Neuropeptide Y levels in microdissected regions of the hypothalamus and in vitro release in response to KCl and prostaglandin E2: effects of castration

Intracerebroventricular administration of neuropeptide Y (NPY) has been shown to modify LH secretion, with the direction of the response dependent on the steroid background. To study further the role of gonadal steroids in the regulation of NPY secretion, the basal and KCl-evoked release of NPY from the medial basal hypothalamus (MBH) of intact and castrated male rats was assessed twice with the use of an in vitro incubation system. In each experiment, the amounts of NPY released in response to a 15-min pulse of KCl (45 mM) were significantly smaller from the MBH of castrated rats than of intact rats (P less than 0.05). Next, to assess the possible effects of prostaglandin E2 (PGE2), the MBH were exposed in a similar manner to two 15-min pulses, 30 min apart, of 0.568 and 56.8 mumol PGE2. Unlike KCl, PGE2 failed to stimulate NPY release from the MBH of either intact or castrated rats. However, a similar 56.8 mumol concentration of PGE2 was effective in stimulating the release of LHRH. We next examined the effects of castration on NPY levels in several microdissected regions of the hypothalamus. Whereas NPY concentrations were unchanged in the medial preoptic area, paraventricular nucleus and dorsomedial nucleus, NPY levels were significantly decreased in the median eminence, arcuate nucleus, and ventromedial nucleus 2 weeks after castration. These studies show that KCl can stimulate NPY release from the MBH in vitro, like that of LHRH, the KCl-induced NPY response is significantly smaller from the MBH of castrated than intact males, castration can significantly reduce the levels of NPY in the median eminence, arcuate nucleus, and ventromedial nucleus, thereby suggesting that testicular secretions may modulate NPY levels and release from the MBH, and because PGE2 stimulated the release of LHRH but not of NPY, separate regulatory neural events may underlie the secretion of these two neuropeptides.     

Testosterone raises neuropeptide-Y concentration in selected hypothalamic sites and in vitro release from the medial basal hypothalamus of castrated male rats

Although neuropeptide-Y (NPY)-containing neurons are widely distributed in the hypothalamus, castration decreased NPY concentrations only in the median eminence (ME), arcuate nucleus (ARC), and ventromedial nucleus (VMN). We have now examined the effects of testosterone (T) replacement in 2-week castrated male rats on NPY levels in hypothalamic and preoptic area regions and in vitro NPY release in three experiments. In the first experiment we studied the effect of T on NPY concentration in castrated rats. Two-week castrated rats were implanted sc with T-filled or empty Silastic capsules 30 mm in length. Ten days later rats were killed, and NPY levels were measured by RIA in microdissected sites. T implants raised serum T levels to the range found in gonad-intact rats and decreased serum LH levels to the basal range. Further, of the six brain sites examined, significant increases in NPY concentrations occurred selectively in the ME, ARC, and VMN of T-implanted rats. In the second experiment, the ability of T to reverse the effect of castration on NPY levels compared to those in intact (sham) rats was assessed. Again, castration decreased NPY levels in the ME, ARC, and VMN only, and replacement of physiological levels of T restored NPY levels approximately 100%, 127%, and 74% in the ARC, VMN, and ME, respectively. In the third experiment, the effect of castration and T implants (30-mm T capsules for 10 days) to 2-week castrated rats on the in vitro release of NPY from medial basal hypothalamus (MBH) was assessed. Basal NPY release was not significantly changed after castration and T replacement. However, in response to a 30-min pulse of KCl (45 mM) NPY release from the MBH of castrated rats was significantly reduced compared to that in intact and T-replaced castrated rats. These studies show that castration decreases and T replacement restores NPY levels selectively in three hypothalamic sites, viz. ME, ARC, and VMN, and KCl-induced NPY release from the MBH in vitro is decreased after castration and restored by T replacement, thereby suggesting that a local subset of androgen-concentrating neurons may regulate NPY levels and release in a site-specific manner. Further, these results are in line with our emerging view that gonadal steroids modulate neurosecretion not only of LHRH, but also of other functionally linked regulatory peptides.     

