Culture Of Human Placental Cells Research Articles

Advanced glycation end products regulate interleukin-1β production in human placenta.

Maternal obesity is a major risk factor for pregnancy complications, causing inflammatory cytokine release in the placenta, including interleukin-1β (IL-1β), IL-6, and IL-8. Pregnant women with obesity develop accelerated systemic and placental inflammation with elevated circulating advanced glycation end products (AGEs). IL-1β is a pivotal inflammatory cytokine associated with obesity and pregnancy complications, and its production is regulated by NLR family pyrin domain-containing 3 (NLRP3) inflammasomes. Here, we investigated whether AGEs are involved in the activation of NLRP3 inflammasomes using human placental tissues and placental cell line. In human placental tissue cultures, AGEs significantly increased IL-1β secretion, as well as IL-1β and NLRP3 mRNA expression. In human placental cell culture, although AGE treatment did not stimulate IL-1β secretion, AGEs significantly increased IL-1β mRNA expression and intracellular IL-1β production. After pre-incubation with AGEs, nano-silica treatment (well known as an inflammasome activator) increased IL-1β secretion in placental cells. However, after pre-incubation with lipopolysaccharide to produce pro-IL-1β, AGE treatment did not affect IL-1β secretion in placental cells. These findings suggest that AGEs stimulate pro-IL-1β production within placental cells, but do not activate inflammasomes to stimulate IL-1β secretion. Furthermore, using pharmacological inhibitors, we demonstrated that AGE-induced inflammatory cytokines are dependent on MAPK/NF-κB/AP-1 signaling and reactive oxygen species production in placental cells. In conclusion, AGEs regulate pro-IL-1β production and inflammatory responses, resulting in the activation of NLRP3 inflammasomes in human placenta. These results suggest that AGEs, as an endogenous and sterile danger signal, may contribute to chronic placental cytokine production.

A time-course regulatory and kinetic expression study of steroid metabolizing enzymes by calcitriol in primary cultured human placental cells

1,25-dihydroxivitamin D3 (calcitriol), is a secoesteroid involved in several placental functions. In particular, we and others showed that calcitriol regulates peptides, proteins, cytokines and hormones production in human trophoblastic cells. On the other hand, calcitriol modifies the activity and expression of some steroidogenic enzymes, a process that is considered tissue-specific. However, the effects of calcitriol on the expression of enzymes involved in the synthesis of sex steroids in placental tissue have not yet been entirely studied. The aim of the present study was to investigate the effects of calcitriol upon gene expression of several steroid enzymes such as cytochrome P450scc (CYP11A1), type 1 3β-hydroxysteroid dehydrogenase(3β-HSDI), 17β-HSD3, 17α-hydroxylase/17,20 lyase (CYP17A1) and aromatase (CYP19A1) in primary cultures of human placental cells. Cell cultures were performed using placentas obtained immediately after delivery by caesarean section from normotensive healthy women and calcitriol effects were evaluated, at level of transcription, by qPCR. The results showed that: 1) from basal expression values of the five genes studied, 3β-HSDI was the most expressed gene (P<0.05); 2) basal expression of all enzymes was significantly higher in cultured syncytiotrophoblast than in cytotrophoblasts (P<0.05); 3) the presence of calcitriol in cultured trophoblast cells generally resulted in a stimulatory effect of CYP11A1, CYP19A1 and 17β-HSD3 gene expression at 3h of treatment whereas 3β-HSDI was induced at 6h (P<0.05). However, a time-dependent variable was also observed; 4) protein expression of CYP11A1 and 3β-HSDI were not modified significantly by calcitriol, however that of CYP19A1 was regulated in similar fashion as gene expression. In conclusion, calcitriol affected in a time-dependent manner the expression of steroids metabolizing enzymes in human placental cell cultures.

