The effect of blockade of dopamine receptors on the inhibition of episodic luteinizing hormone release during electrical stimulation of the arcuate nucleus in ovariectomized rats.

This study examined the possible involvement of dopamine (DA) in mediating the inhibition of episodic LH release that occurs during electrical stimulation of the arcuate nucleus (ARH) in ovariectomized rats. Animals were treated before stimulation with pimozide (1.26--2.0 mg/kg) or d-butaclamol (1 mg/kg), blockers of DA receptors, or l-butaclamol. Apomorphine, which inhibits episodic LH release by activating DA receptors, was given near the end of the experiment to determine if these receptors were blocked. ARH stimulation suppressed pulsatile LH release in six rats when DA receptors were not blocked by pimozide (as well as two in which blockade was not tested). A transient increase occurred in one other animal. When DA receptors were blocked by pimozide, stimulation of the ARH inhibited episodic LH release in nine rats, suggesting that DA may have no role in mediating this inhibition. However, because increased LH release occurred in five additional animals, as well as in one with partial receptor blockade, the possibility remains that DA may perhaps have a minor role in this inhibitory response. Although ARH stimulation increased LH release after DA receptor blockade by d-butaclamol, this effect could not be ascribed to the DA antagonist property of this agent, because elevated blood LH levels also occurred during stimulation in rats treated with l-butaclamol, in which DA receptors were not blocked. d- and l-butaclamol may possess a non-stereospecific action on a non-dopaminergic event, thus reversing the response to ARH stimulation. Finally, whether DA receptors were blocked or not by pimozide, d-, or l-butaclamol, activation of the ventromedial hypothalamic and periventricular nucleus regions suppressed episodic LH release, but did not increase LH secretion. This suggests that the region through which stimulation can inhibit, but not increase, LH release may extend in the hypothalamus to these two areas.

Serotonin mediated inhibition of episodic luteinizing hormone release during electrical stimulation of the arcuate nucleus in ovariectomized rats.

The possible involvement of serotonin (5HT) in mediating the inhibition of episodic LH release produced by electrical stimulation of the arcuate nucleus (ARH) of ovariectomized rats was investigated. Animals were pretreated with p-chlorophenylalanine (PCPA) an inhibitor of 5HT synthesis, or PCPA and 5-hydroxytryptophan (5HTP). Unanesthetized, unrestrained rats were bled continuously through indwelling cannulae prior to the onset of stimulation, and bleeding continued for 3 h during which electrical stimulation was applied for one or two 60 min periods separated by a 60 min nonstimulation period. Whole blood was analyzed for LH by radioimmunoassay. Brain 5HT levels were determined in individual rats by a fluorometric method. PCPA caused a significant depletion in brain 5HT levels by 71h. Administration of 30 or 120 mg/kg 5HTP to PCPA treated rats resulted, respectively, either in a restoration of normal brain 5HT levels, or a 3 1/2-fold increase above controls. Although stimulation of the ARH inhibits episodic LH release in ovariectomized rats, following depletion of brain 5HT levels with PCPA stimulation of the ARH markedly increased LH release. This increase lasted for most if not all of the stimulation period and was followed by a 1 h period of little or no LH secretion. These increases were not seen during stimulation in other hypothalamic areas outside the ARH. Reestablishment of normal, or even further increasing, brain 5HT levels in PCPA treated rats with 5HTP greatly reduced the extent of the increase in LH release seen during ARH stimulation, but only restored inhibition of LH secretion in a few animals. However, a decrease in LH rt 1) a decrease in brain 5HT levels reverses the effect of ARH stimulation on LH release from inhibition to excitation, and 2) repletion of 5HT levels greatly reduces the magnitude of this increased LH release. These data suggest that 5HT may be involved in mediating the inhibition of episodic LH secretion by electrical stimulation of the ARH. The inability of 5HTP to restore completely inhibition of episodic LH release during ARH stimulation suggests that a substance other than 5-HT may also be involved in mediating this response.

The effect of blockade of kappa-opioid receptors in the medial basal hypothalamus and medial preoptic area on luteinizing hormone release during midpregnancy in the rat.