Simultaneous measurement of luteinizing hormone (LH)-releasing hormone, LH, and follicle-stimulating hormone release in intact and short-term castrate rats

The temporal relationship between LHRH release and gonadotropin secretion as well as the effects of castration on LHRH release were investigated in conscious, freely moving male rats. LHRH release was measured in hypothalamic/median eminence perfusates, while levels of pituitary gonadotropins (LH, FSH) were determined in sequential blood samples obtained via atrial catheters. Twenty-four to 26 h before experiments, rats underwent sham surgery or castration. LHRH release in push-pull perfusates from both groups was pulsatile, and nearly all identified LH pulses (83.3%) were temporally associated with LHRH pulses. Of the fewer irregular FSH pulses that were observed, only 43.7% were temporally associated with LHRH pulses. Mean LHRH pulse amplitude and mean LHRH levels were not different in intact and castrate animals. The frequency of LHRH pulses was moderately increased in castrate rats (1.30 pulses/h) compared to that in intact animals (0.83 pulses/h), and this acceleration was accompanied by a significant increase in LH pulse frequency, pulse amplitude, and mean level. It was also noted that the number of silent LHRH pulses (those not associated with LH pulses) was dramatically reduced in castrate animals. Characteristics of gonadotropin release (pulse frequency, pulse amplitude, and mean level) were not significantly different in animals undergoing push-pull perfusion/bleeding procedures from those in rats not receiving push-pull cannula implants. We conclude from these studies that 1) LH pulses show a high concordance with LHRH pulses, providing evidence that the LHRH pulse generator operates as the neural determinant of LH pulses in male rats, 2) FSH secretion is not associated with LHRH release in an obvious and consistent manner, suggesting that LHRH/FSH relationships are not easily discerned in these animals or that a FSH-releasing factor distinct from the LHRH decapeptide may regulate FSH secretion, 3) a modest increase in LHRH pulse frequency occurs 24-30 h after castration, and 4) silent LHRH pulses occur with much greater regularity in intact than in castrate rats. The latter two observations suggest that both hypothalamic and intrapituitary sequelae of castration may be critically important in the development of postcastration increases in LH secretion and the negative feedback of gonadal steroids.     

Neuropeptide-Y innervation of beta-endorphin-containing cells in the rat mediobasal hypothalamus: a light and electron microscopic double immunostaining analysis.

Central administration of neuropeptide-Y (NPY) inhibits pituitary LH release in ovariectomized rats and stimulates LH release in intact and ovariectomized rats pretreated with ovarian steroids. Although the precise neural mechanism of this dual effect of NPY is not known, experimental evidence suggests an underlying interaction between hypothalamic NPY and the inhibitory beta-endorphin (beta END) systems in the neuroendocrine regulation of pituitary LH release in the rat. The present study was undertaken to examine the morphological basis of the interaction between these two peptidergic systems in the hypothalamus. Sections of the mediobasal hypothalamus of colchicine-pretreated female rats were double immunostained for NPY and beta END and examined by light and electron microscopy. The light brown diaminobenzidine reaction was used to visualize beta END cells, while NPY neurons were labeled with a dark blue nickel ammonium sulfate-intensified diaminobenzidine reaction. Under the light microscope, a dense network of NPY-immunoreactive axons and axon terminals was observed in close apposition with beta END-immunoreactive neurons throughout the medial basal hypothalamus. Electron microscopic examination revealed that NPY-immunoreactive boutons formed axosomatic and axo-dendritic synaptic connections with beta END cells. A majority of these synaptic membrane specializations appeared asymmetrical [corrected]. In light of the previous evidence of excitatory and inhibitory effects on LH release and the existence of direct synaptic connections between NPY and LHRH neurons in the hypothalamus, the current results imply that the dual effects of NPY on LH secretion may involve modulation of LHRH secretion, both by the direct route and indirectly through the hypothalamic beta END system.     

Differential response of luteinizing hormone-releasing hormone in the basal hypothalamus and the preoptic area following anterior hypothalamic deafferentation and/or castration in male rats.

