Surgery for endometrial cancers with suspected cervical involvement: is radical hysterectomy needed (a GOTIC study)?

BACKGROUND: Radical hysterectomy is recommended for endometrial adenocarcinoma patients with suspected gross cervical involvement. However, the efficacy of operative procedure has not been confirmed. METHODS: The patients with endometrial adenocarcinoma who had suspected gross cervical involvement and underwent hysterectomy between 1995 and 2009 at seven institutions were retrospectively analysed (Gynecologic Oncology Trial and Investigation Consortium of North Kanto: GOTIC-005). Primary endpoint was overall survival, and secondary endpoints were progression-free survival and adverse effects. RESULTS: A total of 300 patients who underwent primary surgery were identified: 74 cases with radical hysterectomy (RH), 112 patients with modified radical hysterectomy (mRH), and 114 cases with simple hysterectomy (SH). Median age was 47 years, and median duration of follow-up was 47 months. There were no significant differences of age, performance status, body mass index, stage distribution, and adjuvant therapy among three groups. Multi-regression analysis revealed that age, grade, peritoneal cytology status, and lymph node involvement were identified as prognostic factors for OS; however, type of hysterectomy was not selected as independent prognostic factor for local recurrence-free survival, PFS, and OS. Additionally, patients treated with RH had longer operative time, higher rates of blood transfusion and severe urinary tract dysfunction. CONCLUSION: Type of hysterectomy was not identified as a prognostic factor in endometrial cancer patients with suspected gross cervical involvement. Perioperative and late adverse events were more frequent in patients treated with RH. The present study could not find any survival benefit from RH for endometrial cancer patients with suspected gross cervical involvement. Surgical treatment in these patients should be further evaluated in prospective clinical studies.

Activin A is stimulated by tumor necrosis factor-alpha and modulates collagen gene expression in human amniotic cells.

BACKGROUND: Accumulating evidence supports the idea of activin A as a modulator of inflammation. In human pregnancy, elevated activin A concentrations in amniotic fluid are reported in women with intra-amniotic infection and inflammation- induced pre-term birth. AIM: To test the hypothesis that activin A was involved in the pathophysiology of amnionitis, we evaluated the effects of tumor necrosis factor-α and lipopolysaccharide on activin A production in human amniotic epithelial cells, and the effects of activin A on the expression of collagen mRNA in amniotic mesenchymal cells. MATERIALS AND METHODS: Amniotic membranes were obtained from patients without systemic disease, signs of premature delivery or fetal complications, during elective cesarean sections at term. Amniotic epithelial cells and mesenchymal cells were separately obtained by enzymatic digestion and cultured. Activin A was measured by enzyme-linked immunosorbent assay and collagen mRNA levels were assessed by quantitative PCR. RESULTS: Amniotic epithelial cells produced activin A in a cell density- and time-dependent manner. Tumor necrosis factor- α enhanced activin A production in a time-dependent (48-120 h) and dose-dependent (10-300 ng/ml) manner in amniotic epithelial cells. Lipopolysaccharide also stimulated activin A production, but the effect was less prominent. In amniotic mesenchymal cells, the effect of activin A on the expression of type I and type III collagen mRNA was suppressive. CONCLUSIONS: Tumor necrosis factor-α and lipopolysaccharide stimulated activin A production in amniotic epithelial cells, and activin A modulated expression of collagen mRNA in amniotic mesenchymal cells. These results support the idea that activin A is involved in the pathophysiology of amnionitis.

Predicted indoor radon concentrations from a Monte Carlo simulation of 1,000,000 granite countertop purchases.

