A selective retinoid X receptor agonist bexarotene (LGD1069, targretin) inhibits angiogenesis and metastasis in solid tumours.

The present study determined the influence of a retinoid X receptor agonist bexarotene on angiogenesis and metastasis in solid tumours. In the experimental lung metastasis xenograft models, treatment with bexarotene inhibited the development of the lung tumour nodule formation compared to control. In vivo angiogenesis assay utilising gelfoam sponges, bexarotene reduced angiogenesis in sponges containing vascular endothelial growth factor, epidermal growth factor and basic fibroblast growth factor to various extent. To determine the basis of these observations, human breast and non-small-cell lung cancer cells were subjected to migration and invasion assays in the presence of bexarotene. Our data showed that bexarotene decrease migration and invasiveness of tumour cells in a dose-dependent manner. Furthermore, bexarotene inhibited angiogenesis by directly inhibiting human umbilical vein endothelial cell growth and indirectly inhibiting tumour cell-mediated migration of human umbilical vein endothelial cells through Matrigel matrix. Analysis of tumour-conditioned medium indicated that bexarotene decreased the secretion of angiogenic factors and matrix metalloproteinases and increased the tissue inhibitor of matrix metalloproteinases. The ability of bexarotene to inhibit angiogenesis and metastasis was dependent on activation of its heterodimerisation partner peroxisome proliferator-activated receptor gamma. Collectively, our results suggest a role of bexarotene in treatment of angiogenesis and metastasis in solid tumours.

Novel, non-steroidal, selective androgen receptor modulators (SARMs) with anabolic activity in bone and muscle and improved safety profile.

A novel approach to the treatment of osteoporosis in men, and possibly women, is the development of selective androgen receptor modulators (SARMs) that can stimulate formation of new bone with substantially diminished proliferative activity in the prostate, as well as reduced virilizing activity in women. Over the last several years, we have developed a program to discover and develop novel, non-steroidal, orally-active selective androgen receptor modulators (SARMs) that provide improved therapeutic benefits and reduce risk and side effects. In recent studies, we have used a skeletally mature orchiectomized (ORX) male rat as an animal model of male hypogonadism for assessing the efficacy of LGD2226, a nonsteroidal, non-aromatizable, and non-5alpha-reducible SARM. We assessed the activity of LGD2226 on bone turnover, bone mass and bone strength, and also evaluated the effects exerted on classic androgen-dependent targets, such as prostate, seminal vesicles and muscle. A substantial loss of bone density was observed in ORX animals, and this loss was prevented by SARMs, as well as standard androgens. Biochemical markers of bone turnover revealed an early increase of bone resorption in androgen-deficient rats that was repressed in ORX animals treated with the oral SARM, LGD2226, during a 4-month treatment period. Differences in architectural properties and bone strength were detected by histomorphometric and mechanical analyses, demonstrating beneficial effects of LGD2226 on bone quality in androgen-deficient rats. Histomorphometric analysis of cortical bone revealed distinct anabolic activity of LGD2226 in periosteal bone. LGD2226 was able to prevent bone loss and maintain bone quality in ORX rats by stimulating bone formation, while also inhibiting bone turnover. LGD2226 also exerted anabolic activity on the levator ani muscle. Taken together, these results suggest that orally-active, non-steroidal SARMs may be useful therapeutics for both muscle and bone in elderly hypogonadal men through their anabolic activities. Since SARMs both prevent bone loss, and also stimulate formation of new bone, they may have significant advantages relative to currently used anti-resorptive therapies. Coupled with their activity in muscle and their ability to maintain or restore libido, they offer new therapeutic approaches for male and female hormone replacement.

New progestins and potential actions.

Newer, nonsteroidal, orally active, tissue-selective progestins are being developed through a molecular approach to compound selection with human progesterone receptor (hPR) serving as the molecular target. The co-transfection and binding assays are used to test receptor selectivity and cross reactivity with a panel of receptors. Transcriptional products are used to further profile new progestin compounds. Desirable new progestins will suppress estrogen-induced endometrial stimulation, show no or minimal proliferative activity, maintain pregnancy, inhibit ovulation, contain no androgenic, mineralocorticoid or glucocorticoid activity, and possess minimal adverse physiologic effects. Newer progestins that possess many of these desirable properties are in development.

Estrogen activity and novel tissue selectivity of delta8,9-dehydroestrone sulfate in postmenopausal women.

