Initiating egg production in turkey breeder hens: thyroid hormone involvement.

The role of thyroid hormones in the expression of photosensitivity-photorefractoriness in female turkeys was investigated through the use of an antithyroidal agent, 6-n-propyl-2-thiouracil (PTU). In experiment 1, females held continuously from hatch on long day lengths (16L:8D; LD) and fed 0.1% PTU from 0 to 16 wk, began laying eggs at 26 wk of age, peaking at 75% hen-day egg production by 29 wk, whereas controls initiated lay 3 wk earlier but only achieved less than 50% hen-day egg production. In experiment 2, PTU treatment from 10 to 18 wk severely suppressed plasma triiodothyronine and thyroxine, as confirmed by RIA. Egg production of PTU and control hens held on LD from hatch began by 23 wk, with PTU hens reaching a substantially greater rate of lay than controls. Eggs were smaller initially in both treatments but exceeded 75 g by 28 wk. In experiment 3, recycled hens on short day lengths (8L:16D) received PTU for 2 wk before LD and 12 wk thereafter; a subset of these hens was killed after 48 h of LD for immunohistochemical analysis of fos-related antigen (FRA) expression in the tuberal hypothalamus as a marker of photoinduced neuronal activity. The PTU treatment completely forestalled egg production until its withdrawal; egg production then rose sharply to control levels before resuming, along with controls, a typical seasonal decline. The PTU treatment did not impair photoinduced FRA expression. Together, these results demonstrate the following: 1) that a period of pharmacological suppression of triiodothyronine and thyroxine can substitute for short day exposure in conferring photosensitivity on juvenile-aged turkeys (and is actually superior to short day exposure), 2) that reproductive development does not limit egg production of turkey hens photostimulated as young as approximately 20 wk of age, and 3) that effects of thyroid suppression on photostimulation lie downstream of photoinduced FRA expression. Taken together, these results suggest that there is ample physiological potential to substantially advance the age of photoinduced egg production in commercial flocks.

Photostimulated expression of type 2 iodothyronine deiodinase mRNA is greatly attenuated in the rostral tuberal hypothalamus of the photorefractory turkey hen.

For many temperate-zone avian species, termination of breeding occurs when individuals no longer respond to previously stimulatory day lengths, a condition called photorefractoriness. Long day lengths induce significantly greater expression of c-fos and fos-related antigens (FRAs) in the tuberal hypothalamus of the photosensitive hen than that of the photorefractory hen. The tuber is also a site of photoinducible glial expression of type 2 iodothyronine deiodinase (Dio2), which converts thyroxine into its active form, triiodothyronine (T3). T3 induces withdrawal of glial processes from gonadotrophin-releasing hormone (GnRH) I nerve terminals, which is believed to permit the efficient release of GnRH I into the associated portal vasculature. Using a riboprobe, we tested whether long days induce Dio2 mRNA expression in the turkey tuber and, if so, whether this expression is reduced in photorefractory hens. Long days significantly induced rostral and caudal tuberal hypothalamic Dio2 expression in photosensitive hens. Photorefractory hens had reduced expression of Dio2 with most subjects expressing no detectable mRNA in the rostral tuber and variably attenuated amounts throughout the medial and caudal tuber. We also performed double-label immunohistochemistry to identify co-localisation between FRAs and glial fibrillary acidic protein, a glial marker. FRAs were present in the nuclei of a few astrocytes in the median eminence and infundibular nucleus of the tuber. The temporal and spatial coincidence between FRA and Dio2 expression, their mutual association with glia, and the attenuation of their response during photorefractoriness suggests that the two events are linked and that photorefractoriness involves a reduced capacity for photo-inducible gene expression within glia of the tuberal hypothalamus.

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.

Photostimulated fos-like immunoreactivity in tuberal hypothalamus of photosensitive vs. photorefractory turkey hens.

