Changes in the pulsatile pattern of luteinizing hormone secretion during the rat estrous cycle

To ascertain whether changes in the pattern of GnRH release from the hypothalmus occur during the 4-day rat estrous cycle, the pattern of LH release was characterized on each day of the estrous cycle, and the results were interpreted in light of the changes in pituitary responsiveness to GnRH previously described by this laboratory to occur during this time. Blood samples were taken from intact, freely moving rats via venous catheters at 6- to 10-min intervals for 3-4 h. LH pulse height and LH interpulse interval were quantified on each day of the cycle, and the transition on the afternoon of proestrus from tonic LH release to the preovulatory LH surge was detailed. The effects on the pattern of LH release during estrus of small doses of GnRH (0.4 ng) and the continuous infusion of the opioid antagonist naloxone were also examined. Plasma LH concentrations (NIAMDD rat LH-RP-1) were determined with a highly sensitive LH RIA. LH pulses were identified using the PULSAR algorithim. The LH interpulse intervals of 46 +/- 2 min on diestrous-1 day, 49 +/- 4 min on diestrous day 2, and 60 +/- 8 min on proestrus immediately before the LH surge were not significantly different. There were no changes immediately preceding the preovulatory LH surge on the afternoon of proestrus in either the LH interpulse interval or the LH pulse height. Instead, the transition from tonic LH secretion to the preovulatory LH surge was found to occur abruptly. These data suggest that an abrupt increase in GnRH secretion during the afternoon of proestrus initiates the dramatic rise in LH concentrations. The pattern of LH secretion during the day of estrus differed significantly from that on the other days of the cycle in that no LH pulses were observed. However, the administration of small pulses of GnRH elicited physiological elevations in LH release. Furthermore, the continuous infusion of naloxone to estrous rats immediately stimulated a pulsatile pattern of LH secretion, with a LH interpulse of 56 +/- 4 min. These data indicate that the absence of LH pulses during estrus may result from a deficit in GnRH release. Similar modifications in GnRH release during the other days of the cycle were inferred from the observed changes in LH pulse heights. The LH pulse height of 21 +/- 3 ng/ml on diestrous day 2 was significantly less than the LH pulse height of 41 +/- 4 ng/ml on diestrous day 1 or 35 +/- 4 ng/ml on proestrus before the surge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of gestational mastectomy on postpartum gonadotropin releasing hormone and thyrotropin releasing hormone-induced luteinizing hormone and prolactin response in first lactation Holstein cattle

First lactation Holstein cows were divided into two treatment groups to evaluate thyrotropin releasing hormone (TRH, 0.25 microgram/kg body weight) and gonadotropin releasing hormone (GnRH; 200 micrograms) induced secretion of prolactin (PRL) and luteinizing hormone (LH) on days 7 and 16 postpartum. Disregarding treatment, LH response was greater (p less than 0.01) on day 16 than day 7 postpartum (7.5 +/- 0.3 ng/ml on day 7 vs 10.2 +/- 0.3 ng/ml serum on day 16). Mastectomized cattle had similar time for initiation of LH increase, but peak concentrations were achieved later. Peak PRL concentrations were reached 12 to 15 min after injection and returned to baseline within 2.5 h in both groups. However, intact cows had higher (p less than 0.01) mean serum PRL than the mastectomized cows for 1 h following injection. Peak PRL concentration was 83.3 +/- 17.6 ng/ml for mastectomized cows vs 128.0 +/- 24.7 ng/ml for intact cows. It appears that udder removal allows for greater pituitary responsiveness to GnRH but diminishes PRL response to TRH suggesting the mammary gland differentially affects pituitary secretion of LH and PRL.     

Endogenous opioid suppression of luteinizing hormone pulse frequency and amplitude in the ewe: hypothalamic sites of action.

