Abstract
Vasoactive intestinal peptide (VIP) has been implicated in the regulation of avian reproductive activity and appears to act at the level of the hypothalamus and pituitary. This in situ hybridization histochemistry study describes the distribution of VIP receptor mRNA expression in the hypothalamus and the pituitary of reproductively active (laying) and quiescent (nonphotostimulated, incubating, and photorefractory) female turkeys and characterizes the differences observed in VIP receptor gene expression. VIP receptor mRNA, while expressed throughout the hypothalamus, was specifically expressed in areas known to contain GnRH-I neurons in the chicken, i.e., the lateral septum, medial preoptic area, anterior hypothalamus, and paraventricular nucleus. Significant differences in VIP receptor mRNA expression between different reproductive states was observed only within the infundibular nuclear complex. VIP receptor mRNA was markedly less in nonphotostimulated and photorefractory hens as compared with laying and incubating hens. The most dense VIP receptor mRNA was found in the anterior pituitary, where it was 2.4- and 3.0-fold greater in laying and incubating hens, respectively, as compared with that in nonphotostimulated ones. Hens that stopped incubating and became photorefractory displayed pituitary VIP receptor mRNA levels similar to those of nonphotostimulated birds. The changes in pituitary VIP receptor mRNA expression were positively correlated with known changes in pituitary prolactin (PRL) mRNA expression and PRL content and release. These findings indicate that the variations in PRL secretion observed across the turkey reproductive cycle are, in part, regulated by changes in VIP receptors at the pituitary level.
Introduction
Pituitary prolactin (PRL) secretion in birds is tonically stimulated [1, 2], and the releasing factor responsible for this stimulation is vasoactive intestinal peptide (VIP), which is secreted from neurons located in the infundibular nuclear complex (INF) of the caudo-medial hypothalamus [3]. Variations in VIP immunoreactivity, VIP content, and VIP mRNA steady-state levels occurring within the hypothalamus are correlated with changes in the amount of circulating PRL throughout the turkey reproductive cycle [4, 5]. The only exception to this correlation between hypothalamic VIP and circulating PRL is found in photorefractory hens, where plasma PRL levels are extremely low in the presence of high hypothalamic VIP levels [4]. Dopamine (DA) also appears to have a prominent role, acting on the VIPergic system to release PRL [6, 7]. Serotonin and the opiate dynorphin stimulate PRL release and may act along a single pathway with DA to influence VIP secretion and ultimately PRL level [8].
In mammals, VIP is widely expressed throughout the body and has important regulatory effects on the circulatory, immune, reproductive, and gastrointestinal systems [9, 10]. VIP occurs extensively in neurons of both the central and peripheral nervous systems [11, 12], with high concentrations found in the hypothalamus [13, 14] and hypophyseal portal blood [15]. Furthermore, VIP is a potent stimulator of PRL secretion both in vivo [16, 17] and in vitro [18–20].
VIP belongs to a family of regulatory peptides, including secretin, glucagon, growth hormone releasing factor, and pituitary adenylate-activating polypeptide, the actions of which are mediated via interaction with specific receptors that are coupled to adenylyl cyclase and the production of cAMP [21, 22]. The mammalian VIP receptors have been cloned and functionally characterized [23–29]. Pharmacological evidence indicates that there are two VIP receptor subtypes (VIP1 and VIP2) with different but related amino acid sequences. Each receptor is expressed in a tissue-specific manner [27, 29, 30]. It has been suggested that a single VIP receptor is expressed and functions in nonmammalian species [31]. In birds, VIP receptors are present on the surface membranes of the anterior pituitary [32–34], the hypothalamus [35], the small intestine, and the granulosa cells [36]. VIP stimulation increases levels of intracellular cAMP in chicken pituitary cells [37] and in the hypothalamus [38]. Recently, avian VIP receptors were cloned and functionally characterized in the chicken [31] and turkey [39]. The present studies were designed to localize and characterize the VIP receptor within individual areas of the turkey hypothalamus and pituitary. This study attempted to provide further understanding of the effect that the differential expression of VIP receptors has on the regulation of PRL and the changes observed in circulating PRL throughout the turkey reproductive cycle.
