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Endocrinology, doi:10.1210/en.2004-0858
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Endocrinology Vol. 146, No. 3 1403-1410
Copyright © 2005 by The Endocrine Society

Effects of Polypeptide YY3–36 upon Luteinizing Hormone-Releasing Hormone and Gonadotropin Secretion in Prepubertal Rats: In Vivo and in Vitro Studies

R. Fernandez-Fernandez, E. Aguilar, M. Tena-Sempere and L. Pinilla

Department of Cell Biology, Physiology, and Immunology, University of Córdoba, 14004 Córdoba, Spain

Address all correspondence and requests for reprints to: Leonor Pinilla, Physiology Section, Department of Cell Biology, Physiology, and Immunology, Faculty of Medicine, University of Córdoba, Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain. E-mail: fi1agbee{at}uco.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polypeptide YY3–36 (PYY3–36) is a gastrointestinal secreted molecule, agonist of neuropeptide Y (NPY) receptor subtypes Y2 and Y5, recently involved in the control of food intake. Notably, several factors with key roles in energy homeostasis conduct pleiotropic effects upon the reproductive axis. However, whether PYY3–36 is provided with similar biological actions remains so far largely unexplored. To address this issue, expression analyses of neuropeptide Y receptor Y2 and Y5 genes were conducted at the pituitary and the hypothalamus, and functional studies testing the effects of PYY3–36 in vivo and in vitro were implemented, using the prepubertal rat as a model. Expression of the genes encoding Y2 and Y5 receptors was demonstrated, albeit at low levels, in whole hypothalamic and pituitary samples, and challenge of pituitary tissue with increasing doses of PYY3–36 elicited LH and FSH secretion in male and female rats, a response that was persistently observed in the absence of extracellular calcium. Moreover, 10–6 M PYY3–36 enhanced LH and FSH responsiveness to LHRH in vitro. In contrast, systemic ip administration of PYY3–36 over a range of doses (3, 10, and 30 µg/kg) failed to significantly modify serum LH levels in males and females, whereas central (intracerebroventricular) injection of 3 nmol PYY3–36 inhibited LH secretion in vivo, and 10–6 M PYY3–36 decreased LHRH release by hypothalamic fragments in vitro in male but not in female rats. Overall, our data document the complex mode of action of the gut-derived anorexigenic signal PYY3–36 at the hypothalamic-pituitary unit in the control of gonadotropin secretion and evidence that, as is the case for other peripheral factors with key roles in energy balance (as leptin and ghrelin), PYY3–36 might play a role in the neuroendocrine modulation of the reproductive axis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SEQUENCE OF NEUROPEPTIDE Y (NPY), a member of pancreatic polypeptide family, was originally identified by Tatemoto in 1982 (1). Since then, a great body of evidence has demonstrated the pivotal role of NPY in several neuroendocrine functions. In this sense, NPY-producing neurons are located in brain stem and hypothalamic areas (2), and rapid time-dependent changes in NPY levels take place in hypothalamic sites in response to fasting and refeeding (3). Indeed, many experimental approaches have proven the role of NPY, as an orexigenic factor, in the control of food intake (4).

NPY is also involved in the control of pituitary secretion. Intracerebroventricular administration of NPY stimulated LH release in ovariectomized rats primed with ovarian steroids (5). These excitatory effects derive from LHRH stimulation by NPY (6, 7, 8). In addition, NPY facilitates the LHRH-induced LH secretion in intact females at proestrus as well as in ovariectomized estrogen- and progesterone-primed animals (9, 10, 11). The effect of NPY at the pituitary level seems to be mediated by an increase in the effectiveness of LHRH (7, 12).

In addition, NPY has been implicated in the control of physiological reproductive processes such as puberty and ovulation (13, 14). Thus, central administration of NPY advances puberty development (15), whereas immunoneutralization of NPY reduces the magnitude of the LH surge during the afternoon of first proestrus (16). A facilitatory role of NPY on the onset of puberty has been also reported in the female rhesus monkey (17) and chicken (18). Secretion of NPY to the portal vasculature is increased on the afternoon of proestrous and serves to amplify the actions of LHRH in initiating the preovulatory surges of LH and probably FSH (19).

