CHD7 mutations in patients initially diagnosed with Kallmann syndrome--the clinical overlap with CHARGE syndrome.

Kallmann syndrome (KS) is the combination of hypogonadotropic hypogonadism and anosmia or hyposmia, two features that are also frequently present in CHARGE syndrome. CHARGE syndrome is caused by mutations in the CHD7 gene. We performed analysis of CHD7 in 36 patients with KS and 20 patients with normosmic idiopathic hypogonadotropic hypogonadism (nIHH) in whom mutations in KAL1, FGFR1, PROK2 and PROKR2 genes were excluded. Three of 56 KS/nIHH patients had de novo mutations in CHD7. In retrospect, these three CHD7-positive patients showed additional features that are seen in CHARGE syndrome. CHD7 mutations can be present in KS patients who have additional features that are part of the CHARGE syndrome phenotype. We did not find mutations in patients with isolated KS. These findings imply that patients diagnosed with hypogonadotropic hypogonadism and anosmia should be screened for clinical features consistent with CHARGE syndrome. If such features are present, particularly deafness, dysmorphic ears and/or hypoplasia or aplasia of the semicircular canals, CHD7 sequencing is recommended.

Clinical counterpoint: gonadotropin-releasing hormone deficiency: perspectives from clinical investigation.

Advances in our understanding of the pathophysiology of Kallmann syndrome have come from an interdisciplinary approach involving developmental biology, clinical investigation, and molecular biology. It is equally clear that progress to date is but the first chapter of what will be a fascinating biological story. It now seems likely that the full expression of reproductive potential from the neuroendocrine perspective is likely to be as complicated as other aspects of reproduction, such as the multigene control of external genital differentiation. An analogous story may well emerge for the neuroendocrine control of reproduction in which the GnRH gene is encoded on the eighth chromosome, the protein guiding the embryonic journey of the GnRH-producing neuron to the hypothalamus lies on the X chromosome, and many, as yet to be determined, other genetic loci collaborate in the full expression of reproductive potential. Such a detailed study is warranted not only because of the clinical and genetic implications for an individual patient with this disorder, but also from an organizational theme for the evolution of the species (and its potential regulation). Given the pressing nature of world population growth, obtaining such understanding and its applications to fertility and contraception is crucial. These advances will only come from enlightened interactions of clinical investigators, molecular geneticists, and developmental biologists in which interdisciplinary approaches should be fostered. This should be an exciting story to follow given the remarkable nature of the tools at hand to study these clinical conditions.

Hypogonadotropic disorders in men and women: diagnosis and therapy with pulsatile gonadotropin-releasing hormone.

Hypogonadotropic hypogonadism (HH) is a clinical syndrome occurring in both sexes which has long puzzled clinicians due to the apparent paradox of nonfunctioning gonads in the face of normal or only slightly lowered levels of circulating gonadotropins. Using frequent sampling of gonadotropin levels as an index of hypothalamic GnRH secretion, we have examined the hypothesis that this group of disorders represents a spectrum of abnormal patterns of the pulsatile release of endogenous GnRH. After a broad, normative data base was established in both men and women for purposes of comparison, it appears that quantifiable abnormalities of GnRH secretion are discernible in both males and females with HH. These abnormalities include a total absence of GnRH secretion, defects of the amplitude and frequency of its secretion, and altered bioactivity of the gonadotropins released. In addition, physiological regimens of hypothalamic replacement therapy with exogenous GnRH, which are fashioned to mimic the normal frequency of endogenous GnRH secretion, result in complete normalization of reproductive function and fertility in hypogonadotropic subjects of both sexes. Thus, the heterogeneous nature of HH, as well as its favorable clinical and biochemical responses to GnRH, suggest that the basic defect in this family of disorders involves a partial or complete inability to synthesize and/or release GnRH from the hypothalamus in a manner compatible with physiological reproductive function. Conversely, these findings imply that maintenance of a physiological amplitude and frequency of endogenous GnRH secretion appear to be essential for normal reproductive function.

Use of a potent, long acting agonist of gonadotropin-releasing hormone in the treatment of precocious puberty.

