Hormone ontogeny in the ovine fetus. XVIII. The effect of an opioid antagonist on luteinizing hormone secretion

Endogenous opioid-like peptides influence gonadotropin release in adult animals and man; however, the role of these peptides in the regulation of fetal LH secretion is not known. We administered naloxone hydrochloride (1.3 mg/kg iv), a specific opioid receptor antagonist, to 22 chronically catheterized ovine fetuses of gestational ages 94-143 days (term = 147 days). As a control, each fetus also received the vehicle on a separate occasion, the sequence of the studies being randomized. After the administration of naloxone, LH secretion increased from 38.6 +/- 5.8 to 114 +/- 21 ng/h ml-1 (P less than 0.001); LH release was not affected by administration of the vehicle. Morphine (13 mg/kg) and naloxone (1.3 mg/kg) were administered together to three fetuses (gestational age 94-105 days); LH secretion was sharply reduced from 411 +/- 14.3 ng/h ml-1 after naloxone alone to 53 +/- 17.5 ng/h ml-1 after the administration of both naloxone and morphine (P less than 0.01). The response to naloxone varied with gestational age. Fetuses of 94-115 days showed a significantly higher increment in LH secretion when given naloxone (112.3 +/- 30.7 ng/h ml-1) than did older fetuses of gestational age 126-143 days (64.8 +/- 20.8 ng/h ml-1) (P less than 0.02). These findings indicate that, in the ovine fetus endogenous opioid-like peptides exert a tonic suppressive effect on LH secretion at least as early as 94 days gestation. Moreover, the effectiveness of naloxone in augmenting LH release decreases with advancing gestational age. This latter observation supports the concept that, in the ovine fetus, endogenous opioid tone is not the sole factor involved in the dampened fetal LH secretion which is characteristic of late gestation.     

Gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists do not alter endogenous GnRH secretion in short-term castrated rams

The administration of GnRH agonists and antagonists suppresses pituitary LH secretion. However little is known about their effects on endogenous GnRH secretion. To determine if GnRH analogs act on GnRH secretion through a short or ultrashort loop feedback mechanism, experiments were performed to analyze GnRH secretion in hypophyseal portal blood of conscious short-term castrated rams under both agonist or antagonist treatment. In Study 1, six rams were castrated and surgically prepared for portal blood collection on day -7. Portal and peripheral blood were collected simultaneously every 10 min for 14-15 h on day 0. Five hours after the beginning of the portal blood collection, animals were injected im with 5 mg potent GnRH antagonist (Nal-Glu). In Study 2, six rams were treated daily from day -11 to day 0 with the GnRH agonist D-Trp6 GnRH (0.5 mg im). Castration and surgical preparation for portal blood collection were performed on day -7. On day 0 portal and peripheral blood were collected simultaneously every 10 min for 10-11 h. In both studies, to determine whether an increase in GnRH concentration in hypophyseal portal blood can overcome the inhibitory effect of the GnRH analogs, between 5 and 5.5 h after the injection of the analogs, endogenous GnRH secretion was stimulated by Naloxone administration (3 x 100 mg, iv, at 30-min intervals) followed by a bolus of exogenous GnRH (2 x 10 micrograms, iv at 30-min intervals). In Study 1, Nal-Glu administration led to a rapid cessation of pulsatile LH secretion for the duration of blood collection while GnRH pulse frequency and amplitude were not affected. GnRH and LH pulse frequency before and after Nal-Glu administration were, 6.2 +/- 0.6 vs. 5.7 +/- 0.8 (NS) and 5.3 +/- 0.3 vs. 0.3 +/- 0.2 pulses/6 h (P less than 0.001) respectively. In Study 2, peripheral LH secretion was completely suppressed while GnRH secretion (portal blood) remained pulsatile. GnRH pulses frequency and pulse amplitude were 4.3 +/- 0.3 pulses/6 h and 43.0 +/- 4.7 pg/ml, respectively. In both experiments, neither stimulation of endogenous GnRH secretion by naloxone nor administration of exogenous GnRH allowed reinitiation of LH secretion. However, additional studies in two animals of each treatment group (study-III) showed that this was clearly a dose related effect in antagonist treated but not in agonist-treated animals since higher doses of exogenous GnRH (i.e. 100 micrograms or 1000 micrograms) can increase significantly LH levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of gonadotropin secretion in the ovine fetus. II. A sex difference in pulsatile luteinizing hormone secretion after castration