Photorefractoriness in commercial turkey hens can be viewed as a failure of previously sexually stimulatory photoperiods to maintain egg production via activation of cGnRH I neurons, but the neural locus of photorefractoriness, i.e., where in the brain failure occurs, is not known. We used a c-fos antiserum that detects c-Fos and Fos-related antigens to characterize Fos-like immunoreactivity (FLI) as a measure of neuronal activation. FLI was measured in somatically mature, photosensitive hens (held on short photoperiods [8L:16D] for at least 10 weeks) before (non-photostimulated-photosensitive group) and after 48 h of exposure to long photoperiods (16L:8D; photostimulated-photosensitive group). We also measured FLI in hens that had become photorefractory, transferred to short photoperiods for 1 week--an insufficient time period to reverse photorefractoriness--and then exposed to long photoperiods for 48 h (photostimulated-photorefractory group). FLI was nearly absent in the tuberal hypothalamus of non-photostimulated-photosensitive hens but FLI was abundant in photostimulated-photosensitive hens. FLI was greatly reduced (P<0.01) in the rostral tuberal hypothalamus of photostimulated-photorefractory hens. All hens showed variable extra-tuberal FLI in locations associated with stress, e.g., paraventricular nucleus, lateral septal area, and nucleus taenia. Double-label fluorescence immunohistochemistry with c-fos antiserum and anti-Neu-N, a neuron-specific protein, showed that a substantial fraction of tuberal FLI-positive cells in photostimulated-photosensitive hens were neuronal. These results implicate neurons in the rostral tuberal hypothalamus as a potential neural locus of photorefractoriness, as FLI in this region appears coupled with cGnRH I activation in photostimulated-photosensitive but not photostimulated-photorefractory turkey hens.

Apposition of enkephalinergic axons with cGnRH I-containing perikarya in turkey hen brain.

Several lines of evidence support a role of endogenous opioids in the brain's regulation of gonadotropin secretion in birds and mammals, although the neuroanatomic basis of such regulation is not clear. We used double-label fluorescence immunohistochemistry employing polyclonal antisera raised in sheep against chicken gonadotropin-release hormone I (cGnRH I) and in rabbits against met-enkephalin to determine whether the potential exists for synaptic contact between neurons containing these neuropeptides in the preoptic/septal region of domestic turkey breeder hens. The cGnRH I antiserum was highly specific for cGnRH I, while the met-enkephalin antiserum showed some cross-reactivity with leu-enkephalin. We found numerous instances in which enkephalinergic neuronal elements appeared to contact cGnRH I perikarya and axons in and around the diagonal band of Broca, the bed nucleus of the pallial commissure and in the lateral septal nucleus. These appositions, confirmed by confocal scanning microscopy, appear to provide a neuroanatomic basis for how enkephalinergic innervation might influence gonadotropin secretion in turkey hens by directly regulating activity of cGnRH I neurons.

Posthatch oral estrogen exposure impairs adult reproductive performance of zebra finch in a sex-specific manner.

We determined whether short-term, posthatch oral exposure to estradiol benzoate (EB) or the industrial surfactant octylphenol (OP) could impair the reproductive performance of zebra finches. If so, naturally occurring phytoestrogens and xenoestrogens might influence reproduction in wild populations. Chicks were given oral administration of 10 or 100 nmol EB per gram of body mass (earlier work showed the latter to be the minimum oral dose required to maximally masculinize female song nuclei) or an equimolar amount of OP daily from 5 through 11 days of age. Canola oil was used as a vehicle and control. Reproductive testing was done either in individual pair cages or in communal cages that permitted self-selection of mates, N = 10 pairs per group. Pairs consisted of EB-treated males and females, EB-treated males paired with canola-treated females, vice versa, and canola-treated males and females. Posthatch EB treatment produced sex-specific impairments in reproduction that, in some instances, were additive when both sexes were treated. Egg production was reduced and egg breakage was increased in 100 nmol/g EB-treated male and female pairs. The incidence of missing eggs was increased in 10 nmol/g EB-treated male and female pairs. Candled fertility was reduced in both groups containing 100 nmol/g EB-treated males. The number of hatched chicks was severely reduced in all EB-treated groups. No adverse effects of OP treatment were detected. These significant treatment effects (all P < 0.05) show that posthatch EB treatment profoundly disrupts the reproductive performance of zebra finches, suggesting that exposure to estrogens in the wild could impair the reproductive performance of wild populations.

Regulation of brain-derived neurotrophic factor messenger RNA levels in avian hypothalamic slice cultures.

Mechanisms regulating the expression of brain-derived neurotrophic factor, a member of the neurotrophin family, have been extensively studied in the rat cerebral cortex, hippocampus and cerebellum. In contrast, little is known regarding the regulation of this growth factor in the hypothalamus. Here we present an analysis of the regulation of brain-derived neurotrophic factor messenger RNA levels in chick embryo hypothalamic slice cultures following exposure to potassium chloride, glutamate agonists and sex steroids. Following a week in chemically-defined media the tissue was depolarized by exposure to 50 mM potassium chloride for 6h, resulting in a significant 4.2-fold increase in the level of brain-derived neurotrophic factor messenger RNA. This result is consistent with studies of other brain regions. Similar 6-h acute exposures of the hypothalamic cultures to 25 microM N-methyl-D-aspartic acid, 25 microM kainic acid and 25 microM alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid also significantly increased messenger RNA levels 2.5-, 2.1- and 1.4-fold, respectively. It was previously reported that brain-derived neurotrophic factor levels within the rat cerebral cortex, olfactory bulb and hippocampus are altered by exposure to 17beta-estradiol. Here we show that in hypothalamic slice cultures neither acute nor chronic treatments with 10 and 100 nM 17beta-estradiol and 10nM testosterone significantly altered the steady-state level of this growth factor. These findings show that neuronal activity, induced by glutamate agonists and potassium chloride, can regulate brain-derived neurotrophic factor messenger RNA levels within embryonic hypothalamic slice cultures. This regulation could play a critical role in the modulation of programmed cell death and synaptic maturation during development of the hypothalamus.