Recent basic and clinical advances have consolidated the concept of tissue-selective estrogens, i.e. molecules that express different degrees of partial agonist, full agonist or antagonist activity in different tissues or cells. Delta8,9-Dehydroestrone sulfate (delta8,9-DHES) is a conjugated estrogen and a component of conjugated equine estrogens (CEE). It is metabolized in the human in at least a 1:1 ratio to its 17beta form, 17beta-delta8,9-DHES. To evaluate its activity in different clinical and biochemical parameters, a clinical research study was conducted with delta8,9-DHES and estrone sulfate as a comparator in postmenopausal women. Delta8,9-DHES was given orally at a daily dose of 0.125 mg for 12 weeks in a group of 10 women. Two additional groups of women received either estrone sulfate alone (1.25 mg/day) or the combination of delta8,9-DHES and estrone sulfate at the previously specified doses. A significant and consistent suppression of hot flushes (number, severity, and total score) was observed with delta8,9-DHES, reaching more than 95% suppression in all parameters of vasomotor symptoms. This level of activity was equal to that obtained with the much higher dose of estrone sulfate, and it was sustained for the duration of the treatment period (12 weeks). Measurements of a bone resorption marker, i.e. urinary excretion of N-telopeptide, demonstrated that delta8,9-DHES at 8 weeks produced a degree of suppression (40%) similar to that observed with the higher dose of estrone sulfate. Gonadotropin secretion (FSH and LH) was significantly suppressed in women receiving delta8,9-DHES, similar to that observed with estrone sulfate alone or with the combination of the two. Other parameters, such as total cholesterol, low density lipoprotein cholesterol and high density lipoprotein cholesterol were not modified significantly, whereas serum globulins (sex hormone-binding globulin and corticosteroid-binding globulin) showed only marginal increases after delta8,9-DHES administration. Taken together with preclinical data, it is found that delta8,9-DHES is an active estrogen with a distinct pharmacological profile that results in significant clinical activity in vasomotor, neuroendocrine (gonadotropin and PRL) and bone preservation parameters, whereas displaying little or no efficacy, at the dose tested, on other peripheral parameters normally affected by estrogens. Collectively, this information supports the concept that delta8,9-DHES is an integral component of CEE, with distinct tissue selectivity contributing to the CEE's overall clinical activity, and places this estrogen as a distinct member of a novel class of centrally active molecules with unique peripheral tissue selectivity.

Modulating mechanisms of neuroendocrine cell activity: the LHRH pulse generator.

1. Luteinizing hormone-releasing hormone (LHRH), synthesized in specialized neurons in the hypothalamus, is the prime regulator of reproduction. In its absence, reproductive development is arrested and disorders of LHRH secretion result in several reproductive dysfunctions. 2. The LHRH neuronal network plays a paramount role in the regulatory loop controlling gonadal homeostasis. LHRH input to the pituitary gland maintains gonadotropin secretion, which, in turn, is responsible for gonadal trophism. Steroidal and peptidergic hormones from the gonad close the regulatory system by establishing negative (male and females) and positive (females) feedback loops. 3. Interestingly, LHRH input to the pituitary is intermittent rather than continuous. In fact, continuous exposure to LHRH results in paradoxical hypogonadism. Several studies in animals have provided direct evidence for episodic secretion of LHRH into the hypophyseal portal system. However, the nature of the system(s) responsible for the generation of the LHRH pulsatile profile is not currently known. The recent observation that immortalized LHRH neurons secrete LHRH in a pulsatile manner suggests that the pulse generating mechanism resides within the LHRH neuronal network. 4. In this overview, we compile several lines of evidence supporting this notion and put this characteristic of LHRH neurons in perspective with gonadal influences both internal and external to the LHRH neuronal network. Some recent data regarding the site of action of gonadal steroids on the LHRH neuronal system, the functional significance of galanin colocalization with LHRH, and the role of nitric oxide in the pulse generating mechanism are also discussed.

Expression of functional estrogen receptors and galanin messenger ribonucleic acid in immortalized luteinizing hormone-releasing hormone neurons: estrogenic control of galanin gene expression.

