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P M Sluss

Publications and source records attributed to P M Sluss.

At least 19 recordsLinked to original sources
PubMed1996

Adrenal hypoplasia congenita with hypogonadotropic hypogonadism: evidence that DAX-1 mutations lead to combined hypothalmic and pituitary defects in gonadotropin production.

Adrenal hypoplasia congenita (AHC) is an X-linked disorder that typically presents with adrenal insufficiency during infancy. Hypogonadotropic hypogonadism (HHG) has been identified as a component of this disorder in affected individuals who survive into childhood. Recently, AHC was shown to be caused by mutations in DAX-1, a protein that is structurally similar in its carboxyterminal region to orphan nuclear receptors. We studied two kindreds with clinical features of AHC and HHG. DAX-1 mutations were identified in both families. In the JW kindred, a single base deletion at nucleotide 1219 was accompanied by an additional base substitution that resulted in a frameshift mutation at codon 329 followed by premature termination. In the MH kindred, a GGAT duplication at codon 418 caused a frameshift that also resulted in truncation of DAX-1. Baseline luteinizing hormone (LIT), follicle-stimulating hormone (FSH), and free-alpha-subunit (FAS) levels were determined during 24 h of frequent (q10 min) venous sampling. In patient MH, baseline LH levels were low, but FAS levels were within the normal range. In contrast, in patient JW, the mean LH and FSH were within the normal range during baseline sampling, but LH secretion was erratic rather than showing typical pulses. FAS was apulsatile for much of the day, but a surge was seen over a 3-4-h period. Pulsatile gonadotropin releasing hormone (GnRH) (25 ng/kg) was administered every 2 h for 7 d to assess pituitary responsiveness to exogenous GnRH. MH did not exhibit a gonadotropin response to pulsatile GnRH. JW exhibited a normal response to the first pulse of GnRH, but there was no increase in FAS. In contrast to the priming effect of GnRH in GnRH-deficient patients with Kallmann syndrome, GnRH pulses caused minimal secretory responses of LH and no FAS responses in patient JW. The initial LH response in patient JW implies a deficiency in hypothalamic GnRH. On the other hand, the failure to respond to pulsatile GnRH is consistent with a pituitary defect in gonadotropin production. These two cases exemplify the phenotypic heterogeneity of AHC/HHG, and suggest that DAX-1 mutations impair gonadotropin production by acting at both the hypothalamic and pituitary levels.

Adrenal Glands↗
PubMed1996

Activin inhibits basal and androgen-stimulated proliferation and induces apoptosis in the human prostatic cancer cell line, LNCaP.

LNCaP cells, derived from an androgen-sensitive cell line widely employed as an in vitro model of human prostate cancer, have been shown to express activin receptors. Activin is a local regulator of cellular growth, appears to play a key role in mesoderm induction and differentiation during development, and has been implicated in gonadal tumorigenesis. Follistatin, a monomeric glycoprotein that specifically binds and neutralizes activin, is often coexpressed with activin and, thus, modulates the autocrine/paracrine biological activity of this potent growth factor. We tested the hypothesis that LNCaP growth is modulated by the activin/follistatin system. Recombinant human activin A inhibited [3H]thymidine incorporation in a dose-dependent fashion with an ED50 of approximately 0.43 +/- 0.3 nM. Activin (0.1-3 nM) also inhibited dihydrotestosterone (DHT)-stimulated [3H]thymidine incorporation in LNCaP cells. Similarly, recombinant human inhibin A inhibited LNCaP proliferation, but was only 1/100th as potent as activin. Furthermore, activin (3 nM) induced a 3-fold increase in the extent of labeling of low mol wt DNA fragments typical of apoptosis. Activin-induced apoptosis was also indicated by an increase in the number of cells with reduced DNA content, as measured by flow cytometry of activin-treated cells. Both activin-mediated inhibition of cell proliferation and induction of apoptosis could be completely blocked by recombinant human follistatin. Based upon these results using an in vitro model, we speculate that activin functions locally to oppose androgen-driven cell proliferation and, thus, is a key factor controlling prostate growth. Reduced activin biosynthesis, increased follistatin secretion, or signaling defects in the activin receptor system should be further investigated in future studies as potential mechanisms underlying enhanced androgen-independent growth of human prostate cancer cells.

Activin Receptors↗
PubMed1996

Characterization of inhibin/activin subunit, follistatin, and activin type II receptors in human ovarian cancer cell lines: a potential role in autocrine growth regulation.

