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.

Regulation of chick bone growth by leptin and catecholamines.

Leptin and the sympathetic nervous system have a unique role in linking nutritional status to skeletal metabolism in mammals. Such a regulatory mechanism has not been identified in birds but would be beneficial to signal information about energy reserves to an organ system essential for locomotion, reproduction, and survival. To explore this potential role of leptin and the sympathetic nervous system in birds, an ex vivo chick tibiotarsal model was used to test the effects of leptin and sympathetic activity on longitudinal bone growth and the expression of chondrocyte markers. Reverse transcription-PCR analysis revealed the expression of chicken leptin receptor mRNA as well as both alpha-adrenergic (alpha1A, alpha2A, alpha2B, alpha2C) and beta adrenergic (beta1, beta2) receptor subtype mRNA in the whole bone. Incubation with norepinephrine (NE; 0, 10, or 100 microM for 4 d) caused a significant increase in distal condyle length as compared with vehicle-treated, contralateral tibiotarsi. In contrast, no change in condyle length was detected after leptin treatment (0 or 10 nM or 1 microM for 4 d). Analysis of cell proliferation by bromodeoxyuridine incorporation revealed no increase in bromodeoxyuridine-positive cells in the condyles in response to leptin or NE treatments. Real-time PCR analysis showed that NE enhanced type X collagen mRNA expression, a marker of mature hypertrophic chondrocytes, with no effect on type II collagen mRNA, the matrix protein secreted by proliferating chondrocytes. Leptin treatment had no effect on the expression of either matrix protein. Treatment with agonists specific for alpha- or beta-adrenergic receptors indicates that the activation of alpha-adrenergic receptors is most likely responsible for the sympathetic effect on type X collagen gene expression. These results suggest that NE and other sympathetic agonists have positive effects on bone elongation and the changes in critical genes associated with this process. These neurotransmitters may facilitate this by promoting chondrocyte maturation. These studies represent novel evidence suggesting a role of sympathetic tone in the regulation of skeletal growth in avian species.

Photoperiodic modulation of clock gene expression in the avian premammillary nucleus.

The premammillary nucleus (PMM) has been shown to contain a daily endogenous dual-oscillation in dopamine (DA)/melatonin (MEL) as well as c-fos mRNA expression that is associated with the daily photo-inducible phase of gonad growth in turkeys. In the present study, the expression of clock genes (Bmal1, Clock, Cry1, Cry2, Per2 and Per3) in the PMM was determined under short (8 : 16 h light/dark cycle) and long (16 : 8 h light/dark cycle) photoperiods relative to changes associated with the diurnal rhythm of DA and MEL. Constant darkness (0 : 24 h light/dark cycle) was used to assess the endogenous response of clock genes. In addition, light pulses were given at zeitgeber time (ZT) 8, 14 and 20 to ascertain whether clock gene expression is modulated by light pulse stimulation and therefore has a daily phase-related response. In the PMM, the temporal clock gene expression profiles were similar under short and long photoperiods, except that Per3 gene was phase-delayed by approximately 16 h under long photoperiod. In addition, Cry1 and Per3 genes were light-induced at ZT 14, the photosensitive phase for gonad recrudescence, whereas the Clock gene was repressed. Gene expression in established circadian pacemakers, the visual suprachiasmatic nucleus (vSCN) and the pineal, was also determined. Clock genes in the pineal gland were rhythmic under both photoperiods, and were not altered after light pulses at ZT 14, which suggests that pineal clock genes may not be associated with the photosensitive phase and reproductive activities. In the vSCN, clock gene expression was phase-shifted depending on the photoperiod, with apexes at night under short day length and during the day under long day length. Furthermore, light pulses at ZT 14 induced the Per2 gene, whereas it repressed the Bmal1 gene. Taken together, the changes in clock gene expression observed within the PMM were unique compared to the pineal and vSCN, and were induced by long photoperiod and light during the daily photosensitive phase; stimuli that are also documented to promote reproductive activity. These results show that Cry1 and Per3 are involved in the photic response associated with the PMM neuronal activation and are coincident with an essential circadian mechanism (photosensitive phase) controlling the reproductive neuroendocrine system.

Serotonergic and catecholaminergic interactions with co-localised dopamine-melatonin neurones in the hypothalamus of the female turkey.

