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Cells of the choriocarcinoma cell line (JAr) in co-culture with cytotrophoblasts increase the synthesis of CG and hPL
384
Placenta(1991), Vol. 12
significantly different, yet the levels in the umbilical vessels (artery and vein) are nearly the same. In about two-thirds of the cases, the fetal EPO levels were higher than the maternal ones. In the 47 cases with higher maternal levels, there was no significant difference between the umbilical vein and the umbilical artery EPO levels. Although only an indirect indication, these results provide strong evidence that there is a placental barrier to the transfer of erythropoietin.
BIDIRECTIONAL TRANSPORT OF SELENIUM IN THE PERFUSED HUMAN PLACENTA IN VITRO C. J. Eisenmann & R. K. Miller (Department of Obstetrics and Gynecology, Division Toxicology, University of Rochester, Rochester, NY 14642, USA)
of
Selenium (Se), essential for development, is found in many forms. Glutathione in red blood cells (RBC) adds to this diversity by reducing selenite so that selenium binds to RBCs and plasma protein. In humans, cord and maternal (M) blood [Se] are similar, while placental [Se] is about three times blood levels. Little is known about how selenium crosses and accumulates in the placenta. Therefore, transport of selenium given as selenite was studied in the dually perfused human placenta using methods previously described (Contr. Ohs. Gyn. 13, 77,8.5). After 2 h of control perfusion, selenium (with 75Se) was added to maternal (M) (2,20 or 45 nmol/ml) or to fetal (F) (20 nmol/ml) perfusates, and perfusates were recirculated for 4 h. Oxygen transfer to F, monitored to verify M/F circulation overlap, averaged 0.8 ml/min kg perfused tissue. Following selenium addition to M, selenium in the F vein peaked at 121 f 17 min with 5 * 2 per cent of the dose recovered in F in 4 h. Perfused tissue contained eight times M artery [Se]. Following selenium addition to F, selenium in the M vein did not peak in twothirds of the studies, with 20 f 5 per cent of the dose recovered in M, while perfused tissue contained two times F artery [Se]. After 4 h of perfusion, free [Se] was higher in F relative to M for both perfusion types (free M/free F = 2-3). Higher RBC and protein levels in M resulting in selenite reduction, may contribute to higher M selenium protein binding. Like cadmium and inorganic mercury, selenium accumulates in the placenta, but in contrast to these metals, the bidirectional transport of selenium across the placenta is more rapid. (Supported by NIH ES02774; ES01247.)
CELLS OF THE CHORIOCARCINOMA CELL LINE (JAr) IN CO-CULTURE WITH CYTOTROPHOBLASTS INCREASE THE SYNTHESIS OF CG AND hPL T. Eldar-Geva, N. de Groot & A. Hochberg (Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, Jerusalem, Israel) In the human trophoblast two major protein hormones are synthesized, chorionic gonadotropin (CG) and placental lactogen (hPL). Early in culture the cells synthesize only CGa subunits, but after a lag period CG/3 and hPL are produced. JAr cells in culture synthesize CG but do not produce hPL. It was shown previously
4hz.tTucls
385
(Hochberg et al, 1991,J. Biol. Chem., in press) that in a co-culture of cytotrophoblasts and JAr cells the amount of CG and hPL synthesized was considerably higher than the sum of the amounts synthesized by the cells in separate cultures. In order to investigate the level at which the synthesis of CG and hPL are controlled, we isolated RNA from cytotrophoblasts, JAr cells and the co-culture cultivated for 72 h. Equal aliquots of isolated RNAs were electrophorized in agarose gels, blotted on nylon filters and hybridized with labeled cDNA probes corresponding to CGa, CG/? and hPL sequences. The results show that the relative abundances of CGa, CG/3 mRNA was considerably higher in the RNA isolated from the cells of the co-culture than in the RNA isolated from cells grown separately. On the basis of previous results (Hochberg et al) we assume that the increase in the mRNA abundance is due to the cytotrophoblastic element of the co-culture. We calculated that the abundance of CGa, CGP and hPL mRNA in the RNA synthesized in the co-culture was increased 20, 100 and lo-fold respectively as compared to that in RN.4 from cytotrophoblasts grown separately. These changes in the abundance ofthe mRNAs ma) be solely responsible for the increase in CG and hPL synthesis. No increase could be detected in the abundance of a number of other mRNAs tested.
