Molecular Human Reproduction, Vol. 6, No. 7, 661-664,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
Effects of progesterone on prostaglandin E2-induced changes in glycosaminoglycan synthesis by human cervical fibroblasts in culture
1 INSERM U 361, Paris, 2 Department of Obstetrics and Gynaecology, Hôpital Saint-Antoine, Université Pierre et Marie Curie, Paris VI, 3 Department of Obstetrics and Gynaecology, Hôpital Intercommunal de Créteil, Créteil, and 4 Maternité Port-Royal-Baudelocque, Hôpital Cochin, Université René Descartes, Paris V, France
Abstract
Prostaglandins are known to induce cervical ripening and this effect may be mediated by an increase in glycosaminoglycan (GAG) concentration. The aim of this study was to assess the effects of progesterone on prostaglandin E2 (PGE2)-induced changes in GAG synthesis by human cervical cells in culture. Human cervical fibroblasts were obtained by cervical biopsies in hormonally active women and cultured. Cells were submitted to an incubation with progesterone or control medium. A second incubation was then performed with increasing concentrations of PGE2. GAG synthesis by the cervical cells was assayed after extraction, by incorporation of [3H]-glucosamine and [35S]-sulphate into GAGs. It was found that progesterone alone induced a dose-dependent increase in GAG synthesis. After pre-incubation with progesterone, PGE2 further increased [3H]-glucosamine and [35S]-sulphate uptake. However, when expressed as percentage of stimulation, the stimulatory effect of PGE2 on GAG synthesis was inhibited at high progesterone concentrations. Therefore we concluded that, although high concentrations of progesterone increase the overall synthesis of GAG, they may also play a preventative role against PGE2-induced changes in GAG production during pregnancy.
cervix/glycosaminoglycan/progesterone/prostaglandin/parturition
Introduction
It is now widely accepted that the cervix plays an active role in the process of labour and is not just under the control of uterine contractions. Whereas the onset of uterine contractions seems to be a sudden event, the process of cervical ripening is slow under normal conditions and appears to start early in the course of pregnancy (Uldbjerg et al., 1983
). Preterm premature cervical ripening may be responsible for preterm birth and its prevention would require precise knowledge about the intimate mechanisms of cervical maturation.
It has been previously demonstrated that softening of the cervix is associated with changes in glycosaminoglycan (GAG) concentration occurring before parturition (Uldbjerg et al., 1983; Cabrol et al., 1987). The most relevant changes are an increase in total GAGs, a relative decrease in sulphated GAGs (especially dermatan sulphate), an increase in hyaluronic acid (Danforth et al., 1974; von Maillot et al., 1979; Cabrol et al., 1985; Osmers et al., 1993) and tissue water content (Uldbjerg et al., 1983; Cabrol et al., 1985). These changes precede the more dramatic events associated with collagenase activity observed during labour (Uldbjerg et al., 1983).
Progesterone is necessary to maintain pregnancy and to prevent preterm birth in many mammalian species. Contrary to these animal species (i.e. sheep or rat), parturition in humans is not preceded by a drop in maternal plasma progesterone concentration (Anderson et al., 1985; Challis and Olson, 1988). However, the theory of progesterone withdrawal is still pre-eminent, since myometrial changes in hormone and/or receptor concentration (Ferré et al., 1978; How et al., 1995) and antiprogesterone compounds have demonstrated their ability to induce cervical ripening (Carbonne et al., 1995) and to favour labour induction (Cabrol et al., 1990a, 1991; Frydman et al., 1992). On the other hand prostaglandins, particularly prostaglandin E2 (PGE2), are effective agents for labour induction in humans when the cervix is unripe (Rayburn, 1989) and they have been shown to induce some of the biological features of cervical ripening, at least in animal models (Cabrol et al., 1987).
The aim of this study was to assess the effects of progesterone on PGE2-induced changes in GAG synthesis by human cervical cells in culture.
