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In vitro secretory patterns of human chorionic gonadotrophin, placental lactogen and pregnancy-specific β1-glycoprotein
Placenta (I985) , 6, 4 1 7 - 4 2 2
In Vitro Secretory Patterns of Human Chorionic Gonadotrophin, Placental Lactogen and Pregnancy-specific/31-Glycoprotein JANET WINIKOFF & GLENN D. B R A U N S T E I N a Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, 87oo Beverly Boulevard, Los Angeles, CA 9oo48, USA a To whom correspondence should be addressed
INTRODUCTION The control of secretion of the placental protein hormones, human chorionic gonadotrophin (hCG) and human placental lactogen (hPL), and the trophoblastic protein, pregnancy-specific /~l-glycoprotein (SP1) , is not well understood. Unlike the situation for most other large protein hormones, no primary regulatory substance or trophic hormone has been clearly identified for the placental protein hormones. In spite of this, there is some evidence that there is specific regulation of each of these proteins. Secretion of hCG, which is first detected in the maternal circulation approximately six to ten days after fertilization, undergoes an initial rapid rise until it peaks at eight to ten weeks of gestation, then falls to a nadir at about i8 weeks and subsequently plateaus for the duration of pregnancy (Braunstein et al, I976). This occurs in spite of a steady increase in trophoblastic mass, so that it is evident that tissue secretion of hCG is not merely a passive process. In contrast to hCG, hPL and SP 1 levels in serum increase steadily throughout pregnancy and appear to be related to trophoblastic mass (Braunstein et al, 198o). The purpose of this study was to see whether these relationships between hCG, hPL and SP1 observed in vivo are also seen with placental explant cultures in vitro. MATERIALS AND METHODS Placental explants Placental explants were prepared using sterile techniques from placentae obtained following normal vaginal delivery (n = I) or uncomplicated caesarean sections (n = 2). Decidual tissue was dissected from the maternal surface of the placenta, and sections of trophoblastic tissue, approximately one centimetre in thickness, were removed from multiple sites, placed in iced saline in the delivery room, and transferred to the laboratory. T h e tissue was washed until all visible blood and debris had been removed. The tissue sections were divided into explants weighing approximately 75 to I25 mg each (mean = 91.8 mg + 24.2 s.d.) and placed in a Petri dish containing medium MI99 and pre-incubated for several hours at 37~ in 95 per cent air, 5 per cent CO 2. Fifiy-millilitre Falcon flasks were filled with 5 ml of media and weighed. Individual explants were removed from the preincubation dish, blotted dry, and placed in an 417
418
J. Winikoff, G. D. Braunstein
appropriate flask which was reweighed. The difference between the final weight and the initial weight of the flask plus medium was taken as the weight of the tissue explant. Incubation conditions Medium MI99 (Irvine Scientific, Irvine, CA) with io per cent fetal calf serum and antibiotics was used for all experiments. Some explants were cultured in freshly prepared solutions of M 199 containing dibutyryl adenosine 3'5'-monophosphate (dbcAMP) and theophylline, each at a final concentration of ~ mM. Five to twelve explants were used for each treatment in each experiment. The medium in each flask was renewed daily, and the spent medium was collected and frozen for subsequent assay for hCG, hPL and SP r All samples were incubated at 37 ~ in a humidified atmosphere of 95 per cent air, 5 per cent COg. For all experiments, some explants were placed in sealed flasks and frozen or kept at room temperature for the duration of the experiments and used as controls in the viability studies. Viability The viability of each sample was assessed by the incorporation into protein of 3H-leucine (SA = 58 Ci/mole, ICN Radiochemicals, Irvine, CA). At the conclusion of each study, the explants were incubated with 3H-leucine for 3 to 24 h, after which they were rinsed in saline and homogenized in cold I o per cent trichloroacetic acid. The homogenate was collected and rinsed twice with cold trichloroacetic acid and twice with 95 per cent ethanol on a o.45 ~m MiUipore filter. The filters which were covered with the homogenized tissue were placed in i o ml of Bray's solution and counted in a Beckman LS-35o beta scintillation counter. Placental