Although ovarian cancer is the most common gynecological malignancy with a relatively poor 5-yr survival record, the mechanism(s) by which these tumors arise is not well understood. A role for inhibins and activins in regulating this transformation is suggested by the detection of circulating alpha or dimeric inhibin in some patients with ovarian cancer and by the alpha inhibin knockout mouse, in which development of gonadal tumors in 100% of homozygotes is associated with greatly elevated activin levels. To develop diagnostic tools with greater specificity for ovarian cancers, the present study was targeted at characterizing the biosynthetic capacity of the epithelial ovarian cancer cell lines from the American Type Culture Collection with respect to inhibin, activin, the related activin-binding protein follistatin (FS), and activin receptor type II. In addition, the functional capacity of this system was investigated by examining the ability of activin and FS to modulate cellular proliferation. All six cell lines contained abundant messenger RNA (mRNA) for activin receptor type II, but no inhibin alpha-subunit mRNA was detected in any cell line. Two cell lines contained mRNA for activin beta B-subunit (CaOV4 and SKOV3), one cell line contained beta A-subunit mRNA (SW626), and one cell line contained both (ES2); the latter also contained FS mRNA. FS mRNA was detected in another cell line (PA-1) that contained no detectable activin beta-subunit mRNA. Finally, one cell line (CaOV3) contained neither beta-subunit nor FS mRNA. Protein secretion was also examined. Consistent with the mRNA studies, the two cell lines containing FS mRNA secreted FS (PA-1 and ES2 cells), whereas three of the remaining lines secreted activin (A or B). In the cell line containing neither FS nor beta-subunit mRNA, no FS or activin could be detected. Finally, none of the cell lines secreted detectable immunoreactive inhibin. The effects of exogenous activin and FS on cellular proliferation were examined in these cell lines. No response was detected in the two cell lines that secreted FS (PA-1 and ES2). For the four cell lines not synthesizing FS, treatment with activin (1-100 ng/ml) resulted in an increase, whereas FS treatment (1-100 ng/ml) resulted in a decrease in cellular proliferation, as determined by [3H]thymidine incorporation. The response to activin correlated negatively with endogenous activin production, suggesting that autocrine activin production may be involved with cell proliferation. The differential expression and production of inhibin/activin subunits, activin receptors, and follistatin as well as the range of responses to exogenous activin among six ovarian epithelial cancer cell lines suggest that this family of hormones may be important in regulating cell proliferation in the ovary. Whether primary tumors have the same profile and the degree to which these results can be generalized to additional forms of ovarian cancer remain to be determined.

Activin Receptors↗
PubMed1996

Inhibin B secretion in males with gonadotropin-releasing hormone (GnRH) deficiency before and during long-term GnRH replacement: relationship to spontaneous puberty, testicular volume, and prior treatment--a clinical research center study.

To evaluate the physiology of inhibin B in the human male, we measured serum concentrations in normal adult men and men with isolated GnRH deficiency before and during long-term replacement with pulsatile GnRH. At baseline, inhibin B levels in the GnRH-deficient men (n = 31) were significantly lower than normal controls (85 +/- 10 pg/mL vs. 239 +/- 14 pg/mL; P < .01) and correlated positively with pretreatment testicular volume (r = .80, P = .001) and a history of spontaneous puberty, suggesting additional maturational influences on the both testicular volume and inhibin B secretion. Pulsatile GnRH administration was associated with significant increases in inhibin B, with levels averaging 108 +/- 7 pg/mL when serum LH, FSH, and T concentrations had reached the normal adult male range (n = 22; P = .02 vs. baseline). Continued GnRH administration for at least an additional year was not associated with further increases in inhibin B concentrations. Throughout the course of long-term pulsatile GnRH replacement, serum FSH levels were negatively correlated with inhibin B concentrations (e.g. r = -.71, P < 0.01; n = 14 treated 12 months after normalization of T). Although inhibin B concentrations did not correlated with sperm density during therapy, rates of fertility were higher in patients with higher baseline levels (inhibin B > or = 60 pg/mL). Increases in serum concentrations of inhibin B occurring during GnRH replacement demonstrate the gonadotropin regulation of gonadal inhibin B secretion. However, the variation in baseline inhibin B levels before GnRH administration suggests an additional gonadotropin-independent level of modulation. The negative correlation between FSH and inhibin B secretion in GnRH-deficient men receiving long-term GnRH replacement is consistent with a putative role of inhibin B in the negative feedback regulation of FSH, although direct confirmation of this role requires further investigation.

