Biomedical subjects

K M Menon

Publications and source records attributed to K M Menon.

At least 37 records · Page 2Linked to original sources

PubMed1994

Molecular cloning of a novel luteinizing-hormone/human-chorionic-gonadotropin-receptor cDNA. Identification of a long 3' untranslated region and cDNA sequence of the major transcript in rat ovary.

The biological action of luteinizing hormone/human chorionic gonadotropin (lutropin/choriogonadotropin) in the ovary is mediated by interaction with its specific receptor. Lutropin/choriogonadotropin-receptor hnRNA is processed into multiple mRNAs. However, nucleotide sequences for many of the transcripts, including the major form (6.7 kb), have yet to be determined. In an attempt to identify a cDNA encoding the major transcript, we have isolated a 3.5-kb cDNA clone from a rat ovary cDNA library. The 3.5-kb cDNA recognized only two (6.7 kb, 4.4 kb) of the three (6.7, 4.4, 2.6 kb) ovarian lutropin/choriogonadotropin-receptor transcripts when used as a probe. The first 732 nucleotides of the newly identified 3.5-kb cDNA showed 98% identity to the 3' untranslated region (3' UTR) of the previously cloned cDNA corresponding to the 4.4-kb transcript. Southern blot analysis indicated that the 3.5-kb cDNA and the C-terminal domain of the lutropin/choriogonadotropin-receptor originate from the same gene. Oligonucleotide-directed cleavage of the 6.7-kb lutropin/choriogonadotropin-receptor mRNA by RNase H revealed that the newly identified 3.5-kb cDNA is a 3' extension of the 4.4-kb transcript. We propose that the nucleotide sequence of the 6.7-kb lutropin/choriogonadotropin-receptor transcript, the major form found in rat ovary, contains a long 3' UTR, which has not been previously identified.

Animals↗

PubMed1994

Protein kinase C-dependent and -independent pathways mediate epidermal growth factor (EGF) effects in human endometrial adenocarcinoma cell line KLE.

The role of EGF in the proliferation of the poorly differentiated endometrial adenocarcinoma cell line (KLE) was examined. EGF (10 ng/ml) and the tumor promoter, protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA) were potent stimulators (P < 0.01) of DNA synthesis in this cell line as determined by [3H]thymidine incorporation into DNA. Staurosporine, a protein kinase C inhibitor, partially blocked the EGF effects on [3H]thymidine incorporation. Similarly, downregulation of protein kinase C also failed to completely abolish EGF effect on DNA synthesis and cell division. These results suggest that in the KLE cell line EGF stimulation of cell growth is exerted through both protein kinase C-dependent and -independent pathways.

Adenocarcinoma↗

PubMed1994

Expression of high density lipoprotein-binding protein messenger ribonucleic acid in the rat ovary and its regulation by gonadotropin.

The presence of high density lipoprotein-binding protein (HBP) mRNA in rat ovarian cells and its possible regulation by gonadotropin were examined in this study. RNA, isolated from pseudopregnant rat ovaries, was amplified by reverse transcriptase-polymerase chain reaction (PCR) with HBP-specific oligonucleotide primers. A distinct and prominent 576-basepair PCR product was generated. Sequence analysis revealed that the sequence homology of the PCR product and the corresponding human HBP cDNA sequence was greater than 90%. Using Northern blot analysis, we identified two species of HBP mRNA transcripts in the ovarian cells (i.e. a major one of 4.5 kilobases and a minor one of 6.0 kilobases). Cellular localization of HBP mRNA in the ovary was determined by in situ hybridization analysis. Prominent specific hybridization of the labeled antisense probe was observed in all cell types of the follicles and corpora lutea, whereas little hybridization was observed in the stroma and the connective tissues separating corpora lutea and follicles. As hCG has been shown to induce high density lipoprotein-binding activity in the rat ovary, we examined the possible regulation of ovarian HBP mRNA by this hormone. Administration of hCG caused a significant time-dependent increase in the steady state levels of HBP mRNA. The induction of HBP mRNA levels by hCG is specific for the ovary, as pretreatment with hCG had no effect on the HBP mRNA levels of the liver, heart, lung, or kidney. The present study, for the first time, shows conclusively the presence of HBP mRNA in the rat ovary and its induction by hCG, implicating a physiological role for HBP in the ovary.

