Inhibition of follicle-stimulating hormone secretion from gonadotroph adenomas by repetitive administration of a gonadotropin-releasing hormone antagonist

As a preliminary step in searching for a pharmacological treatment for gonadotroph adenomas, we administered the GnRH antagonist analog Nal-Glu GnRH to five patients, four men and a woman, with FSH-secreting gonadotroph adenomas in order to determine its effect on FSH secretion. Administration of a single 10-mg dose of Nal-Glu GnRH to four of the patients produced a significant decrease in the serum FSH concentration in two patients and returned the FSH level to normal in only one. Administration of 5 mg Nal-Glu every 12 h for 7 days, however, produced a significant (P less than 0.001) decrease, and to within the normal range, in four of the five patients (mean +/- SEM, 32.7 +/- 5.6 IU/L during the 3 days before treatment and 9.8 +/- 1.4 IU/L during the last 3 days of treatment). Also, in response to the 7-day treatment, LH fell significantly in all five patients, alpha-subunit fell in three, and testosterone fell in all four men. Administration for 6 weeks of the GnRH agonist analog leuprolide did not decrease the serum FSH concentration of one of the patients whose serum FSH did decrease in response to Nal-Glu GnRH. We conclude that repetitive administration of Nal-Glu GnRH may often inhibit FSH secretion by gonadotroph adenomas and that FSH secretion by gonadotroph adenomas may be dependent on endogenous GnRH secretion.     

Desensitization of luteinizing hormone release in cultured pituitary cells by gonadotropin-releasing hormone

Preincubation of cultured pituitary cells with GnRH caused a marked decrease in subsequent LH release. The rate of desensitization increased when the preincubating concentration of GnRH and the preincubation time were increased. Pituitary cells obtained from male rats were not as sensitive to GnRH as cells obtained from female rats and the extent of desensitization was also smaller in cells from male rats. Densensitization was found to be a long-lasting effects, without any change in the viability of the cells. A superactive analogue of GnRH (D-Phe6-GnRH) caused almost complete desensitization of LH secretion, while a competitive inhibitory analogue of GnRH caused a much smaller decrease in LH response which could be overcome by increasing the concentration of GnRH used for reincubation. These data suggest that the desensitization is closely related to the biological activity of GnRH and does not correlate with receptor binding. High concentrations of potassium also induced desensitization, although to a lower extent than GnRH. Since K+ induces LH release by a different mechanism than GnRH, our data suggest that the desensitization phenomenon cannot be explained only at the receptor level. The time curve of desensitization supports the idea that GnRH action has two-phases: an acute effect which cannot be desensitized, and a secondary phase which can be densensitized.     

Gonadal regulation of pituitary gonadotropin-releasing hormone receptors during sexual maturation in the rat

The number of pituitary GnRH receptors increases during sexual maturation in rats. In females, GnRH receptor content (GnRH-RC, femtomoles bound per gland) rises to a plateau (50 +/- 9 fmol) between 15-30 days of age before increasing further to 107 +/- 19 at 50 days. In males, GnRH-RC rises gradually to 140 +/- 9 fmol at 35 days, then remains stable through 60 days. Administration of estradiol or testosterone to immature females and males, respectively, inhibits the early rise in GnRH-RC. GnRH given for 2 days to steroid-treated immature animals restores receptor content to control levels. Neonatal castration in both sexes rapidly increases GnRH-RC and this response is maintained through 60 days of age. Castrations performed at different ages between 5-60 days showed a sex difference in GnRH-RC responses. Females exhibited a 2-fold increase in GnRH-RC by 5 days post castration at all ages studied. In males a similar increase in GnRH-RC was seen up to 25 days, but later diminished and no receptor response occurred when castration was performed between 30-45 days of age. Orchidectomy after 50 days again resulted in a 2-fold rise in GnRH-RC. GnRH injections (20 micrograms/day in divided doses) increased GnRH-RC in intact males at all ages studied. The same dosage did not increase GnRH receptors in 35-45 day male castrates and 5- to 10-fold higher doses were required to increase GnRH-RC indicating reduced receptor responsiveness to GnRH. Serum gonadotropins increased in response to castration at all ages in both sexes and did not parallel receptor responses in males. These data indicate that pituitary GnRH receptors are modulated by gonadal steroids from day 10 of life in both sexes and that the mechanism involves modification of hypothalamic GnRH secretion. Additionally, factor(s) other than gonadal steroids are operative in males during maturation which alter pituitary receptor responses to GnRH and result in discordant receptor and gonadotropin responses to GnRH.     

