Intracellular molecular species of human chorionic gonadotropin from normal but non-cultured first trimester placental tissues

Intracellular forms of human chorionic gonadotropin (hCG) were analyzed by SDS-polyacrylamide gel electrophoresis, protein blotting and immunological techniques in normal but non-cultured first trimester placentae. Placental cells were found to contain the major components of the 23K and 19K forms of the beta-subunit and the 21K form of the alpha-subunit of hCG which remained sensitive to endoglycosidase H and Con A-Sepharose 4B and small amounts of mature (urinary) subunits. An unknown molecular species of the alpha-subunit (Mr = 17K) that was not bound to Con A-Sepharose 4B was also detected. These intracellular molecular species accumulated in the placentae mainly during the first trimester. These results suggest that hCG subunits accumulate in placental cells as predominant intermediates containing high-mannose oligosaccharides.     

Ontological differences in first compared to third trimester human fetal placental chorionic stem cells

Human mesenchymal stromal/stem cells (MSC) isolated from fetal tissues hold promise for use in tissue engineering applications and cell-based therapies, but their collection is restricted ethically and technically. In contrast, the placenta is a potential source of readily-obtainable stem cells throughout pregnancy. In fetal tissues, early gestational stem cells are known to have advantageous characteristics over neonatal and adult stem cells. Accordingly, we investigated whether early fetal placental chorionic stem cells (e-CSC) were physiologically superior to their late gestation fetal chorionic counterparts (l-CSC). We showed that e-CSC shared a common phenotype with l-CSC, differentiating down the osteogenic, adipogenic and neurogenic pathways, and containing a subset of cells endogenously expressing NANOG, SOX2, c-MYC, and KLF4, as well as an array of genes expressed in pluripotent stem cells and primordial germ cells, including CD24, NANOG, SSEA4, SSEA3, TRA-1-60, TRA-1-81, STELLA, FRAGILIS, NANOS3, DAZL and SSEA1. However, we showed that e-CSC have characteristics of an earlier state of stemness compared to l-CSC, such as smaller size, faster kinetics, uniquely expressing OCT4A variant 1 and showing higher levels of expression of NANOG, SOX2, c-MYC and KLF4 than l-CSC. Furthermore e-CSC, but not l-CSC, formed embryoid bodies containing cells from the three germ layer lineages. Finally, we showed that e-CSC demonstrate higher tissue repair in vivo; when transplanted in the osteogenesis imperfecta mice, e-CSC, but not l-CSC increased bone quality and plasticity; and when applied to a skin wound, e-CSC, but not l-CSC, accelerated healing compared to controls. Our results provide insight into the ontogeny of the stemness phenotype during fetal development and suggest that the more primitive characteristics of early compared to late gestation fetal chorionic stem cells may be translationally advantageous.     

Zebra chorionic gonadotropin: partial purification and characterization

Six samples of pregnant zebra (z) serum from the first and second trimesters of pregnancy were analyzed by RIA and shown to have chorionic gonadotropin levels comparable to that of the mare (0.9-5.3 micrograms/ml); first trimester levels in most cases were higher than second trimester levels. A pool of the sera (10 ml) was fractionated by methods previously employed for the purification of equine (e) and donkey (d) chorionic gonadotropin to achieve a concentration of the zebra chorionic gonadotropin (zCG). A yield of 1.0 mg of glycoprotein was obtained. HPLC analysis of the material indicated the content of zCG to be about 7%. Its molecular size as judged by Ve/Vo values is smaller than eCG, greater than ovine LH, and about the same as equine LH. The zCG was tested in RIAs for LH and eCG, radioreceptor assays (RRA) for LH and FSH, and the rat testis Leydig cell assay for LH. Comparisons were made with equine and donkey chorionic gonadotropin, and equine and zebra LH. The results, preliminary because the preparation is not of high purity, showed that zCG is bioactive as an LH; immunologically similar to eCG, eLH, dCG, and zLH; and competes in RRAs for LH but not FSH receptors. It differs, therefore, from eCG and eLH--which have high levels of intrinsic FSH activity, and is more like dCG, dLH, and zLH--all of which have minimal if any FSH activity.     

Subcellular localization of intracellular forms of human chorionic gonadotropin in first trimester placenta

To determine the subcellular sites for synthesis and processing of human chorionic gonadotropin subunits in cells, first trimester placental cells were fractionated subcellularly on sucrose density gradients. Analysis of the subcellular fractions by immunobinding techniques revealed that the rough endoplasmic reticulum-rich fraction contained only intermediates having high-mannose oligosaccharides, but the Golgi-rich fraction contained not only intermediates but also mature forms which were resistant to endoglycosidase H but sensitive to neuraminidase. These results show that human chorionic gonadotropin subunits are synthesized in the rough endoplasmic reticulum as forms containing high-mannose oligosaccharides, and their maturation occurs in the Golgi apparatus by trimming with endogenous glycosidases. They are then modified by addition of complex oligosaccharides and terminal sialic acid through glycosyltransferases.     