Evidence suggests that endogenous opioid peptides (EOP) inhibit pulsatile luteinizing hormone (LH) secretion during both the luteal and follicular phases of the ovine estrous cycle. Further data from sheep and other species indicate that the hypothalamus is the primary site of action for this EOP inhibition. The purpose of the following experiments was to determine which areas of the hypothalamus are involved in the EOP inhibition of pulsatile LH secretion. Regularly cycling ewes (n = 10) were stereotaxically implanted with guide tubes into the preoptic area (POA) and medial basal hypothalamus (MBH). Implants containing the EOP antagonist WIN 44,441-3 (WIN) were placed into each of these areas. Blood samples were collected at 12-min intervals for 3 h before and during WIN administration in the luteal phase and for 4 h before and during WIN administration in the follicular phase of the estrous cycle. During the luteal phase, WIN implants in either area increased (p less than 0.01) LH pulse frequency (POA 1.4 +/- 0.3/3 h before vs. 3.1 +/- 0.4/3 h during; MBH 1.1 +/- 0.2/3 h before vs. 2.8 +/- 0.5/3 h during). There was no effect on LH pulse amplitude. In contrast, during the follicular phase, WIN implants selectively increased (p less than 0.01) LH pulse frequency when implanted in the POA (3.2 +/- 0.4/4 h before vs. 5.2 +/- 0.6/4 h during) while increasing (p less than 0.05) only LH pulse amplitude when placed in the MBH (0.7 +/- 0.2 ng/ml before vs. 1.4 +/- 0.3 ng/ml during).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of gonadal steroids on gonadotropin secretion in males: studies with perifused rat pituitary cells

The feedback effects of testosterone (T) and estradiol (E2) on FSH and LH secretion were compared in dispersed pituitary cells from adult male rats perifused with pulses of GnRH. Cells were stimulated with 10 nM GnRH for 2 min every 1 h. T (10 nM) pretreatment for 24 h reduced the amplitude of FSH and LH pulses to 77 +/- 4% (mean +/- SE) and 47 +/- 3% of control values, respectively (P less than 0.01), whereas 6-h T treatment was without effect. By contrast, interpulse secretion of FSH was increased after 24 h T to 184 +/- 7% of the control value (P less than 0.01), but interpulse LH release was unchanged (104 +/- 5%). E2 (0.075 nM) treatment of pituitary cells reduced GnRH-stimulated FSH and LH release within 2 h to 75 +/- 2% and 73 +/- 3% of control values, respectively (P less than 0.01). E2 pretreatment for 24 h stimulated (P less than 0.025) GnRH-induced FSH (136 +/- 10%) and LH (145 +/- 8%) release and also increased (P less than 0.01) interpulse FSH (127 +/- 5%) and LH (145 +/- 8%) secretion. These data indicate that the suppression of FSH and LH secretion by T in males is due in part to a direct effect on the pituitary. The findings that T suppresses GnRH-stimulated FSH less than LH, and that T stimulates interpulse FSH, but not LH, provide evidence for differential regulation of FSH and LH secretion by T. The dissimilar actions of T on GnRH-stimulated pulses and interpulse gonadotropin secretion suggest that interpulse secretion is unrelated to stimulation by GnRH, although its physiological significance is unknown. Since E2, in physiological levels for males, increased pituitary FSH and LH secretion, the suppression of gonadotropin secretion by E2 in vivo in males may result from an effect on the hypothalamic pulse generator; however, additional studies are needed before extending these conclusions to higher mammals and men.     

Sex steroid control of gonadotropin secretion in the human male. II. Effects of estradiol administration in normal and gonadotropin-releasing hormone-deficient men

Although prior studies have suggested that estrogens exert their negative feedback effect at the pituitary level in men, these conclusions have been based on models that evaluate changes in LH pulse amplitude and frequency and, therefore, only provide indirect information concerning the site of action of estrogens. To assess whether estradiol (E2) inhibits gonadotropin secretion directly and solely at the pituitary level in men, we determined the pituitary responses to physiological doses of GnRH in six men with complete GnRH deficiency, whose pituitary-gonadal function had been normalized with long term pulsatile GnRH delivery, before and during a 4-day continuous E2 infusion (90 micrograms/day). To deduce whether E2 has an additional inhibitory effect on hypothalamic GnRH secretion, their responses were compared with the effects of identical E2 infusions on spontaneous gonadotropin secretion and the responses to a 100-micrograms GnRH bolus in six normal men. Both groups were monitored with 15 h of frequent blood sampling before and during the last day of the E2 infusion. In the GnRH-deficient men, the first three GnRH doses were identical and chosen to produce LH pulses with amplitudes in the midphysiological range of values in our normal men (i.e. a physiological dose), while the last four doses spanned 1.5 log orders (7.5, 25, 75, and 250 ng/kg). The 250-ng/kg dose was always administered last because it is known to be pharmacological. In the GnRH-deficient men, mean LH and FSH levels as well as LH pulse amplitude all decreased significantly (P less than 0.02) during E2 infusion, demonstrating a direct pituitary-suppressive effect of E2. Mean LH (P less than 0.01) and FSH (P less than 0.05) levels and LH pulse amplitude (P less than 0.01) also decreased significantly in the normal men. The degree of suppression of mean LH (52 +/- 3% vs. 42 +/- 12%) and FSH (49 +/- 10% vs. 37 +/- 10%) levels was similar in the two groups. These results provide direct evidence that E2 inhibits gonadotropin secretion at the pituitary level in men and suggest that the pituitary is the most important, and possibly the sole, site of negative feedback of estrogens in men.     