Corticotropin-releasing hormone-immunopositive nerve elements in apposition to chicken gonadotropin-releasing hormone I-containing perikarya in Japanese quail (Coturnix coturnix japonica) brain.

The neuroanatomic basis of how stress inhibits reproduction in birds is not understood. To address this question we used double-label immunofluorescence histochemistry to determine whether corticotropin-releasing hormone (CRH)-immunoreactive (ir) neuronal elements contact chicken gonadotropin-releasing hormone I (cGnRH I)-ir somata in brains of Japanese quail. The double-label system used a sheep anti-cGnRH I primary antibody with a secondary antibody conjugated to dichlorotriazinylaminofluorescein dihydrochloride for green fluorescence and a rabbit anti-CRH antibody with a secondary conjugated to Texas Red for red fluorescence. Immunohistochemical (IHC) distribution of both peptides resembled that in previous reports using single-label IHC. In four areas of the quail brain in which CRH nerve fibers and cGnRH I somata co-occurred (bed nucleus commissural pallii, nucleus preopticus medialis, nucleus septalis lateralis and nucleus accumbens), numerous instances were found of CRH-ir nerve fibers or terminals in apposition to cGnRH I cell bodies. These interactions provide a potential neuroanatomic route by which CRH may directly inhibit the activity of cGnRH-I-containing neurons, thereby inhibiting gonadotropin output and halting or slowing the progression of reproductive cycles. It remains to be demonstrated by electron microscopy whether these interactions, which appear abundant by IHC, represent instances of synaptic contact, as has been demonstrated to occur in analogous areas in mammalian species.

Reproductive management of captive parrots.

Studies of the behavior of Amazon parrots throughout a reproductive trial indicate that activities such as food gathering, which may occupy large fractions of the activity budget of wild parrots, occupy little time in captivity. This may be one factor contributing to the large percentage of time during which Amazon parrots are generally inactive in typical captive conditions. The extent of inactivity in captive Amazons creates an open time niche wherein enrichment devices might play a role in improving their well being. Studies of the reproductive endocrinology and the behavior of parrots suggest that hand rearing may impair adult fertility and nest box use. Hand rearing may also cause adult Cockatiels to lay eggs on cage floors rather than in nest boxes. However, the use of nest boxes with oversized nest entrances can be very effective in alleviating chronic floor laying in Cockatiels. Another egg-laying problem in Cockatiels, unwanted egg laying, can be prevented by the use of long-acting formulations of the superactive GnRH agonist, leuprolide acetate, which presumably [figure: see text] acts in birds, as in mammals, by down-regulating pituitary GnRH receptors. Manipulations to limit the increases in prolactin normally seen during incubation in poultry can significantly increase egg production. As clutch size in Cockatiels may also be limited by rising prolactin levels, such manipulations may be effective in stimulating egg production in parrots. An alternative approach for increasing flock egg production is to place foster eggs in nests of Cockatiel pairs that are slow to lay. This technique stimulates males to increase their nest-oriented behavior and, subsequently, may stimulate egg laying in some females that might not otherwise have laid eggs. The parental phases of reproduction in Amazon parrots are often a time of heightened aggressiveness towards humans, but low levels of serum testosterone in males during that time suggest that this particular interspecies aggressiveness may not be dependent on elevated testosterone levels. Occasional human handling during the nestling stage may produce a degree of tameness comparable with hand-reared chicks, yet not impair adult reproductive performance. Such handling may also alter the immune status of captive parrots, and possibly reduce the serum corticosterone response to handling. If so, occasional human handling during the nestling stage could improve the adaptation of parrots to captivity.

Multiple forms of GnRH are released from perifused medial basal hypothalamic/preoptic area (MBH/POA) explants in birds.