Gonadal involvement in the control of fetal LH secretion was examined by studying LH pulsatility in 12 chronically catheterized male (9 castrate and 3 sham-control) and 12 female (8 castrate and 4 sham-control) ovine fetuses operated upon in utero at 106-116 days gestation (term = 147 days). Fetuses were studied longitudinally over a 2- to 30-day period in castrates and over a 2- to 37-day period in controls. LH pulsatility was determined from blood samples obtained every 15 min over a standard 3-h observation period and assayed for LH by RIA (NIH LH S16 standard). In female fetuses there was no significant difference in LH pulse frequency between castrates (25 pulses in 32 periods; 1 pulse/3.8 h of observation) compared to controls (15 pulses in 15 periods; 1 pulse/3.0 h). LH pulse frequency was similar in the sham-castrate males (11 pulses in 17 periods; 1 pulse/4.6 h). In contrast, LH pulse frequency was significantly higher in the castrate male group (90 pulses in 42 periods; 1 pulse/1.4 h) compared to that in each of the other 3 groups (P less than 0.005). LH pulse frequency did not vary with gestational age in castrate and control females or in control males. In castrate males, however, LH pulse frequency declined significantly (P less than 0.005) with advancing gestation from 80 pulses in 32 periods (1 pulse/1.2 h) before 130 days compared to 10 pulses in 10 periods (1 pulse/3.0 h) after 130 days. Thus, LH pulse frequency was indistinguishable in castrate vs. eugonadal males after 130 days. The absence of a castration effect on LH pulsatility in male fetuses older than 130 days was confirmed in an additional group of 8 male fetuses (5 castrate and 3 sham-controls) operated upon at 121-130 days gestation and studied over a 2- to 20-day period. Overall, LH pulse amplitude was similar in male [4.7 +/- 0.5 ng/ml (+/- SE)] and female (3.9 +/- 0.5 ng/ml) fetuses and did not vary as a function of gonadal status or gestational age. The postcastration increment in LH pulse frequency in the castrate male fetus from 108-130 days gestation delineates a role of the fetal testis in feedback regulation of LH secretion at this stage of development. The absence of a postcastration rise in LH pulse frequency in the castrate female indicates that the fetal ovary does not play a similar role.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormone ontogeny in the ovine fetus and neonate. XXII. The effect of somatostatin on the growth hormone (GH) response to GH-releasing factor

We postulated that an increase in the biological effectiveness of somatostatin (SRIF) accounts, at least in part, for the decrease in basal and GRF-induced ovine GH (oGH) secretion observed around birth in the ovine fetus and neonate. To test this hypothesis, SRIF (SRIF-14; given as 30 micrograms/kg iv bolus, followed by 2 micrograms/kg.min for 75 min) was infused into chronically catheterized fetal and neonatal lambs, and the oGH response induced by GRF [GRF-(1-44) amide; 1 microgram/kg] in the presence of exogenous SRIF was compared to the oGH response induced by GRF in saline-infused controls. In fetuses of 115-122 days gestation, SRIF had no detectable effect on the oGH response to GRF [peak incremental oGH response (mean +/- SEM), 527 +/- 124 vs. 562 +/- 103 ng/ml in controls]. In neonatal lambs (3-17 days old), SRIF completely suppressed the immediate oGH response to GRF (peak incremental response, 0.8 +/- 1.3 vs. 111 +/- 34 ng/ml in controls; P less than 0.02). In late gestational fetuses (126-139 days old), a transitional pattern was observed (peak incremental oGH response, 207 +/- 56 vs. 324 +/- 30 ng/ml in controls; P less than 0.04). In the second part of this study, we explored, in the neonatal lamb, the hypothesis that SRIF withdrawal plays a role in pulsatile GH secretion and that the amount of GRF to which the somatotrope is exposed before SRIF withdrawal is a major factor in determining the amplitude of GH bursts. SRIF (SRIF-14; a 30 micrograms/kg bolus, followed by 2 micrograms/kg.min) was infused iv for 40 min, GRF [GRF-(1-44) amide; 1 microgram/kg] was injected iv 20 min after starting the SRIF infusion, and the oGH rise after SRIF withdrawal was evaluated. In one series of controls GRF was replaced by saline, and in the other SRIF was replaced by saline. The oGH rise during recovery after SRIF alone was lower than that after the combined administration of SRIF and GRF (peak oGH increment, 8 +/- 3 vs. 38 +/- 12 ng/ml; P less than 0.04). The amplitude of the GH pulse after SRIF and GRF was similar to the immediate oGH response to GRF alone. These studies show that SRIF is unable to suppress the immediate oGH response to GRF in the ovine fetus, and that the suppressive effect of SRIF on the immediate oGH response to GRF increases gradually in late gestation and sharply at birth.(ABSTRACT TRUNCATED AT 400 WORDS)     

Progesterone stimulates luteinizing hormone secretion by acting directly on the pituitary.

To determine if progesterone (P) does affect gonadotropin secretion by acting directly on the pituitary, six women with hypothalamic gonadotropin deficiency were studied. They were treated with 17 beta-estradiol (E2; 2 mg/day, orally) to induce P receptors and maintain constant plasma E2 levels during two 15-day periods separated by 1 month. GnRH was administered iv at a dose of 10 microgram/pulse every 90 min during the last 5 days of E2 treatment. Either P (400 mg/day) or a placebo was administered intravaginally in a cross-over randomized design during the 5 days of pulsatile GnRH therapy. A baseline study of pulsatile LH secretion was performed, with sampling performed every 10 min for 8 h. The sampling was then repeated on day 15 of each study period at the end of pulsatile GnRH administration. Plasma levels of E2 and P were measured every day during the 5 days of either GnRH and P or GnRH and placebo treatment. In the six patients, the observed apulsatile pattern of LH during the baseline study confirmed the diagnosis of complete gonadotropin deficiency. Plasma E2 levels were not significantly different at the time of each pulse analysis (288 +/- 61 vs. 252 +/- 77 pmol/L). The plasma P level achieved with the vaginal pessaries was 22 +/- 5 nmol/L. P treatment resulted in all cases in a significant increase in the mean plasma LH level (5.2 +/- 0.9 vs. 3.6 +/- 0.7 IU/L after GnRH plus placebo; P less than 0.001). Furthermore, LH pulse amplitude was significantly increased by P compared to placebo (3.1 +/- 0.3 vs. 1.4 +/- 0.1 IU/L, respectively; P less than 0.01). Mean plasma FSH levels were significantly increased by GnRH regardless of whether P or placebo was present. In conclusion, these data indicate that a short exposure to physiological levels of P in the range of early luteal phase levels has a stimulatory effect on LH secretion by acting directly at the pituitary level.     

