Suppression Of Pulsatile LH Release Research Articles

Enkephalin-δ Opioid Receptor Signaling Mediates Glucoprivic Suppression of LH Pulse and Gluconeogenesis in Female Rats.

Energy availability is an important regulator of reproductive function at various reproductive phases in mammals. Glucoprivation induced by 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, as an experimental model of malnutrition suppresses the pulsatile release of GnRH/LH and induces gluconeogenesis. The present study was performed with the aim of examining whether enkephalin-δ-opioid receptor (DOR) signaling mediates the suppression of pulsatile GnRH/LH release and gluconeogenesis during malnutrition. The administration of naltrindole hydrochloride (NTI), a selective DOR antagonist, into the third ventricle blocked the suppression of LH pulses and part of gluconeogenesis induced by IV 2DG administration in ovariectomized rats treated with a negative feedback level of estradiol-17 β (OVX + low E2). The IV 2DG administration significantly increased the number of Penk (enkephalin gene)-positive cells coexpressing fos (neuronal activation marker gene) in the paraventricular nucleus (PVN), but not in the arcuate nucleus (ARC) in OVX + low E2 rats. Furthermore, double in situ hybridization for Penk/Pdyn (dynorphin gene) in the PVN revealed that approximately 35% of the PVN Penk-expressing cells coexpressed Pdyn. Double in situ hybridization for Penk/Crh (corticotropin-releasing hormone gene) in the PVN and Penk/Kiss1 (kisspeptin gene) in the ARC revealed that few Penk-expressing cells coexpressed Crh and Kiss1. Taken together, these results suggest that central enkephalin-DOR signaling mediates the suppression of pulsatile LH release during malnutrition. Moreover, the current study suggests that central enkephalin-DOR signaling is also involved in gluconeogenesis during malnutrition in female rats.

Temporary suppression of pulsatile LH release following a single injection of a GnRH agonist (deslorelin) in ovariectomised Holstein dairy cows

The objective of the experiment was to investigate the potential for using a single injection of the GnRH agonist [D-Trp 6, Pro 9−des-Gly 10-NH 2] GnRH-ethylamide (deslorelin) to suppress LH secretion in ovariectomised Holstein cows. Each dose of 10, 100 and 1000 μg deslorelin was injected intravenously into each of four ovariectomised cows on day 0. Blood samples were collected hourly on day 0 to profile the induced LH release. Frequent serial blood samples were collected at 10 min intervals over 4 h on days −3, −1, +2, +4 and +6. The injection of deslorelin induced a surge-like release of LH that begun within 1 h in all cows. There was no difference between deslorelin doses in terms of maximum LH concentration, area under the LH curve (AUC) or log 10(AUC). The average interval from injection to maximum LH concentration was longer for cows receiving 1000 μg than in those receiving 10 μg (3.5 versus 1.5 h; P<0.01), though no different to 100 μg (2.8 h; P>0.1). This relationship was described by a logarithmic function of deslorelin dose in micrograms ( R 2=73.3%, P<0.01). Pre-treatment smoothed mean LH concentration was significantly correlated with peak LH concentration of the induced surge: max_LH=5.37+9.57×pre-amplitude ( R 2=33.2%, P=0.05). Similarly, LH pulse amplitude pre-deslorelin was also correlated with peak LH of the induced surge max_LH=0.07+12.9×pre-amplitude ( R 2=53.7%, P=0.07). Pulsatile release of LH was suppressed only with the 1000 μg dose on day +2. Suppression was characterised by a reduction in mean LH, smoothed mean LH and LH pulse amplitude. By day +4, LH parameters were no different to pre-treatment ones. Pulse frequency was not affected by the treatment, although a small non-significant reduction at day +2 for 1000 μg dose was observed (3.9 versus 2.8, P=0.14). In conclusion, temporary suppression of LH output for at least 48 h occurred following a single intravenous injection of 1000 μg of deslorelin, even though there were similar peak LH concentrations were for the three doses.

Duration of treatment with progesterone and regression of persistent ovarian follicles in cattle.

