Infusion of progestins into the hypothalamus of female New Zealand white rabbits: effect on in vivo luteinizing hormone-releasing hormone release as determined with push-pull perfusion

Previously, we have reported that intermittent infusion of progesterone (P4; 10 ng/ml) into the hypothalamus of conscious unrestrained female New Zealand White rabbits stimulates LHRH release in vivo. To further investigate this phenomenon, in the present studies, the effect of pulsatile (six pulses; 10 min on, 30 min off) and continuous infusion of P4 (Exp I) and 4-pregnen-20 alpha-ol-3-one or 20 alpha-hydroxyprogesterone (20 alpha-OH-P; Exp II) on LHRH release were studied in vivo using push-pull cannulae. Furthermore, the effect of pulsatile infusion (six pulses; 10 min on, 30 min off) of low doses of the following three progestins [5 beta-pregnan-3 beta-ol-20-one (pregnanolone), 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-5 alpha-P), and 20 alpha-OH-P] into the hypothalamus of does using push-pull cannulae were examined in Exp III. In Exp I, continuous infusion of P4 at 10 ng/ml was unable to stimulate mean LHRH release. However, pulses of P4 at 0.01 ng/ml (n = 4) were found to significantly increase the amplitude of the largest LHRH pulse (control period, 1.18 +/- 0.41; versus treatment period, 3.15 +/- 0.75 pg; P less than 0.035) as well as the frequency of LHRH pulses (control period, 0.72 +/- 0.26; treatment period, 1.37 +/- 0.12 pulses/h; P less than 0.035). On the other hand, there was no effect of pulses of P4 at 0.001 ng/ml (n = 4) on the activity of the LHRH neural apparatus. In Exp II, pulses of 20 alpha-OH-P at 10 ng/ml (n = 4) were found to significantly increase the mean LHRH release (control period, 1.24 +/- 0.10; treatment period, 2.07 +/- 0.52 pg/10 min) as well as the amplitude of the largest LHRH pulse (control period, 0.99 +/- 0.36; treatment period, 8.15 +/- 3.75 pg). Interestingly, continuous infusion of 20 alpha-OH-P (10 ng/ml) also significantly increased the mean amplitude (control period, 0.58 +/- 0.34; treatment period, 1.90 +/- 0.29 pg) as well as the amplitude of the largest LHRH pulse (control period, 0.64 +/- 0.37; treatment period, 4.30 +/- 1.97 pg).(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of luteinizing hormone-releasing hormone release advances the onset of female puberty

The juvenile-peripubertal transition period in the female rat is associated with an ovarian-independent afternoon increase in the amplitude of plasma luteinizing hormone (LH) pulses. To determine if the immature pituitary could be activated to cause precocious puberty juvenile female rats were subjected for 4 days to a microprocessor-driven pulsatile intravenous administration of LH-releasing hormone (LHRH) at a dose that produced a peripubertal pattern of LH release. To determine if the LHRH neurons themselves could be prematurely activated to induce such a pattern of plasma LH, and hence lead to precocious puberty, the neuroexcitatory amino acid analog N-methyl-DL-aspartic acid (NMA) was similarly administered. The time of puberty (vaginal opening and first ovulation) was advanced by both the LHRH and NMA treatments, by 5 and 7 days, respectively. Ovarian weight and incidence of corpora lutea at first diestrus were similar in all animals regardless of treatment, but a juvenile body weight was retained by the animals that underwent precocious puberty. Therefore, just as the adenohypophysis can be driven by exogenous LHRH to initiate puberty, the LHRH neuronal system can be precociously activated by the episodic administration of an excitatory amino acid analog that is known to interact with specific brain receptors. It is likely, therefore, that sexual maturation is limited by factors that lie further upstream in the hypothalamo-pituitary axis (e.g., the neuronal circuits that impinge upon LHRH-producing neurons).     