Studies utilizing the administration of GnRH in various GnRH-deficient models have revealed the critical importance of the dose and mode of delivery of this releasing factor in determining the subsequent pituitary response. Chronic administration of long acting GnRH agonists (GnRHa), like continuous infusion of high doses of the native peptide, results in suppression of pituitary gonadotropin secretion. This selective and reversible suppression of gonadotropin secretion suggested several therapeutic applications for these analogs, particularly in the treatment of central precocious puberty (CPP), a disorder for which the previously available therapies lacked uniform efficacy and were associated with potential side effects. In our series, 74 children with CPP have been treated during the last 5 yr with the potent GnRH agonist, [D-Trp6, Pro9-ethylamide(NEt)]GnRH. Having selected a dose and route of administration that produced uniform suppression of spontaneous and stimulated pituitary gonadotropin secretion, GnRHa therapy resulted in a fall of gonadal sex steroid levels into the prepubertal range, a halting or regression of secondary sexual development, and a complete cessation of menses. Growth velocity slowed during therapy, with this slowing more pronounced during prolonged treatment periods and among those patients with more advanced chronological and skeletal ages. Skeletal maturation was retarded to a greater degree than linear growth, with resultant increases in the predictions for adult stature. Moreover, these benefits have been achieved in the absence of significant side effects. Complete reversal of the suppression of gonadarche has followed discontinuation of therapy; however, patterns of growth and skeletal maturation after discontinuation of GnRHa administration remain to be characterized. Thus, the impact of GnRHa therapy on final height must await further longitudinal study. The selective nature of GnRHa suppression of gonadarche also permits an investigation of the natural history of adrenarche and its discrete influences upon skeletal growth and maturation. In addition, GnRHa therapy of CPP provides a unique opportunity to study the effects of gonadal sex steroids on GH secretion and somatomedin-C (Sm-C) generation during sexual maturation. Finally, the detailed characterization of children with precocious puberty has helped to define more precisely a subset of patients whose precocity occurs in the absence of demonstrable gonadotropin secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuroendocrine regulation of the corpus luteum in the human. Evidence for pulsatile progesterone secretion.

The pattern of episodic gonadotropin release was studied in 15 normal female volunteers during the luteal phase of the menstrual cycle with 24 h of blood sampling for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels at 10-min intervals. Six subjects (two in the early, two in the mid-, and two in the late luteal phase) also had each of these specimens processed for progesterone levels. A progressive slowing of LH pulsations was present across the luteal phase with the mean LH pulse frequency declining from 15.2 pulses/24 h in the early to 8.4/24 h in the late luteal phase. A trend towards reduction in the amplitude of LH pulses was also observed (12.3 +/- 2.2 SD mIU/ml in the early vs. 8.6 +/- 3.4 mIU/ml in the late luteal phase; NS). In addition, LH pulses of heterogeneous amplitude were identified during the same 24-h study. The mean +/- SD of the larger and of the smaller LH pulses was 16.9 +/- 4.7 and 2.3 +/- 1.0 mIU/ml, respectively (P less than 0.001). While the slowing of the frequency of all LH pulses correlated well (r = 0.80, P less than 0.001) with the day of the luteal phase and poorly with the actual plasma progesterone levels, the incidence of the small LH pulses was highest in the mid-luteal phase and correlated well with the mean progesterone plasma levels (r = 0.63, P less than 0.01). In the early luteal phase, the pattern of progesterone secretion was stable over the 24-h studies and showed no relationship to episodic LH release. In contrast, in the mid- and late luteal phase, plasma progesterone concentrations rapidly fluctuated during the 24-h studies from levels as low as 2.3 to peaks of 40.1 ng/ml, often within the course of minutes. Progesterone increments closely attended episodes of LH release, as documented by the significant (P less than 0.05) cross-correlation between LH and progesterone levels, at time lags of 25-55 min. The results of this study indicate that in the human luteal phase: (a) the frequency of pulsatile release of LH declines progressively and correlates well with the duration of exposure to progressively and correlates well with the duration of exposure to progesterone; (b) the amplitude of LH pulses varies with the appearance of an increased percentage of smaller pulses correlating well with the acute level of progesterone; (c) in the early luteal phase, the pattern of progesterone secretion is stable; (d) in the mid- and late luteal phase, progesterone secretion is episodic, and correlates with LH pulsatile release; and (e) single progesterone estimations in the mid- and late luteal phase do not accurately reflect corpus luteum adequacy.

Effects of decreasing the frequency of gonadotropin-releasing hormone stimulation on gonadotropin secretion in gonadotropin-releasing hormone-deficient men and perifused rat pituitary cells.