Regulation of CYP27B1 and CYP24A1 gene expression by recombinant pro-inflammatory cytokines in cultured human trophoblasts

Placenta is an important source of endocrine and immunological factors. During pregnancy, calcitriol, the active metabolite of vitamin D, is also metabolized by decidua and placental tissue by means of CYP27B1 and CYP24A1 for synthesis and inactivation of calcitriol respectively. Calcitriol production is regulated by several factors in a tissue-specific manner. However, the association of pro-inflammatory cytokines on calcitriol metabolism has not been studied in human placenta. The aim of the present study was to investigate the effects of TNF-α, INF-γ, IL-6 and IL-1β upon CYP27B1 and CYP24A1 gene expression in primary cultures of human placental cells. Placentas were obtained immediately after delivery by cesarean section from normotensive women. Cytokine effects upon mRNA of CYPs in enriched trophoblastic cell preparations were evaluated by using qPCR. The results showed that incubation of trophoblasts in the presence of each cytokine resulted in a significant increase of both CYPs expression. Interestingly, TNF-α increased significantly the ratio of CYP24A1/CYP27B1 gene expression, while IFN-γ preferentially induced CYP27B1, whereas IL-1β and IL-6 stimulated gene expression of both CYPs in the same proportion. The results suggest that cytokines among other factors regulate calcitriol metabolism in human placenta; specifically, INF-γ may contribute to calcitriol production while TNF-α favors its catabolism.

Effects of leptin on the expression of fatty acid-binding proteins in human placental cell cultures

The present study aimed to evaluate the protein expression of the transmembrane translocase FAT/CD36 and cytoplasmic H-FABP and L-FABP in human trophoblast tissue and evaluate the effects of exogenous leptin upon differential expression of each biomolecule; consequently, it aimed to derive information regarding the effects of leptin upon the expression of proteins implicated in fatty acid metabolism. Protein and total RNA were isolated from 72 samples of trophoblast tissue obtained from chorionic villous sampling. Of these, 36 samples were evaluated for protein (supernatant and pellet fraction separated) and the other 36 for total RNA expression. For each subgroup of samples, 12 were treated immediately and 24 were cultured. Half of the cultured samples were treated with 10 ng/-ml exogenously added leptin and the other half were untreated. Western blotting and PCR techniques were used for the evaluation of biomolecule expression. Our results were obtained from samples at a mean gestational week of 12+5 (n=72; min, 11+0; max, 14+1 gw; SD,0.89). In promptly treated samples we observed the presence of FAT/‌CD36 protein and absence of cytoplasmic FABPs. In the latter, only mRNA transcription of H-FABP was noted. A cytoplasmic pool of FAT/CD36 was also noted in the supernatant fraction of proteins. For cultured samples, when leptin was added, a statistically significant increase in FAT/CD36 protein expression was observed (n=24, p<0.001; mean difference, 0.219; SD, 0.0315; CI, -0.284 to -0.154). In our study we demonstrated the protein and mRNA expression of biomolecules implicated in fatty acid metabolism in human placenta. A cytoplasmic pool of the transmembrane protein FAT/CD36 was noted. Leptin caused the increase in FAT/CD36 protein expression in the cultured samples. Therefore, we conclude that leptin has an immediate effect and plays a role in lipid metabolism in human placenta.

Single Serum Activin A Testing to Predict Ectopic Pregnancy

Most fatal cases of maternal deaths in early pregnancy from ectopic pregnancy (EP) result from delayed diagnosis. Transvaginal sonography (TVS), the method of choice for initial diagnosis, fails to identify 8–31% of women with EP. For these women, either surgical or biochemical assessment (usually by measurement of serum levels of human chorionic gonadotropin (β-hCG) and/or progesterone) is used to make the correct diagnosis. None of these methods, however, has the accuracy, reproducibility, and simplicity to become a universal predictive marker for EP. Levels of activin A, a placental glycoprotein, increase in the maternal circulation throughout pregnancy until delivery. Addition of activin A to primary human placental cell cultures stimulates progesterone production and the release of β-hCG. This prospective observational study assessed the possible role of activin A as a diagnostic marker among 535 pregnant women with unknown pregnancy location (UPL) presenting at a tertiary referral center between 2004 and 2005. Following clinical examination, the women were evaluated with TVS, β-hCG, and progesterone, and blood levels of activin A were measured. The sensitivity and specificity of activin A as a diagnostic test for the detection of EP in women with UPL was assessed with the receiver operating curve (ROC) test. Pregnancy outcomes were evaluated in 155 (28.9%) viable intrauterine pregnancies (IUP), 305 (56.9%) first-trimester spontaneous abortions (SAB), and 76 (14.2%) EP. SAB had lower β-hCG and progesterone concentrations compared to both EP or IUP (P < 0.001 for each). Among women with EP, both β-hCG and progesterone levels were significantly lower than those with IUP or SAB (P < 0.001 for both). In contrast, levels of activin A were lowest among women with EP, being significantly lower in comparison to either SAB or IUP (P < 0.001 for each). Activin A levels were significantly lower in women with IUP than those with SAB (P < 0.001). ROC analysis showed that activin A had the highest accuracy for prediction of EP in women with UPL at the cutoff of 0.37 ng/ml; the sensitivity was 100% with a specificity of 99.6%. These findings suggest that measurement of activin A may be a useful biochemical marker to identify EP in women with UPL.