Materials and methods
Cultures of human cervical fibroblasts
Cervical biopsies were obtained after hysterectomy in hormonally active women for non-malignant lesions of the uterus, not affecting the cervix. Special care was taken in removing exo- and endocervical epithelia. Explants were minced and plated out in 60 mm diameter plastic dishes in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL Life Technologies, Eragny, France) containing 20% inactivated fetal calf serum (FCS), glutamine and penicillin– streptomycin (100 IU/ml-100 mg/ml). Cultures were placed in a 5% CO2 humidified incubator at 37°C. The medium was replaced every third day. After 7 days, cells started growing out from the explants and the medium was then replaced by DMEM containing only 10% FCS. Cells reached confluence after ~3 weeks. Explants were then removed after treatment with 0.1% trypsin/0.005% EDTA, and subcultures were established in DMEM containing 10% FCS.
GAG synthesis assay
Cervical cells were subcultured onto 24-well plastic culture plates at a density of 7x104 cells per well. When cell confluence was obtained, cultures were rinsed with phosphate-buffered saline (PBS) and the culture medium was replaced by serum-free DMEM containing 2.5 mCi/ml D-[1-3H]-glucosamine (Amersham, Les Ulis, France) and 2.5 mCi/ml [35S]-sulphate (Amersham) for 24 h.
GAG extraction was conducted as previously described (Wasteson et al., 1973) and modified by Redini et al. (1991). After 24 h, monolayer cultures were washed with PBS and digested with pronase (1 mg/ml; Boehringer Mannheim; Meylan, France) in 0.1 mol/l Tris–HCl (pH 7.5)/5 mmol/l CaCl2. Proteolysis was continued for 24 h at 56°C. GAGs from media and cell extracts were precipitated with cetylpyridinium chloride (CPC) 1% w/v, in presence of carriers (hyaluronic acid and chondroitin sulphate 100 mg/ml). The complex GAG–CPC was dissolved in 2 mol/l MgCl2. GAGs were then precipitated with ethanol. The final pellet was dissolved in 75 mmol/l NaCl. Radioactivity incorporated into GAGs was measured by liquid scintillation (Beckman LS3801).
Incubations
Before the labelling period, cervical cell cultures were pre-incubated with progesterone (Sigma, France) at concentrations ranging from 10–7 to 10–3 mol/l in serum-free DMEM or with serum-free DMEM alone (control medium) for 48 h at 37°C. All incubation media were oestrogen-free. Then the radiolabelled GAG precursors were added either in the control medium alone, or together with increasing concentrations of PGE2 from 10–10 to 10–5 mol/l for the next 24 h. The media were then removed and GAG extraction was performed as described above. Experiments were conducted simultaneously in 4 wells for each experimental condition.
Statistical analysis
Results were expressed as mean ± SEM in cpm/106cells. Comparisons were performed using analysis of variance (ANOVA). When adequate, post-hoc inter-group comparisons were made using Scheffé test.
Results
Effects of progesterone on GAG production
The effect of pre-incubation with progesterone on cultured cervical cells was a dose-dependent increase in the incorporation of both [35S]-and [3H]-into GAGs (Figure 1). Progesterone concentration in the culture media ranged from 10–7 to 10–3mol/l. Despite high progesterone concentrations (10–3 mol/l), the dose–response curve did not reach a plateau.
|
–) and [35S]-sulphate (- - -
- - -) into newly synthesized glycosaminoglycans. Results are expressed as: (A) cpm/106 cells and (B) percentage of stimulation with 10–7 to 10–3 mol/l progesterone, compared to incubation with control medium. *P < 0.05 when compared with control group (analysis of variance and Scheffé test for inter-group comparisons).When the results were expressed as a percentage of increase in GAG production, the stimulating effect of progesterone alone was proportionally more important on the incorporation of [3H]-than that of [35S]-into newly synthesized GAGs (Figure 1B
). The maximum stimulation, 90% for [3H]-glucosamine and 30% for [35S]-sulphate, observed at 10–3 mol/l progesterone, was significant when compared with incubation in control medium. Higher progesterone concentrations were not tested since they were too far out of the range observed in human plasma during pregnancy (182–690 nmol/l) (Anderson et al., 1985).
