Biphasic response in the secretion of gonadotrophin-releasing hormone in ovariectomized ewes injected with oestradiol

In ovariectomized ewes, an injection of oestrogen initially inhibits the tonic secretion of LH, and then induces a large release of LH similar to the preovulatory surge in intact ewes. The pattern of hypothalamic secretion of gonadotrophin-releasing hormone (GnRH) into the pituitary portal blood during this biphasic response to oestrogen was investigated in conscious, unrestrained, ovariectomized adult Ile-de-France ewes during the breeding season. The ewes were ovariectomized and implanted with cannulae for portal blood collection on the same day. Seven days later, portal and peripheral blood samples were collected simultaneously every 5 min for 25 h. The ewes were injected with oestradiol-17 beta (25 micrograms i.v. and 25 micrograms i.m.) 6.25 h after the start of sampling. GnRH and LH were measured by radioimmunoassay in portal and jugular plasma samples respectively. A clear pulsatile pattern of LH secretion was observed before the oestradiol injection in all the ewes, followed by the typical biphasic decrease (negative feedback) and increase (positive feedback) in mean concentrations. The sampling period was divided, for analysis, into pretreatment, negative feedback and positive feedback phases. Before injection with oestradiol, the GnRH pulses were clearly defined in portal blood and were always synchronized with LH pulses in the peripheral circulation. The frequency was 5.9 +/- 0.6 pulses/6 h (mean +/- S.E.M.), and the amplitude was 31.6 +/- 7.6 pmol/l. During negative feedback, both the frequency (4.2 +/- 0.5 pulses/6 h, P less than 0.01) and amplitude (15.2 +/- 4.6 pmol/l, P less than 0.05) of the GnRH pulses decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteinizing hormone-beta mRNA levels are regulated primarily by gonadotropin-releasing hormone and not by negative estrogen feedback on the pituitary

Long-term effects of gonadotropin-releasing hormone (GnRH) and/or estrogen on pituitary mRNA levels for the beta-subunit of luteinizing hormone (LH-beta) were determined in anterior pituitary glands from ovariectomized (OVX) ewes. The relative roles of these two factors were assessed by studying hypothalamopituitary disconnected (HPD) ewes with appropriate hormonal treatments. Levels of LH-beta mRNA were increased by ovariectomy and substantially reduced by HPD. Treatment of OVX-HPD ewes with pulses of GnRH (250 ng each 2 h) for 1 week restored LH-beta mRNA levels to OVX levels, whereas treatment with estrogen alone did not alter the low levels found in OVX-HPD ewes. Combined GnRH and estrogen treatment for one week produced LH-beta mRNA levels that were similar to those found in OVX-HPD ewes given GnRH alone; plasma LH pulse amplitudes were also similar in these two groups. From these data we conclude that the long-term negative feedback effect of estrogen to reduce LH secretion is due to a primary inhibition of GnRH secretion and is not a pituitary effect of estrogen. Long-term regulation of LH-beta mRNA is thus primarily regulated by GnRH.     

Pattern of gonadotropin-releasing hormone (GnRH) secretion leading up to ovulation in the ewe: existence of a preovulatory GnRH surge

We have previously shown LH surges induced by physiological estradiol levels are invariably accompanied by robust and sustained GnRH surges in the ewe. Such an increase, however, has not been observed consistently during the preovulatory LH surge. In the present study, we examined GnRH secretion in Suffolk and Ile de France ewes during the preovulatory period using a method for pituitary portal blood collection which allows simultaneous portal and jugular blood samples to be taken at frequent intervals for up to 48 h. Ewes were sampled either during the mid-late luteal phase (n = 8) or follicular phase (n = 20). During the follicular phase, a robust increase in GnRH secretion occurred at the onset of the LH surge in 11 of 12 ewes sampled during the LH surge. The GnRH increase in most ewes was a massive surge, reaching values averaging 40-fold greater than baseline and extending well beyond the end of the preovulatory LH surge. In the single ewe not exhibiting a GnRH surge during the LH surge, postmortem inspection indicated blood was probably not sampled from the pituitary portal vessels. In the early follicular phase, GnRH-pulse frequency was greater than that observed in the luteal phase and, within the follicular phase, GnRH-pulse frequency increased further and amplitude decreased as the surge approached. These data demonstrate GnRH secretion leading up to ovulation in the ewe is dynamic, beginning with slow pulses during the luteal phase, progressing to higher frequency pulses during the follicular phase and invariably culminating in a robust surge of GnRH. The LH surge, however, ends despite continued elevation of GnRH.     

