Chronic nitric oxide deficiency is associated with altered leutinizing hormone and follicle-stimulating hormone release in ovariectomized rats

Nitric oxide (NO) synthase (NOS) has been found in the gonadotrophs and folliculo-stellate cells of the anterior pituitary. Previous observations from our laboratory suggest that NO may play a role in regulating gonadotropin secretion. Because estrogen secretion by the ovary can influence gonadotropin secretion, we investigated the hypothesis that chronic in vivo NO deficiency has a direct estrogen-independent effect on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Chronic NO deficiency was induced by adding an NOS inhibitor, N-nitro-L-arginine (L-NNA, 0.6 g/l) to the drinking water of ovariectomized (OVX) rats. The control OVX rats were untreated. After 6-8 weeks, the animals were sacrificed, and the pituitaries were removed and perfused continuously for 4 hr in the presence of pulsatile gonadotropin-releasing hormone (GnRH, 500 ng/pulse) every 30 min. S-Nitroso-L-acetyl penicillamine (SNAP, an NO donor, 0.1 mM) or L-nitro-arginine methyl ester (L-NAME, an NOS inhibitor, 0.1 mM) was added to the media and perfusate samples were collected at 10-min intervals. GnRH-stimulated LH and FSH levels were significantly lower in pituitaries from OVX/NO-deficient pituitaries compared with pituitaries from the OVX control group. The addition of SNAP significantly decreased LH and FSH secretion by pituitaries from OVX control animals, but significantly increased their secretion by pituitaries from the OVX/NO-deficient animals. L-NAME also suppressed LH and FSH secretion by pituitaries from the OVX control animals and stimulated their release by pituitaries from the NO-deficient/OVX animals. Immunohistochemistry of frontal sections through the hypothalamus demonstrated that OVX/NO deficiency is associated with increased GnRH in the median eminence. We conclude that NO has a chronic stimulatory effect on LH and FSH release and the subsequent altered secretory responsiveness to NO agonist or antagonist is the result of chronic NO suppression.     

Relationships among mammalian gonadotropin-releasing hormone, prostaglandins, and sex steroids in the brain of the crested newt, Triturus carnifex.

The present study was carried out to evaluate the in vitro brain release of prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), androgens, and 17 beta-estradiol in male and female crested newt, Triturus carnifex, during three different periods of the annual sexual cycle; in addition, the effects of mammalian gonadotropin-releasing hormone (mGnRH), PGF2 alpha, and PGE2 on prostaglandins and steroids release by the brain were evaluated during the same periods. In brain incubations of both sexes, PGF2 alpha and estradiol were higher during postreproduction, while PGE2 and androgens were higher during reproduction. In both sexes, mGnRH increased PGF2 alpha and estradiol during postreproduction, and PGE2 during reproduction; PGF2 alpha increased estradiol secretion during postreproduction. Only in the male, did both mGnRH and PGE2 increase androgens during reproduction. It could be suggested that in Triturus carnifex, the regulation of the reproductive activity in the central nervous system (CNS) depends on the relationships among mGnRH, prostaglandins and steroids. In particular, PGF2 alpha and PGE2 seem to play different roles in the CNS of the newt: PGF2 alpha is involved in the postreproductive processes, through estradiol secretion, while PGE2 in the reproductive ones (through androgens secretion?).     

Independent actions of gonadotropin releasing hormone upon cyclic GMP production and luteinizing hormone release

Gonadotropin releasing hormone (GnRH) and its potent analog [D-Ser(tBu)6]des-Gly10-GnRH N-ethylamide elevate pituitary cyclic GMP levels while stimulating gonadotropin release in cultured pituitary cells. Addition of mycophenolic acid to pituitary cell cultures decreased basal and GnRH-induced cGMP production to undetectable levels, but did not reduce basal or GnRH-stimulated luteinizing hormone (LH) release. Elevation of endogenous cGMP levels by sodium nitroprusside, or addition of cGMP or its potent derivatives, was also without effect on basal or GnRH-stimulated LH release. These findings demonstrate that the elevation of intracellular cGMP during GnRH action does not mediate the release of LH by pituitary cells.     