Animals↗

PubMed1994

In situ hybridization of luteinizing hormone/human chorionic gonadotropin receptor messenger ribonucleic acid during hormone-induced down-regulation and the subsequent recovery in rat corpus luteum.

Previous studies have shown that injection of a pharmacological dose of hCG to rats primed with PMSG/hCG results in a loss of hCG binding in the luteinized ovary, which is closely coupled with the loss of LH/hCG receptor (LH/hCG-R) messenger RNA (mRNA). The time course of down-regulation of the receptor mRNA reveals that mRNA totally disappears 24 h after the hormone injection, but fully recovers by 72 h. The purpose of this study was to determine by in situ hybridization whether the recovery of the receptor mRNA occurs in preexisting or newly formed corpora lutea. Twenty-one-day-old female rats were treated with 50 IU PMSG, followed 56 h later by a single injection of hCG. On day 5 after the hCG injection, one group of rats was treated with a desensitizing dose of hCG, and a control group received saline. The ovaries were collected 6, 12, 24, 48, 72, and 96 h after the treatments and were processed for in situ hybridization using 35S antisense RNA synthesized from a 750-mer LH/hCG-R complementary DNA. In control ovaries, heavily labeled LH/hCG-R mRNA-containing cells were observed in the numerous corpora lutea. Ribonuclease pretreatment of the sections eliminated the signal, and no specific hybridization was observed when sense strand probe was used. No hybridization to the granulosa cells was seen. Some hybridization occurred to the theca interna, but the intensity was lower than that in the corpora lutea. Time-course studies showed a marked decline in the hCG-R mRNA signals in corpora lutea as early as 6 h after hCG injection, with a maximum loss of receptor mRNA by 24 h. After 48 h, hCG-R mRNA reappeared in preexisting corpora lutea, with the intensity of the hybridization signal equaling that in corpora lutea of controls. New corpora lutea could not be identified at any time after injection of the down-regulating dose of hCG. As down-regulated receptor mRNA recovered in preexisting, not new, corpora lutea, hormone-induced loss of luteal cell receptors would appear to be reversible.

Animals↗

PubMed1993

Identification of a scavenger receptor in rat luteal cells which recognizes chemically modified lipoproteins and mediates the uptake of cholesterol for steroidogenesis.

The presence of the acetyl low density lipoprotein (acetyl-LDL), or scavenger receptor, which binds modified forms of LDL, was examined in rat luteal cells. Acetyl-LDL supported progesterone production by dispersed rat luteal cells at least equal to that of the native LDL under basal conditions and in the presence of human chorionic gonadotropin (hCG). The acetyl-LDL-supported progesterone production was stimulated by hCG and dibutyryl cAMP in a concentration-dependent manner. Studies on acetyl-LDL binding to ovarian plasma membranes revealed a single class of binding sites with high affinity. The binding is specific in that unlabeled acetyl-LDL and fucoidin, a known competitor for binding to scavenger receptor, were effective competitors, while native LDL was not. Furthermore, degradation of 125I-acetyl-LDL by cultured luteal cells was inhibited by unlabeled acetyl-LDL and fucoidin but not native LDL. The experiments on cross-competition between acetyl-LDL and tetranitromethane-modified high density lipoprotein (TNM-HDL) indicated that TNM-HDL is also recognized as a ligand by this receptor. In addition, in vivo pretreatment of rats with hCG resulted in induction of acetyl-LDL binding activity of ovarian plasma membranes in a time-dependent manner when compared to saline injected controls. The increase in binding activity was due to an increase in the number of binding sites rather than to a change in the binding affinity. In conclusion, this study demonstrates that, in addition to receptor-mediated LDL and HDL pathways, rat luteal cells possess a scavenger receptor pathway, which recognizes TNM-HDL as well as acetyl-LDL. This receptor may play an important role in the uptake and utilization of modified lipoprotein-associated cholesterol by luteal cells.

Animals↗

PubMed1993

Epidermal growth factor receptor expression in three different human endometrial cancer cell lines.