The objective of this study was to determine if kappa-opioid receptors present in the medial preoptic area (MPOA) and medial basal hypothalamus (MBH) are involved in opioid peptide suppression of LH secretion during midpregnancy (day 13-16) in the rat. Nor-binaltorphimine (nor-BNI), a selective antagonist of brain kappa-opioid receptors, was applied directly to the MPOA or MBH for 3.5 h by means of push-pull perfusion. Nor-BNI perfusion in the MBH produced a dose-dependent increase in LH pulse frequency as well as increases in blood LH level. The effect on amplitude could not be determined, since too few pulses occurred in cerebrospinal fluid-treated control rats. Nor-BNI perfusion in the MPOA also increased LH pulse frequency. Moreover, in the majority (62%) of rats perfused with nor-BNI in the MPOA, the final 1.5 h of perfusion were unexpectedly characterized by an increase in LH that was of greater magnitude and more prolonged than an LH pulse and produced an elevation in blood LH levels. This delayed LH response did not occur in any rat perfused in the MBH. Perfusion with nor-BNI in the diagonal band of Broca had no significant effect on LH secretion. The LH responses observed during nor-BNI perfusion in the MPOA or MBH were not due to spread to the third ventricle and subsequent diffusion via the cerebrospinal fluid to another brain site, since perfusion with nor-BNI in an area of the ventral thalamus close to the third ventricle had no effect on LH release. These results provide support for the involvement of kappa-opioid receptor-mediated mechanisms in both the MPOA and MBH in the suppression of LH secretion during midgestation in the rat.

Lack of ovarian steroid negative feedback on pulsatile luteinizing hormone release between estrus and diestrous day 1 in the rat estrous cycle.

The object of this study was to determine the roles of ovarian estradiol (E2) and progesterone (P) in regulating pulsatile LH release between estrus and diestrous day 1 (D1) in the rat estrous cycle. Three groups of rats were bled at rates of 75 or 100 microliter whole blood/6 or 8 min, respectively, between 0930-1230 h on estrus or 24 h after either sham ovariectomy (OVX) on estrus (i.e. on D1) or OVX on estrus. There were no differences in plasma E2 and P levels in rats between estrus and early D1. However, after OVX on estrus, plasma levels of both steroids declined and were significantly lower 24 h later than values in D1 controls, indicating an active ovarian secretion of both hormones in this interval of the rat cycle. A significant increase in mean blood LH levels occurred between estrus and D1 due to an increase in LH pulse amplitude and frequency. After OVX on estrus, all parameters of pulsatile LH release also increased within 24 h, but mean blood LH levels as well as LH pulse amplitude and frequency were virtually identical to values in D1 controls, despite the decline in plasma E2 and P levels. Thus, OVX did not augment the increases in LH pulse amplitude and frequency that occur between estrus and D1. This demonstrates that the increase in pulsatile LH release from estrus to early D1 occurs in the absence of ovarian steroid negative feedback; the increases in LH pulse amplitude and frequency are not under negative feedback control by the low plasma levels of E2 and P present at this time. These data stand in direct contrast to the presence of prominent ovarian steroid negative feedback systems operative between D1 and diestrous day 2, and diestrous day 2 and proestrus.

Regional differences in response to electrical stimulation within the medial preoptic-suprachiasmatic region on blood luteinizing hormone levels in ovariectomized and ovariectomized, estrogen-primed rats.

The effect of electrical stimulation of the medial preoptic-suprachiasmatic nucleus (MPOA-SCN) region of the forebrain on blood LH levels was studied in ovariectomized, pentobarbital-anesthetized rats. Animals were either not primed with estrogen or previously given 5 micrograms estradiol benzoate (EB)/100 g BW . day for the 2 days before stimulation. Rats were bled continuously (30, 40, or 50 microliter whole blood/5--6 min) through indwelling right atrial cannulae for 1.5 h before stimulation, 1.5 h during stimulation, and up to 1 h afterwards. Whole blood was analyzed for LH by RIA. In ovariectomized, unprimed rats, electrical stimulation of the ventral MPOA consistently increased LH release. In contrast, stimulation of the peri-SCN region (immediately caudal to the MPOA, and lateral and dorsal to, but not within, the SCN), uniformly inhibited pulsatile LH secretion. Activation of the SCN elevated blood LH levels in most unprimed rats tested, but suppression of episodic LH release occasionally occurred. Pretreatment with estrogen resulted in increased LH secretion in response to stimulation of each of these three regions. Estrogen prolonged the LH increase occurring during MPOA stimulation, completely reversed the inhibitory LH response to peri-SCN stimulation, and either reversed any possible inhibitory response to SCN stimulation or greatly increased the magnitude and duration of the increase in blood LH levels produced in the unprimed rat during activation of this nucleus. In summary, the present study indicates that well defined areas in the MPOA-SCN region can have strikingly different effects on LH secretion in the absence of ovarian estrogen and that this steroid is critically important in determining the direction, magnitude, and duration of the LH response to a localized brain stimulus.

