Pituitary follistatin regulates activin-mediated production of follicle-stimulating hormone during the rat estrous cycle.

Follistatin, an activin-binding protein, plays a key role in the modulation of activin-dependent functions. In the anterior pituitary, activin stimulates the synthesis and secretion of FSH. In the current study, we assessed the roles of locally produced activin and follistatin in the control of FSH gene expression and secretion. The anterior pituitary gland follistatin content was measured at frequent intervals during the rat estrous cycle. Follistatin protein levels were high before the primary gonadotropin surges, decreased by 50% on proestrous evening, and rebounded to a peak at midnight on proestrus before returning to presurge levels on estrus morning. Changes in pituitary follistatin protein content were preceded by parallel changes in pituitary follistatin messenger RNA (mRNA). The trough in follistatin protein content on proestrus coincided with a peak in circulating levels of free activin A (not bound to follistatin) and a sharp rise in FSHbeta mRNA levels, suggesting that decreased pituitary follistatin leads to increased available activin. To quantitate the contribution of pituitary free activin to pituitary expression of FSHbeta mRNA and the primary and secondary serum FSH surges, rats were infused through carotid catheters with saline or recombinant human follistatin (288-amino acid isoform; rhFS-288) at different times during the estrous cycle. Infusion of rhFS-288 on diestrus did not affect FSH production. In contrast, infusion of rhFS-288 during the secondary FSH surge decreased the peaks in FSHbeta mRNA and serum FSH by 63% and 47%, respectively, relative to those in saline-infused control animals. Infusion of rhFS-288 during the primary FSH surge decreased serum FSH to a lesser degree (24%). These data indicate a physiological role for pituitary follistatin in the control of activin-mediated FSH synthesis and secretion during the rat estrous cycle.

Expression of ryanodine receptors in the pituitary gland: evidence for a role in gonadotropin-releasing hormone signaling.

GnRH elicits secretion of LH and FSH from gonadotropes by activating an array of intracellular signals including the generation of inositol triphosphate and the release of intracellular calcium. Given the important role of calcium in the secretory responses to GnRH, we examined the expression and function of the ryanodine receptors, which are known to modulate calcium release from intracellular stores. Using RT-PCR analysis, we found that ryanodine receptor (RyR) types 2 and 3, but not type 1, are expressed in rat pituitaries. Pulses of GnRH were administered to perifused primary rat pituitary cells in the presence or absence of a ryanodine receptor antagonist, ruthenium red, to assess effects on GnRH-mediated LH secretion. Treatment with ruthenium red resulted in a 40% decrease in the spike phase of GnRH-induced LH release and a 35% reduction in the plateau phase. Ruthenium red also inhibited GnRH-mediated transcription of a transfected alpha-LUC reporter plasmid. RyR messenger RNA (mRNA) expression varied during the rat estrous cycle with maximal levels following increases of progesterone. The effects of gonadal steroids on pituitary RyR mRNA levels were examined directly in ovariectomized rats that were treated with estrogen (E), or estrogen and progesterone (P). In this paradigm, E decreased, whereas E + P increased RyR3 mRNA levels. These results indicate that RyR is expressed and hormonally regulated in the rat pituitary and suggest that it might play a role in mediating GnRH-induced gonadotropin synthesis and secretion.

Gonadotropin-releasing hormone regulates follicle-stimulating hormone-beta gene expression through an activin/follistatin autocrine or paracrine loop.