Adolescent↗
PubMed1996

A randomized, controlled trial of estradiol replacement therapy in women with hypergonadotropic amenorrhea.

Premature ovarian failure is classically defined as menopause occurring before age 40 and is associated with elevated serum FSH levels. If elevated FSH levels indicate lack of ovarian feedback and depletion of primordial follicles, women with prematurely elevated FSH levels should have infertility. However, there are many reports of pregnancies in affected women occurring during estrogen therapy leading to the hypothesis that estrogen may have a salutary effect on folliculogenesis and conception. This randomized, controlled trial was designed to investigate whether estrogen replacement therapy offered a significant therapeutic benefit in hypergonadotropic amenorrhea and to evaluate the potential pathophysiologic mechanisms that would explain the reported pregnancies. Thirty seven women, aged 16 to 40, with menstrual dysfunction and documented FSH levels elevated above the 95% confidence limits of the mid-cycle gonadotropin peak of the normal menstrual cycle (> 40 IU/L 2nd IRP hMG in our RIA) on at least two occasions, entered the study. The average duration of their amenorrhea was 15.9 months (range 2-96 months). Subjects were randomized to begin estradiol replacement (micronized estradiol [Estrace TM], 2 mg orally each day) or no therapy for 6 weeks in a 12-week, cross-over design with weekly monitoring by both pelvic ultrasonography and serum hormone levels. Thirty-one women completed the entire randomized study. As expected, estradiol therapy increased mean serum estradiol levels by 98 pg/mL and was associated with a significant decrease in mean LH and FSH levels (LH: 45.4 IU/L 2nd IRP hMG vs. 37.1 IU/L, FSH: 63.4 IU/L vs. 40.6 IU/L, geometric means). However, there was no effect of estradiol replacement on mean ovarian volume, the number or size of new follicles, or the ovulation rate in all subjects or in the subset with no identified cause for their hypergonadotropic hypogonadism (n = 20). Two pregnancies occurred during the randomized trial, one on and one off estradiol. In both arms of the study, the majority of subjects developed cystic ovarian structures by ultrasound that were temporally associated with increasing serum estradiol levels, indicating functional ovarian follicles. Seventy-eight percent of all subjects grew at least one new follicle over 10 mm in diameter and 46% ovulated at least once, as determined by a serum progesterone level more than 4 ng/mL. Although ovulations were significantly more common in the 10 women subjects who had less than 3 months of amenorrhea (all of whom ovulated) than in the 27 with greater than 3 months of amenorrhea (only 7 of whom ovulated (26%), P < 0.001), there was no significant difference in eventual pregnancies (2 of the 10 women with less than 3 months of amenorrhea vs. 3 of the 27 with greater than 3 months of amenorrhea, P = 0.47). We conclude that in hypergonadotropic women with amenorrhea: 1) folliculogenesis occurs often but is less frequently followed by ovulation and rarely by pregnancy, suggesting that elevated FSH is a marker of oocyte dysfunction occurring distinct from and earlier than granulosa cell or follicular dysfunction; and 2) estrogen therapy does not improve the rate of folliculogenesis or ovulation.

Adolescent↗
PubMed1996

Inhibin B in males with gonadotropin-releasing hormone (GnRH) deficiency: changes in serum concentration after shortterm physiologic GnRH replacement--a clinical research center study.

To examine the role of inhibin B in the feedback regulation of FSH secretion in the human male, we determined serial levels in 18 men with idiopathic hypogonadotropic hypogonadism (IHH) during their initial 8 weeks of GnRH replacement. Pulsatile GnRH was administered every 2 h, with the dose increased at 2-week intervals (5-50 ng/kg/bolus). Every 2 weeks, sera were assayed for inhibin B, FSH, LH, and testosterone. Serial comparisons were performed within the IHH group as well as vs. normal men (n = 20). The baseline inhibin B level in IHH patients averaged 68 +/- 11 pg/mL (mean +/- SEM), significantly less than that in normal men (239 +/- 14 pg/mL; P < 0.001). After 8 weeks of pulsatile GnRH, inhibin B levels in the IHH patients increased significantly to 118 +/- 14 pg/mL (P = 0.003). During GnRH replacement, FSH concentrations correlated negatively with inhibin B concentrations at all doses. Patients previously treated with testosterone began with somewhat lower inhibin B levels but demonstrated a significantly greater increase in serum concentrations than patients who had received prior gonadotropin or GnRH therapy. A history of cryptorchidism did not have a significant impact on inhibin B concentrations before or during GnRH replacement. The low inhibin B levels in IHH men at baseline and their prompt increase in response to pulsatile GnRH suggest acute regulation by gonadotropin stimulation of the testis. The variation in inhibin B levels at baseline and in response to GnRH suggest that prior gonadotropin exposure and seminiferous tubular development also modulate inhibin B secretion. The consistent negative correlation between FSH and inhibin B during the induction of sexual maturation with GnRH supports the role of gonadal inhibin B secretion as an important endocrine regulator of FSH in the human male.