Corticotropin-releasing hormone gene expression in primary placental cells is modulated by cyclic adenosine 3',5'-monophosphate.

CRH, the principal neuropeptide regulator of pituitary ACTH secretion, is also expressed in placenta. Placental CRH has been linked to the process of human parturition. However, the mechanisms regulating transcription of the CRH gene in placenta remain unclear. cAMP signaling pathways play important roles in regulating the expression of a diverse range of endocrine genes in the placenta. Therefore, we have explored the effect of cAMP on CRH promoter activity in primary cultures of human placental cells. Both forskolin and 8-bromo-cAMP, activators of protein kinase A, can increase CRH promoter activity 5-fold in transiently transfected human primary placental cells, in a manner that parallels the increase in endogenous CRH peptide. Maximal stimulation of CRH promoter activity occurs at 500 micromol/L 8-bromo-cAMP and 10 micromol/L forskolin. Electrophoretic mobility shift assay and mutation analysis combined with transient transfection demonstrate that in placental cells cAMP stimulates CRH gene expression through a cAMP regulatory element in the proximal CRH promoter region and involves a placental nuclear protein interacting specifically with the cAMP regulatory element.

Peptide transport and metabolism across the placenta

Advances made in the field of DNA and recombinant technology have led to the emergence of peptides and proteins as an important class of therapeutic compounds. While a significant amount of information exists regarding the transport and metabolism of peptides across different barriers (e.g. gastro-intestinal, nasal, and blood–brain barrier), limited attention has been paid towards their transport and metabolism across the placental barrier. The mechanism of placental transport of peptides is of importance in assessing the exposure of these drugs to the fetus, particularly when the drugs potentially may have adverse effects on the developing fetus. The absence of a well accepted, simple and convenient animal model may be a reason for the limited information available on the placental transport and metabolism of peptides. Although several in vivo models have been utilized to study the transport and metabolism of drugs across the placenta, species differences in the placental physiology and anatomy of the animal models with regard to the human placenta have prevented their widespread use. The in vitro human placental cell culture models are morphologically similar to the trophoblasts and often express the enzymes and carrier systems found in the human placenta. They can provide an easy and rapid method to determine the mechanisms of transport and metabolism of drugs across the placental barrier. These in vitro models have been utilized in the determination of transport mechanisms of drugs of abuse across the placenta. This article overviews the available literature on the placental transport of peptides and describes the application of an in vitro cell culture model (BeWo) to determine the mechanisms of transport of opioid peptides and their analogues across the placenta.

Regulation of inhibin secretion in human placental cell culture by epidermal growth factor, transforming growth factors, and activin.

In this study, the effects of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), TGF beta 1, and activin-A on inhibin secretion were investigated in primary culture of human placental cells. Immunoreactive inhibin in the culture medium was measured by immunoenzymatic assay. EGF stimulated testosterone-induced inhibin secretion in placental cells. Although testosterone alone induced only a slight enhancement of inhibin release in the culture, treatment of trophoblast cells with EGF and testosterone caused a significant increase in inhibin secretion, with immunoreactive inhibin levels much higher than those of testosterone or EGF alone. TGF alpha combined with human placental lactogen (hPL) had a stimulatory effect on inhibin secretion in placental cell culture. Simple addition of either TGF alpha or hPL to the culture did not show any effect on inhibin secretion in placental cells. A remarkable augmentation of inhibin secretion was obtained after the trophoblasts were exposed to both TGF alpha and hPL simultaneously. TGF beta 1 and activin-A showed synergistic effects to suppress inhibin secretion in placental cells. TGF beta 1 alone did not show any action on inhibin secretion, and activin-A alone induced a small decrease in inhibin release in the culture. In the presence of activin-A, addition of TGF beta 1 to the culture induced a profound decrease in immunoreactive inhibin levels in the medium. Activin-A could also suppress hCG-induced inhibin secretion in placental cells. Addition of hCG alone resulted in a small, but not significant, increase in inhibin release in the cultured cells, whereas the presence of activin-A combined with hCG in the culture conversely decreased inhibin secretion in the culture, with immunoreactive inhibin levels significantly lower than those in the presence of hCG or activin-A alone. These findings suggest that EGF and TGF alpha, alone or in combination with other hormones, may be stimulators, and TGF beta and activin may act as suppressors of inhibin secretion in human placental cells.

