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This study was conducted to evaluate potential hormonal mechanisms associated with the stress response, thermoregulation, and metabolic changes of broiler chickens exposed to high environmental temperature. Nine hundred 1-day-old male broiler chicks (Ross 708) were placed in floor pens and raised to 24 d. At 24 d, chicks were randomly assigned to 1 of 2 treatments, heat stress (HS) or no HS, and allocated into battery cages in 8 batteries (10 birds per cage, 2 cages per battery). On day 31, blood was collected prior to HS and analyzed using an iSTAT analyzer. Half of the batteries were then moved into 2 rooms with an elevated ambient temperature (35°C) for 8 h. The remaining batteries stayed in the thermoneutral rooms with an ambient temperature of 22°C. Beginning at 5 h after the initiation of HS, blood was collected and analyzed using an iSTAT analyzer, birds were euthanized, and hypothalamus and pituitary samples were collected (16 birds per treatment), flash frozen, and stored at -80°C until RNA extraction. Reverse transcription-quantitative PCR was used to compare mRNA levels of key corticotropic and thyrotrophic genes in the hypothalamus and pituitary. Levels of mRNA for each target gene were normalized to PGK1 (pituitary) and GAPDH (hypothalamus) mRNA. Differences were determined using mixed model ANOVA. HS decreased (P < 0.05) feed intake, BW, bicarbonate, potassium, CO2, and triiodothyronine, while it increased mortality, glucose, pH, plasma thyroxine, and corticosterone. Expression of pituitary corticotropin-releasing hormone receptor 1 was downregulated (P < 0.001), while corticotropin-releasing hormone receptor 2 mRNA levels were higher (P = 0.001) in HS birds. HS increased expression of thyroid hormone receptor β (P = 0.01) (2.8-fold) and thyroid stimulating hormone β (P = 0.009) (1.4-fold). HS did not affect levels of mRNA of genes evaluated in the hypothalamus. Results showed that HS significantly affected both the thyrotropic and corticotropic axes. Understanding the role and regulation of these pathways during HS will allow researchers to better evaluate management strategies to combat HS.

Low-egg-producing hens (LEPH) ovulate less frequently than high-egg-producing hens (HEPH) and exhibit differences in mRNA levels for components of the hypothalamo–pituitary–gonadal (HPG) axis, suggesting differential responsiveness to trophic stimulation. Ovulation frequency is governed by the production of the pituitary gonadotropins and feedback of the ovarian follicle steroid hormones, which are regulated by HPG axis stimulation and inhibition at the hypothalamic level. The pituitary and follicle cells from LEPH and HEPH were subjected to in vitro hormonal treatments to stimulate or inhibit the HPG axis, followed by expression analysis of mRNA levels for HPG axis genes and radioimmunoassays for steroid hormone production. Statistical analysis was performed using the mixed models procedure of SAS. The pituitary cells from HEPH showed upregulation of genes associated with ovulation stimulation, whereas cells from LEPH showed upregulation of genes associated with inhibition of ovulation. High-egg-producing hens’ follicle cells displayed a higher sensitivity and responsiveness to gonadotropin treatment. Level of egg production impacted ovulation-related gene expression in the pituitary cells as well as steroid hormone production in the follicle cells, with HEPH displaying a greater positive response to stimulation. These findings indicate that differences in egg production among turkey hens likely involve differential responsiveness of the cells within the HPG axis.

Variation in egg production exists in commercial turkey hens, with low egg producing hens (LEPH) costing more per egg produced than high egg producing hens (HEPH). Egg production correlates with ovulation frequency, which is governed by the hypothalamic–pituitary–gonadal (HPG) axis. Ovulation is stimulated by a preovulatory surge (PS) of progesterone and luteinizing hormone, triggered by gonadotropin releasing hormone release and inhibited by gonadotropin inhibiting hormone. Differences between LEPH and HEPH were characterized by determining HPG axis plasma hormone profiles and mRNA levels for key genes, both outside and inside of the PS (n = 3 per group). Data were analyzed with a 2-way ANOVA using the mixed models procedure of SAS. In the HPG axis, plasma progesterone levels were not affected by egg production level but were elevated during the PS. In contrast, plasma estradiol levels were higher in HEPH than in LEPH but were not associated with the PS. LEPH exhibited decreased gene expression associated with ovulation stimulation and increased gene expression associated with ovulation inhibition in the hypothalamus and pituitary. In ovarian follicle cells, LEPH displayed decreased gene expression associated with progesterone, androgen, and estradiol production in the F1 follicle granulosa cells, F5 theca interna cells, and small white follicle cells, respectively. Different degrees of stimulation and inhibition within all tissues of the HPG axis were noted between LEPH and HEPH turkey hens, with HEPH showing higher expression of genes related to ovulation and steroidogenesis.