Materials and Methods
Experimental Animals
Hatchmate breeder turkey hens, Nicholas strain and 22 wk of age, were subjected to a daily short-day lighting regimen of 6L:18D. After this, 30 of these birds at a time were introduced into a room with a 15L:9D light regimen (lights on at 0400 h and off at 1900 h) at 34, 40, and 46 wk of age. All birds were then kept in this room and under the same lighting regimen until the end of the experiment. Groups of birds were housed in separate floor pens within this room, and trap nests were available when needed. Nonphotostimulated (NPS) birds remained in a separate room under 6L:18D lighting conditions. This lighting schedule allowed for obtaining hens of the same age and weight (54 wk old, 10–13 kg) but in different reproductive stages. From this entire flock, six birds per treatment group were selected. Laying (LAY) and incubating (INC) hens were used 3–4 wk after the initiation of their reproductive state. Photorefractory (REF) hens were selected after completion of their first molt. Two birds from each treatment were killed (pentobarbital sodium, 6 g/ml; Anpro Pharmaceuticals, Arcadia, CA) together, beginning at 1000 h for 3 consecutive days. A postmortem examination of each hen was performed to confirm its reproductive status [40]. The animal protocols described in this study were approved by the University of Minnesota Institutional Animal Care and Use Committee.
Processing of Tissues for In Situ Hybridization
Immediately after turkeys were killed, the brain and pituitary were removed and frozen on dry ice, then stored at −80°C. Prior to cryostat sectioning, the brain temperature was allowed to equilibrate to −20°C. Brains were sectioned in a cryostat (Bright Instruments, Huntingdon, UK) at a thickness of 15 μm and mounted onto microscope slides (Probe-On; Fisher Scientific, Minneapolis, MN). The slides were stored desiccated at −80°C until processed.
Preparation of VIP Receptor cRNA Probe
A 500 base pair (bp) fragment of a cDNA sequence coding for the turkey VIP receptor was provided [39]. The cDNA was subcloned into XbaI/EcoRI-digested in pBluescript SK(+) vectors (Stratagene, La Jolla, CA) and transformed into XL1-Blue cells according to established methods [41]. Nucleotide sequence analysis of positive clones was performed on both strands by automated DNA sequencing (Advance Genetics Analysis Center, University of Minnesota, St. Paul, MN). To generate the cRNA probe, the in vitro transcription and 33UTP radioactive labeling of the probe was performed as previously described [42] by using a MAXIscript T7/T3 In Vitro Transcription Kit (Ambion Inc., Austin, TX). The cRNA probe was purified to obtain the full-length strand by polyacrylamide gel electrophoresis as previously described [41].
In Situ Hybridization Histochemistry
To localize gene expression of VIP receptor within the brain and pituitary, in situ hybridization (ISH) was performed as previously described [5]. Briefly, tissue sections were thawed to room temperature before use. The slides were then hybridized with 100 μl of hybridization solution containing 33P-UTP-VIP receptor cRNA probe (0.5 × 106 cpm/slide) and incubated at 52°C for 24 h in a humid incubator. The slides were paired together instead of using coverslips before incubation. The hybridization solution contained 50% formamide; 300 mM NaCl; 10 mM Tris HCl, pH 8.0; 1 mM EDTA; 0.02% polyvinylpyrrolidone; 0.02% Ficoll 400; 0.02% bovine serum albumin; 10 mM dithiotreithol; 500 ng/ml yeast tRNA; 1% SDS, and 10% dextran sulfate. After hybridization, the paired sections were separated by soaking in 2× SSC at room temperature and then treated with RNase A solution (RNase A; Boehringer Mannheim, Roche Applied Science, Indianapolis, IN; 20 μg/ml in 10 mM Tris HCl, pH 8.0 and 0.5 M NaCl) for 30 min at 37°C. The slides were washed once with 2× SSC for 5 min at room temperature, once with 2× SSC for 5 min at 55°C, twice with 1× SSC for 20 min at 55°C, twice with 0.5× SSC for 20 min at 55°C, and twice with 0.1× SSC for 20 min at 55°C. After the final wash, the sections were allowed to slowly cool to room temperature in the washing solution. The sections were then dehydrated through graded alcohol (50–100%) and quickly air