Effect of adrenergic agonists on human chorionic gonadotropin release by human trophoblast cells obtained from first trimester placenta

The cultured syncytiotrophoblast cells from human first trimester placenta were used to determine the effect of adrenergic agonists on human chorionic gonadotropin (hCG) production in vitro. Beta-adrenergic agonists isoproterenol, ritodrine and isoxsuprine increased the hCG release during the 2 h incubation period, however, alpha-agonists norepinephrine and phenylephrine and a beta 1-agonist dobutamine had no effect. The effect of isoproterenol was blocked by propranolol and butoxamine, but less efficiently by phentolamine and atenolol. These results indicate that placental hCG production can be modulated by stimulation of beta-, possibly beta 2-adrenoceptors but not by alpha-adrenoceptors.     

Role of calcium in secretion of chorionic gonadotropin by first trimester human placenta.

The role of calcium in regulation of secretion of human chorionic gonadotropin (hCG) by first trimester human placental minces in vitro has been investigated. Depletion of calcium in the medium by addition of EGTA resulted in a drastic decrease in the levels of immunoreactive hCG in the medium with consequent of accumulation of hCG in the tissue. Addition of A 23187 which is a calcium ionophore resulted in a dose dose dependent increase in the hCG in the medium and this stimulatory response could not be observed in the absence of calcium. Use of lanthanum (a calcium antagonist) in place of calcium in the medium used resulted in a significant decrease in the levels of hCG in the medium. Addition of veratridine (a sodium channel activator) stimulated hCG secretion in a dose dependent manner. These results suggest that calcium is essential for normal secretion of hCG by human placenta.     

Synthesis of first trimester placental alkaline phosphatase in cultured human term placental cells

Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta.     

Existence of associated, non-associated, and oligomeric forms of human chorionic gonadotropin subunits in placental extracts

The molecular sizes of human chorionic gonadotropin (hCG) subunits in the native state in normal first trimester placental extracts were determined by gel filtration on Sephacryl S-300, followed by SDS-polyacrylamide gel electrophoresis, protein blotting, and immunobinding analysis using anti-alpha and - beta antibodies. Mature forms of hCG subunits in the extracts were only found in the same fraction as that which contained standard urinary hCG, indicating an alpha beta dimer. On the other hand, immature forms were detected with a wide range of molecular weights, which were higher than that of standard hCG, suggesting oligomerization of associated or non-associated immature subunits. In order to determine the associated state of these subunits, various forms of associated subunits (hCG alpha beta) in placental extracts were immunoprecipitated with anti-hCG antiserum, which only recognized hCG alpha beta, and Protein A-Sepharose. They were then analyzed by SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions, followed by immunobinding assaying. It has been suggested that there are three kinds of hCG alpha beta S (one mature and two immature). To confirm the above results and to clarify the existence of free subunits, placental extracts were subjected to two-dimensional SDS-polyacrylamide gel electrophoresis. With this technique, high molecular weight forms of immature hCG subunits were found to be present in placental cells as an oligomer of not only the alpha beta dimer but of each subunit as well.     

Human placental chorionic renin: production, purification and characterization

Native human renin, produced from the culture of human chorionic trophoblasts, has been purified to homogeneity on a milligram scale using a five-step purification scheme. The chorion cells secrete 50-200 milliGoldblatt Units of trypsin-activatable prorenin per ml into the medium. The pro-enzyme is partially purified by ammonium sulfate fractionation and chromatographies on QAE-Sephadex and cibracon blue-agarose. Following conversion of prorenin to the active enzyme by porcine trypsin, the renin is purified to homogeneity by affinity chromatography and gel filtration. Chorionic prorenin has a molecular weight of 43,000; the active enzyme 40,000. Both proteins exist as a single polypeptide chain as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions. The average specific activity of six different preparations was found to be 1072 Goldblatt Units/mg. The amino acid composition and N-terminal sequence of the active enzyme has been determined and is identical to the human kidney enzyme. Microheterogeneity of chorionic renin was demonstrated by isoelectrofocusing analysis. The physical characterization of chorionic renin is compared with that reported for the human kidney enzyme.     

