Steroid-protein interaction in human placenta

Human placenta produces a large variety of bioactive substances with endocrine and neural competence: pituitary and gonadal hormones, hypothalamic-like releasing or inhibiting hormones, growth factors, cytokines and neuropeptides. The most recent findings indicate that locally produced hormones regulate the secretion of other placental hormones supporting a paracrine/autocrine regulation. In placental endocrinology, a particular relevance is played by steroid hormones. In fact, a specific gonadotropin-releasing hormone (GnRH)-human chorionic gonadotropin (hCG) regulation of placental steroidogenesis has been proposed as a placental internal regulatory system acting on steroids production from human placenta. In addition, activin and inhibin have been proposed as further regulatory substances of the synthesis and secretion of steroids; the addition of activin A to placental culture augments GnRH, hCG and progesterone, and this effect can be significantly reduced by the addition of inhibins. Finally, a steroid-steroid interaction is suggested by the evidence that placental estrogen has a positive role in the regulation of progesterone biosynthesis. Other steroid-protein interactions have been observed in human placenta. In fact, recent data indicate that progesterone inhibits placental corticotropin-releasing factor (CRF) and estrogens act on placental conversion of cortisol to cortisone, activating cortisol secretion by the fetal adrenal and enhancing fetal adrenal function with advancing gestation.     

GnRH-prohormone-containing neurons in the primate brain: immunostaining for the GnRH-associated peptide

The structure of the prohormone for mammalian gonadotropin releasing hormone (proGnRH) includes the GnRH decapeptide followed by a 56 amino acid GnRH-associated peptide (GAP). In this study, we compared immunostaining of brain neurons and fibers for GAP and GnRH in fetal rhesus monkeys and juvenile baboons. We used antisera against different portions of human and rat GAP (proGnRH 14-24, proGnRH 40-53, and proGnRH 52-66) or against GnRH and the PAP technique. Liquid phase absorption with GAP or GnRH confirmed the specificity of these antisera. Major accumulations of GAP immunoreactive (GAP+) perikarya occurred in the medial septal and preoptic areas and the nucleus of the diagonal band of Broca (44.6% in rhesus, 49.6% in baboon), supraoptic region including the area dorsal to the optic tract (21.9% in rhesus, 23.0% in baboon), and the medial basal hypothalamus (15.7% in rhesus, 16.4% in baboon), especially at the infundibular lip. Occasional cell bodies were scattered throughout the hypothalamic and forebrain regions studied. GAP+ fibers were widely distributed, but formed well-defined pathways such as the periventricular and ventral hypothalamic tract. In addition, GAP+ nerve terminals with various densities occurred in the lamina terminalis, the zona externa of the infundibulum, and behind the infundibular stalk. Fetal rhesus macaques had more GAP+ cell bodies, denser fiber networks, and more distinct pathways than juvenile baboons. However, fiber and terminal immunostaining was somewhat less intense for GAP than GnRH in comparable regions. These results indicate that proGnRH (GAP) is present in the same population of neurons as GnRH in the primate brain. They also suggest that post-translational products of proGnRH are present in perikarya, axons and terminals, and that GnRH and GAP and/or further cleavage products are consecreted into hypophysial portal blood in the primate.     

A specific antibody to a new peptide growth factor from human placenta: immunocytochemical studies on its location and biosynthesis

Recently, we isolated a new peptide growth factor of Mr 34 000 from synctial membranes of human placenta. In its polypeptide molecular weight and receptor binding specificity it is unlike several known growth factors. In this paper we described immunocytochemical studies on its cellular location and biosynthesis. A rabbit antiserum was raised against a homogeneous preparation of the placental peptide. The specificity of the antibody was established by immunoprecipitation and immunoblot analyses. The antibody recognized both the native and denatured 34-kilodalton (kDa) peptide but showed no binding to a variety of other growth factors and hormones tested. The antibody was used to investigate the genesis and location of the 34-kDa membranous mitogen. Immunoperoxidase staining of placental tissue slices revealed a restricted localization of the antigen in the cytoplasmic organelles of cytotrophoblasts and in the brush border membranes of syncytiotrophoblasts. No other placental structures contained the antigen. A developmentally regulated appearance of the mitogen was suggested by the fact that first trimester placenta consistently stained far more strongly than term placenta. These studies show that the 34-kDa mitogenic protein originates in placenta from embryo-derived cellular structures and suggest that in its strategic location it may influence trophoblastic growth in an autocrine manner. In other studies we investigated the presence and biosynthesis of the 34-kDa peptide in the A431 vulval carcinoma cell line, which was shown earlier to contain a membrane-associated 34-kDa growth factor. The studies demonstrate that this cell line, as well as some other human carcinomas of breast and bladder origin, actively expresses this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroanatomical localization of cells containing gonadotropin-releasing hormone messenger ribonucleic acid in the primate brain by in situ hybridization histochemistry

