Somatotropin response in vitro to corticosterone and triiodothyronine during chick embryonic development: Involvement of type I and type II glucocorticoid receptors.

Corticosterone (CORT) can stimulate growth hormone (GH) secretion on embryonic day (e) 12 in the chicken. However, CORT failed to induce GH secretion on e20 in a single report, suggesting that regulation of GH production changes during embryonic development. Secretion in response to CORT during embryonic development is modulated by the thyroid hormones triiodothyronine (T(3)) and thyroxine (T(4)). Growth hormone responses on e12 involve both glucocorticoid (GR) and mineralocorticoid receptors (MR); however, involvement of MR has not been evaluated past e12. To further define changes in somatotroph responsiveness to CORT, pituitary cells obtained on e12-e20 were cultured with CORT alone and in combination with T(3) and GH-releasing hormone (GHRH). Growth hormone mRNA levels and protein secretion were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and radioimmunoassay (RIA), respectively. Corticosterone significantly increased GH mRNA and protein secretion on e12; however, mRNA concentration and protein secretion were unaffected on e20. Contributions of GR and MR in CORT responses were evaluated using GR and MR antagonists. Treatment with a GR-specific antagonist effectively blocked the CORT-induced increase in GH secretion on e12. The same treatment on e20 had no effect on GH secretion. These findings demonstrate that GR is directly involved in glucocorticoid stimulation of GH secretion at the time of somatotroph differentiation but is not regulatory at the end of embryonic development. We conclude that positive somatotroph responses to CORT are lost during chicken embryonic development and that GR is the primary regulator of CORT-induced GH secretion.

Exposure duration to long day lengths associated with the expression of photorefractoriness in turkey breeder hens.

In addition to inducing egg production, exposure to long days concomitantly activates processes that eventually result in photorefractoriness (PR) and cessation of egg production. Experiments were conducted to evaluate the duration of exposure to long days that result in these processes. In each of 3 experiments, we subjected Large White turkey breeder hens to long days (16 or 18 h per day) for differing lengths of time from initial photostimulation and then returned them to a photoperiod (12L:12D) that provided sufficient, but decreased, photoperiodic drive to support egg production but not induce PR. Photoresponsiveness was then evaluated by egg production after a return to a longer day length (20 h per day) late in the lay period and beyond the mean onset of PR typical for these turkey hens. Hens that have undergone any reduction in photoresponsiveness should not increase egg production in response to the increased photoperiod. From experiments 1 and 2, exposure to long days for as little as 1 d and as much as 9 wk from initial photostimulation did not result in an alteration in subsequent photoresponsiveness. This was based on an increased egg production response to a change in photoperiod from 12L:12D to 20L:4D after 20 wk of photostimulation that was similar to controls held continuously on 12L:12D and opposite to the response of controls held continuously on 18L:6D. It was clear that PR had been fully programmed by 20 wk of exposure to long days. Exposure to long days for 12 wk (experiment 3) resulted in a partial alteration of subsequent photoresponsiveness. It was concluded that programming of PR during late spring-summer season occurs after 9 wk of long day exposure, is not fully expressed by 12 wk of long days, and can be fully expressed by 20 wk of photostimulation.

Gonadotrophin-inhibitory hormone receptor expression in the chicken pituitary gland: potential influence of sexual maturation and ovarian steroids.