BACKGROUND: The aim of the current study was to establish the characteristics of the epidermal growth factor (EGF) receptor in endometrial cell lines to determine the possible relation of EGF to endometrial cancer. METHODS: Three different cell lines were used: RL95-2 (derived from a moderately differentiated adenosquamous carcinoma), HEC-I-A (from a moderately differentiated adenocarcinoma), and KLE (from a poorly differentiated adenocarcinoma). The binding of (125-I) EGF to these cell lines and the stimulatory effect of EGF on (3H) thymidine incorporation into DNA were examined. RESULTS: EGF receptor was present in all three cell lines. The binding of 125-I-labeled EGF was saturable and of high affinity. Scatchard analysis of the competitive binding data for KLE, HEC-I-A, and RL95-2 revealed linear plots, indicating a single class of binding sites with almost identical equilibrium dissociation constants (0.34 nM, 0.23 nM, and 0.20 nM, respectively). Other peptides, such as insulin-like growth factor (IGF) I and II and insulin, did not compete for the receptor. RL95-2 cells bound significantly more EGF (P < 0.005) than did the HEC-I-A and KLE cell lines. EGF increased 3H-thymidine incorporation in all three cell lines. CONCLUSION: Because EGF receptors are expressed by all three cell lines at markedly different levels and because EGF stimulates 3H-thymidine incorporation into DNA in the three cell lines, the current study suggests that EGF may play a role in the promotion of growth endometrial adenocarcinoma.

Cell Division↗

PubMed1993

Mitogenic activity of growth factors in the human endometrial adenocarcinoma cell lines HEC-1-A and KLE.

Endometrial adenocarcinoma is the most common gynecologic malignancy occurring in the United States. Evidence is accumulating that links peptide growth factors with malignant proliferation. Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) are known mitogens for endometrial adenocarcinoma in vitro. However, the biological activity of other growth factors in this malignancy is unclear. This study was undertaken to determine the influence of growth factors on the mitogenic activity of the human endometrial adenocarcinoma cell lines HEC-1-A and KLE. Incubation with EGF, IGF-I, insulin-like growth factor II (IGF-II), or insulin stimulated a time-dependent mitogenic response in both cell lines, with the peak response occurring at 24 hr for HEC-1-A and 48 hr for KLE. After two doubling intervals, the number of HEC-1-A cells was increased 3.5-fold by EGF (100 ng/ml), 2.7-fold by IGF-I (100 ng/ml), 2.3-fold by IGF-II (100 ng/ml), and 2.2-fold by insulin (1000 ng/ml) when compared to untreated controls (P < 0.05). The number of KLE cells was increased 2.6-fold by EGF (100 ng/ml), 2.3-fold by IGF-I (100 ng/ml), 2.1-fold by IGF-II (100 ng/ml), and 2.0-fold by insulin (1000 ng/ml) when compared to untreated controls (P < 0.05). Similar results were obtained when DNA content was measured. PDGF failed to stimulate any mitogenic response in either cell line at all concentrations tested (0.1-100 ng/ml). These findings suggest that EGF, IGF-I, IGF-II, and insulin may play a regulatory role in the proliferation of endometrial adenocarcinoma.

Adenocarcinoma↗

PubMed1993

Loss of lutropin/human choriogonadotropin receptor messenger ribonucleic acid during ligand-induced down-regulation occurs post transcriptionally.

Previous studies have demonstrated that ligand-induced down-regulation of the LH/hCG receptor in rat corpus luteum results in a loss of ligand-binding activity and a parallel decline in steady state levels of receptor mRNA. The purpose of the present study was to determine whether this loss of receptor mRNA during receptor down-regulation is due to inhibition of transcription or increased degradation of the receptor mRNA. To differentiate between these two possibilities, nuclear run-off assays were performed to study transcription rates of nuclei isolated from control and hCG down-regulated rat ovaries. A 750-mer LH/hCG receptor cDNA probe, spanning the carboxy-terminal region of the peptide and the 3'-untranslated region of the receptor cDNA, was constructed by polymerase chain reaction. RNA transcripts were synthesized from existing nuclear RNA from control and down-regulated nuclei and hybridized to the receptor cDNA probe immobilized on nylon membranes and subjected to autoradiography. Hybridization to actin cDNA was also run alongside as a control. The results of run-off transcription analysis indicated that during down-regulation, the transcription rates of LH/hCG receptor mRNA are not decreased compared to those in saline-treated controls. Although no decrease in the transcription rates of the receptor mRNA was seen in the down-regulated state, the steady state levels of the receptor mRNA showed a decline when assayed by either solution hybridization or Northern blot analysis. Furthermore, hCG induced down-regulation appears to increase the transcriptional activity of the nuclei. Estimates of steady state LH/hCG receptor mRNA turnover rates indicate that the half-life of the message in down-regulated ovaries is markedly reduced compared to that in the control. It is concluded that the loss of steady state LH/hCG receptor mRNA is not due to a decrease in transcription, but probably represents an increased degradation of the receptor mRNA.