CHANGES IN THE RESPONSIVENESS OF LUTEINIZING HORMONE SECRETION TO INFUSION OF THE OPIOID ANTAGONIST NALOXONE THROUGHOUT MALE SEXUAL MATURATION

The present study investigated the time of male sexual maturation during which hypothalamic inhibitory opioid activity can be detected. Normal prepubertal (Tanner stage G 1 (Ts-G1) (n = 4], early pubertal (Ts-G2 (n = 5], pubertal (Ts-G3 (n = 4), and Ts-G4 (n = 2] and adult subjects (Ts-G5 (n = 4] receives a rapid infusion of the selective opiate antagonist nalocone (NAL) (20 mg over 10 min). LH secretion was assessed by frequent (every 10 min for 2 h) venous sampling before and after administration of the opiate blocker, as well as by the LH response to exogenous GnRH. All but one (a Ts-G2 subject) pubertal boys showed aprompt and sustained increase in serum LH concentrations after NAL administration, as disclosed by the areas under the LH curve (aLHc) calculated from samples obtained before and after NAL infusion (aLHc in four Ts-G2 responders, 162 +/- 20 (mean +/- SEM) vs 314 +/- 56 mIU/ml/min before and after NAL respectively, P less than 0.025; Ts-G3, 227 +/- 35 vs 362 +/- 56 mIU/ml/min, P less than 0.025; Ts-G4 and Ts-G5, 432 +/- 77 vs 687 +/- 91 mIU/ml/min, P less than 0.05). In contrast, none of the prepubertal children had significant changes in LH secretion after the NAL challenge (154 +/- 17 vs 154 +/- 9 mIU/ml/min). Although all NAL responders exhibited serum testosterone (T) levels above 5 nmol/l, a positive correlation between individual T values and magnitude of LH responses to NAL was not found. All subjects had significant serum LH increments after GnRH administration. In a second series of studies, additional groups of Ts-G1 subjects were primed during 5 days either with GnRH alone or with GnRH plus sex steroids (ethinyl oestradiol 12.5 micrograms/12 h or testosterone enanthate 1.8 mg/kg body weight (single dose], before NAL administration, to investigate whether hypothalamic opioid activity might be unmasked by additional sex steroids. None of the priming schemes significantly modified the pituitary LH responses to NAL infusion (GnRH-primed group, 145 +/- 48 vs 139 +/- 43 mIU/ml/min before and after NAL, respectively; GnRH plus ethinyl oestradiol-primed group, 124 +/- 42 vs 107 +/- 34 mIU/ml/min; GnRH plus testosterone enanthate-primed group, 64 +/- 10 vs 57 +/- 24 mIU/ml/min). This study suggests that the development and/or maturation of the opioid control of LH secretion is temporally related with the onset of puberty.(ABSTRACT TRUNCATED AT 400 WORDS)     

QUANTITATIVE AND QUALITATIVE CHANGES IN LH SECRETION FOLLOWING PULSATILE GnRH THERAPY IN A MAN WITH IDIOPATHIC HYPOGONADOTROPHIC HYPOGONADISM

The pattern of bioactive and immunoreactive LH secretion before and during pulsatile GnRH therapy (18 micrograms/90 min) in a hypogonadotrophic hypogonadal male has been studied. Before treatment the patient was azoospermic and had low testosterone (1.2 nmol/l) with low and apulsatile immunoreactive LH (1.9 +/- 0.2 IU/l) and FSH (1.4 +/- 1.9 IU/l) levels. There was no detectable LH bioactivity. During the first 24 h of GnRH therapy there was a small increase in immunoreactive (5.4 +/- 0.8 IU/l) and bioactive (6.7 +/- 1.3 IU/l) LH, with an irregular pattern and little effect on testosterone production (2.2 nmol/l). Within 1 week of treatment both bioactive (30.5 +/- 6.8 IU/l) and immunoreactive (13.6 +/- 1.5 IU/l) LH levels were above the normal range and the pattern of secretion was pulsatile. The bioactive to immunoreactive (B:I) LH ratios within the pulses (2.6 +/- 0.3) were higher (P less than 0.01) than between pulses (1.97 +/- 0.1) and the testosterone concentration (17.8 +/- 2.1 nmol/l) was now normal. At one month LH secretion was similar and testosterone pulses of high amplitude were evident corresponding to high-amplitude bioactive LH pulses. By 3 months mature spermatozoa (1.3 x 10(6)/ml) were seen in the patient's semen. The pattern of LH secretion was pulsatile but the levels of bioactive (13.1 +/- 3.6 IU/l) and immunoreactive (9.5 +/- 1.3 IU/l) LH decreased towards the normal range reflecting maturation of the testicular feedback control at the pituitary level. This effect was more pronounced on bioactive rather than immunoreactive LH secretion (57% vs 32% relative decrease). At 6 months LH levels were similar and the sperm count was normal (34 x 10(6)/ml).     

