Dopamine and mesotocin neurotransmission during the transition from incubation to brooding in the turkey.

We investigated the neuroendocrine changes involved in the transition from incubating eggs to brooding of the young in turkeys. Numbers of mesotocin (MT; the avian analog of mammalian oxytocin) immunoreactive (ir) neurons were higher in the nucleus paraventricularis magnocellularis (PVN) and nucleus supraopticus, pars ventralis (SOv) of late stage incubating hens compared to the layers. When incubating and laying hens were presented with poults, all incubating hens displayed brooding behavior. c-fos mRNA expression was found in several brain areas in brooding hens. The majority of c-fos mRNA expression by MT-ir neurons was observed in the PVN and SOv while the majority of c-fos mRNA expression in dopaminergic (DAergic) neurons was observed in the ventral part of the nucleus preopticus medialis (POM). Following intracerebroventricular injection of DA or oxytocin (OT) receptor antagonists, hens incubating eggs were introduced to poults. Over 80% of those injected with vehicle or the D1 DA receptor antagonist brooded poults, while over 80% of those receiving the D2 DA receptor antagonist or the OT receptor antagonist failed to brood the poults. The D2 DA/OT antagonist groups also displayed less c-fos mRNA in the dorsal part of POM and the medial part of the bed nucleus of the stria terminalis (BSTM) areas than did the D1 DA/vehicle groups. These data indicate that numerous brain areas are activated when incubating hens initially transition to poult brooding behavior. They also indicate that DAergic, through its D2 receptor, and MTergic systems may play a role in regulating brooding behaviors in birds.

Rhythmic dependent light induction of gonadotrophin-releasing hormone-I expression and activation of dopaminergic neurones within the premammillary nucleus of the turkey hypothalamus.

Our previous studies using turkey hens have demonstrated that c-fos mRNA (a marker of neuronal activation) is expressed in gonadotrophin-releasing hormone-I (GnRH-I), vasoactive intestinal peptide (VIP) and dopamine (DA) neurones following electrical stimulation in the preoptic area. DA has been shown to have both stimulatory and inhibitory effects on the GnRH-I/luteinising hormone (LH), follicle-stimulating hormone (FSH) and VIP/prolactin (PRL) systems. To identify the DA neurones that mediate the stimulatory influences of photoperiod on the reproductive system, we examined c-fos mRNA induction in DA, GnRH-I, and VIP neurones in the turkey hypothalamus using a dark-interruption experimental design. A 30-min light period was provided to short day (6L : 18D) photosensitive turkeys at times when birds were responsive to light (14 h after first light) and at times when birds were unresponsive to light (8 h and 20 h after first light). The only area where DA neurones were activated when the birds were provided with light was in the nucleus premammillaris (PMM). The number of activated DA neurones was significantly greater when light was provided at 14 h (during the photoinducible phase) than at 8 h or 20 h. At 14 h, there was also an increase in the number of GnRH-I neurones activated in the area of the nucleus commissura pallii (nCPa), as well as an up-regulation of GnRH-I mRNA expression. No expression of c-fos mRNA was observed in VIP neurones in the nucleus infundibularis or up-regulation of VIP mRNA expression in any of the experimental light treatments. These results are the first evidence to demonstrate a relationship between the dopaminergic system in the PMM and the GnRH-I system in the nCPa during the photoinduction of avian reproductive activity.

Rhythm-dependent light induction of the c-fos gene in the turkey hypothalamus.

