Biomedical subjects

A Amsterdam

Publications and source records attributed to A Amsterdam.

At least 73 records · Page 4Linked to original sources

PubMed1996

Removal of proteasomes from the nucleus and their accumulation in apoptotic blebs during programmed cell death.

Apoptosis can be initiated in immortalized cAMP-stimulated rat ovarian granulosa cells by induction of wild-type p53 activity. Immunocytochemical studies using confocal microscopy reveal that in apoptotic, unlike in normal growing cells, the proteasomes are removed from the nucleus and accumulate within the apoptotic blebs at the periphery of the cell. In parallel, a striking reorganization of the actin cytoskeleton is observed which forms a spherical network separating the apoptotic blebs from the cytoplasmic organelles, such as mitochondria and lipid droplets which remain in the perinuclear region. The reorganization of the actin cytoskeleton as well as disappearance of proteasomes from the nucleus suggest possible function of proteasomes in apoptotic regulation.

Animals↗

PubMed1996

Highly efficient germ-line transmission of proviral insertions in zebrafish.

An important technology in model organisms is the ability to make transgenic animals. In the past, transgenic technology in zebrafish has been limited by the relatively low efficiency with which transgenes could be generated using either DNA microinjection or retroviral infection. Previous efforts to generate transgenic zebrafish with retroviral vectors used a pseudotyped virus with a genome based on the Moloney murine leukemia virus and the envelope protein of the vesicular stomatitis virus. This virus was injected into blastula-stage zebrafish, and 16% of the injected embryos transmitted proviral insertions to their offspring, with most founders transmitting a single insertion to approximately 2% of their progeny. In an effort to improve this transgenic frequency, we have generated pseudotyped viral stocks of two new Moloney-based genomes. These viral stocks have titers up to two orders of magnitude higher than that used previously. Injection of these viruses resulted in a dramatic increase in transgenic efficiency; over three different experiments, 83% (110/133) of the injected embryos transmitted proviral insertions to 24% of their offspring. Furthermore, founders made with one of the viruses transmitted an average of 11 different insertions through their germ line. These results represent a 50- to 100-fold improvement in the efficiency of generating transgenic zebrafish, making it now feasible for a single lab to rapidly generate tens to hundreds of thousands of transgenes. Consequently, large-scale insertional mutagenesis strategies, previously limited to invertebrates, may now be possible in a vertebrate.

3T3 Cells↗

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

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.

Adenylyl Cyclases↗

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

Requirements for green fluorescent protein detection in transgenic zebrafish embryos.

We have generated transgenic (Tg) lines of zebrafish in which the green fluorescent protein (GFP)-encoding gfp cDNA is driven by the Xenopus laevis ef1 alpha enhancer/promoter; Tg embryos from most of these lines show detectable fluorescence throughout their body. We have investigated the copy number of the Tg genes in fluorescent and non-fluorescent lines, in order to determine how this affects the production of detectable levels of GFP in the zebrafish embryo. Additionally, we have injected purified recombinant GFP into embryos to determine the intracellular GFP concentration required for detection, both when all of the cells in the embryo contain GFP and when only a few do.

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↗

PubMed1996

High-dose carboplatin, etoposide, and cyclophosphamide for patients with refractory germ cell tumors: treatment results and prognostic factors for survival and toxicity.

PURPOSE: The efficacy and toxicity of high-dose carboplatin, etoposide, and cyclophosphamide with autologous bone marrow transplantation (AuBMT) was investigated in a prospective trial for patients with cisplatin-refractory germ cell tumor (GCT). Prognostic factors for survival and treatment-related toxicity were identified. PATIENTS AND METHODS: Fifty-eight patients with refractory GCT were treated with high-dose carboplatin, etoposide, and cyclophosphamide plus AuBMT. Prognostic factors for toxicity and survival were examined in multivariate analyses. RESULTS: Twenty-three patients (40%) achieved a complete response and 12 (21%) are alive and free of disease at a median follow-up time of 28 months. Myelosuppression was severe and there were seven (12%) treatment-related deaths. Independently predictive factors that resulted in faster blood count recovery were the use of granulocyte colony-stimulating factor (G-CSF) for the number of days to neutrophil count recovery (P = .013) and prior treatment with cisplatin limited to six cycles or less for the number of days to platelet count recovery (P = .0012). Both were predictive for the number of days of hospitalization (P = .04 and .03, respectively). The two independently predictive variables for survival were pretreatment level of HCG; human chorionic gonadotrophin (HCG; < or = 100 times the upper limit of normal [xnl] v > 100 xnl, P = .02) and the presence of retroperitoneal metastases (yes or no, P = .04). Patients grouped by HCG < or = 100 xnl with retroperitoneal metastases, HCG < or = 100 xnl without retroperitoneal metastases, and all patients with HCG more than 100 xnl had median survival times of 14, 11, and 3 months, respectively (P = .04). CONCLUSION: High-dose carboplatin, etoposide, and cyclophosphamide is an effective therapy for patients with refractory GCT, and results in a complete response proportion of 40% and a 2-year survival rate of 31% at a median follow-up time of 28 months. This was accomplished in a group of patients with a dismal prognosis to conventional-dose therapy.

