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D Aharoni

Publications and source records attributed to D Aharoni.

9 recordsLinked to original sources
PubMed1997

Differential effect of components of the extracellular matrix on differentiation and apoptosis.

BACKGROUND: Epithelial cells are closely associated with a basement membrane, but the intimate relationships that affect growth, differentiation and survival remain enigmatic. We have previously reported that granulosa cells adjacent to the basement membrane of the ovarian follicle have a higher degree of differentiation compared with cells located distal to the basement membrane. By contrast, granulosa cells distal to the basement membrane are the first to undergo apoptosis during follicular atresia. Moreover, growth of granulosa cells in vitro on a naturally produced basement-membrane-like extracellular matrix (ECM) enhances progesterone production and the cellular response to gonadotropic hormones by an undefined mechanism. RESULTS: To investigate the effect of the ECM on granulosa cell differentiation and death, primary granulosa cells were cultured on ECMs that lacked or contained bFGF (basic fibroblast growth factor). These otherwise identical ECMs were deposited by HR9 mouse endodermal cells, which do not synthesize bFGF, or by HR9 cells transfected with the bFGF gene. Both ECMs provided protection against apoptosis in serum-free medium, but only the bFGF-containing ECM maintained expression of the steroidogenic P450scc enzyme system and the production of progesterone. Moreover, culturing the cells on this ECM enhanced the expression of the 30 kDa steroid acute regulatory protein which plays a key role in steroid hormone biosynthesis. Laminin, but not fibronectin, was able to replace the ECM in protecting the cells from apoptosis; but not in maintaining steroidogenesis, whereas bFGF was able to enhance steroidogenesis without protecting the cells against apoptosis. Cells cultured on both ECMs or laminin had a well-developed actin cytoskeleton compared with cells cultured on non-coated dishes, which underwent apoptosis. CONCLUSIONS: Cellular responses to ECM are mediated by the combined action of macromolecular constituents and regulatory molecules, such as bFGF, that are sequestered and stored in the ECM. ECM or laminin protects against cell death by interacting with specific integrin receptors and maintaining a well-developed actin cytoskeleton. ECM-bound bFGF provides differentiation signals for granulosa cells, which are in intimate contact with the ECM. Thus, a clear distinction can be made between the survival activity and the differentiation stimulus exerted by the ECM on epithelial cells.

Actins↗
PubMed1997

Apoptosis in steroidogenic cells: structure-function analysis.

Granulosa cells are the main producers of the female sex steroid hormones, progesterone and estradiol, which are responsible for the cyclicity in ovarian function. Programmed cell death in the ovary plays a crucial role in limiting the number of follicles that can ovulate and thus prevents the development of more embryos than can successfully complete pregnancy. Granulosa cell apoptosis is regulated by the concerted action of endocrine, paracrine, and autocrine factors. These factors lead to the developmental decision of whether the steroidogenic cell will luteinize and enter the pathway leading to programmed cell death, or whether the life span of the luteinized cell will be prolonged to continue secretion of progesterone, which is essential for the maintenance of pregnancy. At the level of the individual cell, we find that enhanced steroidogenesis can be maintained during the initial steps of apoptosis as long as the steroidogenic apparatus remains intact. This can be achieved by a unique mechanism of compartmentalization of steroidogenic organelles in the perinuclear region and migration of the multicatalytic proteinase, the proteasome, to the apoptotic blebs. Reorganization of the actin cytoskeleton during apoptosis may provide an efficient barrier between the proteolytic activity and the steroidogenic activity in the apoptotic cell. It is suggested that steroidogenesis can be maintained in the apoptotic cells as long as the steroidogenic organelles bearing the steroidogenic apparatus remain intact.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Fourier analysis of differential light scattering for the quantitation of FSH response associated with structural changes in immortalized granulosa cells.

We have established granulosa cell lines which express constitutively the rat FSH receptors by cotransfection of primary granulosa cells obtained from preovulatory follicles with SV40 DNA, Ha-ras oncogene and a plasmid expressing FSH receptors. These cells respond specifically to ovine and human FSH by cell rounding, intracellular cAMP accumulation, and progesterone secretion in a dose-dependent manner. A new method for the demonstration and quantitation of changes in cell shape-Small Angle Laser Light Scattering (SALLS) analysis-has been utilized for measurement of cell rounding in response to FSH stimulation in these cells. When cells were incubated with increasing doses of either ovine or human FSH, partial rounding of cells was observed at FSH concentrations as low as 24 pM, while complete rounding of cells was observed at a range of 0.24-2.4 nM of FSH. Following aldehyde fixation, hormone-treated cells were examined using the method of SALLS analysis. Histograms obtained by applying SALLS analysis on FSH stimulated GFSHR-17 cells were a reflection of the structural changes induced by the hormone. FSH- and forskolin-incubated cells yielded structured distributions with defined mean size and standard deviations. Moreover, the increase in sharpness of dominant peak in the histogram was correlated with elevated concentration of FSH in a dose dependent manner. In conclusion, cellular response to FSH is correlated with a specific pattern of light scattered in immortalized granulosa cells expressing functional FSH receptors. Therefore, SALLS analysis may serve as a useful tool for in vitro bioassay of the gonadotropic hormone. Moreover, this method may lend itself to in vitro bioassay of any hormone that induces specific morphological changes in target cells.