During the normal embryonic-to-neonatal development, the chicken liver is subjected to intense lipid burden from high rates of yolk-lipid oxidation and also from the accumulation of the yolk-derived and newly synthesized lipids from carbohydrates. High rates of hepatic lipid oxidation and lipogenesis are also central features of non-alcoholic fatty liver disease (NAFLD) in both rodents and humans, but is associated with impaired insulin signaling, dysfunctional mitochondrial energetics and oxidative stress. However, these adverse effects are not apparent in the liver of embryonic and neonatal chicken, despite lipid burden. Utilizing comprehensive metabolic profiling, we identify that steady induction of hepatic mitochondrial tricarboxylic acid (TCA) cycle and lipogenesis are central features of embryonic-to-neonatal transition. More importantly, the induction of TCA cycle and lipogenesis occurred together with the downregulation of hepatic β-oxidation and ketogenesis in the neonatal chicken. This synergistic remodeling of hepatic metabolic networks blunted inflammatory onset, prevented accumulation of lipotoxic intermediates (ceramides and diacylglycerols) and reduced reactive oxygen species production during embryonic-to-neonatal development. This dynamic remodeling of hepatic mitochondrial oxidative flux and lipogenesis aids in the healthy embryonic-to-neonatal transition in chicken. This natural physiological system could help identify mechanisms regulating mitochondrial function and lipogenesis, with potential implications towards treatment of NAFLD.

Dysregulation of the preovulatory surge (PS) leads to lowered egg production. The hypothalamo–pituitary–thyroid (HPT) axis has been shown to influence plasma progesterone levels and follicle ovulation. The presence of thyroid hormone receptors (THR) in the reproductive axis suggests possible effects of thyroid hormone. To further understand the potential role of thyroid hormone on the PS, HPT axis plasma hormone concentrations and gene expression were characterized surrounding the PS in average egg producing hens (AEPH), low egg producing hens (LEPH), and high egg producing hens (HEPH) (n = 3 hens/group). Data were analyzed using the mixed models procedure of SAS, with significance indicated at P < 0.05. Average egg producing hens and HEPH displayed lower levels of triiodothyronine (T3) and higher levels of thyroxine (T4) inside of the PS, whereas LEPH showed inverse T3 and T4 levels relative to the PS. Expression of mRNA for hypothalamic thyrotropin-releasing hormone (TRH), pituitary thyrotropin (TSHB), and the main thyroid hormone metabolism enzyme (DIO2) were downregulated during the PS in AEPH and HEPH. Low egg producing hens displayed higher expression of mRNA for hypothalamic TRH as well as pituitary TSHB and DIO2 compared with HEPH. Average egg producing hens expression of THR mRNAs was upregulated during the PS in the hypothalamus but downregulated in the pituitary. High egg producing hens showed decreased expression of THR mRNAs in both the hypothalamus and pituitary when compared with LEPH. In ovarian follicles, THR mRNAs were more prevalent in the thecal layer of the follicle wall compared with the granulosa layer, and expression tended to decrease with follicle maturity. Minimal differences in follicular THR expression were seen between LEPH and HEPH, indicating that THR expression is unlikely to be responsible for steroid hormone production differences occurring between LEPH and HEPH. Generally, downregulation of the HPT axis was seen during the PS in AEPH and HEPH, whereas upregulation of the HPT axis was seen in LEPH. Further studies will be required to clarify the role of the HPT axis in the regulation of ovulation and egg production rates in turkey hens.