Previous research examining radon exposure from granite countertops relied on using a limited number of exposure scenarios. We expanded upon this analysis and determined the probability that installing a granite countertop in a residential home would lead to a meaningful radon exposure by performing a Monte Carlo simulation to obtain a distribution of potential indoor radon concentrations attributable to granite. The Monte Carlo analysis included estimates of the probability that a particular type of granite would be purchased, the radon flux associated with that type, the size of the countertop purchased, the volume of the home where it would be installed and the air exchange rate of that home. One million countertop purchases were simulated and 99.99% of the resulting radon concentrations were lower than the average outdoor radon concentrations in the US (14.8 Bq m(-3); 0.4  pCi l(-1)). The median predicted indoor concentration from granite countertops was 0.06 Bq m(-3) (1.59 × 10(-3) pCi l(-1)), which is over 2000 times lower than the US Environmental Protection Agency's action level for indoor radon (148 Bq m(-3); 4 pCi l(-1)). The results show that there is a low probability of a granite countertop causing elevated levels of radon in a home.

Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin increases chemosensitivity of CaSki cells to paclitaxel.

Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of pacitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24 h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells.

A SrTiO3 photoanode prepared by the particle transfer method for oxygen evolution from water with high quantum efficiencies.

A photoanode prepared from flux-synthesized Al-doped SrTiO3 by the particle transfer method with a Ta contact layer exhibited a high IPCE of 69% at 320 nm. The photocatalytic activity of SrTiO3 particles was very sensitive to the synthesis method used to make the SrTiO3 particles, while its photoelectrochemical performance was not.

Follicle-stimulating hormone regulation on its receptor messenger ribonucleic acid levels in cultured rat granulosa cells.

Our studies using immature rat granulosa cells cultured in serum-free medium on collagen-coated dishes indicated that FSH receptor mRNA levels do not change for at least 4 days of culture in the absence of hormone treatment. Addition of FSH (30 ng ml[-1]) led to a reduction of FSH receptor mRNA for a short time (6 h), followed by an increase in FSH receptor mRNA levels that reached maximum of around 200% of the initial level within 2-3 days after the addition of FSH. Following the addition of 10 nM PMA, FSH receptor mRNA levels were decreased to 50% of the pretreatment levels. During prolonged exposure to PMA, gradual recovery of the FSH receptor mRNA level was observed, and it was significantly higher than the control level at 48 h. The inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate did not depress FSH receptor mRNA levels. Downregulation of the FSH receptor mRNA was detectable at a PMA concentration of 1 nM. The two predominant FSH receptor mRNA transcripts, ca. 5.5 and 2.4 kb, respectively, appeared to be equally affected by SH and PMA treatments. To examine the role of PKC mediation of the effect of FSH on FSH receptor mRNA levels, granulosa cells were treated with the PKC inhibitor, H-7, and FSH. Although, FSH receptor mRNA levels decreased to 50% of control in the cells treated with FSH alone, the addition of H-7 (0.1 nM) caused no decline in FSH receptor mRNA levels relative to the control in the cells treated with FSH. On the other hand, inhibition of FSH receptor mRNA by FSH was partially suppressed by the PKC-selective inhibitor bisindolylmaleimide. The mRNA turnover experiments showed that the half-life of FSH receptor transcripts was unaffected by PMA exposure.

Regulation of cAMP responsive element binding modulator isoforms in cultured rat ovarian granulosa cells.

A pituitary glycoprotein hormone FSH stimulates ovarian granulosa cells to induce ovarian follicular development. In this study we identified rat ovarian genes that were rapidly induced by FSH in the cultured rat granulosa cells by means of subtraction cloning. Complementary DNA clones encoding cAMP responsive element binding modulator (CREM) were identified as one of the FSH inducible genes. Northern blotting and reverse transcription and polymerase chain reaction (RT-PCR) analyses revealed that only the repressor type of CREM gene products, ICER (inducible cAMP early repressor) isoforms, were induced by FSH treatment in cultured rat granulosa cells. The induction of ICER by FSH was mimicked by reagents known to increase intracellular cAMP levels, indicating that the induction is through cAMP and protein kinase A signal transduction system. Induction of ICER was also confirmed as the protein levels. Electrophoretic mobility shift assay of granulosa cell extracts with a radiolabeled double stranded oligonucleotide corresponding to somatostatin cAMP responsive element also revealed that only the ICER proteins were induced by FSH treatment, whereas levels of CREM proteins were nearly constant regardless of the FSH treatment. Our present study demonstrates that FSH-induced and cAMP-mediated induction and attenuation of transcriptional responses by CREM gene products may be a key mechanistic component for the granulosa cell differentiation and proliferation.