Social status controls LH secretion and ovulation in female marmoset monkeys (Callithrix jacchus)

The suppression of ovulation in subordinate female marmosets was associated with suppressed pituitary LH secretion and reduced pituitary LH response to gonadotrophin-releasing hormone (GnRH). In subordinate females, basal plasma LH concentrations were commonly below 2 IU/l (n = 5) (maximum 10.7 IU/l). Plasma oestrogen concentrations were similarly low (maximum 0.62 nmol/l) and plasma progesterone concentrations of below 30 nmol/l confirmed the anovulatory condition. This infertility condition was rapidly reversed when subordinate females (n = 5) were removed from their social groups and housed singly, when plasma LH (maximum 140.0 IU/l) and oestrogen (maximum 7.84 nmol/l) concentrations increased preceding ovulation. Infertility was rapidly reimposed when these singly housed females were re-introduced to subordinate status in new social groups, when plasma LH concentrations fell to their previous low values within 4 days; no ovulation occurred thereafter. Plasma oestrogen levels also fell, but less dramatically. The luteal phases of three of the subordinate females were shortened following the re-instatement of subordinate status. The maximum LH response of subordinate females to the highest dose of GnRH (200 ng) was only 19.1 +/- 6.7 IU/l (mean +/- S.E.M.; n = 8): this contrasted with that in dominant females in either the follicular phase (40.0 +/- 13.3 IU/l; n = 6) or the luteal phase (126.7 +/- 24.9 IU/l; n = 10) of the ovarian cycle. These results suggest that the social suppression of fertility in subordinate female marmosets is mediated by impaired hypothalamic GnRH secretion. Such an immediate and precise behavioural control of LH secretion and ovulation is without equal in anthropoid primates.     

Gonadotropin-releasing hormone (GnRH) analog suppression renders polycystic ovarian disease patients more susceptible to ovulation induction with pulsatile GnRH

Pulsatile GnRH administration consistently restores normal reproductive hormone levels and ovulation in women with hypogonadotropic hypogonadism, but is less effective in those with polycystic ovarian disease (PCOD). We pharmacologically created a hypogonadotropic condition with a GnRH analog (GnRH-A) in six women with PCOD to investigate the role of deranged gonadotropin secretion in PCOD and to improve the response to pulsatile GnRH ovulation induction. Before GnRH and GnRH-A treatment the women with PCOD had increased LH pulse frequency [one pulse every 55 +/- 2 (+/- SE) min; P less than 0.05] and LH pulse amplitude (10.9 +/- 1.4 U/L; P less than 0.05) compared to normal women in the follicular phase of their menstrual cycle. Each PCOD woman completed one cycle of pulsatile GnRH administration for ovulation induction before (pre-A cycles; n = 6) and one or two cycles after (post-A cycles; n = 9) GnRH-A administration [D-Ser(tBu)6-Des,Gly10-GnRH; 300 micrograms, sc, twice daily for 8 weeks]. Pulsatile GnRH (5 micrograms/bolus) was given at 60-min intervals using a Zyklomat pump. Daily blood samples were drawn during the pulsatile GnRH ovulation induction cycles for the determination of serum LH, FSH, estradiol (E2), progesterone, and testosterone, and pelvic ultrasonography was done at 1- to 4-day intervals. Mean (+/- SE) serum LH levels were elevated during the pre-A cycle (49.2 +/- 3.1 IU/L) and decreased to normal levels during the post-A cycles (19.6 +/- 1.4 IU/L; P less than 0.0001). Mean testosterone concentrations were lower during the post-A cycles [88 +/- 2 ng/dL (3.1 +/- 0.1 nmol/L)] than during the pre-A cycles [122 +/- 3 ng/dL (4.2 +/- 0.1 nmol/L); P less than 0.0001]. In the follicular phase of the post-A cycles E2 levels were significantly lower [81 +/- 5 pg/mL (300 +/- 20 pmol/L) vs. 133 +/- 14 pg/mL (490 +/- 50 pmol/L); P less than 0.0001], preovulatory ovarian volume was smaller (24.6 +/- 2.0 vs. 31.4 +/- 2.4 cm3; P less than 0.01), and the FSH to LH ratio was higher (0.56 +/- 0.03 vs. 0.16 +/- 0.01) than in the pre-A cycle, suggesting more appropriate function of the pituitary-gonadal axis. Excessive LH and E2 responses to pulsatile GnRH administration in the early follicular phase of the pre-A cycle were abolished in the post-A cycles.(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.     