Adolescent↗
PubMed1996

A two-site monoclonal antibody immunoradiometric assay for human follistatin: secretion by a human ovarian teratocarcinoma-derived cell line (PA-1).

The follistatin/activin/inhibin system increasingly appears to have important growth and differentiating effects in a variety of cell types, including cancer. We have developed a two-site immunoradiometric assay for measurement of human follistatin using two monoclonal antibodies against recombinant human follistatin. This cloned protein donor assay is sensitive (0.5 ng/mL), specific for free human follistatin, and precise (<5% within assay coefficient of variation). Using this assay, native human follistatin could be measured in human pituitary extracts, follicular fluid, and granulosa-luteal cell-conditioned medium. To identify and characterize human follistatin secreted by ovarian cancer cells, we screened five human ovarian carcinoma cell lines currently available from the American Type Culture Collection (Rockville, MD). One of these, a cell line derived from a teratocarcinoma (designated PA-1, American Type Culture Collection, CRL1572), secreted large (3 microg/10(6) cells per 24 h) quantities of immunoreactive follistatin constituitively. Increasing volumes of conditioned medium from these cultured cells generated response curves parallel to those of recombinant human follistatin 288 reference protein, human follicular fluid, or culture medium from human granulosa-luteal cells. Secretion of follistatin by PA-1 cells was time and cell-number dependent with 297.9 +/- 15.2, 654 +/- 29.8, and 940 +/- 49.1 ng follistatin secreted over 24 h by 1 x 10(5), 2 x 10(5), and 3 x 10(5) cells, respectively. Western and ligand blot analysis revealed that the immunoreactive follistatin secreted by PA-1 cells and isolated by sulfate-cellufine chromatography was identical to the molecular weight variants (32,000 and 35,000 Mr) of recombinant human follistatin 288. PA-1 cell-conditioned medium suppressed basal secretion of FSH by cultured rat anterior pituitary cells in a dose-dependent fashion. This follistatin bioactivity was completely removed by adsorption with either solid-phase monoclonal antifollistatin or a dextran-sulfate chromatography gel. Because activin suppressed the proliferation of PA-1 cells, secretion of bioactive follistatin may represent an autocrine mechanism opposing activin to maintain the rapid growth rate of PA-1 cells. These observations demonstrate that the ovarian teratocarcinoma cell line, PA-1, secretes considerable amounts of human follistatin that is biologically active, capable of binding human activin, and antigenically similar to recombinant human follistatin 288. The monoclonal antibodies and two-site assay reported herein should be useful in assessing the regulation of follistatin secretion and as a diagnostic tool, especially if follistatin measurements prove to be a marker for some ovarian cancers.

Activins↗
PubMed1996

Homologous in vitro bioassay for follicle-stimulating hormone (FSH) reveals increased FSH biological signal during the mid- to late luteal phase of the human menstrual cycle.

