The neuroendocrine thymus: coexistence of oxytocin and neurophysin in the human thymus.

Immunoreactive oxytocin and neurophysin were identified and measured by radioimmunoassay in human thymus extracts. Serial dilutions of extracts paralleled the appropriate standard curves. Thymus-extracted oxytocin and neurophysin eluted in the same positions as reference preparations on Sephadex G-75. Authenticity of oxytocin was confirmed by biological assay and high-performance liquid chromatography analysis. In most instances, thymus contents of oxytocin and neurophysin were far greater than those expected from known circulating concentrations and declined with increasing age. The molar ratio of oxytocin to neurophysin in thymus was similar to that found in the hypothalamo-neurohypophyseal system, which strongly suggested with the other data a local synthesis of oxytocin. These findings indicate the presence of neurohypophyseal peptides in the human thymus and further support the concept of a neuroendocrine function integrated in an immune structure.

Neuroendocrine mechanism of onset of puberty. Sequential reduction in activity of inhibitory and facilitatory N-methyl-D-aspartate receptors.

In humans and in several animal species, puberty results from changes in pulsatile gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. In particular, the frequency of pulsatile GnRH secretion increases at the onset of puberty, as can be shown by using hypothalamic explants of male rats of 15 and 25 d. Previous observations from us and others suggested that the initiation of puberty could involve a facilitatory effect of excitatory amino acids mediated through N-methyl-D-aspartate (NMDA) receptors. We found that GnRH secretion could be activated through NMDA receptors only around the time of onset of puberty (25 d). The aim of this study was to clarify why this activation did not occur earlier (at 15 d) and could no longer be observed by the end of puberty (at 50 d). We studied GnRH secretion in the presence of MK-801, a noncompetitive antagonist of NMDA receptors or AP-5, a competitive antagonist. We showed that, in the hypothalamus of immature male rats (15 d), a highly potent inhibitory control of pulsatile GnRH secretion in vitro was mediated through NMDA receptors. These data were confirmed in vivo because administration of the antagonist MK-801 (0.001 mg/kg) to immature male rats resulted in early pubertal development. Onset of puberty (25 d) was characterized by the disappearance of that NMDA receptor-mediated inhibition, thus unmasking a facilitatory effect also mediated through NMDA receptors. During puberty, there was a reduction in activity of this facilitatory control which was no longer opposed by its inhibitory counterpart. We conclude that a sequential reduction in activity of inhibitory and facilitatory NMDA receptors provides a developmental basis for the neuroendocrine mechanism of onset of puberty.

Relationship between whole plasma calcitonin levels, calcitonin secretory capacity, and plasma levels of estrone in healthy women and postmenopausal osteoporotics.

The exact role of calcitonin (CT) in the pathogenesis of postmenopausal osteoporosis remains unknown. Whole plasma calcitonin (iCT) basal levels, metabolic clearance rate (MCR), and production rate (PR) of CT were measured in 9 premenopausal and 16 postmenopausal women, including 11 osteoporotics (OP). Basal iCT levels were statistically lower in postmenopausal women than in the premenopausal group (P less than 0.01) and strongly correlated (r = 0.72; P less than 0.001) with estrone circulating levels (E1). MCR were similar in all groups. PR were similar in eugonadal women between 22 (mean +/- SD = 30.9 +/- 9.9 micrograms/d) and 37 yr (mean +/- SD = 25.5 +/- 11.1 micrograms/d) premenopausal women. In healthy postmenopausal women PR were reduced, but not significantly (mean +/- SD = 19.5 +/- 6.95 micrograms/d), whereas osteoporotic patients presented a highly significant reduction of CT PR (mean +/- SD = 9.8 +/- 4 micrograms/d) (P less than 0.01). Because there is a strong relationship between E1 and PR (r = 0.64; P less than 0.001), CT secretory capacity appears to be modulated by estrogen circulating levels. This modulation leads to a menopause-related decrease in iCT. In osteoporotics, an independent impairment of CT production drastically lowers PR and basal iCT levels. CT might be one of the determining factors in the pathogenesis of postmenopausal osteoporosis.

Presence of alpha-lactalbumin, epidermal growth factor, epithelial membrane antigen, and gross cystic disease fluid protein (15,000 daltons) in breast cyst fluid.