NPY conducts its biological effects through interaction with at least five receptor subtypes (20). The presence of mRNA encoding the different NPY receptors has been analyzed in hypothalamus but not in pituitary (21). Development of selective agonists/antagonists for the different receptors and the use of knockout animal models have improved our knowledge of the role of different NPY receptor subtypes in the control of the reproductive axis. However, characterization of the relative contribution of each receptor subtype to the plethora of NPY actions is incomplete. Nevertheless, recent experiments described that the NPY-Y1 receptor exerts an inhibitory action upon the gonadotrope axis (22), and its blockade accelerates the onset of puberty (23). In addition, NPY-Y4 receptors have also been involved in mediating NPY effects on LH release (24).

Proper development and function of the reproductive axis requires a certain (threshold) degree of somatic growth and energy stores, and different gonadal pathologies are associated with alterations in body weight. In the last decade, identification of different molecules, such as leptin, ghrelin, and orexins, with actions on food intake, body weight, and the hypothalamic-pituitary-gonadal axis has helped to establish the hormonal basis for the interaction between energy balance and reproductive function. In this context, the polypeptide YY3–36 (PYY3–36), a hormone that is structurally related to NPY and is an agonist of receptor subtypes Y2 and Y5 (25), has been recently proposed as a putative anorexigenic signal, from gastrointestinal origin, involved in the control of food intake (26). Yet, after initial reporting, conflicting results on the repeatability of the effects of PYY3–36 in terms of body weight control have been very recently published (27, 28, 29). Nonetheless, whether PYY3–36 has additional regulatory effects upon other neuroendocrine functions, including reproduction, remains largely unknown. Because NPY-Y5 receptors have been only pharmacologically identified in pituitary (30), present experiments were undertaken to analyze the pituitary and hypothalamic expression of the genes encoding NPY receptor subtypes involved in PYY3–36 actions and to evaluate the effects of PYY3–36 on LH and FSH secretion using in vitro and in vivo models. Because the effects of NPY on gonadotropin secretion have been proven dependent on the steroid milieu and phase of the ovarian cycle (31), the present experiments were carried out in prepubertal animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and drugs
Wistar rats born in our laboratory were kept under controlled conditions of light (12 h light/12 h darkness, lights on at 0700 h) and temperature (22 C) with free access to pelleted food (Pacsa Sanders, Seville, Spain) and tap water. On d 1 of life, each dam was left with eight pups. Experiments were carried out in 23- to 25-d-old animals. PYY3–36 and LHRH were purchased from Bachem (Barcelona, Spain).

Experimental designs
Experimental procedures were approved by the Córdoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. Experiments were carried out between 1000 and 1200 h. Special caution was taken to avoid any stressing influences upon the experimental animals (all the animals were handled daily for a week before the experiment and killed by the same person, and the different drugs were injected at random). The following experiments were conducted.

Experiment 1.
To identify pituitary and hypothalamic expression of the genes encoding NPY-Y2 and -Y5 receptors, 23-d-old male and female rats (n = 8–10 per group) were killed by decapitation, and the pituitary and whole hypothalamus (excised by a horizontal cut of ~2 mm depth with the following limits: 1 mm anteriorly from the optic chiasm, the posterior border of the mamillary bodies, and the hypothalamic fissures) were immediately dissected out from each animal, snap frozen in liquid nitrogen, and stored at –80 C until use for RNA isolation and analysis.

Experiment 2.
To analyze whether PYY3–36 regulates in vitro LH and FSH secretion under basal and LHRH-stimulated conditions, groups of anterior pituitaries were obtained from 23-d-old male and female rats (n = 10 per group) and placed in scintillation vials in a Dubnoff shaker at 37 C with constant shaking (60 cycles/min) under an atmosphere of 95% O2/5% CO2. Each vial contained 1 ml DMEM solution. After 1 h of preincubation, the medium was replaced by fresh medium alone or medium containing LHRH (10–9 M), PYY3–36 (10–8–10–6 M) or LHRH plus PYY3–36. Samples were collected at 60, 120, and 180 min of the incubation period for hormone determinations.