Day length (photoperiod) is a powerful synchroniser of seasonal changes in the reproductive neuroendocrine activity in temperate-zone birds. When exposed to light during the photoinducible phase, reproductive neuroendocrine responses occur. However, the neuroendocrine systems involved in avian reproduction are poorly understood. We investigated the effect of light exposure at different circadian times upon the hypothalamus and components of the circadian system, using c-fos mRNA expression, measured by in situ hybridisation, as an indicator of light-induced neuronal activity. Levels of c-fos mRNA in these areas were compared after turkey hens (on a daily 6-h light period) had been exposed to a 30-min period of light occurring at 8, 14, or 20 h after the onset of first light of the day (subjective dawn). Non-photostimulated control birds were harvested at the same times. In birds, photostimulated within the photoinducibile phase (14 h), in contrast to before or after, c-fos mRNA was significantly increased in the nucleus commissurae pallii (nCPa), nucleus premamillaris (PMM), eminentia mediana (ME), and organum vasculosum lamina terminalis (OVLT). Photostimulation increased c-fos mRNA expression in the pineal gland, nucleus suprachiasmaticus, pars visualis (vSCN) and nucleus inferioris hypothalami compared to that of their corresponding nonphotostimulated controls. However, the magnitudes of the responses in these areas were similar irrespective of where in the dark period the pulses occurred. No c-fos mRNA was induced in the nucleus infundibulari, in response to the 30-min light period at any of the circadian times tested. The lack of c-fos up-regulation in the pineal gland and vSCN following photostimulation during the photoinducible phase lends credence to the hypothesis that these areas are not involved in the photic initiation of avian reproduction. On the other hand, c-fos mRNA increases in the nCPa, ME, and OVLT support other studies showing that these areas are involved in the onset of reproductive behaviour initiated by long day lengths. The present study provides novel data showing that the PMM in the caudal hypothalamus is involved in the neuronally mediated, light-induced initiation of reproductive activity in the turkey hen.

Identification of dopamine, gonadotrophin-releasing hormone-I, and vasoactive intestinal peptide neurones activated by electrical stimulation to the medial preoptic area of the turkey hypothalamus: a potential reproductive neuroendocrine circuit.

The neural and neurochemical substrates regulating reproduction in birds remain vaguely defined. The findings that electrical stimulation in the medial preoptic area (ES/MPOA) or intracerebroventricular infusion of dopamine (DA) stimulated luteinising hormone (LH) and prolactin (PRL) release in female turkeys, led to the suggestion that ES/MPOA might help to clarify the DA circuitry regulating LH and PRL. We used c-fos mRNA and tyrosine hydroxylase immunoreactivity as measured by double in situ hybridisation/immunocytochemistry (ISH/ICC) to determine which group/subgroup of DA neurones was activated following unilateral ES/MPOA. To establish that the reproductive neuroendocrine system was activated, double ISH/ICC was also conducted on c-fos/gonadotrophin-releasing hormone-I (GnRH-I) and c-fos/vasoactive intestinal peptide (VIP). Changes in circulating LH and PRL were determined by radioimmunoassay. Unilateral ES/MPOA (100 microA, right side) of anaesthetised laying turkeys for 30 min increased circulating LH and PRL levels. It also induced c-fos mRNA expression on the ipsilateral side by all GnRH-I neurones within the septopreoptic region, implying that GnRH-I neurones in this region share similar circuitry. VIP neurones within the nucleus infundibularis were the only VIP group to show c-fos mRNA expression, suggesting their involvement in ES/MPOA induced PRL release. c-fos mRNA expression was also observed in a subgroup of DA neurones in the nucleus mamillaris lateralis (ML). To our knowledge, the present study is the first to show that activation of DAergic cells in the ML is associated with the activation of GnRH-I and VIP neurones and the release of LH and PRL. It is likely that ES/MPOA activated VIP/GnRH-I neurones via activation of DA neurones in the ML, as this was the only DA subgroup that showed c-fos mRNA expression.

Regulation of prolactin gene expression by vasoactive intestinal peptide and dopamine in the turkey: role of Ca signalling.

Our recent work has demonstrated that dopamine, acting through D2 dopamine receptors on pituitary cells, inhibits the stimulatory effects of vasoactive intestinal peptide (VIP) on prolactin release and prolactin gene transcription. It is hypothesised that the stimulatory and inhibitory roles of VIP and dopamine, respectively, on prolactin synthesis and release are mediated by their opposite effects on intracellular Ca2+ concentration ([Ca2+]i) in lactotrophs. The present study aimed: (i) to investigate the effect of VIP and dopamine on [Ca2+]i of cultured turkey anterior pituitary cells and (ii) to examine the role of Ca2+ signalling in mediating the regulatory effects of VIP and dopamine on prolactin mRNA levels and prolactin release. Changes in [Ca2+]i were measured spectrofluorometrically using Fura-2/AM as a fluorescent Ca2+ indicator. Semi-quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to determine prolactin mRNA levels and prolactin release, respectively. VIP or the L-type Ca2+ channel activator, Bay K8644 (Bay) increased [Ca2+]i in a concentration- and time-dependent fashion, an effect abolished by preincubating the cells with R(-)-propylnorapomorphine HCl, a D2 dopamine receptor agonist (D2AG) or Verapamil (VR), a specific L-type Ca2+ channel blocker. Similarly, either VR or D2Ag diminished the VIP/Bay stimulatory effect on prolactin expression and release. On the other hand, pretreatment of pituitary cells with thapsigargin (TG) or neomycin (NEO), to deplete the intracellular Ca2+ stores, showed no effect on basal or VIP-stimulated prolactin mRNA levels; although VIP-induced prolactin release was partially inhibited by NEO but not TG. These results suggest that intracellular Ca2+ represents a common signal transduction pathway through which VIP and dopamine can exert antagonistic control on prolactin synthesis and release in avian lactotrophs.