The frequency of gross cystic breast disease in premenopausal women and its possible association with increased breast cancer risk emphasize the importance of investigations relating to breast cyst fluid composition. In order to contribute to a better analysis of this medium, we have measured four proteins the presence of which in human milk was well documented. Breast cyst fluid specimens from 266 breast cyst disease patients were assayed and compared as to concentrations of alpha-lactalbumin, gross cystic disease fluid protein (GCDFP-15), epidermal growth factor (EGF), and epithelial membrane antigen (EMA). All the analyzed cyst fluids contained GCDFP-15, EMA, and EGF whereas alpha-lactalbumin was detected in only 14.2% of fluids assayed. Positive correlations were observed between GCDFP-15 and EMA concentrations (P less than 0.005), as well as GCDFP-15 and EGF concentrations (P less than 0.0005). The cyst fluid GCDFP-15 and EGF levels were higher when alpha-lactalbumin concentrations were below detection limits. This association was statistically significant for GCDFP-15 (P less than 0.03) and for EGF (P less than 0.001). These results suggest that GCDFP-15 and EMA could be the biochemical expression of apocrine metaplasia and epithelial hyperplasia, respectively, two histopathological features which characterize breast cystic disease. On the other hand, the occasional presence of alpha-lactalbumin in the cyst fluid would reflect the persistence of differentiated cells in the epithelium surrounding the cyst and would be inversely proportional to the degree of cellular proliferation. The omnipresence of EGF in the cyst fluid argues for the hypothesis of its production by the mammary gland. The highly significant relationship between GCDFP-15 and EGF levels in the medium remains to be elucidated but may be related to an androgen sensitivity in the breast epithelium surrounding the cyst.

Radioimmunoassay for glycoprotein gp47 of murine mammary tumor virus in organs and serum of mice and search for related antigens in human sera.

Murine mammary tumor virus main glycoprotein (gp47), prepared by diethylaminoethyl cellulose and hydroxyapatite chromatography of detergent-mercaptoethanol-KCI-disrupted virion, was used as labeled antigen in a highly specific and reproducible radioimmunoassay. Seven other (glyco) proteins of the virus were antigenically distinct from gp47. Serum and organs of unifected C57BL mice did not contain gp47, but sera of infected Swiss and RIII mice did contain the antigen. Despite the high content in the mammary gland, the level of gp47 in other organs was identical in male and female mice. The titer of gp47 in serum was high in tumor-bearing females, but it varied with the mouse strain. Anti-gp47 immunoglobulins could not be detected. The investigation included 314 human sera (107 normal, 65 benign mastopathy, 89 breast vancer, and 53 digestive cancer). None contained an antigen related to gp47. One of 20 human mammary cyst fluids was positive.

Radioimmunoassay for protein p28 of murine mammary tumor virus in organs and serum of mice and search for related antigens in human sera and breast cancer extracts.

The main protein of the core of murine mammary tumor virus, with a molecular weight of 28,000 (p28), was solubilized by deoxycholate treatment of the virus and purified by Ultrogel ACA-54 filtration and hydroxyapatite chromatography. This protein was used as labeled antigen in a highly specific and reproducible radioimmunoassay. Organ extracts of uninfected C57BL mice did not contain p28, but organ extracts of infected RIII mice did contain the antigen. Despite the high content in the mammary gland, the level of p28 in the other organs was identical in male and female mice. Sera of uninfected mice and the majority of the sera of infected mice did not contain the antigen. The investigation included 338 human sera (50, normal; 157, breast cancer; 77, polycystic disease; 32, benign mastopathy; 12, fibroadenoma; 10, at risk of developing breast cancer). None contained an antigen related to p28. Eight of 24 extracts of human breast cancer gave results that appeared weakly positive, possibly as a result of proteolysis. Extracts of healthy breast tissue and the serum from the breast arterial and venous blood of corresponding patients were negative.

Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus.

The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus.

Expression and processing of the activin-A/erythroid differentiation factor precursor: a member of the transforming growth factor-beta superfamily.

The biosynthesis and intracellular processing of the polypeptide precursor of the beta A-chain of the fertility hormone inhibin were assessed by infecting a wide spectrum of cell types with a recombinant vaccinia virus. Most cell lines, including follicular granulosa cells, secrete both prohormone and mature hormone as homodimers (activin) composed of disulfide-linked subunits of 54 kDa (proactivin-A) and 14 kDa (activin-A), respectively, and a small amount of prohormone-mature hormone heterodimers. Mature activin is secreted from mouse pituitary cells (AtT-20), while pig kidney cells [PK(15)] secrete mostly proactivin. More prohormone is secreted in the presence of NH4Cl, suggesting that prohormone processing is facilitated by low pH. Proactivin-A is not a ligand for the mannose-6-phosphate/insulin growth factor-II receptor. The recombinant activin stimulates FSH release from pituitary cells and differentiates erythroleukemia cell lines in vitro.