The FSH beta gene is stimulated by low frequency pulses of GnRH, but is unaffected or suppressed when GnRH is applied at higher frequencies or continuously. The current studies explored the hypothesis that GnRH frequency-dependent regulation of FSH beta may be mediated by pituitary expression of activin, which stimulates FSH beta messenger RNA (mRNA), and follistatin, which blocks activin. Using a system of perifused male rat pituitary cells, a reciprocal relationship was observed between FSH beta and follistatin mRNAs in response to different patterns of GnRH treatment. Pulses of GnRH (5 min; 10 nM) applied every 60 min stimulated FSH beta mRNA 14.0-fold with no change in follistatin mRNA. Pulses of GnRH applied every 30 and 15 min elicited stepwise increases in follistatin mRNA and decreases in FSH beta mRNA, and continuous GnRH stimulated follistatin mRNA 4.1-fold, with no significant increase in FSH beta mRNA. Stimulation of FSH beta mRNA by hourly GnRH pulses (3.7-fold) was blocked in the presence of 30 ng/ml recombinant follistatin (0.8-fold), suggesting that GnRH stimulation of FSH beta mRNA requires endogenous activin. Treatment of plated pituitary cells with continuous GnRH for 24 h confirmed that secretion of follistatin protein rises (1.5-fold) coincident with follistatin mRNA (1.7-fold) under conditions that suppress FSH beta mRNA (9% of the control value). When male rats were infused through arterial cannulas for 6 h with continuous GnRH (100 nM) or recombinant follistatin (5 micrograms/h), continuous GnRH suppressed FSH beta mRNA levels to 50% of the control value, and follistatin decreased expression to 61% of the control value. We conclude that GnRH stimulation of FSH beta mRNA is activin dependent, and pituitary follistatin production is a major pathway by which higher GnRH pulse frequencies suppress FSH beta mRNA. Changes in activin or follistatin tone, therefore, provide a mechanism by which LH and FSH can be differentially regulated by GnRH in a variety of physiological and pathophysiological conditions.

Sexually dimorphic transcriptional responses to gonadotropin-releasing hormone require chronic in vivo exposure to estradiol.

GnRH regulates secretion of the gonadotropins, LH and FSH, in a sexually dimorphic manner. In the present study, we examined GnRH regulation of the gonadotropin alpha-subunit promoter to assess whether sex-dependent hormonal effects are manifest at the transcriptional level. Primary cultures of male or female rat pituitary cells were transfected with a reporter gene containing the alpha-promoter linked to luciferase (-420 alpha-LUC) and then subjected to treatment with GnRH for 24 h. Basal alpha-LUC expression was 4.2-fold greater in pituitary cells from males than in those from females. alpha-LUC activity was stimulated 5.3-fold by GnRH in males, whereas GnRH induced a 148-fold increase in alpha-promoter activity in females. The GnRH responsiveness of the transfected alpha-promoter did not vary in pituitary cells isolated at different stages of the female reproductive cycle, suggesting that acute changes in the hormonal milieu are not sufficient to alter transcriptional responses to GnRH. In males, orchidectomy minimally influenced alpha-LUC activity, indicating that testosterone does not exert a suppressive effect on GnRH responsiveness. In ovariectomized females, basal expression of alpha-LUC increased 3.7-fold, and GnRH stimulation was reduced from 165- to 11-fold, suggesting that an ovarian factor suppresses basal activity and enhances GnRH stimulation. Treatment of ovariectomized females with estrogen suppressed basal activity and restored GnRH stimulation of alpha-LUC, but the estrogen effects required long term treatment (10 days). Addition of progesterone to estrogen or treatment with the progesterone antagonist, RU486, had little effect on GnRH responsiveness. We conclude that estrogen exerts dual effects to suppress basal expression and to dramatically enhance GnRH responsiveness of the alpha-promoter. This model reveals potent actions of estrogen at the level of transcription and should provide new insights into the mechanisms that control estrogen priming of gonadotrope cells.

Gonadotropin-releasing hormone regulation of pituitary follistatin gene expression during the primary follicle-stimulating hormone surge.

Follistatin is produced in the gonadotrope and folliculostellate cells of the pituitary gland and is thought to indirectly regulate FSH biosynthesis and secretion through its ability to bind activin. Recent measurements of follistatin gene expression during the rat estrous cycle revealed a marked increase in pituitary follistatin messenger RNA (mRNA) levels at the time of the preovulatory FSH surge. This finding suggests a role for follistatin in the regulation of FSH at this dynamic time of the cycle. The aim of the present study was to identify the hormonal control mechanisms responsible for stimulating follistatin gene expression on proestrus. In particular, the roles of estrogen (E) and GnRH were assessed using an in vivo ovariectomized (OVX) animal model in which steroid priming results in daily gonadotropin surges. Follistatin mRNA and serum FSH levels were unchanged throughout the day in untreated OVX rats. E priming of OVX rats elicited a 2-fold elevation in follistatin mRNA levels between 1600-2000 h coincident with the peak of the E-induced FSH surge. To determine whether this effect of E on follistatin mRNA levels was the result of the direct or indirect effects of E on the pituitary, follistatin mRNA levels were examined in E-primed OVX rats that had been treated with pentobarbital at 1430 h (to block hypothalamic neurosecretion). Pentobarbital treatment prevented the E-induced increase in follistatin mRNA levels, suggesting that the effects of E are mediated via GnRH or other hypothalamic factors. The effects of GnRH on follistatin gene expression were examined further using an in vitro perifusion model. Proestrous or metestrous pituitaries were perifused for 8 h with pulsatile GnRH (one pulse per h), continuous GnRH, or medium only. Continuous GnRH treatment resulted in a significant elevation in follistatin mRNA levels in both proestrous and metestrous pituitaries, whereas pulsatile GnRH had no effect at either cycle stage. These results suggest that the proestrous GnRH surge is responsible at least in part for the elevation in pituitary follistatin mRNA levels that is associated with the primary FSH surge. GnRH-induced follistatin production on proestrus probably plays a role in the dynamic regulation of FSH at this time of the ovulatory cycle.

Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle.

Recent evidence indicates that the GnRH receptor (GnRH-R) gene is expressed in a number of tissues besides the anterior pituitary gland, suggesting that GnRH may serve other functions in addition to its role as a hypothalamic releasing factor. In particular, high levels of GnRH-R transcripts have been detected in rat and human ovarian granulosa cells. To better understand the role of the GnRH-R in the ovary under physiological conditions and to determine which follicles are potentially responsive to the actions of GnRH, we used in situ hybridization histochemistry and quantitative reverse transcriptase-polymerase chain reaction for measurement of ovarian GnRH-R messenger RNA (mRNA) expression during the rat ovulatory cycle. Reverse transcriptase-polymerase chain reaction analyses revealed that total ovarian GnRH-R mRNA levels were elevated significantly at 1800 h on proestrus and again at 0900 and 1800 h on estrus compared to metestrus 0900 h levels. In situ hybridization analysis of GnRH-R gene expression at different stages of follicular maturation revealed significant variation in GnRH-R mRNA levels with respect to the degree of follicular development as well as the estrous cycle stage. GnRH-R gene expression was greatest in the granulosa cells of Graafian and atretic follicles, with lower levels of expression present in preantral and small antral follicles and corpora lutea. GnRH-R mRNA levels in atretic follicles showed substantial variation across the 4-day rat estrous cycle, with mRNA levels increasing 3-fold on the day of proestrus coincident with the preovulatory gonadotropin surges. A second peak of expression in atretic follicles was observed on the morning of estrus. Levels of GnRH-R gene expression in corpora lutea also varied significantly during the estrous cycle, with gene expression increasing 3-fold between the morning of metestrus and the afternoon of proestrus. These results demonstrate that the level and localization of ovarian GnRH-R mRNAs change significantly during the rat ovulatory cycle. The finding that atretic follicles exhibit the greatest degree of GnRH-R gene expression is consistent with a role for GnRH in the induction of follicular atresia.

Roles of estrogen, progesterone, and gonadotropin-releasing hormone (GnRH) in the control of pituitary GnRH receptor gene expression at the time of the preovulatory gonadotropin surges.

Pituitary GnRH receptor (GnRH-R) messenger RNA (mRNA) levels are regulated dynamically during the rat estrous cycle. GnRH-R mRNA levels increase 3-fold on the morning of proestrus and remain elevated throughout the gonadotropin surges, after which they decline rapidly. Because the day of proestrus is characterized by complex changes in the steroidal milieu and increased release of hypothalamic peptides such as GnRH, a series of in vivo steroid replacement and in vitro perifusion studies was used to assess the relative contributions of estrogen (E), progesterone (P), and GnRH to the induction and decline of GnRH-R gene expression during the gonadotropin surges. Steroid replacement studies in ovariectomized (OVX) E-primed rats demonstrated that GnRH-R mRNA levels were elevated before and during the E-induced LH surge (1000-1800 h). Receptor mRNA levels declined after the peak of the LH surge and were significantly lower by 2000 h. Pentobarbital treatment, which inhibits hypothalamic input and the LH surge, prevented the gonadotropin surge-associated increase in GnRH-R mRNA levels in E-primed OVX rats. Although GnRH-R mRNA levels did not change throughout the day of experiments in OVX unprimed rats, treatment with pentobarbital significantly reduced GnRH-R mRNA expression in these animals. P treatment of E-primed OVX rats had no significant effect on GnRH-R mRNA expression during the LH surge, but significantly reduced mRNA levels immediately after the LH surge (2000 h). Data from in vitro perifusion experiments using either metestrous or proestrous pituitary glands demonstrated that pulsatile GnRH up-regulates the expression of its own receptor mRNA at both estrous cycle stages. Based on these results, we conclude that enhanced GnRH-R mRNA expression observed on the day of proestrus is largely due to the actions of E, exerted indirectly via hypothalamic routes (presumably through enhanced GnRH secretion). Furthermore, preovulatory P secretion may account for the rapid decline in GnRH-R mRNA levels observed on the evening of proestrus.