The activity of estradiol on the LHRH neuronal network is crucial in the regulation of reproduction. In vivo, estradiol induces galanin (GAL) gene expression in LHRH neurons and GAL/LHRH colocalization is sexually dimorphic and neonatally determined by steroid exposure. The effects of estradiol on LHRH neurons, however, are considered to be indirect because estrogen receptors (ER) have not been detected in LHRH neurons in vivo. Using immortalized mouse LHRH neurons (GT1-7 cells), we demonstrated by RT-PCR and Southern blotting that GT1-7 cells express ER messenger RNA (mRNA). Sequencing of the amplification products indicated that GT1-7 ER is of the alpha-subtype (ER alpha). Additionally, estrogen receptors in GT1-7 cells were characterized by competitive radioligand receptor binding and IC50 values for 17beta-estradiol and ICI-182,780 were found to be 0.24 and 4.1 nM, respectively. The ability of endogenous GT1-7 cell ER to regulate transcription was determined in transient transfection studies using a construct that consisted of a luciferase reporter gene that is driven by tandem estrogen response elements (ERE) and a minimal herpes simplex virus thymidine kinase promoter. 17Beta-estradiol was found to enhance luciferase activity by 2.5-fold at physiological concentrations with an ED50 value of 47 pM. This induction was completely inhibited by ICI-182,780 which had an IC50 value of 4.8 nM. Raloxifene, tamoxifen, 4-hydroxytamoxifen, and droloxifene also fully blocked estrogen-mediated luciferase induction with IC50 values of 58.4, 89.2, 33.2, and 49.8 nM, respectively. In addition, GAL mRNA was detected and identified by RT-PCR followed by Southern blotting using a rat GAL complementary DNA (cDNA) probe. The ability of 17beta-estradiol to modulate expression of the endogenous GAL gene in immortalized LHRH neurons was also determined. Quantitative RT-PCR demonstrated that physiological concentrations of estrogen increase GAL gene expression by 2-fold with an ED50 value of 23 pM. ICI-182,780, raloxifene, and droloxifene completely blocked this induction. In summary, our data demonstrate the presence of ER alpha and GAL mRNA in GT1-7 cells. The ER in GT1-7 cells is biologically active because 17beta-estradiol enhances both endogenous GAL gene expression and an ERE-driven reporter gene. These results suggest that estrogenic control of GAL gene expression in immortalized LHRH neurons may be transduced by ER. Thus, hypothalamic-derived LHRH neurons appear to have the capacity to be directly regulated by estrogen.

Ontogeny of gonadotrophin and inhibin secretion in normal girls through puberty based on overnight serial sampling and a comparison with normal boys.

We measured luteinizing hormone (LH) and follicle stimulating hormone (FSH) by immunofluorometric assays and alpha-inhibin by radioimmunoassay in serum sampled every 10 min throughout the night (2100-0500 h) from 44 normal girls. Mean overnight LH values rose log-linearly from a mean of 0.2 IU/l in prepubertal girls to 3.0 IU/l in late pubertal girls. Log2 mean overnight FSH rose rapidly through early puberty and then remained constant; mean FSH rose from 1.0 IU/l in prepubertal girls to approximately 2.8 IU/l in Tanner III-V girls. Mean overnight inhibin increased through puberty, rising from 151 ng/l in prepubertal girls to 432 ng/l in fully pubescent girls. Within each of the first three Tanner stages, LH differed approximately 100-fold between the smallest and largest mean concentrations but differed <10-fold within stages IV or V. Such within-pubertal stage variability was less pronounced for FSH, which differed approximately 16-fold among Tanner I subjects and 4-10-fold at later stages, and for inhibin, which varied approximately 4-fold within each Tanner stage. The frequency of LH pulses during overnight sampling increased significantly during puberty, but the frequency of FSH and inhibin pulses remained constant. We compared the results from girls to those from 50 normal boys [Manasco et al. (1995) J. Clin. Endocrinol. Metab., 80, 20462052]. At each pubertal stage, girls had approximately the same mean overnight LH values as boys; girls had higher mean overnight FSH, particularly during Tanner stages II-IV; and boys had mean overnight alpha-inhibin immunoreactivity approximately 1.5 times that of girls at each pubertal stage. Still, hormone concentrations for individuals of both sexes intergraded at each pubertal stage.

Nitric oxide is involved in the genesis of pulsatile LHRH secretion from immortalized LHRH neurons.