Experiment 3.
Because our in vitro experiments demonstrated that PYY3–36 directly stimulates LH and FSH secretion, we aimed at characterizing the mechanism of action of PYY3–36. As a first step, we evaluated whether the PYY3–36 effect upon gonadotropin secretion is dependent on extracellular calcium influx. To this end, pituitaries obtained from 23-d-old male rats (n = 10 per group) were incubated with PYY3–36 (10–6 M) in calcium-free medium (Eagle’s MEM) (BioWhittaaker Europe, Verviers, Belgium).

Experiment 4.
To analyze whether PYY3–36 regulates in vivo LH and FSH secretion, 23-d-old male and female rats (n = 10 per group) were ip injected with vehicle or different doses of PYY3–36 (3, 10, and 30 µg/kg). The doses of PYY3–36 for in vivo testing were selected on the basis of a previous reference (26). In detail, net doses of PYY3–36 per animal were approximately 0.1 µg/rat (3 µg/kg), 0.3 µg/rat (10 µg/kg), and 1 µg/rat (30 µg/kg), and doses of 0.3 and 3 µg PYY3–36 have been shown to be effective in mice to reduce food intake (26). Animals were decapitated 15, 30, and 120 min later, and trunk blood samples were collected for hormonal determinations.

Experiment 5.
Because previous experiments showed that systemic administration of PYY3–36 did not affect LH or FSH secretion, we evaluated the potential effects of PYY3–36 at the central (hypothalamic) level. To this end, prepubertal (23-d-old) male and female rats (n = 10 per group) were intracerebroventricular (icv) injected with 3 nmol PYY3–36 per animal dissolved in 10 µl vehicle. The procedure of icv injection was as previously described (32). Briefly, animals were implanted on d 21 with icv cannulae under light ether anesthesia. To allow delivery of PYY3–36 into the lateral cerebral ventricle, the cannulae were lowered to a depth of 3 mm beneath the surface of the skull; the insert point was 1 mm posterior and 1.2 mm lateral to bregma. Animals were decapitated 15 min after injection, and trunk blood samples were collected.

Experiment 6.
To further characterize the mechanism of action of PYY3–36 at central levels, its effects upon hypothalamic LHRH secretion were tested using a static incubation system. Briefly, prepubertal (23-d-old) male and female rats were decapitated, and the retrochiasmatic hypothalamus was rapidly dissected, as described in detail elsewhere (33, 34). Tissue specimens were subsequently incubated in 500 µl Krebs-Ringer-bicarbonate glucose buffer (KRB), in a Dubnoff shaker incubator under an atmosphere of 95% O2 and 5% CO2 at 38 C. After a 30-min preincubation, the medium was removed and hypothalamic fragments were challenged for 30 min with PYY3–36 (at 10–8 and 10–6 M) or KRB alone. At the end of the incubation period, medium samples were boiled to inactivate endogenous protease activity and stored at –80 C until used for hormone measurements.

RNA analysis by RT-PCR
Hypothalamic and pituitary expression of NPY-Y2 and -Y5 receptor mRNAs was assessed by semiquantitative RT-PCR. Total mRNA was isolated from tissue samples using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method, followed by DNase I treatment (35). For amplification of the target genes, the following primer pairs were used: NPY-Y2 sense (nt 375–398; 5'-GGT GCC CTA TGC CCA GGG TCT GGC-3') and NPY-Y2 antisense (nt 530–509; 5'-GCG CTG ACA CCC CAC GCC AGG C-3') for amplification of a 156-bp fragment of rat NPY-Y2 receptor cDNA; and NPY-Y5 sense (nt 131–153; 5'-GGT CCT GCT CCT GCC GCC ACC GC-3') and NPY-Y5 antisense (nt 274–253; 5'-CTT GTT AAA ATG CTC CTC AAG C-3') for amplification of a 144-bp fragment of rat NPY-Y5 receptor cDNA. These oligo-primers were synthesized according to the published rat cDNA sequences of NPY-Y2 and NPY-Y5 receptors (GenBank accession no. NM_023968 and NM_012869, respectively). In addition, to provide an appropriate internal control, amplification of a 241-bp fragment of S11 ribosomal protein mRNA was carried out in each sample, using the primer pair S11 sense (nt 11/32; 5'-CAT TCA GAC GGA GCG TG TTA C-3') and S11 antisense (nt 231/250; 5'-TGC ATC TTC ATC TTC GTC AC-3').