The effects of decreasing the frequency of pulsatile gonadotropin-releasing hormone (GnRH) stimulation on pituitary responsiveness were studied in (a) men with isolated GnRH deficiency who had achieved normal sex steroid levels during prior long-term pulsatile GnRH replacement and (b) perifused dispersed pituitary cells from male rats in the absence of sex steroids. In three groups of four GnRH-deficient men, the frequency of GnRH stimulation was decreased at weekly intervals from (a) every 2-3-4 h (group I), (b) every 2-8 h without testosterone replacement (group II), or (c) every 2-8 h with testosterone replacement (group III). In three groups of three columns of perifused dispersed pituitary cells, pulses of GnRH were administered every 2, 4, or 8 h. In groups I and II, mean area under the luteinizing hormone (LH) curve increased (P less than 0.025) and serum testosterone levels fell (P less than 0.035) as the frequency of GnRH stimulation was decreased. In group III, the area under the LH curve also increased (P less than 0.01) although serum testosterone levels were constant, thereby demonstrating that the increase in pituitary responsiveness to slow frequencies of GnRH stimulation occurs independently of changes in the sex steroid hormonal milieu. The area under the LH curve also increased in the perifused dispersed rat pituitary cells when the frequency of GnRH administration was decreased to every 8 h (P less than 0.05), thus demonstrating that the enhanced pituitary responsiveness to slow frequencies of GnRH stimulation is maintained even in the complete absence of gonadal steroids. Nadir LH levels fell in all three groups (P less than 0.01) as the frequency of GnRH stimulation was decreased. In contrast, mean peak LH levels, the rate of LH rise, and the rate of endogenous LH decay were constant as the frequency of GnRH stimulation was decreased. Finally, as the GnRH interpulse interval increased, mean LH levels fell, and mean follicle-stimulating hormone levels were stable or fell. These results indicate that (a) pituitary responsiveness to GnRH increases at slower frequencies of GnRH stimulation in models both in vivo and in vitro, (b) these changes in pituitary responsiveness occur independently of changes in gonadal steroid secretion, and (c) the increases in LH pulse amplitude and area under the curve at slow frequencies of GnRH stimulation are due to decreases in nadir, but not peak, LH levels. Slowing of the frequency of GnRH secretion may be an important independent variable in the control of pituitary gonadotropin secretion.

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.

Adrenarche and skeletal maturation during luteinizing hormone releasing hormone analogue suppression of gonadarche.

During puberty the effects of adrenal androgens upon skeletal maturation are obscured by the influence of gonadal steroids. Suppression of gonadarche with an analogue of luteinizing hormone releasing hormone (LHRHa) affords an opportunity to examine the onset and progression of adrenarche in the absence of pubertal levels of gonadal steroids in a controlled fashion and to explore the relationship between adrenal androgens and the rate of epiphyseal maturation. In 29 children with central precocious puberty, gonadarche was suppressed with LHRHa administration for 1-4 yr. During LHRHa exposure, dehydroepiandrosterone sulfate (DHAS) levels, as an index of adrenal maturation, were constant or increased in an age-expected manner. The change in bone age for change in chronologic age decreased from 1.7 +/- 0.1 to 0.49 +/- 0.05 (P = 0.00005), indicating that the LHRHa-induced return to a prepubertal gonadal steroid environment was associated with a slowing of skeletal maturation. DHAS levels were correlated with the rate of skeletal advancement before (r = 0.57, P = 0.001) and during 12 to 48 mo of exposure to LHRHa (r = 0.52, P = 0.003). A negative correlation of DHAS values with subsequent increases in predicted mature height was observed (r = -0.49, P = 0.007). Thus, in children with central precocious puberty, adrenarche progressed normally during LHRHa suppression of gonadarche. In children with the onset of progression of adrenarche during maintenance of a prepubertal gonadal steroid milieu, there was less evidence than in preadrenarchal children of a restraint upon skeletal maturation. These data suggest that adrenal androgens contribute importantly to epiphyseal advancement during childhood.

Characterizing discrete subsets of polycystic ovary syndrome as defined by the Rotterdam criteria: the impact of weight on phenotype and metabolic features.