One year's treatment of Paget's disease of bone by synthetic salmon calcitonin as a nasal spray.

The effectiveness of synthetic salmon calcitonin (SCT) administered as a nasal spray was assessed via clinical, biological, and radiological variables in 17 previously untreated Pagetic patients over a 1-year course of therapy. The results showed a highly significant decrease of serum alkaline phosphatase (S-ALP) (p less than 0.05 after 1 month of treatment) and of the urinary hydroxyproline/creatinine ratio (OH/Cr) (p less than 0.01 after 1 month of treatment). For the whole group, the mean decrease in S-ALP was 37 +/- 4% (SEM) after 6 months (p less than 0.01) and 31 +/- 5% after 1 year (p less than 0.01). The mean fall in OH/Cr was 35 +/- 6% (SEM) (p less than 0.01) and 37 +/- 7% (p less than 0.01) after 6 and 12 months, respectively. None of the usual side-effects of SCT were reported and local tolerance was excellent throughout the study.

Efficacy and tolerability of a new formulation of oral tiludronate (tablet) in the treatment of Paget's disease of bone.

We sought to assess efficacy and safety of a new oral formulation (tablet) of tiludronate in Paget's disease of bone. We studied 128 patients with Paget's disease in an open-label uncontrolled trial. Patients received a daily dose of 400 mg oral tiludronate (two tablets). Treatment was for 6 months. Serum alkaline phosphatase activity (SAP) and fasting urinary excretion of hydroxyproline/creatine (OH/Cr) were measured every 3 months, as were biochemical parameters reflecting renal, hepatic, and hematologic functions. Analgesic efficacy was self-evaluated from a visual analog scale (VAS). Statistical analysis revealed a significant reduction from baseline in SAP and OH/Cr levels, as well as VAS scores. In the whole population with evaluation under treatment, there was a reduction in initial SAP activity after 3 months (47.2 +/- 2.2%, mean +/- SEM) and 6 months (58.3 +/- 2.3%). In the population with SAP levels above twice the upper limit at inclusion and with evaluation at month 3 and month 6 (n = 96), the reduction in SAP levels was 49.3 +/- 2.4% after 3 months and of 59.5 +/- 2.6% after 6 months (ANOVA time effect, p = 0.0001). Aside from mild gastrointestinal disturbances, as experienced with other oral bisphosphonates, clinical tolerance was good. Exhaustive biochemical investigation failed to reveal significant toxicity of tiludronate tablets at the dose of 400 mg/day. The dose of 400 mg daily of this new formulation appears to be a satisfactory tiludronate regimen for the treatment of Paget's disease of bone.

Long-term (3 years) prevention of trabecular postmenopausal bone loss with low-dose intermittent nasal salmon calcitonin.

The long-term effect of intermittent low-dose nasal salmon calcitonin on trabecular early postmenopausal bone loss was assessed as follow-up to a previously published study. Randomized controlled group comparison was made of 287 healthy women with 6-36 months of natural menopause and no treatment interfering with calcium metabolism at an outpatient clinic for research in bone and cartilage metabolism. The 287 women were randomly allocated to 3 years of treatment with either 500 mg/day, 5 days/week of calcium or the same amount of calcium plus 50 IU/day, 5 days per week of nasal salmon calcitonin. A total of 186 women complied with the study protocol throughout. The main outcome measures were bone mineral density of the lumbar spine (DPA) and biochemical parameters reflecting bone turnover (serum alkaline phosphatases, urinary calcium/creatinine, and hydroxyproline/creatinine ratio). The average changes in bone mineral density after 36 months showed a positive (p < 0.05) outcome (1.8 +/- 5.7%; mean +/- SD) in the group treated with salmon calcitonin and calcium and a significant (p < 0.01) loss (-5.8 +/- 4.8%) in patients receiving calcium alone. The difference between the evolution of the two groups was significantly (p < 0.01) different after 6 months of treatment and remained so until the end of the study. No significant changes were recorded in biochemical parameters reflecting bone turnover. As previously shown during a 1 year follow-up, nasal salmon calcitonin given at low dose and intermittently, in association with calcium, can counteract trabecular postmenopausal bone loss.