Neuronal networks controlling endocrine events present synchronous activity which is required for maintaining physiological functions, including reproduction. Although pulsatile hormone secretion is of paramount importance, the mechanism(s) by which secretory episodes are generated remain largely unknown. Nitric oxide (NO) has become the prototype of a new family of signaling molecules in the body. Nitric oxide diffuses from the source cell and controls activity of neighboring neurons as well as itself as a retrograde messenger. Cells of the luteinizing hormone-releasing hormone (LHRH) neuronal network, the key component in the control of reproduction, are scattered and loosely arranged in the anterior hypothalamus. A diffusible neurotransmitter could provide a means for establishing synchronous activation of the LHRH neuronal network leading to physiologically-relevant pulsatile LHRH secretion. In this study, we demonstrate that immortalized LHRH-producing neurons (GT1-7 cells) express NO synthase (NOS) mRNA and protein. Furthermore, GT1-7 cells are NADPH-diaphorase-positive (a marker of NOS activity) and the histochemical reaction can be abolished by treatment with a competitive NOS blocker. The presence of citrulline in these cells provides further evidence for the biological activity of NOS. These observations indicate that an active NO synthesizing machinery is present in immortalized LHRH neurons. In addition, we show that LHRH secretion is enhanced by NO in a cGMP-dependent manner. Since pulsatile LHRH secretion from immortalized LHRH neurons in vitro is abolished by NOS blockers and NO scavengers, it appears that NO is a unique neurotransmitter that is necessary to set LHRH neurons in phase to establish synchronized pulsatile LHRH secretion.

Responses in the brain of estrogen receptor alpha-disrupted mice.

These studies sought to determine if neurons in the estrogen receptor-alpha knockout (ERalphaKO) mouse brain concentrated 16alpha-[125I]iodo-11beta-methoxy-17beta-estradiol (125I-estrogen), and if so, whether estrogen binding augmented the expression of progesterone receptor (PR) mRNA. Mice were injected with 125I-estrogen and cryostat sections thaw mounted onto emulsion-coated slides. After 30-90 days of exposure, cells with a nuclear uptake and retention of 125I-estrogen were observed in a number of ERalphaKO mouse brain regions including the preoptic nucleus and arcuate nucleus of the hypothalamus, bed nucleus of the stria terminalis, and amygdala, although the number of labeled cells and intensity of nuclear concentration was markedly attenuated when compared with wild-type littermates. Competition studies with excess 17beta-estradiol, diethylstilbestrol, or moxestrol, but not with R5020 or dihydrotestosterone, prevented the nuclear concentration of 125I-estrogen. To determine if the low level of estrogen binding was capable of regulating gene expression, in situ hybridization was used to evaluate PR mRNA in the brain. ERalphaKO and wild-type mice were ovariectomized and treated with vehicle or 17beta-estradiol, and brains were sectioned and hybridized with a PR cRNA probe. Analysis of hybridization signal revealed a similar, low level of PR mRNA in ovariectomized wild-type and homozygous mice, and a marked increase in expression after treatment of ovariectomized animals with 17beta-estradiol, with the level of hybridization signal being significantly higher in wild-type animals when compared with ERalphaKO mice. The results demonstrate that estrogen binds in the ERalphaKO brain and is capable of modulating PR gene expression, thus supporting the presence and functionality of a nonclassical estrogen receptor.

Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms.

Under physiological conditions, FSH is secreted into the circulation as a complex mixture of several isoforms that vary in the degree of glycosylation. Although it is well established that the glycosylation of FSH is important for the serum half-life of the hormone and coupling of the receptor to adenylate cyclase, little is known concerning how physiologically occurring glycosylation patterns of this hormone affect receptor signaling. In this study, we have examined the biological activity of deglycosylated human FSH (DeGly-phFSH), recombinant mammalian-expressed hFSH (CHO-hFSH), and insect cell-expressed hFSH (BV-hFSH, alternatively glycosylated) as compared with that of purified human pituitary FSH (phFSH) using a Chinese hamster ovarian cell line stably expressing the hFSH receptor (3D2 cells). Differentially glycosylated forms of FSH did not bind to the FSH receptor in the same manner as phFSH. Although all hormones showed similar potency in competing for [125I]phFSH binding to the hFSH receptor, competition curves for deglycosylated and insect cell-produced FSH were steeper. Similarly, glycosylation of FSH had a profound effect on bioactivity of the hormone. Purified hFSH produced a sigmoidal dose-dependent stimulation in cAMP production, whereas DeGly-phFSH and BV-hFSH induced biphasic (bell-shaped) dose-response curves. BV-hFSH also elicited biphasic effects on steroidogenesis in primary cultures of rat granulosa cells. The cellular response to BV-hFSH was dependent on the degree of receptor-transducer activation. BV-hFSH bioactivity was strictly inhibitory when combined with the ED80 of phFSH. Lower concentrations of phFSH resulted in a gradual shift from inhibition to a biphasic activity in the presence of the ED20 of phFSH. Biphasic responses to BV-hFSH were attributed to activation of different G protein subtypes, since treatment of 3D2 cells with cholera toxin or pertussis toxin differentially blocked the two phases of BV-hFSH bioactivity. These data suggest that alternative glycosylation of FSH leads to a functionally altered form of the hormone. Functionally different hormones appear to convey distinct signals that are transduced by the receptor-transduction system as either stimulatory or inhibitory intracellular events via promiscuous, glycosylation-dependent G protein coupling. Promiscuity in signaling of the FSH receptor, in turn, may represent a potentially novel mechanism for FSH action, whereby the gonad may respond in diverse ways to complex hormonal signals such as those presented by circulating FSH isoforms.