For amplifications of the targets, RT and PCR were run in two separate steps. Furthermore, to enable appropriate amplification in the exponential phase for each target, PCR amplification of specific signal and S11 ribosomal protein transcripts were carried out in separate reactions with a different number of cycles (see below) but using similar amounts of the corresponding cDNA templates, generated in single RT reactions, as previously described (36, 37). Briefly, equal amounts of total RNA (2 µg) were heat denatured and reverse transcribed by incubation at 42 C for 90 min with 12.5 U avian myeloblastosis virus RT (Promega, Madison, WI), 20 U ribonuclease inhibitor RNasin (Promega), 200 mM deoxy-NTP mixture, and 1 nM specific and internal control antisense primers in a final volume of 30 µl of 1x avian myeloblastosis virus RT buffer. The reactions were terminated by heating at 97 C for 5 min and cooling on ice, followed by dilution of the RT cDNA samples with nuclease-free H2O (final volume, 60 µl). For semiquantitative PCR, 10-µl aliquots of the cDNA samples (equivalent to 650 ng total RNA input) were amplified in 50 µl of 1x PCR buffer in the presence of 2.5 U Taq DNA polymerase (Promega), 200 nM deoxy-NTP mixture, and the appropriate primer pairs (1 nM of each primer). PCR consisted in a first denaturing cycle at 97 C for 5 min, followed by a variable number of cycles of amplification (n = 36 cycles for NPY-Y2 and -Y5 receptors; n = 26 cycles for RP-S11) defined by denaturation at 96 C for 1.5 min, annealing for 1.5 min, and extension at 72 C for 3 min. A final extension cycle of 72 C for 15 min was included. Annealing temperature was adjusted for each target: 58 C for NPY-Y2 receptor and S11 and 61.5 C for NPY-Y5 receptor. Different numbers of cycles were tested to optimize amplification in the exponential phase of PCR. On this basis, the numbers of PCR cycles indicated above were chosen for further semiquantitative analysis of specific targets and RP-S11 internal control.

PCR-generated DNA fragments were resolved in Tris-borate-buffered 1.5% agarose gels and visualized by ethidium bromide staining. Specificity of PCR products was confirmed by direct sequencing (Central Sequencing Service, University of Córdoba). Quantification of intensity of RT-PCR signals was carried out by densitometric scanning, and values of the specific targets were normalized to those of internal controls to express arbitrary units of relative expression. In all assays, liquid controls and reactions without RT were included, yielding negative amplification.

LH, FSH, and LHRH measurements by specific RIAs
After centrifugation (1600 x g at 4 C for 20 min), serum was collected, frozen, and stored at –20 C until use. The concentrations of LH and FSH were measured in 5–50 µl by a double-antibody method using RIA kits supplied by National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Rat LH-I-10 and FSH-I-9 was labeled with 125I by the chloramine T, method and hormone concentrations were expressed using a reference preparation LH-RP3 and FSH-RP2 as standards. Intra- and interassay variations were, respectively, 8 and 10% for LH and 6 and 9% for FSH. The sensitivities of the assay were 75 and 400 pg/ml for LH and FSH, respectively. In addition, LHRH concentrations in the incubation media from retrochiasmatic hypothalamic explants were measured in 100-µl aliquots using a commercial RIA kit purchased from Peninsula Laboratories Inc. (Bachem Group, San Carlos, CA), following the instructions of the manufacturer. The sensitivity of the assay was 1 pg/tube. All samples were measured in the same assay.