Puberty-related increase in episodic LHRH release from rat hypothalamus in vitro.

The retrochiasmatic hypothalamus (RCH) was removed from brains of male rats between 12 and 50 days of age, and immediately studied in vitro. The release of LHRH from the RCH was evaluated by periodic (7.5-min) collections of culture medium and subsequent RIA. With synthetic LHRH in the experimental system, the mean (+/- 1 SD) recovery was 94 +/- 7% with a variation coefficient of 14 +/- 3%. An increase in LHRH release was considered to be significant when it exceeded 6 pg/7.5 min. Biological viability of RCH in vitro was assessed by an increased release of LHRH in response to the depolarizing effect of veratridine. As age increased, from 12 to 50 days, the hypothalamic LHRH content steadily increased. However, a significant increase in veratridine - induced release of LHRH occurred only at 23 days and thereafter. At various ages, single hypothalami were studied during a mean 112-min period to evaluate the spontaneous release of LHRH. In all age groups, the in vitro LHRH release occurred in pulses. However, mean pulse frequency increased significantly with age: in 12- and 17-day-old rats, 0.3 pulse/112 min was observed; at 23, 25 and 27 days, this frequency varied between 1.8 and 3.0 pulses/112 min. At 50 days of age, the observed frequency was within the same range. We conclude that the RCH obtained from rats of various ages may retain in vitro its capacity to release LHRH episodically and that the frequency of these episodic pulses markedly increases with age to the time of the onset of puberty in male rats.

Maturation of the hypothalamic control of pulsatile gonadotropin-releasing hormone secretion at onset of puberty: II. Reduced potency of an inhibitory autofeedback.

At the time of onset of puberty in the male rat, between 15 and 25 days of age, we have reported that pulsatile GnRH secretion by hypothalamic explants showed an increased frequency as indicated by the reduction of the mean interpulse interval from 66 to 40 min (P less than 0.05). This study aimed to evaluate whether these changes in GnRH secretion involved a self-regulatory mechanism. A 7.5-min exposure of explants obtained at 50 days to 0.1 microM superagonist D-TRP6-PRO9-N-Et-GnRH (GnRH-A) resulted in a delay of the next GnRH secretory pulse so that the mean interpulse interval increased from 35 to 67 min (P less than 0.001). In addition, after a 7.5-min exposure to GnRH-A, there was a 15-min period with absent or reduced release of GnRH in response to 50 microM veratridine, a depolarizing agent. A similar refractory period of 15 min was observed using explants obtained at 25 and 50 days whereas, at 15 days, the period of refractoriness lasted for 52.5 min. The inhibitory effect of GnRH-A on the subsequent response to veratridine occurred at similar concentrations of GnRH-A at the three studied ages and the inhibition was prevented using an antagonist of GnRH together with GnRH-A. The involvement of GnRH itself in an autofeedback mechanism was evaluated by studying the period of refractoriness separating two GnRH pulses elicited by 7.5-min exposures to veratridine. The initial responsiveness to veratridine was recovered after a refractory period of 52.5, 22.5, and 15 min when studied at 15, 25, and 50 days, respectively. While refractoriness occurred during repeated depolarization with K+ or veratridine, such an effect was not observed using N-methyl-D-aspartate (NMDA). During exposure to GnRH-A, the NMDA-induced release of GnRH was only reduced by 38% whereas veratridine-induced secretion showed a 94% reduction. Thus, exogenous activation of NMDA-sensitive receptors could bypass the inhibitory autofeedback. We conclude that: 1) pulsatile GnRH secretion is controlled by an inhibitory autofeedback involving NMDA sensitive receptors, 2) the increased frequency of pulsatile GnRH secretion at onset of puberty may be related to a reduced sensitivity of the hypothalamic pulse generator to an inhibitory autofeedback.

Maturation of the hypothalamic control of pulsatile gonadotropin-releasing hormone secretion at onset of puberty. I. Increased activation of N-methyl-D-aspartate receptors.