Serum LH, FSH and LHRH concentrations and the LHRH content in the medial basal hypothalamus (MBH) and the preoptic area (POA) were measured by radioimmunoassay in male rats 33 days after anterior hypothalamic deafferentation (AHD) and/or castration. In castrate rats following AHD, there was a significant decrease in serum LH, FSH, and LHRH concentrations, whereas, in intact rats, serum LH was elevated in AHD over the sham AHD rats. Castration and AHD each caused a significant fall in the LHRH levels in the MBH; the decline was more pronounced in rats undergoing both castration and AHD. In contrast, deafferentation in intact and castrate rats resulted in the accumulation of LHRH activity in the POA. These studies support the suggestion that a) a substantial amount of LHRH normally found in the MBH of intact and castrate male rats originates in the rostral regions and, b) the LHRH-containing neural elements within the MBH have the competence to respond to a loss in the circulating testicular steroids.     

Prostaglandin D2 induces release of luteinizing hormone from the rat pituitary gland without the modulation of hypothalamic luteinizing hormone releasing hormone

The effect of prostaglandin D2 (PGD2) on release of LH and LH releasing hormone (LHRH) was studied in a sequential double-chamber superfusion system using the medial basal hypothalamus (MBH) and the pituitary gland from female rats at dioestrus. Infusion of PGD2 (5.7 or 57 mumol/l) caused a significant (P less than 0.05) increase in LH release to values 40-60% above the preinjection values from the pituitary gland superfused either alone or in series with the MBH. No release of LHRH in response to PGD2 was observed from the superfused MBH. These data demonstrate that PGD2 causes LH release from the pituitary gland not by inducing release of hypothalamic LHRH but by a direct action on the gland.     

The influence of chronic morphine treatment on the negative feedback regulation of gonadotropin secretion by gonadal steroids

The influence of continuous stimulation of opiate receptors with morphine (M) on the negative feedback effects of testosterone (T), 5 alpha-dihydrotestosterone (DHT), and 17 beta-estradiol (E2) on LH and FSH secretion was studied in rats that had been castrated 2 weeks previously. In the absence of gonadal steroids, 4 days of continuous M exposure did not alter LH or FSH levels. Similarly, Silastic capsules containing crystalline T (5 mm) or E2 [5 mm long (75 micrograms E2/ml) to 7.5 mm long (300 micrograms E2/ml)] alone had little effect on LH or FSH release. However, in M-exposed rats, T reduced serum LH by greater than 90%, and E2 reduced LH by more than 75%. Among the doses of DHT evaluated, only the highest dose (7.5-mm Silastic capsules packed with crystalline DHT) reduced LH secretion, and M exposure only slightly enhanced this suppression. M or gonadal steroids alone produced little change in FSH levels in castrated rats. However, the combination of M plus E2 or DHT further reduced FSH levels. Evaluation of pituitary responses to LHRH revealed that when administered alone, T did not alter, DHT reduced, and E2 enhanced the LH response to the decapeptide. Neither M treatment alone nor M plus T or DHT altered the pituitary LH response to LHRH. On the other hand, M appeared to enhance the stimulatory effects of E2 on pituitary responsiveness to LHRH. These findings suggest that the interaction of M and gonadal steroids at the level of the pituitary could not explain the observed marked suppression of gonadotropin secretion by suboptimal T or E2 during opiate receptor stimulation with M. Collectively, these observations are in accord with the view that endogenous opioid peptides may play a role in modulating the sensitivity of the hypothalamus to the negative feedback effects of gonadal steroids.     

Effect of Nitric Oxide Synthase Inhibitors on Preventing Ethanol-Induced Suppression of the Hypothalamic-Pituitary-Gonadal Axis in the Male Rat

