Cortisol reduces gonadotropin-releasing hormone pulse frequency in follicular phase ewes: influence of ovarian steroids

Stress-like elevations in plasma glucocorticoids suppress gonadotropin secretion and can disrupt ovarian cyclicity. In sheep, cortisol acts at the pituitary to reduce responsiveness to GnRH but does not affect GnRH pulse frequency in the absence of ovarian hormones. However, in ewes during the follicular phase of the estrous cycle, cortisol reduces LH pulse frequency. To test the hypothesis that cortisol reduces GnRH pulse frequency in the presence of ovarian steroids, the effect of cortisol on GnRH secretion was monitored directly in pituitary portal blood of follicular phase sheep in the presence and absence of a cortisol treatment that elevated plasma cortisol to a level observed during stress. An acute (6 h) cortisol increase in the midfollicular phase did not lower GnRH pulse frequency. However, a more prolonged (27 h) increase in cortisol beginning just before the decrease in progesterone reduced GnRH pulse frequency by 45% and delayed the preovulatory LH surge by 10 h. To determine whether the gonadal steroid milieu of the follicular phase enables cortisol to reduce GnRH pulse frequency, GnRH was monitored in ovariectomized ewes treated with estradiol and progesterone to create an artificial follicular phase. A sustained increment in plasma cortisol reduced GnRH pulse frequency by 70% in this artificial follicular phase, in contrast to the lack of an effect in untreated ovariectomized ewes as seen previously. Thus, a sustained stress-like level of cortisol suppresses GnRH pulse frequency in follicular phase ewes, and this appears to be dependent upon the presence of ovarian steroids.     

Sleep-associated decrease in luteinizing hormone pulse frequency during the early follicular phase of the menstrual cycle: evidence for an opioidergic mechanism

To investigate the possible role of an opioidergic mechanism(s) in the sleep-associated decrease in LH pulse frequency during the early follicular phase of the menstrual cycle, 10 normal cycling women were studied on days 3 and 4 of their cycles before and after the administration of a specific opiate receptor antagonist, naloxone. Sequential 24-h infusions of naloxone (10-mg iv bolus dose followed by an infusion of 30 micrograms/kg . h) or NaCl were administered randomly. Pulsatile LH activity was assessed for 48 h. Sleep was confirmed by electroencephalogram monitoring during the night hours (2300-0700 h). Significant sleep-associated decreases in LH pulse frequency (P less than 0.01) and mean serum LH levels (P less than 0.01) were found during the NaCl control studies. While naloxone infusion had no effect on LH pulse frequency during the waking hours, it prevented the sleep-associated decrease in pulse frequency and, in fact, significantly (P less than 0.001) increased the LH pulse frequency during the sleeping hours. These observations provide evidence that a diurnal variation of naloxone sensitivity exists in early follicular phase women and that the decrease in LH pulse frequency normally found during sleep is based at least in part on increased opioidergic inhibition.     

Polycystic ovarian syndrome: evidence that flutamide restores sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol and progesterone

Polycystic ovarian syndrome (PCOS) is a complex disorder with multiple abnormalities, including hyperandrogenism, ovulatory dysfunction, and altered gonadotropin secretion. The majority of patients have elevated LH levels in plasma and a persistent rapid frequency of LH (GnRH) pulse secretion, the mechanisms of which are unclear. Earlier work has suggested that the sensitivity of the GnRH pulse generator to inhibition by ovarian steroids is impaired. We performed a study to determine whether antiandrogen therapy with flutamide could enhance feedback inhibition by estradiol (E2) and progesterone (P) in women with PCOS. Ten anovulatory women with PCOS and nine normal controls (days 8-10 of the cycle) were studied on three occasions. During each admission, LH and FSH were determined every 10 min and E2, P, and testosterone (T) every 2 h for 13 h. After 12 h, GnRH (25 ng/kg) was given iv. After the first admission, patients were started on flutamide (250 mg twice daily), which was continued for the entire study. The second admission occurred on days 8-10 of the next menstrual cycle for normal controls and on study day 28 for PCOS patients. Subjects were then given E2 transdermally (mean plasma E2, 106+/-18 pg/mL) and P by vaginal suppository to obtain varied plasma concentrations of P (mean P, 4.4+/-0.5 ng/mL; range, 0.6-9.0 ng/mL), and a third study was performed 7 days later. At baseline women with PCOS had higher LH pulse amplitude, response to GnRH, T, androstenedione, and insulin and lower sex hormone-binding globulin concentrations (P < 0.05). Most hormonal parameters were not altered by 4 weeks of flutamide, except T in controls and E2 and FSH in PCOS patients, which were lower. Of note, flutamide alone had no effect on LH pulse frequency or amplitude, mean plasma LH, or LH responsiveness to exogenous GnRH. After the addition of E2 and P for 7 days, both PCOS patients and normal controls had similar reductions in LH pulse frequency (4.0+/-0.7 and 5.8+/-0.7 pulses/12 h, respectively). This contrasts with our earlier results in the absence of flutamide, where a plasma P level of less than 10 ng/mL had minimal effects on LH pulse frequency in women with PCOS, but was effective in controls. These results suggest that although the elevated LH pulse frequency in PCOS may in part reflect impaired sensitivity to E2 and P, continuing actions of hyperandrogenemia are important for sustaining the abnormal hypothalamic sensitivity to feedback inhibition by ovarian steroids.     

