Effect of morphine on hCG release by first trimester human trophoblast in vitro

Opiate synthesis by human placental cells and the presence of kappa-type opiate binding sites in the syncytiotrophoblast brush border membrane may indicate the possible role of morphine-like substances in the autocrine regulation of trophoblast cell metabolism. This study was undertaken to examine the in vitro effect of morphine on hCG (human chorionic gonadotrophin) and hPL (human placental lactogen) release by 1st and 3rd trimester placental tissue explants. The results have shown that morphine (100 nM) significantly stimulated hCG secretion by 6-8 weeks old trophoblast and was without effect on hPL. Hormone secretion by term placental tissue explants was unaffected by morphine treatment. Based on these results we assume that opiates may have a role in the local (autocrine and/or paracrine) regulation of hCG secretion in early gestation.     

Properties and functions of human placental opioid system.

Human placental villus tissue contains opioid receptors and peptides. Kappa opioid receptors (the only type present in this tissue) were purified with retention of their binding properties. The purified kappa receptor is a glycoprotein with an apparent molecular weight of 63,000. Two opioid receptor mediated functions were identified in trophoblast tissue, namely regulation of acetylcholine and hormonal (human chorionic gonadotrophin and human placental lactogen) release. Placental content of kappa receptors increases with gestational age. Term placental content of kappa receptors correlates with route of delivery (higher in those abdominally obtained). Opioid use and/or abuse during pregnancy affects placental receptor content at delivery, as well as its mediated functions. Opioid peptides identified in placental extracts were beta-endorphin, methionine enkephalin, leucine enkephalin and dynorphins 1-8 and 1-13. Dynorphin 1-8 seem to be the predominant opioid peptide present in placental villus tissue.     

A maternal serum screen for trisomy 18: an extension of maternal serum screening for Down syndrome.

The feasibility of extending second-trimester maternal blood screening for Down syndrome so as to include screening for trisomy 18 was examined using stored maternal serum samples collected for neural tube-defect screening. There were 12 samples from trisomy 18 pregnancies and 390 controls. The median maternal serum concentration of alpha-fetoprotein, free alpha-subunit human chorionic gonadotrophin, free beta-subunit human chorionic gonadotrophin, intact human chorionic gonadotrophin, total estriol, unconjugated estriol, estradiol, human placental lactogen, and progesterone were lowered in those pregnancies affected by trisomy 18 when compared with unaffected pregnancies matched for racial origin, maternal age, gestational age, and sample-storage duration. At an estimated odds risk of 1:400, 83.3% of affected pregnancies were detected using an algorithm which combines the maternal age-related risk with the maternal serum concentrations of unconjugated estriol, free alpha-subunit human chorionic gonadotrophin, free beta-subunit human chorionic gonadotrophin, estradiol, and human placental lactogen. The associated false-positive rate was 2.6%. At high risk odds of 1:10, the detection rate was 58.3%, with an associated false-positive rate of 0.3%. beta-Subunit human chorionic gonadotrophin and unconjugated estriol were the most powerful discriminators. It is possible to incorporate into existing Down syndrome screening programs an algorithm for detecting trisomy 18 with high sensitivity and specificity.     

Effects of glucose, glycerol, 3-hydroxybutyrate, insulin, and leptin on placental growth hormone secretion in placental explants

Placental growth hormone (PGH) is secreted from the syncytiotrophoblast in increasing amounts during pregnancy. The physiology and regulation of PGH is not well known; however, low glucose levels appear to stimulate PGH liberation IN VITRO and IN VIVO. PGH appears to have lipolytic effects, and inverse correlations between maternal body mass index and serum PGH levels have been reported. Therefore, substances related to maternal adipose tissue metabolism could influence PGH secretion. The effect of insulin, glycerol, 3-hydroxybutyrate (3-OHB), and leptin on PGH and human placental lactogen (hPL) secretion from cultured placental explants was studied. In glucose-free media, PGH content increased upto 237.5+/-28.4% of control media (p<0.001, ANOVA). Insulin levels were without effect on PGH secretion, as were 3-OHB, leptin, and glycerol at 0.02 mmol/l. Glycerol at 0.2 mmol/l increased PGH in all of the placental explants studied (n=8; mean increase 27.3+/-7.1%), and this difference was significantly different from the control explants (p=0.004). The liberation of hPL to culture media was different from PGH and was influenced by glucose and insulin. In conclusion, the absence of glucose profoundly increased PGH secretion in cultured placental explants. Addition of glycerol in physiologically relatively high concentrations showed a less pronounced stimulatory effect.     