Effects of intraventricular administration of catecholamines on luteinizing hormone release in morphine-treated rats.

Morphine (M) treatment has been shown to suppress LH release in rats. These studies were undertaken to determine whether a decrease in the response of LHRH neurons to excitatory neurotransmitters may be responsible for the depressed LH secretion in M-treated rats. Ovariectomized rats bearing permanent cannulae in the third ventricle of the brain were primed with estradiol benzoate and progesterone; 3 days later, they received M (20 mg/kg, sc) or saline (controls). The effects of two intraventricular (Ivt) 2-min pulses delivered 80 min apart of vehicle (artificial cerebrospinal fluid), dopamine, norepinephrine, or epinephrine (E) on LH release were assessed. Basal blood LH levels were undisturbed by Ivt administration of vehicle in saline-treated rats. Intraventricular infusions of dopamine (5.3 micrograms/pulse) also failed to evoke LH release in saline-treated rats. However, similar pulse norepinephrine or E infusions (5.3 micrograms/pulse) readily elicited well defined episodes of LH hypersecretion. The magnitude and temporal pattern of LH responses in the control and M-treated rats were quite similar. In another experiment, the progesterone-induced afternoon LH surge was blocked by M treatment of estradiol benzoate-primed rats. In these blocked rats, Ivt administration of E evoked rapid and substantial LH secretion. Thus, our results failed to demonstrate any evidence of diminution in the response of LHRH neurons to excitatory neurotransmitters in M-treated rats. On the other hand, they lend credence to the view that a decreased influx of adrenergic signals in the vicinity of the LHRH neurons may result in the suppression of LH release after M administration.

Further evidence for inhibition of episodic luteinizing hormone release in ovariectomized rats by stimulation of dopamine receptors.

Stimulation of dopamine receptors by apomorphine inhibits episodic LH release in ovariectomized rats. The present study was designed to examine further the role of dopamine in this process. Unrestrained, unanesthetized rats with indwelling right atrial cannulae were bled continuously (30 or 50 microliters of whole blood/5 min for 3-6 h) and whole blood samples analyzed for LH by radioimmunoassay. Animals were treated with various compounds reported to stimulate or block dopamine receptors. ET 495, a long acting dopamine receptor stimulating agent, caused a marked inhibition of episodic LH release (2 1/2-4 h). Control injections of distilled water had no effect. d-Butaclamol, a blocker of dopamine receptors, did not itself alter episodic LH release but prevented the inhibitory effects seen following apomorphine or ET 495. I-butaclamol, a biologically inactive form of butaclamol, had no effect. Measurement of plasma corticosterone levels in these same animals indicated increased values following apomorphine or ET 495 alone (when LH release was inhibited), as well as after apomorphine or ET 495 administration to d-butaclamol-pretreated rats (when LH levels did not change). These data support our previous hypothesis that in ovariectomized adult rats, activation of dopamine receptors is capable of inhibiting episodic LH release, but that dopamine may not play an inhibitory role under normal physiological conditions in the modulation of LH secretion. In addition, the inhibitory action of apomorphine and ET 495 does not appear to be exerted via a stress-induced release of adrenal corticosterone.

Catecholamine involvement in episodic luteinizing hormone release in adult ovariectomized rats.

This study was intended to examine the role of hypothalamic norepinephrine (HNE) and dopamine (HDA) in episodic luteinizing hormone (LH) release in adult ovariectomized rats. Unrestrained, unanesthetized rats with indwelling right atrial cannulae were bled continuously (30, 50, or 100 mul of whole blood/4-6 min for 3-4 hours), and the blood samples were analyzed for LH by radio-immunoassay. In other individual rats, changes in the hypothalamic levels of norepinephrine and dopamine after drug administration were determined by a radioisotopic-enzymatic catechol-O-methyl transferase assay. alpha-Methyl-p-tyrosine significantly decreased HNE and HDA concentrations but failed to alter episodic LH release. Two dopamine-beta-hydroxylase inhibitors (U-14,624 and FLA-63) caused marked reductions in HNE, small but not statistically significant increases in HDA, and an inhibition of episodic LH secretion. Apomorphine, a dopamine receptor stimulator, caused a transient (50-60 min) but marked inhibition of episodic LH release. Saline injection had no effect. Pimozide, a blocker of dopamine receptors, prevented the inhibitory effects seen following apomorphine. Although not studied in detail, pimozide alone did not appear to alter episodic LH secretion. These data suggest that in adult ovariectomized rats norepinephrine may be an excitatory neurotransmitter in the modulation of episodic LH release. The activation of dopamine receptors may be capable of inhibiting this release process. However, the apparent inability of pimozide alone to alter episodic LH discharge suggests that under physiological conditions dopamine may not play a role in the modulation of episodic LH secretion.