Modulation by galanin of growth hormone and gonadotropin secretion from perifused pituitary and median eminence of prepubertal male calves.

Galanin is widely distributed in the peripheral and central nervous system and has been indicated as a putative hypothalamic-hypophysiotropic hormone. This study was performed to investigate the effects of galanin on both growth (GH) and luteinizing hormones (LH) from pituitaries of young male calves. Pituitary slices (P, 500 microm in thickness) were perfused alone or coincubated with median eminence terminals (ME) in DMEM-F12 plus BSA 0.1% and antibiotics. The perifusion chambers were kept in equilibrium for 150 min, and medium samples were collected every 10 min for 240 min and stored at -20 degrees C until the measurement of LH and GH levels. Basal GH release increased up to 60% after galanin infusion (p < 0.01 vs. baseline levels) for 60 min in P alone; in P + ME coincubation, galanin-stimulated GH secretion was further increased by up to 200%. Basal LH release in chambers with P was significantly increased (up to 25%; p < 0.05) for 70 min after galanin infusion; P + ME coincubation showed a galanin-mediated increase in LH release of up to 50%. GH and LH responsiveness to exogenous GH-releasing hormone and gonadotropin-releasing hormone was not significantly modulated by galanin in our experimental model. In conclusion, galanin is demonstrated to have a significant stimulatory role in the secretion of GH and LH, with a combined action at both the hypothalamic and pituitary levels. ......................

Pretreatment with phorbol ester and an LHRH agonist reduces testosterone production and protein kinase C activity in rat Leydig cells challenged with PDBu and LHRH.

We have investigated the role of protein kinase C (PK-C) in luteinizing hormone-releasing hormone (LHRH)-induced testosterone secretion from purified rat Leydig cells (70-80-day old Sprague-Dawley rats) by pretreating the cells in vitro with 200 mM phorbol 12,13-dibutyrate (PDBu) (a known procedure to down-modulate this enzyme in most cell types) and 1 muM [D-Ala6,Des-Gly10]-LHRH ethylamide, an LHRH agonist (LHRH-A). Following pretreatment we measured PK-C activity and secretion of testosterone in response to subsequent challenges with the PK-C activator PDBu (20-2000 nM) and with LHRH (0.001-1.0 muM) and the Ca(2+)-mobilizing secretagogue A23187 (0.1-100 microM) in the same cell preparation. PDBu and LHRH-A pretreatments caused a reduction in testosterone secretion in response to subsequent exposure to PDBu or LHRH. Both pretreatments decreased PK-C activity in crude and purified extracts of the same cells. The magnitude of reduction of the secretory response was greater than that of enzyme activity for both PDBu and LHRH-A pretreatment (68.9% reduction of testosterone secretion vs 54.7% reduction of PK-C activity in PDBu-pretreated cells and 78.6% reduction of testosterone production vs 36.6% reduction of PK-C activity in LHRH-A-pretreated cells). The effect of phorbol ester pretreatment on PDBu- or LHRH-stimulated testosterone secretion and PK-C activity was specific (no measurable effect with 4 alpha-PDBu, an inactive phorbol ester). While PDBu and LHRH-A pretreatment reduced Leydig cell responsiveness to PDBu or LHRH, the secretion of testosterone in response to the Ca2+ -mobilizing secretagogue A23187 was similar in PDBu- and LHRH-A-pretreated and in control (non-pretreated) cells. We conclude that down-modulation of protein kinase C by prolonged exposure of Leydig cells to phorbol esters or LHRH-A results in decreased PK-C activity and testosterone secretion. These results provide the first evidence that pretreatment with LHRH-A, which does not enter the cell, can affect the steroidogenesis and PK-C activity responses to PDBu (the intracellular ligand of PK-C).