Using in situ hybridization histochemistry, we have mapped the anatomic localization of perikarya containing mRNA that codes for GnRH and GnRH-associated protein (GAP) in the forebrain of four male macaques, Macaca fascicularis. DNA oligomers, with sequences complementary to either the GnRH or the GAP portion of the mRNA sequence, were synthesized and hybridized to paraformaldehyde fixed, coronal sections of the basal forebrain and hypothalamus. GnRH mRNA was found in the same population of cells as those containing GAP mRNA. GnRH/GAP mRNA-containing cell bodies were observed consistently in the medial septal nucleus, the diagonal band of Broca, the medial preoptic area, supraoptic nucleus, and ventromedial-infundibular region. We detected the presence of GnRH mRNA and GAP mRNA within the same neuroanatomic regions previously shown to include perikarya containing immunoreactive GnRH. The ventromedial-infundibular region and the medial preoptic region contained the greatest number of GnRH/GAP mRNA-containing perikarya (37.0% and 22.5%, respectively). The diagonal band contained 21.0% and the supraoptic nucleus 13.0% of the cells, while the medial septum contained the fewest number (6.7%). This study demonstrates the feasibility of using in situ hybridization as a strategy to study the developmental and steroidal regulation of GnRH gene expression in the nonhuman primate.     

Regulation of growth hormone secretion in human trophoblastic cells in culture

In this study, we have cultured in vitro purified trophoblastic cells from first-trimester and term human placenta. These cells were obtained by specific enzymatic digestion and centrifugation through a Percoll gradient. Using 2 specific monoclonal antibodies, the pituitary 22-kD growth hormone (GH) and the placental GH variant were assayed in the culture medium by radioimmunoassay. After 48 h of culture, only the placental GH variant was measured in the medium corresponding to first-trimester placenta (3.4 ng/24 h/10(5) cells). Surprisingly, an immunoactivity pattern of pituitary GH type was found in 3 out of 5 media conditioned with term placenta cells, while GH immunoactivity was very low, around the detection level, in the 2 others. These secretions are not modified with the time in culture and the state of differentiation of the cells from cytotrophoblast to syncytiotrophoblast. Neither in early nor in term placenta does the addition of GH-releasing factor (10(-6) M in the culture medium) stimulate the secretion of pituitary 22-kD GH or placental GH variant.     

Regulation of leptin expression by 17beta-estradiol in human placental cells involves membrane associated estrogen receptor alpha

The placenta produces a wide number of molecules that play essential roles in the establishment and maintenance of pregnancy. In this context, leptin has emerged as an important player in reproduction. The synthesis of leptin in normal trophoblastic cells is regulated by different endogenous biochemical agents, but the regulation of placental leptin expression is still poorly understood. We have previously reported that 17β-estradiol (E(2)) up-regulates placental leptin expression. To improve the understanding of estrogen receptor mechanisms in regulating leptin gene expression, in the current study we examined the effect of membrane-constrained E(2) conjugate, E-BSA, on leptin expression in human placental cells. We have found that leptin expression was induced by E-BSA both in BeWo cells and human placental explants, suggesting that E(2) also exerts its effects through membrane receptors. Moreover E-BSA rapidly activated different MAPKs and AKT pathways, and these pathways were involved in E(2) induced placental leptin expression. On the other hand we demonstrated the presence of ERα associated to the plasma membrane of BeWo cells. We showed that E(2) genomic and nongenomic actions could be mediated by ERα. Supporting this idea, the downregulation of ERα level through a specific siRNA, decreased E-BSA effects on leptin expression. Taken together, these results provide new evidence of the mechanisms whereby E(2) regulates leptin expression in placenta and support the importance of leptin in placental physiology.     