Hormone ontogeny in the ovine fetus: XIV. The effect of 17 beta-estradiol infusion on fetal plasma gonadotropins and prolactin and the maturation of sex steroid-dependent negative feedback

17 beta-Estradiol (E2; 100 micrograms/kg X day) was infused iv for 6 days into three chronically catheterized ovine fetuses beginning at 105-106 days of gestation (term, 147 days) and into four younger fetuses for 3 days commencing at 87-92 days. Control studies were performed on three fetuses at each age. At 90 days, E2 had no effect on the basal concentration of either LH or FSH. In both control and E2-infused fetuses, repeated LHRH testing was associated with a decreased LH response, but the decrease was not greater in the E2-infused fetuses. The FSH response was unaffected in either group. At 105 days, E2 caused suppression of the mean basal concentration of plasma LH from 1.2 +/- 0.1 to 0.1 +/- 0.1 ng/ml (P less than 0.01) and of basal FSH from 5.6 +/- 0.5 to 3.3 +/- 0.6 ng/ml (P less than 0.05). The LH response to LHRH administration (50-micrograms iv bolus) also was suppressed by E2. The maximal incremental LH response fell from 7.1 +/- 0.4 to 3.3 +/- 0.5 ng/ml (P less than 0.01); the integrated response decreased from 5.8 +/- 0.2 to 1.8 +/- 0.2 ng/ml X h (P less than 0.01). However, no effect was observed on the rise in FSH concentration evoked by LHRH. In control studies at 105 days, basal and LHRH-stimulated gonadotropin concentrations remained constant during the experiment. These results indicate that the capacity for exogenous E2 to suppress fetal pituitary gonadotropin secretion in the ovine fetus develops between 90 and 105 days of gestation, before the normal ontogenic decrease both in the basal concentration of fetal pituitary gonadotropins and in LHRH-stimulated FSH and LH secretion, which occur later in gestation. We suggest that the development of the E2-sensitive negative feedback mechanism in the fetal hypothalamic-pituitary unit is a major factor in this decrease. At 90 days gestational age, the mean fetal plasma concentration of PRL was not affected by the infusion of E2 into the fetus (5.9 +/- 1.5 ng/ml before E2 infusion 8.1 +/- 3.2 ng/ml during E2 infusion). However, at 105 days, the mean PRL concentration rose from 49.3 +/- 18.2 to 101.6 +/- 26.1 ng/ml (P less than 0.01) after the infusion of E2. The capacity for E2 to stimulate PRL release develops in parallel with the rise in fetal plasma PRL and estrogen concentrations. The results provide evidence that circulating estrogens are a determinant of fetal PRL concentrations in late gestation.     

Control of gonadotropin secretion in the ovine fetus. III. Effect of castration on serum follicle-stimulating hormone levels during the last trimester of gestation

Gonadal involvement in fetal FSH regulation was examined by studying FSH levels in 13 female (7 castrate and 6 sham control) and 13 male (8 castrate and 5 sham control) chronically catheterized ovine fetuses operated upon in utero at 106-115 days gestation (term = 147 days). These fetuses had been studied previously for pulsatile LH secretion every 2-7 days over a 2- to 38-day period until fetal delivery or death. From each study day, 3 1-h spaced blood samples (1.5-2.0 ml) were taken for FSH determination by RIA (NIH FSH-S8 standard), and the results were averaged. The overall mean was then calculated for each fetus. In female fetuses, there was no significant difference in mean serum FSH levels between castrates [53.4 +/- 5.0 ng/ml (+/- SEM)] and controls (52.5 +/- 14.4 ng/ml). In contrast, serum FSH levels in the eugonadal males were significantly (P less than 0.001) lower (23.4 +/- 8.0 ng/ml) than those in castrate males (56.9 +/- 7.1 ng/ml, a value comparable to those observed in both female groups). Mean serum FSH levels declined significantly (P less than 0.001) in castrate fetuses of both sexes after 125 days (61.5 +/- 5.6 vs. 42.4 +/- 7.7 ng/ml in females; 64.1 +/- 6.1 vs. 51.5 +/- 8.6 ng/ml in males). In the males, the FSH decline did not reach sham control levels, which remained unchanged with advancing gestation. Moreover, mean serum FSH levels were significantly higher in a group of 4 male fetuses (62.2 +/- 13.7 ng/ml) castrated at 121-130 days gestation compared to values in 3 age-matched sham castrate controls (22.1 +/- 2.6 ng/ml; P less than 0.001). The increment in serum FSH levels in castrate compared to sham castrate male fetuses demonstrates an important role for the fetal testis in FSH regulation from 106 days gestation until term. The lack of a detectable castration effect on the relatively high serum FSH levels in eugonadal females indicates that the fetal ovary does not play a similar role and suggests that in females, FSH is secreted in a functionally castrate mode. The decline in FSH levels after 125 days in castrate fetuses of both sexes may result at least in part from the previously reported coincident rise in circulating levels of feto-placental sex steroids and/or PRL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitory effect of bromocriptine treatment on luteinizing hormone secretion in polycystic ovary syndrome