Ethanol (EtOH) suppression of the hypothalamic-pituitary-gonadal (HPG) axis results in broad reproductive malfunction. In the HPG axis, the suppressive effects of EtOH are manifested by decreased serum testosterone, reduced testicular luteinizing hormone (LH) receptor numbers, lowered serum LH and pituitary β-LH mRNA levels (in castrated animals), and impaired luteiniring hormone releasing hormone (LHRH) release from the hypothalamus. Increasing evidence has suggested that nitric oxide (NO) plays a role in regulation of the HPG axis. NO was shown to stimulate LHRH secretion from the hypothalamus and to have variable effects on LH release from the pituitary. At the gonadal level, NO is inhibitory to testosterone production. NO may directly inhibit some testicular steroidogenic enzymes. To investigate the effect of EtOH, NO, and their interaction on the male HPG axis, three NO synthase (NOS) inhibitors, N^Q-nitro- l -arginine methyl ester, N^Q-nitro- l -arginine, and 7-nitro indazole were used to study overall HPG function in the presence and absence of EtOH. Animals were given intraperitoneal injections of saline, EtOH, various NOS inhibitors, or EtOH, along with NOS inhibitors 2 hr before sacrifice. Serum testosterone and LH concentrations, pituiiry βLH mRNA levels, hypothalamic LHRH mRNA levels, and LHRH content were determined. It was found that blocking NOS by these NOS inhibitors prevented EtOH-induced suppression of testosterone and, in some cases, serum LH. However, this was not accompanied by concurrent changes with NOS blockade on LHRH mRNA, hypothalamic pro-LHRH or LHRH content or pituitary LH β mRNA levels. It appears that the protective effect of NOS blockade was largely, although not completely, due to a direct effect at the gonadal level.     

Neuropeptide Y enhances the release of luteinizing hormone (LH) induced by LH-releasing hormone

Depending upon the steroid hormonal milieu, centrally administered neuropeptide Y (NPY) exerts differential effects on the release of LH. Ovarian hormones also effect the concentrations of NPY in hypothalamic nuclei, and some of the changes are similar to those caused by LHRH. The present studies tested whether NPY acts directly on the pituitary gland, either alone or in combination with LHRH, to modify LH secretion. Hemipituitary fragments obtained from ovariectomized rats were incubated in medium 199, and the in vitro effects on LH release of LHRH, NPY, or the two peptides together were assessed. As expected, LHRH (10(-9)-10(-7) M) produced a dose-dependent release of LH, whereas NPY alone had a lesser stimulatory effect at concentrations of 10(-7) or 10(-6) M. On the other hand, 10(-6) M NPY significantly enhanced LH release in response to 10(-9) M LHRH. A potentiation by NPY of the LHRH-induced LH response was observed in an anterior pituitary cell culture system. Cells from the pituitaries of ovariectomized rats were dispersed and cultured for 3 days in medium 199 with BSA, gentamicin, horse serum, and fetal calf serum. During a 3-h incubation, NPY alone (10(-9)-10(-7) M) failed to affect LH release, but significantly potentiated the release induced by 10(-9) or 10(-8) M LHRH. These findings are in accord with the hypothesis that hypothalamic NPY neurons may participate in the regulation of LH secretion in the rat and indicate that one of the mechanisms of its action may be to increase the pituitary LH response to LHRH.     

Effects of testosterone implants and hypothalamic lesions on luteinizing hormone regulation in the castrated male rat.

The effects of intrahypothalamic and subcutaneous implants of testosterone (T) and those of hypothalamic lesions on resting levels of circulating LH and pituitary responsiveness to exogenous LHRH were studied in castrated male rats to elucidate hypothalamic and pituitary regulation of LH secretion. Two hundred mug implants of testosterone propionate (TP) in the median eminence region suppressed plasma LH titers before evidence of direct inhibition of pituitary function (as indicated by testing with LHRH) was found. Such implants release appreciable amounts of T into the peripheral circulation in the immediate post-operative period, and SC Silastic (constant release) capsules containing T have similar effects. The findings suggest that, regardless of the site of implant, the initial negative feedback inhibition of LH by T is not dependent on direct action at the pituitary levels but rather appears to be a hypothalamic effect. In the days following exposure to hypothalamic or peripheral implantation of T, however, a progressively developing decline in the response to exogenous LHRH was observed. In order to determine whether this effect results from suppression of endogenous LHRH release, the median eminence-arcuate region was destroyed to remove the source of LHRH. In these animals, the suppression of plasma LH was evident on the first day after the lesion, but pituitary responsiveness to LHRH was unaffected until after one week. When Sialastic capsules were implanted SC into lesioned animals, a more rapid (less than 1 week) inhibition of pituitary responsivity ensued. Suprachiasmatic lesions did not affect basal LH secretion or pituitary responses to LHRH. The data provide evidence for a dual feedback action of T on LH in castrated male rats: an initial inhibitory effect presumably due to hypothalamic inhibition (commencing at around 6h after hypothalamic of SC implantation of T), and a subsequent suppression of pituitary responisveness (after one day) presumably due to direct action of T on the pituitary. In addition to these phenomena, findings in rats bearing median eminence-arcurate lesions suggest that the removal of endogenous LHRH by itself leads to an eventual decline in pituitary responsiveness (greater than one week postoperatively).     