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)     

A surge of gonadotropin-releasing hormone accompanies the estradiol-induced gonadotropin surge in the rhesus monkey.

In several species, the ovulatory LH surge is preceded by a surge of GnRH. Although a role for estradiol in the initiation of the LH surge is well established in the primate, several observations in the rhesus monkey have questioned whether such an estradiol-induced neurosecretory event takes place. We report on GnRH measurements in cerebrospinal fluid (CSF) samples obtained from the third ventricle of intact and ovariectomized (OVX) conscious rhesus monkeys during control periods and throughout the estradiol-induced positive feedback phase. In the first experiment, we measured control GnRH concentrations in CSF collected at 15-min intervals uninterruptedly for a period of 1-5 days in tethered OVX monkeys (n = 4) in their cages without steroid priming. As had been demonstrated previously with the same method in restrained animals, CSF from the third ventricle contained detectable amounts of GnRH. Spontaneous GnRH secretion was pulsatile; overall mean pulse interval was 67.4 (+/- 2.2 SE) min for a total of 177 GnRH pulses. During 2 periods (8 and 6 h) when simultaneous blood and CSF samples were obtained, 14 out of 15 GnRH pulses were accompanied by an LH pulse. To evaluate the effects of an estrogen challenge on GnRH secretion, estradiol benzoate (E2B; 330 micrograms) was given to 4 intact (5 experiments) and to 2 OVX monkeys. CSF collection was initiated 8-24 h before E2B injection and continued for 72-84 h thereafter. E2B administration resulted in a surge of LH and of GnRH in all but one experiment. The mean time of onset of the GnRH surge was 22.0 (+/- 4.0) h after E2B, whereas that of the LH surge was 24.7 (+/- 3.4) h. In contrast to LH, which declined after a peak at 35.2 +/- 3.9 h, the increase in GnRH secretion persisted throughout most of the observation period. The magnitude of the GnRH response differed in the 2 groups; in the intact animals, mean peak GnRH concentration increased 8.9-fold but only 3.8-fold in the OVX monkeys. A similar GnRH surge was observed in 1 OVX monkey, receiving an iv infusion of E2, which produced more physiological concentrations of E2. In this animal, an initial suppression of GnRH concentration in the 24-48 h period after E2 (GnRH control, 14.6 +/- 1.9; post-E2, 4.0 +/- 0.5 pg/ml) preceded the initiation of the GnRH surge which occurred at 54 h after E2.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Pharmacodynamics of gonadotropin-releasing hormone. 1. Effects of gonadotropin-releasing hormone pulse contour on pituitary luteinizing hormone secretion in vivo in sheep

The gonadotropin-releasing hormone (GnRH) waveform arrives at the pituitary gonadotropes via the pituitary portal blood and provides the immediate suprapituitary stimulus to luteinizing hormone (LH) secretion. Despite their importance, nature and influence of the physiological GnRH waveform in vivo have been difficult to study. Recent pharmacological and in vitro studies have suggested the potential importance of the wave contour as a specific and independent factor in the pharmacodynamic effects of GnRH on pituitary gonadotrope LH secretion in vivo, and it has been hypothesized that the steepness of the rising edge of the GnRH wave contour is a specific determinant of pituitary LH secretion. In order to investigate the pharmacodynamic influence of GnRH pulse wave contour on pituitary LH secretion in vivo, variations in plasma LH responses to alterations in GnRH wave contour were measured in chronic ovariectomized, hypothalamopituitary-disconnected sheep undergoing physiological pulsatile GnRH maintenance regimen at a fixed dose (250 ng/pulse) and frequency (interpulse interval 120 min). Variable wave contours were then generated by administration of the same total GnRH pulse dose over various lengths of time from near-instantaneous bolus to increasing lengths of constant-rate infusion time up to 8 min. This model allowed specific examination of pulse wave contour in the absence of concurrent changes in endogenous GnRH or sex steroid secretion and holding constant GnRH pulse dose, frequency, and route of administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The suppression of pulsatile luteinizing hormone secretion during lactation in the rat

