M. E. El Halawani

Vasoactive intestinal peptide is a hypothalamic prolactin-releasing neuropeptide in the turkey (Meleagris gallopavo).

The hypothesis that vasoactive intestinal peptide (VIP) functions as a hypothalamic prolactin (PRL)-releasing peptide in the turkey was tested by determining the effects of hypothalamic VIP immunoneutralization and pituitary VIP receptor blockade on hypothalamic extract (HE)-induced PRL secretion from dispersed anterior pituitaries. Incubation of cells with porcine VIP (pVIP; 0.5 or 10 nM) significantly stimulated PRL secretion. This effect was inhibited in a dose-related manner by 1-hr preincubation of pVIP with a VIP antisera (A/S; 1:500-1:50,000). Likewise, HE (0.3 equivalent)-stimulated PRL secretion was inhibited by preincubation with VIP A/S (P less than 0.0001). A 96-98% reduction in PRL secretion was obtained from cells cultured with HE, that was previously incubated with 1/500 dilution of antiserum. Pretreatment of pituitary cells for 15 min with [4Cl-D-Phe6,Leu17] VIP, a VIP receptor antagonist (10(-5) M), significantly depressed the PRL response to 0.5 nM VIP (9.9 +/- 0.5 micrograms/500,000 cells vs 4.9 +/- 0.1 micrograms/500,000 cells; 22.4 +/- 0.9 micrograms/500,000 cells vs 14.7 +/- 0.4 micrograms/500,000 cells) or 0.3 eq HE (8.8 +/- 0.6 micrograms/500,000 cells vs 5.2 +/- 0.2 micrograms/500,000 cells; 15.3 +/- 0.3 micrograms/500,000 cells vs 8.2 +/- 0.2 micrograms/500,000 cells). These results suggest that hypothalamic stimulation of PRL secretion appears to be mediated by receptors specific for VIP and that VIP is an endogenous hypothalamic PRL-releasing peptide in the turkey.

Melanopsin expression in dopamine-melatonin neurons of the premammillary nucleus of the hypothalamus and seasonal reproduction in birds

Melanopsin (OPN4) is a photoreceptive molecule regulating circadian systems in mammals. Previous studies from our laboratory have shown that co-localized dopamine-melatonin (DA-MEL) neurons in the hypothalamic premammillary nucleus (PMM) are putatively photosensitive and exhibit circadian rhythms in DAergic and MELergic activities. This study investigates turkey OPN4x (tOPN4x) mRNA distribution in the hypothalamus and brainstem, and characterizes its expression in PMM DA-MEL neurons, using in situ hybridization (ISH), immunocytochemistry (ICC), double-label ISH/ICC, and real time-PCR. The mRNA encoding tOPN4x was found in anatomically discrete areas in or near the hypothalamus and the brainstem, including nucleus preopticus medialis (POM), nucleus septalis lateralis (SL), PMM and the pineal gland. Double ICC, using tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis)-and OPN4x antibodies, confirmed the existence of OPN4x protein in DA-MEL neurons. Also, tOPN4x mRNA expression was verified with double ISH/ICC using tOPN4x mRNA and TH immunoreactivity. PMM and pineal gland tOPN4x mRNA expression levels were diurnally high during the night and low during the day. A light pulse provided to short day photosensitive hens during the photosensitive phase at night significantly down-regulated tOPN4x expression. The expression level of tOPN4x mRNA in PMM DA-MEL neurons of photorefractory hens was significantly lower as compared with that of short or long day photosensitive hens. The results implicate tOPN4x in hypothalamic PMM DA-MEL neurons as an important component of the photoreceptive system regulating reproductive activity in temperate zone birds.

DOPAMINE-MELATONIN NEURONS IN THE AVIAN HYPOTHALAMUS CONTROLLING SEASONAL REPRODUCTION

Day length cues are used by temperate zone birds to time seasonal changes in reproductive physiology and behavior. However, the neuronal and neurochemical circuits used to measure day length (photoperiodic time measurement; PTM), transduce light information and activate the reproductive neuroendocrine system have not been definitely established. Recent findings from our laboratory provide data showing dopamine (DA) neurons within the premammillary nucleus (PMM) of the caudal turkey hypothalamus are putative photoreceptive neurons. These neurons reach threshold activation when a brief pulse of light is provided during the photo-inducible phase for photosexual stimulation. To further clarify the role of PMM neurons in coding daylight information, we showed that by using double-label immunocytochemistry (ICC) these neurons are immunoreactive (ir) to both tyrosine hydroxylase (TH; the rate limiting enzyme in DA biosynthesis) and melatonin (MEL). Moreover, we found these neurons to express tryptophan hydroxylase 1 (TPH1; the first enzyme in MEL biosynthesis) and 5-HT N-acetyltransferase (AANAT; a key regulatory enzyme in MEL synthesis) mRNAs but not neuronal tryptophan hydroxylase 2 mRNA (TPH 2; the rate limiting enzyme in 5-HT pathway). Both TH and TPH1 mRNAs were shown to cycle rhythmically, and with opposite phases, in PMM neurons of birds kept under a diurnal illumination cycle (12-h light/dark; LD). These neurons could also generate 24 h TH and TPH1 mRNA expression rhythms with the same phase relationship in constant light (LL) and constant dark (DD). In addition, the expression patterns and amplitudes of TH and TPH1 mRNAs were different between long and short photoperiods. These findings may form the basis for an endogenous dual-oscillator circadian system within PMM DA-MEL co-localized neurons controlling reproductive seasonality in birds.