Gonadotrophin-inhibitory hormone (GnIH), a hypothalamic RFamide, has been found to inhibit gonadotrophin secretion from the anterior pituitary gland originally in birds and, subsequently, in mammalian species. The gene encoding a transmembrane receptor for GnIH (GnIHR) was recently identified in the brain, pituitary gland and gonads of song bird, chicken and Japanese quail. The objectives of the present study are to characterise the expression of GnIHR mRNA and protein in the chicken pituitary gland, and to determine whether sexual maturation and gonadal steroids influence pituitary GnIHR mRNA abundance. GnIHR mRNA quantity was found to be significantly higher in diencephalon compared to either anterior pituitary gland or ovaries. GnIHR mRNA quantity was significantly higher in the pituitaries of sexually immature chickens relative to sexually mature chickens. Oestradiol or a combination of oestradiol and progesterone treatment caused a significant decrease in pituitary GnIHR mRNA quantity relative to vehicle controls. GnIHR-immunoreactive (ir) cells were identified in the chicken pituitary gland cephalic and caudal lobes. Furthermore, GnIHR-ir cells were found to be colocalised with luteinising hormone (LH)beta mRNA-, or follicle-stimulating hormone (FSH)beta mRNA-containing cells. GnIH treatment significantly decreased LH release from anterior pituitary gland slices collected from sexually immature, but not from sexually mature chickens. Taken together, GnIHR gene expression is possibly down regulated in response to a surge in circulating oestradiol and progesterone levels as the chicken undergoes sexual maturation to allow gonadotrophin secretion. Furthermore, GnIHR protein expressed in FSHbeta or LHbeta mRNA-containing cells is likely to mediate the inhibitory effect of GnIH on LH and FSH secretion.

The effect of heat stress on ovarian function of laying hens.

Reproductive failure associated with heat stress is a well-known phenomenon. The mechanism involved in this failure is not clearly understood. In order to test a possible direct effect of heat stress on ovarian function, 36 White Leghorn laying hens were housed in individual cages in 2 temperature- and light-controlled rooms (n = 18). At 31 wk of age, one group was exposed daily for 12 h to high temperature (42 +/- 3 degrees C), and the second group was maintained under thermoneutral conditions (24 to 26 degrees C) and served as control. Body temperature, feed intake, egg production, and egg weight were recorded daily; heparinized blood samples were drawn every 3 d for plasma hormonal level of luteinizing hormone, follicular stimulating hormone, progesterone, 17beta-estradiol, and testosterone. Six days after exposure half of the birds in each group were killed, and the ovary and oviduct were weighed and preovulatory follicles removed and extracted for mRNA of Cytochrome P 450 aromatase, 17-alpha hydroxylase. The same procedure was repeated 9 d later with the rest of the birds. Short and long heat exposure caused significant hyperthermia and reduction of egg production, egg weight, ovarian weight, and the number of large follicles. In addition, a significant reduction in plasma progesterone and testosterone was detected 2 d after exposing the birds to heat stress, and plasma 17beta-estradiol was significantly reduced 14 d after initiation of heat stress. Short exposure to heat stress caused significant reduction in mRNA expression of cytochrome P450 17-alpha hydroxylase, exposing the birds to long-term heat stress caused significant reduction in expression of mRNA of both steroidogenic enzymes. No significant change was found in plasma luteinizing hormone and follicular stimulating hormone levels during the entire experimental period. We suggest a possible direct effect of heat stress on ovarian function.

Seasonal and photoperiodic regulation of secretion of hormones associated with reproduction in Magang goose ganders.

This study examined the reproductive endocrine profile under natural and artificial photoperiods in Magang goose ganders. Group 1 ganders (n=8) served as non-treated controls and were exposed to natural photoperiod throughout the experiment from 13th January to 17th December 2004. Group 2 ganders (n=8) were exposed to 18 h long daily photoperiod for 60 days from 13 January till 15 March 2004 and again to 16 h photoperiod for 75 days till 10th October 2004, and the 11h short photoperiod in the remainder periods of the experiment. In control ganders, plasma LH concentrations were high in normal breeding seasons (August-March) and decreased to low levels in non-breeding season from April to July. Testosterone concentrations changed similarly to that of LH throughout the seasons. Seasonal pattern of PRL concentrations was opposite to those of LH and testosterone, with low values in breeding season and high values in non-breeding season. In artificial photoperiod treated ganders, increasing photoperiod increased PRL and decreased LH and testosterone concentrations, while decreasing photoperiod reversed these changes. There were no seasonal or photoperiod caused changes in plasma T3 concentrations in both control ganders and artificial photoperiod treated ganders. These results demonstrated that in Magang goose ganders that long photoperiod stimulates PRL secretion and decreases LH secretion, which terminates reproductive season in spring and early summer, and short photoperiod stimulates LH secretion and inhibits PRL secretion rendering ganders enter into reproductive season.