The mechanisms of retinoic acid-induced regulation on the follicle-stimulating hormone receptor in rat granulosa cells.

The present study was undertaken to identify the mechanisms underlying the effect of retinoic acid (RA) on follicle-stimulating hormone receptor (FSH-R) in rat granulosa cells. Treatment with FSH produced a substantial increase in FSH-R mRNA level, as was expected, while concurrent treatment with increasing concentrations of RA brought about dose-dependent decreases in FSH-induced FSH-R mRNA, with a maximal inhibition one-third lower than that induced by FSH alone. RA, either alone or in combination with FSH, did not affect intracellular cAMP levels, while it inhibited the effect of 8-Br-cAMP on FSH-R mRNA production. These results suggested that RA diminished the action of FSH on FSH-R expression at sites distal to cAMP generation in the granulosa cells. Whether the effect of RA and FSH on FSH-R mRNA levels was the result of decreased transcription and/or altered mRNA stability was also investigated. The rate of FSH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, was found to decrease by the addition of RA. On the other hand, the decay curves for the 2.4 kb FSH-R mRNA transcript in primary granulosa cells did not alter the slope of the FSH-R mRNA decay curve in the presence of RA. Our data suggests for the first time that the effect of RA on FSH-R expression is possibly mediated by the reduction of the FSH-R mRNA level due to a negative regulation of the FSH-R gene in the presence of FSH. These findings assist in understanding the molecular mechanism underlying the effect of RA on reproductive function in rat granulosa cells.

Molecular cloning and functional expression of chicken luteinizing hormone receptor.

A complementary DNA for chicken luteinizing hormone (LH) receptor containing the entire coding region was isolated from chicken F1 granulosa cell cDNA library. Nucleotide sequence analysis revealed that there are characteristic GC-rich regions around the N-terminal part. Chicken LH receptor consists of a 19-residue signal peptide, a 366-residue extracellular domain, a 267-residue region containing seven transmembrane segments, and a 76-residue cytoplasmic C-terminal tail. The deduced amino acid sequence of the chicken LH receptor shares 67%, 69%, and 69% identity with the human, rat and porcine LH receptor sequences, respectively, and 51% with chicken FSH receptor. However, an insertion of about 30 amino acid residues is found in chicken LH receptor in the extracellular domain about 44 amino acid residues upstream of the first transmembrane segment. In addition, alternative splicing seems likely to occur at the point where the insertion starts (nucleotide position 933), resulting in the truncated forms of chicken LH receptor with only the extracellular domain. Northern blot analysis revealed the presence of multiple transcripts of LH receptor, a major 3.0-kb and minor 7-kb and 1.5-kb bands, in chicken F1 to F3 granulosa cells. The full length chicken LH receptor cDNA was transiently expressed in COS-7 cells and the transfected cells displayed a concentration-dependent increase in cAMP production when exposed to varying concentrations of chicken LH. This clearly indicates that the cloned cDNA encodes a functional chicken LH receptor protein.

Expression of variant luteinizing hormone/chorionic gonadotropin receptors and degradation of chorionic gonadotropin in human chorionic villous macrophages.