In order to investigate a possible common role of central dopaminergic mechanisms in the release of PRL and LH in patients with the polycystic ovary syndrome (PCO), plasma LH pulsatile profiles and the response to GnRH were studied in a group of 12 PCO patients before and after 3 months of treatment with bromocriptine, 2.5 mg twice daily. They were divided into two groups of six patients according to the occurrence or not of hyperprolactinemia (plasma PRL, 30.3 +/- 2.7 (SE) ng/ml vs. 9.5 +/- 0.8 (SE) ng/ml). Integrated LH secretion significantly decreased in hyperprolactinemic [2537 +/- 371 (SE) vs. 907 +/- 102 mIU/ml X min] as well as in normoprolactinemic (2847 +/- 460 vs. 901 +/- 152 mIU/ml X min) patients, but there was no difference in the response of the two groups. The LH increment after a bolus injection of 100 micrograms GnRH was reduced (P less than 0.01) to the same extent in both groups. These results indicate a dopaminergic component in the control of LH release in PCO patients, independent of the mechanism governing PRL secretion. Since bromocriptine reduced LH secretion, it may be useful for the management of this condition.     

Effects of progesterone on pulsatile luteinizing hormone (LH) secretion and LH subunit messenger ribonucleic acid during lactation in the rat

In ovarian-intact lactating rats, removal of the suckling stimulus leads to restoration of pituitary LH beta mRNA levels and pulsatile LH secretion after 72 h, which correlates with a sharp decrease in plasma progesterone concentrations to basal levels. In contrast, in ovariectomized lactating rats, the increase in pituitary LH function is observed by 24 h after pup removal. To determine if progesterone secretion from the ovary participates in the delayed recovery of LH secretion, we treated lactating rats with the progesterone antagonist RU 486 and determined the effects on the time course of recovery of pulsatile LH secretion and LH subunit mRNA after pup removal and on pituitary responsiveness to GnRH. In ovarian-intact lactating rats treated with RU 486, pulsatile LH secretion was observed in about 40% of the rats within 24 h after pup removal (LH interpulse interval, 43.7 +/- 8.3 min) and in about 90% of the rats within 48 h after pup removal (LH interpulse interval, 46.1 +/- 3.6 min). The mean plasma LH level in the RU 486-treated rats was 10.1 +/- 2.2 ng/ml 24 h after removal of pups (control, less than 5 ng/ml) and had increased to 35.1 +/- 6.4 ng/ml 48 h after pup removal (control, 9.1 +/- 2.5 ng/ml). However, RU 486 treatment had no significant effect on LH mRNA subunit levels. To determine whether progesterone acts at the pituitary to block GnRH stimulation of LH secretion, we tested the effects of RU 486 on LH secretion in response to 2- and 5-ng pulses of GnRH. Pituitary responsiveness was tested 24 h after pup removal. We found that both doses of GnRH were effective in stimulating pulsatile LH secretion, and treatment with RU 486 had no significant effect on this response. We conclude from these studies that progesterone secretion from the ovary contributes to the inhibition of LH secretion that occurs after pup removal, since antagonizing progesterone's action resulted in an earlier restoration of pulsatile LH secretion. The increase in LH secretion occurred in the absence of any significant changes in responsiveness of the pituitary to GnRH stimulation or in LH subunit mRNA levels. Therefore, the primary site of action of progesterone would appear to be at the hypothalamus to suppress pulsatile GnRH secretion.     

Leptin regulates pulsatile luteinizing hormone and growth hormone secretion in the sheep

Administration of leptin during reduced nutrition improves reproductive activity in several monogastric species and reverses GH suppression in rodents. Whether leptin is a nutritional signal regulating neuroendocrine control of pituitary function in ruminant species is unclear. The present study examined the control of pulsatile LH and GH secretion in sheep. We determined whether exogenous leptin could prevent either the suppression of pulsatile LH secretion or the enhancement of GH secretion that occur during fasting. Recombinant human met-leptin (rhmet-leptin; 50 microg/kg BW; n = 8) or vehicle (n = 7) was administered s.c. every 8 h during a 78-h fast to estrogen-treated, castrated yearling males. LH and GH were measured in blood samples collected every 15 min for 6 h before fasting and during the last 6 h of fasting. Leptin was measured both by a universal leptin assay and by an assay specific for ovine leptin. During the fast, endogenous plasma leptin fell from 1.49 +/- 0.16 to 1.03 +/- 0.13 ng/ml. The average concentration of rhmet-leptin 8 h after leptin administration was 18.0 ng/ml. During fasting, plasma insulin, glucose, and insulin-like growth factor I levels declined, and nonesterified fatty acid concentrations increased similarly in vehicle-treated and leptin-treated animals. In vehicle-treated animals, LH pulse frequency declined markedly during fasting (5.6 +/- 0.5 vs. 1.1 +/- 0.5 pulses/6 h; fed vs. fasting; P < 0.0001). Leptin treatment prevented the fall in LH pulse frequency (5.0 +/- 0.4 vs. 4.9 +/- 0.4 pulses/6 h; P = 0.6). Neither fasting nor leptin administration altered GH pulse frequency. Fasting produced a modest increase in mean concentrations of circulating GH in control animals (2.4 +/- 0.5 vs. 3.4 +/- 0.6 ng/ml; P = 0.04), whereas there was a much greater increase in GH during leptin treatment (2.7 +/- 0.6 vs. 8.6 +/- 1.6 ng/ml; P = 0.0001). GH pulse amplitudes were also increased by fasting in control (P = 0.04) and leptin-treated sheep (P = 0.007). The finding that exogenous rhmet-leptin regulates LH and GH secretion in sheep indicates that this fat-derived hormone conveys information about nutrition to mechanisms controlling neuroendocrine function in ruminants.     