Potential role of thyroid hormones and prolactin in the programming of photorefractoriness in turkey hens.

The domestic turkey hen is a seasonal breeder, requiring a period of short days to establish photosensitivity and a long day length to initiate egg production. The reproductive season is then limited by the onset of photorefractoriness (PR), which causes a decline, and then termination, of egg laying. In passerine birds, PR is programmed early in the reproductive season by the presence of thyroid hormones and a long photoperiod. High circulating prolactin (PRL) is thought to hasten the onset of PR. In a prior study, we reported that hens destined to have PR exhibited lower levels of thyroxine (T4) and PRL at certain points (weeks) following photostimulation than did hens destined to remain photosensitive (PS), a result opposite to what might be expected. The present study was conducted to further explore the possible relationship between circulating hormone levels and subsequent PR in the commercial turkey hen at times (days) closer to photostimulation than our previous study. Plasma levels of triiodothyronine (T3), T4, and PRL were compared in 2 subpopulations of hens identified retrospectively after 50 wk of egg production: A group of 17 hens that exhibited PR (mean onset = 27 wk of photostimulation) and a group of "good" layers that remained PS (mean production = 210 eggs/50 wk). Results showed no differences between groups in plasma T3 or T4 levels or in the T3:T4 ratio at -6, 0, 1, 3, and 7 d from photostimulation. Plasma PRL levels were significantly higher at 8 and 9 wk after photostimulation in hens that remained PS vs. those that became PR. We conclude that thyroid hormone levels around the time of photostimulation either are not actively related to programming of subsequent PR in turkeys or programming for PR in the turkey hen occurs later in the reproductive cycle than in passerine birds. We further conclude that hens that exhibit PR tend to have lower circulating PRL levels early in the reproductive season than hens that remain PS and lay at a relatively high rate.

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.

Lack of effects of atrazine on estrogen-responsive organs and circulating hormone concentrations in sexually immature female Japanese quail (Coturnix coturnix japonica).

The widely used herbicide, atrazine, has been reported to exhibit reproductive toxicity in rats and amphibians. The present studies investigate toxicity of atrazine in Japanese quail and its ability to influence reproduction in sexually immature females. Atrazine was administered in the diet at concentrations from 0.001 to 1000 ppm (approximately 109 mg kg-1 per day) or systemically via daily subcutaneous injections (1 and 10 mg kg-1) or Silastic implants. Atrazine did not cause overt toxicity in sexually immature female quail (no effects on change in body weight, feed intake, mortality or on circulating concentrations of the stress hormone, corticosterone). It was hypothesized that if atrazine were to have estrogenic activity or to enhance endogenous estrogen production, there would be marked increases in the weights of estrogen sensitive tissues including the oviduct, the liver and the ovary together with changes in gonadotropin secretion. However, atrazine had no effect on either liver or ovary weights. Atrazine in the diet increased oviduct weights at 0.1 and 1 ppm in some studies. These effects were not consistently observed and were not significant when data from studies were combined. Systemic administration of atrazine had no effect on oviduct weights. Dietary (concentrations from 0.001 to 1000 ppm) and systemically administered atrazine had no effect on circulating concentrations of luteinizing hormone (LH). The present studies provide evidence for a lack of general or reproductive toxicity of atrazine in birds.

Thyroid hormone and prolactin profiles in male and female turkeys following photostimulation.