Plasma testosterone and 17 beta-estradiol concentrations, and aromatase activity, during courtship in male Triturus carnifex.

Plasma testosterone and 17 beta-estradiol were monitored during the main phases of male Triturus carnifex courtship. "Inactive" males showed higher levels of testosterone with respect to those found during the various courtship phases. Estradiol was low in "inactive" males, and it reached the highest values at the beginning of the courtship. In addition aromatase activity was higher in the brain of the newts at the beginning of the courtship. These findings seem to support the "aromatization hypothesis" in Triturus carnifex.     

PKC and ERK are differentially involved in gonadotropin-releasing hormone-induced growth hormone gene expression in the goldfish pituitary

Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus and stimulates the synthesis and secretion of gonadotropin hormones. In addition, GnRH also stimulates the production and secretion of growth hormone (GH) in some fish species and in humans with certain clinical disorders. In the goldfish pituitary, GH secretion and gene expression are regulated by two endogenous forms of GnRH known as salmon GnRH and chicken GnRH-II. It is well established that PKC mediates GnRH-stimulated GH secretion in the goldfish pituitary. In contrast, the signal transduction of GnRH-induced GH gene expression has not been elucidated in any model system. In this study, we demonstrate, for the first time, the presence of novel and atypical PKC isoforms in the pituitary of a fish. Moreover, our results indicate that conventional PKC alpha is present selectively in GH-producing cells. Treatment of primary cultures of dispersed goldfish pituitary cells with PKC activators (phorbol ester or diacylglycerol analog) did not affect basal or GnRH-induced GH mRNA levels, and two different inhibitors of PKC (calphostin C and GF109203X) did not reduce the effects of GnRH on GH gene expression. Together, these results suggest that, in contrast to secretion, conventional and novel PKCs are not involved in GnRH-stimulated increases in GH mRNA levels in the goldfish pituitary. Instead, PD98059 inhibited GnRH-induced GH gene expression, suggesting that the ERK signaling pathway is involved. The results presented here provide novel insights into the functional specificity of GnRH-induced signaling and the regulation of GH gene expression.     

Metformin decreases GnRH- and activin-induced gonadotropin secretion in rat pituitary cells: potential involvement of adenosine 5' monophosphate-activated protein kinase (PRKA)

Metformin is an insulin sensitizer molecule used for the treatment of infertility in women with polycystic ovary syndrome and insulin resistance. It modulates the reproductive axis, affecting the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). However, metformin's mechanism of action in pituitary gonadotropin-secreting cells remains unclear. Adenosine 5' monophosphate-activated protein kinase (PRKA) is involved in metformin action in various cell types. Here, we investigated the effects of metformin on gonadotropin secretion in response to activin and GnRH in primary rat pituitary cells (PRP), and studied PRKA in rat pituitary. In PRP, metformin (10 mM) reduced LH and follicle-stimulating hormone (FSH) secretion induced by GnRH (10(-8) M, 3 h), FSH secretion, and mRNA FSHbeta subunit expression induced by activin (10(-8) M, 12 or 24 h). The different subunits of PRKA are expressed in pituitary. In particular, PRKAA1 is detected mainly in gonadotrophs and thyrotrophs, is less abundant in lactotrophs and somatotrophs, and is undetectable in corticotrophs. In PRP, metformin increased phosphorylation of both PRKA and acetyl-CoA carboxylase. Metformin decreased activin-induced SMAD2 phosphorylation and GnRH-induced mitogen-activated protein kinase (MAPK) 3/1 (ERK1/2) phosphorylation. The PRKA inhibitor compound C abolished the effects of metformin on gonadotropin release induced by GnRH and on FSH secretion and Fshb mRNA induced by activin. The adenovirus-mediated production of dominant negative PRKA abolished the effects of metformin on the FSHbeta subunit mRNA and SMAD2 phosphorylation induced by activin and on the MAPK3/1 phosphorylation induced by GnRH. Thus, in rat pituitary cells, metformin decreases gonadotropin secretion and MAPK3/1 phosphorylation induced by GnRH and FSH release, FSHbeta subunit expression, and SMAD2 phosphorylation induced by activin through PRKA activation.     