dried. Tissue sections were apposed to hyperfilm βmax autoradiograph film (Amersham, Arlington Heights, IL) for 2 days at −20°C. X-ray films were developed and evaluated. The sections were then dipped in NTB2 nuclear track emulsion (Kodak, Rochester, NY) diluted 1:1 with distilled water, dried overnight, and stored in a light-proof box at 4°C until developing (4 days). The dipped sections were developed at room temperature with D-19 developer (Kodak) diluted 1:1 with water for 5 min, followed by a 20-sec rinse in distilled water, and fixed in a rapid fixer (Kodak) for 5 min. The sections were then rinsed with distilled water for 5 min. Following the development of autoradiographic grains, the sections were counterstained with the fluorescent dye Hoechst 33258 (0.001% bisbenzimide in 0.2 M KCl-HCl buffer, pH 2.0; Sigma, St. Louis, MO) as described by Schnell and Wessendorf [43] and coverslipped with DPX Mountant (Fluka Chemical, Ronkonkoma, NY). Autoradiographic grains and counterstain were visualized with a fluorescence microscope equipped with a darkfield condenser and ultraviolet excitation using a UG1 filter set for excitation (Schott, Duryea, PA) and a 420-nm longpass emission filter.
Specificity of cRNA Probe Used for In Situ Hybridization
The specificity of the VIP receptor cRNA probe used for in situ hybridization was previously verified by two methods. First, a 32P-UTP-labeled antisense or sense cRNA probe was used to probe the total RNA from the turkey hypothalamus by Northern blot analysis. Second, 33P-UTP-labeled sense and antisense cRNA probes of a VIP receptor-specific fragment were hybridized to the brain sections. The labeled probes were applied to adjacent sections. Only the antisense cRNA probe showed hybridization signals. No labeling on brain sections was observed with the sense cRNA probe (data not shown). The criteria used in the present study have been accepted as sufficient verification of specificity for probes used for in situ hybridization [44–46].
Image Analysis
Data were organized by coronal brain sections, which were taken from approximately 1.0 mm rostral to the optic chiasma to approximately 1.0 mm caudal to the median eminence and pituitary. To aid in the documentation of neuroanatomical results, nomenclature from the atlases of the turkey hypothalamus (unpublished results), the chicken brain [47], and chicken hypothalamus [48] was used.
For ISH, autoradiograms of brain sections were examined as previously described for comparison of the expression of VIP receptor mRNA within different brain areas across reproductive groups. Microscopic images of brain sections were visualized at 20× magnification using a videocamera (CoolCam 2000 color) fitted to a fluorescence microscope (Nikon E800 Eclipe; Nikon, Japan). The images were then captured and stored by Image Pro Plus 1.3 software (Media Cybernetics, Silver Spring, MD) with a fixed setting for the videocapture. Eight microscopic fields on pairs of adjacent sections (four microscopic fields/section) from each bird were chosen for area imaging in each treatment group, corresponding to the lateral septum (LS), medial preoptic nucleus (POM), anterior hypothalamic nucleus (AM), lateral hypothalamus (LHy), paraventricular nucleus (PVN), ventromedial nucleus (VMN), infundibular nuclear complex (INF), and pituitary (PIT). The integrated density of hybridization signals was then analyzed on a pixel-by-pixel basis in the defined area using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Background was measured from eight nonhybridizing tissue fields, averaged, and subtracted from the integrated density of hybridization signals. Values corresponding to the expression of VIP receptor in the defined areas were calculated for each bird by summing the integrated density values from eight microscopic fields from two consecutive sections as described [5].
Statistical Analysis
Results are expressed as mean ± SEM. Statistical analysis for VIP receptor gene expression by ISH was performed employing the General Linear model procedure of the Statistical Analysis System [49]. Significant differences in mean ± SEM among treatment groups were assessed using the Tukey studentized range test at a significance level of α = 0.05.