Effects of PGE2 alone on GAG production
PGE2 alone (i.e. after pre-incubation in control medium without progesterone) induced a significant increase in the incorporation of both [35S]-sulphate and [3H]-glucosamine into GAGs in a dose-dependent manner (Figures 2 and 3). The effect was observed for concentrations from 10–8 to 10–5 mol/l PGE2. The experiments were not pursued above 10–4 mol/l PGE2. A plateau was reached for [3H]-glucosamine and [35S]-sulphate uptake at concentrations above 10–6 mol/l PGE2. The stimulatory effect of PGE2 was most prominent on [3H]-glucosamine uptake since the maximum stimulation of incorporation into GAGs was 150% for [3H]-glucosamine after incubation with 10–5 mol/l PGE2, and only 50% for [35S]-sulphate in the same conditions (Figures 2 and 3).
|
-·-·-) or 10–3 (– –•– –). Results are expressed as: (A) cpm/106 cells and (B) percentage of stimulation, compared to incubation with control medium. *P < 0.05 when compared to control group. §P < 0.05 when compared with other concentrations of progesterone during pre-incubation (analysis of variance and Scheffé test for inter-group comparisons).
|
Effects of progesterone on PGE2-induced production of GAGs
The administration of PGE2, after pre-incubation with progesterone, led to the highest levels of [3H] and [35S] uptake (Figures 2 and 3
). However, when expressed as percentage of stimulation, pre-treatment with progesterone did not alter the stimulation of radiolabelled precursors uptake obtained with PGE2 alone until the highest progesterone concentrations were reached (Figures 2 and 3). The stimulatory effect of PGE2 on [3H]-glucosamine incorporation was significantly inhibited at the highest concentration of progesterone (10–3 mol/l) (Figure 2). The stimulation of [35S]-incorporation into GAGs was also reversed, although less dramatically, at high progesterone concentrations (Figure 3). The effect of progesterone on skin fibroblasts is being investigated in order to exclude a non-specific effect of progesterone. Discussion
In many mammalian species, progesterone is considered a key hormone in the mechanism of uterine quiescence and in the prevention of preterm delivery (Challis and Olson, 1988). However, in our study, pre-incubation with progesterone increased synthesis of GAGs by human cervical cells in culture as shown by incorporation of [3H]-glucosamine and [35S]-sulphate into total GAGs. Thus, progesterone does not prevent, and high concentrations even favoured, changes in GAG production which are usually considered to reflect cervical ripening, i.e. increased total amount of GAG and increased hydrogenation of GAGs, mainly represented by hyaluronic acid (Danforth et al., 1974; von Maillot et al., 1979; Cabrol et al., 1987). Although this result may seem paradoxical, it is noteworthy that progressive changes in cervical consistency and in cervical GAG content can be observed as early as the first trimester of pregnancy (Cabrol et al., 1991; Osmers et al., 1993) despite the increasing concentration of progesterone throughout pregnancy. On the other hand, progesterone is known to favour quiescence of the uterine body and this could play a role in preventing preterm birth (Carbonne et al., 1998). The mechanisms involved in this direct effect of progesterone on GAG production by cervical cells is currently under investigation by reversal studies with the antiprogesterone compound mifepristone.