Electrical stimulation of the arcuate nucleus in ovariectomized rats inhibits episodic luteinizing hormone (LH) release but excites LH release after estrogen priming.

The effect of electrical stimulation of the arcuate nucleus of the hypothalamus (ARH) on the blood levels of luteinizing hormone (LH) was studied in ovariectomized (ovx) rats. Unanesthetized, unrestrained rats were bled continuously (30 or 50 mul whole blood/4-6 min) through indwelling right atrial cannulae for 1 1/2-2h prior to the onset of stimulation. The bleeding was continued for the following three hours, during which electrical stimulation was applied for two 60 min periods separated by a 60 min non-stimulation period. Whole blood was analyzed for LH by radioimmunoassay. Electrical stimulation of the ARH in ovx rats inhibited the episodic LH release characteristic of these animals. The inhibition was rapid in onset, beginning within minutes, and lasted for most if not all of the stimulation period or longer. The blood LH levels returned to control values prior to the second stimulation period during which the inhibitory response was again seen. Stimulation in other hypothalamic areas outside the ARH was generally ineffective, although repeatable inhibition of episodic LH release did occur in 2 of 3 rats stimulated in the periventricular nucleus. In a second study, ovx rats were primed with 5 mug estradiol benzoate/100 g body weight/day for 2 days prior to stimulation. In these rats, stimulation of the ARH or median eminence produced increases in LH release, whereas stimulation in other hypothalamic areas outside the ARH was ineffective. The parameters of electrical stimulation that produced increases in LH release were the same as those that caused the inhibition of episodic LH release. The results demonstrate that electrical stimulation of the ARH inhibits LH release in ovx rats, but increases LH release if the animals have been primed with estrogen.

Effect of methysergide, a blocker of serotonin receptors, on plasma prolactin levels in lactating and ovariectomized rats.

The effect of methysergide (MES, 2.5 mg/100 g body wt), a serotonin antagonist, on prolactin release has been studied in lactating and ovariectomized rats. MES caused significant increases in prolactin release in both animals. Studies in ovariectomized, hypophysectomized rats indicate that this effect is not due to a decrease in the peripheral metabolism of prolactin. In vitro incubations of anterior pituitary fragments with MES failed to demonstrate any increase in prolactin release, suggesting that MES does not act directly on the anterior pituitary. Parachlorophenylalanine (PCPA; 32 mg/100 g body wt) decreased brain serotonin levels in ovariectomized rats 5, 24, and 70 h after its administration, yet did not alter plasma prolactin levels. L-tryptophan (6.3 mg/100 g body wt) given 1 and 1 1/2 h prior to sacrifice increased brain serotonin levels, yet did not affect plasma prolactin levels. Neither PCPA nor L-tryptophan altered MES-induced prolactin release. In lactating rats, suckling caused marked increases in plasma prolactin levels, an effect completely abolished by the administration of MES to the mother rats 3 1/4 h prior to suckling. However, MES-induced prolactin release was not altered by prior treatment with MES, either in lactating or ovariectomized rats. Others have shown that suckling releases prolactin through an excitatory serotonergic mechanism. Therefore, the failure of suckling to release prolactin in MES-pretreated rats suggests that MES can block brain serotonin receptors. However, the ability of methysergide to release prolactin in rats with serotonin receptors presumably blocked, suggests that the serotonin receptor-blocking and the prolactin-releasing actions of MES are not related.

Ovarian steroid regulation of basal pulsatile luteinizing hormone release between the mornings of proestrus and estrus in the rat.