Presentation of data and statistics
Serum hormone determinations were conducted in duplicate, with a total number of at least 10 samples per group. Semiquantitative RT-PCR analyses were carried out in duplicate from at least three independent RNA samples of each experimental group. The results are given as means ± SEM. Differences between groups were analyzed using Student’s t test or repeated one-way ANOVA followed by Tukey’s test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypothalamic and pituitary expression of NPY receptor Y2 and Y5 mRNAs
RT-PCR analysis using specific primer pairs demonstrated expression, albeit at low levels, of the genes encoding the putative receptors for PYY3–36, i.e. NPY receptors Y2 and Y5, at the pituitary and the hypothalamus of prepubertal (23-d-old) male and female rats. In detail, moderate expression levels of NPY-Y2 and -Y5 receptor mRNAs were observed in whole hypothalamic preparations (Fig. 1Go), whereas low (NPY-Y2) to very low (NPY-Y5) levels were detected at the pituitary (Fig. 2Go). No significant differences were observed in the relative expression levels of these signals at the hypothalamus and pituitary between males and females.



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  		FIG. 1. Expression of the genes encoding NPY receptors Y2 and Y5 at the hypothalamus of prepubertal male and female rats. Representative images of ethidium bromide-stained gel electrophoresis of the specific amplicons are presented. Three independent samples from female (F1–F3) and male (M1–M3) rats are shown. Amplification of a fragment of RP-S11 mRNA served as internal control. Semiquantitative values of gene expression levels in the experimental groups are shown, which are the mean ± SEM of at least three independent determinations. Negative controls were run in parallel with specific RT-PCR assays and yielded negative amplification (data not shown).

 


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  		FIG. 2. Expression of the genes encoding NPY receptors Y2 and Y5 at the pituitary of prepubertal male and female rats. Representative images of ethidium bromide-stained gel electrophoresis of the specific amplicons are presented. Two independent samples from female (F1 and F2) and male (M1 and M2) rats are shown. Amplification of a fragment of RP-S11 mRNA served as internal control. Semiquantitative values of gene expression levels in the experimental groups are shown, which are the mean ± SEM of at least three independent determinations. Negative controls were run in parallel with specific RT-PCR assays and yielded negative amplification (data not shown).

 
Direct effects of PYY3–36 on gonadotropin secretion in male and female rats
Analysis of direct effects of PYY3–36 upon pituitary secretion of both gonadotropins was conducted using a static incubation system. Concentrations of LH in the media increased in both sexes during the incubation period (Fig. 3Go). In males, 10–6 M PYY3–36 significantly increased LH secretion at 60 and 120 min of incubation. The effect of 10–8 M PYY3–36 was also significant at 120 min. In pituitary from female rats, 10–6 M PYY3–36 also stimulated LH release at 60 and 180 min of the incubation period. In contrast, 10–8 M PYY3–36 was unable to stimulate LH release in females (Fig. 3Go). Concerning FSH secretion, 10–6 M PYY3–36 significantly stimulated FSH secretion in male and female rats at the times tested, whereas 10–8 M was effective only in male rats at 120 min of the incubation period (Fig. 4Go).



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  		FIG. 3. LH concentrations in the media after incubation of pituitaries from 23-d-old male (left) and female (right) rats. Incubations were carried out in the presence of PYY3–36 (10–8–10–6 M) or DMEM alone. Values are expressed as means ± SEM (n = 10/group). **, P ≤ 0.01 vs. DMEM (ANOVA followed by Tukey’s test).

 


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  		FIG. 4. FSH concentrations in the media after incubation of pituitaries from 23-d-old male (left) and female (right) rats. Incubations were carried out in the presence of PYY3–36 (10–8–10–6 M) or DMEM alone. Values are expressed as means ± SEM (n = 10/group). **, P ≤ 0.01 vs. DMEM (ANOVA followed by Tukey’s test).