Monitoring of the secretory dynamics of FSH during the human menstrual cycle has demonstrated conflicting results between the amounts of FSH measured by dimer-specific immunoassays and previous heterologous in vitro bioassays. These differences suggest somewhat different models of the steroidal and nonsteroidal regulation of FSH secretion and its control of folliculogenesis in the human. We have developed a homologous in vitro bioassay, using the recombinant human FSH receptor cotransfected into Chinese hamster ovary cells with a cAMP-responsive luciferase reporter gene, that overcomes many of the theoretic shortfalls of previous assays and allows reevaluation of the changes in bioactive FSH across the menstrual cycle. Bioactive FSH levels measured across 12 menstrual cycles in 11 normal women ranged from 4-40 IU/L. FSH bioactivity was constant during the menstrual cycle, with elevations noted only during the mid- to late luteal phase. Bioactive FSH levels were similar to immunoactive FSH levels across the cycle as indicated by a ratio of bioactive to immunoreactive FSH (FSH B/I) of 1.10 +/- 0.04 across the follicular and early luteal phases. However, during the mid- to late luteal phase, the mean FSH B/I rose to 1.65 +/- 0.07, which significantly exceeded that during the rest of the cycle (P < 0.001). This change in FSH B/I occurs at a critical time during folliculogenesis when the next cohort of follicles is being recruited and appears to be secondary to a decrease in immunoreactive FSH unaccompanied by a similar decrease in in vitro bioactivity. There was good agreement between immunoassay and bioassay results on the day of the midcycle gonadotropin surge (FSH B/I = 1.07 +/- 0.14), which was not different from that in the follicular phase (days -17 to -2; FSH B/I = 1.06 +/- 0.05) or the FSH B/I measured in postmenopausal women (0.93 +/- 0.2). These observations using a novel homologous human FSH in vitro bioassay indicate that bioactive FSH levels are not declining during the time of active corpus luteum formation and secretory activity. Thus, there is a previously undetected increased biologic signal during the mid- to late luteal phase, suggesting that the influence of elevated FSH on the cohort of developing follicles (including the subsequent dominant follicle) begins earlier during the luteal-follicular transition than previously predicted by FSH immunoreactivity.

Adult↗
PubMed1995

Serum follistatin levels in women: evidence against an endocrine function of ovarian follistatin.

Follistatin is a monomeric protein first identified in and isolated from ovarian follicular fluid. Evidence that follistatin might be an ovarian endocrine hormone functioning in a negative feedback fashion to modulate pituitary FSH production is based primarily on in vitro experiments. To examine the possible role of follistatin as an endocrine agent in vivo, we sought to relate circulating levels of follistatin to ovarian activity in women. Therefore, we developed a specific and sensitive homologous RIA using antiserum generated against recombinant human follistatin for the measurement of total follistatin in the presence or absence of activin. Follistatin was measured quantitatively (106 +/- 6% recovery) using calibration standards ranging from 0.4-25 ng/tube and up to 400 microL/tube serum. Furthermore, all of the endogenous follistatin measured in human serum could be removed by adsorption to activin-coated plates. Using this homologous RIA, human follicular fluid (100-600 ng/mL; n = 75) contained 3-150 times more follistatin than serum (4-35 ng/mL), an observation consistent with the notion that serum follistatin originates from the gonad. However, further studies of follistatin levels across the normal menstrual cycle (mean +/- SE, 8.09 +/- 0.73; n = 72 daily samples from 4 women), in pregnant women (17.49 +/- 1.34; n = 8), in daily samples from 20 women undergoing ovarian stimulation by exogenous FSH (9.90 +/- 0.62; n = 119), in postmenopausal women including two ovariectomized individuals (9.57 +/- 0.43; n = 8), and in GnRH-deficient women (9.85 +/- 0.50; n = 6) failed to support the hypothesis that serum levels of follistatin reflect ovarian activity in women. Levels of follistatin measured in serum collected across normal menstrual cycles did not fluctuate. However, the roughly nanomolar concentrations of follistatin measured suggest a physiological role for this protein. Follistatin at nanomolar concentrations may be capable of binding and inactivating circulating activin and perhaps in this way limiting the biological activity of activin to local autocrine or paracrine mechanisms. Measurement of peripheral levels of follistatin apparently represents only a first, albeit crucial, step in the study of the physiological significance of this protein in human reproduction.

Endocrine Glands↗
PubMed1994

Characterization of unique binding kinetics of follistatin and activin or inhibin in serum.