Evidence for a clathrin-mediated recycling of albumin in human term placenta

During human pregnancy, the trophoblast layer is in direct contact with maternal albumin. In contrast to immunoglobulins, albumin does not cross the placental barrier. However, albumin affects the trophoblast placental lactogen and chorionic gonadotroph secretion. The present study investigated the interaction between albumin and syncytiotrophoblast using human term placental explants. Bovine serum albumin, labeled with either 125I or fluorescein isothio-cyanate, was taken up rapidly by placental explants. This process was temperature-sensitive. The internalized labeled BSA quickly outflowed from the tissue at the maternal side, largely without any major modification in molecular weight. Colchicine (1 mM), which disrupts the microtubule network, or cytochalasin B (40 microM), which disassembles filamentous actin, did not interfere with the placental transmembrane movements of labeled BSA. Megalin, clathrin, and caveolin 1 are three membrane proteins associated with albumin endocytosis in other tissues, but only megalin and clathrin were detected in the syncytiotrophoblast layer by immunohistochemistry. The uptake of labeled BSA into placental explants was not modified by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (1 mM) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM), two pharmacological tools known to disturb megalin-mediated albumin endocytosis. By contrast, methyl-beta-cyclodextrin (10 mM) and chlorpromazine (1.4 mM), both of which disrupt the clathrin-mediated endocytotic system, significantly reduced the uptake of labeled BSA. These data suggest, to our knowledge for the first time, that maternal albumin is actively internalized into the human trophoblast according to an apical recycling pathway. This temperature-sensitive process does not depend on an intact cytoskeleton, but it is associated with a clathrin-mediated endocytotic system.     

An immunogold,cryoultrastructural study of sites of synthesis and storage of chorionic gonadotropin and placental lactogen in human syncytiotrophoblast

Summary The sites of intracellular synthesis and storage of human placental lactogen (hPL) and human chorionic gonadotropin (hCG) are controversial. We have used one of the most sensitive methods, cryoultramicrotomy and immunogold labelling, to localise these hormones at the electron-microscopic level. In both 12-week and term placentas hCG and hPL are present throughout the rough endoplasmic reticulum cisternae, in the Golgi bodies, and in the infrequent small dense granules of the syncytiotrophoblast. Previous assays have shown that hCG is at a higher concentration in early pregnancy and hPL peaks in late pregnancy, and our results corroborate these findings. No significant localisation of either hormone was seen in the cytotrophoblast or villous stroma. The results suggest that both hCG and hPL are synthesised and packaged by the classical secretory pathway, although the level of hormone stored in granules at any one time is small.     

Effect of high oxygen on placental function in short-term explant cultures

Ex situ culture of human gestational tissues has been routinely used as a model to investigate tissue function. The objective of this study was to determine the effect of varying oxygen concentrations on human term placental explants over a 24-h time period. Specifically, the effect of incubating placental explants in oxygen concentrations of 8%, 21% or 95% on tissue viability, metabolism and cell death was measured by assessing glucose consumption, lactate production, release of lactate dehydrogenase, parathyroid hormone-related protein (PTHrP), tumour necrosis factor-alpha (TNF-α) and 8-isoprostane, immunoreactivity for cleaved-caspase-9 and immunohistochemistry for the caspase-3-cleaved cytokeratin-18 neoepitope, M30. Exposure to higher oxygen concentrations significantly increased the rates of glucose consumption and lactate production. Apoptosis was significantly increased under conditions of higher oxygen as evidenced by increased M30 in placental explant sections. Similarly, hyperoxia significantly increased the releases of PTHrP, TNF-α and 8-isoprostane. Thus, incubation of placental explants with oxygen concentrations of 95% and, to a lesser extent, 21% oxygen was associated with the modulation of multiple cellular response pathways including those associated with tissue viability and cell death. These data are consistent with the hypothesis that hyperoxia activates pathways and mechanisms involved in cellular metabolism, necrosis and apoptosis, thereby shifting the balance from a steady state towards cell death.     