Adolescent↗

PubMed1995

Archaebacterial and eukaryotic proteasomes prefer different sites in cleaving gonadotropin-releasing hormone.

Thermoplasma 20 S proteasomes are composed of only two different types of subunits (designated as alpha and beta) but are nearly indistinguishable in their quaternary structure from eukaryotic 20 S proteasomes consisting of 14 distinct subunits. In this study, we compared both the nature and the rate of the proteolytic activities of Thermoplasma and of granulosa cell proteasomes on the neurohormone, gonadotropin-releasing hormone (GnRH), the degradation products of which can be unequivocally identified. Both Thermoplasma and granulosa proteasome degrade the decapeptide GnRH at the Trp3-Ser4, Ser4-Tyr5, Tyr5-Gly6, and Gly6-Leu7 bonds. While the main product of Thermoplasma proteasomes was a GnRH-(1-4) fragment, the main product of granulosa cell proteasome was a GnRH-(1-5) fragment, indicating that the principal degrading activity of Thermoplasma proteasome targets Ser4-Tyr5 bond, while the principal degrading activity of granulosa cell proteasome targets the Tyr5-Gly6 bond of GnRH. These differences in the degradation pattern of the neurohormone were observed when proteasome activities were compared both at 60 degrees C, the optimal temperature for Thermoplasma proteasomal activity, and at 37 degrees C, the optimal temperature of granulosa proteasome proteolytic activity. Although the catalytic mechanism is probably conserved from archaebacterial to eukaryotic proteasomes, our results suggest that there are striking differences in the preferred cleavage site of GnRH. This reflects the changes in the proteasomal subunit repertoire during evolution.

Amino Acid Sequence↗

PubMed1995

The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos.

The green fluorescent protein (GFP) from the cnidarian Aequorea victoria is capable of producing fluorescence without an exogenously added substrate. Here we demonstrate that a cDNA for GFP driven by a Xenopus elongation factor 1 alpha enhancer-promoter can confer fluorescence upon live zebrafish embryos, either as an injected plasmid or as a transgene after passage through the germline. When injected into zebrafish embryos at the one-cell stage, this construct starts to express detectable GFP after about 4 hr of development at 28 degrees C, about 1 hr after the midblastula transition. Fluorescence can be observed in cells of many tissue types in the embryo for at least 3 weeks after injection. We used three different expression constructs, each employing a modified ef1 alpha enhancer-promoter, to generate 12 transgenic lines. Eight of the 12 lines, including 5 of 5 derived from one construct with an intron, express detectable fluorescence in the F1 and, where tested, in the F2 generation. Most expressing lines showed very similar expression patterns. Generally, fluorescence is not seen in the transgenic embryos before 20 hr postfertilization, at which point it appears uniformly throughout the embryo. Fluorescence is most visible between 24-36 hr, and it becomes less visible after this, except that in many lines strong fluorescence remains visible in the eye for at least 5 days. A single inherited copy of the transgene is sufficient to produce detectable fluorescence in hemizygous F1 and F2 embryos.

Animals↗

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↗

PubMed1995

Involvement of p53 expression in cAMP-mediated apoptosis in immortalized granulosa cells.