The aim of this study was to examine the regulation of basal pulsatile LH release by ovarian estradiol (E2) and progesterone (P) during the interval between the mornings of proestrus and estrus in the rat estrous cycle. Pulsatile LH release was studied in six groups of rats bled continuously through jugular venous cannulae between 0930-1230 h at a rate of 50 microliter whole blood/5 min: 1) bled on proestrus; 2) sham ovariectomy (OVX) at 0900-1000 h on proestrus and bled on estrus; 3-6) OVX at 0900-1000 h on proestrus, implanted with either empty or E2-, P-, or E2- plus P-containing Silastic capsules, and bled 24 h after OVX. In our colony, plasma E2 levels peaked at 1300 h, remained high through 1730 h, and then declined. Plasma P values increased between 1300 and 1730 h, peaked at 2000 h, and were rapidly declining by 2400 h. To reproduce the magnitude as well as the temporal pattern for these changes in plasma E2 and P levels, E2 capsules were inserted at the time of OVX on proestrus and removed at 1830 h. P capsules were inserted at 1400 h and removed at 2300 h. Groups of ovariectomized or sham-ovariectomized control animals had empty capsules implanted and removed at comparable times. Capsules producing basal E2 and P levels were not inserted after the removal of the original implant, since mean blood LH levels, pulse amplitude, and frequency were the same in rats sham ovariectomized or ovariectomized at 1830 h on proestrus and bled the next morning between 0930-1230 h. Mean blood LH levels decreased between the mornings of proestrus and estrus due to a reduction in LH pulse frequency as pulse amplitude remained stable. OVX at 0900-1000 h on proestrus increased mean blood LH levels 2.5-fold compared to values on estrus due to increases in both LH pulse frequency and amplitude. Restoration of physiological proestrous levels of only E2 returned LH pulse frequency to estrous values, but did not significantly affect LH pulse amplitude. P alone also had no significant effect on LH pulse amplitude, but slightly reduced pulse frequency, although, unlike E2, not to values seen on estrus. Replacing both E2 and P returned LH pulse amplitude to estrous levels and reduced LH pulse frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro release of lutenizing hormone from anterior pituitary fragments superfused with constant or pulsatile amounts of luteinizing hormone-releasing factor.

Anterior pituitary fragments from ovariectomized rats were superfused with medium to which lutenising hormone-releasing factor (LH-RF) was added for either prolonged or short intervals. Samples of superfusate were collected at 2-min intervals before and after the onset of LH-RF administration. In control experiments without anterior pituitary fragments, infusions of constant amounts of NIAMDD-Rat LH were made into the superfusion chamber. The superfusate was collected in all experiments and luteinizing hormone (LH) levels measured by radioimmunoassay. LH release was increased 100-200% following superfusion with concentrations of LH-RF ranging from 10-9 to 10-5M. The percent coefficient of variation (%CV) was determined for LH to quantitate fluctuations in hormone release. A nonepisodic pattern of LH release was obtained prior to LH-RF administration and during superfusion with constant levels of LH-RF. There was no significant difference in the %CV in these periods as compared to that obtained during infusion of constant amounts of LH into the superfusion chamber. However stimulation by LH-RF for only 2-5 min caused episodes of LH release lasting 30-40 min. The %CV of superfusate LH during these pulsatile stimulation studies was significantly increased compared to that obtained during superfusion with constant levels of LH-RF. These results suggest that the pituitary is not the site of origin of the mechanism underlying episodic release of LH, but can release LH in a pulsatile manner when driven by pulsatile LH-RF stimulation.

Pulsatile luteinizing hormone release during pregnancy in the rat.

The present studies were designed to characterize LH release during pregnancy in the rat. Unanesthetized animals with jugular cannulae were bled for 3 h between 1000-1300 h on days 6-8, 14-16 or 22 of gestation (50 microliters whole blood/5 min). Plasma estradiol and progesterone values both increased from days 6-8 to days 14-16. However, while plasma estradiol levels increased further between days 14-16 and day 22, plasma P levels had declined 86%. The percent coefficients of variation obtained for alterations in blood LH levels at each stage of pregnancy were all significantly greater than intraassay variation, indicating that LH release was pulsatile at each stage. Although there were no significant differences in mean blood LH levels, pulse amplitude, or frequency between days 6-8 and 14-16, the individual patterns of LH release clearly varied between these 2 groups, and most notably within the 14-16 day group. Fifty-three percent (9 of 17) of the LH records in rats on days 14-16 were nonpulsatile compared to only 20% (3 of 15) on days 6-8. However, despite a trend toward an absence of pulsatile LH release on days 14-16, mean frequency at this time did not differ from days 6-8, since on days 14-16 the remaining 8 animals demonstrated 3.5 pulses/3 h, while on days 6-8 the other 12 rats averaged only 2.5 pulses/3 h. On day 22, there was a marked increase in mean blood LH levels compared with either days 6-8 or 14-16. This increase was due to an increase in mean LH pulse frequency. All 15 rats demonstrated pulsatile LH secretion, a significantly greater incidence of pulsatile LH release than on days 14-16 (100% vs. 47%). These data demonstrate that LH release is pulsatile during pregnancy in the rat, and changes in the characteristics of this secretion occur at different stages of gestation.