Molecular cloning and expression of turkey inhibin-alpha and -betaA subunits.

We isolated cDNA encoding turkey inhibin-alpha (tINH-alpha) and -betaA (tINH-betaA) subunits from the turkey ovary using reverse transcription-polymerase chain reaction (RT-PCR). The isolated alpha subunit and betaA subunit included the entire open reading frames encoding 329 and 424 amino acids, respectively. The amino acid sequences of mature tINH-alpha subunit and tINH-betaA subunit (12.6 and 12.9 kDa proteins, respectively), established via DNA sequence analysis, were highly conserved between the chicken and various mammals. Northern blot analysis revealed that the transcripts of tINH-alpha and tINH-betaA subunits were approximately 1.7 and 8.4 kb, respectively. In various stages of follicular development, tINH-alpha mRNA was highly expressed in small white follicles as compared to postovulatory and regressed follicles, whereas tINH-betaA mRNA was predominately expressed in preovulatory F5 follicles.

Active immunization with vasoactive intestinal peptide in turkey hens.

Active immunization of turkey hens against vasoactive intestinal peptide (VIP) has been shown to inhibit incubation behavior and to increase egg production in second-cycle hens. The objective of this study was to compare the effect of VIP immunization on first- and second-cycle turkey hens during a 27-wk production period. First- (25-wk-old) and second- (54-wk-old) cycle hens were intermixed, distributed among 16 pens, and subjected to a photoperiod of 6 h of light and 18 h of darkness for 10 wk. The first-cycle hens were divided into two groups: keyhole limpet hemocyanin (KLH)-immunized controls (n = 16) and VIP-immunized (n = 18). Second-cycle hens were divided into four groups: 1) unimmunized controls (n = 19), 2) KLH-immunized controls (n = 18), 3) VIP-immunized (n = 19), and 4) VIP-preimmunized (immunized during first cycle; n = 16). Each hen received four antigen injections beginning the day of photostimulation (4-wk intervals), except for the preimmunized hens, which received three injections beginning 4 wk after photostimulation. The maximum titer of VIP antibodies in first-cycle, second-cycle, and preimmunized hens was 17.2+/-2.2, 20.9+/-2.9, and 21.7+/-3.2%, respectively. After photostimulation, plasma prolactin of first- and second-cycle control hens peaked between 484 +/-105 and 630+/-118 ng/mL. In contrast, prolactin changed very little in VIP-immunized turkeys. The average number of daily nest visits was less in first- and second-cycle VIP-immunized hens (1.68+/-0.23 and 1.09+/-0.15 visits per hen per day, respectively) than in their respective KLH-immunized controls (2.47+/-0.36 and 2.65+/-0.45 visits per hen per day). Expression of incubation behavior was 50.0 and 52.6% in first- and second-cycle control hens, respectively, upon termination of the study. In contrast, only 11.1% first-cycle and 5.2% second-cycle VIP-immunized turkeys exhibited the hormonal and behavioral characteristics of incubating hens. Average weekly egg production of first- and second-cycle VIP-immunized turkeys was similar (3.58+/-0.19 vs. 3.63+/-0.14 eggs per hen per wk). First- and second-cycle control hens laid 2.63+/-0.25 and 2.41+/-0.20 eggs per hen per wk, respectively. The present results show that comparable egg production was attained in first- and second-cycle hens by active immunization with VIP.

An opioid pathway in the hypothalamus of the turkey that stimulates prolactin secretion.