 
Interactions between LHRH and PYY3–36 on in vitro gonadotropin secretion in male rats
The stimulatory effect of 10–9 M LHRH on LH secretion was observed at 60, 120, and 180 min of the incubation period. PYY3–36 at the dose of 10–6 M significantly increased the effectiveness of LHRH, the effect being statistically significant at 60 and 120 min of the incubation period. In contrast, 10–8 M PYY3–36 failed to alter LHRH-stimulated LH secretion in vitro at any time point tested (Fig. 5Go). In addition, the stimulatory effect of 10–9 M LHRH on FSH secretion was observed at 60, 120, and 180 min of the incubation period. At 10–6 M, PYY3–36 significantly enhanced the FSH-releasing effect of LHRH at 60,120, and 180 min of the incubation period, whereas 10–8 M PYY3–36 did not increase LHRH-induced FSH secretion in vitro at 60 and 180 min, and even a slight but significant inhibition was detected at 120 min of incubation (Fig. 5Go).



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  		FIG. 5. LH and FSH concentrations in the media after 180 min of incubation of pituitaries from 23-d-old male rats. Incubations were carried out in the presence of LHRH, PYY3–36, LHRH plus PYY3–36, or DMEM alone. Values are expressed as means ± SEM (n = 10/group). **, P ≤ 0.01 vs. DMEM; a, P ≤ 0.01 vs. corresponding LHRH-stimulated groups (ANOVA followed by Tukey’s test).

 
Effects of PYY3–36 on pituitary LH secretion in calcium-free conditions
To monitor the dependency of PYY3–36 effects on gonadotropin secretion in vitro on extracellular calcium, challenge of pituitary tissue with PYY3–36 in calcium-free medium was conducted. As shown in Fig. 6Go, the stimulatory effect of PYY3–36 on LH secretion was similarly observed in incubations of pituitary samples in the presence of calcium-containing and calcium-free media.



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  		FIG. 6. LH concentrations in the media after incubation of pituitaries from 23-d-old males. Incubations were carried out in absence (–) or presence (+) of calcium in the incubation medium, and pituitaries were challenged with of PYY3–36 (10–6 M) or medium alone. Values are expressed as means ± SEM (n = 10/group). **, P ≤ 0.01 vs. groups incubated in absence of PYY3–36 (ANOVA followed by Tukey’s test).

 
Effects of systemic administration of PYY3–36 on LH and FSH secretion in vivo
Systemic ip administration of PYY3–36 (at the doses of 3, 10, and 30 µg/kg) did not significantly change serum LH concentrations at 15, 30, and 120 min after injection, both in prepubertal male and female rats (Fig. 7Go). Concerning FSH secretion, a significant stimulation was detected at 15 min only after injection of 10 µg/kg PYY3–36 in male rats, whereas inhibition of FSH secretion was observed at 15 min after ip administration of 10 and 30 µg/kg PYY3–36. No changes in serum FSH levels were detected at 30 and 120 min after ip injection of PYY3–36, either in males or in females, at any of the doses tested (Fig. 7Go).



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  		FIG. 7. Serum LH and FSH levels in prepubertal male and female rats after systemic ip administration of PYY3–36 in a range of doses: 3, 10, and 30 µg/kg. Serum determinations were performed at 15, 30, and 120 min after injection of the peptide. Values are expressed as means ± SEM (n = 10/group). *, P ≤ 0.05; **, P ≤ 0.01 vs. corresponding vehicle-injected groups (ANOVA followed by Tukey’s test).

 
Central effects of PYY3–36 on LH and FSH secretion in vivo and on LHRH release in vitro
In male rats, icv administration of 3 nmol PYY3–36 per animal significantly decreased LH secretion, whereas FSH levels remained unaffected (Table 1Go). In contrast, central administration of PYY3–36 did not change serum concentrations of LH or FSH in prepubertal female rats (Table 1Go). Likewise, challenge of hypothalamic tissue with increasing doses of PYY3–36 in vitro resulted in a dose-dependent inhibition of LHRH secretion, which was detected for 10–6 M (but not for 10–8 M) doses selectively in males, whereas hypothalamic LHRH secretion in vitro was not affected by PYY3–36 in females at any of the doses tested (Fig. 8Go).