Background: High egg producing hens (HEPH) show increased hypothalamic and pituitary gene expression related to hypothalamo-pituitary-gonadal (HPG) axis stimulation as well as increased in vitro responsiveness to gonadotropin releasing hormone (GnRH) stimulation in the pituitary when compared to low egg producing hens (LEPH). Transcriptome analysis was performed on hypothalamus and pituitary samples from LEPH and HEPH to identify novel regulators of HPG axis function. Results: In the hypothalamus and pituitary, 4644 differentially expressed genes (DEGs) were identified between LEPH and HEPH, with 2021 genes up-regulated in LEPH and 2623 genes up-regulated in HEPH. In LEPH, up-regulated genes showed enrichment of the hypothalamo-pituitary-thyroid (HPT) axis. Beta-estradiol was identified as an upstream regulator regardless of tissue. When LEPH and HEPH samples were compared, beta-estradiol was activated in HEPH in 3 of the 4 comparisons, which correlated to the number of beta-estradiol target genes up-regulated in HEPH. In in vitro pituitary cell cultures from LEPH and HEPH, thyroid hormone pretreatment negatively impacted gonadotropin subunit mRNA levels in cells from both LEPH and HEPH, with the effect being more prominent in HEPH cells. Additionally, the effect of estradiol pretreatment on gonadotropin subunit mRNA levels in HEPH cells was negative, whereas estradiol pretreatment increased gonadotropin subunit mRNA levels in LEPH cells.Conclusions: Up-regulation of the HPT axis in LEPH and upstream beta-estradiol activation in HEPH may play a role in regulating HPG axis function, and ultimately ovulation rates. Furthermore, thyroid hormone and estradiol pretreatment impacted gonadotropin mRNA levels following GnRH stimulation, with the inhibitory effects of thyroid hormone being more detrimental in HEPH and estradiol stimulatory effects being more prominent in LEPH. Differential responsiveness to thyroid hormone and estradiol pretreatment may be due to desensitization of target genes to thyroid hormone and estradiol in LEPH and HEPH, respectively, in response general up-regulation of the HPT axis in LEPH and of the HPG axis in HEPH. Further studies will be necessary to identify possible target gene desensitization mechanisms and elicit the full role that the HPT axis and beta-estradiol upstream regulation play in egg production rates in turkey hens.

Background The fasting-refeeding perturbation has been used extensively to reveal specific genes and metabolic pathways that control energy metabolism in the chicken. Most global transcriptional scans of the fasting-refeeding response in liver have focused on juvenile chickens that were 1, 2 or 4 weeks old. The present study was aimed at the immediate post-hatch period, in which newly-hatched chicks were subjected to fasting for 4, 24 or 48 h, then refed for 4, 24 or 48 h, and compared with a fully-fed control group at each age (D1-D4). Results Visual analysis of hepatic gene expression profiles using hierarchical and K-means clustering showed two distinct patterns, genes with higher expression during fasting and depressed expression upon refeeding and those with an opposing pattern of expression, which exhibit very low expression during fasting and more abundant expression with refeeding. Differentially-expressed genes (DEGs), identified from five prominent pair-wise contrasts of fed, fasted and refed conditions, were subjected to Ingenuity Pathway Analysis. This enabled mapping of analysis-ready (AR)-DEGs to canonical and metabolic pathways controlled by distinct gene interaction networks. The largest number of hepatic DEGs was identified by two contrasts: D2FED48h/D2FAST48h (968 genes) and D2FAST48h/D3REFED24h (1198 genes). The major genes acutely depressed by fasting and elevated upon refeeding included ANGTPL, ATPCL, DIO2, FASN, ME1, SCD, PPARG, SREBP2 and THRSPA—a primary lipogenic transcription factor. In contrast, major lipolytic genes were up-regulated by fasting or down-regulated after refeeding, including ALDOB, IL-15, LDHB, LPIN2, NFE2L2, NR3C1, NR0B1, PANK1, PPARA, SERTAD2 and UPP2. Conclusions Transcriptional profiling of liver during fasting/re-feeding of newly-hatched chicks revealed several highly-expressed upstream regulators, which enable the metabolic switch from fasted (lipolytic/gluconeogenic) to fed or refed (lipogenic/thermogenic) states. This rapid homeorhetic shift of whole-body metabolism from a catabolic-fasting state to an anabolic-fed state appears precisely orchestrated by a small number of ligand-activated transcription factors that provide either a fasting-lipolytic state (PPARA, NR3C1, NFE2L2, SERTAD2, FOX01, NR0B1, RXR) or a fully-fed and refed lipogenic/thermogenic state (THRSPA, SREBF2, PPARG, PPARD, JUN, ATF3, CTNNB1). THRSPA has emerged as the key transcriptional regulator that drives lipogenesis and thermogenesis in hatchling chicks, as shown here in fed and re-fed states.