Neuropeptide Y stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamus in vitro: modulation by ovarian hormones

These studies investigated the effects of neuropeptide Y (NPY) on in vitro release of luteinizing hormone-releasing hormone (LHRH) from the medial basal hypothalmus (MBH) and tested whether ovarian steroids modulate the LHRH response to NPY. Ovariectomized rats were implanted with 20-mm-long Silastic capsules containing a low concentration of estradiol (E2) (150 micrograms/ml oil), a high concentration of E2 (250 micrograms/ml oil), or sesame oil vehicle. Additional animals received high-dose E2 capsules plus an injection of progesterone (15 mg) concomitantly. Two days later, individual MBH fragments were incubated in medium alone for a 30-min period to obtain the basal rate of LHRH release, followed by a second 30-min period in medium containing NPY or saline. Exposure to NPY (10(-6) M) increased the release of LHRH from MBH of ovarian hormone-treated, but not from hormonally untreated rats. The LHRH response was most pronounced from the MBH of rats treated with either high-dose E2 or E2 plus progesterone. The increase in LHRH release was also elicited by 10(-7) M, but not by 10(-8) M NPY concentrations, using MBH from E2 plus progesterone-treated rats. In addition, NPY markedly potentiated the KCl-evoked release of LHRH from MBH of ovariectomized, hormonally untreated or low-dose E2-treated rats, under conditions when there was little or no effect of NPY on the basal LHRH release. Further, the release of LHRH stimulated by NPY was not accompanied by increase in the release of norepinephrine or of dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opiate-induced hypersensitivity to testosterone feedback: pituitary involvement

We have examined the mode of morphine's (M) action to increase the sensitivity of castrated male rats to the inhibitory feedback action of testosterone (T) on LH release. In castrated rats, sc implantation of M pellets or 5-mm long T-filled capsules (T5) failed to suppress LH release, but a combination of M and T5 drastically decreased serum LH levels. Likewise, while treatment with a higher dose of T (30-mm long implant, sc) suppressed LH release, combined treatment with M and T30 produced a further suppression of LH levels. We have now assessed the in vitro release rate of LHRH from the medial basal hypothalamus-preoptic area of castrated rats treated with M and/or T as well as the in vivo pituitary LH response to LHRH challenge in similarly treated rats. Interestingly, the in vitro basal and naloxone-induced LHRH release from the medial basal hypothalamus-preoptic area of the six groups of rats was similar, regardless of whether LH levels were in the high castrate or low basal range. On the other hand, M treatment greatly attenuated LH release in vivo in response to LHRH challenge (10pmol-1pmol) [corrected] in T-treated rats. In fact, LH increments in response to 1pmol [corrected] LHRH, seen in control, T5, and T30 groups, were abolished by additional M treatment of T-treated rats. This in vitro assessment of LHRH release suggests that the drastic decrease in LH release in T-plus M-treated rats may not be due to impaired LHRH release, but, rather, be due in part to reduced pituitary responsiveness to intermittent endogenous LHRH signals. The reduced pituitary responsiveness to LHRH in T-plus M-treated rats may be a consequence of either a direct pituitary effect of opiates in conjunction with T or augmented action of hypothalamic neurohumoral agents which may inhibit LH release on their own or antagonize the LH-releasing action of LHRH at the level of pituitary gonadotrophs.     