samples which had been frozen at the beginning of the experiments were thawed and, together with the explants, kept at room temperature to serve as non-viable control tissue. These were treated in a manner similar to that for the experimental explants, and they incorporated negligible amounts of 3H-leucine. Radioimmunoassays HCG, hPL and SP 1 were measured in previously described double antibody radioimmunoassays (Braunstein et al, I98O). Medium MI99 containing io per cent fetal calf serum either alone or with theophylline and dbcAMP did not interfere with these assays. Calculations and statistical analysis The amount of hormone produced daily per gram of tissue was calculated as the product of the volume of media (5 ml) times the measured concentration of hormone divided by the tissue weight in grams. The mean daily hormone production per gram of tissue was calculated by averaging the results each day of all the explants within each experimental condition. The statistical model employed to analyse the data was a mixed model analysis of variance with repeated measures. Within this model, explants were viewed as being nested within treatments (stimulated or control) and within placentae and crossed with time (the repeated measure factor). Additionally, both placentae and explants were treated as random factors with treatment and time being fixed (Winer, I97Q. The variance assumptions required by this statistical model were satisfied by taking the logs (base Io) of the dependent variables, hCG, hPL and SP r The data were analysed using the Statistical Analysis System's general linear model procedure with the type I I I sums of squares option (SAS Institute, Inc, Cary, NC). Analysis of variance effects found to be significant were further analysed using Scheffe's multiple comparison procedure (Winer, i97i ). For all analyses, the o.o5 level of significance was employed.
419
Placental Proteins in Vitro
RESULTS At the end of the incubation periods, explants from each of the three placentae used for this study incorporated into protein at least ten times the amount of 3H-leucine compared with that incorporated by the non-viable control samples. The quantities of hCG, hPL and SP~ per gram of tissue released into the media by explants from three separate placentae at 24, 48 and 72 h of culture, respectively, are presented in Figure i. The levels of hPL and SP~ did not differ between the control or dbcAMP and theophylline stimulated explants at any of the time periods. The hPL and SP 1 concentrations in the media for both the control and stimulated samples decrease significantly with time. In contrast, the hCG secretion into the media from the stimulated explants consistently increased with time. The hCG secretion from the control, unstimulated samples decreased at 48 hours and rose at 72 hours, but remained below the level found at 24 hours. The differences in hCG concentrations between the dbcAMP-stimulated and the control tissues were significant at 48 and 72 hours. There was no loss of immunoreactive hCG, hPL or S P x incubated in medium alone under identical conditions.
DISCUSSION We have shown that the secretion of hCG by placental explants is stimulated by dbcAMP while the secretion of the other placental proteins, hPL and SP1, is not. This suggests that the regulation of hCG secretion is independent of the secretion of hPL and SP v Together with the in vivo data showing that there is a different secretory profile throughout pregnancy for hCG in comparison with the other two proteins (Braunstein et al, 198o), these results support the concept that there are independent physiological regulators for the placental protein hormones. Various substances have been evaluated for their effects on hCG and hPL regulation. These include gonadotrophin-releasing hormone (Khodr and Siler-Khodr, 1978; Rubinstein et al, I978; Butzow, I982; Haning et al, i982a , i982b), dopamine (Macaron et al, 1979; Shu-Rong et al, 1982), epidermal growth factor (Huot et al, I98I), prostaglandins (Genbacev et al, I977; Belleville et al, 1978; Haning et al, I982a, i982b) and progestogens (Wilson, Jawad and Dickson, 198o). In many instances the results have been found only at pharmacological levels of the test substance. The agents which have been shown consistently to stimulate the secretion ofhCG are the derivatives of cyclic AMP and the xanthines (Handwerger et al, 1973; Golander et al, i978 ). Handwerger and associates (1973) and Golander et al (1978) have previously shown that dbcAMP with theophylline causes an increment in hCG without a change in hPL secretion. The present study has extended these observations by showing that SP, is secreted in vitro like hPL but unlike hCG. The viability of our explants was demonstrated by their ability to incorporate 3H-leucine into protein and by the stimulatory response of hCG secretion to dbcAMP and theophylline. This suggests that artefactual leaking of the proteins into the media, due to cell membrane damage, is unlikely. Our data also confirm the work of Golander et al (I 978). They showed that while the amount ofhPL secreted into the media decreased daily, the amount ofhCG secreted into media without dbcAMP or theophylline declined initially and then increased between 24 and 48 hours. This pattern was seen in two of the three placentae described in this study and in three of four additional placental explant experiments carried out under identical conditions. Golander and coworkers (1978) hypothesized that term placental tissue contains an inhibitor of hCG release that is removed in the washes and media" changes in vitro. In parallel experiments we were unable to demonstrate the presence of an inhibitor of placental explant hCG secretion in either
J. Winikoff, G. D. Braunstein
4z o
hCG
3,000-
x<
1,000-
E
500 -
u3
~\\ ~
s~s
100-
50 15,000-
sP~
IO,000-
E
5,000 -
1,500 300,000-
100,000 -
50,000E
10,000-
5,000 214
48
I
72
HOURS OF INCUBATION
Figure 1. Time course of hCG, SP 1 and hPL secretion in vitro. Explants were cultured in MI99 alone ( 9 or Mi99 containing ] mM dbcAMP and i mM theophylline (Q). HCG is shown in the upper panel, SP1 in the middle panel, and hPL in the lower panel. Media (5 ml) were collected and renewed daily, the amount of each hormone measured and expressed as ng/5 ml/gram of placental tissue. Each point presents the average value obtained from three placentae, using at least five explants per placenta for each experimental condition (either stimulated or unstimulated). Bars represent the standard deviations of the log normalized means. Note the difference in scale of the ordinate for the three graphs. * = significantly different from control at P < 0.05.
Placental Proteins in Vitro
42i
first- or third-trimester pregnancy serum which had been stripped of their glycoproteins (including h C G and SP1) by affinity chromatography on concanavalin-A-Sepharose (Winikoff and Braunstein unpublished observations). However, this does not negate the possibility that placental tissue contains a local inhibitor of h C G secretion, or that the inhibitor may be a glycoprotein.
SUMMARY The control of secretion of the placental hormones human chorionic gonadotrophin (hCG) and human placental lactogen (hPL), and the trophoblastic protein pregnancy-specific ]~-glycoprotein (SP~), is not well understood. During pregnancy, the h C G concentrations peak in the first trimester then decrease, while h P L and SP 1increase steadily throughout gestation. In order to determine whether the discordance between h C G secretion and that of hPL and SP a observed in vivo also occur in vitro, we cultured placental explants with and without dibutyryl cyclic A M P (dbcAMP) and theophylline. Between 5 and 12 explants were used for each treatment in each experiment. T h e concentration of the proteins secreted into the media each day was measured by specific radioimmunoassays. T h e quantities of hPL and SP 1 secreted per day declined in a parallel fashion after 24 hours under both basal and dbcAMP-stimulated conditions. T h e hCG output progressively decreased in the unstimulated cultures until 48 hours, at which time an increase in h C G secretion was observed. The dbcAMP-stimulated placentae significantly increased their h C G output at both 4 8 and 72 hours. These data show that hCG secretion is regulated differently from that o f h P L and SP~. T h e results do not negate the possibility that term placental tissue may contain an inhibitor o f h C G release that is removed by experimental manipulation in vitro.
ACKNOWLEDGEMENTS This work was supported in part by an American BioScience Endocrine Fellowship and USPHS Training Grant 5T32AMO4726. The expert technical assistanceof Judith Seliktar and Lynn Kelley, and the helpful suggestionsof Shlomo Melmed, MD, are greatly appreciated. REFERENCES Belleville, F., Lasbemaes, A., Nabet, P. & Paysant, P. (x978) HCS-HCG regulation in cultured placenta. Acta Endocrinologia, 88, I69-xSx.