Commercial selection for meat-type (broiler) chickens has produced economically valuable birds with fast growth rates, enhanced muscle mass, and highly efficient feed utilization. The physiological changes that account for this improvement and unintended consequences associated with them remain largely unexplored, despite their potential to guide further advancements in broiler production efficiency. To identify effects of genetic selection on hormonal signaling in the adrenocorticotropic and thyrotropic axes, gene expression in muscle and liver and post-hatch circulating hormone concentrations were measured in legacy [Athens Canadian Random Bred (ACRB)] and modern (Ross 308) male broilers between embryonic days (e) 10 and e18 and post-hatch days (d) 10 and d40. No interactive effects or main effects of line were observed for adrenocorticotropic gene expression during either developmental period, although age effects appeared for corticosteroid-binding globulin in liver during embryogenesis and post-hatch and glucocorticoid receptor in both tissues post-hatch. There was a main line effect for circulating corticosterone, with levels in ACRB greater than those in Ross. Several thyrotropic genes exhibited line-by-age interactions during embryonic or post-hatch development. In liver, embryonic expression of thyroid hormone receptor beta was greater in ACRB on e12, and deiodinase 3 (DIO3) levels were greater in Ross on e14 and e16. In juvenile liver, deiodinase 2 (DIO2) expression was greater in ACRB on d10 but greater in Ross on d20, while DIO3 was higher in ACRB on d30 and d40. Levels of thyroid hormone receptor alpha mRNA exhibited a main line effect, with levels greater in ACRB juvenile breast muscle. Several thyrotropic genes exhibited main age effects, including DIO2 and DIO3 in embryonic breast muscle, thyroid hormone receptor alpha and thyroid hormone receptor beta in post-hatch liver, and DIO2 in post-hatch breast muscle. Circulating triiodothyronine displayed a main line effect, with levels in Ross significantly reduced as compared to ACRB. These findings suggest that in modern broilers, a decrease in levels of hormones that control basal metabolism triiodothyronine and the stress response circulating corticosterone, as well as altered expression of genes regulating thyroid hormone activity, could contribute to lower heat production, reduced stress response, and altered nutrient partitioning, leading to more efficient feed utilization and faster, more productive growth.

The mesotocinergic (MTergic) and dopaminergic (DAergic) systems have been documented to play pivotal roles in maternal behaviors in native Thai chickens. In native Thai chickens, plasma prolactin (PRL) concentrations are associated with maternal behaviors, which are also controlled by the DAergic system. However, the role of MT in conjunction with the roles of DA and PRL on the neuroendocrine regulation of the transition from incubating to rearing behavior has never been studied. Therefore, the aim of this study was to investigate the association of MT, DA, and PRL during the transition from incubating to rearing behavior in native Thai hens. Using an immunohistochemistry technique, the numbers of MT-immunoreactive (-ir) and tyrosine hydroxylase-ir (TH-ir, a DA marker) neurons were compared between incubating hens (INC; n = 6) and hens for which the incubated eggs were replaced with 3 newly hatched chicks for 3 days after 6, 10, and 14 days of incubation (REC; n = 6). Plasma PRL concentrations were determined by enzyme-linked immunosorbent assay. The results revealed that the numbers of MT-ir neurons within the nucleus supraopticus, pars ventralis (SOv), nucleus preopticus medialis (POM), and nucleus paraventricularis magnocellularis (PVN) increased in the REC hens when compared with those of the INC hens at 3 different time points (at days 9, 13, and 17). On the other hand, the number of TH-ir neurons in the nucleus intramedialis (nI) decreased in the REC13 and REC17 hens when compared with those of the INC hens. However, the number of TH-ir neurons in the nucleus mamillaris lateralis (ML) only decreased in the REC13 hens when compared with the INC13 hens. The decrease in the numbers of TH-ir neurons within the nI and ML is associated with the decrease in the levels of plasma PRL. This study suggests that the presence of either eggs or chicks is the key factor regulating the MTergic system within the SOv, POM, and PVN and the DAergic system within the nI and ML during the transition from incubating to rearing behavior in native Thai chickens. The results further indicate that these two systems play pivotal roles in the transition from incubating to rearing behavior in this equatorial species.