Evidence for decreased luteinizing hormone-releasing hormone pulse frequency in men with selective elevations of follicle-stimulating hormone

To examine the hypothesis that the frequency of endogenous pulsatile LHRH stimulation controls the relative secretion of FSH and LH from the pituitary, we studied men with elevated FSH levels and normal LH levels to determine whether they have an altered frequency of pulsatile LHRH secretion compared to normal men. Because peripheral blood measurements of LHRH do not reflect the pulsatile characteristics of hypothalamic LHRH secretion, and it is generally accepted that the pulse frequency of LH secretion is an index of the frequency of endogenous LHRH pulsation, we used LH pulse frequency as the indicator of LHRH pulse frequency. Frequent blood sampling was performed to characterize LH pulse patterns in five men with selective elevations of FSH and seven age-matched normal men. Beginning at 0800-0930 h, blood samples were obtained every 10 min for 24 h through an indwelling iv catheter. Serum LH and FSH levels were measured by RIA in each sample, and the pattern of LH secretion was determined. Testosterone (T), estradiol, sex hormone-binding globulin, and free T were measured in a pooled serum sample from each man. Men with selective elevations of FSH had fewer LH pulses per 24 h (mean +/- SEM, 10.6 +/- 0.5) than the control group (12.9 +/- 0.6; P less than 0.01). There was no statistically significant difference in LH pulse amplitude (23 +/- 4 vs. 17 +/- 3 ng/ml). There were no statistically significant differences in T (4.9 +/- 0.5 vs. 6.1 +/- 0.5 ng/ml), estradiol (23 +/- 7 vs. 31 +/- 5 pg/ml), sex hormone-binding globulin (7.7 +/- 1.4 vs. 7.7 +/- 1.2 ng bound dihydrotestosterone/ml), or free T (0.16 +/- 0.02 vs. 0.23 +/- 0.04 ng/ml) in these men vs. normal subjects. We conclude that 1) compared to normal men, men with selectively elevated FSH levels have decreased LH pulse frequency, which suggests decreased LHRH pulse frequency; and 2) the relative secretion rates of LH and FSH by the pituitary may be regulated by the frequency of pulsatile LHRH secretion from the hypothalamus.     

Diurnal patterns of pulsatile luteinizing hormone secretion in hypothalamic amenorrhea: reproducibility and responses to opiate blockade and an alpha 2-adrenergic agonist

The pattern of pulsatile GnRH secretion is abnormal in some women with hypothalamic amenorrhea (HA) consequent to previous exercise or weight loss. Both diminished frequency pulsatile LH secretion, and by inference GnRH secretion, and normal LH pulsatility have been reported. We assessed whether the patterns of GnRH secretion varied with time by measuring plasma LH every 15 or 20 min for 24 h on 1-3 occasions during a 10-month period in 14 women with HA (a total of 24 studies). During the day, mean LH pulse frequency [1.0 +/- 0.1 (+/- SE) pulses/8 h] was lower than that in normal women in the early follicular phase of their cycles (5.1 +/- 0.6), and the frequency in individual HA patients was lower than early follicular phase values in 16 of 17 studies. The slow daytime LH pulse frequency also was a consistent finding, in that the values in repeat studies varied by less than 2 pulses/8 h in all but 1 patient. LH pulse frequency (2.0 +/- 0.4 pulses/8 h) was higher and more variable during sleep, and normal early follicular phase frequencies were found in 20% of patients with HA. The mechanisms whereby GnRH pulse frequency is reduced are not known. alpha-Adrenergic agonist drugs stimulate GnRH pulsatile secretion in rodents, but administration of the alpha 2-agonist clonidine (0.15 mg, orally, at 0800 and 2000 h) did not increase the frequency of LH pulses in 7 women (1.7 +/- 0.4 pulses/8 h). In contrast, administration of naloxone (1 mg/m2 X h, iv) for 8 h during the day to 14 patients, increased LH pulse frequency (3.3 +/- 0.5 pulses/8 h). In 8 of these 14 women, LH pulse frequency (4.9 +/- 0.4 pulses/8 h) increased into the range found during the normal early follicular phase, while in the other 6 women pulse frequency was not significantly increased (1.4 +/- 0.4 pulses/8 h). Plasma estradiol levels were similar in naloxone-responsive and unresponsive women, but spontaneous LH pulse frequency was higher at night in naloxone-responsive patients (2.9 +/- 0.6 vs. 1.4 +/- 0.3 pulses/8 h). The presence of nocturnal LH pulses did not predict responsiveness to naloxone, however, and LH pulse frequency was less than 2 pulses/8 h in 4 of the women who responded to naloxone. These data indicate that slow frequency GnRH secretion is a common finding during the day in women with HA. GnRH secretion is more variable at night, suggesting that the mechanisms involved in reducing pulsatile GnRH secretion are less effective during sleep.(ABSTRACT TRUNCATED AT 400 WORDS)     

