Secretion Of Human Placental Lactogen Research Articles

Angiotensin II (Ang II) evoked secretion of the human placental lactogen (HPL) in intrauterine growth retardation: Examination of the relationship with Ang II receptor type 1 (AT1) expression

Angiotensin II (Ang II) and its hemodynamic effects on placental vasculature mediated via Ang II receptor type 1 (AT1) may play significant role in intrauterine growth retardation (IUGR). Placental lactogen (HPL) production directly reflects placental function. We compared influence of Ang II on HPL production in normal and IUGR-complicated pregnancies and correlated this phenomenon with AT1 expression. Basal and Ang II-evoked HPL secretion was examined in perfused placental lobules using ELISA. After immunostaining of placental sections, AT1 expression was estimated using quantitative morphometry. Ang II increased HPL secretion. Ang II-evoked increase in HPL concentration in the perfusion fluid was 27.36±6.4 (%, ±SEM) lower in IUGR (p<0.05) compared to normal-course pregnancies. AT1 expression was significantly decreased in IUGR and was 78.12±8.2 (%, ±SEM) of the mean value of controls. Demonstrating that Ang II-evoked secretion of HPL in preeclampsia-free IUGR is decreased and correlates with down-regulated expression of AT1, we present a new approach to the pathophysiology of IUGR.

Physiological concentrations of albumin stimulate chorionic gonadotrophin and placental lactogen release from human term placental explants.

This study investigates whether albumin, a major plasma protein in direct contact with the trophoblast in vivo, can modulate human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) releases from placental explants. Incubating explants with a near physiological, i.e. 5%, concentration of human or bovine albumin during 30 min increased HCG and HPL release by at least 150%. This albumin effect was not mediated by any difference in hormone adsorption onto glass surfaces. In contrast to the sustained stimulation of hormone releases elicited by the addition of 10 mmol/l extracellular calcium, the albumin-mediated secretory responses were transient. However, the albumin- and calcium-stimulatory effects were abolished at 4 degrees C, depressed by 0.36 mmol/l cycloheximide or 1 mmol/l colchicine and potentiated by 40 micromol/l cytochalasin B. Moreover, the stimulatory effect of albumin on the hormone releases was not modified in the absence of Ca(2+) or in the presence of 1 or 10 mmol/l Ca(2+) in the extracellular milieu. These data suggest that albumin is involved, at physiological concentration, in the secretion of HCG and HPL by human placenta. The cellular mechanism(s) underlying the albumin-mediated secretory responses may be partly different from those involved during the calcium-mediated stimulation.

Expression of human placental D 2-dopamine receptor during normal and abnormal pregnancies

Previous studies have demonstrated the presence of D 2-dopamine binding sites in the human placenta, and that dopamine (DA), via these D 2-like receptors, inhibits both basal- and hormone-stimulated secretion of human placental lactogen (hPL) from trophoblastic cells. However, nothing is known about the ontogenesis of this placental D 2-dopamine receptor (DZR) during pregnancy. Therefore, the aim of this study was to analyse the expression of these receptors throughout gestation in placentae from normal as well as abnormal pregnancies. Western and Northern blot analysis were performed on membrane protein and messenger RNA (mRNA) preparations of human placentae from various weeks of gestation as well as from pregnancies complicated by pre-eclampsia of hydatidiform mole. The autoradiographs of both proteins and rnRNA showed differential expression of placental D 2R during normal pregnancy. When the relative levels of D 2R proteins were analysed throughout pregnancy, there was a significant but transient decrease of ∼ 23 per cent of D 2R content at 9–16 weeks of gestation with a return to baseline levels at 17–18 weeks. An increase in rMRNA levels began at week 19 of gestation and reached a maximum value at term. During the first half of gestation, the relative levels of DZR mRNA (2.5 kb) showed an inverse pattern of expression when compared to D 2R protein content. Specifically, the levels of D 2R mRNA increased by ∼ 26 per cent between weeks 9 and 16 of pregnancy in comparison with the values observed at 7–8 weeks, and returned to baseline levels at 17–18 weeks of gestation. The D 2R relative protein levels subsequently increased from 19 to 30 weeks of gestation, and then remained stable. The autoradiographs of both proteins and mRNA showed significantly decreased expressions in placentae from both pre-eclamptic (∼45 per cent inhibition) and molar (∼0–70 per cent inhibition) pregnancies. Moreover, there was important variability in the expression of placental D 2R from hydatidiform moles. Using immunological and molecular biology techniques, the present study confirms the presence of D 2R in human placenta. The variations of placental D 2R expression during normal and abnormal pregnancies argue for an important role of DA in human placental function, although this remains to be investigated further.