Serum binding proteins (BPs) have been identified for several peptide and protein hormones, and their presence has significant implications for the biological action of the hormone. Follistatin (FS) has been identified as an activin- and inhibin-BP in tissues, serum, and follicular fluid of several species, including humans. In this study, the binding kinetics of FS for activin and inhibin were characterized in human serum using gel filtration chromatography and compared to those of pure recombinant hormones using chromatography and a new solid phase assay. When complexed with radiolabeled activin or inhibin, FS eluted at a volume corresponding to a mol wt range of 67,000-150,000, an elution volume identical to the lower mol wt BP peak observed in serum. Furthermore, kinetic analyses of recombinant FS binding to activin using a solid phase assay revealed that 1) the FS-activin interaction is of high affinity, similar to or exceeding that estimated for activin binding to its receptor; 2) binding to activin is essentially irreversible at physiological pH; and 3) the potency of inhibin is approximately 500- to 1000-fold lower than that of activin in the FS binding assay. The lack of FS-[125I]activin complex reversibility observed in the solid phase assay was confirmed using a modified gel filtration chromatography protocol. Thus, preincubation of pure FS or serum with unlabeled activin for 2 h eliminated all binding of subsequently added labeled activin despite a much longer incubation period. However, when labeled activin was incubated with FS for 2 h, subsequent addition of unlabeled activin or inhibin was unable to displace labeled activin from FS, again demonstrating a lack of reversibility. Finally, to map this high affinity interaction, overlapping synthetic peptides were used to compete with labeled activin for FS binding. Two potential contact sites between FS and activin were identified, one near the N-terminus (amino acids 15-29) and the other near the C-terminus (amino acids 99-116). Given its apparently irreversible nature, high affinity, and ability to neutralize activin's biological activity, FS is quite different from the typical hormone-BP. These unique properties of FS undoubtedly attest to the potency of activin in many physiological and developmental settings and, therefore, to the importance of BPs, such as FS for regulating activin's bioactivity, distribution, and/or clearance.

Activins↗
PubMed1994

Relatively low levels of dimeric inhibin circulate in men and women with polycystic ovarian syndrome using a specific two-site enzyme-linked immunosorbent assay.

The endocrine feedback role of dimeric inhibin on FSH secretion from the pituitary has been well established in many species; however, evidence that inhibin is an important endocrine regulator of FSH in the human is more tenuous. One potential explanation for the equivocal data may be that the inhibin immunoassay used most widely in the human is a heterologous assay with an antiserum that exclusively recognizes the inhibin alpha-subunit in both its monomeric form and in inhibin dimers. The aim of the present study was to quantify serum inhibin levels in a variety of fertile and infertile men and women using a new ultrasensitive enzyme-linked immunosorbent assay that is specific for the dimeric form (alpha/beta) only. The specificity of the present assay was demonstrated by the absence of significant cross-reactivity with Mullerian inhibiting substance, transforming growth factor-beta, activin, FSH, LH, hCG, TSH, and hCG alpha and with the alpha-subunit of inhibin. The assay was sensitive to 1 pg/mL, and serial dilutions of human male and female serum samples paralleled the recombinant 32-kilodalton (kDa) dimeric inhibin standard curve. Complete recovery of exogenous recombinant 32-kDa inhibin added to serum was obtained. Mean serum inhibin levels ranged from a low of 5.7 +/- 0.6 pg/mL in the early follicular phase to 49.0 +/- 11.2 pg/mL in the midluteal phase of the normal menstral cycle and were elevated during ovulation induction (1250 pg/mL) and pregnancy (500 pg/mL). Interestingly, mean levels of dimeric inhibin in women with polycystic ovarian syndrome (PCOS) were indistinguishable from normal follicular phase. The most striking observation was the extremely low mean inhibin levels (< 2 pg/mL) found in normal men, GnRH-deficient men before any pulsatile GnRH treatment, and men with Klinefelter's syndrome, all of which were indistinguishable from levels observed in postmenopausal women. These observations in the male raise the possibly that 1) some forms of circulating and bioactive inhibin in the human are not detected by this assay due to either their conformation or the presence of a unique binding protein for endogenous inhibin that does not bind to recombinant 32-kDa inhibin; or 2) dimeric inhibin is not a major endocrine regulator of FSH in the human male. In addition, the relatively high dimeric inhibin levels at midcycle and in the luteal phase of the normal menstrual cycle, after gonadotropin stimulation, and during pregnancy suggest that dimeric inhibin is predominantly produced by dominant follicles, corpora lutea, and placental tissues.

Adult↗
PubMed1994

Estradiol and progesterone production by cultured granulosa cells cryopreserved from in vitro fertilization patients.