Luteinizing hormone and prolactin secretion in hypophysial-stalk-transected pigs given estradiol and pulsatile gonadotropin-releasing hormone

To determine the sites of action whereby estradiol (E2) induces the preovulatory luteinizing hormone (LH) surge in pigs, hypophysial-stalk-transected (HST) ovariectomized (OVX) gilts (mean +/- SE: 118 +/- 12 kg) received intramuscular injections of E2 benzoate (10 micrograms E2B/kg body weight) on day 0, plus intravenous pulses of 1 microgram gonadotropin-releasing hormone (GnRH) every 45 min either from days -5 to 4, days -5 to 0 or days -5 to 0 then days 2 to 4. A fourth and fifth group received steroid vehicle on day 0 and pulses of GnRH on either days -5 to 4 or days -5 to 0 then days 2 to 4. In stalk-intact OVX gilts, E2B inhibited LH release for 48 h, then induced an LH surge that peaked 60-84 h after steroid treatment. In HST OVX gilts, serum LH increased from undetectable concentrations (less than 0.17 ng/ml) to 0.42 +/- 0.03 ng/ml after 5 days of pulsatile GnRH replacement. In the presence of pulsatile GnRH stimulation, serum LH concentrations were inhibited for 12 h after E2B but then returned to values that were similar to those for HST gilts given GnRH in the absence of steroid treatment. Discontinuing GnRH pulses at the time of E2B injection caused serum LH concentrations to drop to 0.18 ng/ml or less for the remaining 96 h. When GnRH pulses were interrupted for 48 h, beginning immediately after injecting E2B or vehicle to mimic the secretory hiatus of LH and presumably GnRH observed in stalk-intact pigs prior to the surge, resumption of GnRH pulses caused serum LH concentrations to increase progressively over 3 h in E2B-treated gilts but to peak abruptly in controls given vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation of pulsatile cortisol and adrenocorticotropin secretion in fetal sheep during late gestation.

In the fetal sheep, plasma cortisol concentrations gradually increase in the last weeks of gestation and abruptly rise during the final 48-72 h preceding birth. To determine if these changes in mean circulating cortisol concentrations result from increased pulsatile secretion and are driven by changes in ACTH pulses, blood samples from five chronically catheterized fetuses were collected every 5 min for 2 h at 133 days gestation and every 4 days thereafter until delivery at 146 +/- 2 days. Volume was replaced after each blood sample and erythrocytes were returned every 20 min. Plasma cortisol and ACTH secretion were pulsatile in fetuses at all ages. Cortisol pulse frequency increased significantly with gestation from a mean of 2.2 pulses/2 h at 133 days to 4.8 pulses/2 h at 146 days. The interpulse interval (mean +/- SE) decreased between 133 and 146 days from 54 +/- 11 min to 23 +/- 3 min, respectively. Cortisol pulse amplitude increased significantly from 10 +/- 2 ng/ml at 133 days to 44 +/- 13 ng/ml at 146 days. In contrast to cortisol, ACTH pulse frequency (3 +/- 0.6 pulses/2 h) and amplitude (21 +/- 3 pg/ml) were similar at 133 days and 146 days. The coincidence of cortisol and ACTH pulses did not change between 133 and 146 days. Furthermore, the number of coincident pulses failed to exceed random associations (hypergeometric probability analysis) and could have occurred by chance alone (P values ranged from 0.11-0.63). A point by point comparison of cortisol and ACTH concentrations in fetal circulation indicate that only 36% of the variance in cortisol concentrations could be explained by variance in ACTH (cross-correlation analysis). These data suggest that fetal cortisol and ACTH secretion are pulsatile and that, as gestation advances, increases in constitutive cortisol pulse amplitude and frequency may not be predominantly driven by pulsatile changes in ACTH in the ovine fetal circulation near term.     

Acylated ghrelin inhibits spontaneous luteinizing hormone pulsatility and responsiveness to naloxone but not that to gonadotropin-releasing hormone in young men: evidence for a central inhibitory action of ghrelin on the gonadal axis