Prostaglandins are key factors in the mechanism of parturition since they induce both uterine contractions and cervical ripening in vivo as well as in vitro (Challis and Olson, 1988, Rayburn, 1989). In our study, PGE2 alone induced an increase in newly synthesized GAGs in cervical cells in culture. The effect of PGE2 was more important on hydrogenated GAGs than on sulphated GAGs, as observed by Osmers et al. (1993) in cervical biopsies obtained during cervical ripening at different stages of pregnancy. This effect was dose-dependent, reaching a plateau around 10–6 mol/l. Although incubation with progesterone alone also induced an increase in GAG production by cervical cells in culture, this effect was lower than that of PGE2 alone at concentrations usually observed in human peripheral plasma during pregnancy, suggesting a weaker cervical ripening effect of progesterone. When incubations with progesterone and PGE2 were successively performed, we observed an inhibition of PGE2-induced stimulation of GAG production by progesterone at high concentrations (10–3 mol/l). This phenomenon could represent a protection by high concentrations of progesterone against premature cervical ripening and eventually against preterm birth. With this hypothesis, progesterone withdrawal (necessary to allow cervical ripening at term), could be achieved through local changes in progesterone concentration or receptors (Ferré et al., 1978; How et al., 1995; Stjernholm et al., 1996) since plasma progesterone concentrations do not decrease at the end of human pregnancy (Anderson et al., 1985). This hypothesis would also be consistent with the induction of cervical ripening and of parturition by antiprogesterone compounds in humans (Cabrol et al., 1990a; Frydman et al., 1992; Carbonne et al., 1995). The mechanism involved in this effect of progesterone is also under investigation. Similar experiments on skin fibroblasts are being performed in order to exclude a non-specific response to progesterone.
Cervical ripening is a multifactorial process which is only partially controlled by prostaglandins and steroids and the in-vitro model used in this study cannot take into account all its aspects. In particular, the role of inflammatory cells and mediators, involved in the process of cervical ripening and the role of other steroids and praracrine regulations could not be explored by this model. Another activity of progesterone is its ability to inhibit interleukin 8 (IL-8) release by cervical cells in vitro (Denison et al., 1999) and this could play a preventive role on cervical ripening by this indirect mechanism. These results, together with our data, suggest that progesterone could have a weak promoting effect on some aspects of cervical ripening on the one hand while, on the other hand, preventing more dramatic cervical changes induced by inflammatory mediators, e.g. prostaglandins or IL-8.
In conclusion, PGE2 induces increased in-vitro synthesis of sulphated and hydrogenated GAGs. Although pre-incubation with progesterone increases the total amount of newly synthesized GAGs, the stimulatory effect of PGE2 is inhibited at a high progesterone concentration.
Notes
5 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, F-75012 Paris, France. E-mail: carbonne{at}easynet.fr 
References
Anderson, P.J.B., Hancock, K.W. and Oakey, R.E. (1985) Non-protein-bound oestradiol and progesterone in human peripheral plasma before labour and delivery. J. Endocrinol., 104, 7–15.[Abstract]
Cabrol, D., Dubois, P., Sedbon, E. et al. (1987) Prostaglandin E2-induced changes in the distribution of glycosaminoglycans in the isolated rat uterine cervix. Eur. J. Obstet. Gynecol. Reprod. Biol., 26, 359–365.[ISI][Medline]
Cabrol, D., Dubois, C., Cronje, H. et al. (1990a) Induction of labor with mifepristone (RU 486) in intrauterine fetal death. Am. J. Obstet. Gynecol., 163, 540–542.[ISI][Medline]
Cabrol, D., Jannet, D., Le Houezec, R. et al. (1990b) Mechanical properties of the human uterine cervix : Use of an instrument to measure the index of cervical distensibility. Gynecol. Obstet. Invest., 29, 32–36.[Medline]
Cabrol, D., Carbonne, B., Bienkiewicz, A. et al. (1991) Induction of labor and cervical maturation using mifepristone (RU 486) in the late pregnant rat. Influence of a cyclooxygenase inhibitor (diclofenac). Prostaglandins, 42, 71–77.[ISI][Medline]
Carbonne, B., Brennand, J., Maria, B. et al. (1995) Effects of gemeprost and of mifepristone on the mechanical properties of the cervix before first trimester termination of pregnancy. A comparative study. Br. J. Obstet. Gynaecol., 102, 553–558.[ISI][Medline]
Carbonne, B., Cabrol, D., Clerget, M.S. and Germain, G. (1998) Nomegestrol acetate effects on spontaneous and sulprostone-induced uterine contraction in pregnant cynomolgus monkey monitored by telemetry. Am. J. Obstet. Gynecol., 178, 150–155.[Medline]
Challis, J.R.G. and Olson, D.M. (1988) Parturition. In Knobil, E., Neil, J. et al. (eds), The Physiology of Reproduction. Raven Press, New York, USA, pp. 2177–2216.
