Human Reproduction vol.6 no.7 pp.905-909. 1991
The effect of progesterone upon first trimester trophoblastic cell differentiation and human chorionic gonadotrophin secretion
E.R.Barnea1-2-3, D.Feldman and M.Kaplan 'University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical Center, Department Ob/Gyn, Division of Reproductive Endocrinology and Infertility, Camden, NJ, USA and 2 Feto-Placental Endocrinology Unit, Rappaport Institute, Technion, PO Box 9697, Efron Street, Haifa, Israel 3
To whom correspondence should be addressed af 7447 Old York Road, Melrose Park, PA 19126, USA
The effect of progesterone (P) upon first trimester placenta] secretion of human chorionic gonadotrophin (HCG) and cellular differentiation was studied using both static and kinetic methods. At 1 /tM, P inhibited spontaneous episodic secretion of HCG when given in short pulses ( 1 - 4 min) to placental explants in superfusion. Both HCG pulse frequency and amplitude were reduced. At 0.1 — 0.01 /xM P concentrations, the effect of HCG secretion was milder. P also blocked the maximally effective concentration 100 pM of gonadotrophin releasing hormone (GnRH) analogue, a known HCG stimulant, when given together with it for 1 min. This inhibitory effect lasted for 1 h after P administration. Progesterone at 1 /iM, added daily for 1 week blocked HCG secretion by isolated trophoblastic cells in static culture. This inhibitory effect lasted until the fifth day. No effect on differentiation and long-term viability was noticed in P-treated cells. Incubation with 0.1-1.0 /*M P did not affect HCG secretion by explants after 24 h. In contrast, the effect of 1 /tM cortisol or 1 nM oestradiol was stimulatory. In conclusion, P exerts both a rapid and delayed inhibitory effect upon HCG secretion and production. It may do so by counteracting the stimulatory effect of endogenous GnRH on gonadotrophin secretion by the placenta. Key words: cells/culture/HCG/progesterone/superfusion
Introduction In contrast to other placental hormones, human chorionic gonadotrophin (HCG) follows a peculiar pattern, demonstrating an initial exponential rise following implantation until 5 - 6 weeks, a slow mergence into a plateau phase and a gradual decrease towards 11 — 13 weeks, which is maintained until term (Ostandodh and Tuchinsky, 1980). Progesterone (P) is a sex steroid produced mainly by the corpus luteum in early pregnancy. After the seventh gestational week, its production is taken over, to a large degree, by the placenta (Pritchard et al., 1985). This steroid has been shown to have both a stimulatory effect in vivo Oxford University Press
and inhibitory effects in vitro upon placenta] HCG secretion (Yoseph et al., 1984; Maruo et al., 1986). Progesterone was also shown to have a role in pregnancy maintenance and support in cases of threatened abortion (Csapo et al., 1973; Aspillaga et al., 1982; Siiteri et al., 1977; Yen, 1986). Recently, a new method of placental superfusion has been developed, which allows real time measurements of HCG secretion and provides a more physiological approach to the study of HCG regulation (Barnea and Kaplan, 1989; Barnea et al., 1990), since it allows the net effect of test agents on placental function to be tested, by limiting the paracrine and autocrine forms of regulation. We have recently presented preliminary evidence that, in superfusion, 1 min of exposure to P reduces HCG pulsatility (Barnea and Kaplan, 1989). Using the superfusion model, in the present study we have examined in detail the effects of P upon first trimester placenta! HCG secretion, using both static (explants and cells) and dynamic culture methods (explants). The time-dependent effect of P was also tested by superfusion, administering the steroid for a few minutes every hour, or for 24 h to explants and to isolated cells for 7 or more days. In explants, the effect of cortisol and oestradiol was also examined. Finally, the effect of P upon longterm cellular differentiation and viability was examined. We report that P has both a rapid inhibitory effect upon basal HCG secretion and that induced by gonadotrophin releasing hormone analogue (GnRHa), as well as a delayed effect upon HCG production in cell culture, this, however, without affecting trophoblast differentiation.