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  		TABLE 1. Effects of icv administration of 3 nmol PYY3–36 per rat on LH and FSH concentrations in 25-d-old intact male and female rats

 


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  		FIG. 8. LHRH released (pg/fragment) in vitro by hypothalamic fragments from male (top) and female (bottom) rats incubated in the presence of PYY3–36 at 10–8 and 10–6 M concentrations or medium (KRB) alone. Values are expressed as means ± SEM (n = 10/group). **, P ≤ 0.01 vs. corresponding control (KRB) group (ANOVA followed by Tukey’s test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gonadotropin release is primarily controlled by hypothalamic LHRH and peripheral signals. In the last decade, different studies have confirmed that pituitary responsiveness to LHRH is modulated by a number of peptide signals of hypothalamic origin, some of which have been measured in the hypothalamic-pituitary portal blood (38). In this context, NPY was characterized as "a unique member of the family of gonadotropin releasing hormones" (see Ref. 39) because of its multiple effects on hypothalamic LHRH secretion and pituitary responsiveness to LHRH. Thereafter, other hormones with actions on hypothalamic LHRH neurons and pituitary gonadotropes, such as leptin, have been identified (39). The functional relevance of NPY and leptin in the control of LHRH-gonadotropin secretion is further stressed by their concomitant actions on food intake. Thus, NPY and leptin have been proposed as key neuroendocrine integrators for the connection between energy balance and reproductive activity (40, 41, 42), both in physiological and pathological (e.g. obesity and starvation) conditions.

Our present data provide compelling evidence for a stimulatory action of PYY3–36 on LH and FSH secretion directly at the pituitary level. Moreover, this peptide was able to modulate the effects of LHRH upon the secretion of both gonadotropins. Such a stimulatory effect of PYY3–36 on gonadotropin release at the pituitary was apparently independent of the prevailing steroid milieu, because similar responses were detected in our in vitro setting in pituitaries from male and female rats. The mechanisms whereby PYY3–36 conducts such a stimulatory action might involve both NPY-Y2 and -Y5 receptors, because expression of both genes, albeit at low levels, was detected in pituitary tissue. Previous binding studies indicated that Y2 receptors were apparently absent in anterior pituitary (43), although Y5 receptors were present (30). Although evidence for translation into functional receptor proteins is not provided, our current data allow us to speculate that the direct effects of PYY3–36 on LH and FSH release could be mediated by interaction of the peptide with one or both receptors.

Some mechanisms have been proposed to explain the stimulatory effect of NPY on gonadotropes. Specifically, NPY is able to increase extracellular calcium entry (44, 45). Other effects on protein kinase C and adenylate cyclase have also been described (46, 47). At present, the mechanisms for the stimulatory effect of PYY3–36 on gonadotropes remain largely unexplored. Present results suggest that these were not mediated by an increase in the influx of extracellular calcium, because the stimulatory effect was observed when pituitaries were incubated in calcium-free medium. Analyses on the potential involvement of other intracellular signaling mechanisms in the actions of PYY3–36 on gonadotropes are in progress in our laboratory.

Pituitary responsiveness to LHRH is modulated by NPY, although conflicting results have been reported depending on the experimental model used. In ovariectomized females, NPY enhanced the release of LH induced by LHRH (8), whereas a suppressive effect of NPY on LHRH effect was observed in cyclic females in the metestrous phase but not on proestrus (31). An increase of LHRH-stimulated FSH secretion by NPY has been also previously described (48). Present results indicate that PYY3–36 enhanced LHRH effectiveness in terms of LH and FSH secretion. The mechanisms whereby PYY3–36 increases LHRH effectiveness remain unknown. A first possibility is that PYY3–36 could change the LHRH binding to its receptor. In fact, the association between LHRH and its pituitary receptor is stimulated by NPY (49, 50). Two possibilities were advanced to explain this possibility: NPY could bind allosterically to some component of the LHRH receptor, modulating its affinity or, alternatively, NPY could alter LHRH binding consequent to occupation of its own receptor (50). In addition, the possibility that PYY3–36 might enhance LHRH effectiveness at a postreceptor level merits further investigation.