CHORIONIC GONADOTROPIN (hCG) RETARDS TROPHOBLAST INVASION IN VITRO BY DECREASING COLLAGENASE ACTIVITY T. Eldar-Geva, M. Ron, N. de-Groot, A. Milwidsky & S. Yagel (Department of Biological Chemistry, Institute of Life Science and the Department of Obstetrics/Gynaecology, Hadassah mount, Scopus University Hospital, the Hebrew Universiv, Jerusalem, Israel) During implantation, trophoblast cells adhere to the uterine epithelium, degrade the endometrium and penetrate the endometrial basement membrane, however, this invasive capacity seems to be restricted to the fetomatemal interface. We have shown previously that the high invasive ability of first trimester human trophoblast in vitro is dependent on collagenase, activated by plasmin. Plasminogen activator (uPA), the main activator in the proteinase cascade required for trophoblast invasion, hydrolyses plasminogen to plasmin, which forms collagenase from procollagenase. Here we used cultures of invasive first trimester trophoblast cells or isolated cytotrophblasts from term placenta and in vitro amnion invasion or matrigel assays to assess the role of hCG in the invasive process. hCG inhibited trophoblast invasion capacity in a dose-dependent fashion but exerted no effect on the ability of the trophoblasts to attach to basement membrane. The secretion of collagenase (including procollagenase) by the cultured cells (measured by RIA) was downregulated by hCG, again in a dose dependent manner. Similar inhibitory effect of hC(; on the collagenolytic activity of trophoblast conditioned media was shown when SDSpolyacrylamide gels containing collagens were used. In contrast, hCG had no effect on Ihe production of tissue inhibitor of metalloproteinase (TIMP). The hCG effect on collagenase production was not mediated by differences in the concentrations of procollagenase, up.4 or TIMP mRNAs. Incubation of pure uPA with hCG at the same concentration as used in the abole described experiments resulted in a significant reduction of uPA activity in a competitive inhibition fashion. Similar incubation of hCG with pure collagenase has shown no inhibitor?; effect. These observations suggest that hCG may play a role in the regulation of trophoblast invasion by decreasing uPA activity, which is essential for procollagenase activation.
Placenta(1991), Vol. 12
significantly different, yet the levels in the umbilical vessels (artery and vein) are nearly the same. In about two-thirds of the cases, the fetal EPO levels were higher than the maternal ones. In the 47 cases with higher maternal levels, there was no significant difference between the umbilical vein and the umbilical artery EPO levels. Although only an indirect indication, these results provide strong evidence that there is a placental barrier to the transfer of erythropoietin.
BIDIRECTIONAL TRANSPORT OF SELENIUM IN THE PERFUSED HUMAN PLACENTA IN VITRO C. J. Eisenmann & R. K. Miller (Department of Obstetrics and Gynecology, Division Toxicology, University of Rochester, Rochester, NY 14642, USA)
of
Selenium (Se), essential for development, is found in many forms. Glutathione in red blood cells (RBC) adds to this diversity by reducing selenite so that selenium binds to RBCs and plasma protein. In humans, cord and maternal (M) blood [Se] are similar, while placental [Se] is about three times blood levels. Little is known about how selenium crosses and accumulates in the placenta. Therefore, transport of selenium given as selenite was studied in the dually perfused human placenta using methods previously described (Contr. Ohs. Gyn. 13, 77,8.5). After 2 h of control perfusion, selenium (with 75Se) was added to maternal (M) (2,20 or 45 nmol/ml) or to fetal (F) (20 nmol/ml) perfusates, and perfusates were recirculated for 4 h. Oxygen transfer to F, monitored to verify M/F circulation overlap, averaged 0.8 ml/min kg perfused tissue. Following selenium addition to M, selenium in the F vein peaked at 121 f 17 min with 5 * 2 per cent of the dose recovered in F in 4 h. Perfused tissue contained eight times M artery [Se]. Following selenium addition to F, selenium in the M vein did not peak in twothirds of the studies, with 20 f 5 per cent of the dose recovered in M, while perfused tissue contained two times F artery [Se]. After 4 h of perfusion, free [Se] was higher in F relative to M for both perfusion types (free M/free F = 2-3). Higher RBC and protein levels in M resulting in selenite reduction, may contribute to higher M selenium protein binding. Like cadmium and inorganic mercury, selenium accumulates in the placenta, but in contrast to these metals, the bidirectional transport of selenium across the placenta is more rapid. (Supported by NIH ES02774; ES01247.)