GnRH systems of <Emphasis Type="Italic">Cichlasoma dimerus</Emphasis> (Perciformes,Cichlidae) revisited: a localization study with antibodies and riboprobes to GnRH-associated peptides

The distribution of cells that express three prepro-gonadotropin-releasing hormones (GnRH), corresponding to salmon GnRH, sea bream GnRH (sbGnRH), and chicken II GnRH, was studied in the brain and pituitary of the South American cichlid fish, Cichlasoma dimerus. Although the ontogeny and distribution of GnRH neuronal systems have previously been examined immunohistochemically with antibodies and antisera against the various GnRH decapeptides, we have used antisera against various perciform GnRH-associated peptides (GAPs) and riboprobes to various perciform GnRH+GAPs. The results demonstrate that: (1) the GnRH neuronal populations in the forebrain (salmon and sea bream GAPs; sGAP and sbGAP, respectively) show an overlapping pattern along the olfactory bulbs, nucleus olfacto-retinalis, ventral telencephalon, and preoptic area; (2) projections with sGAP are mainly located in the forebrain and contribute to the pituitary innervation, with projections containing chicken GAP II being mainly distributed along the mid and hindbrain and not contributing to pituitary innervation, whereas sbGAP projections are restricted to the ventral forebrain, being the most important molecular form in relation to pituitary innervation; (3) sbGnRH (GnRH I) neurons have an olfactory origin; (4) GAP antibodies and GAP riboprobes are valuable tools for the study of various GnRH systems, by avoiding the cross-reactivity problems that occur when using GnRH antibodies and GnRH riboprobes alone.This work was supported by grants UBACYT X-217, Conicet PIP 0539/R188; NIH-HD-29186 and Fogarty International Fellowship TW00086.     

Differential inhibition of human placental prostaglandin release in vitro by a GnRH antagonist

Previously, we have demonstrated that the production of prostaglandins by human placental tissue varied with gestational age. In addition, we have shown that placental prostaglandin release was affected by GnRH, and that its response was also dependent on the gestational age of the placenta. Thus, we have studied the effect of a GnRH antagonist ([N-Ac-Pro1,D-p-Cl-Phe2,D-Nal(2)3,6-LHRH, Syntex Research, Palo Alto, CA) on basal prostaglandin release from placentas of 6 to 15 weeks' gestation and found that this antagonist (1 microgram/ml) effects an inhibition of the release of prostaglandin E, prostaglandin F, and 13,14-dihydro-15-keto-prostaglandin from placentas of 13 and 15 weeks of gestation. This effect was not overridden by GnRH at 10 times the antagonist concentration in the 13-week placental cultures, but was totally reversed by GnRH (10 micrograms/ml) in the 15-week placental cultures. These data demonstrate that this GnRH antagonist can affect human placental prostaglandin production at 13 to 15 weeks of gestation and indicate that endogenous placental GnRH-like activity may exert a control over placental prostaglandin release at this gestational stage.     

Fish gonadotropin-releasing hormone gene and molecular approaches for control of sexual maturation: development of a transgenic fish model.

The prepro-GnRH gene and mRNA primary structure were fully established from Atlantic salmon (Salmo salar) and partially from rainbow trout (Oncorhynchus mykiss). Results show that the GnRH coding region of 30 base pairs is well conserved during evolution. In contrast, the GnRH-associated peptide (GAP) sequence shows very limited homology when the GnRH genes from mammalian and teleost species are compared. A simple method for selecting transgenic fish after transfer of the firefly luciferase gene was developed. The method involves bioluminescent measurement of live animals in a scintillation counter.     

Effect of sera from women with systemic lupus erythematosus or antiphospholipid syndrome and recurrent abortions on human placental explants in culture

BACKGROUND: Systemic lupus erythematosus (SLE) with or without evidence of antiphospholipid antibodies (aPA) and antiphospholipid syndrome (APS) is associated with a high rate of spontaneous abortions. The placenta is thought to be the site of pathological damage in many of these abortions. To test this hypothesis, we studied the effects of sera obtained from women with SLE with or without treatment on human placental explants in culture. METHODS: We cultured 5.5- to 7.5-week-old human placental explants in a culture medium containing F-12 DMEM and 10% FCS or in 90% human serum obtained from nonpregnant women with SLE prior to or after treatment. Culture was carried out for 96 hr. At the end of the culture period, we studied the secretion of the placental hormones estrogen (E2), progesterone (PGN), and human chorionic gonadotropin (hCG). In addition, we studied the proliferation rate (using PCNA staining) and the rate of apoptosis (using ApoTag) of the trophoblastic cells. RESULTS: Placentae grew better in normal human serum than in a chemically defined medium of F-12 DMEM and 10% FCS. Enhanced growth and higher secretion rates for hCG and estradiol (E2) were manifested in placentae cultured in control sera with no change in PGN secretion. Secretion rates of hCG and PGN (but not of E2 in the treated group) by placental explants were similar to that of controls. However, the serum levels prior to culture were not measured. Further, explants in serum from untreated women with SLE produced a significant decrease in the proliferation rate of the trophoblastic cells and an increase of apoptosis. Treatment significantly reduced the apoptotic rate and increased cell proliferation, but the cell proliferation rate was still lower than that noted in controls. CONCLUSIONS: We conclude that sera from women with SLE may directly damage the developing placenta reducing proliferation and enhancing apoptosis. Successful treatment of the women reduces that damage.     