Histamine-induced paradoxical GH response to TRH/GnRH in men and women: dependence on gonadal steroid hormones

A stimulatory GH response to TRH and GnRH occurs frequently in patients with various pathological conditions, but is absent in normal subjects. We have previously shown that histamine induced a paradoxical GH response to TRH in normal men. Since gonadal steroids influence GH secretion, we investigated whether infusion of histamine might induce a GH response to combined administration of TRH (200 micrograms) and GnRH (100 micrograms) in 6 normal women during the early follicular and luteal phase of the same menstrual cycle and in 7 normal men. Histamine had no effect on basal GH secretion in men or in women during the two phases of the menstrual cycle. However, compared with saline, histamine induced a GH response to TRH/GnRH in men (GH peak: 5.5 +/- 1.0 vs 1.4 +/- 0.3 micrograms/l; p less than 0.01) and in women during the luteal phase (GH peak: 5.2 +/- 1.6 vs 1.5 +/- 0.4 micrograms/l; p less than 0.025), but not during the early follicular phase of the cycle (GH peak: 1.7 +/- 0.5 vs 1.6 +/- 0.3 micrograms/l). In luteal-phase women the GH response to TRH/GnRH correlated with the serum estradiol-17 beta level (GH area/E2: r = 0.98; p less than 0.005) and the serum estrone level (GH area/E1: r = 0.81; p less than 0.05). In men the GH response to TRH/GnRH did not correlate with estrogen or androgen levels. We conclude that high physiological levels of estrogens are pertinent to the activation of a histamine-induced GH response to TRH/GnRH in women, whereas the role of androgens and estrogens for the induction of the response in men seems more complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testosterone administration inhibits gonadotropin secretion by an effect directly on the human pituitary

Testosterone (T) administration slows LH pulse frequency in man, presumably by an effect on the hypothalamic GnRH pulse generator, but it also may have a direct action on the pituitary. To determine if T does indeed affect gonadotropin secretion by acting directly on the pituitary, we studied the effect of T on GnRH-stimulated gonadotropin secretion. Six men with hypogonadotropic hypogonadism were treated with physiological doses of GnRH (5 micrograms every 2 h, sc by automatic infusion pump) for 6 weeks. Once their gonadotropin levels were normal, the men received a supraphysiological dosage of T enanthate (200 mg, im, weekly for 8 weeks) in addition to GnRH. They then received GnRH alone for a final 8-week period. Blood sampling was performed every 10 min for 8 h at the end of each of the three study periods. T administration suppressed the mean serum LH level to about 50% of the value during GnRH alone [18 +/- 2 (+/- SE) vs. 37 +/- 2 micrograms/L; P less than 0.05] and suppressed the mean serum FSH level to about 30% of the value during GnRH alone (39 +/- 6 vs. 128 +/- 28 micrograms/L; P less than 0.05). Eight weeks after stopping T, while continuing GnRH alone, serum LH and FSH levels were similar to those at the end of the first period of GnRH administration. The mean LH response to GnRH was reduced during T administration (17 +/- 3 micrograms/L) compared to that during the initial period of GnRH alone (31 +/- 4 micrograms/L; P less than 0.05). Serum T and estradiol levels were in the low normal range after GnRH alone before T administration (11 +/- 2 nmol/L and 105 +/- 17 pmol/L, respectively) and increased to just above the normal adult ranges after 8 weeks of T administration (36 +/- 5 nmol/L and 264 +/- 49 pmol/L, respectively). These results demonstrate that T and/or its metabolites inhibit LH and FSH secretion by a GnRH-independent mechanism, probably directly on the pituitary gland, in man.     