Braunstein, G. D., Rasor, J. L., Engvall, E. & Wade, M. E. (198o) Interrelationships of human chorionic gonadotropin, human placental lactogen, and pregnancy-specific-B-*-glycoproteinthroughout normal human gestation. American Journal of Obstetrics and Gynecology, I38, Izo5-I213. Bratmstein, G. D., Rasor, J., Adler, D. et al (x976) Serum human chorionicgonadotropinlevelsthroughout normal pregnancy. American Journal of Obstetrics and Gynecology, Iz6, 678-4~I. Butzow, R. (x98u)Luteinizing hormone-releasingfactor increasesreleaseof human chorionicgonadotropinin isolated cell columns of normal and malignant trophoblasts. International Journal of Cancer, 29, 9-I i. Genbacev, O., Ratkovic, M., Kraincanic, M. & Sulovic, V. (x977) Effect of prostaglandin in PGE2 alpha on the synthesis of placental proteins and human placental lactogen (HPL). Prostaglandins, I3, 723-733. Golander, A., Barrett, J. R., Tyrey, L. et al (1978) Differentialsynthesisof human placental lactogen and human chorionic gonadotropin in vitro. Endocrinology, Ioa, 597-6o5. Handwerger, S., Barrett, J., Tyrey, L. & Schomberg, D. (x973) Differential effect of cyclic adenosine monophosphateon the secretionof human placental lactogenand human chorionicgonadotropin.Journal o]'Climcal Endocrinology and Metabolism, 36, x268-I27o. Haning, R. V. Jr, Choi, L., Kiggens, A. J. et al (I98za) Effects of dibutyryl adenosine 3'5'-monophosphate, luteinizing hormone-releasinghormone and aromatase inhibitor on simultaneous outputs of progesterone 17Bestradiol, and hCG by term placental explants. Journal of Clinical Endocrinology and Metabolism, 55, z l 3-218.
422
~. Winikoff, G. D. Braunstein
Haning, R. V. Jr, Choi, L., Kiggens, A. J. et al (t98zb) Effects of dibutyryl cAMP, LHRH, and aromatase inhibitor on simultaneous outputs of prostaglandins Fa, and 13,x4-dihydro-i5-keto-prostaglandin F 2 by term placental extracts. Prostaglandins, a3, 29--4o. Huot, R. !., Foidart, J.-M., Nardone, R. M. & Stromberg, K. 098 0 Differential modulation of human chorionic gonadotropin secretion by epidermal growth factor in normal and malignant placental cultures. Journal of Clinical Endocrinology and Metabolism, 53, lO59-I~ Khodr, G. & Siler-Khodr, T. M. (I978) Effect of iuteinizing hormone-releasing factor on human chorionic gonadotropin secretion. Fertility and Sterility, 30, 3Ol-3O4. Macaron, C., Kynci, M., Famuyiwa, O. et al 0979) In vitro effect of dopamine and pimozide on human chorionic gonadotropin secretion. AmericanJournal of Obstetrics and Gynecology, i35 , 4~9--5o2. Rubinstein, L. M., Pariow, A. F., Derzko, C. & Hershman, J. (1978) Pituitary gnnadotropin response to LHRH in human pregnancy. Obstetrics and Gynecology, 52, 172-175. Shu-Rong, Z., Bremme, K., Eneroth, P. & Nordberg, A. (1982) The regulation in vitro of placental releas6 of human chorionic gonadotropin, placental lactogen, and prolactin: Effects of an adrenergic beta-receptor agonist and antagonist. American Journal of Obstetrics and Gynecology, 143, 444-45o. Wilson, E., Jawad, M. J. & Dickson, L. R. (198o) Suppression of human chorionic gonadotropin by progestational steroids. AmericanJournal of Obstetrics and Gynecology, 138, 7o8-7i 3. Winer, B. J. (197I) Statistical Principles in Experimental Dessgn. and Edition. New York: McGraw-Hill.