The turkey hen, a photosensitive bird, will become photorefractory (PR) during the reproductive cycle and will cease laying despite a stimulatory day length. This response is thought to be "programmed" by hormonal events early in the reproductive cycle. The turkey tom, in contrast, produces semen for extended periods and has not been shown to exhibit PR. We compared hormone profiles following photostimulation of hens and toms to assess differences that might program one, but not the other, for PR. We photostimulated with 16 h light per day and measured plasma prolactin (PRL), thyroxine (T4), and triiodothyronine (T3) weekly for 12 wk, and again at 16 and 22 wk. Hens were fed ad libitum, and toms were moderately feed-restricted. Results showed increasing PRL levels following photostimulation in hens, with peak levels occurring at about the time of peak egg production, and declining thereafter. Toms maintained significantly lower concentrations of PRL (P < 0.0001) than hens after 2 wk of photostimulation. A highly significant sex by time interaction in plasma T3 levels was observed due to extreme fluctuations in males. Similar, often reciprocal, fluctuations in mean T4 concentrations also occurred in males. We recycled the toms and repeated blood collections under identical conditions, but with ad libitum feeding to determine if feed restriction may have produced these unusual results. This study revealed an initial significant decline in plasma T3 levels and an increase in T4 levels immediately following photostimulation, and then steady (T4) or slowly rising (T3) levels through 12 wk photostimulation. We conclude that PRL profiles of toms and hens differ markedly during the reproductive cycle, lending support to the suggestion that rising PRL may mediate the onset of PR. Further study is needed to determine if the low plasma T3 levels in males may be related to delayed PR. The extreme fluctuations in plasma T3 and T4 levels of toms receiving relatively mild feed restriction suggest a need for further study of the metabolic effects of feed restriction in turkeys.

Effect of a single short-term reduction in photoperiod on photorefractoriness in turkey hens.

In a prior study, we reported that a high proportion of hens in a winter-laying flock became relatively photorefractory (rPR) early in the reproductive cyand that successive short-term reductions in photoperiod in such hens each initially depressed egg production but then caused a rebound in rate of lay to briefly exceed that of hens that did not exhibit rPR. The present study was conducted to assess rPR in a summer-laying flock and to determine whether a single short-term reduction in day length early in the reproductive cycle might enhance egg production and delay the onset of absolute photorefractoriness (aPR). Control hens received a photoperiod of 16L:8D throughout the experiment. Experimental hens were photostimulated with 16L:8D, received a reduced (but still stimulatory) photoperiod of 11.5L:12.5D for 2 wk beginning 8 wk after photostimulation, and then were returned to 16L:8D for the remainder of the 23-wk test period. Results showed that a single 2-wk reduction in day length shortly after the hens reached peak egg production did not significantly reduce overall flock egg production, but it also did not improve late-season egg production or retard the onset or incidence of aPR. The incidence of rPR was substantially less in this study than we had observed with a winter-laying flock (32.9 vs. 67.1%), but similar proportions of treated hens exhibited the most severe rPR response (a brief but complete cessation of egg production) in both studies (21.1 vs. 24.0%), and all treated hens that subsequently became aPR had shown this severe rPR response to the test photoperiod. We concluded that a core proportion of hens (approximately one-fifth) exhibited a strong rPR response when presented with a reduced photoperiod early in the reproductive cycle, regardless of season of the year, and that such hens were more likely to subsequently exhibit poor egg production or become aPR than flockmates that did not exhibit rPR. Therefore, some indication of the incidence of rPR early in the lay period may have a predictive value for the overall egg production of the flock.

Sulfamethazine advances puberty in male chicks by effecting a rapid increase in gonadotropins.