Alterations in luteinizing hormone-releasing hormone release from the mediobasal hypothalamus of ovariectomized, steroid-primed middle-aged rats as measured by push-pull perfusion

The spontaneous luteinizing hormone (LH) surge on proestrus as well as the steroid-induced LH surge and hypersecretion of LH following ovariectomy are attenuated in middle-aged female rats when compared to their young counterparts. It is generally assumed that the lower titers of serum LH measured in aging animals result, in part, from age-related alterations in luteinizing hormone-releasing hormone (LHRH) neurosecretion, yet no direct measurements of LHRH release from the hypothalamus of aging females are currently available. The present study utilized the push-pull perfusion technique to characterize and compare in vivo LHRH output from the mediobasal hypothalamus of ovariectomized middle-aged and young females during a steroid-induced LH surge. Twelve-minute perfusates were collected for a period of 6 h from middle-aged and young animals outfitted with push-pull cannulae resting in the mediobasal hypothalamus in close proximity to the median eminence. The overall pattern of LHRH release differed in the two age groups. Mean levels of LHRH measured in brain perfusates from middle-aged females were significantly lower than mean levels measured in young animals. Moreover, mean levels of LHRH detected in perfusates during the 1- and 2-hour intervals prior to and the 1-hour interval after the time of maximal LHRH output were lower in middle-aged animals, and a larger percentage of samples from middle-aged females contained levels of LHRH below the detectability of the assay. The mean amplitude of identified LHRH pulses and maximal LHRH output, however, did not differ in the two age groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous measurement of luteinizing hormone-releasing hormone and luteinizing hormone release in unanesthetized, ovariectomized sheep

The push-pull perfusion technique was used in combination with a sequential bleeding schedule to estimate simultaneously the release patterns of LHRH and LH in unanesthetized ovariectomized sheep and to determine the temporal relationship between the release of these two hormones. Ovariectomized (greater than 30 days) ewes received unilateral push-pull cannula (PPC) implants (od, 0.85 mm) into the median eminence. After at least 6 days of recovery, each ewe was fitted with an indwelling jugular catheter. For push-pull perfusion, a stylette was removed from the outer PPC, and an inner cannula assembly (od, 0.40 mm) was inserted. Artificial cerebrospinal fluid was pushed through the inner cannula and pulled up between the cannulae at 20 microliters/min. Continuous 10-min perfusate fractions were collected, acidified, and stored at -20 C for LHRH RIA. Blood samples were obtained every 10 min via the jugular catheter, each being drawn 5 min after the start of a perfusate collection interval. Plasma LH levels were determined by RIA. The duration of the sampling was 3-7 h. LHRH output was distinctly pulsatile, occurring at a frequency of approximately one pulse every 30-40 min (n = 5 sheep). LHRH pulse amplitude and frequency remained relatively constant throughout each perfusion. Plasma LH values also were pulsatile, and all LH peaks occurred either during the same interval or during the interval after a LHRH pulse. LH pulses always were accompanied or directly preceded by LHRH pulses, but LHRH pulses were not always followed by LH pulses. The amplitudes of LH pulses and corresponding LHRH pulses were highly correlated (r = 0.81; P less than 0.01). Histological examination revealed that detection of LHRH in perfusates depended upon the placement of the PPC tip into either the zona externa of the central median eminence or adjacent areas. These experiments demonstrate that 1) hypothalamic LHRH release in the Ovx ewe occurs in discrete pulses, with a mean interpulse interval of 38.7 +/- 1.5 min, 2) LH pulses invariably are preceded or accompanied by LHRH pulses, and 3) LH pulse amplitude is highly correlated with LHRH pulse amplitude.     

N-methyl D,L-aspartate induces the release of luteinizing hormone-releasing hormone in the prepubertal and pubertal female rhesus monkey as measured by in vivo push-pull perfusion in the stalk-median eminence

The role of the excitatory amino acid glutamate, N-methyl D-aspartate (NMDA) receptor agonist, in stimulating in vivo luteinizing hormone-releasing hormone (LHRH) release in the stalk-median eminence of conscious prepubertal and pubertal female rhesus monkeys was evaluated using push-pull perfusion. In Exp 1, the effects of i.v. bolus injection of N-methyl D,L-aspartate (NMA) on LHRH release were examined. Injection of NMA induced an increase in LHRH release in all maturational stages of monkeys. Although the LHRH response to NMA tended to be larger in the older groups, only the duration of the LHRH response in the midpubertal group was significantly longer than that in the prepubertal group. In Exp 2, the effects of direct infusion of NMA (0.1, 1, and 100 microM) into the stalk-median eminence on LHRH release were similarly examined. NMA infusion stimulated LHRH release in pubertal monkeys, whereas it did not induce any consistent changes in LHRH release in prepubertal monkeys except for the highest dose. These data suggest that: 1) the systemic injection of NMA is more effective than direct infusion of NMA; and 2) the prepubertal LHRH neurosecretory system is capable of responding to NMDA, although the responsiveness may undergo developmental changes. Therefore, stimulation of NMDA receptors may contribute to the pubertal changes in the LHRH neurosecretory activity.     