Circulating prolactin (PRL) levels increase when dynorphin is infused into the turkey brain. This study tested the hypothesis that centrally infused dynorphin requires an intact vasoactive intestinal peptide (VIP) system in order to stimulate turkey PRL secretion. It also investigated the roles of the dopaminergic and serotonergic systems in dynorphin-induced PRL release. Drugs were infused into the third ventricle of anesthetized laying turkeys via stereotaxically guided cannulae and circulating blood was assayed for changes in PRL levels. When a highly selective kappa opioid receptor antagonist was given prior to dynorphin injection, the PRL response to dynorphin was almost totally blocked. The coinfusion of either a serotonin (5-HT) or a D(1) dopamine (DA) receptor antagonist with dynorphin prevented the increase in PRL observed in birds when dynorphin was infused alone. On the other hand, the kappa opioid receptor antagonist failed to prevent the 5-HT-induced release of PRL. In hens actively immunized against VIP, infused dynorphin was unable to increase plasma PRL levels and infused VIP gave a muted PRL rise, while large increases in PRL were seen in nonimmunized birds receiving the same infusions. These data show that: (1) dynorphin stimulates PRL secretion by activating kappa opioid receptors in the avian hypothalamus, and (2) dynorphin, 5-HT, DA, and VIP stimulate avian PRL secretion via a common pathway expressing kappa opioid, serotonergic, dopaminergic, and VIPergic receptors at synapses arranged serially in that functional order, with the VIPergic system as the final mediator (releasing factor).

Expression of vasoactive intestinal peptide/peptide histidine isoleucine in several hypothalamic areas during the turkey reproductive cycle: relationship to prolactin secretion.

The present study examined the changes in vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) gene expression in the hypothalamus and compared their level of expression to prolactin (PRL) secretion throughout the turkey reproductive cycle. Both VIP and PHI expression were determined using in situ hybridization histochemistry and reverse transcription-polymerase chain reaction/Southern blot analysis. Plasma PRL levels were determined by radioimmunoassay. The changes in total hypothalamic VIP and PHI mRNA steady-state levels mirrored the changes seen in plasma PRL across the reproductive stages. In situ hybridization revealed that VIP mRNA was highly expressed throughout the hypothalamus and predominated within the ventromedial nucleus (VMN), inferior hypothalamus, and infundibular nuclear complex (INF). PHI mRNA was expressed in very low abundance within the same areas expressing VIP mRNA. VIP mRNA abundance within the VMN and INF was lowest in nonphotostimulated hens (VMN, 13.8 +/- 1.7; INF, 17.0 +/- 1.8 arbitrary densitometric units (ADU)), intermediate in laying hens (VMN, 29.6 +/- 3.3; INF, 35.4 +/- 4.3 ADU), and highest in incubating hens (VMN, 76.4 +/- 10.2; INF, 119.2 +/- 3.4 ADU). Levels decreased when birds shifted from incubation to photorefractoriness (VMN, 75%; INF, 82%). This relationship was not observed within other areas of the hypothalamus. The expression of PHI mRNA was also highest in the VMN and INF of incubating hens but no correlation between PHI mRNA and the other reproductive states was observed. This study provides additional evidence that VIP is the avian PRL-releasing factor, and suggests that the central site for avian PRL regulation lies within the INF of the hypothalamus.

Vasoactive intestinal peptide stimulates turkey prolactin gene expression by increasing transcription rate and enhancing mRNA stability.

This study evaluates the transcriptional and post-transcriptional regulation of prolactin (PRL) by vasoactive intestinal peptide (VIP). Pituitary nuclei from laying (control), incubating (with enhanced VIP secretion), and VIP-immunized laying turkey hens, and from pituitary cells cultured with or without VIP were used in nuclear run-on transcription assays. Cytoplasmic PRL mRNA was analyzed by slot blot hybridization. PRL transcription was greater in hyperprolactinemic incubating birds (PRL/beta-actin=3.33) than in laying birds (PRL/beta-actin=1.83). VIP-immunoneutralized birds had 47% and 51% decreases in PRL transcription and cytoplasmic PRL mRNA, respectively when compared with laying birds. In primary pituitary cell cultures, VIP significantly increased the transcription rate of PRL (3.8-fold) and cytoplasmic PRL mRNA (3.2-fold) compared with that of non-VIP-treated pituitary cells. The stability of pre-existing PRL mRNA was measured by Northern blot analysis after addition of actinomycin D. PRL mRNA half-lives were calculated using a two-component model, with a first-long component of 18.0+/-1.0 h and a second-short component of 3.7+/-0.7 h in non-VIP-treated pituitary cells. Both half-lives were significantly increased (53. 2+/-6.9 and 26.3+/-4.3 h) in VIP-treated cells. The present data show that VIP acts to stimulate PRL expression by up-regulating the transcription rate of PRL and by enhancing PRL mRNA stability.