A sulfonamide, sulfamethazine (SMZ) has been shown to have a robust, progonadal effect. The mechanism of action of SMZ, however, is unknown. Our hypothesis is that the compound may act centrally and/or at the level of the pituitary. Four experiments were completed to test that hypothesis. Chicks exposed to a continuous photoperiod and fed a diet containing 0.2% SMZ showed an exponential increase in testes size. When 6 weeks of age (5 weeks on the SMZ diet), experimentals had testes weight nine times heavier than controls. Profiles for thyroid and gonadotropin plasma hormones suggested that T(3) was transiently lower in experimentals solely during the first week on treatment, while thyroxine levels were not different from controls. In contrast, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were significantly elevated at the initial 1-week sampling point and remained elevated throughout the entire experiment. In a follow-up study, LH was found significantly higher than controls by 48 h after initially consuming the compound. When T(3) was added to the SMZ diet at 0.5 ppm, the progonadal effect of SMZ was attenuated. Importantly, chronic intake of T(3) delayed but did not block the stimulatory effect of SMZ for increasing plasma LH. We conclude that since one of the primary effects of SMZ is to increase rapidly plasma gonadotropins, data suggest the compound is acting at the level of the brain or pituitary to stimulate early gonadal development in chicks.

Modulation of in vitro DNA synthesis in the chicken ovarian granulosa cell follicular hierarchy by follicle-stimulating hormone and luteinizing hormone.

Folliculogenesis in domestic hens appears to be controlled by numerous factors, particularly the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The involvement of LH in follicular steroidogenesis has been described in some detail; however, the specific role of FSH has remained elusive. In 3 experiments, the effects of ovine (o)- or chicken (c)-derived FSH (oFSH, cFSH) or LH (oLH, cLH) were evaluated on in vitro DNA synthesis [3H-thymidine (3H-TdR) incorporation], indicative of cellular proliferation, of granulosa cells from F1, F3, or F5-6 preovulatory follicles. In experiment 1, oFSH or cFSH stimulated (P < 0.05) and oLH or cLH decreased DNA synthesis by F1 granulosa cells. In experiment 2, oFSH resulted in concentration-related changes in DNA synthesis by F5-6 granulosa cells; however, no significant changes were observed in F1 or F3 granulosa cells. No effect of oLH was observed on granulosa cell proliferation from any of the follicles. Similar to oFSH, cFSH resulted in concentration-related increases in DNA synthesis in granulosa cells from F5-6 follicles with smaller magnitude changes in proliferation of F1 or F3 granulosa cells. Granulosa cells from F5-6 or F3 follicles had small increases in DNA synthesis in response to cLH. These data support the proposed role for FSH in granulosa cell proliferation, possibly contributing to follicle growth, and suggest that in vitro 3H-TdR incorporation by granulosa cells may provide a sensitive and selective bioassay for chicken gonadotropin preparations. Furthermore, data suggest that proliferative responsiveness of granulosa cells to FSH or LH may differ depending on position of follicles in the preovulatory hierarchy.

Photoresponsiveness of turkey breeder hens changes during the egg-laying season: relative and absolute photorefractoriness.

Photosensitive species undergo neuroendocrine changes during a reproductive season that cause them to gradually become unresponsive to a photoperiod that initially stimulated reproduction. They may first become relatively photorefractory (rPR), when they will cease egg laying only if photoperiod is reduced, and then absolutely photorefractory (aPR), when they will cease laying despite long day length. Our objective was to test the photoresponsiveness of breeder turkey hens during egg production at various times following photostimulation and to relate photoresponsiveness to rPR and aPR as well as plasma levels of prolactin (PRL) and luteinizing hormone (LH). Hens were maintained in cages in light-controlled facilities and photostimulated at 31 wk of age (September) with a photoperiod of 16L:8D. At 8, 14, and 20 wk after photostimulation, treated hens received a 2-wk exposure to an 11.5L:12.5D photoperiod and were then returned to 16L:8D. Exposure to the shortened photoperiod at 8 wk of photostimulation resulted in three distinct responses of declining egg production: nonresponders (NR, 32.7% of hens), partial responders (PAR, 43.9%), or full responders (FR, 23.4%). Egg production returned to control levels following return to a 16L:8D photoperiod. This response repeated at the 14- and 20-wk treatment periods but with greater declines in egg production in the NR and PAR groups. The incidence of subsequent aPR in the NR, PAR, and FR groups was 5.7, 8.5 and 24%, respectively, as compared to 23.3% for the controls. Plasma LH and PRL concentrations also declined in response to 11.5L:12.5D and also rebounded following return to 16L:8D. The hormonal responses of NR, PAR, and FR were similar. We conclude that turkey hens exhibit varying degrees of rPR early during the egg laying season and that the incidence and severity of the rPR response increases as the laying season progresses. Further, PRL and LH levels did not reflect the differences in egg production among the responder and nonresponder groups to changes in photoperiod.

