The frequency of gonadotropin-releasing-hormone stimulation differentially regulates gonadotropin subunit messenger ribonucleic acid expression

The hypothalamic decapeptide GnRH is known to regulate the synthesis and secretion of LH and FSH by pituitary gonadotrope cells. The frequency of pulsatile GnRH secretion changes and LH and FSH are differentially secreted in various physiological situations. To investigate the potential role of altered frequency of GnRH stimulation in regulating differential secretion of LH and FSH, we examined the effects of GnRH frequency on expression of the alpha, LH beta, and FSH beta genes. GnRH pulses (25 ng/pulse) were administered to castrate testosterone-replaced rats at intervals of 8-480 min to cover the range of physiological pulsatile GnRH secretion. Fast frequency GnRH pulses (8-min pulse intervals) increased alpha-subunit mRNA concentrations 3-fold above those in saline-pulsed controls (controls, 1.01 fmol cDNA bound/100 micrograms pituitary DNA) and LH beta mRNA by 50% (controls, 0.18 fmol cDNA bound), but FSH beta mRNA was unchanged (controls, 0.38 fmol cDNA bound). GnRH pulses given every 30 min increased all three subunit mRNAs (alpha, 3-fold, LHbeta, 2-fold; FSH beta, 2-fold), and acute LH release and serum FSH concentrations were maximal after this frequency. Slower frequency GnRH stimuli (120- to 480-min pulse intervals) did not change alpha and LH beta mRNA levels, but increased FSH beta mRNA 2- to 2.5-fold, and FSH secretion was maintained. Equalization of the total dose of GnRH given at different intervals over 24 h confirmed the frequency dependence of subunit mRNA expression. Fast frequency GnRH stimuli (8 min) increased alpha mRNA 1.5- to 2.5-fold, while the same total GnRH doses were ineffective when given at slow frequency (480 min). Similarly, LH beta mRNA was only increased by GnRH pulses given at 8-min intervals. In contrast, FSH beta mRNA increased 2-fold after pulses given every 480 min, and the 8-min pulse interval was ineffective. The data show that the frequency of GnRH stimulation can differentially regulate gonadotropin subunit mRNA expression and may be a mechanism that enables a single GnRH peptide to selectively regulate gonadotropin subunit gene expression and hormone secretion.     

Effect of gonadotropin-releasing hormone pulse frequency on gonadotropin secretion and subunit messenger ribonucleic acids in perifused pituitary cells.

Slow frequency GnRH pulses have been proposed to preferentially increase circulating FSH levels by increasing FSH synthesis and pulsatile release. Examination of this proposal using various in vivo models, however, has produced conflicting results. To examine directly the effects of GnRH pulse frequency on the pituitary, we compared the effects of 2.5-nM GnRH pulses administered every 1 h or every 4 h vs. no GnRH, using perifused rat pituitary cells. FSH secretion (total area under the response curve) was 2-fold greater (P less than 0.01) with every hour than with every 4 h GnRH pulses. This difference resulted from the increased number of GnRH pulses and increased (P less than 0.05) interpulse FSH secretion, whereas FSH pulse amplitude was unchanged. FSH beta mRNA levels at the completion of the 11-h perifusion were increased 4.5-fold by GnRH every h (P less than 0.01) and 3.3-fold by GnRH every 4 h (P less than 0.05) above levels in untreated cells. FSH beta mRNA levels were greater (P less than 0.05) at the faster GnRH pulse frequency. Because more frequent stimulation delivered more GnRH during the study, cells were next stimulated with 2.5 nM GnRH every 1 h for nine pulses, 7.5 nM GnRH every 4 h for three pulses to equalize the GnRH dose, or 2.5 nM GnRH every 4 h for three pulses. Interpulse FSH secretion and FSH beta mRNA levels were again greater (P less than 0.05) with every hour than every 4 h GnRH pulses. Interpulse LH secretion, FSH and LH pulse amplitude, and LH beta and alpha-subunit mRNA levels were not different between the groups. GnRH doses of 0.1-10 nM every hour increased FSH and LH pulsatile secretion dose-dependently, but FSH beta, LH beta, and alpha-subunit mRNA levels were similar. In conclusion, our data reveal that reducing the frequency of GnRH pulses from every hour to every 4 h reduces both FSH beta mRNA levels and FSH interpulse secretion, but does not change GnRH-stimulated FSH pulsatile release. We suggest that the finding by others that slow frequency GnRH pulses increase circulating FSH levels under certain experimental conditions in vivo may instead be explained by complex hormonal interactions or changes in FSH clearance.     

Effects of testosterone on gonadotropin subunit messenger ribonucleic acids in the presence or absence of gonadotropin-releasing hormone.