Steroid imprinting and modulation of sexual dimorphism in the luteinizing hormone-releasing hormone neuronal system.

1. Sex differences in the control of gonadotropin secretion and reproductive functions are a distinct characteristic in all mammalian species, including humans. Ovulation and cyclicity are among the most distinct neuroendocrine markers of female brain differentiation, along with sex behavioral traits that are also evident in different species. 2. The luteinizing hormone-releasing hormone (LHRH) neuronal system is the prime regulator of neuroendocrine events leading to ovulation and hormonal changes during the menstrual cycle and, as such, is the potential site where many of these sex differences may be expressed or, at the very least, integrated. However, until recently, no significant differences were seen in LHRH neurons between male and female brains, including cell number, pattern of distribution, and expression of message or peptide (LHRH) levels. 3. Recently, we reported that galanin (GAL), a brain-gut peptide, is coexpressed in LHRH neurons and that this coexpression is sexually dimorphic. When GAL is used as a marker for this neuronal system, it is clear that estradiol as well as progesterone profoundly affects the message and expression of the peptide and that this regulation, at least in rodents, is neonatally predetermined by gonadal steroid imprinting. 4. Changes in GAL expression and message can also be seen at puberty, during pregnancy and lactation, and in aging, all situations that affect the function of the LHRH neuronal system. Using an immortalized LHRH neuronal cell line (GT1) we have recently observed that these neurons express estrogen receptor (ER) and GAL and that estradiol can increase the expression of GAL, indicating functional activation of the endogenous ER.

Role of galanin in the regulation of somatotrope and gonadotrope function in young ovulatory women.

The aim of the study was to elucidate the role of the neuropeptide galanin in the regulation of somatotropic and gonadotropic function in normal women. Thirteen normally ovulating (aged 28 to 40 years), non-obese (body mass index, 18.4 to 27.1 kg/m2) women with infertility due to a tubal or male factor were studied. Each woman underwent three tests: (1) bolus intravenous (IV) injection of growth hormone (GH)-releasing hormone (GHRH) (1-29)NH2 1 microgram/kg plus gonadotropin-releasing hormone (GnRH) 100 micrograms at time 0; (2) IV infusion of porcine galanin 500 micrograms in 100 mL saline from -10 minutes; and (3) bolus IV injection of GHRH(1-29)NH2 1 microgram/kg plus GnRH 100 micrograms at time 0 plus IV infusion of porcine galanin 500 micrograms in 100 mL saline from -10 to +30 minutes. All results are expressed as the mean +/- SEM. GH peak after GHRH was 14 +/- 5 micrograms/L; porcine galanin significantly increased serum GH (GH peak, 7.3 +/- 1.2) with respect to baseline levels. No significant differences were observed between either GH peak or GH absolute values after galanin as compared with GHRH alone. Porcine galanin significantly enhanced GH response to GHRH (peak, 31.4 +/- 4.4 micrograms/L) with respect to either GHRH or galanin alone. Luteinizing hormone (LH)/follicle-stimulating hormone (FSH) peaks after GnRH were 16.5 +/- 5.3 and 17.4 +/- 4 IU/L, respectively. Porcine galanin did not cause significant increases in serum LH and FSH levels with respect to baseline.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of gonadotropin, testosterone, and inhibin secretion in normal boys through puberty based on overnight serial sampling.