In the male rat the timing of puberty can be estimated by the rapid increase in testicular weight occurring between 25-50 days of age. We found that elongated spermatids, the most mature germ cells identified using flow cytometry, were first seen at 25 days (4% of the testicular cells), while an adult proportion (63%) was attained by 45 days of age. We have shown previously that hypothalamic explants could release GnRH in a pulsatile fashion at a frequency increasing around the age of 25 days, thus consistent with the time of onset of puberty. Since pulsatile GnRH secretion could be suppressed by MK-801, a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor activation, we postulated that an increased activation of those receptors could be involved in the neuroendocrine mechanism that activates pulsatile GnRH secretion at the onset of puberty. Such a concept was supported by the NMDA-induced release of GnRH, which was observed using 1 mM NMDA at 25 days, while a dose of 20-50 mM was required at 15 or 50 days of age. MK-801 could provide an index of NMDA receptor activation, since the antagonistic effect of MK-801 is use dependent. This particular property was confirmed by the inability of MK-801 (5 pM) to block the depolarization (veratridine)-induced release of GnRH in the presence of 0.001 mM NMDA, while partial or complete suppression was obtained in the presence of 0.1 and 10 mM NMDA, respectively. Using explants obtained at 5, 10, 15, 20, 25, 30, 35, and 50 days of age, the lowest concentrations of MK-801 that blocked the veratridine-induced release of GnRH were, respectively, 10(7), 10(7), 10(7), 10(3), 10, 10(2), 10(4), and 10(8) pM. In contrast, there was no age-related difference in sensitivity to the inhibitory effect of Mg2+, a noncompetitive NMDA receptor antagonist which is not use dependent. The pulsatile secretion of GnRH occurred at a similar frequency at 25 and 50 days of age (4.7 and 5.4 pulses/3.5 h, respectively) but it was suppressed by a lower MK-801 concentration at 25 days (10(4) pM) than at 50 days (10(8) pM). These data indicate that the NMDA receptors involved in the control of pulsatile GnRH secretion are markedly and transiently activated around the time of onset of puberty in the male rat.

Pulsatile release of gonadotropin-releasing hormone (GnRH) from the rat hypothalamus in vitro: calcium and glucose dependency and inhibition by superactive GnRH analogs.

We have shown previously that the rat hypothalamus retains in vitro its capacity of generating pulsatile release of GnRH. The present study evaluated if pulsatile release of GnRH in vitro was influenced by metabolic conditions (calcium and glucose availability) and the possible self-regulatory role of GnRH in its pulsatile secretion. In the presence of a calcium-chelating agent (EGTA, 20 mM) or a calcium-channel blocker (D-600, 0.1 mM), the release of GnRH induced by a depolarization (veratridine, 50 microM) was markedly and reversibly decreased. In addition, frequency and amplitude of GnRH secretory pulses were significantly reduced (P less than 0.05). When glucose use was inhibited using 2-deoxyglucose (5.6 mM) the release of GnRH induced by veratridine and the frequency of GnRH pulses were also blunted (P less than 0.05). Superactive agonists of GnRH (Buserelin and D-TRP6-PRO9-N-ET, 10 nM) caused a prompt decrease of GnRH release in basal conditions and in the presence of veratridine. A significant inhibition (P less than 0.05) was observed using buserelin concentrations greater than 0.01 nM, whereas two GnRH analogs without biopotency (Leu8-GnRH, Des-gly10-N picolylamide GnRH, 100 nM) did not affect GnRH release. The two agonists of GnRH reduced by 43% to 66% (P less than 0.05) the occurrence of significant GnRH pulses. We conclude that, in vitro, the hypothalamic neuronal circuitry resulting in GnRH pulsatile secretion is dependent on calcium and glucose availability and is sensitive to an ultrashort-loop inhibitory feedback mechanism.

Pulsatile release of gonadotropin-releasing hormone from hypothalamic explants is restrained by blockade of N-methyl-D,L-aspartate receptors.