Animals↗

PubMed1992

Characterization and isolation of a high-density-lipoprotein-binding protein from bovine corpus luteum plasma membrane.

The ovary uses the cholesterol from high-density lipoproteins (HDL) as a substrate source for steroid hormone production. It is not clear, however, how ovarian cells acquire the lipoprotein cholesterol. This study describes the characterization and isolation of a high-affinity-binding protein for apolipoprotein E-free HDL from the plasma-membrane fraction of bovine corpora lutea. Plasma membranes were prepared by differential centrifugation with 5-6-fold enrichment of 5'-nucleotidase activity. The binding of 125I-HDL to the plasma membranes was time-dependent, and there appeared to be a single high-affinity site with a Kd of 6.7 micrograms of HDL/ml of assay buffer. The binding was not affected by high concentrations of low-density lipoproteins or the Ca2+ chelator EDTA, nor by changes in pH in the range 6.5-9.0. The binding was affected by the salt concentration in the buffer, with a dose-dependent increase that reached a maximum at 150-250 mM-NaCl. Binding was increased in the presence of high concentrations of KCl and KBr, and most significantly increased by high concentrations of bivalent metal ions. Ligand-blot analysis under reducing conditions revealed that the binding protein was a single polypeptide of about 108 kDa that was associated with the plasma-membrane fraction. This HDL-binding protein was purified to homogeneity by solubilization with Triton X-100, poly(ethylene glycol) precipitation, DEAE-Sephadex chromatography, and preparative SDS/PAGE. The purified binding protein is a single polypeptide of 108 kDa that retains high affinity and specificity for HDL as assayed by ligand blotting.

Animals↗

PubMed1992

Increased cellular cholesterol upregulates high density lipoprotein binding to rat luteal cells.

Rat luteal cells preferentially utilize cholesterol derived from high density lipoproteins (HDL) as a substrate for steroid hormone synthesis. The uptake of cholesterol from HDL by these cells is in contrast to nonsteroidogenic cells, which export cholesterol to HDL. A previous study demonstrated that HDL binding to luteal cell membranes was increased in conjunction with in vivo cholesterol depletion or cholesterol loading of the ovary induced by pharmacological agents. These results suggest a biphasic regulation of the HDL receptor in luteinized rat ovaries. In the present studies, the in vitro regulation of HDL binding in rat luteal cells by increased intracellular cholesterol was examined. Cultured luteal cells were incubated with increasing doses of low density lipoproteins (LDL) for 2 days after which the cellular sterol content and the effects on progesterone production and HDL binding were measured. As expected, the LDL treatment increased total cellular sterol content in a dose-dependent manner, resulting in a 2.1-fold increase over control at a dose of 1 mg LDL/ml. Increased cellular cholesterol was accompanied by a comparable increase in progesterone secretion. These results suggest that exogenous cholesterol was utilized by these cells. The LDL treatment also increased the binding of HDL to the cells in a dose-dependent manner to a maximum of 2.2-fold over control. The effect of increased cellular sterol on HDL binding was also examined using a more polar cholesterol derivative, 25-hydroxycholesterol. Cells were cultured for 2 days in media containing 0.3-40 micrograms/ml 25-hydroxycholesterol in the presence of 100 micrograms/ml aminoglutethimide, an inhibitor of cholesterol metabolism. The HDL binding to luteal cells exhibited dose-dependent up-regulation by 25-hydroxycholesterol with a 5.8-fold increase in binding at the maximum dose tested. Equilibrium binding studies using cells treated with 10 micrograms/ml 25-hydroxycholesterol revealed a 2.1-fold increase in the number of HDL binding sites on the luteal cells without affecting the binding affinity. From the results of this study, it is concluded that HDL binding in rat luteal cells is up-regulated by an increase in the intracellular cholesterol level.

Aminoglutethimide↗

PubMed1991

Differential responsiveness of luteinized human granulosa cells to gonadotropins and insulin-like growth factor I for induction of aromatase activity.