Impotence associated with low biological to immunological ratio of luteinizing hormone in a man with a pituitary stone

Quantitative reduction in LH secretion resulting from hypothalamic-pituitary dysfunction is a known cause of impotence. Qualitative abnormalities of secreted LH, however, have not been described under these circumstances. During evaluation of a 39-yr-old man with impotence and a calcified pituitary mass (pituitary stone), we detected a qualitative abnormality of LH characterized by a low ratio of bio- to immunoactivity (B:I). Initial work-up revealed basal morning serum testosterone levels of 2.14, 3.18, 3.97, and 3.11 ng/ml on 4 separate days, low to low normal urinary LH (300, 200, and 478 mIU/h), and normal GH, TSH, PRL, and ACTH secretion after provocative testing. The response of impotence to testosterone but not placebo in a double blind trial confirmed the clinical significance of the borderline low androgen levels. These findings prompted a systematic analysis of 24-h LH pulses as well as clomiphene and GnRH responsiveness. By RIA, mean serum LH levels [9.1 +/- 0.3 (+/- SE) mIU/ml] and all other response parameters were normal. In striking contrast, mean serum LH by bioassay was low (9.9 +/- 0.4 mIU/ml vs. 41.4 +/- 5.7 in normal subjects), as were B:I ratios (1.0 +/- 0.03 vs. control values of 3.1 +/- 0.5 to 5.3 +/- 0.3). Only during maneuvers designed to increase GnRH were B:I ratios increased to 3.3 +/- 0.22 (exogenous GnRH) and 1.8 +/- 0.12 (clomiphene). Mean testosterone levels before and after exogenous GnRH treatment were 3.28 +/- 0.24 and 4.76 +/- 0.16, respectively (P less than 0.001). The results suggest an association between the low LH B:I ratio and the anatomical disruption of the hypothalamic-pituitary portal system by the pituitary stone. The increased B:I ratio during GnRH or clomiphene administration indicates a functional link between pituitary GnRH exposure and the greater potency of the LH secreted.     

Pituitary gonadotropin-releasing hormone receptor regulation in mice. II: Females

The regulation of pituitary GnRH receptors (GnRH-R) by gonadal steroids was examined in female mice housed in a constant environment (six to 8 per cage in same room as males). A 60% decrease in GnRH-R occurred 7 days after ovariectomy (OVX) (9.2 +/- 0.9 fmol/pituitary OVX vs. 25 +/- 2 for intact random estrous cycle controls). The receptor affinity (Ka 1.86 X 10(9) M-1) remained constant in intact and OVX female mouse pituitary particles. The pattern of GnRH-R fall after OVX was similar to that found in male mice, except that the GnRH-R decrease began some 6 h later than in males and serum LH also rose more slowly. Serum FSH was significantly elevated 6 h post OVX. In contrast to males, pituitary LH, in spite of a rapid fall (60%) at 12 h, regained the random, estrous cycle control value by 4 days post OVX and then increased to above this level. Pituitary FSH, unlike in males, remained at the intact value (3.1 +/- 0.24 micrograms/pituitary) up to 24 h post OVX and then gradually rose to 7.9 +/- 0.37 micrograms/pituitary on day 4 and 15.5 +/- 0.32 micrograms/pituitary on day 7. Treatment of OVX female mice with estradiol-17 beta (300 ng/day) attenuated the postcastration GnRH-R fall, and was more effective when combined with progesterone (375 micrograms/day). Progesterone alone was ineffective. The GnRH-R fall post OVX persisted for up to 2 months, despite elevated serum and pituitary LH and FSH levels. GnRH-R fell by 40% in lactating mice (20.6 +/- 0.95-lactating vs. 32.4 +/- 1.25 fmol/pituitary-random, estrous cycling females). Serum LH was reduced by 70% in lactating mice. These findings are qualitatively and quantitatively similar to those in lactating rats suggesting that, in this physiological situation, a similar mechanism may account for the receptor fall in both species. In sex reversed (Sxr) mice (genotypic female-phenotypic male) GnRH-R values were about 50% higher than those of intact normal male and normal, random estrous cycling, female values. This was the only situation in mice in which pituitary GnRH-R increases were observed to date. Serum and pituitary LH and FSH values in Sxr mice were elevated, especially when compared with normal, random estrous cycling female controls. The results indicate that pituitary GnRH-R of female mice fall in response to removal of gonadal steroid feedback, in the same way as males.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca+2 dependence of gonadotropin-releasing hormone-stimulated luteinizing hormone secretion: in vitro studies using continuously perifused dispersed rat anterior pituitary cells