Effects of estrogen and progesterone on serum prolactin and luteinizing hormone levels in ovariectomized turkeys (Meleagris gallopavo)

Serum prolactin (PRL) and luteinizing hormone (LH) levels from mature female turkeys were determined following ovariectomy and daily subcutaneous injections for 4 or 9 days of 0.002-2.0 mg/kg estradiol benzoate (EB), 0.05-2.0 mg/kg progesterone (P), or their combinations. Serum PRL levels were increased (P less than 0.05) at the onset of sexual maturity in intact controls, but not in ovariectomized turkeys. Injection of 0.02 mg/kg EB into ovariectomized turkeys resulted in elevated (P less than 0.05) serum PRL levels after 8 treatment days. Higher doses failed to elicit a response of greater magnitude. EB at doses of 0.02-2.0 mg/kg reduced LH levels while 0.002 mg/kg EB increased LH levels above (P less than 0.05) those of ovariectomized controls. The 1.0-mg/kg P dose increased (P less than 0.05) serum PRL within 24-48 hr of administration while the 0.05-, 0.5-, or 2.0-mg/kg P doses failed to elicit a response. When P was injected in doses of 0.5-2.0 mg/kg, LH levels were decreased in a dose-response manner. The injection of EB combined with P had a variable effect on serum PRL levels. The 0.5- or 1.0-mg/kg dose of P facilitated (advanced in time) the rise in serum PRL level induced by 0.02 mg/kg of EB. In contrast, the positive feedback effect of 0.2 mg/kg of EB was blocked when administered with 0.5 mg/kg of P. Serum LH levels were dramatically decreased by all steroid combinations used. These results indicate that daily injections of EB and/or P in ovariectomized turkeys have a variable effect on serum PRL and LH levels depending upon the dose, the duration of treatment, or the ratio between EB and P.

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

Abstract 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 within 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 (quin-pirole) 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. [P.S.E.B.M. 1996, Vol 212]

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.

Plasma LH and prolactin levels during the reproductive cycle of the Cockatiel (Nymphicus hollandicus)

Plasma-luteinizing hormone (LH) and prolactin (Prl) levels were determined using radioimmunoassay during two reproductive cycles in captive cockatiels (Nymphicus hollandicus)--an altricial species in which both parents share incubation and care of young. Birds were stimulated to breed by increasing daylength, light intensity, ambient temperature, and presenting nest boxes. LH levels were elevated during the time of nest inspection in females and peaked during egg laying. In contrast, LH levels were highest in males during nest inspection but were lower during egg laying. In both sexes, LH continued to decline during incubation and care of the young but rose in pairs laying a second clutch. Female and male Prl levels increased during egg laying, peaked during incubation, then declined to egg-laying levels during the nestling stage. Prl continued to decline during the fledgling stage and reached prelaying levels unless a second clutch was begun. In conclusion, in cockatiels, nest inspection and laying are characterized by high LH levels while high Prl levels occur during incubation and feeding of nestlings in both males and females.

SERUM PROLACTIN LEVELS OF TURKEY HENS IN RELATION TO REPRODUCTIVE FUNCTION

Publisher Summary This chapter discusses the serum prolactin levels of turkey hens in relation to reproductive function. Serum prolactin levels of somatically mature, sexually immature photosensitive female turkeys, housed in a 6L: 18D photoperiod are low. In different experiments with birds of varying history mean levels of prolactin have varied from less than 5 ng/ml to as high as 20 ng/ml. The data were obtained from 8 large white strain turkeys. The hens had been held on a 6L:18D photoperiod and were changed to 15L: 9D on the first day of the experiment. Blood was drawn from the birds at 1300–1400 h each day. It appears from the data that the drop in feed consumption follows the behavioral manifestation of broodiness. The time course of changes in feed consumption and serum prolactin suggest that the drop in feed consumption is not a casual factor in increasing the serum prolactin. The composite data suggest that the prolactin levels increase before the increase in nesting. Most birds sampled follow this general pattern, however, one did not. This individual bird showed the behavioral manifestations of broodiness, the concomitant drop in feed consumption, and cessation of egg production but her serum prolactin level did not increase until after the onset of broodiness.

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.

Involvement of catecholaminergic mechanisms in the photoperiodically induced rise in serum luteinizing hormone of Japanese quail (Coturnix coturnix japonica)

Abstract The involvement of brain catecholamines (CAs) in the neuroendocrine mechanisms that control the release of LH has been studied in somatically mature quail with regressed and with recrudescing testes and in sexually mature quail. In quail with recrudescing testes, serum LH was markedly increased 3 days after the onset of photostimulation. Administration of an inhibitor of CA synthesis, α-methyltyrosine (MT), started on the day of photostimulation and continued for 3 days thereafter, decreased brain dopamine (DA), norepinephrine (NE), and epinephrine (E) content and blocked the photoperiodically induced rise in serum LH. Treatment with bis(4-methyl-1-homopiperazinil-thiocarbonil) disulfide (FLA63), an inhibitor of DA-β-hydroxylase, caused a marked reduction of brain NE and E levels and a significant increase in brain DA content, and like MT, blocked the photoperiodically induced rise in serum LH. In sexually mature quail, serum LH and testicular regression were effected differently by MT and FLA63. MT partially reduced LH levels, but FLA63 suppressed them completely (i.e., to those of nonphotostimulated birds). Testicular weights were reduced by both MT and FLA63, but as with LH, the FLA63 was more effectual than MT. Treatment with l -dihydroxyphenylalanine, a direct precursor of DA, augmented DA content in the brain, caused a reduction of LH levels, and induced gonadal regression. The results are consistent with the hypothesis that NE-containing neurons facilitate central mechanisms which release LH in response to neural inputs involved in the photoperiodic stimulus, and that activation of DA-containing neurons is capable of inhibiting this release and inducing testicular regression.