The objective of the present study was to determine the duration of elevated concentrations of progesterone necessary to induce atresia of persistent ovarian follicles. Heifers were administered 25 mg of PGF2alpha on d 6 and 7 (d 0 = d of synchronized estrus) and a norgestomet implant from d 6 to 14. Ovaries were monitored by ultrasonography, and blood samples were collected on d 3, 5, 7, 9, 11, and 12 and daily from d 14 until ovulation. On d 12, heifers received either two progesterone-releasing intravaginal devices (PRID) for 6 h (6-h; n = 5), two PRID for 24 h (24-h; n = 5), or no treatment (CON; n = 5). Blood samples were collected at 15-min intervals from h -6 to 30 (PRID insertion = h 0) and analyzed for concentrations of LH. Characteristics of LH secretion were determined for consecutive 6-h periods (Period 0 to 5). Hourly blood samples, collected from h 0 to 29, were analyzed for concentrations of 17beta-estradiol (estradiol) and progesterone. The dominant ovarian follicles present on d 7 increased in size to 15.4+/-.3 mm on d 12 ("persistent follicle"). Following removal of the PRID and norgestomet implants, atresia of persistent follicles and ovulation of new follicles were induced in one of five and in four of five heifers in the 6-h and 24-h treatments, respectively. Persistent follicles ovulated after withdrawal of norgestomet in all other heifers. Concentrations of progesterone were increased from h 1 to 7 in the 6-h and h 1 to 26 in the 24-h treatment. Frequency of LH pulses was reduced (P < .05) during Periods 1 to 2 in the 6-h and Periods 1 to 5 in the 24-h treatment relative to the CON treatment. By h 10, concentrations of estradiol in the 6-h and 24-h treatments were lower (P < . 05) than in the CON treatment. This suppression continued through h 29 in the 24-h treatment (P < .05), whereas concentrations in the 6-h treatment were intermediate to those of the CON and 24-h treatments after h 14. Suppression of pulsatile LH release and estradiol secretion was evident with 6 and 24 h of treatment with progesterone, but only the 24-h treatment induced atresia of persistent follicles in a majority of the heifers.

Disparate serum free testosterone concentrations and degrees of hypothalamo-pituitary-luteinizing hormone suppression are achieved by continuous versus pulsatile intravenous androgen replacement in men: a clinical experimental model of ketoconazole-induced reversible hypoandrogenemia with controlled testosterone add-back.

To investigate the neuroendocrine mechanisms underlying the negative feedback actions of testosterone on both the pulsatile mode of LH release and the entropy or disorderliness of the LH release process, we blocked testicular androgen biosynthesis using oral high dose ketoconazole treatment with concomitant low dose glucocorticoid replacement for 48 h in six healthy young men. Volunteers were then infused iv with saline or a total of 8.0 mg testosterone base over the second 24 h via either a continuous or a pulsatile (90-min boluses) delivery pattern. Discrete peak detection (Cluster analysis) was applied to obtain a model-independent estimate of the frequency of serum LH concentration peaks, maximal and incremental LH peak amplitudes, peak area, and interpeak nadir serum LH concentrations. Approximate entropy was used to quantify the relative orderliness/disorderliness of the LH release process over 24 h. Ketoconazole treatment markedly lowered 24-h mean serum total and free testosterone concentrations (by 17- and 9-fold respectively), and significantly increased LH pulse frequency, maximal LH peak height, and interpeak nadir serum LH concentrations. Continuous iv testosterone add-back increased 24-h pooled serum free testosterone concentrations 3-fold more and concomitantly reduced mean (24-h) serum LH concentrations by at least 2-fold more than pulsatile delivery of the same total daily amount of androgen. Both modes of testosterone infusion suppressed pulsatile LH release, but the effects were distinguishable; namely, treatment with continuous vs. intermittent androgen add-back, respectively, decreased LH pulse frequency and incremental LH pulse amplitude. Ketoconazole treatment alone also significantly increased approximate entropy values, indicating greater disorderliness of LH release during androgen removal. Approximate entropy/orderliness was restored to baseline by continuous, but not pulsatile, iv testosterone replacement. In conclusion, the present novel testosterone add-back clinical experimental paradigm indicates that 1) remarkably different 24-h mean serum free testosterone concentrations can result from continuous vs. pulsatile testosterone delivery into the bloodstream; 2) androgen negative feedback can exert frequency- as well as amplitude-dependent suppression of pulsatile LH release; and 3) testosterone is required to maintain an orderly 24-h LH release process in young men.