Animals↗
PubMed1996

Cross-talk between cAMP and p53-generated signals in induction of differentiation and apoptosis in steroidogenic granulosa cells.

In each menstrual cycle only very few follicles in the mammalian ovary undergo maturation and ovulation while most of the follicles degenerate in the process of atresia. Moreover, in the absence of pregnancy, the newly formed corpora lutea will degenerate and disappear in the process of luteolysis. Recent studies suggest that ovarian follicular atresia is associated with DNA fragmentation and degeneration of follicular cells, characteristics of programmed cell death (apoptosis). Apoptosis can be induced in vitro, in primary granulosa cell culture, by serum deprivation and by induction of a high intracellular level of cAMP. This induction of apoptosis can be blocked by fibroblast growth factor, suggesting that receptor-medicated activation of a tyrosine kinase can serve as a survival signal. Apoptosis can also be induced in immortalized steroidogenic granulosa cells, transformed by SV40 DNA and Ha-ras oncogene, by overexpression of the wild-type p53 tumor suppressor gene in cAMP-stimulated cells. Omitting the cAMP stimulus prevents the p53-induced apoptosis in these cells, suggesting cross-talk between p53 and c-AMP-generated signals in the induction of apoptosis. Steroidogenic activity in these cells, as well as in nontransformed granulosa cells, does not decline during apoptosis but is rather significantly elevated before total cell collapse occurs. Cytochemical studies using confocal laser microscopy, electron microscopy, and three-dimensional reconstruction reveal a specific reorganization pattern of proteasomes, the most abundant nonlysosomal protease, and of the steroidogenic organelles, such as mitochondria and lipid droplets, in the apoptotic cell. Our results suggest that compartmentalization of intracellular organelles during apoptosis permits proteolysis without interfering with steroidogenesis, characteristic of the differentiated phenotype of the granulosa cell. Moreover, cytoskeletal rearrangement may serve as a barrier between these cellular activities.

Animals↗
PubMed1996

Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells.

Freshly isolated granulosa cells obtained from women undergoing in-vitro fertilization (IVF) become refractory to further gonadotrophin stimulation in culture due to their previous hormonal treatment. However, when precultured for 7 days in gonadotrophin-free medium they regain their response to both human chorionic gonadotrophin (HCG) and follicle stimulating hormone (FSH) with a 10-fold and 5-fold increase in progesterone production respectively, within an additional 7 days of culture. Forskolin, a direct activator of adenylate cyclase, increased progesterone levels 12-fold compared with non-stimulated cultures. Oestradiol formation was also significantly elevated (P < 0.005) following 48 h stimulation with luteinizing hormone (LH), FSH or forskolin. Intracellular cAMP levels rose 1.5-fold, 10-fold and 15-fold after 1 h of FSH, HCG or forskolin treatment. Expression of both cytochrome P450 side chain cleavage enzyme (SCC) and the steroidogenic transcription factor SF1/Ad4BP could be demonstrated by Western blotting. However, elevation of P450 SCC alone was evident following FSH and HCG stimulation. In the presence of serum, the ultrastructure of these cultured cells displayed numerous lipid droplets and well-developed mitochondria, characteristic of highly steroidogenic cells. The proportion of apoptotic nuclei in these cultures was < 30%. Removal of the serum increased apoptotic incidence to 40%, whereas addition of FSH prevented cell death significantly (P < 0.01). HCG and forskolin increased apoptosis to approximately 50%, while treatment with 8Br-cAMP led to 80% cell death. Our data suggest that, after prolonged culture, human granulosa cells can regain cAMP and steroidogenic response to gonadotrophin stimulation. Moreover, our experiments indicate that apoptosis and steroidogenesis can coexist in the same cell population while the interrelationship between these processes can be determined by the intracellular levels of cAMP.

Apoptosis↗
PubMed1995

cAMP-mediated signals as determinants for apoptosis in primary granulosa cells.