We have shown previously that N-methyl-D,L-aspartate (NMDA) and kainate, two neuroexcitatory amino acids acting through distinct receptors, may induce the release of GnRH from hypothalamic explants. However, that effect could have no physiological significance, since very high concentrations (50 mM) of NMDA and kainate were required. Here, using agents blocking the activation of receptors to neuroexcitatory amino acids, we evaluated their possible physiological involvement in the pulsatile release of GnRH from the hypothalamus of 50-day-old male rats in vitro. In control conditions (10 nM glycine and 1 mM mg2+), the release of GnRH in 7.5-min fractions collected for 2-4 h showed an obvious pulsatile pattern. The mean (+/- 1 SD) interval between pulses, identified by PULSAR program, was 34.3 +/- 11.4 min. The stimulation of GnRH release by NMDA (50 mM) added to the medium for 7.5 min could be blocked reversibly in the presence of MK-801 (100 microM) using medium without glycine or enriched with Mg2+ (2 mM). The endogenous pulses of GnRH secretion were abolished in the presence of MK-801 or using increased Mg2+ concentrations as well as in the absence of glycine. In contrast, pulsatile release of GnRH was not affected in the presence of 6,7-dinitroquinoxaline-2,3-dione (0.1 mM), a selective inhibitor of kainate and quisqualate receptors which suppressed the increase in GnRH release induced by kainate (50 mM) without affecting the response to NMDA. These data indicate that the physiological mechanism of pulsatile GnRH secretion in the hypothalamus may involve endogenous neuroexcitatory factors acting through NMDA-sensitive receptors.

Gonadotropin releasing hormone inhibitory autofeedback by subproducts antagonist at N-methyl-D-aspartate receptors: a model of autocrine regulation of peptide secretion.

The secretion of Gonadotropin-releasing Hormone (GnRH) involves activation of N-methyl-D-aspartate (NMDA) receptors. Here, we show that pulsatile GnRH secretion from hypothalamic explants is suppressed by 1-5GnRH, an endogenous breakdown product of GnRH, while 2-10GnRH has no effect. GnRH secretion evoked by NMDA is selectively inhibited by 1-5GnRH and this effect is similar to that of AP-5, a competitive antagonist at NMDA receptors. In addition, 1-5GnRH accounts for a dose-related inhibition of tritiated glutamate binding to hypothalamic membrane preparations. Using GnRH secretion as a model of NMDA-receptor controlled system, the effect of different peptides has been studied. Growth Hormone Releasing Factor (GRF), Insulin-like Growth Factor-I (IGF-I) and Proinsulin result in inhibition of GnRH secretion. Bioactive subproducts of those peptides (1-29GRF, 4-701GF-I and insulin) do not show any effect, suggesting that their classical receptors are not involved. In contrast, GnRH secretion is inhibited by other subproducts (1-37GRF, 1-31GF-I and C-peptide) all terminating in a glutamate residue. These subproducts selectively suppress the NMDA-evoked secretion of GnRH. Protease inhibitors prevent the inhibitory effects of IGF-I on GnRH secretion. This, breakdown products of different peptide hormones are possible endogenous antagonists at NMDA receptors. This effect could account for an autocrine or paracrine limitation of NMDA-receptor-mediated secretion of peptides.

Evidence for a hypothalamic site of action of inhibin to suppress FSH release.

To determine if inhibin has a hypothalamic site of action to suppress FSH release, highly purified inhibin preparations from the rete testis fluid (RTF) of rams were injected into adult male rats which had been orchidectomized (ORDX) 24 hours earlier. Third ventricular (3rd V) injection of a potent inhibin fraction (RTF 38-I) significantly depressed plasma FSH concentrations, without influencing LH, 4-10 h after treatment. A less active preparation of inhibin (RTF 38-II) at the same dose had no effect. A higher dose of another less potent fraction (RTF 1A) significantly reduced FSH 2-6 h following 3rd V administration, accompanied by slight but significant decrements in LH at 2 and 4 only. To determine responsiveness of the pituitary, luteinizing hormone-releasing hormone (LHRH) was injected intravenously at 6 h. It induced similar elevations of FSH and LH in inhibin- and saline-treated groups. Systemic administration of RTF 38-II at a dose 2.5-fold higher than the dose effective centrally failed to modify either FSH or LH levels up to 6 h. These results provide evidence that inhibin from the male can preferentially suppress FSH release by a CNS site of action in addition to its well-known pituitary site of action.

Tumor necrosis factor-alpha antagonizes follicle-stimulating hormone action in cultured Sertoli cells.