CELLS OF THE CHORIOCARCINOMA CELL LINE (JAr) IN CO-CULTURE WITH CYTOTROPHOBLASTS INCREASE THE SYNTHESIS OF CG AND hPL T. Eldar-Geva, N. de Groot & A. Hochberg (Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, Jerusalem, Israel) In the human trophoblast two major protein hormones are synthesized, chorionic gonadotropin (CG) and placental lactogen (hPL). Early in culture the cells synthesize only CGa subunits, but after a lag period CG/3 and hPL are produced. JAr cells in culture synthesize CG but do not produce hPL. It was shown previously
4hz.tTucls
385
(Hochberg et al, 1991,J. Biol. Chem., in press) that in a co-culture of cytotrophoblasts and JAr cells the amount of CG and hPL synthesized was considerably higher than the sum of the amounts synthesized by the cells in separate cultures. In order to investigate the level at which the synthesis of CG and hPL are controlled, we isolated RNA from cytotrophoblasts, JAr cells and the co-culture cultivated for 72 h. Equal aliquots of isolated RNAs were electrophorized in agarose gels, blotted on nylon filters and hybridized with labeled cDNA probes corresponding to CGa, CG/? and hPL sequences. The results show that the relative abundances of CGa, CG/3 mRNA was considerably higher in the RNA isolated from the cells of the co-culture than in the RNA isolated from cells grown separately. On the basis of previous results (Hochberg et al) we assume that the increase in the mRNA abundance is due to the cytotrophoblastic element of the co-culture. We calculated that the abundance of CGa, CGP and hPL mRNA in the RNA synthesized in the co-culture was increased 20, 100 and lo-fold respectively as compared to that in RN.4 from cytotrophoblasts grown separately. These changes in the abundance ofthe mRNAs ma) be solely responsible for the increase in CG and hPL synthesis. No increase could be detected in the abundance of a number of other mRNAs tested.
CHORIONIC GONADOTROPIN (hCG) RETARDS TROPHOBLAST INVASION IN VITRO BY DECREASING COLLAGENASE ACTIVITY T. Eldar-Geva, M. Ron, N. de-Groot, A. Milwidsky & S. Yagel (Department of Biological Chemistry, Institute of Life Science and the Department of Obstetrics/Gynaecology, Hadassah mount, Scopus University Hospital, the Hebrew Universiv, Jerusalem, Israel) During implantation, trophoblast cells adhere to the uterine epithelium, degrade the endometrium and penetrate the endometrial basement membrane, however, this invasive capacity seems to be restricted to the fetomatemal interface. We have shown previously that the high invasive ability of first trimester human trophoblast in vitro is dependent on collagenase, activated by plasmin. Plasminogen activator (uPA), the main activator in the proteinase cascade required for trophoblast invasion, hydrolyses plasminogen to plasmin, which forms collagenase from procollagenase. Here we used cultures of invasive first trimester trophoblast cells or isolated cytotrophblasts from term placenta and in vitro amnion invasion or matrigel assays to assess the role of hCG in the invasive process. hCG inhibited trophoblast invasion capacity in a dose-dependent fashion but exerted no effect on the ability of the trophoblasts to attach to basement membrane. The secretion of collagenase (including procollagenase) by the cultured cells (measured by RIA) was downregulated by hCG, again in a dose dependent manner. Similar inhibitory effect of hC(; on the collagenolytic activity of trophoblast conditioned media was shown when SDSpolyacrylamide gels containing collagens were used. In contrast, hCG had no effect on Ihe production of tissue inhibitor of metalloproteinase (TIMP). The hCG effect on collagenase production was not mediated by differences in the concentrations of procollagenase, up.4 or TIMP mRNAs. Incubation of pure uPA with hCG at the same concentration as used in the abole described experiments resulted in a significant reduction of uPA activity in a competitive inhibition fashion. Similar incubation of hCG with pure collagenase has shown no inhibitor?; effect. These observations suggest that hCG may play a role in the regulation of trophoblast invasion by decreasing uPA activity, which is essential for procollagenase activation.