In the accompanying paper we described the induction of apoptosis by extended cyclic AMP (cAMP)-mediated signals in primary granulosa cells and the reduction in this process in transformed cells expressing SV40 T antigen. In the present work, we examined the effect of overexpression of either wild-type or mutant p53 on cAMP-mediated apoptosis in steroidogenic granulosa cell lines transfected with SV40 DNA together with the Ha-ras oncogene and a temperature-sensitive variant of p53, p53val135. In cell lines expressing low amounts of T antigen and high amounts of p53val135, growth arrest was induced by transferring the cells from 37.5 degrees to 32 degrees C, a temperature which allows the manifestation of the wild-type phenotype of p53 and the induction of the WAF1 gene. While nonstimulated cells showed only a very modest apoptotic process, rapid and massive apoptosis was evident in cells stimulated by forskolin at 32 degrees C. The presence of serum could delay, but not abolish, this phenomenon. Progesterone production in such cells treated with cAMP was significantly higher at 32 degrees C than at 37.5 degrees C, suggesting that wild-type p53 can also enhance granulosa cell differentiation. Furthermore, at least at early stages, apoptosis is correlated with increased cell differentiation. On the other hand, in lines expressing high amounts of T antigen and low amounts of p53, neither an increase in cAMP-induced differentiation nor massive apoptosis was seen at 32 degrees C. These findings demonstrate that wild-type p53 can cooperate with cAMP-generated signals in the induction of steroidogenesis and of programmed cell death in granulosa cells.

Animals↗

PubMed1995

Phase II trial of topotecan in patients with cisplatin-refractory germ cell tumors.

Fifteen patients with advanced, cisplatin-refractory germ cell tumors (GCT) were treated on a phase II trial with topotecan. None of the 14 evaluable patients achieved a complete or partial response. Myelosuppression was the major toxicity. The median nadir leukocyte count was 1.75 cells/mm3, neutrophil count was 1.55 cells/mm3, hemoglobin was 8.75 gm/dl, and platelet count was 20,500 cells/mm3. Topotecan is not efficacious in the treatment of cisplatin-refractory GCT.

Adult↗

PubMed1995

Detection of circulating tumor cells in patients with localized and metastatic prostatic carcinoma: clinical implications.

PURPOSE: To determine the frequency with which prostate-specific antigen (PSA)-positive cells can be detected in the peripheral blood of patients with prostatic cancer in different stages and with different sensitivities to hormonal therapy. PATIENTS AND METHODS: Peripheral blood from 107 men with prostatic cancer and 27 non-prostate cancer controls was analyzed for PSA mRNA using reverse-transcriptase polymerase chain reaction (RT-PCR) and Southern blotting. RESULTS: The lower limit of detection was one PSA-producing cell diluted into 1 x 10(6) blood mononuclear cells. The test detected PSA mRNA in four of 25 patients (16%) with clinically organ-confined (T1-2) disease, three of 10 (30%) with T3-4 or N+ tumors, and 25 of 72 (35%) with distant metastases. None of the control samples were positive. An increase in positivity was observed with increasing PSA levels. Within the subgroup of patients with distant metastases, positivity was observed in six of 16 patients (38%) with normal or undetectable PSA levels after hormonal therapy and, overall, in 37% of patients (21 of 57) with androgen-independent disease. CONCLUSION: An RT-PCR-based assay for PSA mRNA can detect circulating cells in the peripheral blood of patients with prostatic cancer. The frequency of positivity increases with tumor stage. A unique observation was the detection of cells in patients with no measurable PSA on hormonal therapy. This suggests that continued seeding of distant sites may still be occurring in these patients, despite seemingly successful therapy. The relationship between continued seeding, disease progression, and survival will require further study.

Base Sequence↗

PubMed1995

Regulation of follistatin messenger ribonucleic acid in steroidogenic rat granulosa cell lines.