CONTEXT: The Rotterdam criteria for polycystic ovary syndrome (PCOS) defines discrete subgroups whose phenotypes are not yet clear. OBJECTIVE: The phenotypic characteristics of women in the PCOS subgroups defined by the Rotterdam criteria were compared. DESIGN: The study was observational. SETTING: Subjects were studied in an outpatient setting in Boston and Reykjavik. PATIENTS: Four subgroups of subjects with PCOS defined by 1) irregular menses (IM), hyperandrogenism (HA), and polycystic ovary morphology (PCOM, n = 298); 2) IM/HA (n = 7); 3) HA/PCOM (n = 77); and 4) IM/PCOM (n = 36) and a group of controls (n = 64), aged 18-45 yr, were examined. INTERVENTION: Subjects underwent a physical exam; fasting blood samples for androgens, gonadotropins, and metabolic parameters; and a transvaginal ultrasound. MAIN OUTCOME MEASURES: The phenotype was compared between groups. RESULTS: Ninety-seven percent of women with IM/HA had PCOM. Therefore, the groups with and without PCOM were combined. The Ferriman-Gallwey score and androgen levels were highest in the hyperandrogenic groups (IM/HA and HA/PCOM), whereas ovarian volume was higher in all PCOS subgroups compared with controls, as expected based on the definitions of the PCOS subgroups. Body mass index and insulin levels were highest in the IM/HA subgroup. CONCLUSIONS: Subjects with PCOS defined by IM/HA are the most severely affected women on the basis of androgen levels, ovarian volumes, and insulin levels. Their higher body mass index partially accounts for the increased insulin levels, suggesting that weight gain exacerbates the symptoms of PCOS.

Defining constant versus variable phenotypic features of women with polycystic ovary syndrome using different ethnic groups and populations.

CONTEXT: The phenotype of women with polycystic ovary syndrome (PCOS) is variable, depending on the ethnic background. OBJECTIVE: The phenotypes of women with PCOS in Iceland and Boston were compared. DESIGN: The study was observational with a parallel design. SETTING: Subjects were studied in an outpatient setting. PATIENTS: Women, aged 18-45 yr, with PCOS defined by hyperandrogenism and fewer than nine menses per year, were examined in Iceland (n = 105) and Boston (n = 262). INTERVENTION: PCOS subjects underwent a physical exam, fasting blood samples for androgens, gonadotropins, metabolic parameters, and a transvaginal ultrasound. MAIN OUTCOME MEASURES: The phenotype of women with PCOS was compared between Caucasian women in Iceland and Boston and among Caucasian, African-American, Hispanic, and Asian women in Boston. RESULTS: Androstenedione (4.0 +/- 1.3 vs. 3.5 +/- 1.2 ng/ml; P < 0.01) was higher and testosterone (54.0 +/- 25.7 vs. 66.2 +/- 35.6 ng/dl; P < 0.01), LH (23.1 +/- 15.8 vs. 27.6 +/- 16.2 IU/liter; P < 0.05), and Ferriman Gallwey score were lower (7.1 +/- 6.0 vs. 15.4 +/- 8.5; P < 0.001) in Caucasian Icelandic compared with Boston women with PCOS. There were no differences in fasting blood glucose, insulin, or homeostasis model assessment in body mass index-matched Caucasian subjects from Iceland or Boston or in different ethnic groups in Boston. Polycystic ovary morphology was demonstrated in 93-100% of women with PCOS in all ethnic groups. CONCLUSIONS: The data demonstrate differences in the reproductive features of PCOS without differences in glucose and insulin in body mass index-matched populations. These studies also suggest that measuring androstenedione is important for the documentation of hyperandrogenism in Icelandic women. Finally, polycystic ovary morphology by ultrasound is an almost universal finding in women with PCOS as defined by hyperandrogenism and irregular menses.

Divergent responses of gonadotropin subunit messenger RNAs to continuous versus pulsatile gonadotropin-releasing hormone in vitro.

Episodic GnRH input is necessary for the maintenance of LH and FSH secretion. In the current study we have assessed the requirement of a pulsatile GnRH signal for the regulation of gonadotropin alpha- and beta-subunit gene expression. Using a dispersed rat pituitary perifusion system, GnRH (10 nM) was administered as a continuous infusion vs. hourly pulses. Secretion of free alpha-subunit, LH, and FSH were monitored over 5-min intervals for the entire 12-h treatment period before the responses of alpha, LH beta, and FSH beta mRNAs were assessed. Basal release of all three glycoproteins declined slowly over 6-8 h before reaching a plateau. The cells were responsive to each pulse of GnRH, but continuous GnRH elicited only a brief episode of free alpha-subunit, LH, and FSH release, followed by a return to unstimulated levels. Despite the similar patterns of secretion, differences were observed in the responses of gonadotropin mRNAs to the two modes of GnRH. alpha mRNA increased in response to continuous (1.6-fold) or pulsatile (1.7-fold) GnRH. FSH beta mRNA was suppressed to 48% of the control value after continuous GnRH, but was stimulated over 4-fold by the pulses. LH beta mRNA was unresponsive to either treatment paradigm. We conclude that in vitro 1) alpha mRNA levels are increased in response to GnRH independent of the mode of stimulation; 2) under the conditions studied, LH beta mRNA levels are unresponsive to either mode of GnRH input; and 3) the response of FSH beta mRNA to GnRH is highly dependent on the mode of administration, with levels depressed in response to continuous GnRH, but stimulated by pulsatile GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial function in hypothyroidism. Relation to disease severity and response to treatment.

