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Biomedical subjects

W F Crowley

Publications and source records attributed to W F Crowley.

At least 55 records · Page 3Linked to original sources
PubMed1997

A mutation in the follicle-stimulating hormone receptor occurs frequently in human ovarian sex cord tumors.

A subset of ovarian tumors, referred to as sex cord-stromal tumors, produce endocrine manifestations due to the secretion of estrogens or androgens. Because gonadotropins induce the growth, differentiation, and function of the steroid-producing cells of the ovary, we hypothesized that mutations in the FSH receptor (FSH-R) might occur in this group of tumors. Ovarian sex cord tumors (n = 13), small cell carcinomas of the ovary (n = 3), and control DNA specimens (n = 116) were screened for mutations in the transmembrane domains of the FSH-R. A heterozygous T-->C mutation was found at nucleotide 1777 that converts codon 591 from phenylalanine to serine (F591S). This sixth transmembrane domain mutation was found in 9 of 13 (69%) sex cord tumors and 2 of 3 ovarian small cell carcinomas, but it was not present in control specimens, including 5 normal ovaries, 5 nonsex cord ovarian tumors, 16 thyroid tumors, or 90 specimens of peripheral blood leukocyte DNA, suggesting that this nucleotide change is not a polymorphism. The functional effects of identified mutations were assessed by expression of the wild-type or the F591S mutant FSH-R in COS-7 cells. The F591S mutation eliminated FSH-stimulated cAMP production, and a similar effect was observed when this mutation was introduced into the homologous location of the LH receptor. The high prevalence of the F591S mutation in the FSH-R suggests that it plays a role in the development of ovarian sex cord tumors.

Adolescent↗
PubMed1997

Frequency modulation of follicle-stimulating hormone (FSH) during the luteal-follicular transition: evidence for FSH control of inhibin B in normal women.

To isolate the impact of GnRH pulse frequency on FSH secretion and to examine the effect of differing levels of FSH on inhibin B secretion during the luteal-follicular transition, exogenous GnRH was administered to GnRH-deficient women using one of two regimens, and the results were compared to those in normal women. In the GnRH-deficient women, the GnRH pulse frequency was increased from every 4 h in the late luteal phase to every 90 min on the day of menses to mimic normal cycling women (physiological frequency transition; n = 8 studies) or the GnRH pulse frequency was kept constant at a late luteal phase frequency of every 4 h through the first 6 days of the subsequent early follicular phase of cycle 2 (slow frequency transition; n = 6 studies). The differential rise in FSH secretion induced in these studies allowed us to examine the subsequent contribution of varying levels of FSH to inhibin B secretion. A physiological regimen of GnRH during the luteal-follicular transition resulted in a rise in FSH and inhibin B levels that did not differ from that in normal cycling women and a normal follicular phase length. On the other hand, maintaining a luteal frequency of GnRH for 6 days into the subsequent early follicular phase produced FSH levels significantly lower than those in the physiological transition (P < 0.05), with the greatest difference seen on the day after menses (9.1 +/- 1.0 vs. 16.4 +/- 1.4 IU/L for the slow and physiological transition groups, respectively; P < 0.005), but no difference in LH. This slower rise of FSH secretion in the slow frequency group was associated with significantly lower inhibin B levels (43.3 +/- 21.5 vs. 140.0 +/- 24.4 pg/mL, mean days 1, 3, and 5; P < 0.02), a later doubling of estradiol from baseline (day 9.6 +/- 0.9 vs. day 5.6 +/- 0.1; P < 0.02), and a longer follicular phase length (16.0 +/- 1.4 vs. 11.6 +/- 0.9 days; P < 0.05) compared with those in the physiological transition group. In conclusion, during the luteal-follicular transition, the GnRH pulse frequency contributes to but is not solely responsible for the FSH rise that initiates folliculogenesis. Alteration of FSH dynamics induced by changes in GnRH pulse frequency in GnRH-deficient women provides evidence that FSH stimulates inhibin B production in the human. Timely follicular development indicated by both estradiol and inhibin B secretion appears to be dependent on the pattern of increase in FSH during the luteal-follicular transition.

Adolescent↗
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

The continuing dilemma in clinical investigation and the future of American health care: a system-wide problem requiring collaborative solutions.