In Vitro Secretory Patterns of Human Chorionic Gonadotrophin, Placental Lactogen and Pregnancy-specific/31-Glycoprotein JANET WINIKOFF & GLENN D. B R A U N S T E I N a Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, 87oo Beverly Boulevard, Los Angeles, CA 9oo48, USA a To whom correspondence should be addressed
INTRODUCTION The control of secretion of the placental protein hormones, human chorionic gonadotrophin (hCG) and human placental lactogen (hPL), and the trophoblastic protein, pregnancy-specific /~l-glycoprotein (SP1) , is not well understood. Unlike the situation for most other large protein hormones, no primary regulatory substance or trophic hormone has been clearly identified for the placental protein hormones. In spite of this, there is some evidence that there is specific regulation of each of these proteins. Secretion of hCG, which is first detected in the maternal circulation approximately six to ten days after fertilization, undergoes an initial rapid rise until it peaks at eight to ten weeks of gestation, then falls to a nadir at about i8 weeks and subsequently plateaus for the duration of pregnancy (Braunstein et al, I976). This occurs in spite of a steady increase in trophoblastic mass, so that it is evident that tissue secretion of hCG is not merely a passive process. In contrast to hCG, hPL and SP 1 levels in serum increase steadily throughout pregnancy and appear to be related to trophoblastic mass (Braunstein et al, 198o). The purpose of this study was to see whether these relationships between hCG, hPL and SP1 observed in vivo are also seen with placental explant cultures in vitro. MATERIALS AND METHODS Placental explants Placental explants were prepared using sterile techniques from placentae obtained following normal vaginal delivery (n = I) or uncomplicated caesarean sections (n = 2). Decidual tissue was dissected from the maternal surface of the placenta, and sections of trophoblastic tissue, approximately one centimetre in thickness, were removed from multiple sites, placed in iced saline in the delivery room, and transferred to the laboratory. T h e tissue was washed until all visible blood and debris had been removed. The tissue sections were divided into explants weighing approximately 75 to I25 mg each (mean = 91.8 mg + 24.2 s.d.) and placed in a Petri dish containing medium MI99 and pre-incubated for several hours at 37~ in 95 per cent air, 5 per cent CO 2. Fifiy-millilitre Falcon flasks were filled with 5 ml of media and weighed. Individual explants were removed from the preincubation dish, blotted dry, and placed in an 417
418
J. Winikoff, G. D. Braunstein
appropriate flask which was reweighed. The difference between the final weight and the initial weight of the flask plus medium was taken as the weight of the tissue explant. Incubation conditions Medium MI99 (Irvine Scientific, Irvine, CA) with io per cent fetal calf serum and antibiotics was used for all experiments. Some explants were cultured in freshly prepared solutions of M 199 containing dibutyryl adenosine 3'5'-monophosphate (dbcAMP) and theophylline, each at a final concentration of ~ mM. Five to twelve explants were used for each treatment in each experiment. The medium in each flask was renewed daily, and the spent medium was collected and frozen for subsequent assay for hCG, hPL and SP r All samples were incubated at 37 ~ in a humidified atmosphere of 95 per cent air, 5 per cent COg. For all experiments, some explants were placed in sealed flasks and frozen or kept at room temperature for the duration of the experiments and used as controls in the viability studies. Viability The viability of each sample was assessed by the incorporation into protein of 3H-leucine (SA = 58 Ci/mole, ICN Radiochemicals, Irvine, CA). At the conclusion of each study, the explants were incubated with 3H-leucine for 3 to 24 h, after which they were rinsed in saline and homogenized in cold I o per cent trichloroacetic acid. The homogenate was collected and rinsed twice with cold trichloroacetic acid and twice with 95 per cent ethanol on a o.45 ~m MiUipore filter. The filters which were covered with the homogenized tissue were placed in i o ml of Bray's solution and counted in a Beckman LS-35o beta scintillation counter. Placental samples which had been frozen at the beginning of the experiments were thawed and, together with the explants, kept at room temperature to serve as non-viable control tissue. These were treated in a manner similar to that for the experimental explants, and they incorporated negligible amounts of 3H-leucine. Radioimmunoassays HCG, hPL and SP 1 were measured in previously described double antibody radioimmunoassays (Braunstein et al, I98O). Medium MI99 containing io per cent fetal calf serum either alone or with theophylline and dbcAMP did not interfere with these assays. Calculations and statistical analysis The amount of hormone produced daily per gram of tissue was calculated as the product of the volume of media (5 ml) times the measured concentration of hormone divided by the tissue weight in grams. The mean daily hormone production per gram of tissue was calculated by averaging the results each day of all the explants within each experimental condition. The statistical model employed to analyse the data was a mixed model analysis of variance with repeated measures. Within this model, explants were viewed as being nested within treatments (stimulated or control) and within placentae and crossed with time (the repeated measure factor). Additionally, both placentae and explants were treated as random factors with treatment and time being fixed (Winer, I97Q. The variance assumptions required by this statistical model were satisfied by taking the logs (base Io) of the dependent variables, hCG, hPL and SP r The data were analysed using the Statistical Analysis System's general linear model procedure with the type I I I sums of squares option (SAS Institute, Inc, Cary, NC). Analysis of variance effects found to be significant were further analysed using Scheffe's multiple comparison procedure (Winer, i97i ). For all analyses, the o.o5 level of significance was employed.