Suppression of pituitary and testicular function in normal men by constant gonadotropin-releasing hormone agonist infusion

In a trial for male fertility control the effects of constant GnRH agonist (buserelin) infusion on pituitary and testicular function was investigated. The agonist was administered sc for 12 weeks to two groups of normal young men using extracorporeal osmotic minipumps. Seven men received 118 +/- 24 (SD) micrograms/day from pumps changed biweekly and four men received 230 +/- 27 micrograms/day from pumps changed weekly. After an initial rise serum LH, FSH, and testosterone decreased. The decrease occurred faster in the high dose group and these subjects had no LH response to acute GnRH stimulation after 4 weeks of treatment, whereas the response was drastically reduced in the group receiving the low dose. Androgen substitution with testosterone undecanoate (80-120 mg orally daily) was initiated when the subjects complained of decreased libido and/or potency or when serum testosterone fell below 10 nmol/liter on average in the fifth week. Sperm counts decreased significantly and below the lower normal limit of 20,000,000/ml. The nadir was reached in week 12 of treatment in the high dose group, and in week 4 post treatment in the low dose group. Despite desensitization of the pituitary and impaired testicular function azoospermia did not occur. A higher dose of agonist appears to be required to achieve this goal.     

Hypoglycemia does not affect gonadotroph responsiveness to gonadotropin-releasing hormone in rhesus monkeys

Hypoglycemia inhibits gonadotropin secretion in primates by an undefined mechanism. Some evidence suggests that hypoglycemia inhibits gonadotropin secretion independent of gonadotropin-releasing hormone (GnRH) inhibition. To this end, the effect of insulin-induced hypoglycemia on the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) response to graded doses of GnRH (25, 75, and 250 ng/kg) administered at 120-min intervals was determined in rhesus monkeys. A crossover design was employed such that each animal received GnRH under both hypoglycemic and euglycemic conditions. Experiments were performed in the follicular phase. Gonadotroph responsiveness to GnRH was quantified by determining the change in area under the LH (ΔAULHC) and FSH (ΔAUFSHC) curves that occurred in the first 60 min following each GnRH pulse. There was no statistical difference in ΔAULHC between euglycemic and hypoglycemic animals at any GnRH dose (25 ng/kg: p=0.19; 75 ng/kg: p=0.41; 250 ng/kg: p=0.46). Similarly, changes in AUFSHC following GnRH administration were comparable in euglycemic and hypoglycemic animals (25 ng/kg: p=0.59; 75 ng/kg: p=0.90; 250 ng/kg: p=0.33). We conclude that hypoglycemia had no effect on gonadotroph responsiveness to GnRH. These results are consistent with the conclusion that hypoglycemia inhibits gonadotropin secretion by acting primarily at the level of the hypothalamus to reduce GnRH secretion rather than affecting pituitary responsiveness to GnRH.     

Modulation of pituitary gonadotropin response to gonadotropin-releasing hormone by estradiol

The effects of 17beta-estradiol on the responsiveness of the pituitary to gonadotropin-releasing hormone (GnRH) and on the rate of disappearance of GnRH were studied in 15 healthy nulliparous women aged 18-21 years. The women were divided into 3 groups: Group 1 received no estradiol, Group 2 received the amount of estradiol needed to achieve a circulating level comparable with that in the late follicular phase, and Group 3 received enough estradiol to achieve a concentration similar to that at midcycle. Following administration of GnRH, a marked increase in both LH and FSH was seen in Group 1 subjects. A smaller increase in LH level was observed in Group 2, and virtually no LH response occurred in Group 3. There was no significant increase in FSH level in either group treated with estradiol. The infusion of estradiol did not affect the maximal plasma concentration of exogenously administered GnRH or its disappearance rate in 4 women studied.     

Receptor-mediated internalization of fluorescent gonadotropin-releasing hormone by pituitary gonadotropes.