Immunohistochemical localization of chromogranin A in gonadotrophs and somatotrophs of the turkey and chicken pituitary.

In the course of producing monoclonal antibodies to turkey prolactin, three monoclonal antibodies to turkey chromogranin A (CgA) were also produced, apparently arising from minor contamination of the turkey prolactin immunogen with peptide fragments of CgA. The identity of the antigen recognized by these antibodies was established by tandem mass spectrometry de novo sequencing of seven tryptic peptides from a turkey pituitary protein purified by immunoaffinity chromatography. These peptides showed high homology with distinctly separate regions of mammalian and ostrich CgA, and in silico cloned chicken CgA sequences. Chromogranin A immunostaining patterns on Western blots and pituitary tissue sections differed from those of prolactin, growth hormone, or luteinizing hormone (LH). Dual-label fluorescent immunohistochemistry revealed that CgA was co-localized with LH in most avian gonadotrophs in young chickens and turkeys, but not in adult, laying birds. Conversely, CgA was found in a majority of somatotrophs in laying birds but was absent from somatotrophs in young, growing chickens and turkeys. Lactotrophs contained no detectable CgA immunoreactivity in the tissues studied. These results suggest that CgA may modulate hormone secretion by gonadotrophs and somatotrophs in a manner that differs between cell type with age or reproductive state.

Relative and absolute photorefractoriness in turkey hens: profiles of prolactin, thyroxine, and triiodothyronine early in the reproductive cycle.

An experiment was conducted to determine whether a commercial strain of turkey hens exhibits relative photorefractoriness (rPR) during a reproductive cycle and to ascertain whether plasma levels of certain hormones early in the reproductive cycle might be associated with subsequent expression of rPR or absolute photorefractoriness (aPR). Twenty-seven percent of hens maintained on a stimulatory photoperiod of 18L:6D for 19 wk and then given a shorter, but still stimulatory, photoperiod (13L:11D) ceased to lay and their ovaries regressed within 4 wk. These hens were considered rPR. Subsequent exposure to the 18L:6D photoperiod resulted in ovarian recrudescence in 41.7% of these PR individuals, confirming the presence of rPR at 19 wk after photostimulation. Absolute PR was observed in 15.1% of hens during a 27-wk reproductive season. Hens that became rPR or aPR exhibited significantly lower plasma prolactin levels at 8 and 14 wk after photostimulation than did hens that remained photosensitive (PS). Plasma levels of thyroxine were lower at 1 and 2 wk following photostimulation in hens that subsequently became PR than in hens that remained PS. We conclude that turkey hens may exhibit rPR and aPR during a reproductive cycle, whereas flockmates may remain PS for at least 27 wk. The presence of long daylengths, thyroid hormones, and PRL did not assure expression of PR. The expression of PR appears to be associated with reduced plasma throxine levels during a period when programming of PR is thought to occur and with reduced levels of prolactin following peak egg production.

FSH- and LH-cells originate as separate cell populations and at different embryonic stages in the chicken embryo.

