified directly the actual conversion rate of ATP substrate into cAMP. In addition, we purified all the components of the nucleoside-regenerating system in order to improve assay sensitivity. Also, we routinely included a particulate fraction of rat and human corpora lutea as internal controls in all determinations. Using this methodology, we conclude that the human endometrium has the highest capacity of cAMP production compared to other human reproductive tissues including myometria, corpora lutea, Fallopian tubes and implanting trophoblasts (Nulsen et al., 1988; Tanaka et al., 1993). We have also demonstrated that endometrial AC is markedly increased following adequate oestrogen priming and a short time exposure to progesterone (after 2–5 i.m. injections of 100 mg/day progesterone). Since this correlated with a parallel increment of Gs protein, which is a constitutive element of the enzyme, we conclude that progesterone is a physiological inducer of AC regulatory components in human endometria. This finding agrees with in-vitro studies where the addition of progesterone to oestradiol-primed stromal cells induced a marked increase of forskolin-promoted cAMP production (Houserman et al., 1989). The highest stimulation of the catalytic component with forskolin has been achieved after ovulation and during the early secretory phase of the natural cycle (Tanaka et al., 1993). Additionally, higher tissue content of cAMP during secretory changes of human endometrium and Fallopian tubes have been reported (Munemura et al., 1979; Bergamini et al., 1985). The finding that progesterone can modulate AC activity is compatible with the well-known inductive effect played by progesterone on other endometrial enzymes function including 11ß- and 17ß-dehydrogenase, glycogen synthase, glycogen phosphorylase, cyclooxygenase II, phospholipases, sulphatases, sulphotransferase and 15-hydroxy prostaglandin dehydrogenase (Schmidt-Gollwitzer et al., 1978; Tseng and Liu, 1981; Gal et al., 1982; Tseng et al., 1986; Benedetto et al., 1990; Raw and Silvia, 1991; Beck et al., 1995). The induction of AC by progesterone is particularly relevant considering that this enzyme is responsible for the synthesis of one of the most important second cellular messengers capable of regulating phosphorylation and gene transcription of all the above cited endometrial enzymes (Meyer and Habener, 1993). It is also interesting that the upregulation of AC prompted by progesterone occurs early when the pre-secretory luminal and glandular epithelial changes largely prevail over the stromal component of endometrium. This functional acquisition during early luteal phase may be instrumental in attaining adequate uterine receptivity to embryo implantation and proper decidualization of stromal cells. Also this would agree with the concept that cAMP may be implicated in signal amplification of embryo–maternal messages during implantation (Iyengar, 1996). Indeed, recent data suggest that G proteins and cAMP are important for the epithelial cells changes occurring in embryo adhesion and apposition and in luminal surface negativity, glycocalix thickness, lectin affinity and receptor expression (Beck et al., 1995). Also, glandular cAMP may act on stromal cells to induce progesterone receptor expression, prolactin secretion and morphological transformation of stromal into decidual cells (Aronica and Katzenellenbogen, 1991; Tang et al., 1993). On the other side, the embryo and corpus luteum may directly modulate endometrial cAMP production. cAMP is, in fact, the intracellular second messenger of several molecules released by the blastocyst (HCG, prostaglandin E2 and IL-1) (Shirakawa et al., 1988; Turunen et al., 1990; Yee and Kennedy, 1991; Tang and Gurpide, 1993) and corpus luteum (relaxin) (Fei et al., 1990) known to be strong mediators of immunological and vascular responses occurring during implantation. In addition, local cAMP may even control the process of trophoblast invasion by modulating the differentiation of cytotrophoblast and its HCG and progesterone secretion (Ringler et al., 1989; Tao et al., 1995).
Our data show that regardless of the type of endometrium analysed (HRT or ovarian stimulation cycles) the pattern of AC activation was similar. In both cases, a significant upregulation of the enzyme occurred during early luteal phase of the cycle. However, in comparison with artificial cycles, consistently lower levels of enzymatic activity were noted during cycles of ovarian stimulation. This could not be ascribed to any apparent alteration of Gs protein expression, suggesting that other unknown factors should have intercurred. Perhaps, the pre-luteal exposure of endometrium to abnormal concentrations of steroid hormones (oestradiol, progesterone and androgens) and gonadotrophins (FSH and LH) and LHRH analogues may have had a detrimental effect on endometrial membranes by interfering with multiple signal transduction pathways. In human endometrium, a maturational dissynchrony of glands and stroma due to a premature maturation of stroma has been frequently described following medical ovarian stimulation (Forman et al., 1989; Blasco, 1994). Since we found the lowest levels of AC activity in endometria sampled during the delayed secretory changes of endometrial stroma, it is tempting to speculate that during stimulated cycles a premature luteinization of the stroma may have occurred, thereby accounting for the difference of enzymatic function observed in this type of endometrium. From this viewpoint, it seems interesting to investigate whether these biochemical alterations have temporal and morphological parallelism with analogous abnormalities reported for pinopode appearance on the endometrial surface during ovarian stimulation (Kolb and Paulson, 1997). Additional work is needed to determine the impact that these changes in the transmembrane AC system may have upon maturation of the endometrial glands and the window of uterine receptivity to implantation.
In conclusion, human endometrium is capable of producing huge amounts of cAMP and AC activity is critically modulated by hormonal treatment. Evidence for high capacity to produce cAMP strongly supports a regulatory role of this nucleotide in the function of human endometrium. Premature endometrial maturation occurring in cycles of ovarian stimulation may be detrimental for full acquisition of biochemical responsiveness of endometrium to embryo implantation.
Notes
5 To whom correspondence should be addressed at: S. Martino's Hospital, L.go R. Benzi 10, 16132 Genova, Italy 
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Submitted on February 23, 1999; accepted on July 12, 1999.
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