Serotonin and catecholamines (dopamine, norepinephrine, epinephrine) have important roles as neurotransmitters in avian reproduction, but their anatomical relationship to the neuroendocrine circuitry that regulates reproduction is poorly understood. Our previous studies have shown that co-localised dopamine-melatonin (DA-MEL) neurones in the avian premammillary nucleus (PMM) are active during periods of photoresponsiveness and, therefore, are potentially photosensitive neurones. Because serotonergic and catecholaminergic neurotransmitters are important regulators of reproductive function in the female turkey, we hypothesised that the serotonergic/catecholaminergic neurones within the brainstem might interact with PMM DA-MEL neurones and constitute an important circuit for reproductive function. To examine this possible interaction, the retrograde fluorescent tract tracer, 1,1'dioctadecyl-3,3,3'3'-tetramethyleindocarbocyanine perchlorate (DiI) was injected into the PMM, and combined with serotonin, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and phenyl N-methyltransferse (PNMT) immunocytochemistry to reveal neuroanatomical connections. Changes in the activities of serotonergic, dopaminergic, adrenergic and noradrenergic neuronal systems projecting to the PMM were measured at different reproductive states with in situ hybridisation (ISH) techniques, using tryptophan hydroxylase 2 (TPH2) and TH mRNA expression, respectively. Cells labelled with DiI were found in anatomically discrete areas in or near the hypothalamus and the brainstem. Double immunocytochemistry confirmed that there were serotonin, DBH and PNMT fibres in close apposition to DA-MEL neurones. TPH2 mRNA expression in serotonin neurones was found in several nuclei, and its most abundant mRNA expression was seen in the nucleus Locus ceruleus of laying and incubating hens. TH mRNA expression levels in the six catecholaminegic areas labelled with DiI was measured across the different reproductive states. In the nucleus tractus solitarius (adrenergic), the highest level of TH mRNA expression was found in photorefractory hens and the lowest level in incubating hens. These observed patterns of serotonin/catecholamine neuronal distribution and their variable interactions with PMM DA-MEL neurones during different reproductive states may offer a significant neuroanatomical basis for understanding the control of avian reproductive seasonality.

Structural characterization and chromosomal localization of the mouse cDNA and gene encoding the bone tyrosine phosphatase, mOST-PTP.

Tyrosine kinases and phosphatases are regulators of the steady-state levels of phosphotyrosine proteins and, in this way, are key players in determining the functional state of the cell. As a unique member of the protein tyrosine phosphatase (PTP) superfamily, osteotesticular PTP (OST-PTP) is a receptor protein whose expression is highly regulated during osteoblast differentiation and in response to modulators of bone remodeling such as parathyroid hormone and vitamin D3. To explore the molecular mechanisms and signaling pathways important in the regulation of this gene, we characterized the structural organization of the mouse OST-PTP cDNA and gene and determined its chromosomal localization. The mouse cDNA is approximately 5.5 kb including 5.1 kb of coding sequence, 315 bp 5' UTR and 102 bp 3' UTR. It is expressed as a single approximately 5.8 kb transcript in day 8 differentiated MC3T3 osteoblasts. Although highly homologous to the rat OST-PTP cDNA, the mouse cDNA possesses a 74 bp insert in the 5' UTR which contains several potential transcription factor binding sites such as AP-2 and NFkappaB. The mouse OST-PTP (mOST-PTP) gene is a single copy gene encompassing 35 exons and spanning only 20.65 kb. As such, it is the smallest gene of the characterized receptor PTP genes. This is due to the lack of large introns and the conserved spatial organization of exons which encode specific protein motifs in the mOST-PTP molecule. Sequence analysis of the putative mOST-PTP promoter revealed basal elements as well as many potential cis-acting regulatory elements with relevance to gene regulation in bone. Of particular interest is the single osteoblast specific element known as osteocalcin specific element 2 (OSE2) found at position -1867, as well as numerous VDRE and NFkappaB sites found throughout the promoter and the 5' UTR. Fluorescence in situ hybridization studies have shown that mOST-PTP localizes to mouse chromosome 1, region F-G which is syntenic to the segment of human chromosome 1q32-33. This characterization of the mOST-PTP cDNA and gene will facilitate future experiments exploring the mechanisms of regulation of this phosphatase during osteogenesis.

Parathyroid hormone regulates the expression of the receptor protein tyrosine phosphatase, OST-PTP, in rat osteoblast-like cells.