Endometrial stromal cell decidualization inhibits human chorionic gonadotrophin and human placental lactogen secretion by co-cultured trophoblasts.

We have developed an in-vitro co-culture system to examine the interaction between purified first trimester cytotrophoblasts and purified non-pregnant human endometrial stromal cells (ESC). ESC decidualization is an important step in endometrial maturation and may modulate embryo implantation. In order to investigate the effects of ESC decidualization on trophoblast function, we examined human chorionic gonadotrophin (HCG), human placental lactogen (HPL), progesterone and estrogen secretion by trophoblasts co-cultured in contact with ESC, either with or without decidualization induced by progesterone. Decidualized ESC inhibited basal HCG and HPL secretion for 3 days during the culture for HCG, and for 5 days during the culture for HPL (P < 0.01 and P < 0.03 respectively). After 5 days of co-culture, decidual transformation of ESC as indicated by prolactin production occurred in the control cultures due to progesterone and oestradiol secretion by the co-cultured trophoblasts, but no significant differences in HCG or HPL secretion were observed between the two groups. Although the type of trophoblast used in the present study is far from implantation, our results clearly demonstrated that HCG and HPL secretion by trophoblasts was inhibited by the presence of co-cultured decidualized ESC, and suggested that ESC decidualization may regulate trophoblast function at the human fetal-maternal interface.

Endometrial stromal cell decidualization inhibits human chorionic gonadotropin and human placental lactogen secretion by co-cultured trophoblasts

Endometrial stromal cell decidualization inhibits human chorionic gonadotropin and human placental lactogen secretion by co-cultured trophoblasts

Hepatocyte Growth Factor Promotes the Growth of Cytotrophoblasts by the Paracrine Mechanism

Both placental and decidual tissues contained extractable HGF, the former HGF level (31.4 +/- 23.4 ng/mg total protein) being approximately 30 times the latter HGF level. When the localization of HGF protein and HGF mRNA in placental tissues was examined by immunohistological staining and in situ hybridization, HGF protein and HGF mRNA were detected in the mesenchymal cells of the placenta, but were absent in the cytotrophoblast and syncytiotrophoblast. Although c-met protein was expressed in the cytotrophoblast, this receptor was not detectable in the syncytiotrophoblast by immunohistochemical methods. c-met mRNA was detected in placental cell line (tPA30-1) and 4 choriocarcinoma cell lines (BeWo, Jar, Jeg-3, and NUC-1), but HGF mRNA was absent in these cells. When cytotrophoblast cells were cultured in a serum-free medium in the presence of HGF, their DNA synthesis was enhanced depending on the HGF concentration, although human placental lactogen secretion itself was not affected by HGF. These results demonstrated that HGF promotes the growth of the cytotrophoblast by the paracrine mechanism, although it does not serve as a placental differentiation factor.

Involvement of epidermal growth factor (EGF)/EGF receptor autocrine and paracrine mechanism in human trophoblast cells: functional differentiation in vitro.

