Mohamed E. El Halawani

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.

Hormonal changes associated with natural and manipulated incubation in the sex-role reversed Wilson's phalarope

Wilsons phalarope (Phalaropus tricolor) is characterized by intense female intrasexual competition and exclusive male parental care. Females occasionally are polyandrous and no territories are defended. Prolactin (Prl) and testosterone (T) were analyzed from plasma samples obtained from individuals of different reproductive stages. Males tended to have higher plasma Prl levels than females throughout the breeding season. Prolactin levels of males declined posthatch, reaching baseline levels when chicks were approximately 9 days old--the time when chicks approach thermal independence and brooding is minimal. Testosterone levels dropped at the onset of incubation and remained low through the brooding period. Eggs were manipulated such that some males incubated clutches for shortened periods, others for extended periods. Regardless of the length of incubation, Prl levels were determined primarily by environmental events, i.e., incubation, hatching, and brooding.

Three Different Turkey Luteinizing Hormone Receptor (tLH-R) Isoforms I: Characterization of Alternatively Spliced tLH-R Isoforms and Their Regulated Expression in Diverse Tissues

Abstract Using combinations of reverse transcription-polymerase chain reaction (RT-PCR) and 5′- and 3′-rapid amplification of cDNA ends, three different, alternatively spliced, partial turkey LH receptor (tLH-R) cDNA isoforms were characterized from ovarian mRNA. The first cDNA (tLH-Rintact) showed 98% and 72–75% similarity with chicken and mammalian LH-R sequences, respectively. The second cloned cDNA isoform (tLH-Rinsert) contained an in-frame TGA stop codon within an 86-base pair insertion that was located in the extracellular domain of the seven-transmembrane region. The tLH-Rinsert isoform could encode a truncated soluble protein isoform that lacked the transmembrane region. The third cDNA isoform truncated the transmembrane region (tLH-Rtrunc) and was derived by the deletion of the last exon by incomplete splicing. Generation of multiple transcripts by alternative splicing was elucidated by partial characterization of tLH-R genomic sequences. The differentially regulated expression of the tLH-R mRNA isoforms in nongonadal tissues and ovarian stromal tissues during various reproductive stages was quantified and analyzed by Northern blot and/or RT-PCR. Alternatively spliced tLH-R isoforms were differentially expressed in a tissue-specific manner in most of the tissues examined. The steady-state levels of tLH-R mRNA isoforms were relatively high in the hypothalamus and optic nerve and relatively low in the cortex, pituitary, and cerebellum when compared to levels in ovarian follicles. In nongonadal reproductive tissues, the steady-state levels of tLH-R mRNA isoforms were relatively high in the uterus and infundibulum and relatively low in the isthmus, oviduct, and magnum. In addition, in the nongonadal peripheral tissues, the steady-state levels of tLH-R isoforms were relatively high in the thyroid gland and relatively low in the spleen, adrenal gland, kidney, skin, bursa, and muscle. The present study suggests that the alternative splicing of LH-R transcripts occurs in a tissue-specific manner and has been evolutionarily conserved (similar results were obtained in chicken and swine). These results raise fundamental questions as to the function of LH-R isoforms in nongonadal tissues.

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.

Seasonal changes in prolactin and luteinizing hormone in the polyandrous spotted sandpiper, Actitis macularia

The polyandrous spotted sandpiper (Actitis macularia) is a species characterized by female dominance over males and predominant male parental care. Prolactin (Prl) and luteinizing hormone (LH) were analyzed in plasma samples obtained serially from individuals across different stages of the breeding season. The reproductive status of each sampled individual was known in detail. Similar Prl values were obtained independently by two different assays. Males tended to have higher plasma Prl levels than females throughout the breeding season. Prl was significantly elevated in both sexes by the first few days of incubation. This rapid rise in Prl may indicate its role in brood patch development and the onset of incubation behavior. In males Prl continued to rise during incubation, whereas it remained constant in females. Higher levels of Prl in males than females, especially late in incubation, reflects the greater contribution of males to incubation. LH declined markedly in males and females from prelaying to early incubation. There was a significant negative correlation between Prl and LH among males, especially from the prelaying to early incubation phases of the season. There was no such correlation among females.

Expression of D1 and D2 Dopamine Receptors in the Hypothalamus and Pituitary during the Turkey Reproductive Cycle: Colocalization with Vasoactive Intestinal Peptide