Reduction of glucose availability suppresses pulsatile luteinizing hormone release in female and male rats.

Glucose availability controls reproductive activity through modulation of LH secretion. The aim of the present study was to determine whether the glucoprivic suppression is potentiated by gonadal steroids and if glucoprivic suppression of pulsatile LH release is sexually differentiated. Pulsatile LH secretion was examined in rats after peripheral (jugular) administration of the competitive inhibitor of glycolysis, 2-deoxyglucose (2DG). Fourteen days after gonadectomy, blood samples were collected every 6 min for 3 h. One hour after the onset of sampling, 2DG was administered peripherally (200, 400, or 800 mg/kg BW, iv), and food intake was determined after 2DG injection in gonadectomized males and females in the presence or absence of sex steroids (testosterone or estradiol). To test the ability of the pituitary to produce LH under glucoprivic conditions, LHRH was injected every 30 min for 2.5 h in ovariectomized (OVX) rats 30 min after treatment with 400 mg/kg 2DG. At all peripheral doses of 2DG in females and at the middle and high doses of 2DG in males, mean plasma LH and LH pulse frequency decreased (P < 0.05) in the presence of steroids. However, in the absence of sex steroids, the lowest dose in females and the middle dose in males were not effective. Pituitary function appeared normal, because increases in mean plasma LH in response to the exogenous LHRH occurred in OVX rats treated with the middle dose of 2DG. Food intake significantly (P < 0.05) increased after 2DG injection in all groups except estrogen-treated OVX females at the low and high doses of 2DG. These findings suggest that glucoprivic suppression of LH pulses is potentiated by gonadal steroids in both sexes. Moreover, the hypothalamo-hypophyseal axis of the female rat seems to be more sensitive to the decreased glucose availability induced by 2DG than that of the male.

Estrogen feedback needed at the paraventricular nucleus or A2 to suppress pulsatile luteinizing hormone release in fasting female rats

The feedback sites of estrogen within the hypothalamus and lower brain stem involved in the suppression of pulsatile LH secretion during 48-h fasting were examined in ovariectomized rats with local estradiol (E2) implants. The animals were ovariectomized and immediately implanted stereotaxically with stainless steel cannula containing crystal E2 diluted 10 times with crystal cholesterol into the medial preoptic area, paraventricular nucleus (PVN), arcuate nucleus (ARC), locus ceruleus, or A1 or A2 region of the brain. Five days later, animals were deprived of food for 48 h, and blood samples were collected every 6 min for 3 h. Animals were immediately refed for 45 h and bled again as described above. Changes in the mean LH concentrations over the 3-h sampling period and the frequency and amplitude of LH pulses were determined by calculating the differences in these parameters between the first and second blood samplings in each animal. Fasting significantly lowered mean LH concentrations in animals implanted with E2 in the A2. The more potent suppression of pulsatile LH release during fasting was found in rats with E2 implants in the PVN: the mean LH concentrations and LH pulse frequency were significantly reduced by fasting in this group. In the animals with E2 implants in the medial preoptic area, ARC, locus ceruleus, or A1, 48-h fasting did not induce any significant changes in LH pulse parameters compared to those in cholesterol-implanted controls. A decrease in LH pulse amplitude was apparent in refed rats as well as fasted animals only when E2 was implanted in the ARC. These results suggest that the feedback action of estrogen at the PVN and/or A2 is required for fasting-induced suppression of pulsatile LH release, as opposed to the so-called negative feedback action of estrogen, which tonically suppresses LH release in nonfasting rats.

Involvement of the catecholaminergic input to the paraventricular nucleus and of corticotropin-releasing hormone in the fasting-induced suppression of luteinizing hormone release in female rats.