We have studied the expression of epidermal growth factor (EGF) and EGF receptors (EGF-R) in isolated human trophoblast cells at various stages of differentiation and also the biological significance of the EGF/EGF-R autocrine and paracrine mechanism. Cytotrophoblast cells were isolated from human placental tissues of 6-9 weeks of gestation. Trophoblast cells underwent morphological and functional differentiation during in vitro culture. The expression of EGF and EGF-R protein and mRNA was studied in trophoblast cells cultured for 0-5 days, using immunocytochemical staining, and reverse transcription and polymerase chain reaction. Monoclonal antibodies (mAbs) against EGF and EGF-R showed specific staining in trophoblast cells at all stages of differentiation. Both EGF and EGF-R gene transcripts were detected in RNA samples isolated from trophoblast cells at all stages. These data suggest the presence of an EGF/EGF-R autocrine and paracrine mechanism in human trophoblast cells. Next, we examined the biological significance of this mechanism on trophoblast cell differentiation in vitro. EGF added to the culture medium significantly increased human chorionic gonadotrophin-beta (hCG-beta) secretion and, more importantly, anti-EGF neutralizing mAbs significantly reduced both hCG-beta and human placental lactogen secretion from trophoblast cells in culture. All these results suggest that human trophoblast cells express both EGF and EGF-R, and that EGF may play an important role in the functional differentiation of human trophoblast cells.

D 2-dopamine agonists inhibit adenosine 3′ :5′-cyclic monophosphate (cAMP) production in human term trophoblastic cells

We previously reported that dopamine (DA) acted via D 2-dopamine receptors on human trophoblastic cells to inhibit basal and hormone-stimulated secretion of human placental lactogen (hPL). We also described that these DA effects were coupled with inhibition of calcium influx. The present study examines the interaction of placental D 2-dopamine receptor with adenylate cyclase (AC). Incubations of isolated human term trophoblastic cells with R(-)-propylapomorphine (NPA), (±)-PPHT, and bromocriptine (3 different D 2 agonists) led to time- and dose-dependent inhibitions of cAMP production as determined by measuring the conversion of [2- 3H]-ATP into [2- 3H]-cAMP. The maximal inhibition was reached after 15 min of incubation and was 33±1 (SE)%, 29±3% and 31±1% for bromocriptine (10 −5 M), NPA (10 −7 M) and (±)-PPHT (10 −8 M) respectively. However, the time- and dose-dependent curves were biphasic with NPA and (±)-PPHT and the inhibition of cAMP production was abolished at higher agonist concentrations or after time incubations longer than 15 min. These inhibitions were receptor specific since they were reversed by spiperone and haloperidol, two specific-dopamine antagonist, and by butaclamol (mix D 2/D 1-dopamine antagonists) but not by α- and β-adrenergic, D 1- and D 4-dopaminergic, and 5-HT 2-serotonergic antagonists. The results reported here suggest that human placental D 2 receptors interact with AC to inhibit its activity. Also, bromocriptine seems a better agonist for the characterization of dopaminergic effects on human placenta.

Human Macrophage Colony-Stimulating Factor Induces the Differentiation of Trophoblast

When human cytotrophoblastic cells in the early stage of pregnancy were cultured in a serum-free medium in the presence of human macrophage colony-stimulating factor (M-CSF), the cytotrophoblastic cells fused and formed a typical syncytiotrophoblast which had a dense distribution of microvilli revealed under an electron microscope. On the other hand, cytotrophoblasts incubated with anti-M-CSF antibody showed hardly any syncytiotrophoblast formation. Following this finding, we studied the differentiation of chorionic cells from the viewpoint of hormone secretion. When cytotrophoblasts were incubated in the presence of M-CSF, the supernatant of the culture showed an increase in human chorionic gonadotropin and human placental lactogen levels in proportion to the concentration of M-CSF added. When cytotrophoblasts were incubated in the presence of anti-M-CSF antibody or anti-fms antibody, human chorionic gonadotropin and human placental lactogen secretion were suppressed. Thus, M-CSF was morphologically and endocrinologically found to induce the differentiation of chorionic cells.