Decades of intensive genetic selection in the domestic chicken (Gallus gallus domesticus) have enabled the remarkable rapid growth of today's broiler (meat-type) chickens. However, this enhanced growth rate was accompanied by several unfavorable traits (i.e., increased visceral fatness, leg weakness, and disorders of metabolism and reproduction). The present descriptive analysis of the abdominal fat transcriptome aimed to identify functional genes and biological pathways that likely contribute to an extreme difference in visceral fatness of divergently selected broiler chickens., We used the Del-Mar 14 K Chicken Integrated Systems microarray to take time-course snapshots of global gene transcription in abdominal fat of juvenile [1-11 weeks of age (wk)] chickens divergently selected on bodyweight at two ages (8 and 36 wk). Further, a RNA sequencing analysis was completed on the same abdominal fat samples taken from high-growth (HG) and low-growth (LG) cockerels at 7 wk, the age with the greatest divergence in body weight (3.2-fold) and visceral fatness (19.6-fold)., Time-course microarray analysis revealed 312 differentially expressed genes (FDR ≤ 0.05) as the main effect of genotype (HG versus LG), 718 genes in the interaction of age and genotype, and 2918 genes as the main effect of age. The RNA sequencing analysis identified 2410 differentially expressed genes in abdominal fat of HG versus LG chickens at 7 wk. The HG chickens are fatter and over-express numerous genes that support higher rates of visceral adipogenesis and lipogenesis. In abdominal fat of LG chickens, we found higher expression of many genes involved in hemostasis, energy catabolism and endocrine signaling, which likely contribute to their leaner phenotype and slower growth. Many transcription factors and their direct target genes identified in HG and LG chickens could be involved in their divergence in adiposity and growth rate., The present analyses of the visceral fat transcriptome in chickens divergently selected for a large difference in growth rate and abdominal fatness clearly demonstrate that abdominal fat is a very dynamic metabolic and endocrine organ in the chicken. The HG chickens overexpress many transcription factors and their direct target genes, which should enhance in situ lipogenesis and ultimately adiposity. Our observation of enhanced expression of hemostasis and endocrine-signaling genes in diminished abdominal fat of LG cockerels provides insight into genetic mechanisms involved in divergence of abdominal fatness and somatic growth in avian and perhaps mammalian species, including humans.

Previous studies demonstrated that in ovo photostimulation with monochromatic green light increases body weight and accelerates muscle development in broilers. The mechanism in which in ovo photostimulation accelerates growth and muscle development is not clearly understood. The objective of the current study was to define development of the somatotropic axis in the broiler embryo associated with in ovo green light photostimulation. Two-hundred-forty fertile broiler eggs were divided into 2 groups. The first group was incubated under intermittent monochromatic green light using light-emitting diode (LED) lamps with an intensity of 0.1 W\m2 at shell level, and the second group was incubated under dark conditions and served as control. In ovo green light photostimulation increased plasma growth hormone (GH) and prolactin (PRL) levels, as well as hypothalamic growth hormone releasing hormone (GHRH), liver growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-1) mRNA levels. The in ovo photostimulation did not, however, increase embryo's body weight, breast muscle weight, or liver weight. The results of this study suggest that stimulation with monochromatic green light during incubation increases somatotropic axis expression, as well as plasma prolactin levels, during embryonic development.