Materials and methods Reagents DNase and soybean inhibitor, P, cortisol and oestradiol were purchased from Sigma (St Louis, MO). Dulbecco's Modified Eagle's Medium (DMEM) and fetal calf serum (FCS) were purchased from Beit Haemek (Israel). MAIA CLONE kits for measurements of HCG were obtained from Serono (Rehovot, Israel). GnRH analogue (r>Ala6-Met-Leu7-Pro9-A'-ethylamine) was provided by Dr Millar (SA). Placental material A total of 20 placentae were studied. After obtaining appropriate consent, elective pregnancy termination was carried out in the first trimester (7-10 weeks). Patients were healthy, on no medication and were non-smokers. The collected tissues were immediately placed in sterile 0.9% NaCl solution on ice. 905
E.R.Barnea, D.Feldman and M.Kaplan
Preparation of explants The method used has been published previously (Barnea and Fakih, 1985; Barnea et al., 1990). Briefly, explants of 50-70 mg wet weight were dissected out and rinsed in 1 % antibiotic solution (penicillin 10 000 U/ml, streptomycin 10 ;tg/ml and amphotericin 25 /ig/ml). No contamination was evident since the placenta! tissue used was positively identified before processing. For culture, explants were placed in DMEM media with or without steroids. Twelve replicates per test agent or control were plated. After 24 h of incubation at 37°C in an atmosphere of 95% air: 5% CO 2 , the media were collected and stored at -20°C until HCG assay. The tissue was saved for protein analysis. In preliminary experiments, HCG secretion in our system was linear up to 48 h. Therefore in subsequent experiments, the effect of P was tested only until 24 h at the point of a half-maximal increase. Isolated trophoblastic cell (ITC) culture The method used has been previously published with some modifications (Barnea et al., 1986; Barnea et al., 1990). After rinsing in cold 0.9% NaCl solution, the tissue was rinsed several times in DMEM which contained a 2% antibiotic solution. The membranes were trimmed and the tissue was incubated with 0.25% trypsin-EDTA solution containing DNase, 200 Kunitz units/ml, for 15 min with gentle agitation at 37°C. At the end of the incubation period, the cells were filtered through a sterile gauze. Soybean inhibitor was added to the recovered cell suspension to block trypsin activity. Then, 3 ml of the trophoblastic cell suspension was mixed with 7 ml DMEM containing 10% fetal calf serum (FCS). Cells were separated by centrifugation at 500 g for 10 min at room temperature and the supernatant was discarded. For culture, cells were resuspended in medium composed of DMEM, 5% FCS and 1 % garamycin solution 10 g/ml. Cells were counted using a haemocytometer and in each dish approximately 100 000 cells were plated and incubated at 37°C in an atmosphere of 95% air and 5% CO2. After 24 h of culture, cells attached and the media were replaced; 0.1 — 1.0 jtM P was added. The media were collected 48 h after the start of the experiment. This process was repeated daily for 7 days, and every 3 days thereafter until the experiment was terminated after 2 — 3 months. The media were stored at 20°C until assayed. The details of the histochemical studies have been reported previously (Barnea et al.. 1986). Briefly, at various times during culture, the media were removed from control and P-treated dishes and the cells were fixed. Subsequently, they were stained with trypan blue and observed with a light microscope for morphological changes using previously established criteria (Morrish and Siy, 1986). Stained dishes were coded and analysed blind for at least three microscopical fields in three different dishes from each control and P treatment. Superfusion culture The method of placental superfusion has been reported recently (Barnea and Kaplan, 1989; Barnea et al., 1990). Briefly, a superfusion apparatus (Accusyst, Endotronics, St Paul, MN) with a multichannel peristaltic pump and fraction collector (Model 272. ISCO, Durham, NC) were used to study the short-term dynamics 906
of HCG secretion. The explants (200-300 mg wet weight) were placed into the culture chambers and HEPES (18 mM)-buffered DMEM was washed through in an atmosphere of 5 % CO2 and 95% air at 37°C. Experiments were conducted for a 120 min period. A 1 ml sample from the effluent was collected every 2.4 min. In each experiment, one channel served as control and four as experimental channels. At given intervals, a 1 - 4 min pulse of 0.01 - I . O j i M P with or without 100 pM GnRHa was given through a peristaltic pump equipped with a digital flowmeter (ISMATECH DD, Chicago, IL). Assay Beta-HCG assays were carried out with a commercially available radioimmunoassay kit (MAIA CLONE, Serono Diagnostics). All measurements were carried out in the same assay. Intra-assay variability was 1.7% (Barnea and Kaplan, 1989). The protein content was determined according to the Lowry protein assay procedure (Lowry et al., 1951). Statistical analysis was performed using one-way ANOVA and Student's f-test. A probability
