Human Reproduction vol.6 no.6 pp.766—769, 1991
Stress-related hormones affect human chorionic gonadotrophin secretion from the early human placenta in vitro
Joseph Tal2, Marielle Kaplan1, Mordechai Sharf2 and Eytan R.Barnea1 Feto-Placental Endocrine Unit, Rappaport Institute, Technion and Department of Ob/Gyn Bnai-Zion Medical Centre, Haifa, Technion, Israel 2
'To whom correspondence should be addressed at: Feto-Placental Endocrine Unit, Rappaport Institute, Technion, PO Box 9697 Rh Efron, Haifa 34601, Israel
The effect of a physiological range of concentrations of three stress-related hormones, oxytocin (OT), arginine-vasopressin (AVP) and prolactin (PRL) was tested upon human chorionic gonadotrophin (HCG) secretion by placental explants from early pregnancy in static and supervision cultures. In static cultures, OT and AVP significantly increased HCG secretion, whereas PRL had no effect. In superfusion, 1-min pulses of OT induced a significant (two- to 10-fold) rise in HCG pulse amplitude compared to the control. This effect of this neuropeptide was blocked by coadministration of a specific receptor antagonist. AVP also increased the glycoprotein pulse amplitude by two- to five-fold, but only with every second pulse administered. PRL pulses caused a progressive inhibition of spontaneous HCG pulsatility. In conclusion, stressrelated hormones affect placental HCG secretion in vitro. The involvement of these factors in impairing early pregnancy development is suggested. Key words: HCG/oxytocin/prolactin/superfusion/vasopressin Introduction A gonadotrophin releasing hormone (GnRH)-like compound has been identified in the human placenta (Khodr and Siler-Khodr, 1978). It is believed to act on receptors present on the syncytiotrophoblast cell membrane, stimulating human chorionic gonadotrophin (HCG) secretion. Episodic HCG secretion has been demonstrated in the maternal circulation during early pregnancy (Owens et al., 1981). We have found that supervised human placental explants secrete HCG in a pulsatile fashion with pulses occurring every 18-23 min. In the same system, a GnRH analogue had a stimulatory effect on HCG release while the effect of progesterone was inhibitory (Barnea and Kaplan, 1989). More recently, we found that epidermal growth factor stimulates HCG secretion in both static and superfusion cultures (Barnea et al., 1990). Ranta et al. (1980) studied the effect of long- and short-term hyperprolactinaemia on serum HCG levels early in human pregnancy and failed to find any changes compared with controls. 766
There is no information on whether oxytocin (OT) or argininevasopressin (AVP) affect HCG secretion. Both of these stressrelated hormones have not only a pituitary origin but also are produced by the ovary and especially the corpus luteum (Schaeffer et al., 1984). Active secretion of OT into the human ovarian vein during the luteal phase of the cycle has recently been demonstrated (Khan-Dawood et al., 1989). The aim of this study was to investigate the effect of physiological concentrations of the three stress-related hormones, OT, AVP and prolactin (PRL), on HCG secretion from human early trophoblast both in static and superfusion (dynamic) cultures. The present data show that these agents have a significant modulatory effect on HCG secretion in vitro.