A bioactive, fluorescent derivative of gonadotropin-releasing hormone, < Glu-His-Trp-Ser-Tyr-D-Lys(N epsilon-tetramethylrhodamine)-Leu-Arg-Pro-Gly-NH2, was prepared. This peptide retained high-affinity binding (apparent dissociation constant, 3 nM) to the receptor for gonadotropin-releasing hormone and was utilized for microscopic visualization and localization of gonadotropin-releasing hormone receptors in cultured rat pituitary cells. The fluorescently labeled receptors were initially distributed uniformly on the cell surface and formed patches, which subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide to gonadotrope cells. Cluster formation and internalization were markedly reduced in the absence of Ca2+, which is required for gonadotropin secretion. It is possible that cluster formation, microaggregation, and internalization of gonadotropin-releasing hormone receptors may be important in eliciting biological effects or for the observed loss of tissue responsiveness after desensitization due to exposure to the homologous hormone.     

The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors

Gonadotropin-releasing hormone (GnRH) induces both synthesis and release of pituitary gonadotropins, but rapid or slow frequencies of stimulation result in reduced LH and FSH secretion. We determined the effects of frequency of GnRH stimulation on pituitary GnRH receptors (GnRH-R). Castrate male rats received testosterone implants (cast + T) to inhibit endogenous GnRH secretion. GnRH pulses were injected by a pump into a carotid cannula and animals received GnRH (25 ng/pulse) at various frequencies for 48 h. In control animals (saline pulses) GnRH-R was 307 +/- 21 fmol/mg protein (+/- SE) in cast + T and 598 +/- 28 in castrates. Maximum GnRH-R was produced by 30-min pulses and was similar to that seen in castrate controls. Faster or slower frequencies resulted in a smaller GnRH-R response and GnRH given every 240 min did not increase GnRH-R over saline controls. Equalization of the total GnRH dose/48 h (6.6 ng/pulse every 7.5 min or 200 ng/pulse every 240 min) did not increase receptors to the maximum concentrations seen after 30-min (25 ng) pulses. Serum LH responses after 48 h of injections were only present after 30-min pulses, and peak FSH values were also seen after this frequency. Serum LH was undetectable in most rats after other GnRH frequencies, even though GnRH-R was increased. These data show that GnRH pulse frequency is an important factor in the regulation of GnRH-R. A reduction of GnRH-R is part of the mechanism of down-regulation of LH secretion by fast or slow GnRH frequencies, but altered frequency also exerts effects on secretory mechanisms at a site distal to the GnRH receptor.     

Endotoxin inhibits pituitary responsiveness to gonadotropin-releasing hormone

Immune/inflammatory challenges powerfully suppress reproductive neuroendocrine activity. This inhibition is generally considered to be centrally mediated via mechanisms that regulate GnRH secretion. The present study provides two lines of evidence that bacterial endotoxin, a commonly used model of immune/inflammatory challenge, also acts to inhibit pituitary responsiveness to GNRH: In the first experiment, pulsatile secretion of GnRH into pituitary portal blood and LH into peripheral blood were monitored in ovariectomized ewes treated with a low dose of endotoxin. Although this treatment only marginally suppressed GnRH pulsatile secretion, it markedly disrupted LH pulsatility. In extreme cases, the low dose of endotoxin blocked LH pulses without inhibiting endogenous GnRH pulses, thereby uncoupling GnRH and LH pulsatile suppression. In the second experiment, we tested the hypothesis that endotoxin inhibits pituitary responsiveness to exogenous GnRH pulses. Hourly pulses of GnRH were delivered to ovariectomized ewes in which endogenous GnRH secretion was blocked. Endotoxin suppressed the amplitude of GnRH-induced LH pulses. Together, these observations support the conclusion that endotoxin inhibits pituitary responsiveness to GNRH:     

Opioids modulate pituitary receptors for gonadotropin-releasing hormone

The number of pituitary GnRH receptors (GnRH-BC) is stable throughout the day in ovariectomized-estradiol treated rats, but undergo an acute transient reduction prior to the afternoon gonadotropin surge. To investigate the mechanisms controlling GnRH-BC we studied the effects of opioid-active compounds in this model. Morphine, given at 1400h, abolished both the LH surge and the preceding fall in GnRH-BC. Morphine given at 0900h increased GnRH-BC 30 min later, and this effect was abolished by simultaneous administration of naloxone. Naloxone alone produced an acute transient fall in GnRH-BC of similar magnitude to that seen before the spontaneous LH surge. These data suggest that alterations in endogenous opioid activity can modulate GnRH receptors and may form part of the mechanisms which initiate the afternoon gonadotropin surge.     