Heat stress (HS) is devastating to poultry production worldwide, yet its biology and molecular responses are not well defined. Although advances in management strategy have partially alleviated the negative impact of HS, productivity still continues to decline when the ambient temperature rises. Therefore, identifying mechanism-based approaches to decrease HS susceptibility while improving production traits is critical. Recently, we made a breakthrough by applying a surface wetting strategy and showing that it improves growth performance compared with the current conventional cooling system. In the present study, we aimed to further define molecular mechanisms associated with surface wetting in ameliorating HS productivity loss in broilers. Five-week-old broiler chickens were exposed to acute HS (35°C for 2 h) alone or in combination with surface wetting. A control group was maintained at thermoneutral conditions (25°C). Core body temperature (BT) and feed intake were recorded. Blood was collected and hypothalamic tissues (main site involved in the regulation of energy homeostasis) were harvested to determine the expression profile of stress- and metabolic-related genes. Surface wetting prevents HS from increasing BT and plasma corticosterone levels ( < 0.05) and improves feeding and drinking behaviors. At molecular levels, surface wetting blocks the activation of hypothalamic heat shock protein and adenosine monophosphate-activated protein-induced by HS and significantly modulates the expression of feeding-related hypothalamic neuropeptides (agouti-related protein, proopiomelanocortin, orexin, orexin receptor, and leptin receptor). Taken together, our data represent the first evidence that surface wetting alleviates systemic and intracellular stress induced by HS and preserves the intracellular energy status, which, in turn, may result in improved broiler well-being and growth performance.

Low egg producing hens (LEPH) ovulate less frequently than high egg producing hens (HEPH) and exhibit differences in mRNA levels for components of the hypothalamo-pituitary-gonadal (HPG) axis, suggesting differential responsiveness to trophic stimulation. Ovulation frequency is governed by the production and feedback of pituitary gonadotropins and ovarian follicle steroid hormones, which are regulated by HPG axis stimulation and inhibition at the hypothalamic level. Pituitary and follicle cells from LEPH and HEPH were subjected to in vitro hormonal treatments to stimulate or inhibit the HPG axis, followed by expression analysis of mRNA levels for HPG axis genes and radioimmunoassays for steroid hormone production. Statistical analysis was performed using the mixed models procedure of SAS. Pituitary cells from HEPH showed up-regulation of genes associated with ovulation stimulation, whereas LEPH cells showed up-regulation of genes associated with inhibition of ovulation. HEPH follicle cells displayed a higher sensitivity and responsiveness to gonadotropin treatment. Level of egg production impacted ovulation-related gene expression in pituitary cells as well as steroid hormone production in follicle cells, with HEPH displaying a greater positive response to stimulation. These findings indicate that differences in egg production among turkey hens likely involve differential responsiveness of the cells within the HPG axis.

Avian prolactin (PRL) secretion is under stimulatory control by the PRL-releasing factor (PRF), vasoactive intestinal peptide (VIP). The neuroendocrine regulation of the avian reproductive system has been extensively studied in females. However, there are limited data in males. The aim of this study was to elucidate the VIPergic system and its relationship to PRL and testosterone (T) in the male native Thai chicken. The distributions of VIP-immunoreactive (-ir) neurons and fibers were determined by immunohistochemistry. Changes in VIP-ir neurons within the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) areas were compared across the reproductive stages. Plasma levels of PRL and T were determined by enzyme-linked immunosorbent assay and then compared across the reproductive stages. The results revealed that the highest accumulations of VIP-ir neurons were concentrated only within the IH-IN, and VIP-ir neurons were not detected within other hypothalamic nuclei. Within the IH-IN, VIP-ir neurons were low in premature and aging males and markedly increased in mature males. Changes in VIP-ir neurons within the IH-IN were directly mirrored with changes in PRL and T levels across the reproductive stages. These results suggested that VIP neurons in the IH-IN play a regulatory role in year-round reproductive activity in males. The present study also provides additional evidence that VIP is the PRF in non-seasonal, continuously breeding equatorial species.

