Iris Keren-Tal

Steroidogenesis and apoptosis in the mammalian ovary

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrous/menstrual cycle by programmed cell death is essential to maintain the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, plays an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows systematic studies of the mechanisms that control steroidogenesis and apoptosis in granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated up to 24h following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or TNF-alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one not involving mitochondrial Cyt C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and affymetrix DNA microarray technology we discovered that granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells. Thus, the apoptotic signals could bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures cyclicity of estradiol and progesterone release in the estrous/menstruous cycle even during the initial stages of apoptosis.

Establishment of steroidogenic granulosa cell lines expressing follicle stimulating hormone receptors

Follicle stimulating hormone (FSH) plays an important role in the regulation of oogenesis, spermatogenesis and production of steroid hormones. Receptors to FSH, which are uniquely expressed in ovarian granulosa and testicular Sertoli cells, are rapidly lost in tissue culture conditions and upon cell transformation. We have succeeded, by triple transfection of primary rat granulosa cells with SV40 DNA, Ha-ras oncogene and an FSH receptor expression plasmid, to establish stable steroidogenic cell lines expressing FSH receptors. The cell lines respond to rat, ovine and bovine FSH, which stimulate progesterone production at levels comparable to primary granulosa cells obtained from preovulatory follicles. No steroidogenic response is detected upon stimulation with ovine luteinizing hormone or human chorionic gonadotropin. The steroidogenic response is accompanied by de novo appearance of adrenodoxin which serves as a marker for the mitochondrial steroidogenic enzyme system. These cells express approximately 27,000 receptors per cell with a Kd of 100-115 pM. This Kd is close to the value calculated for the native receptor. The ED50 for the steroidogenic response to ovine FSH is 200 pM, suggesting a tight coupling between receptor activation and the steroidogenic response. FSH induces pronounced morphological changes in the established cell lines, which are also characteristic of primary granulosa cells. These FSH responsive cell lines can serve as a useful model for the study of the structure and function of the FSH receptor and the effect of oncogenes on its expression.

Alternative pathways of ovarian apoptosis: death for life.

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrus/menstrual cycle by programmed cell death is essential for maintaining the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, play an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows for systematic studies of the mechanisms that control steroidogenesis and apoptosis of granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated for up to 24hr following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or tumor necrosis factor alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one that does not involve mitochondrial cytochrome C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and Affymetrix DNA microarray we discovered that Granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells, thereby allowing the apoptotic signals to bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures the cyclicity of estradiol and progesterone release in the estrus/menstrus cycle even during the initial stage of apoptosis.

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.

Activation of FSH-responsive adenylate cyclase by staurosporine: role for protein phosphorylation in gonadotropin receptor desensitization

Prolonged stimulation of gonadotropin receptors in granulosa cells leads to desensitization of the cellular response to gonadotropic hormones which is evident by decrease in cAMP formation. In order to explore the mechanism of desensitization and to examine whether protein phosphorylation may play a role in this phenomenon, we have studied the effect of various stimulators and inhibitors of protein phosphorylation on FSH-induced cAMP formation in the FSH-responsive cell line, GFSHR-17, recently established in our laboratory. Both ovine and human FSH activated the hormone sensitive adenylate cyclase in a dose-dependent manner with an ED50 of 0.5 nM. This stimulation was followed by a sharp decrease in cAMP formation after 30 min incubation of the cell with the hormone. When cells were preincubated for 60 min with staurosporine, cAMP accumulation during 20 min of FSH stimulation was elevated about 500%, compared to cells stimulated by FSH alone. Staurosporine alone showed a negligible effect on cAMP accumulation in these cells. In cells stimulated with forskolin, a non-specific activator of adenylate cyclase, or with cholera toxin (CT), an inhibitor of GTPase activity associated with Gs of adenylate cyclase, preincubation with staurosporine increased cAMP formation in these cells by only 50-70 or 80-120%, respectively. Preincubation of cells with the protein kinase C (PKC) inhibitors chelerythrine and GF109203X increased FSH-stimulated accumulation of cAMP by 50 and 30%, respectively. These drugs exhibit a similar effect on forskolin-stimulated cells. Preincubation of cells for 60 min with a PKC stimulator, TPA, suppressed FSH-mediated cAMP response in these cells by 40%. Tyrosine kinase inhibitors such as AG18, AG33 and genistein exhibit a modest inhibitory effect of up to 20% on FSH-stimulated cAMP accumulation. All the above results were obtained both in the presence and absence of IBMX, a potent inhibitor of the cellular phosphodiesterases. Upon prolonged incubation with FSH (3 h) cells pretreated with staurosporine exhibited a much slower rate of decline in intracellular cAMP levels. Moreover, in desensitized cells, following 1 or 2 h of continuous stimulation with FSH, staurosporine could markedly enhance cAMP formation in the presence of FSH. Our data suggest that staurosporine-sensitive phosphorylation of serine or threonine in the FSH receptor-cyclase system may be responsible for desensitization of the FSH coupled activation of cAMP formation, while reactivation of the system can be achieved by protein dephosphorylation at these specific sites. Because specific inhibition of PKC could not mimic the staurosporine effect on FSH-stimulated cAMP formation, nor could activation of kinase C antagonize it, it is suggested that a specific staurosporine-sensitive receptor kinase may be responsible for modulation of the coupling between the gonadotropin receptor and the adenylate cyclase system.