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release.

1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recent in vivo microdialysis, in vitro superfusion, and in situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways. 2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms. 3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results of in vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed. 4. Future directions for study of the LHRH pulse generator are suggested.

Unmasking of neuropeptide-Y inhibitory effects on in vitro gonadotropin secretion from pituitaries of metestrous, but not proestrous, rats.

We have recently demonstrated that GnRH-stimulated gonadotropin secretion is augmented by coadministration with neuropeptide-Y (NPY) in anterior pituitaries removed in the afternoon from proestrous, but not metestrous, rats. To test the hypothesis that these effects of NPY are due to an interaction with progesterone (P4), we conducted another cycle stage experiment using NPY, adding an in vitro treatment with P4. Pituitaries were taken from rats at 0900 h (before the rise of P4 on proestrus) on proestrus or metestrus and were perifused for 8 h, with and without GnRH pulses. P4 was given continuously in half of each of the basal or GnRH-stimulated perifusions. Pulsatile NPY was administered in half of the basal or GnRH-stimulated runs in the presence or absence of P4. P4 alone had a stimulatory effect on GnRH-induced LH secretion only on the day of metestrus. P4 did not confer responsiveness to NPY stimulation on either day. Unexpectedly, NPY reversed the P4 augmentation of GnRH-stimulated LH release on metestrus. With respect to FSH secretion, NPY or P4 alone had a striking suppressive effect on GnRH-stimulated FSH secretion rates from pituitaries of metestrous, but not proestrous, donors. These data suggest that the previous endogenous endocrine environment may be crucial in determining the divergent and interrelated effects of P4 and NPY on gonadotropin secretion. In particular, the metestrous environment appears to promote a suppressive role for NPY.

Dynamic regulation of pituitary follistatin messenger ribonucleic acids during the rat estrous cycle.

FSH synthesis and secretion are regulated by a complex interplay of hypothalamic, gonadal, and pituitary factors. Recent evidence suggests that inhibin, activin, and follistatin, although originally identified as gonadal peptides, are also expressed in the pituitary, where they may be secreted and play an autocrine/paracrine role in the control of FSH beta gene expression. Attempts to study pituitary regulation of the genes encoding these proteins have been hampered by low levels of mRNA expression. Consequently, we developed quantitative reverse transcription-polymerase chain reaction assays for follistatin (FS) and the three subunits (alpha, beta A, and beta B) that comprise activin and inhibin. Expression of activin type II receptor (actRII) mRNA was also analyzed. Reasoning that mRNA levels of these FSH-regulating pituitary peptides might be modulated at times of increased FSH gene expression, two in vivo models were chosen for further investigation. 1) Two weeks after ovariectomy, rat pituitary FS mRNA levels increased substantially (4.09-fold vs. intact) with a modest increase in beta B-inhibin mRNA levels (1.88-fold vs. intact). No changes in alpha-inhibin, beta A-inhibin, or actRII mRNA levels were observed. 2) During the estrous cycle, pituitary FS gene expression varied strikingly, with a peak at 1800 h on proestrus (13.69-fold vs. 0900 h on proestrus), followed by a rapid decline at 2400 h on proestrus (2.10-fold vs. 0900 h on proestrus). A more detailed analysis of expression during proestrus revealed that peak FS mRNA levels preceded peak FSH beta gene expression by 6 h. Levels of the inhibin subunit and actRII transcripts varied minimally across the estrous cycle. We conclude that during the estrous cycle in rats, pituitary FS mRNA levels are regulated dynamically, whereas levels of inhibin/activin subunits and the activin receptor, actRII, very minimally. The observation that FS mRNA levels peak before maximal expression of FSH beta mRNA raises the possibility that FS facilitates, rather than inhibits, FSH biosynthesis in vivo.

Estrous cycle stage-dependent effects of neuropeptide-Y on luteinizing hormone (LH)-releasing hormone-stimulated LH and follicle-stimulating hormone secretion from anterior pituitary fragments in vitro.