Gonadotropin release by clinically nonfunctioning and gonadotroph pituitary adenomas in vivo and in vitro: relation to sex and effects of thyrotropin-releasing hormone, gonadotropin-releasing hormone, and bromocriptine

We studied in vivo hormone levels and in vitro hormone and subunit release in a group of 22 patients who were operated upon because of a clinically nonfunctioning or gonadotroph pituitary adenoma. In vivo, 5 of the 22 patients, all men, had hypersecretion of FSH, LH beta, or alpha-subunit. An elevated ratio of serum alpha-subunit to LH and FSH was found in 6 of 8 women in vivo, although in all 6 women serum LH, FSH, and alpha-subunit levels were low. LH, FSH, alpha-subunit, LH beta, or a combination of these glycoprotein hormones could be demonstrated in 19 of 22 cultured adenomas. We conclude that 1) virtually all clinically nonfunctioning adenomas contain or release gonadotropins or their subunits in vitro; 2) in vivo hypersecretion of these hormones and subunits occurs infrequently, and in this series only in men; 3) an elevated ratio of alpha-subunit to LH and FSH is frequently found in women and may prove to be a useful diagnostic tool; 4) responses to TRH and bromocriptine do not depend on baseline gonadotropin levels, either in vitro or in vivo, implying that the distinction between gonadotroph adenomas and adenomas without hypersecretion of gonadotropins in vivo is absent where hormone dynamics are concerned.     

Neuroendocrinology of pituitary hormone regulation

Hormone secretion from the pituitary gland is dependent on the intimate association between the hypothalamus and pituitary by vascular and neuronal connections. The anatomic organization of these networks and their specific peptide and/or amide mediators are described in detail. Pertinent factors governing hypothalamic regulation of anterior pituitary secretion are also discussed.     

Regulation of pituitary gonadotropin-releasing hormone receptors by androgens in the male rabbit

The regulation of pituitary GnRH receptors was studied in adult male rabbits after castration and androgen replacement with testosterone (T) or 7 alpha-methyl-19-nortestosterone acetate (U-15,614; T analog) supplied by Silastic capsules implanted sc. Castration increased pituitary GnRH receptors significantly, from 99.3 to 329.5 fmol/mg protein within 4 weeks, without a change in the equilibrium association constant. Serum LH concentrations increased from 0.45 to maximum levels of 2.6 ng/ml by day 8 after orchiectomy; these levels persisted throughout the 4 weeks of study. Serum FSH reached maximum levels of 33.6 ng/ml 5 days after castration. T replacement with 250, 500, and 1000 micrograms/kg X day, prevented a postcastration rise in both pituitary GnRH receptor concentrations and gonadotropin secretion, while 100 micrograms/kg X day prevented an increase in GnRH receptors, but did not completely inhibit hypersecretion of gonadotropins. Administration of T analog at doses of 6.25 and 12.5 micrograms/kg X day partially suppressed the castration-induced increase in pituitary GnRH receptor concentrations, while 25, 50, and 100 micrograms/kg X day suppressed GnRH-binding sites to the levels found in intact controls in 15 of 16 rabbits. By contrast, none of the T analog doses was able to prevent completely LH and FSH hypersecretion. The fact that both T and T analog induced dose-dependent stimulation of prostate and seminal vesicle weights indicates that there are tissue-specific differences in the sensitivity to androgens. We conclude that in the male rabbit 1) pituitary GnRH receptors significantly increase after castration; 2) this increase may partially mediate the postcastration hypersecretion of LH and FSH; 3) castration-induced effects can be prevented by androgen replacement. These results are similar to those obtained in rats, where castration increases LHRH receptors, but contrast with results in mice and hamsters, where castration either reduces or does not change receptor levels. This indicates significant species differences in the response of pituitary GnRH receptor concentrations to elimination of the negative feedback effects of androgens.     

Direct inhibition of testicular function by gonadotropin-releasing hormone: mediation by specific gonadotropin-releasing hormone receptors in interstitial cells.