Systolic time intervals (ST) were used to evaluate myocardial function prospectively in 29 hypothyroid patients. The patients were divided into three categories of disease severity: (1) severe hypothyroidism, (2) mild hypothyroidism, and (3) decreased thyroid reserve or "prehypothyroidism." Groups 1 and 2 showed decreased myocardial contractility with a prolonged preejection period (PEP), shortened left ventricular ejection time (LVET), and increase PEP/LVET, compared with normal controls. The STI were more abnormal (P less than .05) in group 1 than in group 2, suggesting that the severity of myocardial dysfunction correlates with the severity of the hypothyroidism. Group 3 had normal STI. Ten patients were restudied when euthyroid and showed complete normalization of their STI, supporting the thesis that hypothyroidism was the sole cause of the initial myocardial dysfunction.

Pulsatile gonadotropin-releasing hormone modifies polyadenylation of gonadotropin subunit messenger ribonucleic acids.

Gonadotropin subunit mRNA levels rise after castration, coincident with a period of increased GnRH input to the pituitary. In addition to increased levels of gonadotropin mRNAs, we observed that the sizes of the alpha and LH beta mRNAs were increased after ovariectomy (OVX) of rats. To determine whether these changes occurred in the 5' (alternate transcriptional start site or splicing)- or 3' (altered polyadenylation)-end of the molecules, mRNAs were cleaved using oligonucleotide-directed RNase-H digestion, and the fragments were analyzed by Northern blot, using probes specific to the 5'- and 3'-segments of each transcript. After OVX, there was no change in the sizes of the 5'-segments of LH beta, FSH beta, and alpha-subunit mRNAs. However, the LH beta and alpha-subunit 3'-fragments were increased in size, indicating a shift to more adenylated forms of the LH beta and alpha transcripts. For FSH beta, the 3'-fragment bands were more diffuse than for LH beta or alpha-subunit, and no alteration in the lengths of FSH beta poly(A) tails were detected. A perifused pituitary cell system was used to determine whether pulses of GnRH were sufficient to cause modifications of polyadenylation. GnRH was administered as hourly 10-nM pulses for 4-12 h. Time-dependent increases in the sizes of LH beta and alpha-subunit mRNAs were observed in GnRH-treated cells compared to cells receiving no GnRH. Changes in the lengths of LH beta and alpha-subunit mRNAs were shown to be due to increased polyadenylation, and there was no observable change in polyadenylation of FSH beta mRNA. In addition, no changes were observed in the size of the 3'-fragments of PRL or beta-actin mRNAs. These data demonstrate that pulsatile GnRH administered in vitro elicits specific increases in the lengths of the LH beta and alpha-subunit mRNA poly(A) tails. Similar changes occur after OVX. Thus, in addition to transcriptional stimulation of the gonadotropin gene, GnRH modifies gonadotropin mRNAs at a posttranscriptional level.

Gonadotropin-releasing hormone-induced stimulation and desensitization of free alpha-subunit secretion mirrors luteinizing hormone and follicle-stimulating hormone in perifused rat pituitary cells.