The roles of the adrenergic projection to the paraventricular nucleus (PVN) and of central CRH in the suppression of pulsatile LH secretion during 48-h fasting were examined in ovariectomized estradiol (E2)-treated rats. The animals were ovariectomized and immediately implanted with Silastic tubing containing E2. One week after ovariectomy and E2 implantation, the animals were implanted stereotaxically with a guide cannula for microinjection into the PVN or intracerebroventricular (icv) injection. One week later, some of the animals were deprived of food for 48 h. The unfasted controls were provided with food ad libitum. At this point, blood samples were collected every 6 min for 3 h. Animals received an injection of 50 micrograms alpha-methyl-p-tyrosine (AMPT), a catecholamine synthesis inhibitor, into the PVN 3 h before the sampling started or an icv injection of 26 nmol alpha-helical CRF-(9-41), a CRH antagonist, after the first hour of blood sampling; control animals were given the vehicle at the equivalent time. The fasted animals injected with AMPT showed a significantly higher mean LH concentration and LH pulse frequency over the 3-h sampling period compared with the vehicle-injected controls. Treatment with AMPT had no significant effect on LH secretion in unfasted animals. The icv injection of alpha-helical CRF-(9-41) reinstated the suppressed LH release in fasted rats, but had no significant effect in unfasted animals. These results suggest that the adrenergic projection to the PVN and central CRH are involved in the suppression of pulsatile LH release during food deprivation. The possibility that fasting activates an ascending adrenergic projection that stimulates CRH release and thus suppresses pulsatile LH secretion is discussed.

Alpha-melanocyte-stimulating hormone antagonizes the neuroendocrine effects of corticotropin-releasing factor and interleukin-1 alpha in the primate.

alpha-Melanocyte stimulating hormone (alpha-MSH), a peptide derived from POMC has previously been shown to antagonize the action of exogenously administered beta-endorphin (beta-EP) on pituitary PRL and LH release in the primate. In this study, we have tested the ability of alpha-MSH to block some of the acute pituitary effects of CRF and interleukin-1 alpha (IL-1 alpha), effects which are thought in part to result from the release of endogenous beta-EP. Experiments were performed in ovariectomized rhesus monkeys bearing a chronically implanted lateral ventricular cannula for peptide infusion. Peripheral blood samples for LH, cortisol, and PRL RIA were obtained at 15-min intervals during a 3-h control period when saline was infused into the ventricle, followed by a 5-h experimental period. CRF (15 micrograms/h) infused alone for 5 h caused a significant suppression of pulsatile LH release; by the fifth hour, LH secretion was reduced to 32.5 +/- 2.4% of the control saline infusion. The CRF-induced suppression of LH was prevented by coinfusion of alpha-MSH (60 micrograms/h); by the fifth hour LH was 89.0 +/- 3.6% of the control (P less than 0.05 vs. CRF alone). alpha-MSH also prevented the CRF-induced decrease in LH pulse frequency (P less than 0.05). IL-1 alpha (4.2 micrograms) was infused alone for 30 min or in combination with alpha-MSH (120 micrograms/h for 2 h). After IL-1 alpha alone, LH decreased to 30.1 +/- 2.4% of baseline at 5 h. This decrease was prevented by alpha-MSH; by 5 h LH was 101 +/- 5.1% of baseline (P less than 0.005 vs. IL-1 alpha alone). IL-1 alpha did not affect LH pulse frequency but pulse amplitude was reduced; this reduction was prevented by alpha-MSH (P less than 0.05). IL-1 alpha also stimulated PRL release. PRL rose from a mean baseline of 3.5 +/- 0.3 ng/ml to a peak of 13.8 +/- 2.7 ng/ml; after coinfusion of alpha-MSH the mean peak PRL response was only 4.4 +/- 1.5 ng/ml (P less than 0.001 vs. IL-1 alpha alone). After CRF infusion, cortisol increased to 136 +/- 7.9% of the mean morning baseline concentration. This increase was not prevented by alpha-MSH coinfusion; after CRF plus alpha-MSH, cortisol increased to 121 +/- 6.0% of baseline. In contrast, alpha-MSH prevented the IL-1 alpha-induced increase in cortisol: 167 +/- 15.5% vs. 91.7 +/- 8.3% (P less than 0.005).(ABSTRACT TRUNCATED AT 400 WORDS)

Involvement of the gastric vagal nerve in the suppression of pulsatile luteinizing hormone release during acute fasting in rats.