The relationship between declining plasma progesterone levels and increasing luteinizing hormone pulse frequency in late gestation in the rat.

The object of this study was to determine whether the increase in LH pulse frequency and mean blood LH levels on day 22 of pregnancy in the rat is due to the precipitous fall in plasma progesterone (P) levels that occurs late in gestation. On day 20 of pregnancy two groups of animals with indwelling jugular cannulae were implanted sc with empty or P-containing Silastic capsules. Blood samples were withdrawn 0.5 h before and 5.5 h postimplantation on day 20 (0800 and 1400 h), at 1400 h on day 21, and at the end of the study between 1200-1300 h on day 22 to follow the time course of changes in plasma P levels over this 2-day period in both groups. These groups were bled on day 22 for 3 h between 0900-1200 h for analysis of pulsatile LH release. A third group not implanted with Silastic capsules was bled on day 20 for 3 h; plasma P levels in these rats bled on day 20 did not differ from the preimplantation values observed in either group of capsule-implanted rats. In empty capsule-implanted animals, plasma P values declined slightly from days 20 to 21 and were dramatically reduced between days 21 and 22. In contrast, after implantation of P capsules, plasma P levels were elevated on day 20 and remained elevated on day 21 compared with preimplantation values. Although these increased plasma P values declined between days 21 and 22, reflecting a decrease in endogenous P secretion, they were nonetheless comparable to day 20 values due to the presence of the P-containing capsules. Plasma estradiol values did not differ significantly between any of the experimental groups. In the empty capsule group bled on day 22, mean blood LH levels and LH pulse frequency were significantly higher compared to day 20 values, at a time when plasma P levels had fallen significantly from day 20 values. However, in the P capsule group, mean blood LH levels and LH pulse frequency on day 22 were significantly lower than values in the empty capsule group and were not different from the low values on day 20. Thus, preventing a decline in plasma P values to the low levels normally found on day 22 prevented the increase in LH pulse frequency and mean blood LH levels normally seen at this time of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of progesterone and estradiol on the acute changes in pulsatile luteinizing hormone release induced by ovariectomy on diestrus day 1 in the rat.

The aim of this study was to determine whether the rapid increases in LH pulse amplitude and frequency that occur within 24 h after ovariectomy (ovx) on diestrus day 1 (D1) were due to the removal of progesterone (P) and/or estradiol (E). Initial studies demonstrated that plasma levels of E and P were 18.2 +/- 1.2 pg/ml and 34.1 +/- 3.2 ng/ml, respectively, between the evening of D1 and the morning of D2 in our colony of intact rats. Immediately after ovx and jugular venous cannulation on the morning of D1, rats were implanted either with empty Silastic capsules or capsules capable of restoring physiological levels of E and P to the control values reported above. These rats were continuously bled (75 microliter/6 min) for 3 h 1 day after ovx for analysis of pulsatile LH release, and then additional plasma samples were gathered for determination of E and P levels. Rats with empty capsules had decreased levels of E and P and increases in mean blood LH levels, LH pulse amplitude, and pulse frequency. Animals with E capsules had physiological levels of E and decreased levels of P, but no suppression of the acute post-ovx increase in pulsatile LH release. In contrast, animals with P capsules had physiological plasma levels of P, decreased levels of E, and a marked reduction in the acute LH response to ovx. This suppression was due entirely to a decrease in LH pulse amplitude, as pulse frequency was not altered. Rats with E and P capsules had physiological levels of these hormones, which resulted in an even greater reduction in the acute LH response to ovx. This suppression was due to decreases in both LH pulse amplitude and pulse frequency. The effect of P on LH pulse amplitude was centrally mediated, since the in vitro response to LHRH of anterior pituitary fragments from P-implanted rats was the same as that of anterior pituitary fragments taken from rats with empty capsules. These studies demonstrate that the acute increase in LH pulse amplitude that occurs within 24 h after ovx on D1 is due to the absence of a central inhibitory effect of ovarian P, while the rapid increase in LH pulse frequency is due to the loss of both ovarian E and P.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of various inhibitors of phenylethanolamine N-methyltransferase on pulsatile release of LH in ovariectomized rats.