419
Placental Proteins in Vitro
RESULTS At the end of the incubation periods, explants from each of the three placentae used for this study incorporated into protein at least ten times the amount of 3H-leucine compared with that incorporated by the non-viable control samples. The quantities of hCG, hPL and SP~ per gram of tissue released into the media by explants from three separate placentae at 24, 48 and 72 h of culture, respectively, are presented in Figure i. The levels of hPL and SP~ did not differ between the control or dbcAMP and theophylline stimulated explants at any of the time periods. The hPL and SP 1 concentrations in the media for both the control and stimulated samples decrease significantly with time. In contrast, the hCG secretion into the media from the stimulated explants consistently increased with time. The hCG secretion from the control, unstimulated samples decreased at 48 hours and rose at 72 hours, but remained below the level found at 24 hours. The differences in hCG concentrations between the dbcAMP-stimulated and the control tissues were significant at 48 and 72 hours. There was no loss of immunoreactive hCG, hPL or S P x incubated in medium alone under identical conditions.
DISCUSSION We have shown that the secretion of hCG by placental explants is stimulated by dbcAMP while the secretion of the other placental proteins, hPL and SP1, is not. This suggests that the regulation of hCG secretion is independent of the secretion of hPL and SP v Together with the in vivo data showing that there is a different secretory profile throughout pregnancy for hCG in comparison with the other two proteins (Braunstein et al, 198o), these results support the concept that there are independent physiological regulators for the placental protein hormones. Various substances have been evaluated for their effects on hCG and hPL regulation. These include gonadotrophin-releasing hormone (Khodr and Siler-Khodr, 1978; Rubinstein et al, I978; Butzow, I982; Haning et al, i982a , i982b), dopamine (Macaron et al, 1979; Shu-Rong et al, 1982), epidermal growth factor (Huot et al, I98I), prostaglandins (Genbacev et al, I977; Belleville et al, 1978; Haning et al, I982a, i982b) and progestogens (Wilson, Jawad and Dickson, 198o). In many instances the results have been found only at pharmacological levels of the test substance. The agents which have been shown consistently to stimulate the secretion ofhCG are the derivatives of cyclic AMP and the xanthines (Handwerger et al, 1973; Golander et al, i978 ). Handwerger and associates (1973) and Golander et al (1978) have previously shown that dbcAMP with theophylline causes an increment in hCG without a change in hPL secretion. The present study has extended these observations by showing that SP, is secreted in vitro like hPL but unlike hCG. The viability of our explants was demonstrated by their ability to incorporate 3H-leucine into protein and by the stimulatory response of hCG secretion to dbcAMP and theophylline. This suggests that artefactual leaking of the proteins into the media, due to cell membrane damage, is unlikely. Our data also confirm the work of Golander et al (I 978). They showed that while the amount ofhPL secreted into the media decreased daily, the amount ofhCG secreted into media without dbcAMP or theophylline declined initially and then increased between 24 and 48 hours. This pattern was seen in two of the three placentae described in this study and in three of four additional placental explant experiments carried out under identical conditions. Golander and coworkers (1978) hypothesized that term placental tissue contains an inhibitor of hCG release that is removed in the washes and media" changes in vitro. In parallel experiments we were unable to demonstrate the presence of an inhibitor of placental explant hCG secretion in either
J. Winikoff, G. D. Braunstein
4z o
hCG
3,000-
x<
1,000-
E
500 -
u3
~\\ ~
s~s
100-
50 15,000-
sP~
IO,000-
E
5,000 -
1,500 300,000-
100,000 -
50,000E
10,000-
5,000 214
48
I
72
HOURS OF INCUBATION
Figure 1. Time course of hCG, SP 1 and hPL secretion in vitro. Explants were cultured in MI99 alone ( 9 or Mi99 containing ] mM dbcAMP and i mM theophylline (Q). HCG is shown in the upper panel, SP1 in the middle panel, and hPL in the lower panel. Media (5 ml) were collected and renewed daily, the amount of each hormone measured and expressed as ng/5 ml/gram of placental tissue. Each point presents the average value obtained from three placentae, using at least five explants per placenta for each experimental condition (either stimulated or unstimulated). Bars represent the standard deviations of the log normalized means. Note the difference in scale of the ordinate for the three graphs. * = significantly different from control at P < 0.05.