There is accumulating evidence that the negative feedback actions of testosterone on the pituitary may contribute to the differential regulation of FSH and LH secretion in males. In the present study we measured steady state levels of the mRNAs encoding the gonadotropin subunits in pituitary cell cultures treated with 10 nM testosterone (T) as well as in T-treated pituitary cells perifused with pulses of GnRH to explore further the direct actions of T on the pituitary. T treatment of pituitary cells in monolayer culture for 72 h increased FSH beta mRNA 1.5-fold (P less than 0.05), decreased alpha-subunit mRNA to 45% of the control level (P less than 0.05), and decreased LH beta mRNA to 75% of the control level (P less than 0.05). FSH and uncombined alpha-subunit secretion were increased and decreased by T, respectively, whereas basal LH secretion was unchanged. Treatment with 0.1 nM estradiol, a physiological concentration for males, did not change gonadotropin secretion or subunit mRNA concentrations. Between days 2 and 5 in culture in the absence of steroid treatment, steady state levels of LH beta and alpha-subunit mRNA declined (P less than 0.01) 52% and 61%, respectively, but FSH beta mRNA levels were unchanged. Pulsatile stimulation with 2.5 nM GnRH every 1 h for 10 h increased FSH beta mRNA 2.8-fold (P less than 0.05) and increased (P less than 0.05) alpha-subunit mRNA to 117% of the control level. When cell cultures were pretreated with T for 48 h and then perifused with pulses of GnRH, FSH beta, LH beta, and alpha-subunit mRNA levels were 66%, 74%, and 70% of the value during GnRH alone (P less than 0.05). T treatment also reduced (P less than 0.01) the amplitudes of FSH, LH, and alpha-subunit secretory pulses by 18%, 26%, and 41%, respectively. These data indicate that a portion of the negative feedback action of T is at the pituitary to regulate gonadotropin subunit gene expression. Our data reveal two opposing effects of T on FSH beta mRNA: a stimulatory action, which is GnRH independent, and an inhibitory effect, which is related to the actions of GnRH. These divergent actions of T represent one mechanism through which FSH and LH are differentially regulated.     

Gonadal regulation of gonadotropin subunit gene expression: evidence for regulation of follicle-stimulating hormone-beta messenger ribonucleic acid by nonsteroidal hormones in female rats

Gonadectomy results in a rise in gonadotropin secretion and subunit gene expression, although the relative contributions of declining gonadal hormones or increasing hypothalamic GnRH secretion are uncertain. To further delineate the roles of the hypothalamus and gonads in regulation of gonadotropin gene expression, male and female rats were castrated and gonadotropin subunit messenger RNA (mRNA) concentrations measured 2, 7, 14, or 21 days (d) later. In males, FSH beta mRNA was maximal (2-fold increase) by 7 d while peak levels of alpha (3-fold) and LH beta (3-fold) were seen by 14 d. Testosterone (T) replacement restored all three subunit mRNA concentrations to intact values. In females, FSH beta mRNA also reached plateau levels (8-fold increase) earlier than alpha (3-fold) or LH beta (11-fold). When female rats ovariectomized 7 days earlier were given estradiol (E2) and progesterone (P) implants for up to 14 d, suppression of alpha and LH beta to intact levels was observed. However, FSH beta mRNA concentrations only decreased to 67% of castrate values, and remained 2- to 3-fold higher than levels in intact female rats. Female rats were also given E2 replacement at the time of ovariectomy. LH beta mRNA was maintained at intact levels for 14 days while alpha and FSH beta showed partial castration responses (2-fold and 3-fold, respectively). Finally, to determine whether E2 and P regulate gonadotropin subunit expression directly or by reducing GnRH secretion, female rats were ovariectomized and immediately replaced with E2, P, or E2 + P in the presence or absence of a GnRH antagonist (A) for 2 d. alpha mRNA was increased (2-fold) by E2 but not by E2 + A suggesting that E2 requires the presence of GnRH to increase alpha mRNA. P alone was ineffective, but both E2 and A prevented the LH beta mRNA response to ovariectomy. The effects of E2 and A were not additive, suggesting that E suppresses LH beta mRNA by inhibiting the increase in GnRH secretion. In contrast, the FSH beta mRNA response to ovariectomy was only partially suppressed by E2, E2 + P, or E2 + P + A. These data indicate that in castrate males, replacement of T suppresses all three subunit mRNAs to intact levels. However, replacement of E2 to ovariectomized females did not prevent the increase in alpha and FSH beta mRNAs. In female rats, LH beta mRNA is predominantly regulated by GnRH. alpha mRNA expression is also mainly regulated by GnRH, and E2 appears to augment GnRH action on alpha mRNA expression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gonadotropin-releasing hormone is required for enhanced luteinizing hormone subunit gene expression in vivo