A pulsatile pattern of hypothalamic GnRH stimulation is necessary for the maintenance of pituitary LH and FSH secretion, with continuous GnRH leading to a decrement in response. Although the physiological pattern of free alpha-subunit secretion closely mimics that of LH, several reports have indicated that free alpha-subunit is not desensitized by continuous GnRH stimulation. To explore the basis of this phenomenon, we have evaluated the responses of all three gonadotrope secretory products to carefully coordinated administration of pulsatile and continuous GnRH in a dispersed rat pituitary perifusion system. Sensitivities (ED50) to GnRH fell within a narrow range for free alpha-subunit (11.5 nM), LH (12.9 nM), and FSH (17.3 nM), although a greater mass of LH than free alpha-subunit or FSH was released after each pulse of GnRH. The response to a standard GnRH pulse (10 nM) administered every 15, 30, or 120 min for 9 h was very stable, with no evidence of priming, summation, or loss of response. LH, FSH, and free alpha-subunit did, however, show significantly (P less than 0.05) higher pulse amplitude with longer interpulse intervals. In contrast to previous observations in vivo, the three gonadotrope secretory products showed parallel desensitization in response to continuous infusions of GnRH. This loss of response was significant (P less than 0.05) after exposure to as little as 0.1 (FSH) to 0.5 nM (LH and alpha-subunit) GnRH for 2 h or to higher concentrations of GnRH (10 nM) for as little as 15 min (LH, FSH, and alpha-subunit). These concentrations and durations of GnRH stimulation are within the range of values measured in vivo. We conclude that 1) free alpha-subunit, LH, and FSH have similar concentration and frequency responses to pulsatile GnRH, although the absolute amount of hormone released is different for each secretory product; 2) the frequency of pulsatile GnRH stimulation can function as an independent determinant of secretion for each of the three products; and 3) in contrast to observations in vivo, free alpha-subunit, LH, and FSH secretion desensitize similarly after exposure to concentrations or durations of GnRH that may occur in vivo. These observations raise the possibility that desensitization plays a role in the physiological regulation of gonadotrope secretion.

Perifusion of rat pituitary cells with gonadotropin-releasing hormone, activin, and inhibin reveals distinct effects on gonadotropin gene expression and secretion.

Gonadotropin biosynthesis and secretion are influenced by pulsatile GnRH derived from the hypothalamus as well as by paracrine factors. In the current studies, we compared the effects of inhibin, activin, and GnRH, alone and in combination, on gonadotropin subunit messenger RNA (mRNA) levels and gonadotropin secretion. A pituitary perifusion system was used to allow GnRH to be administered as pulses and to minimize paracrine effects. FSH beta mRNA levels were increased 25-fold by a maximal concentration of activin (3 ng/ml) and suppressed 83% by a maximal concentration of inhibin (30 ng/ml). When activin and inhibin were perifused together, inhibin attenuated the effects of maximal activin stimulation in a concentration-dependent manner, with a 10-fold excess of inhibin required to block the effects of activin entirely. Whole cell receptor assays using 125I-labeled activin confirmed that the inhibin used in the perifusion experiments competed for activin binding sites, although with a lower affinity. Direct competition at the activin receptor may thus account for part of the activin/inhibin antagonism observed at the level of FSH beta mRNA. Neither activin nor inhibin had a significant effect on levels of LH beta or alpha mRNAs. Hourly pulses of 10 nM GnRH elicited a 2- to 5-fold increase in FSH beta mRNA. This increment was maintained in the presence of activin and inhibin, suggesting separate, but dependent, mechanisms of action for GnRH vs. inhibin and activin. In studies of secretion, continuous activin stimulation (3 ng/ml) elicited only a small (approximately 30%) increase in basal FSH secretion. However, the response of FSH to pulses of GnRH was amplified 3-fold in the presence of activin. A similar enhancement of GnRH-induced, but not basal, LH release was also observed. Inhibin, in contrast, elicited no changes in basal or GnRH-stimulated release of FSH or LH. We conclude that activin and inhibin are the primary regulators of FSH beta mRNA levels, whereas GnRH appears to be the major effector for gonadotropin secretion. There is significant functional overlap, however, and the combined actions of activin, inhibin, and GnRH determine the final level of FSH beta mRNA and the pattern of gonadotropin secretion.

Homologous desensitization of gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone secretion in vitro occurs within the duration of an endogenous GnRH pulse.