Gonadotropin-stimulated steroidogenesis was studied in cultured human granulosa-lutein cells obtained from patients undergoing procedures for in vitro fertilization. The impact of cryopreservation on cell function in vitro was studied. Granulosa cells obtained from in vitro fertilization patients were cultured in serum-supplemented medium or cryopreserved at -135 degrees C for 2-22 months. Fresh (unfrozen) cells (10(5) produced estradiol at a rate of 1320 pmol/l (over 72 h) and progesterone at about 2500 nmol/l. Estradiol production by either fresh or cryopreserved granulosa cells in culture was unaffected by physiological concentrations of follicle-stimulating hormone (7 IU/l). Adding testosterone (10(-7) mol/l) to the medium increased estradiol secretion approximately sixfold. In contrast, progesterone production was not affected by follicle-stimulating hormone or testosterone. No significant differences were observed in cultures of cryopreserved granulosa cells compared to cultures of unfrozen cells with respect to estradiol secretion, the effects of follicle-stimulating hormone or testosterone on estradiol secretion, or progesterone production. Progesterone production by fresh and cryopreserved cells was stimulated by human chorionic gonadotropin. These data indicate that cryopreservation offers the potential to facilitate prospective studies utilizing large numbers of human granulosa-lutein cells in culture.

Aromatase↗
PubMed1993

Insulin-like growth factor I: action and receptor characterization in human prostate cancer cell lines.

The role of insulin-like growth factor I (IGF-I) in the growth and development of prostate cancer was studied using established human prostate cancer cell lines. Under steroid and growth factor-free culture conditions, IGF-I significantly stimulated the androgen-independent cell lines PC-3 and DU-145 to incorporate [3H]thymidine into DNA, while the androgen-dependent cell line, LNCaP, was not affected. However, in the presence of dihydrotestosterone (DHT), DNA synthesis of LNCaP cells was stimulated by IGF-I in a dose-dependent manner. None of the cell lines tested secreted an immunoreactive level of IGF-I into their conditioned medium. Characterization of receptors by ligand binding assays revealed that all prostate cancer cell lines tested express specific binding sites for IGF-I with similar dissociation constants (0.23-0.39 nM). Crosslinking studies supported the suggestion that 125I-IGF-I was bound to a receptor on these cells. The IGF-I receptor concentrations of androgen-independent cell lines were significantly higher than those of the androgen-dependent cell line. Androgen appeared to affect neither the expression of IGF-I receptors nor the secretion of IGF-I. The results suggest that IGF-I may play an important role in stimulating the growth and progression of prostate cancer.

Binding, Competitive↗
PubMed1993

Characterization of insulin-like growth factor I binding sites in human bladder cancer cell lines.

The role of insulin-like growth factor I (IGF-I) in the growth and development of bladder cancer cells was investigated using cultured human cell lines representing differentiated (RT-4, 5637) or undifferentiated (T-24, J-82, TCC-SUP) transitional cell carcinoma (TCC). In the presence of 2% serum, IGF-I significantly stimulated the growth of all cell lines. The proliferation of T-24, 5637, and RT-4 cells was more sensitive to IGF-I than that of J-82 and TCC-SUP cells. [125I]IGF-I binding to 5637 and J-82 cells was significantly higher than that to T-24 and TCC-SUP cells (P < 0.001). RT-4 cells possessed the lowest binding capacity among the cell lines tested. Scatchard analysis of [125I]IGF-I binding to four of the five cell lines indicated a single binding site for IGF-I, with apparent dissociation constants (Kd) of 1.27, 1.18, 1.34, and 1.39 nmol/l for TCC-SUP, J-82, 5637, and T-24, respectively. Therefore, the difference observed in [125I]IGF-I binding among the bladder cancer cell lines was attributed to the difference of IGF-I binding sites and not to a change in receptor binding affinity. Cross-linking studies supported the suggestion that [125I]IGF-I was bound to a receptor on these cells. The results indicate that cultured human bladder cancer cells contain functional IGF-I receptors. A differentiated cell line, RT-4, possesses significantly fewer IGF-I receptors than other cell lines. This suggests that the overexpression of IGF-I receptor may reflect the malignant potential of bladder cancer cells.

Binding Sites↗
PubMed1993

Multiple molecular weight forms of immunoreactive alpha-inhibin in human seminal plasma.