Transcriptional changes in hypothalamic vasoactive intestinal peptide during a photo-induced reproductive cycle in the turkey.

To characterize further vasoactive intestinal peptide (VIP) as the prolactin-releasing factor in avian species, the present study examined hypothalamic VIP transcription and plasma prolactin (PRL) levels during the turkey reproductive cycle. The contribution of transcription to hypothalamic VIP mRNA steady-state levels and VIP content in response to gonadal stimulating photoperiod was also investigated. Nuclear run-on transcription assays were performed using nuclei isolated from hypothalami. Cytoplasmic VIP mRNA levels, and VIP content in the median eminence and plasma PRL levels were determined by Northern blot analysis and radioimmunoassays respectively. The alterations in VIP transcription mirrored the changes in cytoplasmic VIP mRNA and VIP content during the reproductive stages. VIP transcription, cytoplasmic VIP mRNA level and VIP content were lowest in non-photostimulated birds, higher (P<0.05) in laying hens, and greatest (P<0.05) in incubating birds. These increases paralleled the changes in circulating plasma PRL levels. Changes in VIP transcription (P>0.05) were not observed during the transition from incubation to photorefractoriness, even though there was a sharp decline in circulating plasma PRL levels (P<0.05). Following photostimulation, VIP transcription, cytoplasmic VIP mRNA levels, and VIP content increased as the hens progressed towards sexual maturity (P<0.05), and these increases were correlated with an increased plasma PRL level. These results suggest that VIP is regulated in large part at the transcriptional level during the turkey reproductive cycle and that this transcriptional regulation is coupled to the photo-induced increase in PRL secretion.

Regulation of prolactin secretion by dopamine and vasoactive intestinal peptide at the level of the pituitary in the turkey.

This study investigated the capability of dopamine (DA) to prevent avian prolactin (PRL) secretion by antagonizing the PRL-releasing factor, vasoactive intestinal peptide (VIP), at the level of the pituitary. To test this hypothesis, combined intracranial and intrapituitary infusions of DA, DA agonists, and VIP, plus electrical stimulation of the medial preoptic area (ES/POM), were used to characterize the actions of DA on PRL secretion in anesthetized laying turkey hens. Infused into the third ventricle at the rate of 10 nmol/min, DA induced a 2.8-fold increase in circulating PRL levels (63.8 +/- 15.1 to 181.3 +/- 44.3 ng/ml, p < 0. 05), similar to the 3.1-fold PRL increase induced by ES/POM (65 +/- 10.3 to 199.1 +/- 57.3 ng/ml, p < 0.05). Infused into the anterior pituitary at the rate of 15 ng/min, VIP induced a 2.2-fold increase in PRL (78.6 +/- 22.9 to 173.6 +/- 39.5 ng/ml, p < 0.05). When DA (10 nmol/min) was infused into the anterior pituitary it completely blocked both ES/POM- and VIP-induced PRL secretion. The D2 DA receptor agonist R- (-) -Propylnorapomorphine HCl inhibited VIP-induced PRL secretion at the level of the anterior pituitary, allowing only an insignificant rise in PRL (54.8 +/- 14.3 to 73.9 +/- 21.6 ng/ml, p > 0.05), while the D1 DA receptor agonist (+/-)-SKF-38393 HCl failed to prevent VIP-induced PRL release, allowing PRL to rise 2.5-fold (49.1 +/- 10.8 to 121.0 +/- 34.8 ng/ml, p < 0.05). Pituitary infusion of DA, DA agonists or vehicle alone caused no change in PRL levels. The data showed that DA prevented avian PRL secretion by blocking the action of VIP at the level of the anterior pituitary. DA effected this blockade of PRL via D2 DA receptors residing within the anterior pituitary. The data also suggested that there were no stimulatory D1 DA receptors related to PRL secretion in the avian anterior pituitary.

Vasoactive intestinal peptide secretion by turkey hypothalamic explants.