Exogenous progesterone: influence on ovulation and hormone levels in the cyclic hamster

The oestrous cycle of the hamster is lengthened by 2 to 3 days when 2-5 or 5-0 mg progesterone are injected on day 1 of the cycle (day of ovulation). Antral follicles develop on day 2 but their growth is significantly retarded in progesterone-treated hamsters. Administration of progesterone is followed within 6 h by an abrupt decline in serum FSH and LH concentration but by day 3 the level of FSH is higher than normal. Serum LH is slower to recover in hamsters receiving 5 mg progesterone but normal levels are also restored by days 3 or 4. Increased serum levels of progesterone are maintained until days 3-5 in animals injected with progesterone. Despite the presence of antral follicles on day 2 and the fairly prompt restoration of normal levels of gonadotrophins, the serum concentration of oestradiol is suppressed for several days in the progesterone-treated hamster. This suggests that progesterone, in addition to affecting the hypothalamic-pituitary system, may also directly inhibit oestrogen secretion by the antral follicles.     

The relative role of gonadal sex steroids and gonadotropin-releasing hormone pulse frequency in the regulation of follicle-stimulating hormone secretion in men

OBJECTIVE: Our objective was to determine the importance of testosterone (T), estradiol (E(2)), and GnRH pulse frequency to FSH regulation in men. DESIGN: This was a prospective study with four arms. SETTING: The study was performed at the General Clinical Research Center. PATIENTS OR OTHER PARTICIPANTS: There were 20 normal (NL) men and 15 men with idiopathic hypogonadotropic hypogonadism (IHH) who completed the study. Intervention: Medical castration and inhibition of aromatase were achieved using ketoconazole x 7 d with: 1) no sex steroid addback, 2) T addback starting on d 4, and 3) E(2) addback starting on d 4. IHH men in these arms received GnRH every 120 min. In a further six IHH men receiving ketoconazole with no addback, GnRH frequency was increased to 35 min for d 4-7. Blood was drawn every 10 min x 12 h at baseline, overnight on d 3-4 and 6-7. MAIN OUTCOME MEASURES: Mean FSH was calculated from the pool of each frequent sampling study. RESULTS: In NL men FSH levels increased from 5.1 +/- 0.7 to 8.7 +/- 1.3 and 9.7 +/- 1.5 IU/liter (P < 0.0001). T caused no suppression of FSH. E(2) reduced FSH from 12.4 +/- 1.8 to 9.3 +/- 1.3 IU/liter (P < 0.05). In IHH men on GnRH every 120 min, FSH levels went from 6.0 +/- 1.6 to 9.0 +/- 3.0 and 11.9 +/- 4.3 (P = 0.07). T caused no suppression of FSH. E(2) decreased FSH such that levels on d 6-7 were similar to baseline. Increasing GnRH frequency to 35 min had no impact on FSH. CONCLUSIONS: The sex steroid component of FSH negative feedback in men is mediated by E(2). Increasing GnRH frequency to castrate levels has no impact on FSH in the absence of sex steroids. When inhibin B levels are NL, sex steroids exert a modest effect on FSH.     