American medicine faces a paradox: on the one hand, decades of basic science research have produced fundamental insights into disease mechanisms. On the other, there has rarely been a more difficult environment for the training and employment of clinical investigators, who perform the research that translates basic biomedical knowledge into practical advances in patient care. The authors explain the historical roots of this crisis and the lack of data about specific workforce needs for clinical investigators, discuss long-standing difficulties of recruiting, training, and retaining these scientists (e.g., time-consuming training, inadequate emphasis on clinical research in medical school, fewer role models) and why these processes are becoming more difficult (e.g., a coming flattening of federal support for research; the impact of managed care on academic health centers). In confronting this problem, the authors stress the importance of (1) carefully defining the major subtypes of clinical investigation (i.e., physiologic investigation, outcomes research, and clinical trials) and noting which are and which are not endangered; (2) understanding the potential solutions to the problem that have been offered in the past; and (3) defining and hopefully marshaling a coalition of those institutions whose resources are currently available to address the problem and that have an important stake in its solution: academic health centers, the National Institutes of Health, health care providers, foundations and educational societies, medical schools, and industry. The authors stress that finding solutions to the current problem are a shared responsibility that must be carried out, for without well-trained and innovative clinical investigators, the social contract of biomedical research-to keep society well-cannot be fulfilled.

Academic Medical Centers↗
PubMed1996

Contour of the GnRH pulse independently modulates gonadotropin secretion in the human male.

GnRH pulse frequency, amplitude, and interpulse interval have all been demonstrated to regulate gonadotropin secretion individually. We tested the hypothesis that the contour of the GnRH pulse also modulates gonadotropin output in 10 men with isolated GnRH deficiency in whom a fixed GnRH dose was administered at a constant physiologic frequency by either instantaneous bolus or by 1-, 5-, or 30-min infusions. LH, FSH and free alpha subunit (FAS) responses were also compared to spontaneous gonadotropin secretion in normal adult men. While the LH and FAS pulses following the instantaneous bolus and 1-min infusion of GnRH were indistinguishable, further increases in the duration of gonadotrope stimulation by GnRH were associated with progressive decreases in all parameters of gonadotropin secretion (mean levels, amplitude, peak levels, AUC). FSH secretion was also decreased following variations in the contour of the GnRH pulse, although overall changes were less dramatic than for LH and FAS. The LH pulses following the bolus GnRH stimulation were indistinguishable from spontaneous LH pulses occurring in normal men whereas those stimulated by the 1-, 5-, and 30-min infusions of GnRH became progressively blunted with the lowest levels of secretion occurring after the longest infusion. In sharp contrast, FAS pulse parameters in the GnRH-deficient subjects greatly exceeded those of normal men regardless of the contour of the GnRH stimulus, whereas mean FSH levels were all modestly (although significantly) higher than those of normal adult men. These results demonstrate that the pituitary is sensitive to subtle changes in the contour of the GnRH stimulus, with a more prolonged duration of GnRH stimulation resulting in a diminished pituitary response. Alterations of the contour of endogenous GnRH secretion may represent an additional mechanism for altering gonadotrope function and provide additional evidence for the differential regulation of LH, FAS, and FSH by GnRH. However, the previously reported elevated levels of FAS secretion in GnRH-deficient men undergoing long-term GnRH replacement are not explained by abnormalities of GnRH contour.

Adult↗
PubMed1996

Follistatin-activin complexes in human serum and follicular fluid differ immunologically and biochemically.

Follistatin (FS) is the principle high affinity activin-binding protein in tissues such as the pituitary and ovary as well as in serum. In addition, the activin-binding peaks identified after gel filtration of serum or human follicular fluid (hFF) exhibited high affinity and low reversibility binding kinetics, with higher concentrations in hFF than serum. This extremely low reversibility was also observed for recombinant human follistatin 288 (rhFS288) under a variety of incubation conditions, further supporting the identification of the serum and hFF activin-binding proteins as FS. Using enhanced resolution gel filtration, immunoprecipitation with monoclonal antibodies to rhFS288, and sulfated carbohydrate binding, activin-FS complexes in hFF and serum differed. The activin-FS complex in hFF elutes at approximately 200-300 kDa, is immunoprecipitated by anti-hFS288 monoclonal antibodies, and binds to sulfate Cellufine matrix, all characteristics similar to those of recombinant human FS288. In contrast, the activin binding peak in human serum elutes at an apparent Mr of 60-70 kDa, is no precipitated by anti-rhFS288 monoclonal antibodies, and is weakly bound by sulfate Cellufine matrix, characteristics shared by rhFS315 conditioned medium. As the forms of FS that bind sulfate-containing matrices also bind to cell surface proteoglycans, the molecular differences reported here for serum and hFF activin-binding proteins have implications for potential tissue-specific forms of FS that may well have distinct biological functions.