Materials and methods Reagents OT, AVP and PRL were purchased from Sigma (St Louis, MO). Dulbecco's modified Eagle's medium (DMEM) was purchased from Beit Haemek (Israel). MAIAclone kits for measurements of HCG were obtained from Serono (Rehovot, Israel). The CAPOT receptor antagonist was received as a gift from Dr Per Melin, Ferring, Sweden. Placental material After obtaining appropriate consent, elective pregnancy terminations were carried out in the first trimester (7—10 weeks). The patients were healthy, on no medication and were non-smokers. Tissues collected were immediately placed into sterile 0.9% NaCl solution on ice. Explant preparation The method used was as previously described (Barnea and Fakih, 1985; Barnea et al., 1990). Briefly, explants, 5 0 - 7 0 mg wet weight, were dissected out and rinsed in 1 % antibiotic solution (penicillin, 10 000 U/ml, streptomycin 10 /tg/ml and amphotericin B, 25 jig/ml). For culture, explants were placed in DMEM medium with or without test agents. Six to 12 replicates per test agent or control (vehicle only) were plated at 37 °C in an atmosphere of 95% air and 5% CO2. After 24 h of incubation, the media were collected and stored at — 20°C until assay. The tissue was saved for protein analysis. In the preliminary experiments, we found that HCG secretion was linear up to 48 h; therefore in subsequent experiments effects of stress hormones were tested until 24 h at the half-maximal increase point. © Oxford University Press
Effect of stress hormones on HCG secretion
Supervision culture The method for placental superfusion was recently report! (Barnea and Kaplan, 1989; Barnea et al., 1990). Briefly, a supe fusion apparatus (ACCUSYST, Endotronics, St Paul, MN) wi a multichannel peristaltic pump and a fraction collector (ISC( model 272, Omaha, NE) were used to investigate the effect stress hormones on the short-term dynamics of HCG secretio Explants, 200-300 mg wet weight, were placed into the cultu chambers and a HEPES (18 mM)-DMEM solution was wash* through in an atmosphere of 95% air and 5% CO2 at 37°C Experiments were conducted over a 4 h period and the efflue was collected every 2.4 min (1 ml/sample). In each experimer one channel served as control and four served as experiment channels. At given intervals, a 1-min pulse of stress hormon was given through a peristaltic pump, equipped with a digit flowmeter (ISMATECH DD, Chicago, IL). Assay
control
pg/ml
Fig. 1. Effect of OT on HCG secretion in static culture. The black 3ar is the control: hatched bars are the study group explants. * = P < 0.01 compared with the control.
(3-HCG was measured using a commercially available radi immunoassay (RIA) kit with an intrassay variability of 1.7 (Barnea and Kaplan, 1989). All measurements were carried o in the same assay. Protein content was determined according the Lowry protein assay procedure (Lowry et al., 1951). Da 5 were expressed as mlU HCG/mg protein. I Statistics
50 O/vtocin
lack
100 -
i
Statistical analysis was performed using one-way analysis i variance (ANOVA) and Student's Mest, and P < 0.05 w; considered to be statistically significant. In superfusic experiments, the area under the graph was determined on personal computer using Simpson's rule. This program was ab to calculate a function that best fits the series of points we ha\ given, and then to determine the area under the curve using the integral of the function. Data of superfusion experiments represented a single channel run in duplicate in four different placentas yielding similar results. Data from the explants show the mean ± SEM of 4 - 6 individual placentas. In each placenta, 6-12 dishes per test agent or control group were run. Results Static cultures Figure 1 demonstrates the stimulatory effect of OT on HCG secretion by early placental explants. The 5 pg/mJ and 50 pg/mJ concentrations of OT increased HCG secretion significantly compared with controls, while at the low concentration of 0.5 pg/ml, OT had no significant effect, indicating a dosedependent response. AVP had a significant stimulatory effect on HCG secretion by placental explants in the 0.3-30 pg/ml range of concentrations (Figure 2). In the concentration range of 2-200 ng/ml PRL had no effect upon HCG secretion (data not shown). Superfusion Pulses of 5 pg/mJ of OT (the maximally effective concentration in static culture), given for 1 min, caused a rapid and significant
40 • 20
Fig. 2. Effect of AVP on HCG secretion in static culture. The black bar is the control; hatched bars are the study group explants. * = P < 0.01 compared with the control.
rise in HCG secretion in superfusion, which was two- to tenfold higher in amplitude than the baseline secretory pattern in either the OT or control channels (Figure 3A). Figure 3B, shows the blunting of HCG secretion from explants in superfusion induced by a 1-min pulse of 5 pg/ml OT when 10 pg/ml OT receptor antagonist was added simultaneously. Pulses of AVP, 3 pg/ml (the maximally effective concentration in static culture), given for 1 min induced a significant rise in HCG levels. However, this was evident only with every second pulse, as if a lag-time was needed for the explants to recover and respond (Figure 4). The amplitude of the induced HCG peaks was two- to five-fold higher than in the control channel. Figure 5 shows a gradually developing inhibitory effect on pulsatile HCG secretion as pulses of prolactin, 20 ng/ml were given every 30 min for 1 min. This became evident by the third pulse. Discussion Stress influences various reproductive functions and a common example is hypothalamic amenorrhoea. Prolactin secretion from the anterior pituitary is increased during stress (Speroff et al., 767
J.Tal el at.