Progesterone inhibition of the hypothalamic gonadotropin-releasing hormone pulse generator: evidence for varied effects in hyperandrogenemic adolescent girls

Compared with normal women, adults with polycystic ovarian syndrome (PCOS) require higher progesterone (P) concentrations to inhibit GnRH (LH) pulse frequency, which contributes to persistently rapid GnRH pulses and elevated LH levels in PCOS. To explore the origin of this abnormality, we assessed hypothalamic sensitivity to P feedback in nine normal controls and 11 hyperandrogenemic (HA) adolescents. Subjects first underwent frequent blood sampling for 11 h to assess baseline LH pulse frequency. Thereafter, oral estradiol and micronized P were given for 7 d to achieve mean estradiol and P levels of 143 +/- 16 pg/ml (524 +/- 60 pmol/liter) and 7.8 +/- 0.7 ng/ml (24.9 +/- 2.3 nmol/liter), respectively. LH pulse frequency was then reassessed. On d 7, the slope of the percent reduction of LH pulses per 11 h as a function of the d 7 P concentration was less in the HA group compared with controls (P = 0.02) despite similar P levels. LH pulse frequency was suppressed in all NC (mean, 7.0 to 3.4 pulses/11 h), but was unchanged in six of the HA girls (mean, 8.3 to 7.5 pulses/11 h). In contrast, in the other five HA adolescents, P induced similar slowing of LH pulses to that seen in NC (mean, 10.0 to 5.0 pulses/11 h). Baseline free testosterone levels were similar in both HA groups; the only observed difference between these HA groups is that the P-suppressible subjects were all of Hispanic descent. These data suggest that hyperandrogenemia during adolescence is variably associated with decreased sensitivity to P, which may have a partially genetic basis.     

Muscarinic involvement in the regulation of gonadotropin-releasing hormone in the cyclic rat.

The release of gonadotropin-releasing hormone (GnRH) from the median eminence (ME) in cyclic rats was stimulated to a significant extent by the selective muscarinic antagonists 11[(2)(diethylamino)methyl][-1-piperidinyl]-acetyl-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine, and to a lesser extent also by other ligands selectively antagonistic to m1 and m3 receptors. Such stimulation was estrous-cycle-dependent and was not achieved by muscarinic agonists. We suggest that the effect is induced via the m4 receptor subtype. Attempts to block the muscarinic-antagonist-induced stimulation of GnRH release with a variety of drugs were successful only in the presence of prazosin, an antagonist to alpha 1-adrenergic receptors. One possible explanation for this muscarinically mediated stimulation of GnRH release is that it results from cross-talk between the muscarinic and the alpha 1-adrenergic receptors, i.e., muscarinic agonists might inhibit the release induced by alpha 1-agonists, and muscarinic antagonists, by cancelling this inhibitory effect, might thus allow the endogenous alpha 1-agent, norepinephrine, to induce the release of GnRH.     

The roles of estradiol and progesterone in decreasing luteinizing hormone pulse frequency in the luteal phase of the menstrual cycle

During the luteal phase of the menstrual cycle, plasma progesterone (P) and estradiol (E2) concentrations are elevated, and LH (and by inference GnRH) pulse frequency is slow. In contrast, LH pulse frequency increases during the early follicular phase when plasma E2 and P are lower. To examine the mechanism(s) responsible for the slower GnRH pulse frequency in the luteal phase, we maintained plasma P, E2, or both at midluteal concentrations from the midluteal phase to the time of the next early follicular phase and measured the effects on LH secretion. Thirteen normal women with regular menstrual cycles were studied during two or three cycles. Blood was obtained every 10 min during 10-h studies. Control cycle luteal and early follicular studies were followed by a second control study in the luteal phase of the treatment cycle. P (six women), E2 (seven women), or both (five women) then were given twice daily by im injection for 6-12 days until the day corresponding to the early follicular study of the control cycle (EF + P, EF + E2, or EF + E2 + P). A final study was performed 1 week after the injections were discontinued (F). LH pulse frequency was low in the midluteal phase [3.2 +/- 0.2 (+/- SE) pulses/10 h] and increased by the early follicular phase (8.0 +/- 0.8 pulses/10 h) in the control cycles. The increase in LH pulse frequency was not significantly inhibited by administration of P (6.7 +/- 0.7 pulses/10 h; EF + P). However, during both E2 alone and E2 + P, LH pulse frequency remained low (EF + E2, 3.6 +/- 0.8; EF + E2 + P, 2.0 +/- 0.7 pulses/10 h). The mean plasma FSH concentrations paralleled changes in LH pulse frequency, increasing from the luteal to the early follicular phase in the control cycles and during P injections and remaining low during E2 and E2 + P injections. We conclude that continued exposure to P alone does not maintain GnRH pulse frequency at midluteal phase values and that any effect of P requires the presence of E2. As E2 alone maintained lower LH pulse frequency, E2 may act directly to decrease the pulsatile GnRH secretion or it may potentiate the effects of low (less than 3.2 nmol/L) P concentrations.     