Lipolytic and antilipolytic effects of human growth hormone, its 20-kilodalton variant, a reduced and carboxymethylated derivative, and human placental lactogen on chicken adipose tissue in vitro

The lipolytic and antilipolytic effects of human growth hormone (22K-hGH), its 20-kilodalton variant (20K-hGH), a reduced and S-carboxymethylated derivative (RCM-hGH), and human placental lactogen were examined using chicken adipose tissue explants in vitro. Lipolysis, as determined by glycerol release, was stimulated by 22K-hGH (biosynthetic and pituitary derived), 20K-hGH (pituitary derived), and RCM-hGH (modified biosynthetic). These growth hormone preparations also exhibited similar antilipolytic activity (i.e., transient inhibition of glucagon-induced lipolysis). However, unlike human growth hormone, human placental lactogen neither stimulated lipolysis nor inhibited glucagon-stimulated lipolysis. Some augmentation of glucagon-stimulated lipolysis was observed in the presence of human placental lactogen. These results indicate that the disulfide bridges (Cys53----Cys165; Cys182----Cys189) and amino acid residues 32-46 of hGH are not required for lipolytic or antilipolytic activities of human growth hormone on chicken adipose tissue.     

Immunofluorescent localization of ovine placental lactogen

Summary The localization of ovine placental lactogen (OPL) was studied using the immunofluorescence method. The placentome sections were treated by an indirect technique with anti-OPL antibodies obtained from rabbits injected with purified hormone. OPL was located in large cells of the monostratified epithelium of chorionic villi. These cells are mono- or binucleated and PAS-positive. The immunological reaction was inhibited by the specific antigen (OPL, 400g/ml undiluted anti-OPL antiserum) but not by ovine prolactin, bovine growth hormone, human placental lactogen nor by any other polypeptidic hormone tested. Reciprocally, the localization of OPL-secreting cells was unsuccessful with antibodies raised against these control hormones.     

Super-active forms of placental lactogen and prolactin

Both placental lactogen and prolactin can be converted into super-active forms. These super-active hormones, in combination with insulin and hydrocortisone, stimulate accumulation of α-lactalbumin and increase RNA synthesis in explants from mouse mammary glands to an extent greater than the maximal level obtained with the native hormones. Also, they are able to stimulate RNA synthesis by suspensions of mammary epithelial cells which have lost the ability to respond to native lactogen and prolactin.     

Biochemical evidence that human placental lactogen and human chorionic gonadotropin are not stored in cytoplasmic secretion granules

The intracellular storage sites for the human placental hormones placental lactogen (hPL) and chorionic gonadotropin (hCG) are unknown. To determine whether hPL and hCG are stored in cytoplasmic secretion granules, we have compared the localization of hPL and hCG in placental homogenates following differential and density-gradient centrifugations to those of prolactin (PRL) and luteinizing hormone (LH) in human and rat pituitary homogenates. In the differential centrifugation studies, 93.1 +/- 4.1% (mean +/- SE) of the hPL and 79.4 +/- 6.0% of the hCG were detected in the postmicrosomal supernatant of placental homogenates. In contrast, 95-98% of the hPRL and hLH in the pituitary homogenates were detected in particulate fractions. Following centrifugation on sucrose-density gradients, particulate hPL and hCG were distributed diffusely throughout the gradients, while greater than 90% of the pituitary hormones sedimented as single peaks with densities of 1.22 g/cm3. When human placental and rat pituitary tissues were homogenized together prior to differential and density-gradient centrifugations, similar marked differences were observed between the distribution of the placental and pituitary hormones. These results strongly suggest that the placental hormones hPL and hCG, unlike pituitary PRL and LH, are not stored in large secretory granules. Differences in the intracellular storage sites of the hormones may explain, in part, differences in the regulation of peptide hormone secretion by placental and pituitary tissues.     

Extracellular sodium is required for methacholine-induced secretion of mucus glycoconjugates from canine tracheal explants

Extracellular sodium is known to influence secretion by certain secretory cells, possibly by mobilizing calcium from cellular stores or by altering intracellular pH via regulation of a Na(+)-H+ antiport system. Using canine tracheal explants, we determined whether agents which alter sodium fluxes are capable of modulating basal or cholinergically-induced secretion of mucus glycoconjugates. Methacholine, a cholinergic agonist, increased mucus secretion from explants incubated in the presence or absence of calcium, but had no effect on secretion when incubated in sodium-deficient media, indicating (a) that cholinergically-induced secretion can be mediated by mobilization of cellular calcium and (b) that extracellular sodium was required for this stimulatory effect. Several agents which increase intracellular sodium were tested for their effect on mucus secretion. Ouabain, a sodium pump inhibitor, and veratridine, a sodium channel activator, did not significantly affect control or methacholine-induced secretion; gramicidin, a sodium ionophore, also had no effect on basal release. Tetrodotoxin, a sodium channel inhibitor, was also without effect on basal or methacholine-stimulated mucus release. Agents which alter intracellular pH were also examined for their effects on basal or methacholine-induced glycoconjugate secretion. Amiloride, which decreases intracellular pH by inhibiting Na(+)-H+ exchange, produced a 19 per cent increase in basal secretion (not statistically significant), but had no effect on methacholine-induced secretion. An agent, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which decreases intracellular pH by inhibiting HCO3(-)-Cl- exchange, elicited decreases in both basal and methacholine-induced secretion, but the inhibition did not reach statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of rat serum on gonadotrophin release in vitro