Danforth, D.N., Veis, A., Breen, M. et al. (1974) The effect of pregnancy and labor on the human cervix: changes in collagen, glycoproteins and glycosaminoglycan. Am. J. Obstet. Gynecol., 120, 641–649.[ISI][Medline]
Denison, F.C., Calder, A.A. and Kelly, R.W. (1999) The action of prostaglandin E2 on the human cervix : stimulation of interleukin 8 and inhibition of secretory leukocyte protease inhibitor. Am. J. Obstet. Gynecol., 180, 614–620.[ISI][Medline]
Ferré, F., Janssens, Y., Tanguy, G. et al. (1978) Steroid concentrations in human myometrial and placental tissues at 39 weeks of pregnancy. Am. J. Obstet. Gynecol., 131, 500–502.[Medline]
Frydman, R., Lelaidier, C., Baton-Saint-Mleux, C. et al. (1992) Labor induction in women at term with mifepristone (RU 486): a double blind, randomized, placebo-controlled study. Obstet. Gynecol., 80, 972–975.
How, H., Huang, Z.H., Zuo, J. et al. (1995) Myometrial estradiol and progesterone receptor changes in preterm and term pregnancies. Obstet. Gynecol., 86, 936–940.[Abstract]
Osmers, R., Rath, W., Pflanz, A. et al. (1993) Glycosaminoglycans in cervical connective tissue during pregnancy and parturition. Obstet. Gynecol., 81, 88–92.
Rayburn, W.F. (1989) Prostaglandin E2 gel for cervical ripening and induction of labor: a critical analysis. Am. J. Obstet. Gynecol., 160, 529–534.[ISI][Medline]
Redini, F., Daireaux, M., Mauviel, A. et al. (1991) Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-ß (TGF-ß). Biochim. Biophys Acta, 1093, 196–206.[Medline]
Stjernholm, Y., Sahlin, L., Akerberg, S. et al. (1996) Cervical ripening in humans: potential roles of estrogen, progesterone, and insulin-like growth factor-I. Am. J. Obstet. Gynecol., 174, 1065–1071.[ISI][Medline]
Uldbjerg, N., Ekman, G., Malstrom, A. et al. (1983) Ripening of the human uterine cervix related to changes in collagen, glycosaminoglycans, and collagenolytic activity. Am. J. Obstet. Gynecol., 147, 662–666.[ISI][Medline]
Von Maillot, K., Stuhlsatz, H.W., Mohanaradhakrishnan, V. and Greiling, H. (1979) Changes in the glycosaminoglycans distribution pattern in the human uterine cervix during pregnancy and labor. Am. J. Obstet. Gynecol., 135, 503–506.[ISI][Medline]
Wasteson, A., Uthne, K. and Westermark, B. (1973) A novel assay for the biosynthesis of sulphated polysaccharide and its application to studies on the effects of somatomedin on cultured cells. Biochem. J., 136, 1069–1074.[ISI][Medline]
Submitted on September 9, 1999; accepted on May 9, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Schmitz, M.J. Leroy, E. Dallot, M. Breuiller-Fouche, F. Ferre, and D. Cabrol Interleukin-1{beta} induces glycosaminoglycan synthesis via the prostaglandin E2 pathway in cultured human cervical fibroblasts Mol. Hum. Reprod., January 1, 2003; 9(1): 1 - 8. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Schmitz, E. Dallot, M.J. Leroy, M. Breuiller-Fouche, F. Ferre, and D. Cabrol EP4 receptors mediate prostaglandin E2-stimulated glycosaminoglycan synthesis in human cervical fibroblasts in culture Mol. Hum. Reprod., April 1, 2001; 7(4): 397 - 402. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||