Partial hydatidiform moles have impaired differentiated function (human chorionic gonadotropin and human placental lactogen secretion) in response to epidermal growth factor and 8-bromo-cyclic adenosine monophosphate

The null hypothesis is that partial hydatidiform moles have normal differentiated function (human chorionic gonadotropin and human placental lactogen secretion) in response to epidermal growth factor and 8-bromo-cyclic adenosine monophosphate. Two complete moles, 10 partial hydatidiform moles, and 19 normal first-trimester placentas in monolayer culture were exposed to 10 ng/ml epidermal growth factor, 1 mmol/L 8-bromo-cyclic adenosine monophosphate plus 1 mmol/L theophylline, or control. Human chorionic gonadotropin and human placental lactogen secretion was measured. Frequency of response to stimuli was compared by chi 2 analysis, and hormone secretion was compared by analysis of variance. Partial moles demonstrated reduced frequencies of response of human chorionic gonadotropin and human placental lactogen to epidermal growth factor (partial moles 2/8 and 2/8, respectively; normal placentas 16/19 and 7/18, respectively; p less than 0.025) and of human chorionic gonadotropin to 8-bromo-cyclic adenosine monophosphate (partial moles 3/5, normal placentas 13/16; p less than 0.005). Partial hydatidiform moles demonstrate impaired human chorionic gonadotropin and human placental lactogen secretory responsiveness to epidermal growth factor and cyclic nucleotides in comparison with normal first-trimester trophoblast.

Patterns of spontaneous pulsatile secretion of human chorionic gonadotropin and pregnancy specific β1 glycoprotein by superfused placental explants in first and last trimester. Lack of episodic human placental lactogen secretion

We have recently reported that during superfusion of placental explants, human chorionic gonadotropin secretion is episodic. In the present work we have examined, using the superfusion model, the pattern of secretion of other glycoprotein hormones, pregnancy specific beta 1 glycoprotein and human placental lactogen, in the first trimester and term placenta. This was done by evaluating the pulsatile pattern by two different computerized programmes. By using different sampling intervals (6-0.5 min) of the effluent, two distinct patterns of hCG secretion were detected in the first trimester: a short one, occurring every 8-9 min and the other every 18-25 min. In contrast, at term the only episodic pattern detected was every 40-50 min, with a low amplitude, 20-30% above baseline, and a declining baseline. In two out of seven placentas, no pulsatility was detected. The secretion of pregnancy specific beta 1 glycoprotein was pulsatile, occurring every 14-15 min in the first trimester and every 6-7 min at term. Finally, the secretion of human placental lactogen at term was not pulsatile. The levels of this glycoprotein in the first trimester placenta were below detection limits. In conclusion, the patterns of the three glycoproteins during superfusion are different, suggesting different paracrine/autocrine control mechanisms. The dynamic superfusion model also enables identification of thus far not reported gestational age-dependent differences in the secretion pattern of hCG and pregnancy specific beta 1 glycoprotein.

Interrelated calcium ion and cyclic AMP inhibition of placental lactogen secretion by cultured human term trophoblast

Calcium ion has been shown to have a negative regulatory effect on the secretion of human placental lactogen (hPL). We report an inhibitory effect of cyclic AMP on hPL secretion in a model system of cultured human term trophoblast. Similar results were obtained by modulation of cyclic AMP-phosphodiesterase. The effects of ionophore A23187 and cyclic AMP were non-additive. In searching for the sequence of the common pathway, we found that the ionophore A23187 reduced cyclic AMP concentration and that the inhibitory effect of cyclic AMP was abolished by verapamil, a calcium channel blocker, and by trifluoperazine, a calcium-calmodulin inhibitor. Cyclic AMP seems to mobilize intracellular calcium, and the latter, by way of a calcium-calmodulin complex, modulates the secretion of hPL.