We recently demonstrated that neuropeptide Y (NPY) potentiates LH-releasing hormone (LHRH)-stimulated LH secretion in vivo, and that these actions of NPY are exerted only under the endocrine conditions leading to preovulatory LH surges, viz. the proestrous or estrogen- and progesterone-primed ovariectomized animal. The present experiments tested the hypothesis that NPY's facilitatory actions are exerted directly at the level of the anterior pituitary gland and depend on in vivo exposure of gonadotropes to the preovulatory endocrine milieu. Animals were killed at 1600 h on either metestrus or proestrus. Anterior pituitary glands (APs) were rapidly removed, cut into eighths, and placed into perifusion chambers. APs were perifused with medium 199 for a total of 8 h, and perfusate samples were collected every 5 min. After 30 min of equilibration, APs received hourly pulses of LHRH alone (10(-8) M), NPY alone (10(-6) M), or LHRH plus NPY. Basal runs consisted of perifusion with medium 199 for 8 h with no peptide treatments. Calculations of total hourly LH and FSH responses revealed that whereas LHRH significantly stimulated gonadotropin secretion from both metestrous and proestrous pituitaries, NPY significantly enhanced LHRH-stimulated LH and FSH secretion only from proestrous pituitaries, i.e. from tissue exposed to the endogenous endocrine milieu under which preovulatory gonadotropin surges are generated. NPY had no facilitatory effect on LHRH-induced gonadotropin secretion from metestrous APs. These results are consistent with our previous in vivo findings and demonstrate that the facilitatory actions of NPY on LHRH-stimulated gonadotropin secretion in vitro are limited to the endocrine conditions under which preovulatory gonadotropin surges are generated.

RU486 administration blocks neuropeptide Y potentiation of luteinizing hormone (LH)-releasing hormone-induced LH surges in proestrous rats.

We previously demonstrated that NPY potentiates LHRH-induced LH secretion specifically under endocrine conditions in which preovulatory LH surges are generated. The present study was designed to test the hypothesis that NPY's facilitatory actions are dependent upon preovulatory progesterone secretion. In Exp 1, female rats were fitted with atrial catheters on diestrus. On proestrus, hourly blood samples were collected from 1100-2100 h. At 1230 h, rats received a sc injection of the progesterone receptor antagonist RU486 (6 mg/kg BW) or oil. At 1330 h, rats received pentobarbital (40 mg/kg BW), to block hypothalamic LHRH release, or saline. Every 30 min from 1400-1800 h, pentobarbital-treated rats received iv pulses of LHRH (15 ng/pulse) or saline along with concurrent pulses of NPY (5 micrograms/pulse), or saline. In Exp 2, rats received jugular catheters on diestrus, but were sampled every hour throughout the morning (0700-1600 h), rather than the afternoon, of proestrus. In these morning groups, pentobarbital was injected at 0830 h, and peptides (LHRH or combined LHRH and NPY solutions) were administered as pulses at 30-min intervals between 0900-1300 h. Results from Exp 1 were as follows: administration of RU486 to rats given an ip injection of vehicle at 1330 h and pulses of saline from 1400-1800 h completely blocked the endogenous LH surge. In oil-treated pentobarbital-blocked rats, concurrent administration of NPY with LHRH significantly (P < 0.01) potentiated the ability of LHRH to restore LH surges. However, NPY was without any potentiating effects in animals pretreated with RU486 at 1230 h. RU486 also attenuated the ability of LHRH alone to restore LH surges in pentobarbital-blocked rats. In Exp 2, NPY was without effect on LHRH-induced LH secretion during the morning hours of proestrus. Our results demonstrate that 1) NPY facilitates LHRH-induced LH surges on the afternoon of proestrus; 2) presumptive progesterone receptor blockade by RU486 completely prevents NPY's potentiating effects; and 3) NPY is without effect on the morning of proestrus, before the afternoon surge of progesterone. These findings are entirely consistent with the idea that one function of preovulatory progesterone secretion is to up-regulate pituitary sensitivity to the facilitatory actions of NPY. It is hypothesized that these actions of progesterone together with an increase in NPY neurosecretion mediate the acute increase in pituitary sensitivity to LHRH that occurs just before the LH surge.

Dynamic regulation of gonadotropin-releasing hormone receptor mRNA levels in the anterior pituitary gland during the rat estrous cycle.