Pre- and postcastration changes in LH beta and common alpha mRNAs were correlated with pituitary and serum LH levels in two different species after abolition of pituitary stimulation by GnRH. A GnRH antagonist (GnRH-ANT) was used to block gonadotroph GnRH receptors in male rats, and a GnRH antiserum (GnRH-AS) was used to inhibit GnRH stimulation of female and male mouse and male rat pituitaries. The postcastration increases in LH beta and common alpha mRNA levels (2- and 3.5-fold, respectively) were abolished in male rats after 7 days of continuous GnRH-ANT infusion. The postcastration increases in LH beta and common alpha mRNA in female (1.9- and 2.2-fold respectively) and male mice (1.4- and 3.6-fold, respectively) were also prevented after daily sc injection of GnRH-AS, as were the rises in LH beta (3-fold) and common alpha (4-fold) in castrated male rats. The pituitary LH content (postgonadectomy) was no different from intact control levels in all experimental animals regardless of treatment, while the increase in serum LH concentration in rats (7- and 8-fold) and in female (4.8-fold) and male mice (9.8-fold) was prevented by both GnRH-ANT and GnRH-AS administration. In intact rats treated with GnRH-ANT the LH beta mRNA level decreased (57%) while the common alpha mRNA level was unaffected after 7 days. Neither pituitary nor serum LH levels were altered in intact rats or mice after appropriate treatments. We conclude that endogenous GnRH is required for the postcastration rise of both LH beta and common alpha-subunit mRNA levels in rats and mice.     

Simultaneous effect of gonadotropin-releasing hormone (GnRH) on the expression of two gonadotropin beta genes by passive immunization to GnRH

In order to reveal the action of gonadotropin-releasing hormone (GnRH) on the synthesis of gonadotropins in the pituitary gland of castrated rats, passive immunization to GnRH designed to block the activity of GnRH was performed. The levels of prolactin mRNA in castrated and rabbit anti-GnRH serum (RAGnRH)-treated rats decreased, whereas TSH beta mRNA showed no statistically significant change. In contrast, mRNAs encoding common alpha, LH beta and FSH beta were increased 2.7-, 1.7- and 1.5-fold, respectively, by castration. These elevated mRNA levels of gonadotropin subunits in castrated rats well explain the increased hormone levels in serum and in the pituitary. Two days later, a single administration of RAGnRH to the castrated rats significantly suppressed the mRNA levels to 2.0-fold for alpha, 1.2-fold for LH beta and 1.1-fold for FSH beta relative to the respective control values. These results showed that the two gonadotropin beta genes respond more rapidly to GnRH action that the common alpha gene.     

Testosterone differentially modulates gonadotropin subunit messenger ribonucleic acid responses to gonadotropin-releasing hormone pulse amplitude.

Testosterone (T) inhibits GnRH secretion and can also modulate the effects of GnRH on gonadotropin synthesis and secretion. To assess the effect of T on GnRH stimulation of alpha, LH beta, and FSH beta mRNA expression, we replaced T at three levels to reproduce low (1.5 +/- 0.5 ng/ml), medium (3.5 +/- 0.3 ng/ml), and high (6.2 +/- 0.6 ng/ml) physiological plasma concentrations. Additionally, as peripheral conversion to dihydrotestosterone (DHT) or estradiol (E2) may mediate T action, the effects of GnRH pulses in the presence of DHT and E2 were also studied. Male rats were castrated, and steroids were replaced via implants containing either T (three doses) or DHT or E2 (two doses each). GnRH pulses (10-250 ng/pulse) were administered iv at 30-min intervals for 48 h. Pituitary subunit mRNA concentrations, gonadotropin content, and LH and FSH secretion were determined. The patterns of alpha, LH beta, and FSH beta mRNA responses to increasing GnRH pulse amplitude were similar at all concentrations of plasma T. Alpha mRNA concentrations were increased 2- to 4-fold by GnRH pulses. At the same plasma T concentration, all doses of GnRH produced similar increases in alpha mRNA, but the response tended to be lower at the higher (6.2 ng/ml) levels of T. LH beta mRNA showed a clear dependence on GnRH pulse amplitude, with the maximum responses (2- to 3-fold) occurring after 10- to 25-ng GnRH pulses. At the higher (3.5 and 6.2 ng/ml) T concentrations, the dose-response curve was shifted to the left. The lowest GnRH pulse dose (10 ng) produced maximum responses, and LH beta mRNA increments in response to the higher GnRH doses were suppressed. FSH beta mRNA concentrations were increased by T in saline-pulsed controls. FSH beta mRNA responses were similar (2- to 3-fold) after all GnRH doses and at all concentrations of T. Increasing GnRH pulse doses reduced the pituitary content of both LH and FSH at all levels of T. Acute LH secretion was maximal after 10- and 25-ng pulses of GnRH when plasma T was low, but increased progressively with GnRH dose at the highest plasma T concentrations. Plasma FSH did not show any differential responsiveness to GnRH pulse dose or to increasing plasma T. Thus, LH synthesis and secretion are affected more than those of FSH by changing plasma concentrations of T. T may modulate posttranslational events in LH secretion. The higher GnRH doses effected LH release without increasing LH beta mRNA in the presence of higher physiological concentrations of T.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of recombinant human inhibin and testosterone on gonadotropin secretion and subunit mRNA in superfused male rat pituitary cell cultures stimulated with pulsatile gonadotropin-releasing hormone.