Context: Recent evidence suggests that ghrelin exerts a negative modulation on the gonadal axis. Ghrelin was reported to suppress LH secretion in both animal and human models. Moreover, acylated ghrelin (AG) also decreases the LH responsiveness to GnRH in vitro. Objective: The objective of the study was to evaluate the effects of AG infusion on spontaneous and stimulated gonadotropin secretion. Design, Participants, and Intervention: In seven young healthy male volunteers (age mean +/- sem 26.4 +/- 2.6 yr), we evaluated LH and FSH levels every 15 min during: 1) iv isotonic saline infusion; 2) iv saline followed by AG; LH and FSH response to GnRH (100 mug iv as a bolus), 3) alone and 4) during AG infusion; LH and FSH response to naloxone (0.1 mg/kg iv as a slow bolus), 5) alone and 6) during AG infusion. Results: Significant LH but not FSH pulses were recorded in all subjects under saline infusion. AG infusion inhibited LH levels [area under the curve((240-480)): 415.8 +/- 69.7 mIU/ml.min during AG vs. 744.6 +/- 120.0 mIU/ml.min during saline, P < 0.02] and abolished LH pulsatility. No change in FSH secretion was recorded. The LH and FSH responses to GnRH during saline were not affected by AG administration. However, AG inhibited the LH response to naloxone [area under the curve ((120-210)): 229.9 +/- 39.3 mIU/ml.min during AG vs. 401.1 +/- 44.6 mIU/ml.min during saline, P < 0.01]. FSH levels were not modified by naloxone alone or in combination with AG. Conclusions: AG inhibits both spontaneous LH pulsatility and the LH response to naloxone. Because AG does not affect the LH response to GnRH, these findings indicate that the ghrelin system mediates central inhibition of the gonadal axis.     

Gonadotropin-releasing hormone pulsatile administration restores luteinizing hormone pulsatility and normal testosterone levels in males with hyperprolactinemia

Hyperprolactinemia in men is frequently associated with hypogonadism. Normalization of serum PRL levels is generally associated with an increase in serum testosterone (T) to normal. To determine the mechanism of the inhibitory effect of hyperprolactinemia on the hypothalamic-pituitary-gonadal axis, we studied the effect of intermittent pulsatile GnRH administration on LH pulsatility and T levels in four men with prolactinomas. All patients had high PRL values (100-3000 ng/ml), low LH (mean +/- SEM, 2.2 +/- 0.1 mIU/ml), and low T values (2.3 +/- 0.3 ng/ml), with no other apparent abnormality of pituitary function. GnRH was administered iv using a pump delivering a bolus dose of 10 micrograms every 90 min for 12 days. No LH pulses were detected before treatment. Pulsatile GnRH administration resulted in a significant increase in basal LH levels (6.7 +/- 0.6 mIU/ml; P less than 0.001) and restored LH pulsatility. In addition, T levels increased significantly to normal values in all patients (7.8 +/- 0.4 ng/ml; P less than 0.001) and were normal or supranormal as long as the pump was in use, although PRL levels remained elevated. These data, therefore, suggest that hyperprolactinemia produces hypogonadism primarily by interfering with pulsatile GnRH release.     

Hormone ontogeny in the ovine fetus. XXVI. A sex difference in the effect of castration on the hypothalamic-pituitary gonadotropin unit in the ovine fetus

The detection of pulsatile ovine LH (oLH) secretion in the sheep fetus by 81 days gestation (term 147 days), the suppression of fetal gonadotropin secretion by chronic administration of an LH-releasing factor agonist or antagonist, and the capacity of N-methyl; D-aspartate (a neuroexcitatory amino acid analog) to evoke a fetal oLH pulse strongly support a functional LH-releasing factor pulse-generator in the ovine fetus. In light of the sex difference in fetal gonadal function and gonadotropin secretion before day 114, we postulated that fetal castration would have a discordant effect on the pattern of gonadotropin secretion in males and females. Fetal sheep were either castrated (male = 11; female = 9) or sham operated (male = 9; female = 6) at 110-115 days gestation. Chronic indwelling arterial and venous catheters were implanted, and animals were studied for up to 30 days. During each study period fetal arterial blood samples were drawn every 15 min for 5 h and the plasma assayed for oFSH and oLH by specific RIAs. Multiple studies were performed on each fetus. In all fetuses (both intact and castrated) a decrease in oLH pulse frequency occurred after day 130. In female fetuses before day 130, castration had no effect on mean oLH pulse frequency (sham, 0.72 +/- 0.19 pulses/5 h; castrate, 0.50 +/- 0.13 pulses/5 h; P greater than 0.05). After day 130, pulsatile oLH secretion decreased in both intact and castrated female fetuses to undetectable levels during the sampling period. In contrast, castration significantly (P less than 0.001) increased mean oLH pulsatility in males before and after day 130 (less than 130 days, sham, 1.06 +/- 0.24 pulse/5 h; castrate, 2.70 +/- 0.22 pulse/5 h; greater than 130 days, sham, 0.18 +/- 0.12 pulses/5 h; castrate, 1.65 +/- 0.26 pulses/5 h). Mean oLH pulse amplitude was increased by castration only in the male fetuses (sham, 3.89 +/- 0.87 ng/ml; castrate, 6.02 +/- 0.39 ng/ml; P less than 0.05). oFSH pulses were infrequent in both sexes and not influenced by castration. The mean plasma concentration of oFSH was greater in intact female fetuses than in intact males (female, 5.65 +/- 1.15 ng/ml vs. male, 2.07 +/- 0.45 ng/ml; P less than 0.01). Castration increased the mean value for plasma oFSH in males (4.40 +/- 0.43 ng/ml; P less than 0.001) but had no effect in females (3.83 +/- 0.64 ng/ml; P greater than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Galanin is a physiological regulator of spontaneous pulsatile secretion of growth hormone in the male rat.