The inhibition of LH secretion during lactation may be the consequence of a pituitary insensitivity to GnRH stimulation and/or an inhibition of GnRH release from the hypothalamus. To assess the contribution that these mechanisms may make to the suppression of LH secretion during lactation, we described the pattern of LH secretion in lactating rats and the magnitude of LH secretion in response to a GnRH stimulus. We assessed the effect of the strength of the suckling stimulus (two and eight pups), the length of lactation (5 and 10 days), and the presence of the ovaries on the pattern of LH secretion. We also examined the pattern of LH secretion after removal of a large suckling stimulus. In the intact rat, the pattern of LH secretion during lactation was uniformly nonpulsatile, despite significant differences between animals suckling two and eight pups in pituitary responsiveness to GnRH. In intact rats suckling two pups during day 10 of lactation, significant LH secretion was stimulated by 0.4-ng pulses of GnRH every 50 min, while animals with eight pups secreted little LH in response to the same stimulus. It was concluded that a two-pup suckling stimulus was sufficient to completely suppress pulsatile GnRH release without affecting pituitary function, whereas an eight-pup suckling stimulus also depressed pituitary sensitivity to GnRH. In ovariectomized (ovx) rats suckling two pups, seven of nine animals showed no postcastration rise in LH secretion or evidence of pulsatile LH secretion during day 5 of lactation. In the remaining two animals, a castrate pattern of pulsatile LH secretion was observed, with a LH interpulse interval of 31 +/- 6 min. By day 10 of lactation, all animals suckling two pups had castration patterns of LH secretion, with a LH interpulse interval of 35 +/- 2 min, which was significantly different from the LH interpulse interval of 26 +/- 1 min observed in ovx animals without pups. Therefore, a two-pup suckling stimulus is capable of retarding the increase in LH pulse frequency characteristically seen in the rat after castration. In ovx rats suckling eight pups, the postcastration rise in LH secretion was completely inhibited in all animals examined on days 5 and 10 of lactation, and the pattern of LH secretion was uniformly nonpulsatile. A consistent pattern of pulsatile LH secretion was not reinitiated until 72 h after removal of the suckling stimulus (LH interpulse interval, 31 +/- 2 min).(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct pituitary effects of estrogen and progesterone on gonadotropin secretion in the ovariectomized ewe

The direct pituitary effects of estrogen and progesterone on the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were studied in ovariectomized (OVX) ewes in which the pituitary had been disconnected surgically from the hypothalamus (hypothalamo-pituitary disconnection, HPD). Gonadotropin secretion was restored with hourly pulses of 500 ng gonadotropin-releasing hormone (GnRH) via intra-atrial cannulae. Intramuscular injections of 50 micrograms estradiol benzoate (EB) to 5 sheep initially caused reductions (approximately 50%) in plasma LH baseline, peak values and LH pulse amplitude. Thereafter all parameters of plasma LH concentration increased 2- to 3-fold above starting values. After these 5 sheep had received 2 subcutaneous progesterone implants (mean +/- SEM plasma levels 5.3 +/- 1.5 nmol/l), the biphasic LH response to EB was still apparent and increases in LH peak values (267 +/- 19%) and LH pulse amplitudes (262 +/- 23%) were greater (p less than 0.05) than those seen with EB alone (195 +/- 11 and 172 +/- 14%, respectively). The presence of 2 progesterone implants alone did not change plasma LH baseline, peak values or pulse amplitude, or plasma FSH values. In the second experiment, where 4 OVX-HPD ewes were given 4 progesterone implants (plasma progesterone 27.7 +/- 3.4 nmol/l), there were no effects on basal plasma LH or plasma FSH values. The LH responses to EB were more marked in 4 OVX-HPD ewes given 4 progesterone implants than in the animals given EB alone. Also, the estrogen-induced LH surge occurred earlier in the ewes given 4 progesterone implants than in those given estrogen alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diurnal and estradiol-dependent changes in gonadotropin-releasing hormone neuron firing activity

A robust gonadotropin-releasing hormone (GnRH) surge is a prerequisite signal for the luteinizing hormone (LH) surge that triggers ovulation. In rodents, the GnRH surge is initiated by elevated estradiol and a diurnal switch in estrogen action from negative to positive feedback. The ability of constant estradiol treatment to induce daily LH surges was tested in adult mice that were ovariectomized (OVX) or OVX and treated with estradiol implants (OVX+E). LH in OVX mice showed no time-of-day difference. In contrast, OVX+E mice showed a large LH surge (8- to 124-fold relative to the a.m.) in p.m. samples on d 2-5 post-OVX+E. Targeted extracellular recordings were used to examine changes in firing activity of GnRH neurons in brain slices. There was no time-of-day difference in cells from OVX mice. In contrast, OVX+E cells recorded in the p.m. showed an increased mean firing rate and instantaneous firing frequency, which could increase GnRH release, and decreased duration of quiescence between bouts of firing, possibly reflecting increased pulse frequency, compared with cells recorded in the a.m. In the a.m., OVX+E cells showed changes in GnRH neuron firing reflecting negative feedback compared with OVX cells, whereas in the p.m., OVX+E cells exhibited changes suggesting positive feedback. These data indicate that differences in pattern and level of individual GnRH neuron firing may reflect the switch in estradiol action and underlie GnRH surge generation. The persistence of altered GnRH neuron activity in slices indicates that this approach can be used to study the neurobiological mechanisms of surge generation.     