Acute fasting has been shown to impair gonadotropin secretion in an estrogen-dependent manner in rats. The present study examined whether the vagus nerves have some role in the suppression of pulsatile LH release after 48-h fasting in ovariectomized estradiol-treated rats by performing complete subdiaphragmatic cutting of the nerves or selective sectioning of its branches (gastric, hepatic, or celiac) after the first hour of a 3-h blood sampling period. Vagotomy, alone, did not cause any changes in LH release in the unfasted animals. However, complete and gastric vagotomies restored pulsatile LH release that was suppressed before the nerves were cut in fasted animals. Neither hepatic nor celiac vagotomy restored pulsatile LH release, which remained suppressed throughout the sampling period in fasted animals. These results suggest that some mechanism involving the afferent vagal nerves, particularly the gastric vagus, has a major role in the suppression of pulsatile LH release during acute fasting in ovariectomized estradiol-primed rats.

Involvement of ovarian steroids and endogenous opioids in the fasting-induced suppression of pulsatile LH release in ovariectomized rats

The participation of the ovarian steroids and opioid peptides in the suppression of pulsatile LH release during acute fasting was examined in rats. Ovariectomized rats bearing silicone elastomer implants of oestradiol and/or progesterone were fasted for 48 h and subsequently blood samples were taken every 6 min for 3 h. Pulsatile LH release was suppressed after 48 h of fasting in the ovariectomized rats implanted with oestradiol but not in the oil-implanted controls. This suppression was enhanced after the administration of progesterone together with oestradiol. In a second experiment, ovariectomized rats bearing implants of oestradiol or oil were fasted for 48 h and injected s.c. (2.5 mg/kg body weight) with an opioid antagonist, naloxone hydrochloride, immediately before blood sampling. In the fasted oestradiol-treated ovariectomized rats, naloxone was able to prevent the suppression of pulsatile LH release. In the absence of oestradiol, however, naloxone was without effect on LH release in either the fasted or unfasted animals. These experiments indicate that the suppression of pulsatile LH release after 48 h of fasting is dependent upon oestradiol and that endogenous opioids are involved in the suppression.

Medial preoptic-anterior hypothalamic area involvement in the suppression of pulsatile LH release by a mu-opioid agonist in the ovariectomized rat

The objective of this study was to examine whether specific activation of mu-opioid receptors at the level of the medial preoptic-anterior hypothalamic area (MPOA-AHA) could suppress pulsatile LH release. The experiments were done using rats that had been ovariectomized (OVX) 24 hr before on diestrus 2, animals in which we have previously demonstrated an active endogenous opioid peptide suppression of pulsatile LH release (2). DAGO, DPDPE, or U50488H, specific agonists of mu-, delta- and kappa-opioid receptors, respectively, were continuously applied directly to the MPOA-AHA by means of push-pull perfusion. Perfusion of the MPOA-AHA with 0.5 μg DAGO/hr suppressed LH pulse amplitude. This effect of DAGO was not due to spread to the third ventricle and subsequent diffusion via the CSF to another CNS site, since push-pull perfusion with this dose of DAGO in the region just dorsal to or in the posterior hypothalamus was ineffective in altering LH pulse amplitude. The response to DAGO was dose-dependent since a higher dose (4.8 μg/hr) markedly suppressed both LH pulse amplitude and frequency. The same doses of DPDPE and U50488H (0.5 and 4.8 μg/hr) had no effect on pulsatile LH secretion, providing support for mu receptor involvement in the DAGO-induced suppressive action. These data demonstrate MPOA-AHA involvement in the suppression of pulsatile LH release by a mu-opioid agonist in the OVX rat.