Regulation of inhibin secretion in human placental cell culture by epidermal growth factor, transforming growth factors, and activin

Regulation of inhibin secretion in human placental cell culture by epidermal growth factor, transforming growth factors, and activin

Prostaglandins stimulate the secretion of inhibin from human placental cells.

In this study, the effects of prostaglandins (PGs) on inhibin secretion were investigated in primary culture of human placental cells. Isolated trophoblast cells were cultured for 2-5 days with PGE1, PGE2, 6-keto-PGF1 alpha, PGF2 alpha, and thromboxane B2. Inhibin levels in the culture medium were measured by immunoenzymatic assay. PGs significantly stimulated inhibin secretion in the cell culture. The addition of PGs (1 nM to 10 microM) to the culture resulted in dose-dependent increases in inhibin levels in the medium. At a dose of 10 microM, inhibin levels in the medium were increased by 35.2-172.5% compared to the control value. Compared with the efficacies of these PGs, PGE2 and PGF2 alpha enhanced inhibin secretion more potently than other tested PGs. A close correlation between the effects of PGs and the number of trophoblasts seeded in the culture was observed, with an optimal response of the cells to PGs at 1.0-2.0 x 10(6) cells/well. Time-course studies showed that a significant increment in inhibin levels in the PG-treated culture occurred after 48-120 h, but not during the first 24 h, of the culture, indicating a possible involvement of PGs in inhibin biosynthesis in the cells. Epidermal growth factor (EGF) could enhance PG-stimulated inhibin releases in the cell culture. Treatment with EGF (100 ng/mL) increased inhibin in the medium by 83.3% (ED50) in the presence of PGE2 or by 70.3% (ED50) in the presence of PGF2 alpha compared to the control values. The addition of 8-bromo-cAMP (100 microM) and cholera toxin (1000 ng/mL) to the culture also enhanced basal and PG-induced inhibin secretion, but the addition of the 4-bromo-calcium ionophore A23187 (1 microM) did not alter inhibin releases, suggesting that the stimulatory effects of PGs on inhibin secretion in placental cells may be mediated through the cAMP pathway. In the cell cultures treated with PGE and PGF2 alpha, there was no change in cell growth or intracellular DNA content, as measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay and DNA determination with fluorescence spectrophotometry. Morphological studies showed that PGE2 alpha (1 microM), alone or combined with EGF (100 ng/mL), significantly accelerated the process of differentiation from cytotrophoblasts to syncytiotrophoblasts, indicating that the actions of PGs may be related to their effects on syncytial transformation, rather than cellular proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of ethanol and progesterone on monoamine oxidase activity in cultured cells of human term placenta

The aim of this study was to evaluate the effect of ethanol and progesterone on the monoamine oxidase activity in cultured human term placental cells. Human placental cells were prepared from normal human term placentas by enzymatic dispersion in Dulbecco's modified Eagle medium. The viability of placental cells prepared by our method was 90%, and the yield of placental cells was 0.6 x 10(6) cells per gram of wet placental tissue. Five milliliters of Dulbecco's modified Eagle medium containing 3 x 10(5) placental cells was plated in a 25 cm2 flask and cultured for 8 days in an incubator at 37 degrees C under an atmosphere of 5% carbon dioxide and 95% oxygen with a saturated humidity. During the culture period the culture medium was replenished every 2 days. A confluent monolayer condition was achieved after 8 days in culture. The cultured placental cells were treated with different concentrations of ethanol (0, 34.6, and 69.2 mmol/L) and progesterone (0, 16, and 32 mumol/L) on day 8 of culture for 48 hours. At the end of treatment placental cells from control and treated flasks were harvested for the analysis of monoamine oxidase activity by spectrophotometry. The effects of ethanol and progesterone on cultured placental cells were statistically analyzed by one-way analysis of variance followed by Duncan's multiple comparisons procedure. A human placental cell culture system has been established from normal human term placentas. The monoamine oxidase activity in 8-day-cultured human term placental cells was significantly higher than that of freshly prepared placental cells. Ethanol concentrations at 34.6 and 69.2 mmol/L significantly increased and progesterone concentration at 32 mumol/L significantly decreased the monoamine oxidase activity. These results suggest that the cultured human term placental cells can be used to examine the in vitro effects of ethanol and progesterone on monoamine oxidase activity. However, the physiologic significance of progesterone's inhibitory effect and the stimulatory effect of ethanol monoamine oxidase activity in the in vivo system have yet to be further investigated.