The number of gonadotropin-releasing hormone (GnRH) receptors on pituitary gonadotropes varies substantially during the rat estrous cycle and may modulate pituitary responsiveness to GnRH. The present studies were undertaken to determine to what extent these changes in GnRH receptor number reflect a change in GnRH receptor mRNA expression in the anterior pituitary gland. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), pituitary GnRH receptor mRNA levels were measured at various timepoints throughout the rat estrous cycle. There was a three-fold increase in GnRH receptor mRNA levels on the afternoon of proestrus (PRO) when compared to levels observed on the morning of metestrus (MET). This rise preceded the onset of the LH surge by 6h (1200h). GnRH receptor mRNA levels remained elevated through 2100h PRO, after which they dropped dramatically, and by 2400h PRO were not significantly different from levels observed at 0900h MET. A two-fold increase in GnRH receptor mRNA expression was also observed during the early stages of the estrous cycle (0900h to 1800h MET), and this increase was sustained until 1800h on diestrus, at which time mRNA levels decreased to levels observed at 0900h MET. These results demonstrate that pituitary GnRH receptor mRNAs are dynamically regulated during the rat estrous cycle, with receptor mRNA expression being greatest on the afternoon of PRO, the time of the estrous cycle at which gonadotropes are most sensitive to GnRH stimulation.

Neuropeptide Y gene expression in the arcuate nucleus: sexual dimorphism and modulation by testosterone.

Neuropeptide Y (NPY) peptide concentrations in the arcuate nucleus have recently been shown to be modulated by gonadal steroids in the male rat. The present study was designed to determine whether NPY messenger RNA (mRNA)-synthesizing cells in the arcuate nucleus (Arc) of the male rat are regulated by testosterone (T) and whether there is a sexual dimorphism in the expression of the NPY gene in this region. In situ hybridization and quantitative autoradiography were used to assess the level of NPY gene expression in the Arc. In the first experiment, NPY mRNA levels were measured in the Arc of intact, castrated, and castrated male rats treated with T to maintain physiological (1.3 +/- 0.1 ng/ml) and supraphysiological (5.3 +/- 0.4 ng/ml) plasma levels of T. A 2-week castration produced a modest but significant decrease in NPY mRNA levels in the Arc (P < 0.05). Replacement with either physiological or supraphysiological levels of T prevented the effect of castration on NPY gene expression, and there was no further potentiation of NPY gene expression in those animals that received high levels of T. In the second experiment, NPY gene expression was compared throughout the Arc between intact male and female rats at 1800 h on the afternoon of proestrus. Comparison of NPY gene expression throughout the rostro-caudal extent of the Arc showed that male rats had significantly more NPY mRNA-containing cells than female rats (P < 0.01). This difference was most strikingly observed in the caudal portions of the nucleus (3.80 mm caudal to bregma). No difference was detected in the mean levels of NPY gene expression in the Arc between male and female rats. These data demonstrate that 1) NPY gene expression throughout the arcuate nucleus is modulated by T in male rats, and 2) a marked regional sex difference exists in the distribution of NPY mRNA-containing cells in the caudal extremity of the Arc. It is hypothesized that gonadal hormones may exert both organizational and activational effects upon NPY neurons in the Arc.

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.

Neuropeptide Y gene expression in the arcuate nucleus is increased during preovulatory luteinizing hormone surges.

Recent studies have suggested that neuropeptide Y (NPY) plays an important role in the induction of the preovulatory LH surge. The present study was performed in order to determine if a change in NPY gene expression within arcuate nucleus NPY neurons is associated with the generation of the preovulatory LH surge. In Exp 1, in situ hybridization was used to measure NPY messenger RNA (mRNA) levels in the arcuate nucleus of female rats at 0900 h and every 2 h from 1400-2200 h on the day of proestrus (PRO). Comparisons between groups showed a clear, stepwise increase in NPY gene expression throughout the day of PRO. At 1600 h, when LH values were significantly greater than 0900 h values, NPY mRNA labeling intensities in the arcuate nucleus were significantly greater than 0900 h levels (P < 0.01). By 1800 h, the time at which the LH surge peaked, NPY mRNA levels also peaked and were nearly 3-fold greater than levels observed at 0900 h (P < 0.01). NPY mRNA levels at 2000 h and 2200 h remained elevated above 0900 h levels (P < 0.01) but by 2000 h had decreased significantly from 1800 h levels (P < 0.05). In Exp 2, NPY mRNA levels were measured once again at 0900 h and 1800 h on PRO, and then at 0900 h and 1800 h on metestrus (MET), in order to determine if the change in gene expression seen in Exp 1 was unique to the day of PRO, or if it simply reflected a daily rhythm of gene expression in the nucleus. Analysis of mRNA levels showed no difference in NPY mRNA levels between 0900-1800 h on MET. Also, NPY mRNA levels at 0900 h and 1800 h on MET were significantly less than levels at 1800 h on PRO (P < 0.01). These results are consistent with the hypothesis that NPY neurons participate in the generation of LH surges through increased production of NPY and subsequent potentiation of the release and/or actions of LHRH.