Results
VIP receptor mRNA was expressed throughout the turkey hypothalamus and pituitary, as revealed by single label in situ hybridization (Table 1; Figs. 1 and 2). The greatest density of VIP receptor mRNA for all areas measured was found within the pituitary. In the hypothalamus, the highest density of VIP receptor mRNA-expressing cells were observed in the POM, AM, LHy, VMN, and INF. The least amount of VIP receptor mRNA was found in cells in the LS and PVN.
The expression of VIP receptor mRNA within individual areas of the turkey hypothalamus and pituitary at different reproductive stages. See Figure 1 legend for abbreviations.*
| Area | Reproductive group | |||
|---|---|---|---|---|
| NPS | LAY | INC | REF | |
| LS | 2.9 ± 1.1a | 3.1 ± 1.1a | 4.1 ± 3.3a | 2.9 ± 1.7a |
| POM | 11.1 ± 2.9a | 13.2 ± 3.8a | 14.7 ± 3.5a | 9.0 ± 2.2a |
| AM | 10.2 ± 2.8a | 8.8 ± 2.5a | 14.9 ± 4.3a | 8.0 ± 0.9a |
| LHy | 9.8 ± 3.2a | 9.3 ± 2.2a | 12.6 ± 3.6a | 8.5 ± 0.5a |
| PVN | 3.1 ± 1.1a | 4.6 ± 1.7a | 5.9 ± 1.5a | 4.6 ± 0.6a |
| VMN | 13.3 ± 5.0a | 12.4 ± 3.4a | 13.3 ± 3.6a | 10.3 ± 2.6a |
| INF | 8.3 ± 1.4b | 12.9 ± 3.6a | 15.1 ± 3.9a | 7.7 ± 1.5b |
| PIT | 8.9 ± 2.8b | 21.4 ± 2.7a | 26.4 ± 3.7a | 6.7 ± 1.4b |
| Area | Reproductive group | |||
|---|---|---|---|---|
| NPS | LAY | INC | REF | |
| LS | 2.9 ± 1.1a | 3.1 ± 1.1a | 4.1 ± 3.3a | 2.9 ± 1.7a |
| POM | 11.1 ± 2.9a | 13.2 ± 3.8a | 14.7 ± 3.5a | 9.0 ± 2.2a |
| AM | 10.2 ± 2.8a | 8.8 ± 2.5a | 14.9 ± 4.3a | 8.0 ± 0.9a |
| LHy | 9.8 ± 3.2a | 9.3 ± 2.2a | 12.6 ± 3.6a | 8.5 ± 0.5a |
| PVN | 3.1 ± 1.1a | 4.6 ± 1.7a | 5.9 ± 1.5a | 4.6 ± 0.6a |
| VMN | 13.3 ± 5.0a | 12.4 ± 3.4a | 13.3 ± 3.6a | 10.3 ± 2.6a |
| INF | 8.3 ± 1.4b | 12.9 ± 3.6a | 15.1 ± 3.9a | 7.7 ± 1.5b |
| PIT | 8.9 ± 2.8b | 21.4 ± 2.7a | 26.4 ± 3.7a | 6.7 ± 1.4b |
Values represent mean ± SEM of arbitrary densometric units.
Values with different superscripts on the same line are significantly different (P < 0.05, n = 6).