Considering that PYY3–36 stimulates LH and FSH secretion directly at the pituitary level, it was surprising that systemic administration was ineffective in altering LH secretion and induced only slight but significant FSH responses. A potential explanation for this observation is that the dose of systemic (ip) PYY3–36 was not high enough to modify the function of pituitary gonadotropes. It has to be noted, however, that doses lower than those used in the present experiments were fully effective to decrease food intake (26). Alternatively, it remains possible that systemically delivered PYY3–36 may reach and/or activate only a subset of NPY receptors. In this sense, it is noticeable that central (icv) administration of PYY3–36 induced a significant and specific inhibition in LH release in male rats (Table 1Go). Thus, although ip administration of PYY3–36 can activate NPY receptors mainly at the arcuate nucleus, an area where the blood-brain barrier is relatively permeable, icv administration of the peptide might exert its action on all the NPY receptors located in hypothalamic and extrahypothalamic areas. This anatomical consideration has also been proposed to explain how ip-delivered PYY3–36 decreases food intake and increases c-fos expression in the arcuate nucleus, whereas injection in the paraventricular nucleus is ineffective (28). Overall, present data suggest that in addition to direct pituitary actions, PYY3–36 is able to inhibit LH secretion through central (hypothalamic) mechanisms. These include inhibition of LHRH release by hypothalamic neurons, as evidenced by experiments using static incubation of hypothalamic fragments.

Inhibition of LH secretion after central injection of PYY3–36 and decreased LHRH release after challenge with the peptide in vitro was observed in prepubertal (23 d old) male but not female rats. Moreover, comparative analysis of the effects of systemic (ip) and central (icv) administration of PYY3–36 on LH and FSH secretion provides evidence that some aspects of the gonadotropic responses to PYY3–36 are sexually dimorphic, because serum concentrations of FSH increased in males and decreased in females after ip administration of the peptide (Fig. 7Go). Such sex differences might be linked, at least partially, to the fact that 23-d-old male and female rats are at partially different stages of pubertal maturation (51). Nevertheless, our data strongly suggest that, as is the case for different signals such as serotonin, GABA, and excitatory amino acids (52, 53, 54), PYY3–36 is able to elicit (partially) sexually dimorphic endocrine responses during pubertal development, which opens up the possibility of a potential contribution of PYY3–36 in the control of pubertal development.

Chronic neuropeptide Y5 receptor stimulation suppresses reproduction in virgin female and lactating rats (55), but present results indicated that in female rats neither systemic nor icv administration decreases gonadotropin release. It is possible that chronic activation of Y5 receptors may elicit a different gonadotropin response than that observed after acute stimulation. Because in the aforementioned study serum concentrations of gonadotropins were not measured (55), it is possible that activation of Y5 receptors may affect reproductive function throughout modification of other pituitary hormones, specifically prolactin. In this sense, we have recently reported that PYY3–36 inhibits prolactin release in prepubertal male and female rats (56).

In conclusion, present experiments demonstrate that 1) genes encoding NPY receptors Y2 and Y5 are expressed not only at the hypothalamus but also, albeit at low levels, at the pituitary gland; 2) PYY3–36 stimulates in vitro basal and LHRH-stimulated gonadotropin secretion, an effect that was not mediated by an increase in calcium entrance from extracellular fluid; and 3) systemic administration of PYY3–36 was unable to modify serum levels of LH and only marginally changed serum FSH levels, whereas central (icv) administration of this peptide induced a significant decrease in LH secretion in male rats. Moreover, PYY3–36 selectively inhibited, in a dose-dependent manner, LHRH release by hypothalamic fragments from male rats in vitro. Overall, our present data suggest a potential role of PYY3–36 in the control of gonadotropin secretion. On this basis, the possibility that PYY3–36 might participate in the networks linking energy balance and reproductive function merits further investigation.


   Acknowledgments
 
We are indebted to V. M. Navarro and M. L. Barreiro for outstanding assistance during conduction of some of the experimental designs.


   Footnotes
 
This work was supported by Grant BFI 2002-00176 from Ministerio de Ciencia y Tecnología, Spain.

First Published Online November 24, 2004

Abbreviations: KRB, Krebs-Ringer-bicarbonate glucose buffer; NPY, neuropeptide Y; PYY3–36, polypeptide Y3–36.

Received July 6, 2004.

Accepted for publication November 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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