This study was undertaken to investigate whether hypothalamic adrenaline is involved in pulsatile LH release in rats. Various inhibitors of phenylethanolamine N-methyltransferase (PNMT), the enzyme which catalyses the conversion of noradrenaline to adrenaline, were administered to freely moving ovariectomized rats bearing an atrial cannula. Blood samples were taken continuously from 09.00 to 11.00 h and from 14.30 to 17.00 h. The drugs were administered either at 11.00 h only or at both 11.00 and 14.00 h. The various treatments with the vehicle or an inhibitor of peripheral PNMT, SKF 29661, produced no decrease in any parameter of pulsatile LH release. A single injection of one of the central PNMT inhibitors, SKF 64139 or LY 134046, at 11.00 h had no effect on LH release, but when given at both times the drugs suppressed the mean LH level, pulse frequency and amplitude. The effect of these drugs on the level of dopamine, noradrenaline and adrenaline in the hypothalamus (including preoptic area) was assessed. There was no effect on the concentration of the catecholamines after SKF 29661. None of the treatments with SKF 64139 or LY 134046 resulted in a change in the level of dopamine or noradrenaline; the double dose did, however, produce a significantly greater depletion of adrenaline than that which was achieved with the single injection. These results suggest that the maintenance of normal LH pulses is dependent upon the presence of a sufficient level of hypothalamic adrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Kappa-opioid receptor involvement in the regulation of pulsatile luteinizing hormone release during early pregnancy in the rat.

Abstract The object of this study was to gain further insight into endogenous opioid peptide suppression of pulsatile luteinizing hormone (LH) release in early gestation in the rat by examining whether selective blockade of mu -, delta -, or kappa-opioid receptor(s) results in stimulation of pulsatile LH secretion at this time. Previous reports demonstrated stimulation of pulsatile LH release during early gestation by intravenous infusions of naloxone, an endogenous opioid peptide receptor antagonist whose binding is not specific to a single class of opioid peptide receptors. In the present study, naloxone infused intraventricularly similarly stimulated an increase in pulsatile LH release on Days 7 to 8 of gestation. Antagonists of specific opioid peptide receptor subtypes were thus given by this route. Administration of nor-binaltorphimine, an antagonist of kappa-opioid receptors, but not beta-funaltrexamine or ICI 174, 864, antagonists of mu- and delta-opioid receptors, respectively, exerted a stimulatory action on both LH pulse amplitude and frequency similar to that of naloxone, indicating involvement of this opioid peptide receptor subtype in the endogenous opioid peptide suppression of pulsatile LH release in early gestation in the rat.

Ovarian steroid regulation of pulsatile luteinizing hormone release during early gestation in the rat.

Abstract The object of this study was to examine ovarian regulation of pulsatile luteinizing hormone (LH) secretion during early gestation. This was done primarily by analyzing pulsatile LH release in rats that were either sham ovariectomized (OVX) on Day 7 of pregnancy, implanted with empty Silastic capsules, and bled on Day 8, or OVX on Day 7, immediately implanted with Silastic capsules producing plasma levels of estradiol and/or progesterone characteristic of Day 7 to 8 of pregnancy, and bled on Day 8. In addition, the role of progesterone in regulating pulsatile LH secretion was also examined by administration of the progesterone receptor antagonist, RU486, on Day 7 and examining pulsatile LH release on Day 8 of pregnancy. OVX caused a marked increase in LH pulse amplitude and frequency within 24 h. Replacement with physiological plasma levels of estradiol or progesterone alone had no suppressive effect on this OVX-induced increase in pulsatile LH secretion. Restoration of physiological plasma levels of both estradiol and progesterone returned LH pulse amplitude to values seen in sham OVX controls, and prevented the OVX-induced increase in LH pulse frequency. The group mean LH pulse frequency tended to be less in estradiol + progesterone-treated rats than in sham OVX controls, but this difference was not statistically significant. RU486 blocked uterine progesterone receptors as evidenced by endometrial hemorrhaging. In agreement with the OVX + steroid replacement data, RU486 administration also resulted in increases in LH pulse amplitude and frequency. These data demonstrate that the frequency and amplitude of LH pulses on Day 8 of gestation are held in check by negative feedback signals coming from the ovary. Neither steroid alone exerts any suppressive influence over pulsatile LH secretion during early gestation, but both steroids acting together exert a prominent negative feedback regulation on the pulsatile LH release process.

Ovarian regulation of pulsatile luteinizing hormone secretion during late gestation in the rat*.