To investigate hormonal changes occurring in male puberty, we measured LH, FSH, testosterone, and alpha-inhibin immunoactivity in serum samples drawn every 10 min for 8 h (2100-0500 h) from each of 50 normal prepubertal and pubertal boys, aged 8.4-18.8 yr. We measured gonadotropins with ultrasensitive immunofluorometric assays, and testosterone and alpha-inhibin with RIAs. Unlike previous studies, which indexed pubertal development with Tanner stages, we used testicular volume, a more finely graduated indicator of development, to reveal patterns that were obscured when subjects were grouped by Tanner stage. The overnight mean concentration of each hormone increased with testis volume, but the rate of increase on a logarithmic scale slowed as testes grew. Log LH rose precipitously in the late prepubertal and early pubertal periods and plateaued during mid- and late puberty. Based on fitted regression curves, LH increased about 20-fold (from 0.11 IU/L) between testis volumes of 1 and 10 mL, but only an additional 1.5-fold by 30 mL. The developmental trajectory of log testosterone was like that of log LH, but rose less steeply early in puberty. From 0.14 micrograms/L at a testis volume of 1 mL, testosterone increased about 8.5-fold by 10 mL and an additional 3-fold by 30 mL. In contrast, logarithms of overnight mean FSH and alpha-inhibin concentrations rose at a more nearly constant rate throughout puberty. From 0.62 IU/L at a testis volume of 1 mL, the FSH concentration doubled by 10 mL and increased an additional 1.7-fold by 30 mL. From 270 ng/L at a testis volume of 1 mL, inhibin increased 1.5-fold by 10 mL and an additional 1.3-fold by 30 mL. Overnight pulse amplitudes exhibited developmental trajectories similar to those of the corresponding overnight mean concentrations. The number of LH and testosterone pulses during the sampling period averaged 2.2 and 2.1, respectively, at Tanner stage 1 and increased to 4.5 and 3.2, respectively, at Tanner stage 5. The number of FSH and inhibin pulses remained constant throughout puberty, averaging 3.3 and 3.5, respectively. Pairwise correlations among hormone concentrations were strong, reflecting common increasing trends through puberty; however, after accounting for developmental trends, FSH, LH, and testosterone concentrations remained correlated, whereas inhibin was uncorrelated with each of the other three hormones. Measuring gonadotropins with ultrasensitive assays and analyzing the results on a logarithmic scale as a function of testis volume made clear the dramatic hormonal changes that begin before the clinical changes of puberty.

Sexual dimorphism in copackaging of luteinizing hormone-releasing hormone and galanin into neurosecretory vesicles of hypophysiotrophic neurons: estrogen dependency.

Hypophysiotrophic neurons projecting to hypophyseal portal vessels in the median eminence of the hypothalamus maintain the operation of the master gland, the pituitary, by secreting releasing and release-inhibiting hormones into the bloodstream. LHRH, synthesized in neurons of the rat prosencephalon, is one of the key substances that governs the anterior pituitary-gonadal axis. Recently, it has been shown that the peptide galanin (GAL) is coproduced in a subpopulation of LHRH neurons and is a potent modulator of central processes regulating reproduction. A better understanding of the secretory mechanisms involved in pulsatile hormone release from LHRH axons of the median eminence requires exploration of the organelle domain that displays the cosynthesized peptides in terminal boutons. This study shows that LHRH- and GAL-immunoreactive axons overlap heavily in the lateral part of the median eminence. Double fluorescent labeling revealed colocalization of the peptides at the level of single axon terminals. By means of dual colloidal gold immunolabeling, LHRH and GAL were detected in the same secretory vesicles at the ultrastructural level. The incidence of colocalizing vesicles was high in the female (45%) and low in the male (3%) rat. Ovariectomy resulted in a dramatic decline in the number of LHRH/GAL-coexpressing vesicles (23%), which was reversed (55%) by the administration of estradiol. The observations indicate a sex-related difference in the packaging of LHRH and GAL and suggest that the events are estrogen dependent. Furthermore, the simultaneous release of GAL and LHRH from the colocalizing vesicles provides a mechanism that might ensure the potentiating effect of GAL on LHRH by synchronizing events at the receptor sites in the anterior pituitary.

Arachidonic acid and its metabolites effects on testosterone production by rat Leydig cells.

Arachidonic acid (AA) seems to play an important role in testicular steroidogenesis, although controversial data exist in the literature. In the present study AA induced a dose related increase of testosterone (T) formation and, at the highest dose, stimulated the production of prostaglandin E2 (PGE2), leukotrienes B4 (LTB4) and C4 (LTC4) by purified rat Leydig cells. The contemporary addition of the prostaglandin synthesis blocker, indomethacin (IND), and AA further increased T formation, decreased PGE2 levels and did not modify LTB4 and LTC4 concentrations. The addition of a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA, 5 microM), did not influence the stimulatory effect of AA on T and PGE2 formation while it decreased the output of LTB4 and LTC4. When 20 microM NDGA was used in addition to AA the expected reduction of leukotrienes release was observed together with a surprising impairment of T and PGE2 secretion. PGE2 and PGF2 alpha did not modify basal T production but reduced HCG-stimulated T secretion at the 10 nM dose. When 5-12- and 15-HETE were tested an enhancement of basal T formation was observed at the 10nM dose. 5-HETE (10nM) stimulated HCG-induced T production. LTA4, LTB4 and LTE4 did not influence basal T output while LTC4 and LTD4 inhibited it. LTC4 (10nM) induced a decrease of HCG-stimulated T production. These findings suggest that: 1) exogenous AA stimulates T secretion; 2) conversion of AA to cycloxygenated and lipoxygenated metabolites is not required for its steroidogenic effect; 3) cycloxygenated and lipoxygenated compounds play a diverse modulatory role on testicular steroidogenesis.