The histological distribution of gonadotrophs containing either LH or FSH, but not both gonadotropins, has been demonstrated before in the juvenile and adult chicken throughout the caudal and cephalic anterior pituitary lobes. In the present investigation, the distribution of FSH- and/or LH-containing gonadotrophs was further investigated in the chicken embryo by use of the same homologous antibodies as used in our earlier study. Fluorescent dual-labeling immunohistochemistry revealed that during embryogenesis LH and FSH reside exclusively in separate gonadotrophs, as has been described before in the post hatch bird. LH-immunoreactive cells were observed for the first time at day 9 of embryogenesis. This is as much as 4 days earlier than the FSH-immunoreactive cells, which appeared at day 13 of embryogenesis. Our results confirm that FSH- and LH-containing gonadotrophs are distributed throughout both lobes of the anterior pituitary. No conspicuous differences were observed between the sexes in any of the aspects investigated. The described situation is unique in that it seems to imply the existence of separate cell lineages for FSH- and LH-producing cells, as opposed to the single gonadotrope lineage described in all other species studied so far, with the exception of bovine. Our data indeed raise the question as to which signaling and/or transcription factors may cause the unique dichotomy observed in the chicken gonadotrophs.

The turkey transcription factor Pit-1/GHF-1 can activate the turkey prolactin and growth hormone gene promoters in vitro but is not detectable in lactotrophs in vivo.

The transcription factor Pit-1/GHF-1 plays an important role in regulating the prolactin (Prl) and growth hormone (GH) genes in mammals. In this study, the role that Pit-1 plays in regulating the prolactin and growth hormone genes in avian species was examined by cotransfection assays and immunofluorescence staining of pituitary sections. In cotransfection assays, turkey Pit-1 activated the turkey Prl, turkey GH, and rat Prl promoters 3.8-, 3.7-, and 12.5-fold, respectively. This activation was comparable to rat Pit-1 activation of these same promoters. A point mutation in the turkey Pit-1 cDNA, which changed leu-219 to ser-219, resulted in a 2-, 2-, and 10-fold reduction in the activation of the turkey Prl, turkey GH, and rat Prl promoters, respectively. Unexpectedly, coexpression of tPit-1 (leu-219) and tPit-1(ser-219) activated turkey Prl and rat Prl promoters 9.4- and 35.9-fold, respectively, but had no effect on the turkey GH promoter. Dual-label immunofluorescence analysis of turkey pituitary sections revealed that Pit-1 was not detectable in prolactin-staining cells but was detectable in GH-staining cells. Taken together, these data indicate that in the domestic turkey, Pit-1 can activate the turkey Prl promoter in vitro, but does not appear to play a role in regulating Prl gene expression in vivo. Pit-1, however, still likely plays a role in regulating GH gene expression.

Increased proliferative activity and programmed cellular death in the turkey hen pituitary gland following interruption of incubation behavior.

Incubation behavior or broodiness in turkey hens is characterized by ovarian regression, hyperprolactinemia, and persistent nesting. Nest-deprivation of incubating turkey hens results in disruption of broodiness accompanied by a precipitous decline in plasma prolactin (PRL) concentrations. The objective of the present study is to examine cellular changes in the pituitary gland associated with nest-deprivation for 0, 1, 2, 3, 4, or 7 days. Bromodeoxyuridine (BrdU) was administered prior to kill to study proliferative activity. Pituitary tissue sections were immunostained using turkey growth hormone (GH) antibody, and/or chicken PRL peptide antibody, and BrdU antibody. Plasma PRL concentrations declined significantly following nest-deprivation for 1 or more days. The midsagittal pituitary area immunoreactive (ir) to GH was significantly increased while that of PRL was significantly decreased following nest-deprivation for 2 or more days. Terminal deoxy-UTP nick end labeling and PRL-immunostaining revealed an abundance of apoptotic nuclei in both cephalic and caudal lobes of the anterior pituitary gland, suggestive of programmed cellular death of lactotrophs in the pituitary gland of hens nest-deprived for 2 or more days. Mammosomatotrophs were abundant in hens nest-deprived on Day 0 but were absent in hens nest-deprived for 1 or more days. Proliferating (BrdU-ir) cells were significantly abundant in the pituitary cephalic and caudal lobes following nest-deprivation for 1 or more days but were absent on Day 0 or in laying hens. Dual-labeling studies indicated that most of the BrdU-ir nuclei in the caudal lobe were not colocalized in somatotrophs in hens nest-deprived for 1-4 days but did colocalize with GH following 7 days of nest-deprivation. In conclusion, nest-deprivation of incubating turkey hens results in 1) a precipitous decline in plasma PRL concentration, 2) programmed cell death of lactotrophs, 3) disappearance of mammosomatotrophs, 4) increased proliferative activity of pituitary cells, and 5) recruitment of somatotrophs arising primarily from mitosis of nonsomatotrophic cells.