Differentiation and luteinization of granulosa cells are induced by gonadotrophic hormones and other substances elevating intracellular levels of cyclic AMP (cAMP). We have investigated the correlation between the potency of these substances to enhance steroidogenesis and to induce apoptosis in primary granulosa cell cultures obtained from rat preovulatory follicles. The cAMP analog, 8-Br cAMP, induced apoptosis in more than 90% of the cell population within 15 h of incubation at 37 degrees C in serum-free medium. The physiological stimulants of these cells, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which caused a moderate cAMP response in these cells, followed by a desensitization period, increased progesterone production by fourfold with no apparent effect on cell death. In contrast, forskolin, a potent activator of adenylate cyclase, stimulated both the cAMP and steroidogenic response by an order of magnitude greater than the gonadotropin stimulation, concomitantly with a pronounced increase in cell death (25%). Moreover, blocking of the cellular phosphodiesterase activity in forskolin-stimulated cells by isobutylmethylxanthine (IBMX), which maintains high levels of intracellular cAMP, led to further enhancement of cell death following 40 h of incubation (50%). Basic fibroblast growth factor (bFGF) and gonadotropin-releasing hormone (GnRH), which stimulated steroidogenesis in these cells in a cAMP-independent manner, did not promote cell death. Moreover, costimulation of the cells with forskolin and bFGF led to a substantial decrease in the incidence of apoptosis relative to forskolin alone. In order to examine whether the expression of tumor suppressor genes is involved in granulosa cell differentiation and apoptosis induced by cAMP, we examined the effect of cAMP in SV40 transformed granulosa cells, in which T-antigen expression is expected to block the activity of p53 as well as of the retinoblastoma gene product (pRB) and its related proteins. Cultures of three different cell lines established by SV40 transformation demonstrated resistance to 8-Br-cAMP- or forskolin plus IBMX-induced apoptosis, in contrast to the severe apoptotic response in primary cells. We suggest that stimulation of primary granulosa cells by high levels of cAMP catalyzes programmed cell death, while stimulation of the cells by gonadotropic hormones, which result in a moderate cAMP response, followed by desensitization to further stimulation, can prolong the lifespan of the luteinized granulosa cells. Moreover, one or more tumor suppressor proteins may mediate the cAMP generated signal leading to cell death.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Plasticity of cell organization during differentiation of normal and oncogene transformed granulosa cells.

Granulosa cells, which nurse the oocyte and serve as a major source for estradiol and progesterone production, undergo major morphological changes which correlate very well with modulation of their steroidogenic capacity. These include changes in intercellular contacts and communication, in cell membrane receptors, and in the development and organization of organelles associated with steroidogenesis (i.e., mitochondria, smooth endoplasmic reticulum, lipid droplets, and lysosomes). These biochemical and morphological changes can also be obtained in primary cultures as well as in oncogene transformed granulosa cell lines established recently in our laboratory. A growing body of evidence suggests that plasticity of the cytoskeleton plays a major role in the biochemical and morphological differentiation of granulosa cells as well as in other steroidogenic cells.

Animals↗
PubMed1993

Cross-talk between adenylate cyclase activation and tyrosine phosphorylation leads to modulation of the actin cytoskeleton and to acute progesterone secretion in ovarian granulosa cells.

Steroidogenesis in granulosa cells can be stimulated by gonadotropic hormones and substances elevating cAMP. This cAMP-dependent metabolic event can be enhanced by peptide growth factors such as insulin, insulin-like growth factor, and epidermal growth factor, but the mechanism of cooperation between these two different signaling pathways is not yet clear. We have tested whether enhancement of tyrosine phosphorylation by vanadate, which blocks tyrosine phosphatases, is able to mimic the effect of growth factors on cAMP-induced steroidogenesis and investigated the cellular components involved in such modulation. Ortho- and metavanadate at 0.1-1.0 mM, when added to primary granulosa cell cultures, stimulated by gonadotropic hormones or forskolin, enhanced progesterone production by 1.5- to 9.0-fold within 120 min. Pervanadate showed a similar effect on steroidogenesis at a concentration one order of magnitude lower than ortho- or meta-vanadate. Phenylarsine-oxide, another blocker of tyrosine phosphatase, stimulated forskolin-induced steroidogenesis by 2.5-fold at 30 microM. In contrast, okadaic acid and calyculin A, which block specifically serine and threonine phosphatase, had no effect on steroidogenesis, when used at concentrations of 1 microM and 10 nM, respectively. The stimulation by vanadate was associated with a pronounced change in cell shape and total collapse of the actin network, which retracts to form a few large actin aggregates of 1-7 microns in diameter in the perinuclear region as revealed by visualization of actin by rhodamine-phalloidin staining under the fluorescent microscope. Steroidogenesis is not affected in cells treated with vanadate alone; the effect of vanadate on the actin cytoskeleton is much less pronounced. Electron microscopy of ultra-thin sections showed massive breakdown of thin filament cables in cells stimulated with vanadate together with gonadotropic hormone or forskolin. Massive clustering of lipid droplets and mitochondria as well as sharp increase in the electron-density of mitochondrial matrix was also observed in the stimulated cells. The action of vanadate in cAMP-stimulated cells leads to massive tyrosine phosphorylation of intracellular proteins in the range of 22-200 kilodaltons. It is suggested that the cross-talk between the cAMP pathway and tyrosine phosphorylation, which leads to enhanced steroidogenesis may be mediated by phosphorylation of cytoskeleton or associated proteins. The marked changes in lipid droplet-mitochondria interaction suggests that this enhanced steroidogenesis is due in part to mobilization of cholesterol into mitochondria in cells costimulated with vanadate and gonadotropins.

Actins↗