OBJECTIVE: To examine the in vitro responsiveness of cultured luteinized human granulosa cells over time to insulin-like growth factor 1 (IGF-1), human follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) for the induction of aromatase activity. DESIGN: Granulosa cells were retrieved from preovulatory follicles in patients undergoing in vitro fertilization. Cells were cultured for a period of 72 hours or 10 days. The ability of hCG, human FSH, and/or IGF-I to induce aromatase activity was assayed by the stereospecific release of tritium from [1B-3H]androstenedione. RESULTS: Short-term cultures (72 hours) demonstrated a marked rise in aromatase activity in response to human FSH and IGF-I, whereas a smaller response to hCG was observed. In contrast, 10-day cultures demonstrated responsiveness predominantly to hCG rather than human FSH for the induction of aromatase activity with no remarkable effect of IGF-I. CONCLUSION: Luteinized human granulosa cells undergo a transformation from an initial human FSH and IGF-I responsive state to an hCG responsive state in long-term cultures.

Androstenedione↗

PubMed1991

Evidence that human chorionic gonadotropin/luteinizing hormone receptor down-regulation involves decreased levels of receptor messenger ribonucleic acid.

Injection of pseudopregnant rats with pharmacological doses of hCG leads to a characteristic decrease in LH/hCG binding by the isolated luteal cells. The steady state levels of LH/hCG receptor mRNA were determined in rat ovaries during hCG-induced down-regulation of the receptor. Northern blots were performed using a 20-mer probe corresponding to a guanine cytosine-rich carboxyl-terminal untranslated region of the LH/hCG receptor cDNA. The hybridization of the probe to LH/hCG receptor mRNA was highly specific, since the probe hybridized only to rat luteal cell RNA fraction, with no signal detected in nontarget tissues. The LH/hCG receptor level was quantitated by [125I]hCG binding to the isolated membrane fractions from the corresponding treatment and control groups. Examination of mRNA levels of the receptor during hCG-induced down-regulation showed a steady decrease from 0-24 h, followed by a gradual increase to control levels from 24-72 h corresponding to days 8-9 of pseudopregnancy. The [125I]hCG-binding activity during down-regulation paralleled the mRNA profile in both the experimental and control groups. Examination of the levels of mRNA for alpha-actin showed no change during this period, suggesting that the loss of LH/hCG receptor mRNA at 24 h was not due to a general loss of mRNA in luteal cells. These results suggest that hCG-induced down-regulation of the LH/hCG receptor in luteal cells involves regulation of the receptors at the message level.

Animals↗

PubMed1991

Studies on rat luteal cell response to insulin-like growth factor I (IGF-I): identification of a specific cell membrane receptor for IGF-I in the luteinized rat ovary.

Insulin-like growth factor-I (IGF-I) has been shown to stimulate biosynthesis of progesterone and other differentiated functions of granulosa cells. The presence of the IGF-I receptor messenger RNA and IGF-I receptor as well as the role of IGF-I in the amplification of gonadotropin action in luteinized rat ovarian cells were assessed in the present study. Rat ovarian luteal tissue were obtained on day 6 of pseudopregnancy. After collagenase dispersion, luteal cells were cultured with or without IGF-I for 72 h in a serum-free medium in the absence or presence of human CG (100 ng/ml), 8-bromo-cAMP (1 mM), and 25-OH cholesterol (30 micrograms/ml). IGF-I alone increased (P less than 0.01) progesterone secretion 47% over controls. When combined with hCG the increase in progesterone secretion was enhanced 6-fold (P less than 0.001) over controls. The effects of 8 bromo-cAMP on progesterone secretion also was amplified (P less than 0.001) when IGF-I was present in the medium. When 25-OH cholesterol, a diffusable substrate for steroidogenesis, was included in the incubation medium along with IGF-I, progesterone secretion was enhanced significantly (P less than 0.01) over controls and either treatment alone. Binding studies performed with ovarian luteal cell membranes in the ovary revealed a dissociation constant of 0.94 nM for the IGF-I receptor. Autoradiograms from affinity labeling studies revealed a band corresponding to 132 Kd which is characteristic for the alpha-subunit of the type I IGF receptor. Northern blot analysis showed the presence of a major transcript at approximately 11 kilobases, which agrees with the size of the IGF-I receptor messenger RNA. In conclusion, we provide evidence for the fact that IGF-I regulates differentiated functions of the corpus luteum through interaction with specific high affinity IGF-I receptors.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

Characterization of epidermal growth factor receptor in normal and neoplastic human endometrium.