Effects of recombinant human inhibin (rh inhibin) and testosterone on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion and mRNA levels of gonadotropin subunits were investigated in superfused male rat pituitary cell cultures. During superfusion, the cells were stimulated with gonadotropin-releasing hormone (GnRH) pulses (10 nM, 6 min/h) and exposed to rh inhibin (2 ng/ml) and/or testosterone (10 nM) for up to 20 h. The concentrations of FSH and LH were measured in effluent media by radioimmunoassay (RIA), and subunit mRNAs were determined by Northern blot hybridizations using rat FSH beta, LH beta and alpha genomic and cDNA probes. Rh inhibin suppressed the secretion of FSH (30-40% of control) and the secretion of LH to 50-60% of control, but inhibited only FSH beta mRNA (to non-detectable levels). Testosterone alone suppressed the release of LH to 50% of control, whereas FSH release was increased to 130-160% (P less than 0.05) of control. This increase was due to higher interpulse values without significant changes in the pulse amplitude. Also FSH beta mRNA level was increased (1.5-fold, P less than 0.05) but only after 17-20 h of treatment. On the other hand, testosterone had no effect on LH beta and alpha subunit mRNA levels. Testosterone in combination with rh inhibin showed an inhibitory effect on LH beta mRNA; however, the pattern of LH release was not significantly different from that observed with rh inhibin or testosterone alone. Combined effects of testosterone and rh inhibin on FSH secretion and FSH beta mRNA were similar to those observed with rh inhibin alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone regulates follicle-stimulating hormone beta-subunit gene expression in the male rat

Since the role of GnRH in the control of FSH release and synthesis is controversial, we have examined the effect of elimination of GnRH action on gonadotropes on FSH beta gene expression, FSH release, and synthesis. GnRH stimulation of the pituitary was abolished by continuous infusion of either a GnRH antagonist or a GnRH antiserum. We also examined the effects of gonadotrope desensitization, using a continuous infusion of GnRH or GnRH agonist analog. FSH beta mRNA levels were determined by dot blot hybridization using rat FSH beta cDNA, and changes were related to pituitary and serum FSH concentrations. FSH beta mRNA levels increased after orchidectomy and correlated well with serum FSH concentrations. Overall FSH synthesis was increased after castration, as judged by elevated serum FSH and unchanged pituitary FSH content. In orchidectomized rats, continuous GnRH antagonist infusion prevented the postcastration rise in FSH beta mRNA levels and serum FSH. Pituitary FSH content was reduced at 7 days, but not at 14 days. In intact rats, GnRH antagonist infusion for 7 days had no effect on FSH beta mRNA levels, but after 14 days, there was a 33% reduction, and serum FSH was suppressed. Pituitary FSH content was decreased after GnRH antagonist treatment for 7 or 14 days. Daily injection of GnRH antiserum for 6 days abolished the increases in FSH beta mRNA levels and serum FSH in orchidectomized rats, but pituitary FSH content was unaffected. In intact rats, GnRH antiserum treatment reduced FSH beta mRNA levels by 38%, suppressed serum FSH, and decreased pituitary FSH content. When gonadotropes were desensitized by a continuous infusion of GnRH for 14 days or GnRH agonist analog for 28 days, FSH beta mRNA levels and pituitary FSH content were markedly reduced, and serum FSH was suppressed to undetectable levels. We concluded that 1) endogenous GnRH is required for the maintenance of FSH beta mRNA levels in both intact and orchidectomized rats; 2) FSH beta mRNA levels are coupled to the level of FSH biosynthesis, indicating the physiological importance of this pretranslational regulation; 3) desensitization is more effective at inhibiting FSH beta gene expression and FSH synthesis than preventing gonadotrope stimulation using the GnRH antagonist or antiserum; and 4) the actions of GnRH on FSH beta mRNA levels are paralleled by its effects on LH beta mRNA levels, suggesting that GnRH provides a common primary stimulus for the induction of both beta-subunit genes in vivo. These data provide further evidence for the crucial stimulatory role of GnRH in the control of FSH synthesis.     