A
B 20-, 1816-
Q_
12-
200c
14-
• oxytocin ± antagonist
150-
E
10-
100-
13
I
6-
hCG
E o o SI
• control
•
cn
en
E
•
Stress-related hormones affect human chorionic gonadotrophin secretion from the early human placenta in vitro
Joseph Tal2, Marielle Kaplan1, Mordechai Sharf2 and Eytan R.Barnea1 Feto-Placental Endocrine Unit, Rappaport Institute, Technion and Department of Ob/Gyn Bnai-Zion Medical Centre, Haifa, Technion, Israel 2
'To whom correspondence should be addressed at: Feto-Placental Endocrine Unit, Rappaport Institute, Technion, PO Box 9697 Rh Efron, Haifa 34601, Israel
The effect of a physiological range of concentrations of three stress-related hormones, oxytocin (OT), arginine-vasopressin (AVP) and prolactin (PRL) was tested upon human chorionic gonadotrophin (HCG) secretion by placental explants from early pregnancy in static and supervision cultures. In static cultures, OT and AVP significantly increased HCG secretion, whereas PRL had no effect. In superfusion, 1-min pulses of OT induced a significant (two- to 10-fold) rise in HCG pulse amplitude compared to the control. This effect of this neuropeptide was blocked by coadministration of a specific receptor antagonist. AVP also increased the glycoprotein pulse amplitude by two- to five-fold, but only with every second pulse administered. PRL pulses caused a progressive inhibition of spontaneous HCG pulsatility. In conclusion, stressrelated hormones affect placental HCG secretion in vitro. The involvement of these factors in impairing early pregnancy development is suggested. Key words: HCG/oxytocin/prolactin/superfusion/vasopressin Introduction A gonadotrophin releasing hormone (GnRH)-like compound has been identified in the human placenta (Khodr and Siler-Khodr, 1978). It is believed to act on receptors present on the syncytiotrophoblast cell membrane, stimulating human chorionic gonadotrophin (HCG) secretion. Episodic HCG secretion has been demonstrated in the maternal circulation during early pregnancy (Owens et al., 1981). We have found that supervised human placental explants secrete HCG in a pulsatile fashion with pulses occurring every 18-23 min. In the same system, a GnRH analogue had a stimulatory effect on HCG release while the effect of progesterone was inhibitory (Barnea and Kaplan, 1989). More recently, we found that epidermal growth factor stimulates HCG secretion in both static and superfusion cultures (Barnea et al., 1990). Ranta et al. (1980) studied the effect of long- and short-term hyperprolactinaemia on serum HCG levels early in human pregnancy and failed to find any changes compared with controls. 766
There is no information on whether oxytocin (OT) or argininevasopressin (AVP) affect HCG secretion. Both of these stressrelated hormones have not only a pituitary origin but also are produced by the ovary and especially the corpus luteum (Schaeffer et al., 1984). Active secretion of OT into the human ovarian vein during the luteal phase of the cycle has recently been demonstrated (Khan-Dawood et al., 1989). The aim of this study was to investigate the effect of physiological concentrations of the three stress-related hormones, OT, AVP and prolactin (PRL), on HCG secretion from human early trophoblast both in static and superfusion (dynamic) cultures. The present data show that these agents have a significant modulatory effect on HCG secretion in vitro.