Bone remodeling requires regulated tyrosine phosphorylation mediated by specific protein tyrosine kinases, such as c-src and c-fms, and to date, unknown protein tyrosine phosphatases (PTPs). We previously reported the isolation of a novel bone-specific receptor PTP, named osteotesticular PTP (OST-PTP), which is regulated during osteoblast differentiation and after exposure to PTH. To determine the relevance of this PTH regulation, we characterized the PTH-induced increase in OST-PTP messenger RNA (mRNA) in UMR 106 cells in comparison with PTH effects on a related receptor PTP and a PTH regulated gene, rat collagenase. Treatment of cells with rat PTH 1-34 (rPTH) resulted in a dramatic concentration and time-dependent increase in OST-PTP mRNA with a threshold at 4 h (= or < 1nM rPTH) and maximal response of 6- 10-fold above control levels at 8 h (100 nM rPTH). An increase in collagenase mRNA was detectable 2 h earlier at 100 pM rPTH with a maximal response at least 5-fold greater than that observed for OST- PTP. Levels of mRNA for the structurally similar PTP, rat leucocyte antigen-related molecule, were unaffected by rPTH treatment. Administration of cycloheximide (5-100 microM) abolished the OST-PTP and collagenase responses to PTH. The cAMP analogs, CPT-cAMP (0.01-1mM; 8 h) or Sp-cAMP (0.1 and 0.5 mM) were equal or greater in their effectiveness to enhance both OST-PTP and collagenase mRNA as compared with rPTH. In contrast, phorbol esters, calcium ionophore, bovine PTH (3-34), or human PTHrP (7-34) had no effect on either transcript. Interestingly, 36 h of pretreatment of cells with epidermal growth factor (10 ng/ml), a growth factor known to modulate PTH's actions, resulted in a significant decrease in the abundance of OST-PTP mRNA after rPTH exposure. These studies suggest that regulation of OST-PTP mRNA is a secondary response to PTH stimulation that is dependent on protein synthesis and that may be primarily by activation of the protein kinase A pathway. This specific modulation of a bone receptor PTP may prove to be a critical component in the PTH modulation of osteoblast function.

Identification of a hormonally regulated protein tyrosine phosphatase associated with bone and testicular differentiation.

Absence of the tyrosine kinase activity of c-src and c-fms results in impairment of bone remodeling. Such dysfunction underscores the importance of tyrosine phosphorylation, yet the role of protein tyrosine phosphatases in bone metabolism remains unexamined. We have isolated the cDNA for a novel receptor-like tyrosine phosphatase expressed in bone and testis named osteotesticular protein tyrosine phosphatase (OST-PTP). The deduced 1711-residue protein possesses an extracellular domain with 10 fibronectin type III repeats and a cytoplasmic region with two catalytic domains. In primary rat osteoblasts, the 5.8-kilobase OST-PTP transcript is up-regulated in differentiating cultures and down-regulated in late stage mineralizing cultures. In addition, a presumed alternate transcript of 4.8-5.0 kilobases, which may lack PTP domains, is present in proliferating osteoblasts, but not detectable at other stages. Parathyroid hormone, a modulator of bone function, as well as cyclic AMP analogues, increase OST-PTP mRNA 5-8-fold in UMR 106 cells. In situ hybridization of adult rat testis revealed stage-specific expression of OST-PTP. OST-PTP may function in signaling pathways during bone remodeling, as well as serve a broader role in cell interactions associated with differentiation in bone and testis.

'Zip codes' direct intracellular protein tyrosine phosphatases to the correct cellular 'address'.

The transmembrane and intracellular protein tyrosine phosphatases (PTPs) play an essential role as signal transduction proteins involved in various cellular processes including division, proliferation and differentiation. As such, their activity must be strictly regulated to avoid nonspecific tyrosine dephosphorylation of cellular proteins. The intracellular PTPs possess a diversity of protein sequences outside the catalytic domain that appear to serve as 'zip codes' specifically 'addressing' these proteins to defined subcellular compartments. These localization strategies are proposed to function as a regulatory mechanism, defining the substrate specificity and function of the intracellular PTPs.

Effects of reproductive status, ovariectomy, and photoperiod on vasoactive intestinal peptide in the female turkey hypothalamus.