Growth factors, including epidermal growth factor (EGF), have been implicated in the growth of several types of cancer. This study compares EGF receptors in normal and neoplastic endometrium. Membrane fractions were isolated from surgical specimens. Radioreceptor assays demonstrated the presence of receptors with a dissociation constant of 0.64 nmol/l in normal endometrium. Affinity cross-linking revealed receptor molecular weight of 150 to 170 kiloDaltons (KD). A survey of samples (n = 37) revealed progressive decrease of EGF receptors in cancers of increasing grade: Grade 1-2 adenocarcinoma decreased 34% from control (n = 6, P less than 0.01), whereas Grade 3 adenocarcinoma decreased 90% (n = 7, P less than 0.01) and sarcoma decreased by 72% (n = 3, P less than 0.01). The dissociation constant and molecular weight of the receptor in neoplastic endometrium did not differ significantly from normal. The inverse relationship with grade suggests receptor alteration or down regulation by hormones and/or growth factors.

Adenocarcinoma↗

PubMed1990

Insulin-like growth factor I receptors in normal and neoplastic human endometrium.

Insulin-like growth factor I (IGF-I) binding sites were characterized in normal and neoplastic endometrium. The characteristics of the endometrial IGF-I receptor are similar to those reported for other tissues. The binding of 125I-IGF-I to the endometrial membranes is saturable and time, temperature, and pH dependent. The 125I-IGF-I binding activity to the membranes obtained from differentiated and undifferentiated adenocarcinoma as well as sarcoma of the endometrium was significantly higher (P less than 0.05) when compared to the binding activity of the membranes obtained from normal endometrium. The Scatchard analysis of the competitive binding data of both normal and neoplastic endometrium revealed linear plots. This indicated a single class binding site for IGF-I with equilibrium dissociation constants (Kd) of 5.0, 6.8, 6.94, and 6.88 nM for normal, differentiated, and undifferentiated adenocarcinoma, and sarcoma of the endometrium, respectively. Therefore, the differences observed in 125I-IGF-I binding between normal and neoplastic endometrial membranes was due to an increase in the number of IGF-I binding sites and not to a change in receptor binding affinity. Autoradiograms from affinity labelling studies revealed a band corresponding to Mr 132,000 subunit of the receptor which is characteristic of the type I receptor reported for other tissues. A dimer of the alpha subunit (Mr 263,000) was also observed in all four categories of endometrial tissue. Additionally, autoradiograms obtained from sarcoma of the endometrium revealed a Mr 40,000 band that was only displaced by IGF-I and IGF-II peptides but not by the monoclonal antibody alpha IR-3 to the type I receptor. These suggest that the band is representative of the IGF-I or IGF-II binding protein. A similar band was not observed in the other tissues. The results show that the human endometrium contains high affinity IGF-I or IGF-II binding sites. The fact that IGF-I binding activity was significantly higher for neoplastic endometrium suggests that IGF-I may play an important role on supporting the growth of this neoplastic tissue.

Affinity Labels↗

PubMed1990

In vitro induction of prolactin production and aromatase activity by gonadal steroids exclusively in the stroma of separated proliferative human endometrium.

Prolactin production by decidualized human endometrium has been demonstrated in organ explant cultures and presumed to be of stromal origin by immunocytochemistry and the presence of simultaneous stromal histologic changes. To test the hypothesis that endometrial prolactin production is exclusively of stromal origin, late proliferative human endometrium was separated into glands and stroma and cultured for 3 days. Confluent cultures were incubated with progesterone, 50 ng/ml; estradiol, 5 ng/ml; or both progesterone and estradiol for 4, 8, and 15 days. Prolactin concentration was measured at 2-day intervals by specific radioimmunoassay throughout a 15-day culture in all samples. Aromatase activity at 15 days was assayed by the production of tritiated water from 1 beta 3H-androstenedione. Possible contamination of gland cultures by stromal cells was controlled by the substitution of D-valine in the culture media. No prolactin was detected in the D-valine-treated gland cultures. Stromal cell cultures demonstrated increasing prolactin with continuous incubation with progesterone (15-day control: 11.28 +/- 0.91 ng/100 micrograms deoxyribonucleic acid versus 15-day progesterone: 245.8 +/- 4.24 ng/100 micrograms). Prolactin levels increased after progesterone was removed at day 4 and 8, reaching peak production 4 days later, followed by a steady decline. Estradiol induced a sustained increase in prolactin production even after withdrawal of steroids. Aromatase activity in gland cells exposed to steroids exhibited no change with time. Steroid-treated stromal cell cultures showed an increase in aromatase activity in all 15-day (controls: 21.6 +/- 0.7 pmol substrate converted per mg of deoxyribonucleic acid per 24 hours versus progesterone: 43.4 +/- 4.2 versus estradiol: 34.2 +/- 3.5 versus progesterone and estradiol: 47.5 +/- 2.4) but not in 4- or 8-day incubations. These studies support these conclusions: (1) nongestational endometrial prolactin is of stromal, not glandular origin; (2) stromal prolactin production is induced and maintained by progesterone: (3) stromal prolactin production is induced by estradiol but does not require continued exposure for maintenance; (4) aromatase activity is increased by long-term exposure to progesterone and estradiol in stroma but not in glands.

