Regulation of steroidogenesis of infant and adult rhesus monkey granulosa cells in vitro

The present studies have demonstrated that infant monkey granulosa cells, like the adult ones, have the potential of responding markedly in vitro to human FSH, cyclic-AMP and forskolin, resulting in the increase of progesterone and estrogen production. Exogenous hCG was also capable of increasing FSH-stimulated progesterone biosynthesis in both infant and adult granulosa cells, but did not stimulate the infant granulosa cells to secrete estrogen. Addition of a synthetic estrogen, diethylstilbestrol, to the culture of monkey granulosa cells enhanced the FSH-stimulated progesterone and estrogen production. The steroidogenesis of monkey granulosa cells was also dramatically stimulated by a gonadotropin-releasing hormone agonist. Monkey granulosa cells, unlike the other animal cells, secrete measurable amount of estrogen in the absence of androgen substrate. The findings reported here are significant in regard to understanding of the mechanism of hormonal regulation of primate ovarian function.     

PARADOXICAL EFFECT OF A GnRH AGONIST ON STEROIDOGENESIS IN CULTURED MONKEY GRANULOSA CELLS

In this study we demonstrate: (i) The GnRH agonist exerts a direct dose-dependet stimulative effect on the aromatase activity and progesterone production in cultured monkey granulosa cells; (ii)the stimulative effect on steroidogenesis can be completely blocked by concomitant treatment with a GnRH antagonist, suggesting that the actions of GnRH are mediated through stringent stereospecific recongnition sites; (iii) in addition to the stimulative effect, the GnRH agonist in the presence of gonadotropins also exerts an inhibitory effect, even though the peptide by itself is more effective in the stimulation of steroidogenesis, and the stimulation of gonadotropin on steroidogenesis could be gradually restored by decreasing the concentration of the GnRH agonist in the culture; and (iv) paradoxical effect can also be observed in the presence of cAMP-inducing agents, suggesting that the inhibitory action of the peptide on gonadotropin-induced steroidogenesis is localized at a step distal to the stringent reco     

Paradoxical effect of a GnRH agonist on steroidogenesis in cultured monkey granulosa cells.

In this study we demonstrate: (i) The GnRH agonist exerts a direct dose-dependent stimulative effect on the aromatase activity and progesterone production in cultured monkey granulosa cells; (ii) the stimulative effect on steroidogenesis can be completely blocked by concomitant treatment with a GnRH antagonist, suggesting that the actions of GnRH are mediated through stringent stereospecific recognition sites; (iii) in addition to the stimulative effect, the GnRH agonist in the presence of gonadotropins also exerts an inhibitory effect, even though the peptide by itself is more effective in the stimulation of steroidogenesis, and the stimulation of gonadotropin on steroidogenesis could be gradually restored by decreasing the concentration of the GnRH agonist in the culture; and (iv) paradoxical effect can also be observed in the presence of cAMP-inducing agents, suggesting that the inhibitory action of the peptide on gonadotropin-induced steroidogenesis is localized at a step distal to the stringent recognition sites.     

Study on reproductive endocrinology of human placenta (II)--Hormone secreting activity of cytotrophoblast cells.

The capability of cytotrophoblast cells to produce hCG, progesterone, estrogen, cGnRH and beta-endorphin in vitro has been demonstrated in serum-free culture medium. Before experiment, a 24-h preculture was carried out in order to remove the endogenous hormones of the tissue. During a period of 8 days' culture, the cytotrophoblast cells could constantly produce a small amount of hCG. The production of progesterone rose rapidly and became doubled within six days. The estrogen secretion showed a similar pattern in the presence of androstenedione, a precursor of estrogen, indicating the elevation of aromatase activity in the cells. The elevation of the enzyme activity has been further demonstrated not to be induced by androstenedione. In both cytotrophoblast and syncytiotrophoblast cell cultures, cGnRH was only detected in the culture of cytotrophoblast cells, with a value up to 4 pg/10(5) cells/24 h. However, beta-endorphin was identified both in cytotrophoblast and syncytiotrophoblast cells. Its content increased significantly in the medium of cytotrophoblast cell culture from the 4th to 6th days, but declined in the medium of syncytiotrophoblast cell culture. The results demonstrate clearly that the cytotrophoblast cells are the sole origin of GnRH in human placenta and are also able to synthesize beta-endorphin and steroid hormones. The findings indicate that there is no such a sharp functional demarcation existing between these two kinds of trophoblast cells as suggested before. The data are of significance for a better understanding of the mechanism of hormonal regulation in placenta.     