The objective of this study was to culture turkey hypothalami and examine vasoactive intestinal peptide (VIP) release during the turkey reproductive cycle. The release of VIP was studied employing a computer-guided perifusion system. Hypothalami were perifused with Krebs-Ringer bicarbonate medium for 10 or 15 h at a flow rate of 40 microliter/min, and perifusate was collected at 5-min intervals. Basal VIP secretion increased (p < 0.05) over time, and no differences in release rate were noted between reproductive stages. Basal VIP release during perifusion was episodic throughout each experimental period. Perifusion with dopamine (DA; 10 and 100 nmol/min) in incubating hens stimulated VIP release in a dose-dependent manner. There were no significant differences (p > 0.05) in VIP release in response to DA stimulation between hypothalamic fragments obtained from nonphotostimulated and incubating birds. The data suggest that 1) a VIP pulse generator appears to be located within the turkey hypothalamus, on the basis of the observed pulsatile release of VIP; 2) hypothalamic secretion of VIP is augmented by removal of inhibitory factors residing outside of the hypothalamus, or by the loss of negative feedback mechanisms that inhibit VIP release; and 3) mechanisms responsible for altering VIP release during different reproductive conditions may lie external to the hypothalamus.

Dopamine receptors influence vasoactive intestinal peptide release from turkey hypothalamic explants.

Vasoactive intestinal peptide (VIP) is a significant prolactin-releasing factor (PRF) in avian species, and dopamine (DA) exhibits both a stimulatory and inhibitory influence upon this prolactin (PRL) secretion. The stimulatory effect of DA upon PRL release appears to be mediated by VIP. This study investigated DAergic actions upon VIP release using turkey hypothalamic explants perifused with DA and its agonists or antagonists. VIP release was stimulated by DA in a dose-dependent manner (10 nmol DA/min, from 67.2 +/- 3.9 to 164.3 +/- 3.1 pg/5 min; 100 nmol DA/min, from 70.1 +/- 3.2 to 291.0 +/- 7.5 pg/5 min; 1,000 nmol DA/min, from 72.0 +/- 4.8 to 501.0 +/- 24.7 pg/5 min). The D1 DA receptor antagonist (R+)-SCH-23390 HCl completely negated the stimulatory effect of DA (100 nmol/min) upon VIP release. Perifusion with the D2 DA receptor antagonist S(-)-eticlopride HCl by itself stimulated VIP release from the hypothalamic explants, increasing VIP from 38.1 +/- 5.3 to 161.9 +/- 9.7 pg/5 min, where release stabilized until perifusion was terminated. The D1 DA agonist (+)-SKF-38393 HCl increased VIP release from 52.7 +/- 4.6 to 192.6 +/- 16.9 pg/5 min, and this stimulated release was partially inhibited by the D2 DA receptor agonist R(-)-NPA HCl (from 192.6 +/- 16.9 to 139.7 +/- 13.8 pg/5 min). These results suggest that VIP secretion is in part regulated by possible opposite actions between stimulatory D1 and inhibitory D2 DA receptors in the turkey hypothalamus.

Photoperiod mediates the ability of serotonin to release prolactin in the turkey.

Photostimulation (PS) of turkeys increases the number of hypothalamic vasoactive intestinal peptide (VIP)-immunoreactive neurons, the number of anterior pituitary VIP binding sites, and prolactin (PRL) secretion. Serotonin (5-HT) was recently shown to stimulate PRL secretion through VIP. This study tested the hypothesis that 5-HT's ability to induce PRL secretion is mediated by reproductive status and/or photoperiod in normally cycling turkey hens. Initially, saline or 5-HT was infused into the third ventricle of nest-deprived, previously incubating (ND) hens for 60 min at rates of 0.1, 1.0, or 10 nmol/min. The results led to use of the 10 nmol/min infusion rate for the remaining 5-HT infusions. Next, 5-HT was infused into short-day (SD;6), laying (6), ND (5), and photorefractory (P/R;6) hens. Plasma PRL was elevated in all groups except for the SD hens (P < 0.05). In Experiment 3, VIP was infused into the median eminence of SD (6), laying (5), and P/R (5) hens, increasing circulating PRL levels in all three groups (P < 0.05). Finally, SD hens were photostimulated for 0, 3, or 10 days and then infused with 5-HT. Only the birds which were photostimulated for 10 days exhibited elevated plasma PRL (P < 0.05). In conclusion, PS regulates PRL secretion at the hypothalamic level and more than 3 days of PS are required for 5-HT-ergic stimulation of PRL secretion.