Adult↗

PubMed1990

Regulation of epidermal growth factor and insulin-like growth factor I receptors by estradiol and progesterone in normal and neoplastic endometrial cell cultures.

Growth factors are polypeptides which regulate cell proliferation through binding to specific receptor proteins. Normal and neoplastic human endometrium have been shown to express epidermal growth factor (EGF) and insulin-like growth factor I (IGF-1) receptors. Endometrial cell cultures were used to test modulation of EGF and IGF-1 receptors in response to steroid hormones. Endometrial gland and stroma cells were separated by enzymatic dispersion and were incubated in medium containing estradiol (10, 100, or 1000 pg/ml) or progesterone (1, 10, or 100 ng/ml) followed by radioligand assays. Normal endometrial cultures (n = 6) treated with estradiol demonstrated 40% less EGF binding than control cultures (P less than 0.05), while IGF-1 binding was unaffected. Stromal cells treated identically decreased in only one treatment group. Progesterone treatment stimulated a significant increase in EGF and IGF-1 receptors in gland cultures. Cultures derived from adenocarcinoma (n = 2) demonstrated decreased EGF binding compared with normal endometrium (P less than 0.05). Carcinoma cells treated with progesterone resulted in a dose-dependent increase in EGF binding over control (P less than 0.05). These data illustrate effects of steroid hormones upon growth factor receptors in human endometrium, and suggest involvement of growth factors in the regulation of normal and neoplastic endometrial growth.

Adenocarcinoma↗

PubMed1990

Detection of a 58-kilodalton high density lipoprotein-binding protein in the membrane fraction of luteinized rat ovaries.

The membrane fraction from the ovaries of pseudopregnant rats exhibits specific, high affinity binding of high density lipoproteins (HDL). Previous studies have indicated that HDL binding in this tissue is up-regulated by hCG and may be involved in supplying cholesterol as substrate for steroid hormone production. To characterize the HDL-binding activity, we solubilized the membrane proteins using 40 mM beta-octylglucoside and then separated them by electrophoresis on a 7% sodium dodecyl sulfate-polyacrylamide gel. The separated proteins were transferred to nitrocellulose sheets and subsequently incubated in the presence of [125I]apolipoprotein-E-free HDL. Autoradiography of the nitrocellulose revealed that the labeled HDL was bound to a single major band, with an apparent mol wt of 58,000 daltons. Neither reduction with beta-mercaptoethanol nor heat denaturation before separation on the gel affected the molecular size of the band, which indicates that it is probably a single polypeptide chain. The band was up-regulated in this tissue by in vivo treatment with 25 IU hCG in a time-dependent manner similar to the up-regulation of [125I]HDL-binding activity. In contrast to the binding of low density lipoprotein (LDL) to its receptor, the binding of HDL is independent of Ca+2. Incubation of the transferred proteins in the presence of [125I] Incubation of the transferred proteins in the presence of [125I]apolipoprotein-E-free HDL and either 5 mM CaCl2 or 15 mM EDTA had no effect on the appearance of the 58-kDa band. Furthermore, ligand blotting in the presence of a 100-fold excess of LDL did not affect the appearance of the band, whereas a 100-fold excess of apoliprotein-E-free HDL caused the disappearance of the band, indicating specificity for binding of HDL. Treatment of the sample with trypsin before electrophoresis also caused the band to disappear, revealing the protein nature of the band. These experiments indicate that the HDL receptor in luteinized rat ovaries is a 58,000-dalton protein.

Animals↗