Pulsatile administration of gonadotrophin-releasing hormone stimulates, in oestrogen-treated anaesthetized ovariectomized ewes, a surge release of LH qualitatively and quantitatively different from that induced by oestradiol in conscious ovariectomized ewes

The nature of the gonadotrophin-releasing hormone (GnRH) stimulus of the pituitary necessary for the oestrogen-induced plasma LH surge was studied in ovariectomized ewes. The sheep were treated with oestradiol benzoate (50 micrograms i.m.) at 0 h, and the hypothalamic contribution to the LH surge was blocked by pentobarbitone anaesthesia over the time during which the surge was expected (11-31 h). Pituitary responsiveness to exogenous GnRH (100 ng) administered i.v. in a pulsatile mode (once per hour or once per 20 min) over the period 15-30 h was assessed from plasma concentrations of LH. Neither of the GnRH treatments induced patterns of LH secretion similar to those seen in conscious ovariectomized ewes given oestrogen only. Plasma LH secretion in response to hourly GnRH pulses was less (P less than 0.01) than that associated with oestrogen-induced plasma LH surges in conscious control ewes. With pulses of GnRH administered every 20 min the amount of LH released was greater (P less than 0.05) than that in oestrogen-treated conscious control ewes. In contrast to the single surge induced by oestradiol in conscious ewes, GnRH pulses given every 20 min elicited phasic patterns of LH secretion consisting of two or three distinct surges. The failure of GnRH treatment to elicit an LH surge similar to an oestrogen-induced surge could reflect inappropriate GnRH treatment regimens, and/or inadequate priming of the pituitary with GnRH after induction of anaesthesia but before GnRH treatment.     

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)     

The oestrogen-induced surge of LH requires a 'signal' pattern of gonadotrophin-releasing hormone input to the pituitary gland in the ewe

Two experiments were conducted with ovariectomized and hypothalamo-pituitary disconnected (HPD) ewes to ascertain the pattern of inputs, to the pituitary gland, of gonadotrophin-releasing hormone (GnRH) necessary for the full expression of an oestrogen-induced LH surge. The standard GnRH replacement to these sheep was to give pulses of 250 ng (i.v.) every 2h; at the onset of experimentation, pulses were given hourly. In experiment 1, groups of sheep (n = 7) were given an i.m. injection of 50 micrograms oestradiol benzoate, and after 10 h the GnRH pulse frequency or pulse amplitude was doubled. Monitoring of plasma LH concentrations showed that a doubling of pulse frequency produced a marked increase in baseline values, whereas a doubling of amplitude had little effect on the LH response. In a second experiment, ovariectomized HPD sheep that had received hourly pulses of GnRH for 16 h after an i.m. injection of oil or 50 micrograms oestradiol benzoate were given either a 'bolus' (2.25 micrograms GnRH) or a 'volley' (500 ng GnRH pulses 10 min apart for 30 min, plus a 500 ng pulse 15 min later). Both groups then received GnRH pulses (250 ng) every 30 min for the next 13 h. Oestrogen enhanced the LH responses to the GnRH treatments, and the amount of LH released was similar in ovariectomized HPD ewes given oestrogen plus bolus or volley GnRH treatments and ovariectomized hypothalamo-pituitary intact ewes given oestrogen. These results suggest that the oestrogen-induced LH surge is initiated by a 'signal' pattern of GnRH secretion from the hypothalamus.     

Gonadotrophin-releasing hormone secretion (GnRH) in anoestrous ewes and the induction of GnRH surges by oestrogen

Anoestrous ewes were studied to determine the pattern of secretion of gonadotrophin-releasing hormone (GnRH) in the resting state and following a single i.m. injection of 50 micrograms oestradiol benzoate. In three out of four untreated ewes, two or three GnRH pulses were observed over a 6-h sampling period. In the fourth sheep the GnRH pulse frequency was higher (six pulses/6 h), but GnRH pulse amplitudes were lower. Following oestrogen treatment, GnRH pulses continued until the occurrence of an LH surge 12 h later. In five out of six sheep sampled during the oestrogen-induced LH surge a marked rise in GnRH secretion was seen. In the sixth ewe a large pulse of GnRH was seen at the start of the LH surge followed by increased GnRH secretion. It is concluded that GnRH pulse frequency is lower, generally, during anoestrus than during the mating season, and that oestrogen treatment of anoestrous ewes causes a surge in GnRH secretion unlike that seen in similarly treated ovariectomized ewes or the natural cyclic preovulatory changes in GnRH secretion.     