Immunoelectron microscope observations on secretion of human placental lactogen (hPL) in the human chorionic villi.

Immunoelectron microscope localization of human placental lactogen (hPL) was investigated in the chorionic villi from week 7 to full-term gestation with the protein A-gold technique. With specific antiserum against hPL, immuno-reactive gold particles were found to be preferentially located in Golgi-derived, electron-dense small granules of 80-180 nm in the syncytiotrophoblast. Our electron micrographs indicate that these small granules increase in number in the course of gestation and are released by exocytosis from the apical cell surface. The present study reveals that hPL is segregated from the Golgi apparatus, stored in the syncytiotrophoblast as secretory granules, and released into the maternal blood.

Demonstration of post-translational secretion of human placental lactogen by a mammalian in vitro translation system.

This study demonstrates the post-translational translocation across the rough endoplasmic reticular membrane of a mammalian secretory protein, human preplacental lactogen. In the rabbit reticulocyte lysate, human preplacental lactogen biosynthesis is arrested by addition of cycloheximide prior to supplementation with dog pancreatic microsomal membranes, which have previously been shown to translocate and process nascent secretory proteins in a cotranslational manner. Twenty-five percent of the precursor protein is consistently converted to its mature form under these post-translational conditions. The resulting mature hormone is resistant to proteolytic degradation by added proteases, thus indicating that it is translocated across the microsomal membrane and sequestered within the lumenal space of the microsomal vesicles. Approximately one-half of the precursor protein synthesized is associated with the ribosomes. Only the ribosome-associated fraction is secreted in this in vitro system, suggesting that the process of post-translational secretion requires ribosomes for protein interaction with the elements of a subcellular secretory apparatus.

The modulation of placental lactogen secretion by calcium: studies with cultured human term trophoblast

Previous reports have documented the negative regulatory effect of calcium ion on the secretion of human placental lactogen by the placenta. Human term trophoblasts were dispersed from term placenta and maintained in monolayer cell culture. Incubation of the cultured trophoblast with EGTA produced a dose-dependent stimulation of placental lactogen secretion. The calcium ionophore A23187 inhibited hormone secretion. This inhibitory effect was eliminated by the addition of the calcium-calmodulin complex inhibitor trifluoperazine. It is concluded that calcium exerts a negative regulatory effect on the secretion of placental lactogen by a mechanism which requires the action of the calcium-calmodulin complex.

The synthesis and secretion of human placental lactogen (hPL) in cultured term placenta

We studied the in vitro synthesis and secretion of hPL by human term placental tissue incubated in organ culture. Placental tissue maintains a constant pool of hPL. The synthesis of hPL may be the driving force for its secretion. The de novo synthesis and secretion rates of hPL were also investigated. The higher specific radioactivities of the secreted hPL than those found in the tissue may suggest that newly synthesized hPL is preferentially released. The intracellular distribution of hPL was also compared.

Studies on the secretion of human placental lactogen from human trophoblastic tissues (author's transl)