Melatonin action in neonatal gonadotrophs

Neonatal pituitary cells express MT1 and MT2 subtype of melatonin receptors that are coupled to pertussis toxin-sensitive G proteins. Their activation by melatonin leads to a decrease in cAMP production and activity of protein kinase A, and attenuation of gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion. Single cell calcium and electrophysiological recordings have revealed that a reduction in gonadotropin release results from melatonin-induced inhibition of GnRH-stimulated calcium signaling. Melatonin inhibits both calcium influx through voltage-dependent calcium channels and calcium mobilization from intracellular stores. Inhibition of calcium influx, probably in a cAMP/protein kinase C-dependent manner, and the accompanying calcium-induced calcium release from ryanodine-sensitive intracellular pools by melatonin results in a delay of GnRH-induced calcium signaling. Melatonin-induced attenuation of GnRH-induced and inositol (1,4,5)-trisphosphate-mediated calcium release from intracellular pools attenuates the amplitude of calcium signal. The potent inhibition of GnRH-induced calcium signaling and gonadotropin secretion by melatonin provides an effective mechanism to protect premature initiation of pubertal changes that are dependent on plasma gonadotropin levels. During the development, such tonic inhibitory effects of melatonin on GnRH action gradually decline due to a decrease in expression of functional melatonin receptors. In adult animals, melatonin does not have obvious direct effects on pituitary functions, whereas the connections between melatonin release and hypothalamic functions, including GnRH release, are preserved, and are critically important in synchronizing the external photoperiods and reproductive functions through still not well characterized mechanisms.     

Steroidal modulation of in vitro gonadotropin secretion in prepubertal and adult male Siberian hamsters

The effects of exogenous gonadal steroids, testosterone (T), and 17beta-estradiol (E(2)) upon the hypothalamo-pituitary-gonadal axis were reported to be different between prepubertal and adult Siberian hamsters. Utilizing an in vitro static culture system, we investigated if age-related differences in steroid responsiveness occurs at the pituitary. Prepubertal (20 days old) or adult (140 days old) male Siberian hamsters were implanted with 1 mm silastic capsules containing undiluted T, E(2) or cholesterol (Ch, control). After 15 days, pituitaries were removed, incubated in vitro, and subjected to the following treatments: two baseline measurements, one challenge with 10ng/ml of D-Lys(6)-gonadotropin-releasing hormone (GnRH), and three post-challenge washes. Fractions were collected every 30 minutes and measured for follicle-stimulating hormone (FSH) and luteinizing hormone (LH). T and E(2 )reduced basal secretion of LH and FSH in juveniles but not adults. In juveniles, E(2) increased GnRH-induced FSH and LH secretion, while T augmented GnRH-induced FSH secretion but attenuated GnRH-induced LH secretion. Steroid treatment had no effect on GnRH-stimulated LH or FSH release in adults. The only effect of steroid hormones upon adult pituitaries was the more rapid return of gonadotropin secretion to baseline levels following a GnRH challenge. These data suggest both basal and GnRH-induced gonadotropin secretion are more sensitive to steroid treatment in juvenile hamsters than adults. Further, differential steroidal regulation of FSH and LH at the level of the pituitary in juveniles might be a mechanism for the change in sensitivity to the negative effects of steroid hormones that occurs during the pubertal transition.     

Spontaneous and receptor-controlled soluble guanylyl cyclase activity in anterior pituitary cells

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.     