A continuously perifused dispersed rat anterior pituitary cell system was used to determine the importance of calcium (Ca+2) on the release of LH by GnRH. In response to continuous exposure to 10 nM GnRH, LH was released in a biphasic fashion; arbitrarily, phase I was defined as being the LH released during the initial 40 min and phase II as the subsequent release. Withdrawal of Ca+2 from the perifusion medium during phases I or II of LH release led to a rapid inhibition of the LH secretion. Cells were exposed to GnRH for 2.5 min, washed with medium for 30 min, and then reexposed to GnRH for 30 min. This sequence was repeated 1 h later under identical conditions in the presence of a Ca+2 blocking agent; D600 (20 or 100 microM). D600 inhibited both the 2.5- and the 30-min GnRH-stimulated LH release. The results were expressed as the ratio obtained by dividing the total LH released during the second GnRH exposure (either 2.5 or 30 min) by the total LH released during the respective initial GnRH exposure of same duration. For the cells perifused with 20 microM D600 the ratios +/- SE (D600 vs. control) were 0.48 +/- 0.06 vs. 1.28 +/- 0.13 (P = 0.0001) and 0.29 +/- 0.05 vs. 1.01 +/- 0.08 (P less than or equal to 0.0001) for the 2.5- and 30-min exposures, respectively. For the cells perifused with 100 microM D600 the ratios +/- SE (D600 vs. control) were 0.18 +/- 0.05 vs. 1.28 +/- 0.13 (P less than or equal to 0.00001) and 0.12 +/- 0.03 vs. 1.01 +/- 0.08 (P = 0.002) for the 2.5- and 30-min exposures, respectively, revealing an even more profound inhibitory effect of D600 on GnRH stimulated LH secretion. Our data both confirm previous reports that Ca+2 is involved in LH release and demonstrate that Ca+2 is an essential requirement during both phases of GnRH-stimulated LH release in perifused dispersed rat anterior pituitary cells.     

Effects of epinephrine, norepinephrine, and dopamine on gonadotropin-releasing hormone-induced secretion of luteinizing hormone in vitro

Recent evidence indicates that catecholamines may directly alter anterior pituitary function. In the present study, an in vitro perifusion system was used to investigate whether catecholamines affect the gonadotrope. Pituitary tissue from castrated ram lambs was incubated in the presence of epinephrine (EPI), norepinephrine (NE), or dopamine (DA). During a 2-h treatment period, neither DA (10(-8) M), NE (10(-7) M), nor EPI (10(-7) - 10(-9) M) significantly affected basal LH secretion. In contrast, the LH response to a subsequent 10(-10) M GnRH challenge was significantly potentiated by NE and EPI. NE increased the amount of LH secreted in response to GnRH 14 +/- 1.1% (P less than 0.01). Likewise, 10(-7), 10(-8), and 10(-9) M EPI resulted in 22 +/- 1.4% (P less than 0.001), 13 +/- 1.2% (P less than 0.001), and 6 +/- 1.3% (P less than 0.03) increases, respectively. The stimulatory effect of 10(-7) M EPI was blocked by pretreatment with propranolol (a beta-adrenergic blocker), but not with phentolamine (an alpha-adrenergic blocker). The beta-adrenergic agonist isoproterenol enhanced GnRH-induced LH secretion 46 +/- 1.5% (P less than 0.001), but had no effect on basal LH release. DA had no effect on LH secretion; however, it inhibited PRL release 24 +/- 0.9% (P less than 0.001). Neither NE, EPI, nor isoproterenol had any effect on PRL secretion. These results suggest that EPI, acting by a beta 2-adrenergic receptor, modulates the pituitary gonadotrope's response to GnRH.     

Evidence for a spontaneous nitric oxide release from the rat median eminence: influence on gonadotropin-releasing hormone release

The involvement of nitric oxide (NO) as a gaseous neurotransmitter in the hypothalamic control of pituitary LH secretion has been demonstrated. NO, as a diffusible signaling gas, has the ability to control and synchronize the activity of the neighboring cells. NO is secreted at the median eminence (ME), the common termination field for the antehypophysiotropic neurons, under the stimulation of other signaling substances. At the ME, NO stimulates GnRH release from neuroendocrine terminals. The present studies were undertaken to determine whether NO is secreted spontaneously from ME fragments ex vivo and whether its secretion is correlated to GnRH release. To accomplish this, female rats were killed at different time points of the day and/or of the estrous cycle. The spontaneous NO release was monitored in real time, with an amperometric probe, during 4 periods of 30 min, from individual ME fragments (for each time point, n = 4). GnRH levels were measured in parallel for each incubation-period by RIA. The results revealed that NO was released in a pulsatile manner from female ME fragments and, unambiguously, that the amplitude of NO secretion varied markedly across the estrous cycle. Indeed, though the NO pulse period (32 +/- 1 min, n = 36) and duration (21 +/- 2 min, n = 36) did not vary significantly across the estrous cycle, the amplitude of this secretion pulse was significantly higher on proestrus (Pro; 39 +/- 3 nM, n = 20), compared with diestrus (16 +/- 1 nM, n = 8) or estrus (23 +/- 3 nM, n = 8, P < 0.05). The GnRH levels in the incubation medium were positively correlated to NO secretion across the estrous cycle (r = 0.86, P < 0.003, n = 9), confirming that NO and GnRH release are coupled. Furthermore, 5 x 10(-7) M L-N(5)-(1-iminoethyl)ornithine (L-NIO), a NO synthase inhibitor, succeeded in inhibiting the strong NO-GnRH secretory coupling and GnRH release on PRO: Because at this concentration, L-NIO selectively inhibits endothelial NO synthase, the results further demonstrate that the major source of NO involved in GnRH release at the ME is endothelial in origin. Additionally, the induction of a massive NO/GnRH release in 15-day ovariectomized rat treated with estradiol benzoate strongly suggested that estradiol is participating in the stimulation of NO release activity between diestrus II and PRO: The present study is the first demonstrating that ME can spontaneously release NO and that NO's rhythm of secretion varies markedly across the estrous cycle. This pulsatile/cyclic ME NO release may constitute the synchronizing link to anatomically scattered GnRH neurons.     