Effect of epidermal growth factor on inhibin secretion in human placental cell culture.

The effect of epidermal growth factor (EGF) on inhibin secretion was investigated in a primary culture of human placental cells. Dissociated cells were cultured with EGF, FSH, 8-Br-cAMP, and two agents known to increase intracellular cAMP. Inhibin level in the culture medium was measured by immunoenzymatic assay. Addition of EGF (0.1-1000 ng/ml) in the cell culture induced a dose-dependent increase of inhibin levels in the medium after 2 days of culture. Greater response of placental cells to EGF in the inhibin secretion occurred at the doses of 10-1000 ng/ml, where inhibin levels in the medium increased by 84.9-111.5% compared to the control (P < 0.01). FSH stimulated the inhibin secretion in the placental cells. EGF combined with FSH resulted in a greater response of placental cells in inhibin secretion. Addition of FSH (30 ng/ml) and EGF (0.1-1000 ng/ml) in the culture induced inhibin levels significantly higher than that of either FSH alone or EGF alone (P < 0.01). The effect of EGF on inhibin secretion was closely correlated with the seeding density of trophoblasts and the time course of culture. Obvious effect of EGF was found at the number of 1-2 x 10(6) cells per well and after 36-48 h of culture. Addition of 8-Br-cAMP, cholera toxin, or forskolin in the culture increased the inhibin levels more than 6-fold, 5-fold, and 2-fold, compared to the controls, respectively. When EGF combined with one of these agents was added in the culture, the inhibin in the medium increased to a level higher than those with the individual agents alone. EGF resulted in an increase in basal and cAMP induced human CG secretions in the trophoblasts in a similar manner as in the inhibin secretion. However, the effect of EGF on the proliferation of trophoblasts was not observed by measurements of the cell growth with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and DNA content in the cells with fluorescence spectrophotometry. Morphological study showed that EGF induced trophoblasts to differentiate and form syncytium. These data suggest that EGF stimulates inhibin secretion in human placental cells in vitro. EGF and its interaction with other hormones or growth factors may play an important role in the complicated hormonal regulation during human pregnancy.

Effect of epidermal growth factor on inhibin secretion in human placental cell culture

The effect of epidermal growth factor (EGF) on inhibin secretion was investigated in a primary culture of human placental cells. Dissociated cells were cultured with EGF, FSH, 8-Br-cAMP, and two agents known to increase intracellular cAMP. Inhibin level in the culture medium was measured by immunoenzymatic assay. Addition of EGF (0.1-1000 ng/ml) in the cell culture induced a dose-dependent increase of inhibin levels in the medium after 2 days of culture. Greater response of placental cells to EGF in the inhibin secretion occurred at the doses of 10-1000 ng/ml, where inhibin levels in the medium increased by 84.9-111.5% compared to the control (P < 0.01). FSH stimulated the inhibin secretion in the placental cells. EGF combined with FSH resulted in a greater response of placental cells in inhibin secretion. Addition of FSH (30 ng/ml) and EGF (0.1-1000 ng/ml) in the culture induced inhibin levels significantly higher than that of either FSH alone or EGF alone (P < 0.01). The effect of EGF on inhibin secretion was closely correlated with the seeding density of trophoblasts and the time course of culture. Obvious effect of EGF was found at the number of 1-2 x 10(6) cells per well and after 36-48 h of culture. Addition of 8-Br-cAMP, cholera toxin, or forskolin in the culture increased the inhibin levels more than 6-fold, 5-fold, and 2-fold, compared to the controls, respectively. When EGF combined with one of these agents was added in the culture, the inhibin in the medium increased to a level higher than those with the individual agents alone. EGF resulted in an increase in basal and cAMP induced human CG secretions in the trophoblasts in a similar manner as in the inhibin secretion. However, the effect of EGF on the proliferation of trophoblasts was not observed by measurements of the cell growth with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and DNA content in the cells with fluorescence spectrophotometry. Morphological study showed that EGF induced trophoblasts to differentiate and form syncytium. These data suggest that EGF stimulates inhibin secretion in human placental cells in vitro. EGF and its interaction with other hormones or growth factors may play an important role in the complicated hormonal regulation during human pregnancy.