INDOMETHACIN INHIBITS hCG AND GnRH AGONIST-INDUCED SECRETION OF PLASMINOGEN ACTIVATOR BY GRANULOSA AND THECA INTERSTITIAL CELLS OF HYPOPHYSECTOMIZED RATS

Considerable data have shown that plasminogen activator (PA) may play an important role in the mechanism of ovulation. A recent report argued that PA is not a primary proteoIytic enzyme for follicular rupture because a dose of indomethacin that blocked ovulation did not inhibit ovarian PA content. To further clarify the specific role of PA in the ovulation, we have carefully examined the effect of indomethacin on the secretion of PA in granulosa and theca-interstitial (TI) cells following hCG or GnRH agonist administration in PMSG-primed hypophysectomized immature rats. We have also studies in vitro action of this compound on PA secretion in granulosa cells. The results indicate that indomethacin can only suppress hCG- and GnRH agonist-induced PA secretion, but not suppress the ovarian content of these enzymes.     

Multiplexed immunoassays for the analysis of breast cancer biopsies

Within the last decade, protein microarray technology has been successfully used for the simultaneous quantification of target proteins from minimal amounts of samples in basic and applied proteome research. The robustness and appropriate sensitivity of these miniaturized assays have been demonstrated and thus the transfer to routine and high-throughput applications is now possible. In this study, multiplexed bead-based sandwich immunoassays were used to determine the concentrations of 54 protein analytes, including HER 2 and the estrogen receptor, from ultrasound-guided large-core needle biopsies (LCNBs) from breast cancer patients. Expression levels for HER 2, estrogen receptors and progesterone receptors were also assessed by immunohistochemical routine staining, performed in the clinic on corresponding biopsy samples. The high concordance of the data sets generated with the bead-based protein arrays and by conventional immunohistochemical assessment of HER 2 and the estrogen receptor expressed by breast cancer cells present in the biopsies was demonstrated.     

Protective role of estrogen against excessive erythrocytosis in Monge’s disease

Monge’s disease (chronic mountain sickness (CMS)) is a maladaptive condition caused by chronic (years) exposure to high-altitude hypoxia. One of the defining features of CMS is excessive erythrocytosis with extremely high hematocrit levels. In the Andean population, CMS prevalence is vastly different between males and females, being rare in females. Furthermore, there is a sharp increase in CMS incidence in females after menopause. In this study, we assessed the role of sex hormones (testosterone, progesterone, and estrogen) in CMS and non-CMS cells using a well-characterized in vitro erythroid platform. While we found that there was a mild (nonsignificant) increase in RBC production with testosterone, we observed that estrogen, in physiologic concentrations, reduced sharply CD235a⁺ cells (glycophorin A; a marker of RBC), from 56% in the untreated CMS cells to 10% in the treated CMS cells, in a stage-specific and dose-responsive manner. At the molecular level, we determined that estrogen has a direct effect on GATA1, remarkably decreasing the messenger RNA (mRNA) and protein levels of GATA1 (p < 0.01) and its target genes (Alas2, BclxL, and Epor, p < 0.001). These changes result in a significant increase in apoptosis of erythroid cells. We also demonstrate that estrogen regulates erythropoiesis in CMS patients through estrogen beta signaling and that its inhibition can diminish the effects of estrogen by significantly increasing HIF1, VEGF, and GATA1 mRNA levels. Taken altogether, our results indicate that estrogen has a major impact on the regulation of erythropoiesis, particularly under chronic hypoxic conditions, and has the potential to treat blood diseases, such as high altitude severe erythrocytosis.Subject terms: Haematopoietic stem cells, Myeloproliferative disease     