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-stimulated LH surges in pentobarbital-blocked proestrous rats.

Recent evidence suggests that hypothalamic neurosecretion of neuropeptide Y (NPY) may be required for the preovulatory LH surge in female rats. Results of immunoneutralization and portal blood collection studies have suggested that NPY may serve to enhance the response of gonadotropes to the stimulatory action of LHRH. To directly test this hypothesis, the effects of NPY on LHRH-stimulated LH secretion were assessed in proestrous rats that were anesthetized with pentobarbital (PB) to block endogenous LHRH neurosecretion. Female rats were fitted with atrial catheters on diestrus. On proestrus, hourly blood samples were collected from 0900-2100 h. At 1330 h, rats received PB (40 mg/kg BW) or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15, 150, or 1500 ng/pulse) or saline along with concurrent pulses of NPY (1 or 10 micrograms/pulse). Plasma samples were analyzed by LH RIA. In PB-treated rats receiving vehicle pulses only, LH surges were completely blocked. Pulsatile LHRH treatments at 15, 150, and 1500 ng/pulse produced subphysiological, physiological, and supraphysiological LH surges, respectively. Simultaneous administration of NPY pulses with 15 ng/pulse LHRH produced significant dose-related potentiations of LHRH-stimulated LH surges (P less than 0.0001). Administration of NPY pulses with 150 ng LHRH/pulse also significantly enhanced LHRH-induced LH surges (P less than 0.05). NPY RIA of plasma confirmed NPY increments after treatments. These results demonstrate that NPY administration can potentiate pituitary responsiveness to LHRH stimulation, and are consistent with the hypothesis that one function of NPY is to operate as a neurohormonal modulator at the level of the gonadotrope during generation of the preovulatory LH surge.

Neuroendocrine regulation of the luteinizing hormone-releasing hormone pulse generator in the rat.

We have analyzed the mechanisms by which several known regulators of the LHRH release process may exert their effects. For each, we have attempted to determine how and where the regulatory input is manifest and, according to our working premise, we have attempted to identify factors which specifically regulate the LHRH pulse generator. Of the five regulatory factors examined, we have identified two inputs whose primary locus of action is on the pulse-generating mechanism--one endocrine (gonadal negative feedback), and one synaptic (alpha 1-adrenergic inputs) (see Fig. 29). Other factors which regulate LHRH and LH release appear to do so in different ways. The endogenous opioid peptides, for example, primarily regulate LHRH pulse amplitude (Karahalios and Levine, 1988), a finding that is consistent with the idea that these peptides exert direct postsynaptic or presynaptic inhibition (Drouva et al., 1981). Gonadal steroids exert positive feedback actions which also result in an increase in the amplitude of LHRH release, and this action may be exerted through a combination of cellular mechanisms which culminate in the production of a unique, punctuated set of synaptic signals. Gonadal hormones and neurohormones such as NPY also exert complementary actions at the level of the pituitary gland, by modifying the responsiveness of the pituitary to the stimulatory actions of LHRH. The LHRH neurosecretory system thus appears to be regulated at many levels, and by a variety of neural and endocrine factors. We have found examples of (1) neural regulation of the pulse generator, (2) hormonal regulation of the pulse generator, (3) hormonal regulation of a neural circuit which produces a unique, punctuated synaptic signal, (4) hormonal regulation of pituitary responsiveness to LHRH, and (5) neuropeptidergic regulation of pituitary responsiveness to LHRH. While an attempt has been made to place some of these regulatory inputs into a physiological context, it is certainly recognized that the physiological significance of these mechanisms remains to be clarified. We also stress that these represent only a small subset of the neural and endocrine factors which regulate the secretion or actions of LHRH. A more comprehensive list would also include CRF, GABA, serotonin, and a variety of other important regulators. Through a combination of design and chance, however, we have been able to identify at least one major example of each type of regulatory mechanism.