Pituitary response to luteinizing hormone-releasing hormone in women with variant luteinizing hormone

OBJECTIVE: To assess the LH response of the pituitary gland to GnRH stimulation in healthy women with a mutant beta-subunit (Trp8 to Arg8 and Ile15 to Thr15). DESIGN: Clinical study. PATIENTS: We studied 40 healthy non-pregnant Japanese women of known zygosity for the LH beta-subunit gene (3 homozygotes for the mutant gene, 17 heterozygotes, and 20 homozygotes for the wild type). All women had normal ovulatory cycles. MEASUREMENTS: Serum LH status was determined by comparing LH immunoassays results using a monoclonal antibody recognizing only wild-type LH with those from a polyclonal antibody assay recognizing both variant and wild-type LH. The ratio of monoclonal to polyclonal immunoassay results determined the serum LH status. LH secretion in response to a GnRH stimulation test was measured. RESULTS: All women with the wild-type LH showed a normal response of LH to GnRH according to both assays. Over the time course of the response, the ratios in women with wild-type LH showed no remarkable changes. The response curves in women heterozygous for the mutant peaked 15-30min after GnRH injection; their response patterns included a statistically significant decrease in the rates of response at 15min after injection. CONCLUSIONS: There are the differences in circulatory kinetics between the two LH forms and in regulation of the two types of LHbeta genes. The maximal response of the variant LH to pituitary stimulation with GnRH appears to be greater than that of wild-type LH.     

Functional characteristics of the luteinizing hormone-releasing hormone pulse generator in conscious unrestrained female rabbits: activation by norepinephrine

We probed the activity of the neural LHRH apparatus by means of push-pull cannulae implanted in the hypothalamus of conscious intact anestrous unrestrained New Zealand White female rabbits raised under natural light. The data revealed that the neuropeptide LHRH is released in a pulsatile manner (one pulse per 46.4 +/- 3.9 min; amplitude, 1.63 +/- 0.39 pg; n = 7) from a rather extensive area within the basal hypothalamus of the rabbit covering the rostral, medial, and posterior regions in a rostro-caudal direction and about 1 mm lateral. A remarkable change in mean LHRH output was observed during the year, and two distinct types of LHRH output were detected: low (0.72 +/- 0.07 pg/10 min; n = 7) when these reflex ovulators were perfused during late spring to early summer and high (2.92 +/- 0.29 pg/10 min; n = 5) when perfused during late summer, early fall to spring months. This 4-fold annual change in LHRH output was mainly due to changes in the amplitude of the LHRH signal and was not correlated with the position of the push-pull cannula within a restricted area in the hypothalamus. These changes may be related to the state of the ovaries, since rabbits with high LHRH output showed larger ovaries with well developed follicles than rabbits with low LHRH output. Norepinephrine, a hypothalamic neurotransmitter known to be involved in the mounting-evoked ovulation reflex in this induced ovulator, elicited a 2-fold greater mean LHRH release (P less than 0.05) when infused in doses of 10(-7) - 10(-6) M directly into the hypothalamic perfusion area. The data indicate that the activity of the LHRH pulse generator in this species presents remarkable annual variations in its mean LHRH output mainly due to a greater amplitude of the LHRH signal and that norepinephrine is a potent stimulator of LHRH release in conscious unrestrained female rabbits.     

Stimulation of luteinizing hormone release from chicken pituitary cells by analogues of luteinizing hormone-releasing hormone.