In order to study the secretory mechanism of human placental lactogen (hPL:hCS) from trophoblastic tissues, tissue culture and new placental perifusion systems were developed and used to clarify the effect of various substances on the secretion of hPL. These substances were (1), metal ions([Ca2+], [K+], [mg2+], [Na+]); (2) growth hormone and prolactin releasing or inhibiting factors (arginine, TRH, somatostatin, dopamine); (3) LH-RH, dibutyryl cyclic-AMP which stimulates hCG secretion; (4) prostaglandins F2 alpha and E2, bradykinin; (5) EGF and insulin which have the receptors in the placenta; (6) glucose. It was found that most of the substances examined had no effect on the secretion of hPL, except dopamine and glucose. The effect of dopamine in the tissue culture system is dose-dependent. At high concentrations dopamine slightly inhibited hPL secretion(5mM: 38.6 +/- 15.0 and 10mM; 35.7 +/- 19.0 micrograms/g wet tissue) compared with the control (63.2 +/- 29.8 microgram/g wet tissue). However, these effects may be due to the deviation of pH in the medium from the direct addition of dopamine hydrochloride. At a low concentration(1mM) it was observed to have a rather stimulatory effect (125.7 +/- 18.0 micrograms/g wet tissue, p less than 0.05), but in the perifusion system, this effect could not be observed. The addition of glucose in the perifusion system gave a slightly higher hPL secretion than that of the control. Perhaps this resulted from increased cell activity rather than a stimulatory effect. an incorporation experiment of [3H] leucine was also carried out to study the biosynthesis of hPL. Newly synthesized ([3H]-labelled) hPL was secreted into the medium within two hours. Furthermore, a labelled larger molecular weight substance together with the tritiated hPL was also observed on a Sephadex G-100 gel chromatography. These labelled substances were immunoprecipitable using an anti-hPL serum, indicating that the substances contain the same immunological determinants. This result indicates that the larger molecular substance may represent the biosynthetic precursor or the aggregate of hPL. These data indicate that the secretion of hPL has a unique mechanism, different from GH and PRL, and may be self regulated.

Inappropriate secretion of human placental lactogen and β-subunit of human chorionic gonadotropin by cancer of the uterine cervix

Human placental lactogen (hPL) and the β-subunit of human chorionic gonadotropin (β-hCG) were studied in serum samples of (a) 45 patients with cancer of the uterine cervix, (b) 30 normal men, and (c) 30 normal nonpregnant women. Sera of 27 of 45 (60%) and 13 of 32 (41%) patients with cervical cancer were positive for hPL and β-hCG, respectively. hPL and β-hCG were not detectable in sera of normal subjects mentioned above. Serum hPL was detected with similar incidence in patients with cancer of cervix of grades II, III, and IV (FIGO classification) whereas β-hCG was found with increasing incidence in association with increasing clinical grades. That Serum hPL was not detectable in normal or nonneoplastic conditions and its high incidence in cervical cancer patients indicate its possible use as marker in cancer of cervix.

Studies of human chorionic gonadotrophin, placental lactogen, and progesterone secretion in vitro

The relative ability of explants of villous tissue from 8- to 10-week placenta to synthesize and release human chorionic gonadotrophin (HCG), human placental lactogen (HPL) and progesterone in culture was investigated. It was found that the synthesis and release of all three hormones was two to four times greater than the initial tissue content in the first 24 hours in culture. Between 24 and 48 hours of the secretion of HCG, HPL and progesterone was reduced to about 40 per cent, 20 per cent and 60 per cent respectively of that produced in the previous 24 hours. These results support previous findings that the production of HPL is more labile than HCG in culture, and suggest that progesterone secretion under the present conditions is least affected. When azastene (4, 4, 17 alpha-trimethylandrost-5-eno (2,3-d) isoxazol-17-ol), an inhibitor of gonadal and placental 3 beta-hydroxy steroid dehydrogenase conversion of pregnenolone to progesterone in rhesus monkeys, was added to the culture medium at a concentration of 0.3-150 mumol/l, no marked effect on the secretion of progesterone was observed. This indicates that the dehydrogenase in human placental tissue is either less accessible to azastene or that the inhibitory activity in the rhesus monkey was due to a metabolite of azastene.

Somatostatin in cytotrophoblast of the immature human placenta: localization by immunoperoxidase cytochemistry.

Using the technique of immunoperoxidase cytochemistry, we have demonstrated the presence of immunoreactive somatostatin in the cytotrophoblast. This is contrasted by the immunocytochemical staining of human placental lactogen in the syncytotrophoblast in the adjacent section of the villus tissue of the immature human placenta. It is considered that the somatostatin may be involved in the secretion of human placental lactogen within the two layers of trophoblastic cells.