Materials and methods Reagents OT, AVP and PRL were purchased from Sigma (St Louis, MO). Dulbecco's modified Eagle's medium (DMEM) was purchased from Beit Haemek (Israel). MAIAclone kits for measurements of HCG were obtained from Serono (Rehovot, Israel). The CAPOT receptor antagonist was received as a gift from Dr Per Melin, Ferring, Sweden. Placental material After obtaining appropriate consent, elective pregnancy terminations were carried out in the first trimester (7—10 weeks). The patients were healthy, on no medication and were non-smokers. Tissues collected were immediately placed into sterile 0.9% NaCl solution on ice. Explant preparation The method used was as previously described (Barnea and Fakih, 1985; Barnea et al., 1990). Briefly, explants, 5 0 - 7 0 mg wet weight, were dissected out and rinsed in 1 % antibiotic solution (penicillin, 10 000 U/ml, streptomycin 10 /tg/ml and amphotericin B, 25 jig/ml). For culture, explants were placed in DMEM medium with or without test agents. Six to 12 replicates per test agent or control (vehicle only) were plated at 37 °C in an atmosphere of 95% air and 5% CO2. After 24 h of incubation, the media were collected and stored at — 20°C until assay. The tissue was saved for protein analysis. In the preliminary experiments, we found that HCG secretion was linear up to 48 h; therefore in subsequent experiments effects of stress hormones were tested until 24 h at the half-maximal increase point. © Oxford University Press
Effect of stress hormones on HCG secretion
Supervision culture The method for placental superfusion was recently report! (Barnea and Kaplan, 1989; Barnea et al., 1990). Briefly, a supe fusion apparatus (ACCUSYST, Endotronics, St Paul, MN) wi a multichannel peristaltic pump and a fraction collector (ISC( model 272, Omaha, NE) were used to investigate the effect stress hormones on the short-term dynamics of HCG secretio Explants, 200-300 mg wet weight, were placed into the cultu chambers and a HEPES (18 mM)-DMEM solution was wash* through in an atmosphere of 95% air and 5% CO2 at 37°C Experiments were conducted over a 4 h period and the efflue was collected every 2.4 min (1 ml/sample). In each experimer one channel served as control and four served as experiment channels. At given intervals, a 1-min pulse of stress hormon was given through a peristaltic pump, equipped with a digit flowmeter (ISMATECH DD, Chicago, IL). Assay
control
pg/ml
Fig. 1. Effect of OT on HCG secretion in static culture. The black 3ar is the control: hatched bars are the study group explants. * = P < 0.01 compared with the control.
(3-HCG was measured using a commercially available radi immunoassay (RIA) kit with an intrassay variability of 1.7 (Barnea and Kaplan, 1989). All measurements were carried o in the same assay. Protein content was determined according the Lowry protein assay procedure (Lowry et al., 1951). Da 5 were expressed as mlU HCG/mg protein. I Statistics
50 O/vtocin
lack
100 -
i
Statistical analysis was performed using one-way analysis i variance (ANOVA) and Student's Mest, and P < 0.05 w; considered to be statistically significant. In superfusic experiments, the area under the graph was determined on personal computer using Simpson's rule. This program was ab to calculate a function that best fits the series of points we ha\ given, and then to determine the area under the curve using the integral of the function. Data of superfusion experiments represented a single channel run in duplicate in four different placentas yielding similar results. Data from the explants show the mean ± SEM of 4 - 6 individual placentas. In each placenta, 6-12 dishes per test agent or control group were run. Results Static cultures Figure 1 demonstrates the stimulatory effect of OT on HCG secretion by early placental explants. The 5 pg/mJ and 50 pg/mJ concentrations of OT increased HCG secretion significantly compared with controls, while at the low concentration of 0.5 pg/ml, OT had no significant effect, indicating a dosedependent response. AVP had a significant stimulatory effect on HCG secretion by placental explants in the 0.3-30 pg/ml range of concentrations (Figure 2). In the concentration range of 2-200 ng/ml PRL had no effect upon HCG secretion (data not shown). Superfusion Pulses of 5 pg/mJ of OT (the maximally effective concentration in static culture), given for 1 min, caused a rapid and significant
40 • 20
Fig. 2. Effect of AVP on HCG secretion in static culture. The black bar is the control; hatched bars are the study group explants. * = P < 0.01 compared with the control.
rise in HCG secretion in superfusion, which was two- to tenfold higher in amplitude than the baseline secretory pattern in either the OT or control channels (Figure 3A). Figure 3B, shows the blunting of HCG secretion from explants in superfusion induced by a 1-min pulse of 5 pg/ml OT when 10 pg/ml OT receptor antagonist was added simultaneously. Pulses of AVP, 3 pg/ml (the maximally effective concentration in static culture), given for 1 min induced a significant rise in HCG levels. However, this was evident only with every second pulse, as if a lag-time was needed for the explants to recover and respond (Figure 4). The amplitude of the induced HCG peaks was two- to five-fold higher than in the control channel. Figure 5 shows a gradually developing inhibitory effect on pulsatile HCG secretion as pulses of prolactin, 20 ng/ml were given every 30 min for 1 min. This became evident by the third pulse. Discussion Stress influences various reproductive functions and a common example is hypothalamic amenorrhoea. Prolactin secretion from the anterior pituitary is increased during stress (Speroff et al., 767
J.Tal el at.
A
B 20-, 1816-
Q_
12-
200c
14-
• oxytocin ± antagonist
150-
E
10-
100-
13
I
6-
hCG
E o o SI
• control
•
cn
en
E
•