Heat stress (HS) has been reported to alter fat deposition in broilers, however the underlying molecular mechanisms are not well-defined. The objectives of the current study were, therefore: (1) to determine the effects of acute (2 h) and chronic (3 weeks) HS on the expression of key molecular signatures involved in hepatic lipogenic and lipolytic programs, and (2) to assess if diet supplementation with dried Noni medicinal plant (0.2% of the diet) modulates these effects. Broilers (480 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to two environmental conditions (heat stress, HS, 35°C vs. thermoneutral condition, TN, 24°C) and fed two diets (control vs. Noni) in a 2 × 2 factorial design. Feed intake and body weights were recorded, and blood and liver samples were collected at 2 h and 3 weeks post-heat exposure. HS depressed feed intake, reduced body weight, and up regulated the hepatic expression of heat shock protein HSP60, HSP70, HSP90 as well as key lipogenic proteins (fatty acid synthase, FASN; acetyl co-A carboxylase alpha, ACCα and ATP citrate lyase, ACLY). HS down regulated the hepatic expression of lipoprotein lipase (LPL) and hepatic triacylglycerol lipase (LIPC), but up-regulated ATGL. Although it did not affect growth performance, Noni supplementation regulated the hepatic expression of lipogenic proteins in a time- and gene-specific manner. Prior to HS, Noni increased ACLY and FASN in the acute and chronic experimental conditions, respectively. During acute HS, Noni increased ACCα, but reduced FASN and ACLY expression. Under chronic HS, Noni up regulated ACCα and FASN but it down regulated ACLY. In vitro studies, using chicken hepatocyte cell lines, showed that HS down-regulated the expression of ACCα, FASN, and ACLY. Treatment with quercetin, one bioactive ingredient in Noni, up-regulated the expression of ACCα, FASN, and ACLY under TN conditions, but it appeared to down-regulate ACCα and increase ACLY levels under HS exposure. In conclusion, our findings indicate that HS induces hepatic lipogenesis in chickens and this effect is probably mediated via HSPs. The modulation of hepatic HSP expression suggest also that Noni might be involved in modulating the stress response in chicken liver.

Mesotocin (MT), a homolog of oxytocin (OT) in mammals, is a nonapeptide neurohypophysial hormone that is mainly synthesized in specific neuronal groups within the hypothalamus and released from the posterior pituitary gland in amphibian, reptilian, and avian species. MT is associated with the neuroendocrine regulation of reproductive cycle and maternal behaviors in female native Thai chickens. Male birds exhibit parental behaviors as well. However, there are limited data regarding the role(s) of the MTergic system in males. Thus, the objective of this study was to elucidate the localization of the MT neuronal groups in the brain of male native Thai chickens. The distributions of MT-immunoreactive (-ir) neurons and fibers in the brain were studied utilizing immunohistochemistry technique. The results revealed that MT-ir neurons and fibers were distributed throughout the brain and extensively in the diencephalon. MT-ir neurons and fibers were predominantly located within the nucleus supraopticus, pars ventralis (SOv), nucleus preopticus medialis (POM), nucleus ventrolateralis thalami (VLT), nucleus paraventricularis magnocellularis (PVN), and regio lateralis hypothalami (LHy), suggesting that MT neurons in these nuclei might be involved in the reproductive activities and/or parental behavior in the male chickens. In addition, the numbers of MT-ir neurons within the SOv and POM were significantly higher than those of the VLT, PVN, and LHy. More importantly, the number of MT-ir neurons in the SOv was high in the male brain when compared with the female brain, indicating that the MTergic system in the SOv might play a significant role in male reproductive activities in this equatorial species.

Avian mesotocin (MT) is homologous to oxytocin in mammals. Native Thai chickens ( Gallus domesticus ) strongly express maternal behaviors including incubation and rearing. However, the role of MT during incubation behavior has never been studied. The objective of this study was to determine the physiological function(s) of the MTergic system in incubation behavior in native Thai chickens. The brains were collected from incubating (INC) and nest-deprived (ND) hens at different time points (days 3, 6, 8, 10, 14, 18, and 21; n = 6). Immunohistochemistry technique was used to compare the numbers of MT-immunoreactive (-ir) neurons between the INC and ND hens within the Nucleus supraopticus, pars ventralis (SOv), Nucleus preopticus medialis (POM), and Nucleus paraventricularis magnocellularis (PVN). The results revealed that the numbers of MT-ir neurons within the SOv, POM, and PVN remained high during the incubating stage. The number of MT-ir neurons in the SOv was lower than that of the POM and PVN. Disruption of incubation behavior by nest deprivation caused the numbers of MT-ir neurons within the SOv, POM, and PVN to decrease throughout the observation periods. For the first time, this study demonstrates that the MTergic system within the SOv, POM, and PVN may be involved with incubation behavior. In addition, these results further suggest that the MTergic neurons in these nuclei are not only regulated by rearing behavior but also might have a role in the initiation and maintenance of incubation behavior in this tropical species.