Agonist analogs of gonadotropin-releasing hormone (GnRH) have been shown to exert antigonadal effects in male and female animals. In hypophysectomized male rats treated with follicle-stimulating hormone, administration of a potent GnRH agonist caused depletion of luteinizing hormone and prolactin receptors and marked suppression of serum testosterone levels. The possibility that such direct effects of GnRH agonists on testicular function could be expressed through specific receptors located in the interstitial cells of the testis was supported by the selective concentration of a 125I-labeled GnRH agonist by the testis in vivo. Specific receptors for the releasing hormone were demonstrated in testis particles and dispersed interstitial cells by direct binding analysis with the 125I-labeled GnRH agonist. The binding affinity (Ka = G X 10(9) M-1) and peptide specificity of the testicular GnRH binding sites were similar to those of anterior pituitary and ovarian GnRH receptors. The presence of GnRH receptors in the testis indicates that these sites mediate the direct inhibitory actions of GnRH agonists upon testicular endocrine function.     

Cell-specific transcriptional regulation of follicle-stimulating hormone-beta by activin and gonadotropin-releasing hormone in the LbetaT2 pituitary gonadotrope cell model

FSH is secreted by gonadotropes of the anterior pituitary and plays a crucial role in mammalian reproduction. However, little is known about FSH gene regulation due to the lack of a gonadotrope cell line that synthesizes FSH. The LbetaT2 mouse pituitary cell line, isolated by targeted tumorigenesis in transgenic mice, has the characteristics of a mature gonadotrope, including expression of GnRH receptor, steroidogenic factor 1, and both the alpha- and beta-subunits of LH, but was thought not to express FSH. Using RT-PCR, we show that these cells synthesize FSH beta- subunit messenger RNA, which is induced by activin and inhibited by follistatin. Furthermore, in transient transfections an ovine FSHbeta 5'-regulatory region (5.5 kb) confers LbetaT2 cell-specific expression to a reporter gene compared with other pituitary and nonpituitary cell lines. This FSHbeta regulatory region responds to activin specifically in LbetaT2 cells, an effect that is blocked by follistatin. The LHbeta, alpha-subunit, and GnRH receptor regulatory regions are induced by activin and blocked by follistatin. Furthermore, LbetaT2 cells express the components of the activin system, and addition of follistatin alone reduces FSHbeta gene expression, demonstrating that an endogenous activin autocrine loop regulates FSH in these cells. In addition, GnRH stimulates both the FSHbeta and LHbeta regulatory regions, specifically in LbetaT2 cells. Surprisingly, GnRH induction is reduced by follistatin, suggesting its dependence on endogenous activin. As the mouse GnRH receptor promoter is inhibited by follistatin, reduction of GnRH receptor levels might be one mechanism by which follistatin interferes with GnRH induction of gonadotropin genes. In summary, LbetaT2 cells exhibit the characteristics of fully differentiated gonadotropes, including the expression of LH, FSH, GnRH receptor, and components of the activin/follistatin system, as well as display the appropriate responses to activin and GNRH:     

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.     

The role of calcium in gonadotropin-releasing hormone induction of follicle-stimulating hormone release by the pituitary gonadotrope

Binding of gonadotropin-releasing hormone (GnRH) to the pituitary gonadotrope induces activation of a membrane associated calcium channel, resulting ultimately in luteinizing hormone release. The role of calcium mobilization in GnRH-induced follicle-stimulating hormone (FSH) release was explored using anterior pituitary glands from female rats in a perifusion tissue culture system. While perifusion with GnRH (10 ng/ml) induced a constant level of gonadotropin release, the calcium channel blocker verapamil (10(-4)M) depressed FSH release, as did dantrolene (10(-4)M), an antagonist of intracellular calcium mobilization. When the calcium ionophore A23187 (10(-5) M) was substituted for GnRH, FSH release was not only maintained but increased. Antagonism of the activity of calmodulin (CAM) with trifluoperazine (10(-4)M), however, did not depress FSH release. Cellular content of cAMP and cGMP increased in response to GnRH. When FSH secretion was ionophoretically induced by A23187, however, little cAMP was detected. These results support a role for calcium mobilization in the second messenger cascade underlying GnRH-induced FSH release. The role for calcium in the disparate release of FSH and LH were further discussed in the context of these data.