A pulsatile GnRH signal is required for the maintenance of LH and FSH secretion. Studies in animals and in perifused pituitary cells have shown that continuous exposure to GnRH leads to decreased gonadotropin secretion and a blunted secretory response to subsequent pulses of GnRH, a process referred to as homologous desensitization. In the current study, we demonstrate that the duration of continuous GnRH exposure required to desensitize the gonadotrope in vitro is less than the durations of most in vivo GnRH pulses. Perifused male rat pituitary cells were tested with 20-sec pulses of 100 nM GnRH at 5-min intervals before, immediately upon termination of, and after GnRH infusions of varying concentration and duration. Desensitization in response to a GnRH infusion was calculated as the decrease in the LH response to the pulse of GnRH immediately after the infusion (Dsn pulse) compared to the mean LH response to GnRH pulses before and after the infusion. Gonadotropes were completely desensitized after a 2-min infusion of 10 nM GnRH (P < 0.05), the shortest duration tested. Endogenous GnRH pulses, by contrast, average more than 5 min in length. When the duration of GnRH infusion was held constant at 4 min, a concentration response for GnRH-induced desensitization was observed. Gonadotropes were desensitized by GnRH concentrations as low as 1 nM (P < 0.05), and maximal desensitization was observed with 5 nM GnRH. To determine the recovery period for GnRH-induced desensitization, a second series of experiments was performed. Experiments were conducted as described above, except the cells were perifused with medium that did not contain GnRH (recovery) for varying periods between the GnRH infusion and the Dsn pulse. A small response (16% of control) to the Dsn pulse of GnRH was observed after 1 min of recovery, and the response was not different from the control value (P > 0.05) after a 3-min recovery period. This recovery period is consistent with the ability to respond to endogenous GnRH pulses, which rarely exceed two per h. We conclude that GnRH-induced secretory desensitization and recovery occur within endogenous GnRH pulse durations and interpulse intervals, respectively. These data raise the possibility that homologous desensitization occurs under some in vivo conditions, providing an unexpected mechanism for physiological regulation of gonadotropin secretion.

Heterogeneous expression of the potassium-chloride cotransporter KCC2 in gonadotropin-releasing hormone neurons of the adult mouse.

In mature central neurons, chloride extrusion mediated by the K-Cl cotransporter KCC2 appears to be largely responsible for the Cl(-) driving force that allows gamma-aminobutyric acid(A) (GABA(A)) receptor activation to trigger a hyperpolarization. In its absence, GABA's effect is typically depolarizing and often excitatory. We examined the colocalization of KCC2 and GnRH in adult male and female mice using a combined in situ hybridization-immunofluorescence procedure. We found that KCC2 was localized to approximately 34% of GnRH neurons. This proportion was similar in females and males. However, females exhibited a marked rostrocaudal gradient of colocalization that was not seen in males. By contrast, KCC2 was localized to nearly all vasopressin neurons of the supraoptic nucleus. These results indicate that a substantial fraction of GnRH neurons may be depolarized and excited by GABA(A) receptor activation throughout life, supporting the existence of functionally heterogeneous subpopulations.

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.

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.

Dynamic regulation of follicle-stimulating hormone-beta messenger ribonucleic acid levels by activin and gonadotropin-releasing hormone in perifused rat pituitary cells.

Maintenance of FSH biosynthesis requires ongoing exposure to pulsatile GnRH. Recent data demonstrate that activin also stimulates FSH biosynthesis. We used a perifused pituitary system to examine regulation of FSH beta mRNA levels by pulsatile GnRH and activin. Hourly pulses of 10 nM GnRH increased FSH beta mRNA levels by 3-fold. In the same experiment, continuous infusion of 50 ng/ml activin elicited a 50-fold increase in FSH beta mRNA. This magnitude of response to activin in perifusion was unexpected, as only a 2.7-fold increase in FSH beta mRNA was observed when activin was administered to pituitary cells that were cultured in dishes. Since perifusion columns, unlike culture dishes, are exposed to a continuous supply of fresh medium, we examined the possibility that endogenous factors produced by pituitary cells cultured in dishes were stimulating the cells in a paracrine fashion, thereby precluding the full response to exogenously added activin. The kinetics of FSH beta mRNA expression were examined immediately after pituitary dispersion and at different times after culturing the cells in plates. FSH beta mRNA levels fell rapidly after dispersion to 8% of initial levels and remained low over 8 h. Thereafter, FSH beta mRNA levels increased slowly and exceeded initial levels by the second day of culture. In a parallel set of experiments, when medium conditioned by exposure to plated cells was applied to the perifusion system, FSH beta mRNA levels were selectively stimulated (6-fold). These data suggest the removal during dispersion and subsequent accumulation in culture of pituitary-derived factors that are important for the maintenance of FSH beta mRNA levels. We conclude that activin plays a greater role in the regulation of FSH beta mRNA levels than was suggested by previous experiments employing static culture systems in which autocrine or paracrine stimulation may have obscured the effects of exogenously added activin.