Hypophyseal actions of pulsatile gonadotropin-releasing hormone in the ewe: development and application of a new experimental model

Ovariectomy of ewes during seasonal anestrus and immediate replacement with subcutaneous Silastic progesterone implants which maintained a midluteal-phase level of circulating progesterone obliterated pulsatile luteinizing hormone (LH) secretion for up to 2 weeks without preventing a normal response of the pituitary to exogenous pulses of gonadotropin-releasing hormone (GnRH). Consideration was given to the possibility that such 'progesterone-suppressed ewes' would be useful as an animal model for isolating the pituitary from pulsatile GnRH secretion, and for testing the hypophyseotropic actions of exogenous GnRH. Two experiments were conducted using this progesterone-suppressed ewe as an animal model. In the first, the amplitude of LH pulses elicited by episodic delivery of GnRH was found to depend upon the frequency of exogenous GnRH pulses. Hourly frequency produced larger LH pulses than a 30-min frequency of GnRH. In the second experiment, LH surges were induced in progesterone-suppressed ewes by a combined treatment of estradiol and GnRH in patterns designed to approximate those secreted in the follicular phase of the estrous cycle. Our findings suggest that the progesterone-suppressed ewe is a suitable animal model for studying the hypophyseotropic actions of GnRH. Further, they are consistent with two hypotheses concerning the regulation of the tonic and surge modes of LH secretion. (1) The inverse relationship between LH pulse frequency and amplitude observed in a number of situations can be accounted for, at least in part, by a differential response of the pituitary to GnRH. (2) Progesterone can block the LH surge by an action on the brain and an inhibition of pulsatile GnRH release.     

Progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by inhibiting the surge of gonadotropin-releasing hormone.

Previous studies indicate an elevation of circulating progesterone blocks the positive feedback effect of a rise in circulating estradiol. This explains the absence of gonadotropin surges in the luteal phase of the menstrual or estrous cycle despite occasional rises in circulating estradiol to a concentration sufficient for surge induction. Recent studies demonstrate estradiol initiates the LH surge in sheep by inducing a large surge of GnRH secretion, measurable in the hypophyseal portal vasculature. We tested the hypothesis that progesterone blocks the estradiol-induced surge of LH and FSH in sheep by preventing this GnRH surge. Adult Suffolk ewes were ovariectomized, treated with Silastic implants to produce and maintain midluteal phase concentrations of circulating estradiol and progesterone, and an apparatus was surgically installed for sampling of pituitary portal blood. One week later the ewes were allocated to two groups: a surge-induction group (n = 5) in which the progesterone implants were removed to simulate luteolysis, and a surge-block group (n = 5) subjected to a sham implant removal such that the elevation in progesterone was maintained. Sixteen hours after progesterone-implant removal (or sham removal), all animals were treated with additional estradiol implants to produce a rise in circulating estradiol as seen in the follicular phase of the estrous cycle. Hourly samples of pituitary portal and jugular blood were obtained for 24 h, spanning the time of the expected hormone surges, after which an iv bolus of GnRH was injected to test for pituitary responsiveness to the releasing hormone. All animals in the surge-induction group exhibited vigorous surges of GnRH, LH, and FSH, but failed to show a rise in gonadotropin secretion in response to the GnRH challenge given within hours of termination of the gonadotropin surges. The surges of GnRH, LH, and FSH were blocked in all animals in which elevated levels of progesterone were maintained. These animals in the surge-block group, however, did secrete LH in response to the GnRH challenge. We conclude progesterone blocks the estradiol-induced gonadotropin discharge in the ewe by acting centrally to inhibit the surge of GnRH secreted into the hypophyseal portal vasculature.     

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.     