Gonadotrophin release from a monolayer of cultured pituitary cells from the rat was increased by the addition of fresh rat serum or rat serum albumin to the culture medium. Similar effects were obtained when horse, chicken, lamb or porcine serum was used. Fetal bovine serum or bovine serum albumin showed a stimulatory effect on FSH and LH release but, unlike the case with rat serum, a dose-response release could not be demonstrated. In addition, fresh rat serum caused the accumulation of secretory granules in the gonadotrophs. The addition of LHRH to the culture medium also stimulated gonadotrophin release in a dose-response dependent manner. The dependence of the gonadotrophs on LHRH was markedly increased by the addition of 50% fresh rat serum to the culture medium. These results indicate that fresh rat serum contains some unknown factor(s) which 1) stimulates gonadotrophin release, 2) causes an accumulation of secretory granules, and 3) is important for maintaining secretory capacity of gonadotrophs.     

Characterization of super-active insulin,prolactin and placental lactogen

Complexes between insulin, prolactin or placental lactogen and cyanogen bromide-activated Sepharose release hormone-like materials when treated with bovine serum albumin (BSA) (1). These materials have enhanced biological activities, and are, presumably, N₁N₂-disubstituted guanidines in which the hormones and BSA are the substituents. The present studies show that ammonium bicarbonate can substitute for BSA in the generation of the super-active hormones. Super-activity of the released, guanidinated hormones, therefore, can be manifested in the absence of the BSA substituent. The key derivatized amino acid residues have not yet been identified, but it appears that guanidination of lysine is either unnecessary or insufficient. Several operational considerations which are important for the demonstration of these enhanced activities are discussed.     

Implantation and placental development in somatic cell clone recipient cows

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.     

The effects of insulin-like growth factor-I and insulin on placental lactogen production by human term placental explants

The effects of insulin-like growth factor (IGF)-I and insulin on placental lactogen production (hPL) by term human placental explants were studied. The hPL content in medium and explant decreased rapidly after first 24 hours of culture. The decrease thereafter was gradual and reached a plateau by day 4 of culture. The decrease of HPL content in placental culture has previously been suggested being due to the depletion of a rapidly secreting preformed pool of hPL. Addition of IGF-I (0.1-10 micrograms/ml) and insulin (1-20 micrograms/ml) stimulated the decreased level of hPL in tissue and medium after 24 hours in culture. IGF-I was 10 times more potent than insulin in stimulating hPL. These findings suggest that IGF-I and insulin effects the production of hPL by placenta. The lower potency of insulin may indicate that the effect of insulin on hPL production is via IGF-I receptor.     

Isolation and identification of a cDNA clone of rat placental lactogen II

The developing rat placenta expresses two placental lactogens at different stages of pregnancy: rat placental lactogen I from Days 11 to 13 of pregnancy and rat placental lactogen II (rPLII) from Day 12 to term. In this paper, we describe cDNA clones for rPLII, which have been isolated from a Day 18 rat placental cDNA library. The rPLII clones hybrid-select a mRNA which translates in vitro to a protein of 25,000 daltons. This protein is processed by dog pancreatic microsomes to a 22,000-dalton form, identical in size to rPLII isolated from pregnant rat serum. Both forms are precipitated by an anti-rPLII antiserum and an anti-ovine prolactin antiserum. The mRNA for rPLII is first expressed in Day 12 placenta and reaches a maximum at about Day 18 of pregnancy, in parallel with the appearance of the hormone in serum. Sequencing of the cDNA shows that, unlike human placental lactogen which is 85% homologous to human growth hormone at the amino acid level, rPLII is much more closely related to the prolactins. Thus, rPLII is 52% homologous to rat prolactin at the amino acid level, but only 34% related to rat growth hormone. This is the second placental lactogen to be fully characterized, and in the rat this hormone appears to have evolved by a route quite different from that which produced placental lactogen in humans.     