Modulation of the estradiol-induced luteinizing hormone surge by progesterone or antiestrogens: effects on pituitary gonadotropin-releasing hormone receptors

The present study examined the question of whether modulation of estradiol-induced LH surges by progesterone or antiestrogens in the immature rat might be related to changes in the concentration of pituitary GnRH receptors (GnRH-R). Rats (28 days old) that received estradiol implants at 0900 h had LH surges approximately 32 h later. Administration of progesterone or nafoxidine (U-11,100 A; 1-(2-[P-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]pyrrolidine hydrochloride) concomitantly with estradiol led to blockade of these LH surges (progesterone or nafoxidine inhibition), while progesterone treatment 24 h after estradiol brought about premature and enhanced LH release (progesterone facilitation). GnRH-R-binding capacity was determined by saturation analysis in homogenates of single pituitaries from immature rats treated with estradiol and progesterone or nafoxidine and controls treated only with estradiol using [125I]iodo-(D-Ala6,Des-Gly10)GnRh ethylamide. The affinity of GnRH-R for this analog ranged from 8.2-15.1 X 10(9) M-1 and was not affected by in vivo steroid or antiestrogen treatment. The number of GnRH-R in gonadotrophs from untreated 28-day-old rats (57.2 +/- 2.6 fmol/pituitary or 177 +/- 11 fmol/mg protein) was comparable to values previously reported for 30 day-old females. GnRH-R levels were first measured 1, 8, 24, 32, and 48 h after estradiol treatment. The pituitary content of GnRH-R paralleled changes in total pituitary protein (nadir at 24 h, rebound at 32 h, continued increase at 48 h), while their concentration (femtomoles per mg protein) was highest at 8 h. Next, GnRH-R levels were examined at 1200 h and at hourly intervals (1400-1800 h) on the afternoon of the LH surge. While GnRH-R concentrations were significantly lower at 1400 and 1700 h than at 1200 or 1800 h in animals treated with estradiol in the progesterone facilitation model, they did not change over time in the other two paradigms. There was no significant difference in pituitary content or concentration of GnRH-R at any time between immature rats treated with estradiol and progesterone or nafoxidine and their respective estradiol-treated controls. These results suggest that changes in GnRH-R levels in pituitary gonadotrophs do not play a major role in enhancement of LH surges by progesterone or in their suppression by progesterone or nafoxidine in the immature rat; therefore, these compounds may affect the pituitary at a site distal to the GnRH receptor.     

Pulsatile gonadotropin secretion in women with hypothalamic amenorrhea: evidence that reduced frequency of gonadotropin-releasing hormone secretion is the mechanism of persistent anovulation

Hypothalamic amenorrhea (HA) is a clinical disorder of unknown etiology. The diagnosis is made by exclusion of known abnormalities of pituitary and ovarian function. To determine if abnormalities of GnRH secretion could account for the anovulation and amenorrhea, we measured plasma gonadotropins every 20 min for 10- to 24-h periods in 19 women with HA. Ovarian steroids and gonadotropin responses to an iv bolus dose of GnRH (25 ng/kg) were also measured. The results were compared to those obtained during the early follicular (EF) and late luteal (LL) phases of ovulatory cycles in normal women. Plasma estradiol was lower (mean +/- SE, 52 +/- 5 pg/ml) than either cycle stage in normal women. Mean plasma LH was lower than EF values and FSH was higher than LL values. The amplitude of LH pulses in HA was similar to that in normal women. LH pulse frequency was the same as that present during the LL, but lower than that during the EF (HA, 4.7 pulses/12 h; EF, 7.7 pulses/12 h; P less than 0.05). In addition to the similar frequency, the patterns of LH secretion in HA resembled that of LL in that the amplitude of LH pulses was highly variable and pulses occurred at irregular intervals. Consistent changes in diurnal gonadotropin secretion were not found, and LH secretion was greater at night in 9 studies and during the day in 5 studies. Repeat studies in three patients (5-13 months later) revealed that LH pulse frequency was variable, being unchanged in 1, increased in 1, and decreased in the third patient. Thus, LH pulse frequency and, by inference, GnRH pulse frequency are similar in HA to those in the normal luteal phase despite a different steroid milieu. GnRH pulse frequency increases from the luteal to the follicular phases of normal cycles and may be important in the initiation of ovarian follicular maturation. These data suggest that the absence of cyclical gonadotropin secretion and anovulation in HA result from a decreased frequency and irregular amplitude of GnRH secretion and consequent absence of ovarian follicular maturation.     