Mechanism of action of interleukin-1β in increasing corticotropin-releasing factor and adrenocorticotropin hormone release from cultured human placental cells

The present study evaluated the possible effect and mechanism of action of interleukin-1 beta in regulating the release of corticotropin-releasing factor and adrenocorticotropin hormone from human cultured placental cells. With the use of a primary monolayer culture of human placental cells at term, the addition of interleukin-1 beta increased the release of immunoreactive corticotropin-releasing factor with a dose- and time-dependent effect. The intracellular concentration of both cyclic adenosine monophosphate and cyclic guanosine monophosphate increased in the presence of interleukin-1 beta. The addition of indomethacin, a prostaglandin synthesis inhibitor, partially reversed the effect of interleukin-1 beta. The same doses of interleukin-1 beta stimulated the release of adrenocorticotropin hormone and this effect was partially reversed by the addition of a synthetic corticotropin-releasing factor antagonist or by indomethacin. This study showed that interleukin-1 beta increases the release of corticotropin-releasing factor and adrenocorticotropin hormone from cultured placental cells. This effect is associated with increased intracellular cyclic nucleotide concentrations and is in part reversed by a prostaglandin synthesis inhibitor.

Regulation of human renin expression in chorion cell primary cultures.

The human renin gene is expressed in the kidney, placenta, and several other sites. The release of renin or its precursor, prorenin, can be affected by several regulatory agents. In this study, primary cultures of human placental cells were used to examine the regulation of prorenin release and renin mRNA levels and of the transfected human renin promotor linked to chloramphenicol acetyltransferase reporter sequences. Treatment of the cultures with a calcium ionophore alone, calcium ionophore plus forskolin (that activates adenylate cyclase), or forskolin plus a phorbol ester increased prorenin release and renin mRNA levels 1.3- to 6-fold, but several classes of steroids did not affect prorenin secretion or renin RNA levels. The transfected renin promoter (584 or 100 base pairs of 5'-flanking DNA) initiated at the correct start site in these cells and forskolin increased its expression 2.5- to 4-fold. Constructs containing renin 5'-flanking DNA linked to a heterologous promoter cotransfected into HeLa cells with either glucocorticoid or estrogen receptor expression vectors were not regulated by dexamethasone or 17 beta-estradiol. These results suggest that (i) the first 584 base pairs of the renin gene 5'-flanking DNA do not contain functional glucocorticoid or estrogen response elements, (ii) placental prorenin release and renin mRNA are regulated by calcium ion and by the combinations of cAMP with either C kinase or calcium ion, and (iii) the first 100 base pairs of the human renin 5'-flanking DNA direct accurate initiation of transcription and can be regulated by cAMP. Thus, some control of renin release in the placenta (and by inference in other tissues) occurs via transcriptional influences on its promoter.

Inhibin and activin modulate the release of gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone from cultured human placental cells.

Although it is clear that human chorionic gonadotropin (hCG) and progesterone play fundamental roles in pregnancy, the regulation of placental production of these hormones remains to be defined. Recent evidence suggests that the human placenta expresses proteins related to inhibin (alpha beta subunits) or activin (beta beta subunits). Inhibin and activin (follicle-stimulating hormone-releasing protein) possess opposing activities in several biological systems including pituitary follicle-stimulating hormone (follitropin) secretion, erythroid differentiation, and gonadal sex-steroid production. The actions of purified inhibin and activin on hormonogenesis by primary cultures of human placental cells were studied. The addition of activin increased gonadotropin-releasing hormone (GnRH) and progesterone production and potentiated the GnRH-induced release of hCG. Inhibin by itself did not modify placental immunoreactive GnRH, hCG, and progesterone secretion but reversed the activin-induced changes. Neither inhibin nor activin influenced the release of human placental lactogen. Furthermore, transforming growth factor beta, structurally related to inhibin/activin, did not significantly influence hormone release from cultured placental cells. These results support the hypothesis that inhibin and activin may play a role in regulating the release of GnRH, hCG, and progesterone from placenta and implicate inhibin-related proteins in the endocrine physiology of human pregnancy.