The purpose of these studies was to determine whether the total immunoreactive alpha-inhibin protein concentration in seminal plasma correlated with serum gonadotropin levels or semen characteristics and to identify the forms of alpha-inhibin present in seminal plasma. Thirty-eight serum samples from men being evaluated for infertility were selected for study based on their serum hormone profiles and semen parameters. Serum LH and testosterone levels were normal, but FSH levels ranged from normal to hypergonadotropic (> 20 IU/L). Most semen parameters were within normal ranges, but germ cell numbers ranged from normal to azoospermic. Thus, seminal plasma from these men provided a unique opportunity to examine the antigenic forms of alpha-inhibin in individuals in whom strong correlations between inhibin and FSH levels might be predicted because of the observed ranges of FSH levels and germ cell numbers. Seminal plasma alpha-inhibin was characterized by RIA or Western blotting, using an antiserum directed against the N-terminal of the alpha-subunit of mature [32,000 mol wt (M(r))] inhibin. The antiserum recognized the alpha-subunit of dimeric inhibin as well as free alpha-inhibin and alpha-inhibin precursor proteins. Total immunoreactive alpha-inhibin ranged from 8.21-43.99 nmol/L in seminal plasma. However, alpha-inhibin levels were not statistically correlated with serum FSH levels or any of the measured semen parameters (including germ cell number). In contrast, the immunoreactive alpha-inhibin concentration in seminal plasma was negatively correlated (P < 0.01) with the serum LH level. Western blot analyses revealed that multiple forms of immunoreactive alpha-inhibin are present in seminal plasma. The majority of immunoreactivity was associated with monomeric proteins (ranging from 58,000-95,000 M(r)) that were larger than the alpha-subunit (21,000 M(r)) predicted for mature dimeric human inhibin (32,000 M(r)). The relative amounts of individual forms of immunoreactive alpha-inhibin varied among the patients studied, but could not be correlated with other serum or seminal parameters measured. Our observations demonstrate that various monomeric alpha-inhibin proteins are present in human seminal plasma. It is unlikely that these proteins alone or combined with inhibin beta-subunit proteins have identical biological activities. Thus, until assays specific for each of the various forms of immunoreactive alpha-inhibin are developed, their role as well as that of inhibin in the endocrine or local modulation of testicular function cannot be deduced from RIA data alone.

Blotting, Western↗
PubMed1992

Changes in urinary magnesium, citrate, and oxalate levels due to cola consumption.

The serum concentration and urinary excretion of four potential stone constituents: calcium, oxalate, phosphate, and uric acid; and of two potential inhibitors of urinary stone formation, citrate and magnesium, were studied before and during ingestion of a carbonated cola beverage. Four participants, after baseline serum determinations, abstained for forty-eight hours from cola consumption and then attempted to drink 3 quarts per day of cola in the following forty-eight hours. One participant failed to drink more than two quarts per day of cola. In all 4 participants twenty-four-hour urinary excretion of magnesium decreased by an average of 2.6 mg. In the 3 participants who succeeded in drinking 3 quarts of cola per day, twenty-four-hour urinary excretion of oxalate increased an average of 8.3 mg and excretion of citrate decreased an average of 122 mg. These results demonstrate changes in urine constituents which could contribute to enhanced kidney stone formation in patients who drink large quantities of cola-flavored carbonated beverages.

Adult↗
PubMed1992

Suramin inhibits gonadotropin action in rat testis: implications for treatment of advanced prostate cancer.

Suramin is being evaluated for the treatment of metastatic prostate cancer based on its inhibition of growth factor action. In addition, suramin may inhibit the endocrine control of androgen production, which was explored herein. Adult Sprague-Dawley rats were injected (i.p.) daily with varying doses of suramin. At a cumulative dose of 200 mg., suramin significantly depressed serum testosterone (p less than 0.05), and follicle stimulating hormone (p less than 0.002) levels. In vitro studies showed that suramin-mediated suppression of androgen production might be secondary to inhibition of gonadotropin action. In MA-10 cell cultures, suramin inhibited a maximum stimulatory dose of human chorionic gonadotropin with an ED50 of 4.4 microM. Studies in rat Sertoli cell cultures showed that follicle stimulating hormone action was also inhibited by suramin, with an ED50 of 8.6 microM. Using receptor binding assays with calf testis membrane, we showed that suramin inhibited 125I-hFSH binding to receptor in a dose dependent fashion with an ED50 of 10.4 microM; comparable to the ED50 of suramin inhibition of follicle stimulating hormone action in Sertoli cell culture cells. Thus the mechanism of suramin's suppression of androgen production may involve multiple sites of action, including inhibition of gonadotropin binding to its receptor and suppression of pituitary gonadotropin levels in serum. This inhibition of androgen production may be useful in the treatment of advanced prostate cancer.

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