Pituitary follistatin and activin gene expression, and the testicular regulation of FSH in the adult Rhesus monkey (Macaca mulatta).

In rats, FSHbeta gene expression and FSH secretion are increased and decreased, respectively, by pituitary activin and follistatin. Because little information is available on the paracrine control of FSH secretion in the primate, follistatin and activin/inhibin beta(B) messenger RNA (mRNA) levels were measured in pituitaries of adult male rhesus monkeys 6 weeks after castration or sham surgery (n = 5/group). Follistatin mRNA was determined by quantitative RT-PCR assay using oligonucleotide primers designed to span exons 3-5 of the human follistatin gene. Activin/inhibin beta(B) mRNA levels were measured by ribonuclease protection. Orchidectomy resulted in a 100-fold increase in plasma FSH concentrations and a 60-fold rise in those of LH. In castrated monkeys, levels of mRNA encoding FSHbeta, LHbeta, alpha- subunit, and GnRH receptor (GnRH-R) were increased 21-, 2.1-, 1.7-, and 1.7-fold, respectively (P < 0.01). Levels of pituitary follistatin and activin/inhibin beta(B) mRNAs, however, were similar in castrated and intact animals. These data suggest that the paracrine control of FSH secretion in the male differs substantially in primates and rodents. Specifically, the relatively greater postcastration rise in FSHbeta gene expression and FSH secretion in the adult male monkey may result because in this species pituitary follistatin gene expression does not increase after orchidectomy, as it does in the rat.     

Inhibition of pituitary gonadotropin secretion by the gonadotropin-releasing hormone antagonist antide. I. In vitro studies on mechanism of action

The GnRH antagonist antide is among the most promising "third generation" compounds available for clinical evaluation. In primates, antide manifests prolonged (several weeks) and reversible inhibition of pituitary gonadotropin secretion after a single high dose injection. In the present study, we have examined the effects of antide on pituitary gonadotropin secretion in vitro. Dispersed anterior pituitary cells from adult female rats were plated (48 h; 5 x 10(5) cells/well), washed, and exposed to increasing concentrations of antide for up to 48 h. Media were removed, and cells were washed twice and then incubated with GnRH (1 x 10(-8) M) plus antide for 4 h. Media and cell lysates were assayed for LH/FSH by RIA. Antide had no effect on basal LH/FSH secretion at any dose tested (10(-6)-10(-12) M). In contrast, GnRH-stimulated LH/FSH secretion was inhibited by this GnRH antagonist in a dose- and time-dependent manner. When incubated simultaneously, antide blocked GnRH-stimulated gonadotropin secretion, with a maximal effect at 10(-6) M (ED50, 10(-7) M). Preincubation of pituitary cells with antide for 6-48 h before GnRH exposure shifted the dose-response curve to the left; the maximally effective dose was 10(-8) M; the ED50 was 10(-10) M antide after 48-h preincubation. Intracellular LH/FSH levels increased concomitant with the decrease in secreted gonadotropins. Total LH/FSH levels (secreted plus cell content) remained unchanged. The inhibition of LH secretion by antide was specific for GnRH-stimulated gonadotropin secretion; antide had no effect on K(+)-stimulated LH secretion. Moreover, antide had little or no residual effect on LH secretion; full recovery of GnRH responsiveness in vitro occurred within 4 h after removal of antide. Lineweaver-Burke analysis of antide inhibition of GnRH-stimulated LH secretion indicated that antide is a direct competitor of GnRH at the level of the pituitary GnRH receptor. In summary, antide is a pure antagonist of GnRH stimulation of gonadotropin secretion; no agonistic actions of antide were manifest in vitro. Moreover, antide has no apparent noxious or toxic effect on pituitary cells in culture; the actions of antide are immediately reversible upon removal of antide from pituitary gonadotropes. We conclude that the long term inhibition of gonadotropin secretion by antide in vivo is not due to deleterious effects of this compound at the level of the pituitary gonadotrope.     

The effects of bromocriptine, thyrotropin-releasing hormone, and gonadotropin-releasing hormone on hormone secretion by gonadotropin-secreting pituitary adenomas in vivo and in vitro

The characteristics and dynamics of hormone secretion in vivo and in vitro were investigated in six patients with gonadotropin-secreting pituitary adenomas. All six tumors secreted and contained FSH and different combinations of LH, beta-LH, and alpha-subunit. In addition, immunohistochemical examination of the pituitary tumor tissue showed staining with both LH and FSH in three and either LH or FSH in the other three tumors. TRH and GnRH stimulated hormone secretion in vivo and in vitro, and they also increased the hormone content of the cultured tumor cells. Bromocriptine significantly inhibited hormone release and reduced the hormone content of the tumor cells. In vivo, 2.5 mg bromocriptine significantly suppressed plasma hormone levels; the inhibiting effect on alpha-subunit concentrations was in general more marked than that on LH and FSH. We conclude that hormone release by gonadotropin-secreting pituitary adenomas can be stimulated by TRH and GnRH and inhibited by bromocriptine. Most of these tumors synthesize FSH, but there is a wide variation in the production of LH, beta-LH, and alpha-subunits. The sensitivity of hormone release to bromocriptine suggests that chronic therapy with this drug might have a beneficial effect on pituitary tumor size.     

Differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone pulse amplitude in female rats

The role of GnRH in regulating gonadotropin subunit gene expression was examined in adult female rats. Animals were ovariectomized, estradiol implants inserted sc, and jugular cannulae placed into the right atria. On the next day, animals were given GnRH pulses (saline to controls) every 30 min for up to 48 h and alpha, LH beta, and FSH beta mRNA levels measured by hybridization to cDNA probes. To determine the effects of GnRH treatment duration, rats received GnRH pulses (25 ng at 30-min intervals) for 6, 12, 24, and 48 h. FSH beta mRNA was increased (by 92%) after 6 h of pulses and remained elevated through 48 h. alpha mRNA was not increased until 12 h (27% increase) and rose further (57%) by 48 h. LH beta mRNA levels were only transiently increased at 12 h (67%) and values were not different from saline controls after 24 or 48 h. To examine whether the rise in serum PRL which is characteristic of the ovariectomized-estradiol animal model was responsible for the decrease in LH beta mRNA responsiveness to GnRH over longer durations, studies were repeated in bromocriptine-treated animals (0.6 mg sc, twice daily). The results showed similar response patterns for all three subunit mRNAs including the decrease in LH beta after 48 h. A third experiment examined the effect of varying GnRH pulse amplitude (0.5-250 ng/pulse at 30-min intervals) over 12 h. alpha mRNA levels were increased by all GnRH doses greater than 5 ng with maximum responses after 250 ng pulses. LH and FSH beta mRNAs were both elevated by GnRH pulse doses of 0.5-25 ng (P less than 0.05 vs. saline controls). Maximal increases (2-fold) were seen after 5 ng pulses for LH beta and after 15-ng pulses for FSH beta mRNA. These results show that pulsatile GnRH increases FSH beta mRNA more rapidly than alpha or LH beta mRNAs in female rats. In addition, high amplitude GnRH pulses increase only alpha mRNA, whereas both LH beta and FSH beta mRNAs show maximum responses to lower doses. The data suggest that alterations in the amplitude of the GnRH pulsatile signal can exert differential effects on gonadotropin gene expression.     

Chronic inhibitory effect of follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin on activin- and gonadotropin-releasing hormone-stimulated FSH synthesis and secretion in cultured rat anterior pituitary cells

The effects of bovine FSH-suppressing protein (FSP) or follistatin on activin- and GnRH-stimulated FSH synthesis and secretion have been studied using cultured pituitary cells from adult male Sprague-Dawley rats. Exposure to FSP (0.001-10 nM) for 3 days dose-dependently suppressed basal FSH secretion (IC50 = 146 +/- 21 pM., mean +/- SE), cellular content (IC50 = 269 +/- 8 pM) and total FSH (IC50 = 181 +/- 25 pM), with no effect on LH. Activin (0.3 nM) increased FSH secretion 2.1-fold, cellular content 1.3-fold, and total FSH 1.9-fold during a 3-day incubation, but these increases were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP (0.1-3 nM), with complete inhibition occurring at concentrations between 1 and 3 nM. The 31- and 39-kDa forms of bovine FSP also antagonized the actions of activin. GnRH (1 nM) increased FSH secretion 1.8-fold and total FSH 1.6-fold during a 3-day incubation, effects that were dose-dependently inhibited by concomitant treatment with 35-kDa bovine FSP. The highest tested concentration of FSP (3 nM) suppressed GnRH-stimulated FSH secretion and total FSH to 59 and 57%, respectively, of the levels found in untreated cultures. All three forms of bovine FSP produced a significant inhibition of FSH secretion and total FSH stimulated by GnRH. FSP also suppressed FSH secretion and total FSH in response to activators of protein kinase C including 100 nM phorbol 12-myristate 13-acetate (43 and 59%, respectively) and 100 nM mezerein (40 and 60%, respectively). Finally, treatment of cultured pituitary cells with 35-kDa FSP at 1 and 3 nM for 3 days resulted in 21 and 24% decreases in GnRH binding sites, respectively. It is concluded that (i) FSP inhibits not only the secretion but also the synthesis of FSH induced by activin and GnRH in long-term culture, and (ii) FSP may cause its inhibitory effects on GnRH by suppression of the protein kinase C system, and possibly by reduction of GnRH binding sites.     