Vasoactive intestinal peptide (VIP) appears to be a physiologically relevant prolactin (PRL)-releasing factor during the avian reproductive cycle, yet little is known of the factors involved in modulating the hypothalamic concentrations of this neuropeptide. A heterologous chicken VIP radioimmunoassay was developed to examine the effects of reproductive status, ovariectomy, and photoperiod on hypothalamic VIP immunoreactivity in the female turkey. VIP concentrations were highest in the median eminence/infundibular nuclear complex (ME/INF) relative to other subregions of the hypothalamus and changed only in this region during the reproductive cycle. Quiescent, nonphotostimulated hens subjected to stimulatory photoperiod exhibited a 1.6-fold increase in VIP in the ME/INF (quiescent 59.9 +/- 6.0 vs photostimulated 95.8 +/- 7.1 pg/microgram protein). An additional 2-fold increase in ME/INF VIP concentrations was observed in laying hens (183.0 +/- 28.5 pg/microgram protein). Coincident increases in plasma PRL were also observed. In contrast, during incubation and the photorefractory stage, a dissociation between hypothalamic VIP and plasma PRL occurred. No changes were observed in VIP in incubating hens, yet a 6-fold increase in PRL was noted, compared to layers. In addition, ME/INF VIP concentrations exhibited no change during the photorefractory stage, whereas a 28-fold decrease in plasma PRL occurred. VIP concentrations in the ME/INF of laying hens were unaffected by ovariectomy, whereas exposure to short photoperiod reduced VIP by 44%. The inhibitory effects of short photoperiod could not be reversed by administration of exogenous steroids, while steroid treatment reduced VIP concentrations by 45% in the ovariectomized hens. These results provide additional correlative evidence for a modulatory role of VIP in PRL secretion and suggest that the expression of this neuropeptide in the INF may serve as a neural link between photoperiodic mechanisms and PRL release during the avian reproductive cycle.

Enhanced vasoactive intestinal peptide-induced prolactin secretion from anterior pituitary cells of incubating turkeys (Meleagris gallopavo).

During incubation, female turkeys exhibit elevated circulating prolactin (PRL) which may be the result of enhanced pituitary responsiveness to vasoactive intestinal peptide (VIP). This hypothesis was tested by comparison of spontaneous and porcine VIP-induced PRL secretion from anterior pituitary cells of hens in various reproductive conditions. The effect of VIP and luteinizing hormone releasing hormone (LHRH), alone and in combination, on luteinizing hormone (LH) secretion was also examined. Incubation with pVIP (10(-10) to 10(-6) M) significantly stimulated PRL secretion at all incubation times tested (1-5 hr). This increase was greatest in cells from incubating hens, with those from laying, photorefractory, and quiescent (nonphotostimulated) hens secreting successively less PRL. These responses were obtained when spontaneous PRL secretions were compared. VIP induced approximately a similar 1.5-fold increase in LH secretion, in all reproductive groups. Also, VIP enhanced LHRH-induced LH secretion (1.2- to 1.6-fold; P less than 0.0001). It is concluded that PRL secretion in vitro by pituitary cells from turkey hens in various reproductive stages reflects the circulating levels of PRL at these stages.

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.

Alterations in hypothalamic vasoactive intestinal peptide-like immunoreactivity are associated with reproduction and prolactin release in the female turkey.

Vasoactive intestinal peptide (VIP) has potent PRL-releasing activity, but its physiological role in the regulation of PRL release during the avian reproductive cycle is not known. We used indirect immunofluorescence to determine if changes in hypothalamic VIP are associated with the shifts in circulating PRL during the reproductive cycle of the domestic turkey. In the naturally hyperprolactinemic incubating hen, the majority of VIP immunoreactivity (VIP-IR) existed within neurons of the infundibular nuclear complex (INF) and fibers in the external layer of the median eminence. Within the INF, the numbers of VIP-IR cells increased during the cycle, paralleling increases in serum PRL. In the reproductively inactive, nonphotostimulated hen with low serum PRL, essentially no positive cells were noted, whereas the incubating hen exhibited 32.1 +/- 2.2 cells/pair of adjacent sections in the anterior INF and 59.6 +/- 2.0 cells in the posterior INF. Exposure of inactive hens to a stimulatory photoperiod resulted in a 2.6-fold increase in serum PRL with the appearance of VIP-IR cells in the INF. During laying and incubation, further increases were observed in the number of positive cells in the INF and serum PRL as well as a greater fiber density in the median eminence. To further examine the association between changes in VIP-IR and serum PRL, circulating PRL was artificially lowered by depriving incubating hens of their nests for 0, 2, 5, and 10 days. On day 2 of nest deprivation, serum PRL declined markedly to 12% of day 0 levels, with VIP-IR cell numbers at 64% and 46% in the anterior and posterior INF, respectively. By day 10, birds exhibited cell numbers in the INF averaging 20% of those observed in the day 0 incubating hens, with serum PRL at 6% of day 0 levels. The results of these studies indicate a possible causal relationship between hypothalamic VIP and changes in PRL secretion during the avian reproductive cycle, providing a basis for further research on the importance of this peptide as well as factors responsible for the modulation of its expression in hypothalamic INF neurons.