Identification of Immunoreactive Neuropeptide-γ in Human Placenta: Localization, Secretion, and Binding Sites*

The aim of the present study was to evaluate the possible presence of immunoreactive neuropeptide-gamma (irNPY) in human placenta. Acidic extracts of human placental tissue collected at term pregnancy contained high irNPY concentrations. The extracted irNPY eluted from HPLC with the same retention time as synthetic NPY. The presence of the peptide in placental cells was confirmed by immunohistochemical findings showing numerous cells of the cytotrophoblast layer positively staining for NPY. Further supporting local production of the peptide, primary cultures of human placental cells released irNPY into the culture medium and the addition of high K+ concentrations increased the release of the peptide. The finding of irNPY in human placenta stimulated the characterization of binding sites of NPY in the same tissue. Using autoradiographic techniques we showed specific binding of [125I]NPY in human placental tissue. The binding of [125I]NPY to the placental receptors was saturable and widely distributed within the placental tissue. Finally, the addition of NPY to the medium of cultured placental cells increased the release of immunoreactive CRF, suggesting a possible role of NPY in placental hormone production. The effect of NPY was dose related and augmented by the addition of norepinephrine (10 nM). These results showed that human placenta produces and secretes irNPY and that NPY receptors are present in placental tissue. Moreover, the evidence that NPY stimulated the release of immunoreactive CRF from cultured placental cells suggests an action of NPY in placental hormonogenesis.

Evidence for local stimulation of ACTH secretion by corticotropin-releasing factor in human placenta

The hypothalamic-pituitary-adrenocortical axis is activated in pregnancy and parturition. Levels of immunoreactive corticotrophin releasing factor (irCRF), immunoreactive adrenocorticotropic hormone (irACTH) and cortisol concentrations in maternal plasma are elevated throughout gestation, increase further during labour and fall precipitously after parturition. The placenta contains biologically active CRF and ACTH and it has been suggested that the placenta produces these peptides during pregnancy. Here we show that irCRF is located in the cytotrophoblast cells of placenta collected at term. Using a monolayer primary culture of human placental cells we have found that CRF stimulates secretion of peptides containing the ACTH sequence in the placenta in a dose-dependent manner, as it does in the pituitary. This effect is reversed by a CRF antagonist and is mimicked by dibutyryl cyclic AMP and forskolin. Glucocorticoids, which suppress the secretion of pituitary ACTH, were found to have no influence on release of irACTH by the placenta. Oxytocin and prostaglandins stimulate irACTH and irCRF secretion from cultured placental cells and the irACTH-releasing activity of two prostaglandins is partially reversed by a CRF antagonist. Thus CRF may be involved in the paracrine regulation of placental irACTH secretion.

Identification of cytotrophoblast colonies in cultures of human placental cells using monoclonal antibodies

The morphological appearance of four distinctive types of colony in human placental cultures is described. Using a panel of monoclonal antibodies, one of these colonies is found to comprise cells with the phenotypic characteristics which allow them to be defined as cytotrophoblast with a reasonable degree of certainty. A second type of colony also contains cells of epithelial origin, but the trophoblast lineage of these cells is more difficult to ascertain. The remaining two types of colony are derived from mesenchymal elements representing either macrophages or fibroblasts. It is hoped that the precise identification of cytotrophoblast colonies will provide a useful yardstick to assess the efficiency of any future techniques which are devised for the selective growth and propagation of human trophoblast in vitro.

Insulin-induced receptor regulation in early gestation and term human placental cell cultures

Human placentae of different gestational ages have been used to investigate the binding and degradation of insulin as well as the regulation of insulin membrane receptors. Bacitracin was found to be an effective inhibitor of insulin degradation in human early gestation and term placental cell cultures. In the presence of bacitracin, [125I]-insulin bound rapidly and reversibly: maximal binding occurred at 4 degrees C, with a sharp pH optimum at 7.5, and exhibited a high degree of specificity. The extent of binding was proportional to cell protein and [125I]-insulin concentrations. Term (38 to 41 weeks; n = 25) placental cell cultures possessed receptors for insulin that were increased three-fold compared to early gestation (8 to 18 weeks; n = 17). This was due to an increase in receptor number with no significant alteration in affinity. A decrease in insulin binding in both early gestation and term placental cells was related to both the insulin and bacitracin concentrations present during 12 to 20 h of preincubation at 37 degrees C. The receptor loss was due to a decrease in the number of receptors per mg cell protein with no apparent change in their affinity. We conclude that our in vitro system, which utilizes human placental cells in monolayer culture, will permit a more direct study of the metabolic effects of insulin in both early gestation and term placentae.