Follicle-stimulating hormone-inhibin B interactions during the follicular phase of the primate menstrual cycle revealed by gonadotropin-releasing hormone antagonist and antiestrogen treatment

The aim was to determine the pattern of inhibin A and inhibin B secretion during the ovulatory cycle of the macaque and to explore the effects of manipulating follicular phase FSH on inhibin B secretion by: 1) blocking the early follicular phase rise in FSH with GnRH antagonist treatment; 2) administering FSH in GnRH antagonist-treated animals; and 3) preventing the midfollicular phase decline in FSH by a specific antiestrogen. Treatment with GnRH antagonist, starting on day 25 of the cycle, abolished the early follicular phase rise in FSH and the associated increase in inhibin B. The same treatment, followed by exogenous FSH, restored the secretion of inhibin B. Treatment with antiestrogen, commencing during the midfollicular phase, induced a supraphysiological rise in FSH, followed by a marked stimulation of inhibin B and estradiol secretion. Despite continued antiestrogen treatment, FSH secretion declined before peak values of inhibin B and estradiol were attained, implying a potential endocrine role for inhibin B, in addition to estradiol, in the negative feedback regulation of FSH. These results show that follicular phase FSH is the major stimulus for inhibin B secretion.     

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.     

Increased luteinizing hormone pulse frequency during sleep in early to midpubertal boys: effects of testosterone infusion

Gonadotropin secretion is pulsatile in prepubertal and early pubertal boys, and the onset of puberty is characterized by a sleep-associated rise in LH pulse amplitude. To determine whether an augmentation in LH pulse frequency as well as amplitude occurs at the onset of puberty, we studied gonadotropin secretion in 21 early to midpubertal boys. Blood samples were taken every 20 min (every 15 min in 4 boys) for LH determinations. A 2-fold increase in LH pulse frequency occurred during the nighttime sampling period (2200-0400 h) compared to that in the hours when the boys were awake (1000-2200 h). The maximum frequency (0.7 pulses/h) occurred between 2400 and 0200 h. The mean plasma LH concentration increased during the night from 2.3 +/- 0.2 (+/- SE) mIU/mL (2.3 +/- 0.2 IU/L) between 2000-2200 h to a maximum of 6.2 +/- 0.4 (6.2 +/- 0.4 IU/L) between 0200-0400 h. The mean plasma LH decreased to 5.5 +/- 0.4 mIU/mL (5.5 +/- 0.4 IU/L) between 0400-0600 h and to 4.2 +/- 0.5 (4.2 +/- 0.5 IU/L) between 0600-0800 h. Plasma testosterone rose during the night to a mean maximum value of 2.4 +/- 0.5 (+/- SE) ng/mL (8.3 +/- 1.7 nmol/L). This finding suggested that the rise in testosterone might play a role in decreasing LH secretion during the later hours of sleep (after 0400 h). To address this question and to study further the effects of testosterone in early puberty, we measured plasma LH concentrations every 10 min from 2000-0800 h in 8 early to mid-pubertal boys before and during short term testosterone administration. Saline or testosterone at a concentration of 9.33 micrograms/mL (32 mumol/L) was infused at a rate of 10 mL/h from 2100-1200 h to shift the nighttime testosterone rise 3 h earlier than would occur spontaneously. Blood samples were obtained every 10 min for LH and every 30 min for testosterone determinations from 2000-0800 h. Pituitary responsiveness was assessed by administering sequential doses of synthetic GnRH (25 and 250 ng/kg) at 1000 and 1200 h, respectively. The nighttime increase in LH pulse frequency and mean plasma LH concentration occurred between 2300 and 0200 h despite testosterone infusion. However, testosterone infusion was associated with significantly lower mean plasma LH concentrations from 0200-0800 h compared to those on the night of the saline infusion. Pituitary responsiveness to synthetic GnRH was unaltered by testosterone administration.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of a gonadotropin-releasing hormone agonist on ovulation and steroidogenesis during perfusion of rabbit and rat ovaries in vitro