The effect of progesterone upon first trimester trophoblastic cell differentiation and human chorionic gonadotrophin secretion
E.R.Barnea1-2-3, D.Feldman and M.Kaplan 'University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical Center, Department Ob/Gyn, Division of Reproductive Endocrinology and Infertility, Camden, NJ, USA and 2 Feto-Placental Endocrinology Unit, Rappaport Institute, Technion, PO Box 9697, Efron Street, Haifa, Israel 3
To whom correspondence should be addressed af 7447 Old York Road, Melrose Park, PA 19126, USA
The effect of progesterone (P) upon first trimester placenta] secretion of human chorionic gonadotrophin (HCG) and cellular differentiation was studied using both static and kinetic methods. At 1 /tM, P inhibited spontaneous episodic secretion of HCG when given in short pulses ( 1 - 4 min) to placental explants in superfusion. Both HCG pulse frequency and amplitude were reduced. At 0.1 — 0.01 /xM P concentrations, the effect of HCG secretion was milder. P also blocked the maximally effective concentration 100 pM of gonadotrophin releasing hormone (GnRH) analogue, a known HCG stimulant, when given together with it for 1 min. This inhibitory effect lasted for 1 h after P administration. Progesterone at 1 /iM, added daily for 1 week blocked HCG secretion by isolated trophoblastic cells in static culture. This inhibitory effect lasted until the fifth day. No effect on differentiation and long-term viability was noticed in P-treated cells. Incubation with 0.1-1.0 /*M P did not affect HCG secretion by explants after 24 h. In contrast, the effect of 1 /tM cortisol or 1 nM oestradiol was stimulatory. In conclusion, P exerts both a rapid and delayed inhibitory effect upon HCG secretion and production. It may do so by counteracting the stimulatory effect of endogenous GnRH on gonadotrophin secretion by the placenta. Key words: cells/culture/HCG/progesterone/superfusion
Introduction In contrast to other placental hormones, human chorionic gonadotrophin (HCG) follows a peculiar pattern, demonstrating an initial exponential rise following implantation until 5 - 6 weeks, a slow mergence into a plateau phase and a gradual decrease towards 11 — 13 weeks, which is maintained until term (Ostandodh and Tuchinsky, 1980). Progesterone (P) is a sex steroid produced mainly by the corpus luteum in early pregnancy. After the seventh gestational week, its production is taken over, to a large degree, by the placenta (Pritchard et al., 1985). This steroid has been shown to have both a stimulatory effect in vivo Oxford University Press
and inhibitory effects in vitro upon placenta] HCG secretion (Yoseph et al., 1984; Maruo et al., 1986). Progesterone was also shown to have a role in pregnancy maintenance and support in cases of threatened abortion (Csapo et al., 1973; Aspillaga et al., 1982; Siiteri et al., 1977; Yen, 1986). Recently, a new method of placental superfusion has been developed, which allows real time measurements of HCG secretion and provides a more physiological approach to the study of HCG regulation (Barnea and Kaplan, 1989; Barnea et al., 1990), since it allows the net effect of test agents on placental function to be tested, by limiting the paracrine and autocrine forms of regulation. We have recently presented preliminary evidence that, in superfusion, 1 min of exposure to P reduces HCG pulsatility (Barnea and Kaplan, 1989). Using the superfusion model, in the present study we have examined in detail the effects of P upon first trimester placenta! HCG secretion, using both static (explants and cells) and dynamic culture methods (explants). The time-dependent effect of P was also tested by superfusion, administering the steroid for a few minutes every hour, or for 24 h to explants and to isolated cells for 7 or more days. In explants, the effect of cortisol and oestradiol was also examined. Finally, the effect of P upon longterm cellular differentiation and viability was examined. We report that P has both a rapid inhibitory effect upon basal HCG secretion and that induced by gonadotrophin releasing hormone analogue (GnRHa), as well as a delayed effect upon HCG production in cell culture, this, however, without affecting trophoblast differentiation.