Human gonads and non-gonadal organs/tissues express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors. This study aimed to identify the LH/CG receptors and to clarify their function in human placental chorionic villous macrophages. Macrophages as well as syncytiotrophoblasts of human chorionic villous tissues were immunohistochemically positive for LH/CG receptor throughout gestation. By reverse transcription-nested polymerase chain reaction methods, villous macrophages were shown to express a variant type of LH/CG receptor, the sequencing of which revealed a deletion of exon 9. For experiments in vitro, a monocyte-macrophage cell line, THP-1, was transfected with vector alone, wild-type LH/CG receptor, and exon 9-deleted LH/CG receptor after phorbol 12-myristate 13-acetate (PMA) treatment. Non-PMA-treated THP-1 cells transfected with vector alone were also examined. THP-1 cells expressed exon 9-deleted LH/CG receptor after treatment with PMA. After the cells of the four groups were cultured in medium containing intact human CG (hCG), the concentrations of hCG and its beta-core fragment (beta-CF) were measured in the supernatant of the culture medium and in the cell cytosol. Time-dependent hCG uptake was observed in both non-PMA-treated and PMA-treated THP-1 cells, suggesting that the variant receptor is not directly involved in the ingestion of hCG. The degradation of hCG and excretion of beta-CF were progressed in PMA-treated cells but not in the un-treated cells. In the cell cytosol, the ratio of beta-CF and hCG concentrations (beta-CF/hCG) was significantly higher in the PMA-treated cells than in non-PMA-treated cells; however, it did not differ between the PMA-treated cells transfected with exon 9-deleted receptor and those transfected with vector alone. Macrophages may express the variant receptor in order to recognize the intracytoplasmic hCG and transport it to the lysosome. Among the two PMA-treated cells, the ratio was lower in those transfected with wild-type receptor. The expression of the variant receptor may modulate the degradation of hCG but be reduced by expression of the wild-type receptor in its lacking macrophages. Our data suggest a potentially important role for exon 9-deleted LH/CG receptors expressed in human placental villous macrophages in the local metabolism of hCG.

Expression of gonadotropin and activin receptor messenger ribonucleic acid in human ovarian epithelial neoplasms.

Activin receptors (ActRs) and gonadotropin receptor mRNA expression were investigated in 18 human ovarian epithelial neoplasms. Northern blot analysis showed the presence of 3.0-kb type Ia ActR, 6.0- and 3.0-kb type IIa ActR, and 5.0-kb type IIb ActR mRNA transcripts in total RNA prepared from the cancer tissues. One carcinoma showed two major transcripts of a follicle-stimulating hormone receptor (FSH-R) gene, 4.1 and 2.4 kb, whereas the other two carcinomas showed two major transcripts of the luteinizing hormone/human chorionic gonadotropin receptor (LH-R) gene, 5.4 and 2.4 kb. These results were further analyzed by studying the corresponding PCR-amplified FSH and LH-R cDNA obtained by reverse transcription of total RNA. Expression of FSH-R mRNA was confirmed in about half of the cancer tissues. The size of the FSH-R reverse transcription-PCR product was the same as in normal ovarian follicles. Similarly, expression of LH-R mRNA was also detected in about half of the cancers. Normal ovaries and cancer tissues were homogenized, and activin concentrations were measured in extracts. Activin levels in normal ovarian tissue were around 0.59 +/- 0.01 ng/mg protein (mean +/- SE; n = 5), and activin production was detected in every cancer tissue, except one--serous adenocarcinoma. The findings in this study demonstrated that activin and ActRs are present in and synthesized by human ovarian epithelial neoplasms. Thus, activin seems to be available as an autocrine/paracrine factor in epithelial neoplasms and may contribute to the expression of FSH-R, although the roles of activin and gonadotropin in tumorigenesis has yet to be defined.

Cloning and characterization of gonadotropin-inducible ovarian transcription factors (GIOT1 and -2) that are novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family.