A COMPARISON OF MAMMALIAN CELL SENSITIVITY TO EITHER 254 nm OR ARTIFICIAL "SOLAR" SIMULATED RADIATION

The sensitivity of six mammalian cell strains to either germicidal (254 nm) or artificial "solar" simulated radiation was tested. The solar simulator used had an output similar, in some respects, to natural sunlight. Cellular capacity for Herpes simplex virus production was used as the assay procedure. The tested cells were a strain of African green monkey kidney cells and five human skin fibroblast cell strains. The latter included a "normal" cell strain, and four photosensitive cell strains; three of which were strains of xeroderma pigmentosum cells, and one strain of Bloom's syndrome cells. When comparing the D¹⁰ values, the different cell strains varied by a factor of six in response to germicidal radiation, but only by a factor of two to artificial "solar" simulated radiation. The relative sensitivity of the cells to either type of radiation also varied from 1.7 to 10.9. Large variations in response occurred even among the xeroderma pigmentosum cell strains. These responses suggest that mammalian cell sensitivity to 254 nm radiation may not be a true indicator of a cell's responses to natural sunlight.     

A factor from granulosa cells can stimulate oocyte tissue plasminogen activator activity

Several studies indicate that substances synthesized by granulosa cells are capable of regulating oocyte activity. We have studied the effect of factors synthesized by granulosa cells on tPA activity of denuded oocytes using a co-culture system. The results show that an FSH-dependent factor(s) synthesized by granulosa cells (but not by theca-interstitial cells) is capable of stimulating tPA activity of denuded oocytes. This finding is important for understanding hormonal regulation of oocyte tPA activity by mediators synthesized in granulosa cells.     

PHOTODYNAMIC TREATMENT OF HERPES SIMPLEX VIRUS INFECTION IN VITRO

Abstract— The effects of photodynamic action on in vitro herpes simplex virus infections of CV-1 monkey kidney fibroblasts or human skin fibroblasts were determined using proflavine sulfate and white fluorescent lamps. Photodynamic treatment of confluent cell monolayers prior to virus infection inactivated cell capacity, i.e. the capacity of the treated cells to support subsequent virus growth as measured by plaque formation. The capacity of human cells was more sensitive to inactivation than the capacity of monkey cells when 6 μM proflavine was used. Treated cell monolayers recovered the capacity to support virus plaque formation when virus infection was delayed four days after the treatment. Experiments in which the photodynamically treated monolayers were infected with UV-irradiated virus demonstrated that this treatment induced Weigle reactivation in both types of cells. This reactivation occurred for virus infection just after treatment or 4 days later. A Luria-Latarjet-type experiment was also performed in which cultures infected with unirradiated virus were photodynamically treated at different times after the start of infection. The results showed that for the first several hours of the virus infection the infected cultures were more sensitive to inactivation by photodynamic treatment than cell capacity. By the end of the eclipse period the infected cultures were less sensitive to inactivation than cell capacity. Results from extracellular inactivation of virus grown in monkey cells at 6 μM proflavine indicated that at physiological pH the virus has a sensitivity to photodynamic inactivation similar to that for inactivation of cell capacity. The combined data indicated that photodynamic treatment of the cell before or after virus infection could prevent virus growth. Thus, photodynamic inactivation of infected and uninfected cells may be as important as inactivation of virus particles when considering possible mechanisms in clinical photodynamic therapy for herpes simplex.     

Simultaneous Determination of Estrogen and Progesterone Receptors Using Human Uterus as a Standard

Abstract

Numerous reports have shown a high degree of correlation between the presence of estrogen and progesterone receptors in cancerous breast tumors and response to endocrine therapy. There are several methods in use for measurement of these receptors, but all are time consuming and require large amounts of tumor tissue. Our laboratory needed a faster and simpler method to determine receptor concentration. We describe here an assay which utilizes human uterus extract with known receptor concentration as a standard to determine receptor concentration of tumor tissues. This method is simple, fast and will allow determination of estrogen and progesterone receptor in up to ten patient samples at one time.     