Evidence for a hypothalamic site of action of clomiphene citrate in women

To examine the site of action of clomiphene citrate (CC), LH and FSH pulsatile amplitude, frequency, and responsiveness to GnRH (10 micrograms, iv) were studied in 11 women during the early follicular phase of the menstrual cycle. Six women received CC (150 mg/day) on cycle days 2, 3, and 4, while 5 women received placebo tablets. Blood samples were drawn at 10-min intervals for 8 h before and after the treatment regimen on cycle days 2 and 5, respectively. All women treated with CC had multiple follicular development, as determined by ultrasound. Peripheral levels of estradiol did not change after CC treatment, while progesterone levels decreased slightly. Mean levels of LH increased from 7.5 +/- 0.9 (+/- SEM) to 10.7 +/- 1.4 mIU/ml (P less than 0.05), and FSH increased from 6.7 +/- 0.9 to 10.1 +/- 0.9 mIU/ml (P less than 0.01). After exposure to CC, the pulse frequency of LH during an 8-h period increased significantly (3.3 +/- 0.7 on day 2 vs. 6.8 +/- 0.8 on day 5; P less than 0.01), while the pulse frequency of FSH increased from 3.8 +/- 0.6 to 5 +/- 1.4, as determined by computer pulse analyses. The pulse amplitude of LH and FSH was not significantly altered. In the placebo-treated group, neither pulse amplitude nor pulse frequency changed significantly between cycle days 2 and 5. Pituitary sensitivity to exogenous GnRH did not change after CC treatment. Since the pulsatile frequency of LH is governed by hypothalamic influences, these findings provide compelling evidence for a hypothalamic site of action for CC, probably by inducing an increase in the frequency of GnRH secretion.     

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.     

Follicle stimulating hormone-secreting pituitary adenoma: inappropriate secretion and effect of pulsatile luteinizing hormone releasing hormone analogue (buserelin) administration.

A patient with an FSH secreting pituitary adenoma is reported. Elevated FSH and serum free alpha-subunit (SU) with low levels of LH and testosterone (T) were found. Immunostaining showed the presence of alpha-SU, FSH-beta and LH-beta subunits. LHRH analogue (buserelin) was administered in a pulsatile manner, by portable computerized infusion pump sc for ten days. During the first 24 h of treatment FSH, LH (p less than 0.001) and T (p less than 0.01) rose significantly. Ten days later, the expected desensitization phenomenon did not occur, but further increases of T (8.4 +/- 2.6, mean +/- SD, vs 17.4 +/- 4.1 nmol/l, p less than 0.001) and FSH (58.9 +/- 9.6 vs 70.7 +/- 3.8 mlU/ml, p less than 0.001) were registered. LH decreased (12.5 +/- 2.4 vs 7.1 +/- 0.6 mlU/ml, p less than 0.001) at day 10, but remained higher than basal level (5.0 +/- 0.6, p less than 0.001). Free alpha-SU also rose (2.8 +/- 0.4 vs 4.4 +/- 1.7 mlU/ml, p less than 0.001) after ten days of treatment. The chronic stimulatory effect of analogue on LH with a lack of desensitization suggests tumorous secretion despite a partially preserved negative feedback of testosterone. Low basal LH levels, in some patients with FSH secreting tumors may not be due to tumor mass effect, but rather may be the consequence of altered LH production and/or secretion by the tumor. Although buserelin may not have a therapeutic effect, it is of use in differential diagnosis of hypergonadotropinemia.