Gonadotropins are essential for ovarian follicular development and differentiation. To identify genes that are rapidly induced by gonadotropin in the immature rat ovary, ovarian genes were screened by a subtraction cloning procedure. cDNA clones encoding novel members of the (Cys)(2)-(His)(2)-type zinc finger protein family GIOT1 and -2 (gonadotropin-inducible transcription factor 1 and 2), were identified. Two isoforms of GIOT2 (GIOT2 alpha and 2 beta), which are probably produced by alternative splicing, also exist. Nucleotide sequence analysis revealed that GIOT1, but not GIOT2, contains the krüppel-associated box-A domain at the NH(2) terminus. RNA analyses revealed that these mRNAs were rapidly and temporarily induced by gonadotropins in the rat testis as well as in the ovary. In situ hybridization study revealed that expression of GIOT1 was induced in theca interna cells in the ovary and Leydig cells in the testis. Interestingly, the gene expression of GIOT1 is restricted to the pituitary, adrenal, testis, and ovary, while GIOT2 gene is expressed ubiquitously. A functional analysis of GIOT1 and -2 by a GAL4-based mammalian one-hybrid system revealed that GIOT1, but not GIOT2, is a transcriptional repressor and that the krüppel-associated box-A domain of GIOT1 is responsible for the transcriptional repressor activity. A GAL4-based yeast two-hybrid system was also used to identify proteins that interact with the rat GIOT1. We cloned genes encoding rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta, both of which are transcription-regulatory proteins. Interaction of these proteins with GIOT1 was directly demonstrated by GST pull-down assay. Our data strongly suggest that GIOT1 may function as a novel transcriptional repressor by working with rat homologs of human I-mfa domain containing protein and transcriptional intermediary factor 1 beta proteins and may play a significant role at the transcription level in the folliculogenesis.

Plasma des-acyl and acyl ghrelin in patients with eating disorders.

A recently recognized peptide, ghrelin, increases appetite and energy retention in human. Previous reports have shown higher plasma level in eating disorder (ED) patients and correlations with body mass index (BMI). This study examined these findings by measuring active (N-RIA) and total (C-RIA) levels of plasma ghrelin. Multipurpose assessments of symptoms were conducted for 11 ED patients and 5 control females. Results revealed significant differences of C-RIA between the groups. The BMI did not correlate with ghrelin, but demonstrated reversal correlation with the ratio of N-RIA and C-RIA (NC ratio) according to the ED or control group. The NC ratio also tended to be associated with a self-rating score. The NC ratio might be related to specific characteristics of ghrelin secretion or clearance in ED patients. Further basic and clinical investigations are necessary.

Luteinizing hormone-releasing hormone stimulates arachidonic acid release in rat granulosa cells.

The effect of LHRH on arachidonic acid release was studied in rat granulosa cells in primary culture. In cells prelabeled with [3H]arachidonic acid, LHRH caused an increase in the level of [3H]arachidonic acid released in the culture medium, to 125-150% of control levels at the end of a 60-min incubation period. In subsequent time-course and dose-response experiments, a significant effect on [3H]arachidonic acid release could be observed as early as 15 min after LHRH addition, and the lowest effective dose was 10(-8) M LHRH. Addition of LH, FSH, prostaglandin F2 alpha, or (Bu)2cAMP was without effect. Likewise, an agonistic LHRH analog (LHRHa, 10(-8) M) also markedly stimulated [3H]arachidonic acid from cultured granulosa cells, and the effects of both LHRH and LHRHa were blocked by concomitant presence of a potent LHRH antagonist. In addition to [3H]arachidonic acid release in the culture medium, the effect of LHRH on the level of radiolabel present in cellular phospholipids was also examined. In granulosa cells prelabeled with [3H] arachidonic acid, LHRH significantly depleted the level of radioactivity previously incorporated into cellular phosphatidylinositol, as early as 5 min after its addition, to 85% of control levels. The level of radiolabel found in other major phospholipids such as phosphatidylserine/phosphatidylcholine and phosphatidylethanolamine, as well as the intracellular level of unesterified [3H]arachidonic acid, were not significantly affected by LHRH. The effect of LHRH on [3H]arachidonic acid release from prelabeled phospholipids as well as the LHRH-induced loss of radioactivity previously incorporated into phosphatidylinositol could be reversed by verapamil, suggesting a possible calcium dependency. Taken together, these data support the notion that arachidonic acid liberation from phospholipids may be associated with the mechanism of action of LHRH on ovarian cells.

Effect of activin on luteinizing hormone-human chorionic gonadotropin receptor messenger ribonucleic acid in granulosa cells.