Activins↗
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

The genetic and clinical heterogeneity of gonadotropin-releasing hormone deficiency in the human.

Despite recent advances in the understanding of the pathophysiology of Kallmann's syndrome (KS), the patterns of inheritance in the majority of cases of GnRH deficiency in human subjects remain unclear. To define further the genetic and phenotypic variability of this syndrome, detailed family histories were reviewed in 106 cases of GnRH deficiency with or without anosmia [i.e. KS or idiopathic hypogonadotropic hypogonadism (IHH)]. The great majority of cases appeared to be sporadic, with only 19 probands (18%) having at least 1 family member with GnRH deficiency. However, of the families in which the proband was the sole member affected by KS or IHH, 9 had individuals with isolated anosmia, and 8 had a strong history of delayed puberty. If these phenotypes were considered as alternative manifestations of the same genetic defect that presented as KS or IHH in the proband, 34% of the cases in the present series could be considered familial. In these families, the most likely modes of transmission were assessed in several ways, including analysis of probands with KS as a distinct subset, and separate determinations based upon whether the phenotypes of isolated anosmia and/or delayed puberty were considered relevant to the inheritance of KS or IHH. The proportion of familial cases that could be attributable to an X-linked mode of inheritance was no greater than 36% in any of these analyses. We conclude that 1) most cases of GnRH deficiency in humans are sporadic and, thus, could represent new mutations; 2) the X-linked form is the least common among familial cases of KS or IHH; 3) defects in at least two autosomal genes can results in GnRH deficiency; and 4) associated clinical defects may well represent clues to the nature and/or location of these autosomal genes.

Female↗
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↗
PubMed1996

The gonadotropin genes: evolution of distinct mechanisms for hormonal control.

The glycoprotein hormones (TSH, FSH, LH, CG) are structurally related proteins with diverse physiologic functions. This family of hormones offers an opportunity to address fundamental questions concerning how gene expression is regulated in a cell-specific manner and in response to different hormones. For example, the alpha-subunit gene is expressed in several different pituitary cell types (gonadotropes and thyrotropes) as well as in the placenta. Because it must be coordinantly expressed with the different beta-subunit genes, the alpha-gene provides an interesting model for multihormonal control which varies in a cell-type specific manner. Many of the promoter regulatory DNA sequences and cognate transcription factors in the alpha-gene have been identified. These studies reveal a remarkable series of composite regulatory elements that interact with families of transcription factors that are still being characterized. In contrast, the beta-subunit genes are notable for restricted cell-type expression and more limited hormonal regulation that reflects their individual physiologic roles. The TSH beta gene is expressed only in thyrotropes where, in conjunction with the alpha-gene, it is subject to transcriptional repression by thyroid hormone. The FSH beta gene is expressed in gonadotropes where its expression is controlled primarily by activin and inhibin, with additional regulation by GnRH. The LH beta gene is also expressed in gonadotropes, but it is more dependent upon GnRH input and its expression is unaffected by the activin/inhibin system. The CG beta gene evolved recently from the LH beta gene and in the process, the CG beta promoter acquired new regulatory elements that favor its expression in the placenta rather than the pituitary gland. Less is known about the regulatory elements in the beta genes, in part because highly differentiated cells are required for their normal regulation. This chapter reviews the regulation of this family of genes with an emphasis on recent studies from our laboratory involving the gonadotropins (LH, FSH, CG). Concomitant with our advancing understanding of how the gonadotropin genes are regulated, we are also learning about genetic causes of gonadotropin deficiency syndromes.

Base Sequence↗