Simultaneous induction of neuropeptide Y and gonadotropin-releasing hormone release in the rabbit hypothalamus

Neuropeptide Y (NPY) can induce the release of endogenous mediobasal hypothalamic gonadotropin-releasing hormone (MBH-GnRH) and pituitary gonadotropins, especially LH. In these studies, we monitored changes in endogenous NPY concentrations at 20-min intervals for 6-8 h during push-pull perfusion (PPP) in both the mediobasal hypothalamus (MBH) and the third cerebroventricle (3VT) of ovarian intact, conscious rabbits. Because previous studies had shown that copper ion can induce hypothalamic GnRH release, cupric acetate (CuAc) was administered either intravenously or intraventricularly during the PPP to manipulate changes in NPY concentrations. Our results show that NPY concentrations in both MBH and 3VT PPP samples were detectable by radioimmunoassay. Administration of CuAc sharply increased hypothalamic NPY release within the same time interval as that for induction of hypothalamic GnRH release. The results are consistent with the hypotheses that NPY may act as a neuromodulator for hypothalamic GnRH secretion, or that common mechanisms drive secretion of these two neuropeptides.     

Progesterone-induced changes in hypothalamic luteinizing hormone-releasing hormone and catecholamines: differential effects of pentobarbital

The effects of pentobarbital (Pnt) treatment on the progesterone (P)-induced afternoon increase in the medial basal hypothalamic (MBH) LHRH and serum LH and FSH levels in ovariectomized estradiol benzoate-primed rats were studied. Pnt injection before P blocked the afternoon rise in serum gonadotropins but failed to alter the increase in the MBH LHRH levels. Moreover, when Pnt was injected 150 min after P, the MBH LHRH content continued to rise to levels 25-37% above those seen in control rats. Analyses of LHRH concentrations in discrete hypothalamic nuclei revealed that the Pnt-induced accumulation was confined mainly to the median eminence, with a small increase in the suprachiasmatic nuclei region. P administration increased the MBH norepinephrine activity and concurrently decreased dopamine activity. Pnt was ineffective in suppressing the MBH LHRH response in these rats, but drastically reduced norepinephrine and accelerated dopamine turnovers in the MBH. These studies show 1) no definitive cause and effect relationship of the increments in MBH LHRH either with LH release (or LHRH release) or with changes in hypothalamic catecholamines induced by P treatment, and 2) that the striking rise in the MBH LHRH levels in estradiol benzoate-primed rats may represent formation of new immunoreactive LHRH predominantly in the median eminence region.     

Effects of LH-releasing hormone antagonist or lesions of the medial basal hypothalamus on periovulatory gonadotrophin release in female rats

Follicle-stimulating hormone release on the morning of oestrus was examined by using two different techniques which eliminate LH-releasing hormone (LHRH) stimulation of the pituitary gland. Cyclic female rats were given a potent LHRH antagonist (ALHRH) or were subject to electrolytic lesions of the medial basal hypothalamus (MBH) before or after the pro-oestrous phase of FSH release. Administration of ALHRH at 14.00, 15.30 and 17.00 h or lesioning of the MBH between 11.30 and 13.00 h on pro-oestrus entirely blocked the preovulatory LH surge and both phases of FSH release. Ovulation was abolished in all of these animals. However, when ALHRH was given at 20.30, 22.00 and 23.30 h or lesions of the MBH made between 20.00 and 21.30 h on pro-oestrus after the pro-oestrous FSH and LH surges had occurred, the oestrous phase of FSH release was indistinguishable from that of saline-treated control rats. Ovulation occurred in all of these animals, and the mean number of ova shed was eight/rat. The conclusions are that (1) the pro-oestrous phase of FSH release is dependent upon the hypothalamic hormonal stimulation by LHRH and (2) the oestrous phase of FSH release is entirely independent of direct LHRH stimulation, or any hypothalamic stimulus.     