Follistatin is a 35-kilodalton monomer isolated from follicular fluid that acts on pituitary gonadotropes to suppress the production of FSH. Transfection of rat granulosa cells with specific oncogenes, such as simian virus 40 (SV40) DNA and Ha-ras oncogene, leads to their immortalization concomitant with preservation of their capacity for inducible steroidogenesis. Experiments were designed to investigate the regulation of follistatin messenger RNA (mRNA) accumulation upon stimulation with forskolin, 2-O-tetradecanol-phorbol-13-acetate (TPA), FSH, and hCG in four different granulosa cell lines. Granulosa cells were transfected with SV40 DNA alone (POGS5); with SV40 DNA and Ha-ras oncogene (POGRS1); with SV40 DNA, Ha-ras oncogene, and LH/CG receptor (GLHR15); or with FSH receptor (GFSHR17) expression plasmid. Cells were cultured to reach confluence and then stimulated for 6 or 24 h with ovine FSH (oFSH; 0.004-4 nM), hCG (9 nM), forskolin (50 microM), and TPA (50 nM), alone or in combination. In the POGS5 cell line, forskolin caused a 15-fold stimulation of follistatin mRNA after 24-h incubation. The POGRS1 cell line showed a time-dependent stimulation of follistatin gene expression induced by both forskolin (5.7-fold) and TPA (9.4-fold). In the GFSHR17 cells, forskolin, oFSH, and TPA induced an increase in follistatin mRNA. When oFSH (1.6 nM) was added to cells treated with forskolin (50 microM) or TPA (50 nM), no additional stimulation was observed. The GLHR15 cell line treated with hCG showed a 2.7-fold increase in follistatin mRNA accumulation within 6 h. Our data demonstrate that 1) follistatin mRNA is detectable and induced by forskolin and TPA in transformed granulosa cell lines that do not express the FSH or LH receptors; 2) in the GFSHR17 cell line, FSH, forskolin, and TPA caused a time- and dose-dependent regulation of the gene; and 3) follistatin gene expression is up-regulated by hCG in the GLHR15 cell line. We conclude that these transformed steroidogenic cell lines can serve as a useful model to study the regulation of follistatin gene expression, a peptide known to regulate pituitary and ovarian hormone secretion and differentiation of granulosa cells by its activin-binding action. Moreover, this gene can be regulated in immortalized granulosa cells by both the protein kinase A and protein kinase C pathways, although these cells express the large T antigen and the Ha-ras oncogenic proteins.

Animals↗

PubMed1994

Sequential degradation of the neuropeptide gonadotropin-releasing hormone by the 20 S granulosa cell proteasomes.

The decapeptide gonadotropin-releasing hormone (GnRH) is degraded by the 20 S multicatalytic proteinase complex (proteasome EC 3.4.99.46), purified from ovarian granulosa cells, at the Tyr5-Gly6 bond and to a lesser extent at the Gly6-Leu7 bond, when incubated for 2 h at 37 degrees C. Further cleavage, at Trp3-Ser4 and Ser4-Tyr5 bonds of the neurohormone occurs only subsequently to the appearance of the initial N-terminal degradation products, (1-5)GnRH and (1-6)GnRH. Our results suggest that the sequential degradation of GnRH can serve as an important mechanism for the rapid termination of its biological activity in target cells.

Amino Acid Sequence↗

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↗

PubMed1994

Role of phospholipase-D and phosphatidic acid in mediating gonadotropin-releasing hormone-induced inhibition of preantral granulosa cell differentiation.

The activation of phospholipase-D (PLD) was previously implicated in mediating the differentiative action of GnRH on preovulatory granulosa cells. The activation of PLD and the action of its product, phosphatidic acid (PA), were further studied in preantral granulosa cells, where GnRH exerts an antidifferentiative effect. A GnRH receptor agonist (GnRH-A) activated PLD in the cells, causing a sustained elevation of phosphatidylethanol and a transient increase in cellular PA levels. PLD was also activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). Both GnRH-A and TPA inhibited FSH-induced production of progesterone, a marker of granulosa cell differentiation. D,L-Propranolol, which elevates cellular PA by inhibiting its degradation, mimicked the antidifferentiative action of GnRH-A and TPA in a dose-dependent manner. Addition of PA similarly inhibited FSH-induced progesterone production in a dose-dependent manner. The effect of forskolin, which mimics the steroidogenic effect of FSH by elevating intracellular levels of cAMP, could also be suppressed by GnRH-A and PA. FSH- and cAMP-induced differentiation of preantral granulosa cells is characterized by cell rounding and breakdown of actin filament bundles. This effect is inhibited by GnRH-A and TPA as well as PA. It is concluded that activation of PLD and the resultant production of PA could mediate the antidifferentiative action of GnRH in preantral granulosa cells. Moreover, GnRH-induced PLD-generated signals counteract the FSH-induced cAMP-dependent signals that modulate the organization of the actin cytoskeleton characteristic of steroidogenic cells.

Actins↗