Contraceptive progestins and gonadotropin secretion in vitro

In an in vitro bioassay using rat pituitary cell cultures the effect of contraceptive progestins was tested on basal and gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in vitro. Progestins diminished gonadotropin release in pituitary cells stimulated with GnRH, but did not alter basal values. This inhibitory effect was dose dependent in a range of 10(-10)-10(-5) M tested and the inhibitory action of most of the progestins examined was more potent than that of progesterone. The maximal reduction of LH and FSH values was by 60% of GnRH-induced control levels. Progestins also caused a shift in sensitivity of cells to GnRh (10(-12)-10(-6) M). When time dependence was investigated, some progestins potentiated GnRH effect on gonadotropins in pituitary cell cultures pre-incubated for a short time (4 h) with steroids. More prolonged pre-incubations from 23 to 71 h resulted in a progressive suppression of LH and FSH response to GnRH (10(-7) M). In order to examine intracellular effects, cells were pretreated with progestins and inositol phosphate metabolism was investigated. The data obtained in pituitary cells give evidence that polyphosphoinositide breakdown is potentially an early step in the action of GnRH on gonadotropin secretion by providing diacylglycerol and inositol phosphates. Addition of gonadotropin-releasing hormone to myo-2[3H]inositol-prelabeled rat pituitary cells in primary culture evoked a dose-dependent increase of the accumulation of [3H]inositol phosphates with a rise of inositol triphosphate, inositol diphosphate and inositol monophosphate within 1 min. Using one contraceptive progestin, gestoden, inositol phosphate production was inhibited by 80% compared to controls of GnRH-treated cells without the addition of steroids. The data obtained in this study suggest that this in vitro bioassay using rat pituitary cells is a useful tool in testing progestational compounds regarding their potency on gonadotropin release. In addition, these results show that one possible site of interference of progestins with GnRH-induced gonadotropin secretion may involve polyphosphoinositide breakdown.     

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.     

Pituitary adenylate cyclase-activating polypeptide stimulates nitric-oxide synthase type I expression and potentiates the cGMP response to gonadotropin-releasing hormone of rat pituitary gonadotrophs

Nitric-oxide synthase type I (NOS I) is expressed primarily in gonadotrophs and in folliculo-stellate cells of the anterior pituitary. In gonadotrophs, the expression and the activity of NOS I are stimulated by gonadotropin-releasing hormone (GnRH) under both experimental and physiological conditions. In the present study, we show that pituitary adenylate cyclase-activating polypeptide (PACAP) is twice as potent as GnRH at increasing NOS I levels in cultured rat anterior pituitary cells. The action of PACAP is detectable after 4-6 h and maximal at 24 h, this effect is mimicked by 8-bromo-cAMP and cholera toxin and suppressed by H89 suggesting a mediation through the cAMP pathway. Surprisingly, NADPH diaphorase staining revealed that these changes occurred in gonadotrophs exclusively although PACAP and cAMP, in contrast to GnRH, have the potential to target several types of pituitary cells including folliculo-stellate cells. There was no measurable alteration in NOS I mRNA levels after cAMP or PACAP induction. PACAP also stimulated cGMP synthesis, which was maximal within 15 min and independent of cAMP, however, only part resulted from NOS I/soluble guanylate cyclase activation implying that in contrast to GnRH, PACAP has a dual mechanism in cGMP production. Interestingly, induction of NOS I by PACAP markedly enhanced the capacity of gonadotrophs to produce cGMP in response to GnRH. The fact that PACAP may act on gonadotrophs to alter NOS I levels, generate cGMP, and potentiate the cGMP response to GnRH, suggests that cGMP could play important cellular functions.     

Developmental programming: impact of prenatal testosterone excess on pre- and postnatal gonadotropin regulation in sheep