In the present study, we have tested the effect of tumor necrosis factor-alpha (TNF alpha) on FSH action in cultured purified Sertoli cells isolated from immature porcine testes. FSH action was evaluated through three different parameters (aromatase activity, lactate production, and alpha-inhibin production). TNF alpha was shown to reduce (about 40-60% decrease) FSH-stimulated but not basal aromatase activity (evaluated through the conversion of testosterone into estradiol) in a dose- and time-dependent manner. The maximal and half-maximal (IC50) effects were observed with 6 ng/ml (3.5 x 10(-10) M) and 0.6 ng/ml (3.5 x 10(-11) M), respectively, after a long-term (72 h) treatment. TNF alpha (20 ng/ml) also inhibited Sertoli cell aromatase activity when stimulated with 8-bromo-cAMP (0.01-3 mM, 72 h) instead of FSH, suggesting that the antigonadotropin action of the cytokine is probably exerted at a step located beyond cAMP formation. The inhibitory effect of TNF alpha was not limited to the action of FSH on aromatase activity but also extended to the gonadotropin action on lactate and inhibin-alpha chain production in Sertoli cells. As for FSH-induced aromatase activity, TNF alpha reduced FSH-stimulated lactate accumulation with an IC50 of 0.6 ng/ml, after a long-term (72 h) treatment. Again, the cytokine reduced lactate production stimulated by 8-bromo-cAMP, suggesting that TNF alpha antagonistic action against FSH is exerted at post-cAMP levels. Finally, TNF alpha exerted a more pronounced inhibitory effect (> 90% inhibition) on alpha-inhibin than on inhibin heterodimer production. These inhibitory effects of TNF alpha on the gonadotropin action are probably exerted directly on Sertoli cells, since TNF alpha high affinity binding sites (dissociation constant approximately 5.3 x 10(-10) M) are present in primary cultures of purified porcine Sertoli cells. Altogether, the present findings show that TNF alpha antagonizes FSH action on Sertoli cell functions such as aromatase activity and lactate and alpha-inhibin production. Such an inhibitory effect is probably exerted at a biochemical step(s) located beyond cAMP generation.

Sites of interaction between epidermal growth factor and transforming growth factor-beta 1 in the control of steroidogenesis in cultured porcine Leydig cells.

The present study examines how the hormonal action of gonadotropin is modulated by transforming growth factor-beta 1 (TGF beta 1) and epidermal growth factor (EGF) in primary cultures of purified porcine Leydig cells. Although TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) individually enhanced hCG-stimulated testosterone formation, the effects of EGF were more pronounced than those of TGF beta 1. When studied in combination, the effects of maximal concentrations of TGF beta 1 and EGF were additive on gonadotropin hormonal action. In the present study we demonstrate that their additive effects resulted from a complex interaction occurring at the levels of cholesterol substrate availability in the mitochondria and of 3 beta-hydroxysteroid dehydrogenase/isomerase activity (3 beta HSDI). First, TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) were, respectively, shown to reduce and enhance dehydroepiandrosterone (DHEA) formation (evaluated in the presence of 10(-5) M WIN 24540, an inhibitor of 3 beta HSDI) in Leydig cells when acutely (3 h) stimulated with hCG (0.01-1 ng/ml), but not when incubated with 22R-hydroxycholesterol (3 micrograms/ml). Such findings indicate that TGF beta 1 and EGF did not affect cholesterol side-chain cleavage cytochrome P450 activity, but, respectively, decreased and increased cholesterol substrate availability for this enzyme in the mitochondria. Furthermore, when Leydig cells were treated with the combined factors, the formation of delta 5-steroid intermediates (such as DHEA) in untreated (control) and EGF-plus TGF beta 1-treated cells was not significantly different whether the cells were acutely stimulated with the gonadotropin or incubated with 22R-hydroxycholesterol. Such findings indicate that the effects of EGF and TGF beta 1 on cholesterol substrate availability in the mitochondria are antagonistic. Second, EGF, TGF beta 1, and EGF plus TGF beta 1 significantly (P less than 0.001) increased delta 5-steroid intermediate (i.e. pregnenolone and DHEA), but not delta 4-steroid intermediate (i.e. progesterone and androstenedione), conversion into testosterone, indicating that the growth factors increased, individually or in combination in an additive manner, 3 beta HSDI activity (respectively, 90.7 +/- 0.6%, 80.6 +/- 2.6%, and 164 +/- 4.5% increase in the presence of EGF, TGF beta 1, and EGF plus TGF beta 1). Together, the reciprocal suppression of the effects of TGF beta 1 and EGF on the mitochondrial cholesterol substrate availability coupled to their stimulatory additive actions on 3 beta HSDI activity provide an explanation of the additive actions of the two growth factors on gonadotropin-induced testicular androgen formation.