A FACTOR FROM GRANULOSA CELLS CAN STIMULATE OOCYTE TISSUE PLASMINOGEN ACTIVATOR ACTIVITY

Several studies indicate that substances synthesized by granulosa cells are capableof regulating oocyte activity. We have studied the effect of factors synthesized by gran-ulosa cells on tPA activity of denuded oocytes using a co- culture system. The resultsshow that an FSH- dependent factor(s) synthesized by granulosa cells (but not by theca-interstitial cells) is capable of stimulating tPA activity of denuded oocytes. This findingis important for understanding hormonal regulation of oocyte tPA activity by mediatorssynthesized in granulosa cells.     

STUDY ON REPRODUCTIVE ENDOCRINOLOGY OF HUMAN PLACENTA--CULTURE OF HIGHLY PURIFIED CYTOTROPHOBLAST CELL IN SERUM-FREE HORMONE SUPPLEMENTED MEDIUM

A new method of long-term culture of cytotrophoblast cells in serum-free medium has been developed. Cytotrophoblast cells were isolated with cold trypsin and purified by unit gravity sedimentation through BSA density gradients. The cells were cultured in the FD medium with supplement of EGF, insulin, transferrin and sodium selenite. They could survive over three weeks. The results showed that both EGF and insulin stimulated hCG and progesterone secretion and that sodium selenite elevated hCG output but not progesterone secretion. Transferrin produced synergistic effect with EGF and insulin on hCG and progesterone secretion but it was ineffective when used alone. This study demonstrates that the four growth factors mentioned above are essential for the survival of cytotrophoblast cells in vitro. It is therefore suggested that EGF, insulin and selenium may possibly be involved in the regulation of hCG and progesterone secretion in the human placenta.     

Inhibition by protease inhibitors of binding of adrenal and sex steroid hormones.

Binding of steroid hormones is inhibited by protease inhibitors and substrates. The protease inhibitors phenylmethyl sulphonylfluoride, tosyl-lysine chloromethyl ketone, and tosylamide-phenylethyl-chloromethyl ketone and the protease substrates tosyl arginine methyl ester and tryptophan methyl ester eliminate specific binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen, and progesterone to their respective receptors. These protease inhibitors and substrates also inhibit binding of progesterone to the 20,000 molecular weight mero-receptor formed from the progesterone receptor in chick oviduct. The binding of estradiol to rat alpha-fetoprotein is inhibited by the protease inhibitors and substrates but not by tryptophan or tryptophan amide, indicating the importance of an ester structure in the inhibition of steroid binding. Our results suggest that all steroid hormone receptors have a site with both common structural features and a role in the regulation of steroid hormone binding.     

TIME-RESOLVED FLUORESCENCE OF NITROBENZOXADIAZOLE-AMINOHEXANOIC ACID: EFFECT OF INTERMOLECULAR HYDROGEN-BONDING ON NON-RADIATIVE DECAY

The fluorescence kinetics of the nitrobenzoxadiazole (NBD) chromophore were studied at low concentrations in solvents with varying polarity and hydrogen-bonding donor strength. The emission decay was essentially single exponential in all solvents studied. While the absorption and fluorescence solvatochromism is determined largely by the solvent polarity, the S1 state decay kinetics are strongly modulated by the solvent H-bonding capacity. The NBD emission lifetime, generally approximately 7-10 ns in the aprotic solvents, is reduced to 0.933 ns in water. The solvent deuterium isotope effect on the fluorescence decay is substantial in D2O and in methanol-d4, but is insignificant in DMSO-d6. These results are consistent with acceleration of S1----S0 internal conversion through an accepting vibrational mode created by intermolecular hydrogen-bonding of the NBD chromophore to an H atom-donating solvent. This work bears on the practically of using NBD as a fluorophore in assays for estrogen and progesterone receptors.     