The luteinizing hormone (LH) releasing activities of luteinizing hormone-releasing hormone (LH-RH) and four related analogues were compared using isolated chicken anterior pituitary cells. The analogues, des-Gly¹⁰-LH-RH and Phe⁵-LH-RH, exhibited a greater potency than LH-RH (150 and 237%, respectively), whereas LH-RH(OH) was much less active (1.1%). The potency of Phe⁵-LH-RH was reduced to 0.9% by the insertion of a tyrosine molecule at position 11, indicating that chain length is a significant feature of the biological activity of the molecule. des-Gly¹⁰-LH-RH and Phe⁵-LH-RH were more active in the present system, than is indicated by available information for the rat.     

Interleukin-1 inhibits the ovarian steroid-induced luteinizing hormone surge and release of hypothalamic luteinizing hormone-releasing hormone in rats

Interleukin-1 (IL-1), a polypeptide cytokine secreted by activated macrophages, has been postulated as a chemical messenger between the immune and endocrine systems. IL-1-immunopositive neurons and fibers have been visualized in the human and rat hypothalamus, and IL-1 receptors are present in the rat brain. We have examined the effects of human recombinant IL-1 (alpha- and beta-subtypes) on LH release in vivo and hypothalamic LHRH release in vitro. Ovariectomized rats were primed with estradiol benzoate, and progesterone was injected 48 h later to elicit a LH surge in the afternoon. IL-1 alpha and IL-1 beta were injected either intracerebroventricularly (icv) via a preimplanted cannula in the third ventricle of the brain or iv. Systemic injection of IL-1 alpha or IL-1 beta (58.8 pmol at 1300 and 1500 h) failed to influence the afternoon LH surge seen in saline-injected control rats. However, IL-1 beta (1.76 pmol) administered icv at 1300 and 1500 h or a single icv injection at 1300 h blocked the progesterone-induced LH surge. Similar icv injections of IL-1 alpha also significantly suppressed the afternoon LH surge compared to that in saline-injected control rats. However, IL-1 alpha was relatively less effective than the beta-subtype, since the LH surge was detected in some rats. To ascertain whether suppression of the LH surge was due to inhibition of LHRH release, the medial basal hypothalamus-preoptic area of estradiol benzoate-progesterone-treated ovariectomized rats was incubated with and without IL-1. Both IL-1 alpha and IL-1 beta, at concentrations of 0.1 nM and higher, significantly suppressed LHRH release in vitro from the medial basal hypothalamus-preoptic area. In contrast, IL-1 (10 nM) was completely ineffective in suppressing LHRH release from the microdissected median eminence. These results demonstrated an overall inhibitory effect of icv IL-1 on the LHRH-LH axis and suggest that suppression of the steroid-induced LH surge by IL-1 may primarily be due to inhibition of LHRH release at hypothalamic sites located within the blood-brain barrier.     

Estrogen induces the release of luteinizing hormone-releasing hormone in normal cyclic women

The plasma concentrations of immunoreactive LRH, LH, and FSH were determined by RIA every 6 h until 72 h after iv administration of conjugated estrogens during the midfollicular phase. The percentage change in LH from the preinjection level showed a biphasic pattern after the injection of conjugated estrogens, i.e. significant suppression (-70%) from 6-42 h after the injection, followed by a rebound increase with a peak (+150%) at 56 h. Plasma FSH after the injection also showed a biphasic pattern. The plasma immunoreactive LRH levels were unchanged until 32 h after the injection, but then increased significantly (P less than 0.02) to 160% of the preinjection level at 42 h and then decreased rapidly. These data indicate that 1) estrogen administration results in increases in plasma immunoreactive LRH and LH, and the peak of plasma LRH precedes that of gonadotropin; and 2) the negative feedback effect of estrogen on gonadotropin secretion may not be mediated through LRH.     