The synthesis and secretion of human chorionic gonadotropin by tissue slices from first trimester placentas

Placental tissue slices from first trimester placentas synthesize and secrete proteins labeled by radioactive glucosamine are preferentially secreted as compared to proteins in general. One of the proteins synthesized and secreted is hCG. Processing and secretion of proteins, including hCG, by the tissue slices need a two-hour period. Both secretion and glycosylation of the protein can take place independently of protein synthesis. A method was developed for the specific determination of newly synthesized radioactive hCG in placental tissue.Department of Reproductive Biology, Case Western Reserve University, Cleveland, Ohio, USA     

Gonadotropin-releasing hormone effects on placental hormones during gestation: I. Alpha-human chorionic gonadotropin, human chorionic gonadotropin and human chorionic somatomammotropin

The release of alpha-human chorionic gonadotropin (alpha hCG), gonadotropin human chorionic gonadotropin (hCG) and human chorionic somatomammotropin (hCS) in vitro from placentas of different gestational ages was studied. In addition, the effect of gonadotropin-releasing hormone (GnRH) on these hormonal releases, as related to the gestational age of the placenta cultured and the dose of GnRH, was determined. The basal release of alpha hCG and hCG was greatest at 9-13 wk of gestation (1000-1500 ng/mg and 250-350 ng/mg, respectively). Lowest release rates were at term (28 ng/mg and 20 ng/mg, respectively). Hormonal release declined with extended culture, except from the cultures of 13- and 15-wk placentas, in which the initially high release continued throughout the 8 days of culture. The initial release of hCS was low at 6 wk, increased to maximum rates by 15 wk, and was similar to the initial rate of release at term. Gonadotropin-releasing hormone stimulated the release of alpha hCG and hCG most dramatically in cultures of 16-wk and 17-wk placentas, where as much as a 400- and 250-fold increase, respectively, on Day 6 of culture was observed (p less than 0.0001). In term placenta cultures after 6 days in vitro, a 20-fold stimulation of alpha hCG and a 10-fold increase of hCG was effected by GnRH (p less than 0.001). The largest responses of alpha hCG and hCG to GnRH were observed when estrogen levels were low. Dose-related responses were observed in some placentas, yet in some instances, maximal effects were attained with all doses utilized in these studies (0.2 to 50 micrograms/ml). These data demonstrate that human placentas of different gestational ages have varying hormonogenic capabilities in vitro. The data also establish that synthetic GnRH is capable of stimulating alpha hCG and hCG production, but the degree and pattern of response to GnRH stimulation are related to the gestational age of the placental tissue and its time in culture. The most responsive period to exogenous GnRH stimulation of alpha hCG and hCG release was on Days 5 and 6 of culture, when basal estrogen release was very low. These data support the hypothesis that hCG release might be controlled by a chorionic GnRH stimulation and suggest that local steroid levels may modulate the hCG response to GnRH stimulation.     

Gonadotropin releasing hormone (GnRH) stimulates both secretion and synthesis of human chorionic gonadotropin (hCG) by first trimester human placental minces in vitro

An in vitro system using the minces of placental villi from first trimester human pregnancy (6-10 weeks) has been validated to examine the effect of addition of GnRH and its analogues on hCG secreted into the medium. Addition of low concentration of GnRH or its analogues (1 X 10(-8) M to 1 X 10(-6) M) resulted in an increase in the quantity of hCG in the medium, while addition of high concentrations of GnRH resulted in an inhibitory effect. Of the analogues tested, Buserelin was highly effective in exerting an inhibitory effect. A significant increase in 35S-methionine incorporation into immunoprecipitable hCG was noticed in the presence of GnRH. These results suggests that GnRH stimulates both synthesis and secretion of hCG by first trimester human placenta.     

Regulation of 5 alpha-reductase activity in cultured immature leydig cells by human chorionic gonadotropin

The present studies examined the hormonal regulation of 5 alpha-reductase activity in cultured immature rat Leydig cells. Within the testis 5 alpha-reductase was concentrated in the interstitial cell compartment, and among interstitial cells, the enzyme was localized primarily in Band 3 of Percoll density gradients, which contains the majority of Leydig cells. Among various factors reported previously to stimulate testicular 5 alpha-reductase activity when administered in vivo to immature rats (LH/hCG, FSH, luteinizing hormone releasing hormone or prolactin), only LH/hCG directly stimulated 5 alpha-reductase activity of cultured immature Band 3 cells. Neither growth hormone which was reported previously to stimulate hepatic 5 alpha-reductase activity, nor insulin, insulin-like growth factor-I, or epidermal growth factor, which have been reported to modulate Leydig cell function, had any effect on 5 alpha-reductase activity of Band 3 cells. These studies suggest that the major factor directly stimulating 5 alpha-reductase activity in Leydig cells during early maturation is LH. However, it is possible that other factors acting indirectly may modulate the maturational rise in 5 alpha-reductase activity.