Regulation of luteinizing hormone-releasing hormone and luteinizing hormone secretion by hypothalamic amino acids.

1. The present review discusses the proposed roles of the amino acids glutamate and GABA in the central regulation of luteinizing hormone-releasing hormone (LHRH) and in luteinizing hormone (LH) secretion. 2. Descriptions of the mechanisms of action of these neurotransmitters have focused on two diencephalic areas, namely, the preoptic-anterior hypothalamic area where the cell bodies of LHRH neurons are located, and the medial basal hypothalamus which contains the nerve endings of the LHRH system. Increasing endogenous GABA concentration by drugs, GABA agonists, or blockade of glutamatergic neurotransmission by selective antagonists in rats and non-human primates prevents ovulation and pulsatile LH release, and blunts the LH surges induced by estrogen or an estrogen-progesterone combination. In contrast, glutamate and different glutamate agonists such as NMDA, AMPA and kainate, can increase LHRH/LH secretion. 3. The simultaneous enhancement of glutamatergic activity and a decrease of GABAergic tone may positively influence the maturation of the pituitary-gonadal system in rats and non-human primates. Administration of glutamate receptor agonists has been shown to significantly advance the onset of puberty. Conversely, glutamate antagonists or increased endogenous GABA levels may delay the onset of puberty. The physiological regulation of LHRH/LH secretion may thus involve a GABA-glutamate interaction and a cooperative action of the various types of ionotropic glutamate receptors. 4. The inhibitory actions of GABA on LH release and ovulation may be exerted at the level of afferent nerve terminals that regulate LHRH secretion. A likely candidate is noradrenaline, as suggested by the synaptic connections between noradrenergic nerve terminals and GABAergic interneurons in the preoptic area. Recent experiments have provided complementary evidence for the physiological balance between inhibitory and excitatory transmission resulting in modulation of the action of noradrenaline to evoke LHRH release.

Regulation of luteinizing hormone-releasing hormone and luteinizing hormone secretion by hypothalamic amino acids

1. The present review discusses the proposed roles of the amino acids glutamate and GABA in the central regulation of luteinizing hormone-releasing hormone (LHRH) and in luteinizing hormone (LH) secretion. 2. Descriptions of the mechanisms of action of these neurotransmitters have focused on two diencephalic areas, namely, the preoptic-anterior hypothalamic area where the cell bodies of LHRH neurons are located, and the medial basal hypothalamus which contains the nerve endings of the LHRH system. Increasing endogenous GABA concentration by drugs, GABA agonists, or blockade of glutamatergic neurotransmission by selective antagonists in rats and non-human primates prevents ovulation and pulsatile LH release, and blunts the LH surges induced by estrogen or an estrogen-progesterone combination. In contrast, glutamate and different glutamate agonists such as NMDA, AMPA and kainate, can increase LHRH/LH secretion. 3. The simultaneous enhancement of glutamatergic activity and a decrease of GABAergic tone may positively influence the maturation of the pituitary-gonadal system in rats and non-human primates. Administration of glutamate receptor agonists has been shown to significantly advance the onset of puberty. Conversely, glutamate antagonists or increased endogenous GABA levels may delay the onset of puberty. The physiological regulation of LHRH/LH secretion may thus involve a GABA-glutamate interaction and a cooperative action of the various types of ionotropic glutamate receptors. 4. The inhibitory actions of GABA on LH release and ovulation may be exerted at the level of afferent nerve terminals that regulate LHRH secretion. A likely candidate is noradrenaline, as suggested by the synaptic connections between noradrenergic nerve terminals and GABAergic interneurons in the preoptic area. Recent experiments have provided complementary evidence for the physiological balance between inhibitory and excitatory transmission resulting in modulation of the action of noradrenaline to evoke LHRH release.