Activin (the dimer of inhibin beta-subunit) is involved in the modulation of granulosa cell function. Recent reports have indicated that activin had an effect on LH/human CG (hCG) receptor induction and steroidogenesis in granulosa cells. To characterize the regulation inducing LH/hCG receptor by activin, we investigated messenger RNA (mRNA) levels, the expression of the LH/hCG receptor, and intracellular cAMP accumulation in cultured rat granulosa cells. Northern blot analysis showed an increase in the LH/hCG receptor mRNA level with FSH (30 ng/ml) and activin (100 ng/ml) cotreatment, whereas activin alone could not augment LH/hCG receptor mRNA at all. After the addition of actinomycin D to the culture medium, LH/hCG receptor mRNA was more stable in the presence of FSH plus activin than in the presence of FSH alone. Similarly, a receptor binding assay revealed that the cotreatment with FSH and activin induced more LH/hCG receptor than FSH alone 96 h after exposure to hormone, but that activin (100 ng/ml) alone could not induce the LH/hCG receptor. Since the primary, if not the sole, second messenger mediating the action of FSH in granulosa cells has been shown to be cAMP, intracellular cAMP accumulation was measured in granulosa cells in the presence of FSH (30 ng/ml) and/or activin (100 ng/ml). Although FSH-stimulated cAMP accumulation reached a peak 15 min after incubation, activin did not significantly alter cAMP accumulation in either control nor FSH-stimulated granulosa cells, indicating that the effects of activin on the LH/hCG receptor in granulosa cells are not mediated by the increase in cAMP. These results demonstrate that activin enhances the FSH-induced LH/hCG receptor mRNA, LH/hCG receptor mRNA stability, and LH/hCG binding sites not due to the stimulation of the adenylate cyclase system. Although the signal pathway from the activin receptor has not been elucidated upon yet, activin is capable of increasing LH/hCG receptor levels through the accumulation of LH/hCG receptor mRNA levels.

Expression and effect of adrenomedullin on rat granulosa cell.

Adrenomedullin (AM) is a potent hypotensive peptide recently discovered in extracts of human pheochromocytoma. To elucidate the regulation of AM production in the ovary, the effect of gonadotropin on the production of AM was studied in the cultured rat granulosa cells. A Northern blot analysis of the LH receptor and adrenomedullin yielded a major hybridizing band at 5.4 kb and 1.6 kb, respectively. In our culture system of rat granulosa cells, without any stimulus, the LH receptor mRNA was undetectable and the AM mRNA level was stably expressed for 6 days. FSH significantly induced LH receptor mRNA and suppressed AM mRNA for 4 days of culture and with the addition of hCG after 2 days of pretreatment with FSH, AM mRNA levels were markedly suppressed. FSH and 8-Br-cAMP significantly increase LH receptor mRNA and suppress AM mRNA in a dose-dependent manner. These data indicated that the differentiation of granulosa cells mediated by gonadotropins were associated by suppression in AM expression through a cAMP-dependent mechanism. On the other hand, AM stimulated a rapid rise in intracellular cAMP levels, which peaked within 15 min of addition, in a dose-dependent manner with a maximal response seen at 100 nM. Additionally, AM enhanced the effects of FSH, acting additionally to produce cAMP in the cells. AM may play a role in the process of granulosa cell differentiation as a local regulator through an autocrine/paracrine mechanism.

Induction of polyphosphoinositide breakdown in rat corpus luteum by prostaglandin F2 alpha.