Effect of inhibin on activators of protein kinase-C and calcium-mobilizing agents which stimulate secretion of gonadotropins in vitro: implication of a postgonadotropin-releasing hormone receptor effect of inhibin on gonadotropin release

To further characterize the subcellular mechanisms by which inhibin suppresses GnRH-stimulated gonadotropin release, anterior pituitary cells from adult male Sprague-Dawley rats were treated on day 2 of culture with or without purified 31-kDa bovine inhibin (1-300 pM) for a further 3 days. On day 5, the pretreated cells were washed and incubated in the absence or presence of various secretagogues for 4 h. At the end of the stimulation, the media were saved, and cells were lysed for measurement of both extracellular and intracellular FSH and LH by specific RIAs. Released hormone was expressed as the proportion of total (released plus intracellular) hormone that was available for release in each case. This manipulation of the data corrects for the differential effect of the inhibin pretreatments to suppress intracellular FSH before the stimulation period. Pretreatment for 3 days with inhibin suppressed the proportions of FSH and LH released during 4 h in response to 1) phorbol 12-myristate 13-acetate (100 nM), an activator of protein kinase-C, by maxima of 48% and 53% with inhibin median inhibitory concentrations (IC50) of 17 and 18 pM, respectively; 2) mezerein (100 nM), another type of activator of protein kinase-C, by maxima of 49% and 50% with inhibin IC50 of 19 and 20 pM, respectively; 3) high extracellular K+ (60 mM) by 42% (P less than 0.01) and 38% (P less than 0.01), respectively, with 130 pM inhibin; 4) the calcium ionophore, A23187 (100 microM) by maxima of 54% and 56% with IC50 of 18 and 17 pM, respectively; and 5) GnRH (10 nM) by maxima of 52% and 53% with IC50 of 18 and 19 pM, respectively. However, inhibin had no effect on the proportional release of gonadotropin induced by melittin, an activator of phospholipase-A2. Finally, inhibin had no effect on ACTH release either under basal conditions or in response to CRF (10 nM), phorbol 12-myristate 13-acetate (100 nM), or A23187 (100 microM). We conclude that inhibin suppresses the stimulated release of hormones from gonadotrophs in part by a mechanism common to both gonadotropins that is independent of the previously described inhibitory effect of inhibin on the GnRH receptor. The results are consistent with an action at a site(s) beyond the GnRH receptor, such as protein kinase-C and calmodulin.     

Sodium fluoride provokes gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and gonadotrope sensitization to A23187: evidence for multiple G proteins in GnRH action.

Pretreatment of pituitary cell cultures with GnRH causes altered gonadotrope responsiveness to LH secretagogues. The precise mechanism by which this occurs is not understood. Because a G protein appears to be activated after GnRH stimulation of the gonadotrope, a role for this moiety in GnRH-stimulated alterations in gonadotrope responsiveness was assessed. We show that 3 h pretreatment of pituitary cell cultures with 10 mM NaF (a G protein activator), resulted in decreased gonadotrope responsiveness to subsequent GnRH treatment (3 h, 100 nM; 34.4 +/- 1.6% vs. 23.4 +/- 1.5% of total cellular LH). NaF-provoked gonadotrope desensitization to GnRH also occurred in the presence of 3 mM EGTA and in cells which had been depleted of protein kinase C. Desensitization to GnRH did not occur in response to pretreatment with (Bu)2cAMP (8 h, 1 mM). In addition, neither GnRH nor NaF stimulated inositol phosphate production above basal levels after the NaF pretreatment. GnRH receptor binding also decreased by 30% with NaF pretreatment. In contrast, 3 h NaF (10 mM) pretreatment enhanced responsiveness of the gonadotrope to the Ca2+ ionophore A23187 in a protein kinase C- and cAMP-dependent manner. Responsiveness to the phorbol ester, phorbol 12-myristate 13-acetate, was also increased, whereas responsiveness to the Ca2+ channel activator maitotoxin was unchanged. These data suggest that G protein activation by NaF provokes gonadotrope desensitization to GnRH stimulation by both decreasing receptor numbers and by uncoupling of the receptors from inositol phosphate production. In addition, a distinct G protein action appears to be involved in sensitizing the gonadotrope to A23187 and phorbol 12-myristate 13-acetate.     