The regulation of avian prolactin (PRL) secretion and PRL gene expression is influenced by hypothalamic vasoactive intestinal peptide (VIP), the PRL-releasing factor in avian species. Recent evidence indicates that D1 and D2 dopamine (DA) receptors play a pivotal role in VIP and PRL secretion. The differential expression of DA receptors located on hypothalamic VIP neurons and anterior pituitary cells may affect the degree of prolactinemia observed during the turkey reproductive cycle. The relative expression of D1D and D2 DA receptor subtype mRNA was quantitated using in situ hybridization histochemistry (ISH). D1D and D2 DA receptor mRNA was found expressed throughout the hypothalamus and pituitary. The expression of D1D DA receptor mRNA in the hypothalamus was found to be 6.8-fold greater than that of D2 DA receptor mRNA. Higher D1D DA receptor mRNA content was found in the anterior hypothalamus (3.6-fold), the ventromedial nucleus (2.0-fold), the infundibular nuclear complex (INF; 1.9-fold), and the medial preoptic nucleus (1.5-fold) of laying hens as compared to that of reproductively quiescent non-photostimulated hens. The levels seen in incubating hyperprolactinemic hens were essentially the same as in laying hens, except for the INF where levels were 52% higher. During the photorefractory stage (hypoprolactinemia), the D1D DA receptor mRNA was at its lowest level in all areas tested. No differences were observed in hypothalamic D2 DA receptor mRNA abundance throughout the reproductive cycle, except for an increase in D2 DA receptor mRNA within the INF of photorefractory hens. Also, a marked reduction in D2 DA receptor mRNA was observed in the pituitary of incubating hens. Pituitary D1D DA receptor levels did not change when birds entered the incubating phase. Double ISH revealed that D1D and D2 DA receptor mRNAs were co-expressed within neurons expressing VIP mRNA, predominantly within the lateral hypothalamus and INF. D1D DA receptor mRNA was more highly expressed than D2 DA receptor mRNA. The present findings clearly demonstrate that the expression of stimulatory D1 DA receptor mRNA in the hypothalamus increases in hyperprolactinemic incubating hens, whereas inhibitory D2 DA receptor mRNA increases in the pituitary of hypoprolactinemic photorefractory hens.

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.

The turkey prolactin-encoding gene and its regulatory region ☆

Overlapping prolactin (Prl) lambda clones were isolated from a turkey genomic library. The 6.7-kb turkey Prl gene consists of five exons. Major transcription start points were located by primer extension 51-53 nucleotides upstream from the Met start codon. No estrogen response element (ERE) was found, but two regions similar to mammalian Pit-1/GHF-1-binding sites were identified by computer analysis. This suggests that transcription of the turkey Prl gene may be regulated by Pit-1/GHF-1, and not by the estrogen receptor.

Dopamine–melatonin neurons in the avian hypothalamus and their role as photoperiodic clocks

A timing mechanism in the brain governs reproduction in seasonally breeding temperate zone birds by triggering gonad development in response to long days in the spring. The neural mechanism(s) responsible for the timing and induction of reproductive activity by this clock are unknown. Utilizing in situ hybridization, immunocytochemistry and reverse transcriptase-polymerase chain reaction techniques, a group of dopamine (DA) neurons in the premammillary nucleus (PMM) of the caudal turkey hypothalamus that synthesize and colocalize both DA and melatonin (MEL) were identified. In addition, these neurons are found to express clock genes and the circadian photoreceptor melanopsin. DA-MEL neurons reach threshold activation (c-fos expression) when a light pulse is given during the photosensitive phase. This is associated with increases in the number of gonadotropin releasing hormone-I (GnRH-I) neurones activated, as well as an up-regulation of GnRH-I mRNA expression. The expression of tyrosine hydroxylase (TH; the rate limiting enzyme in DA biosynthesis) and tryptophan hydroxylase 1, (TPH1; the first enzyme in MEL biosynthesis) and consequently DAergic-MELergic activities are associated with the daily light-dark cycle. TPH1 mRNA expression shows low levels during the light phase and high levels during the dark phase of the light/dark illumination cycle and is 180 degrees out of phase with the rhythm of TH mRNA expression. Hypothalamic DA-MEL neurons may constitute a critical cellular process involved in the generation and expression of seasonal reproductive rhythms and suggests a previously undescribed mechanism(s) by which light signals gain access to neural targets.

Regulation of prolactin secretion by dopamine at the level of the hypothalamus in the turkey.

Avian prolactin (PRL) secretion is regulated by vasoactive intestinal peptide (VIP) neurons residing in the infundibular nuclear complex (INF) of the hypothalamus. This VIPergic activity is modulated by stimulatory dopaminergic inputs. Dynorphin, serotonin (5-HT), dopamine (DA) and VIP all appear to stimulate PRL secretion along a hypothalamic pathway, expressing ĸ opioid, serotonergic, dopaminergic and VIPergic receptors in succession, with the VIPergic system as the final mediator. Electrical stimulation (ES) within the turkey hypothalamus at the level of the medial preoptic area (POA), the ventromedial hypothalamic nucleus (VMN), the INF or the median eminence (ME) results in the release of PRL. When the selective D1 DA receptor antagonist SCH-23390 HCl was infused intraventricularly at the rate of 10 nmol/min, ES in the POA or VMN was unable to increase PRL levels, while ES in the INF and ME did increase PRL to the same level as that of controls. These results were interpreted to suggest that the D1 DA receptors involved in PRL release lie caudally to the VMN and dorsally to the INF. Bilateral microinjections (50 ng) of the D1 DA receptor agonist SKF-38393 HCl into the POA or VMN failed to produce any increase in PRL, while similar microinjections in the INF increased PRL significantly within 15 min. Bilateral microinjections of the D1 DA antagonist (50 ng) into the INF blocked the rise in PRL associated with ES in the POA. Bilateral microinjections of a D2 DA antagonist (50 ng) into the INF failed to block PRL secretion induced by ES in the POA. Tract tracing, using double-label immunocytochemistry, revealed the presence of a monosynaptic dopaminergic pathway projecting from the POA to the INF. These data imply that the only hypothalamic D1 DA receptors involved in the regulation of avian PRL secretion are those residing within the INF in the same region as the VIP neurons known to be involved in PRL secretion.