Abstract The object of this study was to examine the influence of both estradiol (E(2)) and progesterone (P) alone or in combination on luteinizing hormone (LH) pulse amplitude and frequency during the interval between Days 21 and 22 of gestation. This was done by analyzing pulsatile LH release in rats bled on Days 21 and 22 of gestation, and in animals ovariectomized (OVX) on Day 21, implanted with silastic capsules producing plasma levels of E(2) and/or P characteristic of the Day 21 to 22 interval, and bled on Day 22 Pulsatile LH release increased between Days 21 and 22 due to an increase in pulse frequency and a small elevation in pulse amplitude. OVX produced no further increase in pulse frequency but markedly enhanced the small change in pulse amplitude. Preventing either the decline in plasma P that normally occurs between Days 21 and 22, or just the small additional decrease in plasma P levels produced by OVX, had no suppressive effect on pulse amplitude or frequency, although Day 22 levels of P alone augmented the normal increase in pulse frequency occurring between Days 21 and 22. Restoration of physiological plasma E(2) levels had no effect on the normal increase in pulse frequency, but partially attenuated the OVX-induced increase in pulse amplitude. Replacement of physiological Day 22 levels of both E(2) and P also decreased LH pulse amplitude, although amplitude was not significantly different from that seen following E(2) replacement alone, and was still greater than the normal Day 22 value. In contrast, restoration of physiological plasma levels of E(2)+ P caused a suppression of LH pulse frequency below that normally seen on Day 22. While E(2)+ P did not completely prevent the OVX-induced increase in pulse amplitude, administration of charcoal-extracted porcine follicular fluid to rats OVX on Day 21, and in which physiological plasma levels of E(2)+ P were restored, caused a further reduction in pulse amplitude. These data demonstrate that 1) marked increases in LH pulse amplitude are prevented from occurring between Days 21 and 22 of gestation by ovarian steroids, notably E(2), and that this suppression is enhanced by a non-steroidal factor present in porcine follicular fluid, 2) neither E(2) or P alone suppresses LH pulse frequency on Day 22 of gestation; LH pulse frequency increases on Day 22 because the plasma level of one of these steroids, P, markedly declines, and 3) restoration of physiological plasma levels of both steroids in the absence of the ovary produces an unphysiological suppression of pulse frequency, i.e. results in a lower pulse frequency than normally occurs in the presence of these same plasma steroid levels in animals with their ovaries intact. One hypothesis consistent with the latter observation is that at the end of gestation in the rat the ovary may produce a factor which 'protects' the frequency of the LH pulse generator from the negative feedback action of ovarian steroids. This allows an increase in LH pulse frequency and mean blood LH levels, and thereby facilitates ovarian follicular development and the normal progress of the first postpartum estrous cycle.

Adrenergic and noradrenergic regulation of pulsatile luteinizing hormone release.

Abstract The object of this study was to further define the roles of both norepinephrine (NE) and epinephrine (EPIN) in regulating pulsatile luteinizing hormone (LH) release in 4-day ovariectomized rats, in particular to examine the effect of decreasing NE synthesis on pulsatile LH secretion in animals with already greatly depleted levels of brain EPIN. Rats were injected ip with vehicle or drug at -27, -20, -5 and - 3 h relative to the onset of a 3-h blood sampling period. Hypothalamic-preoptic area (HPOA) levels of NE and EPIN were determined by high-performance liquid chromatography. Compared to controls, FLA-63 (25 mg/kg, a dopamine-ss- hydroxylase inhibitor), given at - 3 h, produced 50% and 22% declines in HPOA-NE and EPIN, respectively, and reductions in pulse amplitude and frequency. LY134046 (50 mg/kg, a phenylethanolamine N-methyltransferase inhibitor), given at - 27, - 20 and - 5 h, or -27, -20, -5 and -3 h, produced no change in NE, 88% and 86% declines in EPIN, respectively, and reductions in pulse frequency only. Each LY134046 treatment protocol produced the same decline in EPIN and pulse frequency. Thus, EPIN levels were maximally decreased by three LY134046 injections. When rats were given LY134046 at -27, -20 and -5 h, and FLA-63 at -3 h, compared to rats treated with LY134046 alone, there was no further decrease in HPOA-EPIN (82% decline), a 46% decline in NE, a further reduction in pulse frequency and a reduction in pulse amplitude. This further suppression of LH release must be due to a reduction in HPOA-NE levels since no further decrease in EPIN levels occurred. These data demonstrate within the same animal that NE and EPIN are both stimulatory to pulsatile LH release. NE stimulates the amplitude and frequency, and EPIN stimulates the frequency of pulsatile LH secretion.