The expression of VIP receptor mRNA within individual areas of the turkey hypothalamus and pituitary at different reproductive stages. See Figure 1 legend for abbreviations.*
| Area | Reproductive group | |||
|---|---|---|---|---|
| NPS | LAY | INC | REF | |
| LS | 2.9 ± 1.1a | 3.1 ± 1.1a | 4.1 ± 3.3a | 2.9 ± 1.7a |
| POM | 11.1 ± 2.9a | 13.2 ± 3.8a | 14.7 ± 3.5a | 9.0 ± 2.2a |
| AM | 10.2 ± 2.8a | 8.8 ± 2.5a | 14.9 ± 4.3a | 8.0 ± 0.9a |
| LHy | 9.8 ± 3.2a | 9.3 ± 2.2a | 12.6 ± 3.6a | 8.5 ± 0.5a |
| PVN | 3.1 ± 1.1a | 4.6 ± 1.7a | 5.9 ± 1.5a | 4.6 ± 0.6a |
| VMN | 13.3 ± 5.0a | 12.4 ± 3.4a | 13.3 ± 3.6a | 10.3 ± 2.6a |
| INF | 8.3 ± 1.4b | 12.9 ± 3.6a | 15.1 ± 3.9a | 7.7 ± 1.5b |
| PIT | 8.9 ± 2.8b | 21.4 ± 2.7a | 26.4 ± 3.7a | 6.7 ± 1.4b |
| Area | Reproductive group | |||
|---|---|---|---|---|
| NPS | LAY | INC | REF | |
| LS | 2.9 ± 1.1a | 3.1 ± 1.1a | 4.1 ± 3.3a | 2.9 ± 1.7a |
| POM | 11.1 ± 2.9a | 13.2 ± 3.8a | 14.7 ± 3.5a | 9.0 ± 2.2a |
| AM | 10.2 ± 2.8a | 8.8 ± 2.5a | 14.9 ± 4.3a | 8.0 ± 0.9a |
| LHy | 9.8 ± 3.2a | 9.3 ± 2.2a | 12.6 ± 3.6a | 8.5 ± 0.5a |
| PVN | 3.1 ± 1.1a | 4.6 ± 1.7a | 5.9 ± 1.5a | 4.6 ± 0.6a |
| VMN | 13.3 ± 5.0a | 12.4 ± 3.4a | 13.3 ± 3.6a | 10.3 ± 2.6a |
| INF | 8.3 ± 1.4b | 12.9 ± 3.6a | 15.1 ± 3.9a | 7.7 ± 1.5b |
| PIT | 8.9 ± 2.8b | 21.4 ± 2.7a | 26.4 ± 3.7a | 6.7 ± 1.4b |
Values represent mean ± SEM of arbitrary densometric units.
Values with different superscripts on the same line are significantly different (P < 0.05, n = 6).
![Schematic coronal sections showing the distribution of VIP receptor mRNA (circles) throughout the hypothalamus of the laying turkey hen. Coronal illustrations were redrawn from Mauro et al. [4] using an unpublished turkey atlas (personal communication) with nomenclature from Kuenzel and van Tienhoven [48]. The following abbreviations are used in the figure legends: AM, anterior hypothalamus; AME, anterior median eminence; BPC, bed nucleus of pallial commissure; CO, optic chiasma; DHA, dorsal hypothalamic area; DM, dorsomedial nucleus; DS, supraoptic decussation; EM, ectomammilary nucleus; GLv, lateral geniculate nucleus; INF, infundibular nuclear complex; LFB, lateral forebrain bundle; LHy, lateral hypothalamus; LS, lateral septum; OM, tractus occipitomesencephalicus; Ov, ovoid nucleus; PD, pituitary; PME, posterior median eminence; POM, medial preoptic nucleus; PP, paleostriatum primitivum; PVN, paraventricular nucleus; PVO, paraventricular organ; Rt, rotund nucleus; TrO, optic tract; TSM, septomesencephalic tract; V, ventricle; VLT, ventrolateral thalamus; VMN, ventromedial nucleus of the hypothalamus](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/biolreprod/70/3/10.1095_biolreprod.103.022715/1/m_bire-70-03-09-f01.jpeg?Expires=1712769158&Signature=HNUyZ4g66RTMnRZOTFEv8rBXNxUTEAiMsB0nhFhMowcg7s-a1uBpY3jhNnJUiIURAq7ck45MEJPUrIr76GD-LJrIaAiq~cBLJ5NHiUTiEaePV-LhkgVX3YfZjKFHFHJJMa6hgxlNpTqU~vvAO-JvlO6rsHqQYY1lWyjYfcOxZ4JQqyZWrqmNCFnfNa~4cCzFRZnq8wcpn~EX-0GJQieyKnZNe2BcJuKqIMEBGhO6jb6~UJZcWynvy~gdHW-F~A7MPm1vysMqI263bD4urSrmDoT-PRhiCI792~0S0Bak2ULp3xfBK7FYvN-Fn2NUwFZl8v6jdoo9K8SaBAIQ0Tq95A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Schematic coronal sections showing the distribution of VIP receptor mRNA (circles) throughout the hypothalamus of the laying turkey hen. Coronal illustrations were redrawn from Mauro et al. [4] using an unpublished turkey atlas (personal communication) with nomenclature from Kuenzel and van Tienhoven [48]. The following abbreviations are used in the figure legends: AM, anterior hypothalamus; AME, anterior median eminence; BPC, bed nucleus of pallial commissure; CO, optic