Long-term effects of a gonadotrophin-releasing hormone agonist ([D-Ser(But)6]GnRH(1-9)nonapeptide-ethylamide) on gonadotrophin secretion from human pituitary gonadotroph cell adenomas in vitro

We have studied the effects of TRH and native gonadotrophin-releasing hormone (GnRH), and of a GnRH agonist (Buserelin; [D-Ser(But )6]GnRH(1-9) nonapeptide-ethylamide), on LH, FSH, alpha subunit and LH-beta subunit secretion from three human gonadotrophin-secreting pituitary adenomas in dispersed cell culture. During a 24 h study, treatment with 276 nmol TRH/l resulted in a significant (P less than 0.05) stimulated release of FSH and alpha subunit from all three adenomas, and LH from the two adenomas secreting detectable concentrations of this glycoprotein; treatment with 85 nmol GnRH/l significantly (P less than 0.05) stimulated the release of alpha subunit from all three, but FSH from only two and LH from only one adenoma. During a long-term 28-day study, basal FSH and alpha subunit concentrations were maintained, but secretion of LH, and LH-beta (detectable from one tumour only), declined with time from two of the three adenomas. Addition of Buserelin to the cultures resulted in the continuous (P less than 0.05) stimulation of alpha subunit secretion from all three adenomas, and of LH and FSH from two, whilst a transient stimulatory effect on LH and FSH secretion was seen from a third adenoma, with subsequent secretion rates declining towards control values. These data show that human gonadotrophin-secreting adenomas demonstrate variable stimulatory responses to hypothalamic TRH and GnRH, and that during chronic treatment with a GnRH agonist the anticipated desensitizing effect of the drug was not observed in two out of the three adenomas studied. The mechanisms for this is not clear, but such drugs are unlikely to be of therapeutic value in the management of gonadotrophin-secreting tumours. The data also suggest that GnRH and GnRH agonists have a differential effect on the in-vitro release of intact gonadotrophins and the common alpha subunit.     

Distribution and Regulation of Proconvertases PC1 and PC2 in Human Pituitary Adenomas

Pituitary adenomas are members of the family of neuroendocrine cells and tumors which have secretory granules containing chromogranins/secretogranins and other proteins. Pituitary adenomas express the neuroendocrine specific proconvertases PC1 (also known as PC3) and PC2, which are important for the proteolytic processing of chromogranins/secretogranins molecules. We examined the distribution of PC1 and PC2 in primary cultures of 20 pituitary adenomas and analyzed the regulation of the proconvertase mRNAs and proteins by various secretagogues including hypothalamic hormones and phorbol ester to determine the role of PC1 and PC2 in CgA processing in pituitary adenomas. Although PC2 was present in all adenomas, there was a differential distribution of PC1 with PRL adenomas expressing lower levels of PC1 compared to other adenoma types by RT-PCR analysis, in situ hybridization and immunostaining. Treatment of primary cultures of pituitary adenomas with phorbol 12-myristrate 13-acetate (PMA) resulted in an increase in pancreastatin (PST) secretion in most pituitary adenomas and increased PC1 mRNA and protein expression in gonadotroph adenomas, but not in other types of adenomas. Analysis of a human pituitary adenoma cell line, immortalized by recombinant defective adenovirus (HP75), which expressed chromogranin A, FSH, PC1 and PC2 showed that PST was secreted by these immortalized cells. Treatment with TGF1 resulted in an increase in PST secretion and in PC1 mRNA and protein. These results indicate that a) there is a differential distribution of PC1 in human pituitary adenomas with PRL adenomas expressing very little PC1 mRNA and protein and b) that PC1 expression in gonadotropin hormone-producing adenomas is regulated by PMA and TGF1. These findings support the observation that chromogranin A is a substrate for the endoproteinase PC1 in human pituitary adenoma cells.     

Enhanced responsiveness of gonadotropes after protein kinase-C activation: postreceptor regulation of gonadotropin releasing hormone action

GnRH stimulates the release of LH from pituitary gonadotropes in a Ca2+-and calmodulin-dependent manner. Although GnRH also appears to activate protein kinase-C in gonadotropes, the role of this enzyme in GnRH action remains undetermined. In the present work we have assessed the effect of pretreatment of pituitary cell cultures with a protein kinase-C-activating phorbol ester on gonadotrope responsiveness to GnRH. Pretreatment for 6 h with phorbol 12-myristate 13-acetate (PMA) reduced the EC50 for GnRH-stimulated LH release approximately 8-fold without altering the maximum proportion of total cellular LH release. This increase in the potency of GnRH occurred in the absence of any measurable change in receptor affinity. Subsequent studies revealed that PMA pretreatment did not alter the EC50 for GnRH-stimulated [3H]inositol phosphate accumulation (an indicator of phosphoinositide hydrolysis), but did cause a modest reduction (approximately 2-fold) in the EC50 for LH release in response to the Ca2+ ionophore A23187 and the Ca2+ channel-activating compound maitotoxin. These observations demonstrate that the efficiency of coupling of the GnRH receptor to LH release can be regulated at a postreceptor locus by activation of protein kinase-C and that an increased responsiveness of Ca2+-regulated effector systems to mobilized Ca2+ appears to contribute to the observed effect.