Interaction between β-Endorphin and a-Melanocyte- Stimulating Hormone in the Control of Prolactin and Luteinizing Hormone Secretion in the Primate*

The ability of alpha MSH, a POMC-derived peptide, to antagonize the effects of beta-endorphin (beta EP) on PRL and LH secretion was studied in the primate. Seven ovariectomized rhesus monkeys bearing chronic indwelling third ventricular catheters for peptide infusion were used for these studies. Peripheral blood samples for PRL and LH RIA were obtained every 15 min during a 3-h control period when saline was infused into the ventricle, followed by a 5-h period of peptide infusion at a rate of 25 microliters/h. When beta EP was infused at a dose of 5 micrograms/h, plasma PRL rose from a mean baseline of 3.5 +/- 0.7 ng/ml to a peak of 21.3 +/- 2.2 ng/ml. When the same animals were infused with 20 micrograms alpha MSH together with 5 micrograms beta EP, the peak concentration of PRL was reduced to 8.2 +/- 1.7 ng/ml (P less than 0.001). When a higher dose of beta EP (20 micrograms/h) was infused, PRL rose to a peak of 38.2 +/- 1.8 ng/ml. This response was again markedly blunted, and the peak PRL response was reduced to 7.3 +/- 2.2 ng/ml when 20 micrograms beta EP were infused together with 80 micrograms alpha MSH (P less than 0.001). Analysis of the area under the plasma PRL concentration curves demonstrated a significant reduction in area during the 5-h infusion with beta EP plus alpha MSH compared to that during infusion of beta EP alone. The mean area was reduced from 3480 +/- 570 ng min/ml after 5 micrograms beta EP alone to 1030 +/- 200 after 5 micrograms beta EP plus 20 micrograms alpha MSH and from 6230 +/- 990 after 20 micrograms beta EP to 1020 +/- 320 ng min/ml after 20 micrograms beta EP plus 80 micrograms alpha MSH (P less than 0.01). Des-acetyl alpha MSH (80 micrograms) was also effective in reducing the PRL response to 830 +/- 380 ng min/ml (P less than 0.05). The suppression of pulsatile LH release by beta EP was also attenuated by alpha MSH. During the 5-h infusion of beta EP, total LH secretion was reduced to 65.9 +/- 3.8% of that measured during the 3-h saline infusion compared to 87.2 +/- 2.7% after infusion of beta EP plus alpha MSH (P less than 0.001) or 91.7 +/- 4.1% after beta EP plus des-acetyl alpha MSH (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Neurotransmitter involvement in naloxone-induced stimulation of pulsatile LH release on day 8 of pregnancy in the rat

Naloxone, an endogenous opioid peptide (EOP) receptor antagonist, increases LH pulse frequency and amplitude in early gestation in the rat (7). The object of this study was to further explore the suppression of pulsatile LH release by EOPs on day 8 of pregnancy by examining whether inhibition of norepinephrine (NE) or epinephrine (EPIN) synthesis, or stimulation of μ-aminobutyric acid (GABA)-B receptors, modified the ability of naloxone infusion (0.5 mg/kg/hr for 3.5 hr) to stimulate pulsatile LH secretion. Blood sampling (50 μl whole blood/5 min) began 0.5 hr after the onset of infusion. Three studies were conducted. 1) LY 134046 (PNMT inhibitor, 50 mg/kg IP), given at − 27, − 20, and − 3 hr relative to the onset of a 3-hr blood sampling period, produced no change in hypothalamic-preoptic area (HPOA) levels of NE, and a 76% decline in HPOA-EPIN levels, as determined by HPLC. Although basal LH pulse frequency was reduced, this treatment had no effect on the stimulatory action of naloxone on pulsatile LH release. 2) FLA-63 (DBH inhibitor, 25 mg/kg IP) given at − 3 hr produced a 72% decline in HPOA-NE levels, a 44% decrease in HPOA-EPIN values, and blocked the stimulatory action of naloxone on LH pulse frequency. Since depletion of HPOA-EPIN by LY 134046 did not compromise the LH response to naloxone, this effect of FLA-63 is due to depletion of HPOA-NE levels. 3) While saline had no effect on the increased pulsatile LH release caused by naloxone, administration of baclofen (GABA-B receptor agonist, 6 mg/kg IV) suppressed the pulsatile LH secretory response to naloxone infusion. These data demonstrate that inhibition of NE but not EPIN synthesis, or stimulation of GABA-B receptors, are both capable of interfering with naloxone-induced stimulation of pulsatile LH release on day 8 of pregnancy in the rat.