Both chicken gonadotropin-releasing hormones I and II (cGnRH I and II) were detected in abundant quantity by radioimmunoassay of extracts of Japanese quail medial basal hypothalamic/preoptic area (MBH/POA) fragments that included the median eminence (ME) region. However, in radioimmunoassayed extracts of Japanese quail ME alone, the concentration of cGnRH I greatly exceeded that of cGnRH II (approximately 450 pg/ME vs < 10 pg/ME). Likewise, cGnRH I and II were released into perifusates from quail an turkey MBH/POA explants maintained in short-term perifusion. Release of both forms occurred whether or not explants included the ME region, i.e., from quail POA explants did not include the ME or from turkey MBH/POA explants from which the ME region had been dissected out. This indicates that neuropeptides released from areas other than the ME can be a major source of neuropeptides detected in perifusates. Further, release of cGnRH I was altered following the addition of norepinephrine to perfusion media, whereas cGnRH II release was unaffected, again, whether or not explants included the ME. These results demonstrate that the release of neurohemoral substances from perifused explants cannot be assumed to represent regulated secretion from the ME.

Cellular localization of neuropeptide Y mRNA and peptide in the brain of the Japanese quail and domestic chicken.

Neuropeptide Y (NPY) has been implicated in the control of a number of physiological functions in birds including food intake and reproduction. In the present study, sites of NPY synthesis were localized in the brains of Japanese quail and domestic chickens by in situ hybridization histochemistry using a digoxigenin-labelled riboprobe. NPY mRNA was detected in three main cell groups in both species. The most prominent group was associated with structures in the lateral thalamus including the anterior lateral thalamic nucleus, lateral forebrain bundle, rotund nucleus, pretectal nucleus and occipitomesencephalic tract. Other major cell groups were detected in the hippocampus, and in the caudal linear nucleus and raphe nucleus of the brainstem. NPY mRNA was also present in the piriform cortex and taenial nucleus. Double-labelling of NPY mRNA and peptide was demonstrated in individual cells of the hippocampal, thalamic and brainstem cell groups, suggesting that NPY is synthesized and stored in these areas. However, the identity of other cell groups, notably in the hyperstriatal, archistriatal and neostriatal regions of the telencephalon, which exhibit NPY-immunoreactive cell bodies but no NPY mRNA, remains to be determined.

Egg production of cockatiels (Nymphicus hollandicus) is influenced by number of eggs in nest after incubation begins.

Cockatiel eggs hatch asynchronously because most eggs are laid after continuous incubation begins. We manipulated numbers of eggs in the nest after cockatiels began incubating to determine whether the number of eggs in the nest influenced clutch size. We also measured plasma prolactin 2, 7, 12, and 17 days after egg laying began to determine whether clutch size covaried with prolactin concentration. We found that total egg production was stimulated in pairs for which the rate of egg accrual was reduced by egg removal, while total egg production was inhibited in pairs for which the egg accrual rate was increased by egg addition. Female, but not male, plasma prolactin concentrations correlated negatively with egg production on day of lay 17 but not before. We conclude that egg production in incubating cockatiels is influenced by feedback from the number of eggs in the nest and that clutch termination may be signaled by rising prolactin levels in females.

Distribution of chicken-II gonadotropin-releasing hormone in mammalian brain.

Brains of nonmammalian vertebrates typically contain multiple forms of gonadotropin-releasing hormone (GnRH). Until recently, only the mammalian form of GnRH (mGnRH) had been isolated in placental mammals. Biochemical and histological data show that both mGnRH and chicken-II GnRH (cGnRH-II) are present in a primitive placental mammal, the musk shrew (Suncus murinus). Similar to the case in nonmammalian species, in the musk shrew, neurons that express cGnRH-II are located in a discrete cluster in the midbrain. We have used a combination of radioimmunoassay and immunocytochemistry, analyzed at the light level and with electron microscopy, to describe the distribution of cGnRH-II cell bodies and fibers in the musk shrew brain. All cGnRH-II-immunoreactive (ir) neurons reside in the midbrain, and this area contains the greatest concentration of cGnRH-II peptide in the brain. At the light and electron micrographic levels, we have identified synaptic terminals containing dense core vesicles that are immunoreactive for cGnRH-II in the medial habenula. Radioimmunoassay reveals that this region contains the second greatest concentration of cGnRH-II in the brain. Widely scattered cGnRH-II-ir fibers are present throughout the forebrain, particularly in the medial septum, hypothalamus, and midbrain central gray. Scant cGnRH-II fibers are present in the median eminence, arcuate nucleus, and infundibular stem, and only low concentrations of the peptide are detected in these areas. Finally, intravenous administration of mGnRH is ten times more effective than cGnRH-II in promoting ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentration of chicken gonadotropin-releasing hormones I and II in microdissected areas of turkey hen brain during the reproductive cycle.