To determine whether galanin (GAL), a 29-amino acid neuropeptide, plays a role in the physiological regulation of the pulsatile secretion of GH and PRL in the male rat, secretory patterns of both hormones were studied in freely moving animals after GAL passive immunoneutralization. Adult male Sprague-Dawley rats were equipped with iv and intracerebroventricular catheters. After 7 days, 3 microliters of a specific GAL antiserum (GAL-AS) or normal rabbit serum (NRS; controls) were infused in the third ventricle of 10 rats, 25 and 1 h before the animals were bled every 15 min for 6 h (1000-1600 h). Plasma GH and PRL concentrations were measured by RIA, and the hormonal secretory patterns were analyzed by the PULSAR program. Control rats, treated with NRS, displayed typical GH secretion, with pulses of high amplitude (167 +/- 27 ng/ml) and low frequency (2.4 +/- 0.2 pulses/6 h), separated by periods of low trough levels (3.8 +/- 0.6 ng/ml). Rats treated with GAL-AS had altered pulsatile GH secretion. Pulse height was markedly reduced (77 +/- 15 ng/ml; P less than 0.01 vs. controls), and peak frequency was higher (3.6 +/- 0.5 pulses/6 h; P less than 0.05), while GH baseline levels and integrated GH secretion over the 6-h sampling period remained unaltered. Injection of rat GH-releasing hormone (1 microgram/rat, iv) caused a similar GH stimulation in both groups of rats, as determined by the peak GH response at 5 min (368 +/- 112 vs. 342 +/- 81 ng/ml) or by the integrated GH response over 1 h (5.13 +/- 1.30 vs. 4.77 +/- 1.15 micrograms.min/ml in NRS- and GAL-AS-treated rats, respectively; P less than 0.05). In contrast to GH, pulsatile secretion of PRL was not affected by the GAL-AS treatment. These results indicate that GAL is a physiological regulator of spontaneous pulsatile secretion of GH, but not PRL, in the male rat. The influence of GAL on GH secretion appears to be exerted within the hypothalamus, mainly by a stimulation of GRF secretion. However, the changes in GH pulse frequency observed after GAL immunoneutralization suggest that GAL might also influence the somatostatin inhibitory tone.     

Naloxone increases the frequency of pulsatile luteinizing hormone secretion in women with hyperprolactinemia

The ability to change the frequency and amplitude of pulsatile GnRH secretion may be an important mechanism in maintaining regular ovulatory cycles. Hyperprolactinemia is associated with anovulation and slow frequency LH (GnRH) secretion in women. To assess whether the slow frequency of LH (GnRH) secretion is due to increased opioid activity, we examined the effect of naloxone infusions in eight amenorrheic hyperprolactinemic women (mean +/- SE, serum PRL, 160 +/- 59 micrograms/L). After a baseline period, either saline or naloxone was infused for 8 h on separate days, and LH was measured in blood obtained at 15-min intervals. Additional samples were obtained for plasma FSH, PRL, estradiol, and progesterone. Responses to exogenous GnRH were assessed at the end of the infusions. LH pulse frequency increased in all subjects from a mean of 4.0 +/- 0.5 pulses/10 h (mean +/- SE) during saline infusion to 8.0 +/- 1.0 pulses/10 h during naloxone infusion (P less than 0.01). LH pulse amplitude did not change, and mean plasma LH increased from 7.4 +/- 0.8 IU/L (+/- SE) to 11.2 +/- 1.5 IU/L during naloxone (P less than 0.01). A small but significant increase was seen in mean plasma FSH. Plasma PRL, estradiol, and progesterone were unchanged by naloxone infusion. These data suggest that elevated serum PRL reduces the frequency of LH (GnRH) secretion by increasing hypothalamic opioid activity and suggest that the anovulation in hyperprolactinemia is consequent upon persistent slow frequency LH (GnRH) secretion.     

Ontogeny of pulsatile secretion of gonadotropin-releasing hormone in the bull calf during infantile and pubertal development

During the infantile period of development in the bull calf (birth to 6 weeks of age), there is a virtual absence of episodic secretion of LH. Transition from infancy to the prepubertal period (6-10 weeks of age) is characterized by the onset of episodic LH release. This study was conducted to characterize the ontogeny of episodic GnRH release during these developmental periods. During the primary experiment, calves at 2, 5, 8, and 12 weeks of age (n = 4/age) were surgically fitted with cannulae for the collection of mixed hypophyseal portal and cavernous sinus blood. Hypophyseal portal and cavernous sinus and jugular blood samples were collected over a 9- to 12-h period at 10 min intervals. No pulses of LH were observed in calves at 2 or 5 weeks of age. At 8 and 12 weeks of age, pulsatile LH release became evident with a mean of 1.0 +/- 0.3 and 2.20 +/- 0.7 pulses/10 h, respectively. Unlike LH secretion, calves at both 2 and 5 weeks of age released GnRH in a pulsatile manner (3.5 +/- 0.2 and 5.0 +/- 0.6 pulses/10 h, respectively). The frequency of pulsatile GnRH release increased from 7.9 +/- 0.4 pulses/10 h at 8 weeks of age to 8.9 +/- 0.7 pulses/10 h at 12 weeks of age. These findings demonstrate the presence of pulsatile secretion of GnRH during the infantile period of development. Furthermore, the postnatal ontogeny of pulsatile LH release in this species is associated with an increase in the frequency of pulsatile GnRH secretion.