The present study examines the possibility that, in the rat corpus luteum, an initial action of prostaglandin F2 alpha (PGF2 alpha) is to induce a ligand-stimulated breakdown of membrane inositol phospholipids. Luteal cells in primary culture were prepared from immature rats after PMSG and human CG priming. In 32P-prelabeled cells, PGF2 alpha caused a rapid decrease in the level of radiolabel found in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, as early as 20 sec after addition of the hormones. At 1 and 2.5 min, the effect of 10(-6) M PGF2 alpha on phosphatidylinositol 4,5-bisphosphate was significantly greater than that caused by 10(-6) M LHRH in identical cell cultures. By contrast, the levels of the 32P-prelabeled phosphatidylinositol and phosphatidic acid were increased at 5 min by PGF2 alpha or LHRH. Concomitant with the alterations in cellular levels of 32P-prelabeled phospholipids, PGF2 alpha markedly enhanced the accumulation of 3H-labeled inositol phosphates, i.e. inositol 1-phosphate, inositol diphosphate, and inositol triphosphate, during a 5-min incubation. A significant increase of radiolabeled inositol diphosphate was seen as early as 1 min after the addition of either PGF2 alpha or LHRH; PGF2 alpha was more effective than LHRH in this regard. The stimulatory effect of LHRH on inositol phosphate accumulation could be blocked completely by the concomitant presence of a potent LHRH antagonist, and at the concentration used (10(-6) M) the effects of PGF2 alpha and LHRH were not additive. Interestingly, the addition of an exogenous phospholipase C also caused a similar enhancement of inositol phosphate accumulation in identical cell cultures. For the first time, these data suggest that, at the level of the corpus luteum, hydrolysis of phosphoinositides may immediately follow PGF2 alpha (and to a lesser extent LHRH) receptor binding, and this in turn may lead to the generation of 1,2-diacylglycerol and inositol phosphates, resynthesis of phosphatidic acid and phosphatidylinositol, and mobilization of Ca2+.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of follicle-stimulating hormone receptors during cell differentiation in cultured granulosa cells.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is a common environmental pollutant causing public concern. Using a cell culture system derived from rat granulosa cells that provides unique advantages for studying the molecular mechanisms underlying the action of TCDD, the influences of TCDD on FSH receptor (FSH-R) induction were examined. The treatment with FSH produced, as expected, a substantial increase in specific FSH-R expression, whereas concurrent treatment with the environmental amount of TCDD (10 pM) resulted in a significant decrease in FSH-R after being cultured from 24-72 h. Cotreatment with FSH (30 ng/ml) and increasing doses of TCDD inhibited the levels of FSH-induced FSH-R messenger RNA (mRNA) in a dose-dependent manner. Treatment with 8-Br-cAMP (1 mM) produced a significant increase in FSH-R mRNA; concurrent treatment with TCDD (10 pM) produced a significant attenuation of 8-Br-cAMP action. These findings suggest that the ability of TCDD to interfere with FSH action, as regards the induction of FSH-Rs, is exerted at sites distal to those involved in cAMP generation. Because a single transcript of 5.2 kb was seen for the Ah receptor in this granulosa cell system, the effects of TCDD may be mediated by this specific receptor. The rates of FSH-R mRNA gene transcription, assessed by nuclear run-on transcription assay, were decreased by the addition of TCDD. The effect of TCDD on FSH-R mRNA stability was determined by measuring the decay of FSH-R mRNA under conditions known to inhibit transcription. The decay curve for the 2.4-kb FSH-R mRNA transcript was not significantly changed after the addition of TCDD. These findings showed that the effect of TCDD on FSH-R mRNA was, at least in part, the result of decreased transcription.

Effect of prolactin on the expression of luteinizing hormone receptors during cell differentiation in cultured rat granulosa cells.