Hormonal Regulation of Expression of Tissue Type Plasminogen Activator and Plasminogen Activator Inhibitor Type 1 in Cultured Rat Granulosa Cells

In the present study, gonadotropin and gonadotropin-releasing hormone (GnRH) regulation of tPA and PAI-1 expression in PMSG-primed granulosa cells has been investigated, (i) Addition of go-nadotropins (FSH and LH) and GnRH agonist (GnRHa) or PMA to the culture increases tPA activity) FSH (or LH) plus GnRHa (or PMA) in the culture further enhances the enzyme production to such an extent that a more obvious effect than the additive effect caused by these hormones used alone has been observed; (ii) in contrast, FSH and LH decrease PAI-1 activity, whereas GnRHa and PMA alone markedly increase PAI-1 mRNA level and PAI-1 activity. Because FSH and LH stimulate tPA production and have no significant effect on PAI-1 mRNA induction, the observed inhibition of PAI-1 activity by gonadotropins may be due to the occurrence of neutralization of PA and PAI-1 proteins in the conditioned media by the formation of complexes between PA and PAI-1 ; (iii) increases in PAI-1 mRNA level and activity by GnRH and PMA are complet     

Proteome analysis of conditioned medium from cultured adult hippocampal progenitors

It is known that proliferation and survival of neural stem/progenitor cells in vitro not only depend on exogenous factors, but also on autocrine factors secreted into the conditioned medium. It is also well known that the identification of bioactive proteins secreted into the conditioned medium poses a substantial challenge. Recently, neural stem/progenitor cells were shown to secrete a survival factor, cystatin C, into the conditioned medium. Here, we demonstrate an approach to identify other low molecular weight proteins in conditioned medium from cultured adult rat hippocampal progenitor cells. A combination of preparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry was utilized in the analysis. We were able to identify a number of proteins, which include Rho-guanine nucleotide dissociation inhibitor 1, phosphatidylethanolamine binding protein (PEBP), also termed Raf-1 kinase interacting protein, polyubiquitin, immunophilin FK506 binding protein 12 (FKBP12) and cystatin C. The presence of PEBP and FKBP12 in conditioned medium was confirmed immunologically. All nestin-positive progenitor cells showed immunoreactivity for antibodies against PEBP and FKBP12. To our knowledge we are the first to use this preparative proteomic approach to search for stem cell factors in conditioned medium. The method could be used to identify novel bioactive proteins secreted by stem/progenitor cells in vitro. Identification of bioactive proteins in vitro is of potential importance for the understanding of the regulatory mechanisms of the cells in vivo.     

UV-ENHANCED VIRUS REACTIVATION IN MAMMALIAN CELLS: EFFECTS OF METABOLIC INHIBITORS*

Abstract—The induction process of UV-enhanced reactivation of UV-irradiated herpes simplex virus was investigated in CV-1 monkey kidney cells. A protein synthesis inhibitor, cycloheximide (0.5–5 μg/m/), present in the culture medium For 24 h between cell irradiation and virus infection decreased the enhanced virus survival normally found in UV-irradiated cultures. The enhanced virus reactivation became essentially resistant to the addition of cycloheximide by 6–8 h after cell irradiation, indicating that the cycloheximide-sensitive process necessary for enhanced reactivation was complete by that time. Since cycloheximide not only inhibits protein synthesis, but DNA synthesis as well, we investigated the effect of a DNA synthesis inhibitor, hydroxyurea. Hydroxyurea did not decrease UV-enhanced virus survival, but resulted in enhanced virus survival even in unirradiated cells. Therefore, the cycloheximide-caused inhibition of UV-enhanced reactivation did not arise from inhibition of DNA synthesis. The combined results indicate that (1) UV-enhanced virus reactivation in monkey kidney cells requires de novo protein synthesis during the first 6–8 h after cell irradiation and that (2) DNA synthesis inhibition may be the initiating event.     

THE WAVELENGTH DEPENDENCE OF 8-METHOXYPSORALEN PHOTOSENSITIZATION OF HOST CAPACITY INACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract— The addition of 8-methoxypsoralen to cultures of African green monkey cells (CV-I) sensitized the inactivation by near UV radiation (302–370 nm) of the ability of the cells to host herpes simplex virus. No sensitizing effect by drug addition was noted for far UV radiation (232–297 nm). An action spectrum for the photosensitized inactivation of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum of 8-methoxypsoralen.