chiasma; DHA, dorsal hypothalamic area; DM, dorsomedial nucleus; DS, supraoptic decussation; EM, ectomammilary nucleus; GLv, lateral geniculate nucleus; INF, infundibular nuclear complex; LFB, lateral forebrain bundle; LHy, lateral hypothalamus; LS, lateral septum; OM, tractus occipitomesencephalicus; Ov, ovoid nucleus; PD, pituitary; PME, posterior median eminence; POM, medial preoptic nucleus; PP, paleostriatum primitivum; PVN, paraventricular nucleus; PVO, paraventricular organ; Rt, rotund nucleus; TrO, optic tract; TSM, septomesencephalic tract; V, ventricle; VLT, ventrolateral thalamus; VMN, ventromedial nucleus of the hypothalamus
Darkfield illumination photomicrographs of coronal sections illustrating the distribution of VIP receptor mRNA in the hypothalamus and pituitary of the laying turkey. The specific hybridization binding of VIP receptor cRNA probe was observed within the LS (A), POM (B), AM (C), LHy (D), PVN (E), VMN (F), INF (G), and PIT (H). All photographs are printed at the same magnification; bar = 100 μm. See Figure 1 for abbreviation definitions
The expression of VIP receptor mRNA between reproductive groups was significantly greater in the INF (Fig. 3) and pituitary of laying and incubating hens (Table 1, P < 0.05). VIP receptor mRNA was markedly less in reproductively quiescent nonphotostimulated hens and in photorefractory hens when compared with laying and incubating ones (Table 1, P < 0.05). In all other areas examined, the expression of VIP receptor mRNA was essentially the same for all reproductive groups (Table 1; Fig. 4, P > 0.05).
Darkfield illumination photomicrographs of coronal sections demonstrating the distribution of VIP receptor mRNA within the infundibular nuclear complex (INF) of the turkey at different reproductive stages. Nonphotostimulated (NPS) (A), laying (LAY) (B), incubating (INC) (C), photorefractory (REF) (D). All photographs are printed at the same magnification; bar = 100 μm
Darkfield illumination photomicrographs of coronal sections demonstrating the distribution of VIP receptor mRNA within the medial preoptic nucleus (POM) of the turkey at different reproductive stages. Nonphotostimulated (NPS) (A), laying (LAY) (B), incubating (INC) (C), photorefractory (REF) (D). All photographs are printed at the same magnification; bar = 100 μm
The most dense VIP receptor mRNA was found in the pituitary, where content was 2.4- and 3.0-fold greater in laying and incubating hens, respectively, as compared with that of nonphotostimulated ones (Fig. 5). In photorefractory birds, pituitary VIP receptor mRNA levels were comparable with those of nonphotostimulated birds (Table 1, P < 0.05).
Darkfield illumination photomicrographs of coronal sections showing the distribution of VIP receptor mRNA within the turkey pituitary (PIT) at different reproductive stages. Nonphotostimulated (NPS) (A), laying (LAY) (B), incubating (INC) (C), photorefractory (REF) (D). All photographs are printed at the same magnification; bar = 100 μm
Discussion
The present survey of VIP receptor expression in the hypothalamus and pituitary of the turkey hen demonstrates that VIP receptor mRNA is expressed throughout the hypothalamus and pituitary, with the major expression found in the pituitary. These data also show differential expression of VIP receptors within the pituitary and in specific areas of the hypothalamus and reveal that this differential expression is correlated with changes in the turkey reproductive cycle. The data also suggest that the prominent role that VIP plays in avian PRL secretion is regulated through VIP receptor gene expression in the pituitary.