Effect of pulsatile infusion of progesterone on the in vivo activity of the luteinizing hormone-releasing hormone neural apparatus of awake unrestrained female and male rabbits

Eight female and eight male New Zealand White rabbits were outfitted with push-pull cannulae aimed at the tuberal region of the hypothalamus and were used in 19 perfusion experiments. Animals were treated under 2 conditions: a control condition in which female (n = 4) and male (n = 4) rabbits were perfused only with artificial medium for 5-9 h, and an experimental condition in which female (n = 4) and male (n = 5) rabbits were subjected to perfusion with artificial medium, followed by 6 pulses of progesterone (P4) (10 min on, 30 min off; 10 ng/ml) during a 240-min period. Two female rabbits were also subjected to perfusion with artificial medium, followed by 6 pulses of cholesterol (10 min on, 30 min off; 10 ng/ml) during a 240-min period. The LHRH concentration in perfusates collected every 10 min was measured by RIA. In the 4 females undergoing 6- to 7-h control push-pull perfusions, spontaneous pulses were observed, with about 1 pulse every 60 min, and a variable amplitude of the LHRH signal, with a mean release of about 0.91 pg/10 min. In the 4 does treated with pulsatile P4 at 10 ng/ml, the mean LHRH release rate increased significantly from 0.91 +/- 0.13 to 1.66 +/- 0.20 pg/10 min (P less than 0.035), primarily due to an increase in the amplitude of the LHRH pulses, which were significantly greater than controls. This response occurred with an apparent mean latency of about 50 min. Cholesterol pulses did not affect the spontaneous activity of the LHRH neural apparatus. In the 4 males undergoing 5- to 9-h control push-pull perfusions, spontaneous LHRH pulses were observed with about 1 pulse every 60 min, and the mean release increased from 1.25 +/- 0.56 to 1.54 +/- 0.55 (P less than 0.035, by Wilcoxon's matched pairs signed rank test) in the late afternoon primarily due to an increase in the amplitude of the pulses. P4 pulses did not affect the spontaneous activity of the LHRH neural apparatus compared to that in the control animals. Overall, these results clearly demonstrate that although spontaneous pulsatile LHRH release from the hypothalamus of awake unrestrained female and male rabbits is similar, pulses of P4 can activate only the female LHRH neural apparatus, with an apparent latency to peak LHRH release of approximately 50 min. In addition, there appears to be an increase in LHRH mean release levels in the late afternoon to early evening in male rabbits.     

Involvement of transforming growth factor alpha in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus.

Little is known about the presence of trophic factors in the hypothalamus and the role they may play in regulating the functional development of hypothalamic neurons. We have investigated the ability of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) to affect the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls reproductive development. We have also determined whether the genes encoding EGF and TGF-alpha are expressed in the prepubertal female hypothalamus. Northern blot analysis of poly(A)+ RNA utilizing a single-stranded EGF cDNA probe failed to reveal the presence of EGF mRNA in either the hypothalamus or the cerebral cortex at any age studied (fetal day 18 to postnatal day 36). In contrast, both a complementary RNA probe and a double-stranded TGF-alpha cDNA recognized in these regions a 4.5-kilobase (kb) mRNA species identical to TGF-alpha mRNA. The abundance of TGF-alpha mRNA was 3-4 times greater in the hypothalamus than in the cerebral cortex. Both EGF and TGF-alpha (2-100 ng/ml) elicited a dose-related increase in LHRH release from the median eminence of juvenile rats in vitro. They also enhanced prostaglandin E2 (PGE2) release. The transforming growth factors TGF-beta 1 and -beta 2 were ineffective. Only a high dose of basic fibroblast growth factor was able to increase LHRH and PGE2 release. Blockade of the EGF receptor transduction mechanism with RG 50864, a selective inhibitor of EGF receptor tyrosine kinase activity, prevented the effect of both EGF and TGF-alpha on LHRH and PGE2 release but failed to inhibit the stimulatory effect of PGE2 on LHRH release. Inhibition of prostaglandin synthesis abolished the effect of TGF-alpha on LHRH, indicating that PGE2 mediates TGF-alpha-induced LHRH release. The results indicate that the effect of EGF and TGF-alpha on LHRH release is mediated by the EGF/TGF-alpha receptor and suggest that TGF-alpha rather than EGF may be the physiological ligand for this interaction. Since in the central nervous system most EGF/TGF-alpha receptors are located on glial cells, the results also raise the possibility that--at the median eminence--TGF-alpha action may involve a glial-neuronal interaction, a mechanism by which the trophic factor first stimulates PGE2 release from glial cells, and then PGE2 elicits LHRH from the neuronal terminals.     