Chronic and transient hyperprolactinemia has been associated with luteal phase dysfunction. Recently, evidence has emerged to suggest that elevated PRL may exert its antigonadal effects through reducing available ovarian LH receptors. We have now examined the influences of PRL on LH receptor induction in cultured granulosa cells. Basal specific LH binding was negligible and remained unchanged in response to treatment with PRL by itself. Whereas treatment with FSH produced, as expected, a substantial increase in specific LH binding, concurrent treatment with PRL resulted in no significant change during the first 4 days of culture, followed by a significant decrease in LH binding on days 5 and 6 as well as an approximately 50% inhibition of FSH effect on day 6. Scatchard plot analysis showed that concurrent treatment with PRL resulted in inhibition of the granulosa cell LH binding capacity, whereas no difference could be detected in the binding affinity of LH to its receptor. Treatment with 8-bromo-cAMP produced a significant increase in specific LH binding; concurrent treatment with PRL (30 ng/ml) produced a significant attenuation of 8-bromo-cAMP action. In addition, treatment with FSH increased the intracellular accumulation of cAMP, and concurrent treatment with PRL did not result in inhibition of the FSH action, as assessed by the generation of intracellular cAMP. Taken together, these findings suggest that the ability of PRL to interfere with FSH action with regard to the induction of LH receptors is exerted at sites distal to those involved in cAMP generation. The effect of PRL on LH receptor messenger RNA (mRNA) levels was not significant during the increase in receptors, whereas after the maximal level of receptor expression was reached, the effect of PRL was apparent. Cotreatment with FSH (30 ng/ml) and increasing doses of PRL inhibited the levels of FSH-induced LH receptor mRNA in a dose-dependent manner, whereas PRL did not inhibit the effect of FSH on the FSH receptor mRNA. To investigate the hormonal regulation of the 5'-flanking region, we analyzed the effect of FSH on 1379 bp of LH receptor promoter in rat granulosa cells. Treatment with FSH (1-100 ng/ml) significantly enhanced the activity of 1379 bp of the LH receptor 5'-flanking region in dose-dependent manner. Treatment with 30 ng/ml PRL alone did not significantly influence the activity of the LH receptor promoter and did not affect the increased promoter activity induced by FSH. In addition, the rates of LH receptor mRNA gene transcription assessed by nuclear run-on transcription assay increased by the addition of FSH and were not affected by the addition of PRL in the presence of FSH. These data showed that PRL might not effect LH receptor gene transcription in the regulation of LH receptor mRNA. Next, an attempt was made to determine the effect of PRL on LH receptor mRNA stability by measuring the decay of LH receptor mRNA under conditions known to inhibit transcription. However, inhibitors of transcription were found to have a stabilizing effect on the LH receptor mRNA, thus potentially masking the effect of PRL. According to the expression of LH receptor mRNA, PRL might not affect the maximum level induced by FSH, but thereafter the maximum levels of LH receptor mRNA decreased faster than those of the control. Therefore, it may be possible that PRL acts to stimulate labile LH receptor mRNA-destabilizing factors.

A role of insulin-like growth factor I in luteinizing hormone receptor expression in granulosa cells.

The present study was undertaken to identify the mechanisms underlying the effect of insulin-like growth factor (IGF-I) on LH receptor in rat granulosa cells. Treatment with FSH, as expected, produced a substantial increase in LH receptor messenger RNA (mRNA) level, and concurrent treatment with increasing concentrations of IGF-I brought about dose-dependent increases in FSH-induced LH receptor mRNA, with a maximal response 2.5-fold greater than that induced by FSH alone. IGF-I, either alone or in combination with FSH, did not affect intracellular cAMP levels, whereas it enhanced the effect of 8-bromo-cAMP on LH receptor mRNA production. We then investigated whether the effects of IGF-I and FSH on LH receptor mRNA levels are the results of increased transcription and/or altered mRNA stability. To determine whether the LH receptor 5'-flanking region plays a role in directing LH receptor mRNA expression, the proximal area of the LH receptor 5'-flanking regions were inserted into a transient expression vector, pGL-Basic, which contains luciferase as the reporter gene, and the resulting plasmids were transiently transfected into rat granulosa cells. Our studies show that the FSH-induced luciferase activity varied dependent upon the length of the 5'-flanking region sequence in the reporter gene. In addition, FSH (30 ng/ml) significantly enhanced the activity of 1379 bp of the LH receptor 5'-flanking region, but treatment with 10 ng/ml IGF-I alone did not significantly influence the activity of the LH receptor promoter or affect the increased promoter activity induced by FSH. The rates of LH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, were not increased by the addition of IGF-I. On the other hand, the decay curves for LH receptor mRNA transcript in primary granulosa cells showed a significant increase in the half-life after the addition of IGF-I. These data suggest a possible role for changes in LH receptor mRNA stability in the IGF-I-induced regulation of LH receptor in rat granulosa cells. This interface between circulating hormones and paracrine/autocrine systems could provide an important mechanism to amplify the effects of gonadotropic hormones at the local level.