Oncogene-transformed granulosa cells as a model system for the study of steroidogenic processes

Highly steroidogenic granulosa cell lines were established by transfection of primary granulosa cells from preovulatory follicles with SV40 DNA and Ha-ras oncogene. Progesterone production in these cells was enhanced to levels comparable to normal steroidogenic cells, by prolonged (> 12 h) stimulation with 8-Br-cAMP, forskolin and cholera toxin, which elevate intracellular cAMP. The steroidogenic capacity of individual lines correlated with the expression of the ras oncogene product (p21) and the morphology of the cells. Formation of the steroid hormones was associated with de novo synthesis of the mitochondrial cytochrome P450scc system proteins. Since cholesterol import into mitochondria is essential for steroidogenesis, the expression of the peripheral benzodiazepine receptor (PBR) and the sterol carrier protein 2 was characterized in these cells. The induction of the expression of the genes coding for both proteins appeared to be mediated, at least in part, by cAMP. Stimulation of the PBR by specific agonists enhanced progesterone production in these cells. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically suppressed the cAMP-induced steroidogenesis, in spite of enhanced intracellular cAMP levels, suggesting that TPA can modify the effects of cAMP. cAMP stimulation suppressed growth of transformed cells concomitantly with induction of steroidogenesis. The transformed cells lacked receptors for the native stimulants, the gonadotropic hormones. After transfection of the cells with a lutropin (LH) receptor expression plasmid, the LH and hCG response was reconstituted. In these newly established cell lines gonadotropins were able to stimulate the formation of cAMP and progesterone in a dose-dependent manner with an ED₅₀ characteristic of the native receptor. High doses caused desensitization to gonadotropins as observed in normal cells. These newly established oncogene-transformed granulosa cell lines can serve as a useful model to study inducible steroidogenesis and the effect of oncogene expression on this process.

Association of Ad4BP/SF-1 transcription factor with steroidogenic activity in oncogene-transformed granulosa cells.

Adrenal binding protein 4 (Ad4BP) known also as steroidogenic factor 1 (SF-1) is a cell specific transcription factor regulating all steroidogenic P450 genes and is present exclusively in steroidogenic tissues. In this study, we examined whether Ad4BP expression is affected by oncogene-induced cell transformation. Using a gel shift assay we report here that nuclear extracts of steroidogenic granulosa cell lines, transformed by SV40 DNA and the Ha-ras oncogene show specific binding activity towards an Ad4 recognition sequence oligonucleotide. In contrast, nuclear extracts obtained from granulosa cells transformed with SV40 alone, which lost their steroidogenic activity, did not exhibit any binding to the Ad4 oligonucleotide. Using a specific antibody to Ad4BP, it was demonstrated that only the steroidogenic cell lines, i.e. transfected with SV40 + Ha-ras, expressed significant amount of the protein. No binding activity to the Ad4 oligonucleotide was evident in fibroblasts transformed with the same oncogenes (SV40 + Ha-ras). Steroidogenic activity in SV40 + Ha-ras transformed granulosa cells was markedly elevated following forskolin or follice stimulating factor (FSH) and further augmented by incubation of the cells with dexamethasone. However, no change in Ad4BP expression and binding activity was observed following such stimulations. It is suggested that Ha-ras expression in SV40 transformed granulosa cells can play an important role in restoring Ad4BP expression and activity, which are required for their steroidogenic function. Thus, expression of Ad4BP is essential for steroidogenesis both in primary and in oncogene transformed granulosa cells.

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.