Identification of growth-hormone- and prolactin-containing neurons within the avian brain.

Prolactin (PRL)- and growth-hormone (GH)-containing perikarya and fibers independent of the anterior pituitary gland have been reported to exist in the central nervous system of several mammalian species. The specific distributions of PRL- or GH-like neurons in the avian forebrain and midbrain, however, have not been reported. The objective of the study was to identify GH- and PRL-containing neurons in the hypothalamus and a few extrahypothalamic areas of two avian species. Brain and peripheral blood samples were collected from laying and broody turkey hens and ring doves. Broody turkey hens and doves had significantly higher plasma PRL concentrations compared with laying hens. Coronal brain sections were prepared and immunostained using anti-turkey GH and anti-chicken synthetic PRL antibodies. In turkey hens, the most dense GH-immunoreactive (ir) perikarya and fibers were found in hippocampus (Hp), periventricular hypothalamic nucleus, paraventricular nucleus, inferior hypothalamic nucleus, infundibular hypothalamic nucleus, medial and lateral septal area, and external zone of the median eminence (ME). In the ring dove, a similar pattern of distribution of GH-ir neurons was noticed at the brain sites listed above except that GH-ir fibers and granules were found only in the internal zone of ME and not in the external zone. In both turkeys and doves, the most immunoreactive PRL-ir perikarya and fibers were found in the medial and lateral septal area, Hp (turkey only), and bed nucleus of the stria terminalis pars magnocellularis. There were no apparent differences in the staining pattern of GH- or PRL-ir neurons between the laying and broody states in either species. However, the presence of GH-ir- and PRL-ir perikarya and fibers in several hypothalamic nuclei indicates that GH and PRL may influence parental behavior, food intake, autonomic nervous system function, and/or reproduction.

Influence of continuous growth hormone or insulin-like growth factor I administration in adult female chickens.

A series of studies was conducted to determine whether growth hormone (GH) exerts effects on adult female chickens. Recombinant chicken GH (rcGH) was administered continuously via osmotic minipumps. No consistent effects of rcGH treatment were observed on reproductive indices. Hens receiving rcGH treatment for 10 days exhibited hepatomegaly and showed a tendency (P < 0.1) for increased spleen and thymus weights. Moreover, there were increases in the circulating concentrations of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGF-BPs) (22-kDa IGF-BP after 2, 5, and 10 days; 28-kDa IGF-BP after 5 and 10 days; and 36-kDa IGF-BP after 10 days) with rcGH treatment. To determine whether the changes in IGF-BPs were due directly to GH or indirectly via IGF-I, the effects of the continuous administration of rcGH or recombinant human IGF-I (rhIGF-I) were compared. While rcGH again elevated the circulating levels of 28- and 36-kDa IGF-BPs, no such effect was observed with rhIGF-I treatment. However, both treatments exerted similar effects in depressing pituitary GH mRNA levels and elevating plasma concentrations of IGF-I. It is concluded that GH directly elevates circulating concentrations of IGF-I and IGF-BPs, but the negative feedback effect on GH synthesis is mediated via IGF-I.