Materials and methods Reagents DNase and soybean inhibitor, P, cortisol and oestradiol were purchased from Sigma (St Louis, MO). Dulbecco's Modified Eagle's Medium (DMEM) and fetal calf serum (FCS) were purchased from Beit Haemek (Israel). MAIA CLONE kits for measurements of HCG were obtained from Serono (Rehovot, Israel). GnRH analogue (r>Ala6-Met-Leu7-Pro9-A'-ethylamine) was provided by Dr Millar (SA). Placental material A total of 20 placentae were studied. After obtaining appropriate consent, elective pregnancy termination was carried out in the first trimester (7-10 weeks). Patients were healthy, on no medication and were non-smokers. The collected tissues were immediately placed in sterile 0.9% NaCl solution on ice. 905
E.R.Barnea, D.Feldman and M.Kaplan
Preparation of explants The method used has been published previously (Barnea and Fakih, 1985; Barnea et al., 1990). Briefly, explants of 50-70 mg wet weight were dissected out and rinsed in 1 % antibiotic solution (penicillin 10 000 U/ml, streptomycin 10 ;tg/ml and amphotericin 25 /ig/ml). No contamination was evident since the placenta! tissue used was positively identified before processing. For culture, explants were placed in DMEM media with or without steroids. Twelve replicates per test agent or control were plated. After 24 h of incubation at 37°C in an atmosphere of 95% air: 5% CO 2 , the media were collected and stored at -20°C until HCG assay. The tissue was saved for protein analysis. In preliminary experiments, HCG secretion in our system was linear up to 48 h. Therefore in subsequent experiments, the effect of P was tested only until 24 h at the point of a half-maximal increase. Isolated trophoblastic cell (ITC) culture The method used has been previously published with some modifications (Barnea et al., 1986; Barnea et al., 1990). After rinsing in cold 0.9% NaCl solution, the tissue was rinsed several times in DMEM which contained a 2% antibiotic solution. The membranes were trimmed and the tissue was incubated with 0.25% trypsin-EDTA solution containing DNase, 200 Kunitz units/ml, for 15 min with gentle agitation at 37°C. At the end of the incubation period, the cells were filtered through a sterile gauze. Soybean inhibitor was added to the recovered cell suspension to block trypsin activity. Then, 3 ml of the trophoblastic cell suspension was mixed with 7 ml DMEM containing 10% fetal calf serum (FCS). Cells were separated by centrifugation at 500 g for 10 min at room temperature and the supernatant was discarded. For culture, cells were resuspended in medium composed of DMEM, 5% FCS and 1 % garamycin solution 10 g/ml. Cells were counted using a haemocytometer and in each dish approximately 100 000 cells were plated and incubated at 37°C in an atmosphere of 95% air and 5% CO2. After 24 h of culture, cells attached and the media were replaced; 0.1 — 1.0 jtM P was added. The media were collected 48 h after the start of the experiment. This process was repeated daily for 7 days, and every 3 days thereafter until the experiment was terminated after 2 — 3 months. The media were stored at 20°C until assayed. The details of the histochemical studies have been reported previously (Barnea et al.. 1986). Briefly, at various times during culture, the media were removed from control and P-treated dishes and the cells were fixed. Subsequently, they were stained with trypan blue and observed with a light microscope for morphological changes using previously established criteria (Morrish and Siy, 1986). Stained dishes were coded and analysed blind for at least three microscopical fields in three different dishes from each control and P treatment. Superfusion culture The method of placental superfusion has been reported recently (Barnea and Kaplan, 1989; Barnea et al., 1990). Briefly, a superfusion apparatus (Accusyst, Endotronics, St Paul, MN) with a multichannel peristaltic pump and fraction collector (Model 272. ISCO, Durham, NC) were used to study the short-term dynamics 906
of HCG secretion. The explants (200-300 mg wet weight) were placed into the culture chambers and HEPES (18 mM)-buffered DMEM was washed through in an atmosphere of 5 % CO2 and 95% air at 37°C. Experiments were conducted for a 120 min period. A 1 ml sample from the effluent was collected every 2.4 min. In each experiment, one channel served as control and four as experimental channels. At given intervals, a 1 - 4 min pulse of 0.01 - I . O j i M P with or without 100 pM GnRHa was given through a peristaltic pump equipped with a digital flowmeter (ISMATECH DD, Chicago, IL). Assay Beta-HCG assays were carried out with a commercially available radioimmunoassay kit (MAIA CLONE, Serono Diagnostics). All measurements were carried out in the same assay. Intra-assay variability was 1.7% (Barnea and Kaplan, 1989). The protein content was determined according to the Lowry protein assay procedure (Lowry et al., 1951). Statistical analysis was performed using one-way ANOVA and Student's f-test. A probability