Dopaminergic control of prolactin secretion in the turkey.

The stimulatory and inhibitory effects of dopamine (DA) upon avian prolactin (PRL) secretion suggest that, in birds, these actions are mediated by multiple DA receptors. To test this hypothesis, combined intracranial infusions of DA and selective D1 or D2 DA receptor blockers, plus electrical stimulation (ES) of the brain and vasoactive intestinal peptide (VIP) immunoneutralization, were used to characterize the actions of DA on PRL secretion in the turkey. Blockade of D1 DA receptors prevented the increase in circulating PRL observed in response to infusion of stimulatory concentrations of DA or to ES. Stimulatory infusions of DA also failed to increase circulating PRL in birds immunized against VIP. Results from infusion of the D2 DA receptor antagonist were unclear. Low concentrations had no effect, while the highest concentration (100 nmol/min) produced an increase in plasma PRL. At the high concentration the drug may be affecting PRL secretion by (1) acting nonspecifically, (2) acting as a partial DA agonist on D1 DA receptors, or (3) diffusion to the pituitary and blockade of D2 receptors there. These data suggest that avian PRL secretion is mediated by D1 DA receptors within the brain and that the stimulatory effect of DA upon PRL secretion requires an intact VIPergic system.

Active immunization against vasoactive intestinal peptide prevents photo-induced prolactin secretion in turkeys.

Photostimulation initiates and maintains the rise in circulating prolactin (PRL) observed during the reproductive cycle of the female turkey. Vasoactive intestinal peptide (VIP) is the principal PRL-releasing factor. This study tested the hypothesis that gonadal stimulatory photoperiods stimulate PRL secretion by releasing hypothalamic VIP. Therefore, an experiment was designed to determine if VIP immunoneutralization altered photo-induced PRL secretion. Reproductively quiescent female turkeys were divided into two groups comprising turkeys actively immunized with synthetic VIP conjugated to keyhole limpet hemocyanin (VIP-KLH; immunized; n = 48) or KLH alone (control; n = 48). The first immunization was administered 6 weeks before photostimulation. Blood samples were collected at frequent intervals prior to and following photostimulation, and plasma PRL concentrations were determined. Vasoactive intestinal peptide antibody titer was estimated from the percentage of 125I-labeled VIP bound to plasma diluted 1:1000. At the onset of photostimulation (Day 0), plasma PRL levels were similar for immunized and control turkeys (9.1 +/- 0.3 versus 8.9 +/- 0.3 ng/ml, respectively). Plasma PRL of control birds increased (P < 0.05) by Day 16 of photostimulation, reaching a peak value of 724.9 +/- 90.1 ng/ml on Day 84. In contrast, plasma PRL remained essentially unchanged in immunized birds. Titer of anti-VIP antibodies expressed as 125I-VIP bound by plasma in immunized birds was 10.9 +/- 1.5% on the day of photostimulation. Incubation behavior was blocked in immunized birds, whereas 75% of controls exhibited incubation behavior. The control group laid 1.83 eggs/ week/hen compared to 3.40 eggs/week/hen in immunized hens. These findings suggest that photoperiodic modulation of PRL secretion in the turkey is influenced by hypothalamic VIP neuronal system.

Vasoactive intestinal peptide stimulates prolactin mRNA expression in turkey pituitary cells: effects of dopaminergic drugs.

It is well documented that vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor and that dopamine (DA) is an inhibitory neurotransmitter in avian species. However, the roles of VIP and DA in the regulation of PRL gene expression are unclear. In this study, primary anterior pituitary cells cultured from laying turkeys were utilized to investigate the influence of VIP and dopaminergic D1 and D2 receptors on PRL secretion, PRL mRNA, and PRL synthesis. Incubation of pituitary cells with VIP increased PRL secretion up to 3.5-fold within 3 hr. Prolactin mRNA was undetectable during the first 2 hr of pituitary cell treatment; thereafter, the PRL mRNA content response to VIP increased with 24-48 h (P < 0.05). Total PRL content (media + cellular) increased over time in the presence of VIP. The response of cells incubated in the presence of a dopaminergic D1 receptor agonist (SKF38393) was variable and inconclusive. However, cells incubated with a dopaminergic D2 receptor agonist (quinpirole) inhibited VIP-induced PRL secretion (P < 0.05) and PRL mRNA levels (P < 0.05) in a dose-related fashion without effect on the basal levels of PRL release and PRL mRNA. These observations suggest that VIP, in addition to acting as a PRL-releasing peptide, also plays a role in the regulation of PRL gene expression. Moreover, the results of this study also indicate that a drug that can selectively stimulate dopamine D2 receptors can also regulate PRL secretion and PRL mRNA in turkey pituitary cells in culture.