Differential inhibition of human prostaglandin release in vitro by a GnRH antagonist

Previously, we have demonstrated that the production of prostaglandins by human placental tissue varied with gestational age. In addition, we have shown that placental prostaglandin release was affected by GnRH, and that its response was also dependent on the gestational age of the placenta. Thus, we have studied the effect of a GnRH antagonist ([N-Ac-Pro¹, D-p-Cl-Phe², D-Nal (2)^3,6-LHRH, Syntex Research, Palo Alto, CA) on basal prostaglandin release from placentas of 6 to 15 weeks' gestaton and found that this antagonist (1 μg/ml) effects an inhibition of the release of prostaglandin E, prostaglandin F, and 13, 14-dihydro-15-keto-prostaglandin from placentas of 13 and 15 weeks of gestation. This effect was not overridden by GnRH at 10 times the antagonist concentration in the 13-week placental cultures, but was totally reversed by GnRH (10 μg/ml) in the 15-week placental cultures. These data demonstrate that this GnRH antagonist can affect human placental prostaglandin production at 13 to 15 weeks of gestation and indicate that endogenous placental GnRH-like activity may exert a control over placental prostaglandin release at this gestational stage.     

Immunocytochemical demonstration of GAP-like immunoreactive neuronal elements in the human hypothalamus and pituitary

GnRH-associated peptide (GAP)-like immunonreactive elements located in the human hypothalamus were investigated by PAP immunocytochemistry using specific antiserum against [pro-GnRH (14-69) OH]. Immunoreactive neuronal perikarya were distributed in the MPOA, PVN and infundibular nucleus, with the largest numbers of GAP-like immunoreactive perikarya found in the infundibular nucleus. We also detected the coexistence of GAP-like and GnRH-like immunoreactivities in the same neuronal perikarya in the MPOA by using a double immunolabelling procedure. In addition to the above regions immunoreactive neuronal perikarya were present in the region dorsal to the medial mammillary nucleus. GAP-like immunoreactive fibers were distributed in same areas that immunoreactive perikarya were observed. Many immunoreactive terminals were found adjacent to capillaries in the infundibulum. Immunoreactive dots, presumably terminals, were observed in the posterior pituitary and these were particularly evident along the margin adjacent to the anterior pituitary. The distribution pattern and density of GAP-like immunoreactive neuronal elements are compared with those of other mammalian species. We also compared GAP-like immunoreactive elements with that of GnRH as has been previously observed in the human hypothalamus.     

Immunocytochemical demonstration of GAP-like immunoreactive neuronal elements in the human hypothalamus and pituitary

Summary GnRH-associated peptide (GAP)-like immunoreactive elements located in the human hypothalamus were investigated by PAP immunocytochemistry using specific antiserum against [pro-GnRH (14–69) OH]. Immunoreactive neuronal perikarya were distributed in the MPOA, PVN and infundibular nucleus, with the largest numbers of GAP-like immunoreactive perikarya found in the infundibular nucleus. We also detected the coexistence of GAP-like and GnRH-like immunoreactivities in the same neuronal perikarya in the MPOA by using a double immunolabelling procedure. In addition to the above regions immunoreactive neuronal perikarya were present in the region dorsal to the medial mammillary nucleus. GAP-like immunoreactive fibers were distributed in same areas that immunoreactive perikarya were observed. Many immunoreactive terminals were found adjacent to capillaries in the infundibulum. Immunoreactive dots, presumably terminals, were observed in the posterior pituitary and these were particularly evident along the margin adjacent to the anterior pituitary. The distribution pattern and density of GAP-like immunoreactive neuronal elements are compared with those of other mammalian species. We also compared GAP-like immunoreactive elements with that of GnRH as has been previously observed in the human hypothalamus.     

Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources

Human ras GTPase-activating protein (GAP) is a cytoplasmic factor that stimulates the GTPase activity of normal N-ras p21 while having no stimulatory effect on the GTPase activity of oncogenic variants of N-ras p21. We have purified two forms of native ras GAP from human placental tissue. In addition to the Mr = 120,000 type I GAP reported previously (1), an equivalent amount of an Mr = 95,000 molecule with GAP activity was recovered and shown to have the N-terminal sequence expected for type II GAP. The two GAP forms in placental extracts were resolved by molecular sieve chromatography and appeared to have a monomeric native structure. Human recombinant type I GAP was produced intracellularly in Sf9 insect cells using a baculovirus expression vector, and 10-mg quantities were purified to homogeneity in three steps. Comparison of the purified native and recombinant GAP molecules revealed that all three displayed similar biological specific activities in an in vitro GAP assay. A polyclonal antibody to purified recombinant GAP was prepared and shown to neutralize the activity of both native and recombinant GAPs. The antibody was also highly specific for the detection of native GAP by Western blot. Type I and II GAP species were detected in approximately equal amounts in cytoplasmic extracts of human placenta, but only type I GAP was observed when other human tissues were examined.     

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.     

Heterogeneous X inactivation in trophoblastic cells of human full-term female placentas

In female mammalian cells, one of the two X chromosomes is inactivated to compensate for gene-dose effects, which would be otherwise doubled compared with that in male cells. In somatic lineages in mice, the inactive X chromosome can be of either paternal or maternal origin, whereas the paternal X chromosome is specifically inactivated in placental tissue. In human somatic cells, X inactivation is mainly random, but both random and preferential paternal X inactivation have been reported in placental tissue. To shed more light on this issue, we used PCR to study the methylation status of the polymorphic androgen-receptor gene in full-term human female placentas. The sites investigated are specifically methylated on the inactive X chromosome. No methylation was found in microdissected stromal tissue, whether from placenta or umbilical cord. Of nine placentas for which two closely apposed samples were studied, X inactivation was preferentially maternal in three, was preferentially paternal in one, and was heterogeneous in the remaining five. Detailed investigation of two additional placentas demonstrated regions with balanced (1:1 ratio) preferentially maternal and preferentially paternal X inactivation. No differences in ratio were observed in samples microdissected to separate trophoblast and stromal tissues. We conclude that methylation of the androgen receptor in human full-term placenta is specific for trophoblastic cells and that the X chromosome can be of either paternal or maternal origin.     

Human immunodeficiency virus type 1 infection of human placenta: potential route for fetal infection.

To determine the potential role of the placenta in transmission of human immunodeficiency virus (HIV) from mother to fetus, the ability of human placental tissue to support HIV type 1 (HIV-1) infection was examined. HIV-1-seronegative first-trimester placentas were maintained in culture and infected with HIV-1. Virus production, measured by HIV-1 antigen release into the supernatant, and HIV-1 DNA, identified by polymerase chain reaction, were detected for at least 12 days postinfection. Western immunoblot analysis showed Gag proteins, precursor p55, and cleavage products p24 and p17 in HIV-1-infected tissues. Double labeling of placental villi with antibodies to CD4 and placental trophoblast-specific alkaline phosphatase indicated that trophoblasts express CD4 antigen. Additionally, immunostaining of HIV-1-infected tissues with anti-p24 antibodies demonstrated HIV-1 protein expression in placental trophoblasts. Evaluation of human chorionic gonadotropin and progesterone production by the placental cultures indicated that there was a 90% decrease in human chorionic gonadotropin and a 70% decrease in progesterone production in HIV-1-infected cultures in comparison with controls. These data demonstrate that trophoblastic cells of human placenta tissue express CD4 and are susceptible to HIV-1 infection; also, placental endocrine function is decreased by HIV-1 infection. Thus, the placenta may serve as a reservoir of HIV-1 infection during pregnancy contributing to infection of the fetus, and decreased placental hormone production may result in impaired fetal development.     

Co-localization of P2Y1 receptor and NTPDase1/CD39 within caveolae in human placenta

Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39) is the dominant ecto-nucleotidase of vascular and placental trophoblastic tissues and appears to modulate the functional expression of type-2 purinergic (P2) G-protein coupled receptors (GPCRs). Hence, this ectoenzyme could regulate nucleotide-mediated signalling events in placental tissue. This immunohistochemical and immuno-electron microscopic study demonstrates the expression of NTPDase1/CD39, P2Y1 and P2Y2 receptors in different cell types of human placenta. Specifically P2Y1 has an exclusive vascular distribution whereas P2Y2 is localized on trophoblastic villi. Co-localization of P2Y1 and NTPDase1/CD39 are observed in caveolae, membrane microdomains of endothelial cells. The differential localization of these P2 receptors might indicate their unique roles in the regulation of extracellular nucleotide concentrations in human placental tissues and consequent effects on vascular tone and blood fluidity.