Effects of neuropeptide Y on the in vitro release of gonadotropin-releasing hormone, luteinizing hormone, and beta-endorphin and pituitary responsiveness to gonadotropin-releasing hormone in female macaques

The objectives of these studies were to examine the release of gonadotropin-releasing hormone (GnRH) and beta-endorphin-like activity (beta-EP) from macaque hypothalami, and the release of luteinizing hormone (LH) and GnRH-induced LH from macaque anterior pituitaries in response to neuropeptide Y (NPY) treatment. Anterior hypothalamic (AH) and mediobasal hypothalamic (MBH) blocks of tissues and the adenohypophysis were bisected along the midline into two equal-sized fragments. Fragments were superfused with medium for 3 h, followed by 3 h of either NPY (80 nM) or medium alone. In a separate experiment, adenohypophyseal (AP) fragments were superfused in accordance with the same protocol (3 h medium - 3 h NPY or medium) except that exogenous GnRH (352 nM) was added for 30 min at the beginning of hour 3 and again at the beginning of hour 6. Immunoactive GnRH, beta-EP, and LH levels were measured in superfusate samples (400 microliters) collected at 10-min intervals. GnRH levels rose within 20-30 min of initiation of NPY treatment, and elevated GnRH release was sustained for the duration of NPY exposure of both AH and MBH fragments from ovarian intact (INT) rhesus (Macaca mulatta: n = 8; p less than 0.05) or Japanese (Macaca fascicularis; n = 4; p less than 0.01) macaques. NPY treatment had no effect on either AH or MBH fragments isolated from ovariectomized (OVX) rhesus macaques (n = 4 for AH, and n = 5 for MBH). In AP fragments isolated from INT rhesus macaques (n = 8), NPY stimulated LH release within 1 h of treatment (p less than 0.05), whereas NPY had no effect on pituitaries from OVX animals (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential responses of hypothalamic LHRH-I and -II to castration and gonadal steroid or tamoxifen treatment in cockerels

Changes in the hypothalamic contents of LHRH-I and LHRH-II were determined in intact and castrated cockerels injected i.m. with gonadal steroids or tamoxifen. An increase in the plasma concentration of LH after castration was accompained by a significant increase in the content of LHRH-I in the posterior hypothalamus (including the mediobasal hypothalamus and median eminence) which was reversed by oestradiol benzoate given on days 14 and 15 after castration. Under similar circumstances, testosterone propionate did not modify the hypothalamic content of LHRH-I, even though both steroids reduced the plasma concentrations of LH to levels below those of intact cockerels. Treatment of intact cockerels with oestradiol benzoate significantly increased the content of LHRH-I in the posterior hypothalamus, whilst testosterone propionate was again without effect. Tamoxifen significantly raised the plasma concentration of LH in intact cockerels and partially antagonized the suppressive effect of oestradiol benzoate and testosterone on LH secretion in castrated cockerels. However, an anti-oestrogenic effect of tamoxifen on the hypothalamic content of LHRH-I was not demonstrated. There was no evidence of any changes in the hypothalamic content of LHRH-II after castration, with or without gonadal steroid replacement. A change in the hypothalamic content of LHRH-I in response to manipulation of the steroid environment would imply an involvement of this peptide in the mechanism by which gonadal steroids regulate the release of LH. The absence of changes in the hypothalamic content of LHRH-II in the same circumstances suggest that it is not directly involved in the control of LH secretion by the gonadal steroid negative feedback loop.     

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-induced LH secretion only under conditions leading to preovulatory LH surges.