Effect of maternal passive immunization against vasoactive intestinal peptide on prolactin secretion in turkey poults.

The purpose of this study was to examine the role of vasoactive intestinal peptide (VIP) in prolactin (PRL) release in young turkey poults. Poults obtained from hens immunized with keyhole limpet hemocyanin (KLH)-chicken VIP (cVIP-KLH) conjugate or with KLH alone were used in this study. Plasma VIP antibody was tested by means of monoiodinated cVIP. Plasma samples were prediluted (1:100) in 0.05 EDTA PBS and used as a source of primary antibody in a cVIP-binding assay. Antibody levels averaged 50.8 +/- 10.3% at hatch and then declined with age to a level of 5.0 +/- 2.1% after 3 wk. Plasma PRL concentration was lower in cVIP-KLH-immunized poults (p < 0.05) than in KLH-immunized birds. Exogenous VIP administration at 7 days of age (7.8 micrograms/kg) increased plasma PRL level (p < 0.05) to a peak value of 359 +/- 32 ng/ml in the KLH-immunized birds. A smaller increase (p < 0.05) was obtained when the KLH-immunized poults received injections at 2 and 3 wk of age. The PRL response to cVIP administration was not observed in 1- and 2-wk-old poults maternally immunized with cVIP-KLH. Similarly, electrical stimulation of the ventromedial nucleus of the hypothalamus, at 1 and 8 days of age, induced a significant increase in the plasma PRL level of maternally KLH-immunized birds but not in maternally cVIP-KLH-immunized birds. These findings suggest that cVIP is a PRL-releasing factor in young turkey poults, similar to the finding in adult turkey hens.

Protein kinase-C mediates chicken vasoactive intestinal peptide stimulated prolactin secretion and gene expression in turkey primary pituitary cells.

This set of experiments investigated the role of protein kinase-C (PKC) as a second messenger in vasoactive intestinal peptide (VIP)-stimulated prolactin (PRL) secretion and PRL mRNA abundance. Dispersed anterior pituitary cells (5 x 10(5) or 10(6) cells/tube) were isolated from laying turkeys and incubated in 1.0 ml of M-199. In Experiment 1, 10(-7) M VIP increased PRL secretion three- to fivefold. Prolactin mRNA abundance was higher in VIP-treated cells (11.45 +/- 2.11 arbitrary optical unit; AOU) than control cells (4.59 +/- 1.2 AOU). In Experiment 2, the addition of 10(-12), 10(-10), 10(-8), and 10(-6) M phorbol 12-myristate 13-acetate (PMA; PKC agonist) increased PRL release from 8.5 +/- 0.7 to 14.9 +/- 1.1, 17.2 +/- 1.3, 18.1 +/- 2.2, and 18.7 +/- 2.8 micrograms/10(6) cells, respectively. PRL mRNA abundance was significantly (P < 0.01) increased in only 10(-6) M PMA treatment. In Experiment 3, PKC desensitization decreased VIP-stimulated PRL release from 10.0 +/- 2.3 to 4.2 +/- 0.6 micrograms/5 x 10(5) cells and PMA-induced release from 7.1 +/- 1.3 to 2.7 +/- 0.3 micrograms/5 x 10(5) cells. VIP and PMA up-regulated PRL mRNA abundance was decreased two- to fourfold by PKC desensitization. In Experiment 4, 10(-6) M staurosporine (ST; PKC antagonist) decreased both 10(-7) M VIP-stimulated PRL secretion from 7.86 +/- 2.9 to 2.43 +/- 0.5 micrograms/5 x 10(5) cells and 10(-8) M PMA-stimulated PRL secretion from 4.26 +/- 0.2 to 2.23 +/- 0.3 micrograms/5 x 10(5) cells (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)