The ability of a GnRH agonist (GnRHa) to exert direct effects on rat and rabbit ovaries was examined in vitro. Ovaries of estrous rabbits and immature, PMSG-primed rats were surgically removed and perfused with a defined medium via an aortic cannula. In this system, the ovary remains viable and capable of undergoing ovulation in response to LH. Samples of perfusion medium were taken for steroid measurements and the number of ovulations determined by direct observation (rabbit) or oocyte recovery (rat). Follicles of ovaries perfused with medium alone rarely ovulated. GnRHa (0.1 micrograms/ml) induced ovulations in 6 of 7 rat ovaries (4 to 22 ovulations per ovulating ovary) and this effect was blocked by a GnRH antagonist. In contrast, a much higher dose of the agonist (10 micrograms/ml) induced ovulations in only 7 of 15 rabbit ovaries. GnRHa caused small but significant increases in progesterone levels in the perfusion medium in both species in comparison to no treatment. Mean estradiol levels also tended to be higher in the GnRHa groups in comparison to controls but the differences were not significant. GnRHa appears to act directly on both the rabbit and rat ovary but the rat ovary is much more sensitive to its ovulation-inducing effects.     

Further evidence that thyrotropin-releasing hormone participate in the regulation of growth hormone secretion in the rat

Effects of thyrotropin-releasing hormone (TRH) on growth hormone (GH) secretion were investigated in vivo (on intact or mediobasal hypothalamic lesioned rats tested under either anesthesia or free moving conditions) as well as in vitro (in incubation or perifusion systems of anterior pituitary tissue). The peptide induced a rapid, dose-dependent increase of plasma GH levels in free moving animals bearing an extensive lesion of the mediobasal hypothalamus including the median eminence. Under comparable conditions, TRH was ineffective in intact animals. After chloral hydrate anesthesia a GH response to TRH was recorded in both groups, but lesioned rats exhibited a better responsiveness to all doses tested. In vitro TRH increased GH release from incubated or perifused pituitaries sampled from both intact and lesioned rats in a transient and concentration-dependent manner. A similar effect was obtained with the (3 Me His2) analogue of TRH. These findings indicate that TRH can affect GH secretion at the pituitary level under specific experimental conditions and support the hypothesis that either peripheral hormones or other, still unidentified hypothalamic neurohormones may modulate this effect.     

Desensitization to native molecular forms of gonadotropin-releasing hormone in the goldfish pituitary: dependence on pulse frequency and concentration.

Homologous desensitization of gonadotropin-releasing hormone (GnRH) was investigated using goldfish pituitary fragments in vitro. The two native GnRH peptides, sGnRH [( Trp7, Leu8]-GnRH) and cGnRH-II [( His5, Trp7, Tyr8]-GnRH) were administered either continuously or in pulsatile fashion at different frequencies and concentrations. Continuous treatment (60 min) with either sGnRH or cGnRH-II at 10(-7), 10(-8), and 10(-9) M resulted in desensitization of goldfish pituitary in a biphasic fashion, characterized by an initial rapid peak of GTH release (phase 1), followed by a lower sustained release of GTH remaining at a stable concentration above the basal level (phase 2). Pititary fragments were then washed for 60 min and further treated continuously (60 min) with the same concentrations of sGnRH or cGnRH-II (second treatment). Total sGnRH- or cGnRH-II-induced GTH release during the second treatment period was significantly lower than that observed during the initial treatment period, depending upon the concentration of the peptides. The second phase of GTH release was more pronounced at lower concentrations compared to that observed following 10(-7) M treatment, especially for sGnRH. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses of 10(-7), 10(-8), and 10(-9) M given every 20 min) resulted in significant desensitization of the pituitary GTH release. Reduction of pulse frequency to 2 min treatment every 60 min resulted in a lower degree of desensitization; little or no desensitization was observed following treatment with 10(-8) and 10(-9) M cGnRH-II or 10(-9) M sGnRH. A further reduction in frequency to 2-min pulses of sGnRH or cGnRH-II (10(-7) or 10(-8) M) given every 90 min did not result in desensitization of the pituitary GTH release. In summary, the present study demonstrates that GnRH-induced desensitization is dependent on both pulse frequency and concentration in the goldfish pituitary. These findings support the hypothesis that pulsatile secretion of the native GnRH peptides may be essential for maintenance of normal pituitary GTH release in goldfish.