The goal of this study was to explore mechanisms that mediate hypersecretion of LH and progressive loss of cyclicity in female sheep exposed during fetal life to excess testosterone. Our working hypothesis was that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH (but not FSH) secretion and, thus, hypersecretion of LH in adulthood, and that this results from altered developmental gene expression of GnRH and estradiol (E2) receptors, gonadotropin subunits, and paracrine factors that differentially regulate LH and FSH synthesis. We observed that, relative to controls, females exposed during fetal life to excess testosterone, as well as the nor-aromatizable androgen dihydrotestosterone, exhibited enhanced LH but not FSH responses to intermittent delivery of GnRH boluses under conditions in which endogenous LH (GnRH) pulses were suppressed. Luteinizing hormone hypersecretion was more evident in adults than in prepubertal females, and it was associated with development of acyclicity. Measurement of pituitary mRNA concentrations revealed that prenatal testosterone excess induced developmental changes in gene expression of pituitary GnRH and E2 receptors and paracrine modulators of LH and FSH synthesis in a manner consistent with subsequent amplification of LH release. Together, this series of studies suggests that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH response, leading to LH hypersecretion and acyclicity in adulthood, and that this programming involves developmental changes in expression of pituitary genes involved in LH and FSH release.     

Dependence of secretory responses to gonadotropin-releasing hormone on diacylglycerol metabolism. Studies with a diacylglycerol lipase inhibitor, RHC 80267

The role of diacylglycerol (DG) as a source of arachidonic acid during gonadotropin-releasing hormone (GnRH) stimulation of gonadotropin secretion was analyzed in primary cultures of rat anterior pituitary cells. An inhibitor of DG lipase (RHC 80267, RHC) caused dose-dependent blockade of GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The DG lipase inhibitor did not alter gonadotropin responses to arachidonic acid, and addition of arachidonic acid reversed its inhibition of GnRH-stimulated LH and FSH release. In [3H]arachidonic acid-prelabeled cells, incubation with RHC increased the accumulation of [3H]DG. These results suggest that DG lipase participates in GnRH action and that arachidonic acid mobilization from DG is involved in the mechanism of gonadotropin release. Gonadotropin responses to tetradecanoyl phorbol acetate and dioctanoyl glycerol were not altered by RHC, and the addition of these activators of protein kinase C (Ca2+- and phospholipid-dependent enzyme) did not prevent the inhibition of GnRH-induced gonadotropin release by RHC. Activation of phospholipase A2 by melittin increased LH and FSH secretion, whereas blockade of this enzyme by quinacrine reduced GnRH-stimulated hormone release. However, RHC did not diminish the gonadotropin response to melittin. The inhibitory actions of RHC and quinacrine were additive and were reversed by concomitant treatment with arachidonic acid. Ionomycin also increased LH and FSH release, and the gonadotropin responses to the ionophore were unaltered by RHC but were reduced by quinacrine. Incubation of cells in Ca2+-depleted (+/- [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) medium reduced but did not abolish the LH and FSH releasing activity of GnRH. Treatment with RHC also reduced the gonadotropin responses to GnRH under Ca2+-depleted conditions. These observations indicate that RHC inhibition of GnRH action is not due to nonspecific actions on Ca2+ entry, protein kinase C activation and actions, nor phospholipase A2 enzyme activity. The results of this study provide further evidence for an extracellular Ca2+-independent mechanism of GnRH action, and suggest that GnRH causes mobilization of arachidonic acid by two distinct lipases, namely, phospholipase A2 and DG lipase, during stimulation of gonadotropin secretion.     

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.     

Leptin effect on nitric oxide and GnRH-induced FSH secretion from ovine pituitary cells in vitro.

The secretion of gonadotrophins from anterior pituitary cells can be modulated by leptin and signals originating from the immune system, among others, by nitric oxide (NO). There are some studies that have demonstrated a role for leptin and NO in the regulation of FSH in rodents, however, no similar data are available in regards to ewes. Therefore, the objective of the present study was to analyse the leptin effect on GnRH-induced FSH secretion from the ovine anterior pituitary cells in vitro. Additionally, the influence of leptin on NO release and its role in the GnRH and leptin-modulated secretion of FSH from pituitary gland of ewes was investigated. The obtained results show that the influence of leptin on FSH secretion is biphasic. Leptin in concentration 10(-8) and 10(-7) M/l significantly enhances, whereas 10(-6) and 10(-5) M/l of leptin suppresses FSH secretion from the pituitary cells in comparison to the control. The secretion of FSH and NO release under the influence of leptin are in very high positive correlation (r=0.77). The inhibition of NO synthesis with L-NAME., instead, disables leptin from the stimulation of FSH secretion.     