A modulatory role for luteinizing hormone-releasing hormone in nociceptive responses of female rats

We have shown that responsiveness to noxious stimuli change after gonadal steroid treatment and during the estrous cycle. In the present study, we evaluate the role of LHRH in modulating nociceptive responses in female rats. In ovariectomized (OVX) rats, an LHRH agonist ([ Des-Gly10] LHRH ethyl amide; 1 ng/rat/microliters), given intraventricularly (icv) at either 90, 60, or 30 min before a hot-plate test caused a time-dependent, significant increase in sensitivity to the noxious thermal stimulus (hyperalgesia) vs. saline-treated controls. Further, the LHRH agonist (1 ng/rat/microliters; icv) attenuated morphine (5 mg/kg, sc)-induced antinociception. The injection of an LHRH antagonist, [D-Phe2,Pro3,D-Phe6] LHRH, to OVX rats in doses of 0.1, 1, or 10 ng/rat 30 min prior to morphine, enhanced and prolonged morphine-induced antinociception in a dose-dependent manner. Moreover, the hyperalgesia observed in OVX rats treated with naloxone (1 mg/kg, sc) was reversed by preinjection of either the LHRH antagonist (0.1 ng/rat, icv) or LHRH antiserum. OVX rats primed with estradiol benzoate (EB) and progesterone (P) were less sensitive to the antinociceptive effect of morphine than OVX rats. When EBP-treated rats received the LHRH antagonist prior to morphine, a twofold increase in morphine-induced antinociception was observed. A similar effect was observed in EBP-treated rats after the injection of LHRH antiserum. In conclusion, LHRH may interact with central opioid systems causing an increased sensitivity to nociceptive stimulation (hyperalgesia) and reduction of the antinociceptive effect of morphine.     

Dissociation of the porcine anterior pituitary: the kinetics of luteinizing hormone release in response to luteinizing hormone-releasing hormone.

A method has been developed for disaggregating porcine anterior pituitary tissue and for providing dissociated preparations which have good, stable viability in culture. The secretory capacity for these preparations in terms of their ability to release luteinizing hormone in response to luteinizing hormone-releasing hormone has been documented in detail. In following the short-term kinetics of LH secretion a biphasic pattern of release has been demonstrated in which a sharp initial peak, maximal at 2 min, is followed by a second, more prolonged phase of release reaching a maximum between 10 and 20 min.     

Inhibitory pathways and the inhibition of luteinizing hormone-releasing hormone release by alcohol

In this research we examined the mechanisms by which ethanol (EtOH) inhibits luteinizing hormone-releasing hormone (LHRH) release from incubated medial basal hypothalamic explants. EtOH (100 mM) stimulated the release of two inhibitory neurotransmitters: gamma-aminobutyric acid (GABA) and beta-endorphin. EtOH also inhibited NO production, indicative of a suppression of nitric oxide synthase (NOS) activity. This inhibition was reversed by naltroxone (10(-8) M), a micro-opioid receptor blocker, indicating that the inhibition of NOS by EtOH is mediated by beta-endorphin. EtOH also blocked N-methyl-d-aspartic acid-induced LHRH release, but the blockade could not be reversed by either the GABA receptor blocker, bicuculline (10(-5) M), naltroxone (10(-8) M), or both inhibitors added together. However, increasing the concentration of naltrexone (10(-6) M) but not bicuculline (10(-4) M) reversed the inhibition. When we lowered the concentration of EtOH (50 mM), the EtOH-induced blockade of LHRH release could be reversed by either bicuculline (10(-5) M), naltroxone (10(-8) M), or the combination of the two blockers. Therefore, GABA is partially responsible for the blockade of N-methyl-d-aspartic acid-induced LHRH release. The block by GABA was exerted by inhibiting the activation of cyclooxygenase by NO, because it was reversed by prostaglandin E(2), the product of activation of cyclooxygenase. Because the inhibition caused by the higher concentration of EtOH could not be reduced by bicuculline (10(-4) M) but was blocked by naltroxone (10(-6) M), the action of alcohol can be accounted for by stimulation of beta-endorphin neurons that inhibit LHRH release by inhibition of activation of NOS and stimulation of GABA release.