Analysis of estradiol-independent and -dependent endogenous opioid peptide suppression of pulsatile LH release between the mornings of diestrus 2 and proestrus in the rat estrous cycle

The aim of this study was to analyze possible estradiol (E 2)-independent and -dependent endogenous opioid peptide (EOP) suppression of pulsatile LH release between the mornings of diestrus 2 (D2) and proestrus by examining the LH response to naloxone infusions in the presence or absence of proestrous levels of E 2. Pulsatile LH secretion remained unchanged between D2 and proestrus but mean blood LH levels, pulse amplitude and frequency increased within 24 hr following ovariectomy on D2. This increase was due in large part to the loss of E 2 negative feedback, since restoration of physiological proestrous E 2 levels returned LH pulse frequency to proestrous a.m. levels and greatly reduced pulse amplitude. In ovariectomized rats lacking E 2 negative feedback, continuous infusion of the EOP receptor antagonist naloxone (0.5 and 2 mg/kg/hr) caused a further increase in pulse amplitude and frequency. This naloxone-induced increment in pulsatile LH release was exerted via centrally located EOP receptors since naloxone did not alter pituitary responsiveness to LHRH, and its stimulatory action on pulsatile release was diminished by simultaneous infusion with morphine. Naloxone also increased pulsatile LH release in E 2-treated animals. The naloxone-induced increments in LH pulse amplitude were the same in the presence or absence of E 2 negative feedback. Moreover, the increments in amplitude produced by naloxone in E 2-treated rats were significantly less than those resulting from the combination of ovariectomy plus naloxone infusion in empty capsule-implanted rats. These data indicated that naloxone infusion in E 2-implanted animals blocked an E 2-independent EOP suppression of this parameter of pulsatile release. In contrast, with both naloxone doses tested, there was a trend for naloxone-induced increments in pulse frequency to be larger in ovariectomized rats previously implanted with E 2 rather than empty capsules. Although these differences were not statistically significant, the naloxone-induced increments in pulse frequency in E 2-treated rats (unlike those in pulse amplitude) were large enough so that they were also significantly different from the overall increases in pulse frequency caused by the combination of ovarian steroid removal coupled with blockade of EOP receptors during naloxone infusion in empty capsule-implanted animals. This raises the possibility that naloxone infusions in E 2-treated rats compromised both an E 2-independent EOP suppression of pulse frequency and a small, E 2-dependent component as well. Overall, the data demonstrate that between the mornings of D2 and proestrus, EOPs act independent of E 2 to suppress both LH pulse amplitude and frequency, and the negative feedback of E 2 on LH pulse amplitude is not mediated by EOPs whose receptors are antagonized by naloxone. The data also suggest the possibility that E 2 suppression of pulse frequency may be mediated, at least in part, by EOPs.

Evidence that noradrenergic activity does not mediate the inhibition of luteinizing hormone secretion caused by vasoactive intestinal peptide

We have recently shown that intracerebroventricular (i.c.v.) infusion of vasoactive intestinal peptide (VIP) in ovariectomized (OVX) rats causes a pronounced inhibition of pulsatile luteinizing (LH) secretion. Since there is increasing evidence for an interaction between VIP- and norepinephrine-secreting systems in the brain, we tested the hypothesis that disruption of central noradrenergic activity would attenuate VIP-induced suppression of pulsatile LH release. Seven days prior to blood sampling, we administered either 6-hydroxydopamine (30 μg) or vehicle into the third cerebral ventricle of long-term OVX rats. This treatment reduced hypothalamic norepinephrine concentrations to 36% of control values. On the day of blood sampling, serial 300-μl blood samples were taken from conscious, unrestrained animals at 5-min intercals for a tota; of 4 h. Each sample was replaced with an equal volume of a blood replacement mixture. Immediately following a control bleeding period of 2 h, VIP was administered i.c.v. to each animal for 2 h (at a dose of 1.8 nmol/h). The suppression of pulsatile LH secretion caused by VIP infusion was virtually identical in the 6-hydroxydopamine- and vehicle-pretreated groups. These results indicate that a substantial depletion of hypothalamic norepinephrine does not attenuate VIP-induced suppression of pulsatile LH secretion. This provides evidence that centrally administered VIP inhibits pulsatile LH secretion by a mechanism independent of noradrenergic transmission.