We recently demonstrated that neuropeptide Y (NPY) potentiates the ability of pulsatile LHRH infusions to restore LH surges in pentobarbital (PB)-blocked, proestrous rats. In the present study we determined if specific endocrine conditions are necessary for the expression of these direct pituitary effects of NPY. Facilitatory actions of NPY were examined in the absence of gonadal feedback [ovariectomy (OVX)], in the presence of negative gonadal feedback (metestrus), after estrogen priming of the pituitary gland [OVX plus 30 micrograms estradiol benzoate (EB) 2 days before experiments], and after treatments which evoke preovulatory-like LH surges (OVX plus EB and 5 mg progesterone or P the morning of experiments). Rats received jugular catheter implants the day before experiments. On the day of experiments, hourly blood samples were taken from 1100-2100 h. At 1330 h, rats received injections of PB to block endogenous LHRH release, or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15 ng/pulse) or saline, along with concurrent pulses of NPY (1 or 5 micrograms/pulse) or saline. Plasma samples were analyzed by LH RIA. In all cases, pulsatile administration of 15 ng LHRH resulted in plasma LH levels that were significantly elevated above saline-treated, PB-blocked controls. Only in the case of EB+P-treated rats did coadministration of 5 micrograms NPY along with LHRH significantly enhance LHRH-stimulated LH secretion (P < 0.001). NPY had no effect on LHRH-stimulated LH secretion in OVX, OVX + EB-treated, or metestrous rats. Pulsatile administration of either dose of NPY alone did not stimulate LH release in any of the four groups examined. These results demonstrate that the facilitatory effects of NPY on LHRH-stimulated LH secretion can be manifest only under the endocrine conditions required to produce full, preovulatory-like LH surges, i.e. after estrogen and P treatment.     

Relation of gonadal hormones to differential LH response to naloxone in prepubertal male and female rats

Naloxone (NAL) has been shown to induce LH release in female but not in male rats 10-25 days of age. The purpose of this study was to examine the role of neonatal gonadal hormones on NAL-induced LH release in male and female rats 15, 25, and 35 days of age. On each of these days rats received a s.c. injection of either NAL (5 mg/kg) or physiological saline, and blood was collected 30 min later by decapitation. At 15 days of age, NAL induced LH release in intact and ovariectomized (OVX) female rats, and in male rats castrated (CAST) on the 1st day of life (neonate CAST males). Injection of 10 micrograms estradiol benzoate (EB) 24 h prior to NAL administration blocked NAL-induced LH release in these rats. NAL had no effect on LH release in 15- or 25-day-old intact and CAST male rats or in female rats given 2 mg testosterone propionate at 3 days of age (androgenized female rats). At 35 days of age, NAL induced LH release in intact, OVX, and OVX-EB treated female rats, and in neonate CAST and neonate CAST-EB treated male rats. NAL had no effect on serum LH levels in androgenized female rats. NAL induced LH release in intact and CAST 35-day-old male rats, but pretreatment with estrogen prevented NAL from eliciting LH release. These results indicate that neonatal exposure to androgen is responsible for the sex difference in the LH response to NAL observed in prepubertal male and female rats before 30 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

The actions of prostaglandin E2, naloxone and testosterone on starvation-induced suppression of luteinizing hormone-releasing hormone and luteinizing-hormone secretion. In vitro and in vivo studies

In man and other mammals, starvation is accompanied by a severe suppression of luteinizing hormone-releasing hormone (LHRH) and luteinizing-hormone (LH) secretion, which is caused by unknown alterations in hypothalamic functions. Prostaglandin E2 (PGE2), endorphins and testosterone (T) are know to be strongly involved in the regulation of LHRH release. The present study examined whether the influence of these substances on LHRH and LH secretion was affected by starvation. In vitro experiments checked the release of PGE2 and LHRH from median eminences (ME) of fed male rats and ones starved for 5 days. Stimulation with potassium (80 mM) induced an equally strong release of PGE2 and LHRH from the MEs of both fed and starved rats. When PGE2 (10(4) M) was added to the superfusion medium, the potassium-stimulated release of LHRH was significantly enhanced in both groups of animals. The results clearly showed that in the terminal region of the hypothalamic LHRH system the release of this hormone and the action of PGE2 were not altered by starvation. In vivo experiments tested whether the effects of LHRH, PGE2, naloxone (NAL), or T on LH secretion were different in intact or castrated male rats fed or starved for 3 and 5 days. LHRH (250 ng/kg) stimulated the same amount of LH secretion in fed and starved rats. The starvation-induced LH suppression was not due to a dysfunction at the pituitary level. The stimulatory action of PGE2 (1 mg/kg) on LH was gradually reduced throughout the starvation period. NAL (5 mg/kg) had little, respectively, no effect on LH release on the 3rd or 5th day of starvation.(ABSTRACT TRUNCATED AT 250 WORDS)