These results correspond well with VIP binding site studies in the avian hypothalamus and pituitary showing high levels of VIP receptor mRNA in the hypothalamus and are consistent with the function of VIP as a neuroendocrine factor or neurotransmitter [32, 35]. They are also in accordance with recent studies, using Northern blot hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses, which report that major VIP receptor gene expression was found within the avian hypothalamus and pituitary [31, 39].
Changes in pituitary VIP receptor mRNA were observed across the reproductive stages. Increased VIP receptor mRNA in the pituitary was observed in hens with intermediate (laying) or high PRL secretion (incubating), while much less VIP receptor mRNA was observed in the pituitary of hypoprolactinemic nonphotostimulated and photorefractory hens. These results are in good agreement with studies indicating variations in VIP immunoreactivity and VIP content in the INF and median eminence [4, 5], in VIP mRNA steady-state levels in the INF, where VIP acts as the PRL-releasing factor [3], and in VIP concentrations in turkey hypophysial portal blood [40]. In addition, these results correspond with VIP binding studies [32] demonstrating that hypoprolactinemic nonphotostimulated birds exhibit the fewest number of VIP high-affinity binding sites and that these sites increase after photostimulation to reach maximal levels in hyperprolactinemic incubating hens and then decrease in number again when the birds become photorefractory. VIP receptor mRNA expression in the anterior pituitary of hens in different prolactinemic states may account for the alterations in PRL gene transcription observed during the reproductive cycle and in response to VIP stimulation [50, 51]. These results, present and past, provide additional evidence that VIP is the avian PRL-releasing factor and strongly suggest that VIP receptors at the pituitary level play a prominent role in the regulation of PRL secretion.
Decreased VIP mRNA receptor expression was noted in the INF area of nonphotostimulated and photorefractory hens. These findings are difficult to explain at this time because the identity of the neurons expressing VIP receptors in the INF are not known. It has been proposed that VIP neurons within the INF area are encephalic photoreceptors [52] and lesions in this area block photoperiod-induced gonadal recrudescence [53]. No correlation was found between VIP receptor mRNA expression in other areas of the hypothalamus (LS, POM, AM, LHy, PVN, and VMN) and the reproductive phases. Considering the many functions attributed to VIP [54], it is likely that VIP within these areas may be serving a neurotransmitter/neuromodulator role. In the chicken, VIP neurons within the PVN and INF are structurally adjacent to other peptidergic neurons, such as somatostatin, methionine-enkephalin, and β-endorphin, suggesting complex modulation processes [55]. In mammals, about 40% of GnRH neurons contain VIP2 receptor immunoreactivity and processes containing VIP are seen in close apposition to a significant number of VIP2 receptor-positive GnRH neurons [56, 57]. It is not known if GnRH neurons express VIP receptors in birds, but the abundance of VIP receptor mRNA expression in hypothalamic areas (LS, POM, AM, PVN, and VMN; the present study) known to contain GnRH-I immunoreactive neurons and VIP fibers [58–62] suggests a role for VIP in the regulation of the GnRH/gonadotropin system. Central infusion of VIP into laying turkeys lowers their circulating luteinizing hormone and terminates egg-laying activity [63]. VIP immunoneutralization upregulates pituitary LH-β and FSH-β subunit mRNA content and increases egg-laying activity [64, 65]. Active immunization against VIP delays the onset of photorefractoriness and extends reproductive activity in starlings [66].
In summary, VIP receptor mRNA is found to be expressed throughout the hypothalamus and pituitary of the turkey hen, and this expression varies with the areas examined. However, only the differential expression of VIP receptor mRNA in the INF and the pituitary is associated with reproductive changes. In reproductively inactive photorefractory or nonphotostimulated hens, VIP receptor mRNA expression is low. As circulating PRL increases during laying and reaches its apex during incubation, VIP receptor mRNA expression in the INF and pituitary correspondingly increases. This suggests that VIP receptors in the INF are functionally involved in avian reproductive activity. And because the greatest amount of VIP receptor expression product is confined to the pituitary, it further suggests that avian PRL secretion is, at least partially, regulated by VIP receptors at the pituitary level.
References
Author notes
This research was supported by USDA grant 00-35203-9157.