The role of calcium in insulin release from the human fetal pancreas

Previous experiments have established that the human fetal pancreas is relatively unresponsive to glucose as regards insulin release, but will secrete this hormone when exposed to agents which increase levels of cAMP or which activate protein kinase C. The current experiments were designed to establish which role another major stimulus, calcium, had in the release of insulin from this organ. For this purpose, cultured explants of human fetal pancreas were exposed to stimuli either in static or dynamic stimulation. The data show that insulin release is enhanced in the presence of 10 mM Ca2+, as well as the calcium ionophores A23187 and ionomycin, the latter agent being effective only if extracellular Ca2+ was present. A biphasic response was seen for Ca2+ but only a second phase response for A23187. Voltage-dependent calcium channels were shown to be present by the ability of the calcium channel blocker, verapamil, to inhibit insulin release caused by an agent that depolarizes membranes, potassium. The essential role of extracellular calcium in the insulinogenic effect of agents which increase cAMP levels--theophylline--and which activate protein kinase C--12-O-tetradecanoylphorbol-13-acetate--was demonstrated by showing (a) partial inhibition of insulin secretion by calcium channel blockers, (b) no enhancement of insulin release in the absence of extracellular calcium and (c) greater enhancement of insulin release in the presence of the calcium channel activator BAY-K-8644, which caused no stimulation by itself. These data put into better perspective our understanding of the mechanisms involved in insulin release from the human fetal pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine 3':5'-cyclic monophosphate (cAMP) is not the mediator of kappa opiate effect on human placental lactogen release.

We previously reported that kappa opiates stimulated the release of human placental lactogen (hPL) from human placental cells. In this study, we investigated the role of adenylate cyclase as a potential cellular mediator of such an effect. Incubations with ethylketocyclazocine (EKC) led to a time- and dose-dependent inhibition of adenylate cyclase activity. The maximal inhibition was 45 +/- 5% of control value after 15 min exposure to 10(-7)M EKC. This inhibition was reversed by opiate antagonist naloxone and was specific to kappa opiate type. Preincubation of human trophoblastic cells with 0.1 microgram/ml Islet-Activating-Protein (IAP; also called pertussis toxin) did not modify basal adenylate cyclase activity but abolished the inhibition of adenylate cyclase activity by EKC, indicating that the effect of opiates on cAMP production was mediated by an IAP-sensitive GTP binding protein. Also, IAP stimulated basal hPL release; the control levels were 22.4 ng/ml and 46.5 ng/ml without and with IAP respectively. However, the EKC-stimulated hPL levels were unchanged by preincubation with IAP. This difference in cAMP and hPL response in IAP-treated cells suggested that the opiate receptors are not directly coupled to adenylate cyclase. This hypothesis was confirmed by 1) experiments on placental membranes showing that in absence of the cytoplasmic elements (membranes only), EKC had no effect on membrane adenylate cyclase and 2) experiments on placental cells showing that dibutyryl-cAMP (dbcAMP) stimulated hPL release.     

Identification of placental transforming growth factor-beta and bikunin metabolites as contaminants of pharmaceutical human chorionic gonadotrophin preparations by proteomic techniques

A contaminant protein complex found in pharmaceutical urinary human chorionic gonadotrophin preparations is reported to have anti-human immunodeficiency virus-associated Kaposi's sarcoma activity. The aim of this study was to isolate and characterize this protein complex by proteomic approaches. Size exclusion chromatography was used in the isolation of these human chorionic gonadotrophin-associated fragments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of a protein complex that dissociated into two protein bands under reducing conditions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of this complex showed three polypeptides at approximately 6.2, 11.4, and 15.8 kDa. Peptide mass mapping and N-terminal amino acid sequencing identified two polypeptides as metabolites of placental transforming growth factor-beta (11.4 kDa) and bikunin (15.8 kDa). Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of the anti-human immunodeficiency virus-associated Kaposi's sarcoma active preparations CG-10 (Sigma), Pregnyl (Organon), and Profasi (Serono) revealed the presence of metabolites of placental transforming growth factor-beta in all three; no other non-human chorionic gonadotrophin-related protein species were observed in these preparations. Our findings present evidence that urinary human chorionic gonadotrophin preparations are contaminated with metabolites of placental transforming growth factor-beta, which may have transforming growth factor-beta agonist actions, and metabolites of bikunin, which is a protease inhibitor. In combination these molecules may be responsible for the anti-human immunodeficiency virus-associated Kaposi's sarcoma activity demonstrated for these urinary human chorionic gonadotrophin preparations.