Chicken gonadotropin-releasing hormones I and II (cGnRH I and II) were measured by radioimmunoassay (RIA) in extracts of microdissected regions of turkey hen brain (preoptic area [POA], region of periventricular nuclei [PHN], septum [SEP], hippocampus [HP], dorsomedial thalamus/habenula [DMT], midbrain central gray [MCG], and caudal lateral hypothalamus [LH]) at five stages of the reproductive cycle: before photostimulation, during egg laying, during incubation, during photorefractoriness, and after return to short daylengths. The highest concentration of cGnRH I occurred in PHN, followed by POA, SEP, DMT, HP, LH, and MCG, in decreasing order, whereas the highest concentration of cGnRH II occurred in SEP, followed by POA, DMT, HP, MCG, PHN, LH. These results agree, with some exceptions, with the distribution of fibers and cells as determined by immunohistochemistry. cGnRH II was from 1.3 to 24 times as abundant as cGnRH I in different brain areas. During incubation, cGnRH I concentrations were significantly elevated in the POA and cGnRH II levels were significantly elevated in HP; few other significant differences were detected. Correlation analysis detected occasional significant positive and negative correlations between cGnRH I and II concentrations in forebrain areas and MCG of laying birds and in PHN and LH of incubating birds. These results demonstrate an approximate correspondence between hormone concentrations measured in tissue extracts by RIA and immunohistochemistry and indicate an abundance of cGnRH II as compared with cGnRH I. cGnRH I and II concentrations did not, however, change in parallel in all brain areas, suggesting that these peptides do not function in an exactly parallel fashion. Thus, an extent to which cGnRH II is involved in gonadotropin release remains unresolved.

Brain content of cGnRH I and II during embryonic development in chickens.

We used specific radioimmunoassays to measure chicken gonadotropin-releasing hormones I and II (cGnRH I and II) in extracts of chicken brain as a first step in determining whether these peptides may function identically during embryonic development in birds. In three experiments chicken embryo brains were removed at various times between Day 6 of incubation and hatching. In Experiment 3, the sex of embryos was identified by means of polymerase chain reaction amplification of a W chromosome-specific DNA sequence. Brain concentrations of cGnRH I increased sharply from Day 6 of incubation to Day 8, then decreased until Day 10 to Day 12, followed by an increase beginning on approximately Day 17 and continuing until hatch. In contrast, cGnRH II concentration remained low until about Day 14 of incubation, then increased progressively until, at hatch, brain content of cGnRH II was approximately 9 to 11 times that of cGnRH I. The difference in development pattern and total content of these peptides supports the view that any physiologic function they may have might be differentiated as early as Day 7 of incubation.

Immunohistochemical localization of chicken gonadotropin-releasing hormones I and II (cGnRH I and II) in turkey hen brain.

The distribution of cells and fibers immunoreactive (ir) for either chicken gonadotropin-releasing hormone I (cGnRH I; [Gln8]GnRH) or II ([His5,Trp7,Tyr8]GnRH) was determined in brains of turkey hens to reveal whether these peptides occur in separate neuronal systems. ir-cGnRH I cells were located: along the medial aspect of the ventriculus lateralis, nucleus accumbens, and bed nucleus of the stria terminalis; ventral to the tractus septomesencephalicus and extending medially to the third ventricle, and caudally into the lateral hypothalamic area; and in a diffuse band extending from the nucleus preopticus medialis to the nucleus dorsomedialis anterior thalami. cGnRH I fibers were evident in these areas in addition to the hippocampus, nucleus subhabenularis medialis, nucleus ventromedialis hypothalami, and median eminence. Two groups of ir-cGnRH II cells were observed: a magnocellular group lying between the substantia grisea centralis and the nucleus ruber; and a parvicellular group lying medial to the nucleus of the basal optic root and extending into the lateral hypothalamic area. ir-cGnRH II fibers were prominent in limbic structures (cortex piriformis, lateral to nucleus taeniae, hippocampus); olfactory areas (tuberculum olfactorium, nucleus subhabenularis lateralis, nucleus septalis lateralis); areas that in other avian species have steroid-concentrating cells or receptors (medial edge of lobus parolfactorius, nucleus septalis medialis, nucleus periventricularis magnocellularis, nucleus dorsomedialis posterior thalami); and areas containing ir-GnRH I cells or fibers but not in median eminence. These results suggest that cGnRH I and II occur in separate neuronal systems and that cGnRH II does not directly promote pituitary gonadotropin secretion.