Placental Proteins in Vitro
42i
first- or third-trimester pregnancy serum which had been stripped of their glycoproteins (including h C G and SP1) by affinity chromatography on concanavalin-A-Sepharose (Winikoff and Braunstein unpublished observations). However, this does not negate the possibility that placental tissue contains a local inhibitor of h C G secretion, or that the inhibitor may be a glycoprotein.
SUMMARY The control of secretion of the placental hormones human chorionic gonadotrophin (hCG) and human placental lactogen (hPL), and the trophoblastic protein pregnancy-specific ]~-glycoprotein (SP~), is not well understood. During pregnancy, the h C G concentrations peak in the first trimester then decrease, while h P L and SP 1increase steadily throughout gestation. In order to determine whether the discordance between h C G secretion and that of hPL and SP a observed in vivo also occur in vitro, we cultured placental explants with and without dibutyryl cyclic A M P (dbcAMP) and theophylline. Between 5 and 12 explants were used for each treatment in each experiment. T h e concentration of the proteins secreted into the media each day was measured by specific radioimmunoassays. T h e quantities of hPL and SP 1 secreted per day declined in a parallel fashion after 24 hours under both basal and dbcAMP-stimulated conditions. T h e hCG output progressively decreased in the unstimulated cultures until 48 hours, at which time an increase in h C G secretion was observed. The dbcAMP-stimulated placentae significantly increased their h C G output at both 4 8 and 72 hours. These data show that hCG secretion is regulated differently from that o f h P L and SP~. T h e results do not negate the possibility that term placental tissue may contain an inhibitor o f h C G release that is removed by experimental manipulation in vitro.
ACKNOWLEDGEMENTS This work was supported in part by an American BioScience Endocrine Fellowship and USPHS Training Grant 5T32AMO4726. The expert technical assistanceof Judith Seliktar and Lynn Kelley, and the helpful suggestionsof Shlomo Melmed, MD, are greatly appreciated. REFERENCES Belleville, F., Lasbemaes, A., Nabet, P. & Paysant, P. (x978) HCS-HCG regulation in cultured placenta. Acta Endocrinologia, 88, I69-xSx.
Braunstein, G. D., Rasor, J. L., Engvall, E. & Wade, M. E. (198o) Interrelationships of human chorionic gonadotropin, human placental lactogen, and pregnancy-specific-B-*-glycoproteinthroughout normal human gestation. American Journal of Obstetrics and Gynecology, I38, Izo5-I213. Bratmstein, G. D., Rasor, J., Adler, D. et al (x976) Serum human chorionicgonadotropinlevelsthroughout normal pregnancy. American Journal of Obstetrics and Gynecology, Iz6, 678-4~I. Butzow, R. (x98u)Luteinizing hormone-releasingfactor increasesreleaseof human chorionicgonadotropinin isolated cell columns of normal and malignant trophoblasts. International Journal of Cancer, 29, 9-I i. Genbacev, O., Ratkovic, M., Kraincanic, M. & Sulovic, V. (x977) Effect of prostaglandin in PGE2 alpha on the synthesis of placental proteins and human placental lactogen (HPL). Prostaglandins, I3, 723-733. Golander, A., Barrett, J. R., Tyrey, L. et al (1978) Differentialsynthesisof human placental lactogen and human chorionic gonadotropin in vitro. Endocrinology, Ioa, 597-6o5. Handwerger, S., Barrett, J., Tyrey, L. & Schomberg, D. (x973) Differential effect of cyclic adenosine monophosphateon the secretionof human placental lactogenand human chorionicgonadotropin.Journal o]'Climcal Endocrinology and Metabolism, 36, x268-I27o. Haning, R. V. Jr, Choi, L., Kiggens, A. J. et al (I98za) Effects of dibutyryl adenosine 3'5'-monophosphate, luteinizing hormone-releasinghormone and aromatase inhibitor on simultaneous outputs of progesterone 17Bestradiol, and hCG by term placental explants. Journal of Clinical Endocrinology and Metabolism, 55, z l 3-218.
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~. Winikoff, G. D. Braunstein
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