Effect of cortisol or adrenocorticotropic hormone on luteinizing hormone secretion by pig pituitary cells in vitro

The effect of cortisol or adrenocorticotropic hormone (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-induced secretion of luteinizing hormone (LH) was studied in vitro using dispersed pig pituitary cells. Pig pituitary cells were dispersed with collagenase and DNAase and then grown in McCoy's 5a medium containing 10% dextran charcoal-pretreated horse serum and 2.5% fetal calf serum for 3 days. Cells were preincubated with cortisol or ACTH before GnRH was added. When pituitary cells were incubated with 400 micrograms cortisol/ml medium for 6 h or longer, increase basal secretion of LH was observed. However, GnRH-induced LH release was reduced by cortisol. The degree of this reduction was dependent on cortisol, and a concentration of cortisol higher than 100 micrograms/ml was needed. Cortisol also inhibited the 17 beta-estradiol-induced increase in GnRH response. ACTH-(1-24), ACTH-(1-39), or porcine ACTH had no influence on GnRH-induced LH secretion. Our results show that cortisol can act directly on pig pituitary to inhibit both normal and estradiol-sensitized LH responsiveness to GnRH.     

PGF2 alpha, PGE2, and sex steroids from the abdominal gland of the male crested newt Triturus carnifex (Laur.).

Prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), progesterone, androgens, and 17 beta-estradiol in vitro release by the abdominal gland of the crested newt, Triturus carnifex (Laur.), was studied during the prereproductive, reproductive and postreproductive periods. In addition, the in vitro effects of the PGF2 alpha and/or PGE2 on progesterone, androgens and estradiol release by the abdominal gland were evaluated. PGF2 alpha, PGE2 and progesterone release was higher during the reproductive period, and in the same period, PGE2 treatment induced a progesterone increase. PGF2 alpha induced an increase of abdominal gland estradiol release at the end of the reproductive period. These results seemed to confirm the pheromonal role assigned to progesterone, and suggested a PGE2 stimulatory role in inducing progesterone release, even if pheromonal activity of PGF2 alpha and PGE2 cannot be excluded. In addition, PGF2 alpha-dependent estradiol increase at the end of reproduction could be interpreted as a mechanism for interruption of the abdominal gland activity.     

Gonadotropin releasing hormone activates the lipoxygenase pathway in cultured pituitary cells: role in gonadotropin secretion and evidence for a novel autocrine/paracrine loop.

The formation and role of arachidonic acid (AA) and its metabolites during gonadotropin releasing hormone- (GnRH-) induced gonadotropin secretion were investigated in primary cultures of rat pituitary cells. Prelabeled cells ([3H]AA) responded to GnRH challenge with increased formation (about 2-fold) of the leukotrienes LTC4, LTD4, and LTE4 as well as 5- and 15-eicosatetraenoic acids (5- and 15-HETE) as identified by HPLC. Formation of leukotrienes and 15-HETE was further verified by specific radioimmunoassays. No significant increase in the formation of 12-HETE or of the cyclooxygenase products prostaglandin E (PGE) and thromboxane A2 by GnRH was noticed. Addition of physiological concentrations of LTC4 enhanced basal LH release, while subphysiological concentrations of LTC4 (10(-15)-10(-12) M) inhibited GnRH-induced LH release by about 35% (p less than 0.02). Using specific lipoxygenase inhibitors L-656,224 and MK 886, we found inhibition of GnRH-induced LH release by about 40% at concentrations known to specifically inhibit the 5-lipoxygenase pathway. The peptidoleukotriene receptor antagonist ICI 198,615 inhibited LTC4- and LTE4-induced LH release and surprisingly also the effect of GnRH on LH release by 40%. The data strongly suggest a role for AA and its lipoxygenase metabolites in the on/off reactions of GnRH upon LH release. The data also present a novel amplification cycle in which newly formed leukotrienes become first messengers and establish an autocrine/paracrine loop.