Decrease in gonadotropin-releasing hormone (GnRH) pulse frequency with aging in postmenopausal women

Increasing evidence suggests that aging is associated with dynamic changes in the hypothalamic and pituitary components of the reproductive axis that are independent of changes in gonadal hormone secretion. This study was designed to determine the effect of age on GnRH pulse frequency in women in the absence of gonadal feedback using gonadotropin free alpha-subunit (FAS) and LH as neuroendocrine markers of endogenous GnRH secretion. All studies were performed in healthy, euthyroid postmenopausal women (PMW) during daytime hours. The impact of sampling interval and duration on assessment of pulse frequency in PMW was first examined in 10 women with a mean age of 61.6 +/- 8 yr (mean +/- SD), in whom blood was sampled every 5 min for 12 h. Each 5-min series was then reduced to simulate a 10-min series and then a 15-min series for pulse analysis, and the effect of 8 h compared with 12 h of sampling was determined. To define the changes in the frequency and amplitude of pulsatile hormone secretion with aging, 11 younger (45-55 yr) and 11 older (70-80 yr) PMW were then studied over 8 h at a 5-min sampling interval. In the initial series, the mean interpulse intervals (IPIs) for FAS were 53.8 +/- 3.6, 69.2 +/- 3.9, and 87.6 +/- 7.3 min at sampling intervals of 5, 10, and 15 min, respectively (P < 0.0005). The LH IPI also increased progressively with sampling intervals of 5, 10, and 15 min (54.4 +/- 2.5, 70.4 +/- 2.3, and 91.1 +/- 4.4 min; P < 0.0001). At the 5-min sampling interval, the calculated number of pulses/24 h was not different between a 12-h series compared with an 8-h series for either FAS or LH. In the second series of studies, the older PMW had lower gonadotropin levels (LH, 86.5 +/- 8.8 vs. 51.3 +/- 7.7 IU/L, P < 0.01; FSH, 171.6 +/- 16.9 vs. 108.2 +/- 10.5 IU/L, P < 0.005; FAS, 1021.5 +/- 147.4 vs. 425.6 +/- 89.6 ng/L, P < 0.005, in younger and older PMW, respectively) despite no differences in estrone or estradiol levels. The older PMW also demonstrated a slower FAS pulse frequency compared with their younger counterparts, as reflected in an increased FAS IPI (52.6 +/- 3.1 and 70.6 +/- 5.9 min; P < 0.002). The difference in IPIs between younger and older PMW was not statistically significant for LH (65.4 +/- 5.6 and 71.8 +/- 6.6 min for younger and older PMW, respectively). FAS pulse amplitude was decreased in older PMW compared with younger PMW (431.7 +/- 66.2 vs. 224.6 +/- 81.9 ng/L; P < 0.01), whereas the decrease in LH pulse amplitude with age was of borderline statistical significance (23.2 +/- 3.1 vs. 15.9 +/- 2.1 IU/L; P = 0.09). In conclusion: 1) the use of a 5-min sampling interval and measurement of FAS as the primary marker of GnRH pulse generator activity indicate that GnRH pulse frequency in younger PMW is faster than previously reported, but not increased over that seen in the late follicular phase and midcycle surge in women with intact ovarian function; and 2) the marked decrease in FAS pulse frequency with age provides evidence of age-related changes in the hypothalamic component of the reproductive axis that are independent of changes in gonadal function.     

Dynamics of gonadotropin-releasing hormone (GnRH) secretion during the GnRH surge: insights into the mechanism of GnRH surge induction.

Recent studies demonstrate unequivocally that a preovulatory surge of GnRH is secreted into pituitary portal blood during the estrous cycle of the ewe and that this surge is induced by the follicular phase rise in estradiol. These data, obtained at 10-min intervals, suggested the surge results from a continuous elevation of GnRH rather than from a sequence of discrete pulses. This study examines the dynamics of GnRH secretion in more detail to determine if the surge results from strictly episodic release of the decapeptide. Our approach was to monitor GnRH secretion into pituitary portal blood at very frequent intervals during several "windows" of the GnRH surge induced using a physiological model for the estrous cycle. Samples of portal blood were obtained at either 2-min intervals (6 ewes), or 30-sec intervals (12 ewes) at several times during the surge; at other times portal blood was sampled less often to monitor progression of the GnRH surge. All ewes had an unambiguous GnRH surge; amplitude ranged from 100- to 500-fold over pressure levels. Regardless of sampling interval, our results provide no convincing evidence to indicate the enhanced secretion of GnRH is strictly episodic; values remained continuously elevated in portal blood. Our findings are consistent with the hypothesis that the GnRH surge is not composed entirely of discrete synchronous secretory events, and they raise the possibility that one action of estradiol in inducing the GnRH surge may be to switch the pattern of GnRH secretion into portal blood from episodic to continuous.