Further studies on dopamine-induced suppression of pulsatile LH release in ovariectomized rats.

Third ventricular administration of dopamine (DA) in ovariectomized rats suppresses pulsatile LH secretion in a dose-dependent manner (Neuroendocrinology 29: 149, 1979). Since infusion of norepinephrine (NE) also produces a similar response, and DA can be taken up by noradrenergic fibers and converted to NE, an experiment was carried out to determine whether DA acted by directly activating DA receptors. This study also examined which parameters of pulsatile LH secretion were affected during DA-induced suppression of this release process, i.e., the LH interpulse interval and/or the magnitude of increase in blood LH levels produced by LH pulses. Infusion of DA in control rats pretreated with tartaric acid or /-butaclamol decreased mean blood LH levels. This effect is due entirely to a marked increase in the LH interpulse interval, since no change was seen in the magnitude of increase in blood LH levels produced by those LH pulses that did occur during DA infusion. Blockade of DA receptors with pimozide prevented the decrease in blood LH levels in response to DA and significantly reduced the change in the LH interpulse interval. These data indicate that DA suppresses pulsatile LH release solely by affecting the periodicity of this rhythm, and that this effect is mediated primarily through an activation of DA receptors. Pretreatment with d-butaclamol, another DA receptor antagonist, had no effect on the response to DA. As in control rats, DA-induced suppression of pulsatile LH release was again due entirely to a lengthening of the LH interpulse interval. d-Butaclamol does block the inhibition of pulsatile LH release due to peripheral administration of the DA receptor agonist, apomorphine (Endocrinology 100: 796, 1977). However, when concentrations of apomorphine equimolar to DA were infused into the third ventricle in d- or l-butaclamol-treated rats, comparable suppressions of pulsatile LH release occurred. This suggests that the failure of d-butaclamol to prevent the suppression of pulsatile LH release by intraventricular DA administration was apparently due to an ineffective level of DA receptor blockade.

Effect of manipulation of brain dopaminergic or serotoninergic systems on basal pulsatile LH release and perisuprachiasmatic-induced suppression of pulsatile LH release in ovariectomized rats.

This study examined the possible involvement of dopamine (DA) and/or serotonin (5-HT) in regulating basal pulsatile LH release, and in mediating perisuprachiasmatic (peri-SCN)-induced suppression of pulsatile LH secretion in ovariectomized rats. Unanesthetized animals were bled continuously through jugular vein cannulae for a 1½-h prestimulation period, 1½ h of stimulation, and up to 3/4 h after stimulation. Control rats were untreated or given <i>d</i>-butaclamol, the inactive stereoisomer of <i>d</i>-butaclamol. Other groups were pretreated either with pimozide or <i>d-</i>butaclamol to block DA receptors, parachlorophenylalanine (PCPA) or metergoline to inhibit 5-HT synthesis or block 5-HT receptors, respectively, or PCPA + pimozide to affect both 5-HT and DA systems. Prior to stimulation, regardless of whether DA and/or 5-HT systems were interfered with, no significant differences existed in mean blood LH levels, LH interpulse intervals, or the magnitude or rate of increase in blood LH levels for individual LH episodes. This suggests that DA and/or 5-HT may not be essential parts of the neurotransmitter systems regulating basal pulsatile LH secretion. During peri-SCN stimulation significant decreases in mean blood LH levels occurred in all groups. This was due entirely to a lengthening in the interval between LH pulses, since no group showed a decrease in the magnitude or rate of increase in blood LH levels for those LH pulses that did occur during stimulation. These results indicate that peri-SCN-induced suppression of pulsatile LH release is not mediated by known DA and/or 5-HT neuronal inputs to this region, and suggest that the peri-SCN area may be one brain region that influences, in a suppressive manner, only the periodicity of the pulsatile LH release process in the rat.