International audience; In mammals, insulin regulates blood glucose levels and plays a key regulatory role in appetite via the hypothalamus. In contrast, chickens are characterized by atypical glucose homeostasis, with relatively high blood glucose levels, reduced glucose sensitivity of pancreatic beta cells, and large resistance to exogenous insulin. The aim of the present study was to investigate in chickens the effects of 5 h fasting and 5 h insulin immuno-neutralization on hypothalamic mRNA levels of 23 genes associated with food intake, energy balance, and glucose metabolism. We observed that insulin immune-neutralization by administration of anti-porcine insulin guinea pig serum (AI) significantly decreased food intake and increased plasma glucose levels in chickens, while 5 h fasting produced a limited and non-significant reduction in plasma glucose. In addition, 5 h fasting increased levels of NPY, TAS1R1, DIO2, LEPR, GLUT1, GLUT3, GLUT8, and GCK mRNA. In contrast, AI had no impact on the levels of any selected mRNA. Therefore, our results demonstrate that in chickens, food intake inhibition or satiety mechanisms induced by insulin immuno-neutralization do not rely on hypothalamic abundance of the 23 transcripts analyzed. The hypothalamic transcripts that were increased in the fasted group are likely components of a mechanism of adaptation to fasting in chickens.

Within the anterior pituitary gland, glucocorticoids such as corticosterone (CORT) provide negative feedback to inhibit adrenocorticotropic hormone secretion and act to regulate production of other hormones including growth hormone (GH). The ontogeny of GH production during chicken embryonic and rat fetal development is controlled by glucocorticoids. The present study was conducted to characterize effects of glucocorticoids on gene expression within embryonic pituitary cells and to identify genes that are rapidly and directly regulated by glucocorticoids. Chicken embryonic pituitary cells were cultured with CORT for 1.5, 3, 6, 12, and 24 h in the absence and presence of cycloheximide (CHX) to inhibit protein synthesis. RNA was analyzed with custom microarrays containing 14,053 chicken cDNAs, and results for selected genes were confirmed by quantitative reverse transcription real-time PCR (qRT-PCR). Levels of GH mRNA were maximally induced by 6 h of CORT treatment, and this response was blocked by CHX. Expression of 396 genes was affected by CORT, and of these, mRNA levels for 46 genes were induced or repressed within 6 h. Pathway analysis of genes regulated by CORT in the absence of CHX revealed networks of genes associated with endocrine system development and cellular development. Eleven genes that were induced within 6 h in the absence and presence of CHX were identified, and eight were confirmed by qRT-PCR. The expression profiles and canonical pathways defined in this study will be useful for future analyses of glucocorticoid action and regulation of pituitary function.

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5′-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5′-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

Pit1, pituitary-specific transcription factor 1, regulates differentiation of cells of the Pit1 lineage in the anterior pituitary and the synthesis of peptide hormones by these cell types, including GH. Pit1 is characterized by an N-terminal transactivation domain and a C-terminal POU domain. Alternative forms of Pit1, differing from each other in the N-terminal domain, have been reported in several species, but the functional implication of having multiple isoforms is not known. Several PIT1 mRNA transcripts exist in chickens that have not been characterized. This study was conducted to determine which, if any, of the chicken Pit1 isoforms regulate the chicken GH (cGH) promoter. During the course of this work, Pit1β2, a novel isoform of chicken Pit1, was discovered. Effects of known and novel isoforms (Pit1α, Pit1β1, Pit1β2, and Pit1γ) on cGH promoter activity were characterized in chicken Leghorn male hepatoma cells. Three of the isoforms, Pit1α, Pit1β1, and Pit1β2, activated the cGH promoter, whereas Pit1γ did not. Results from gel-shift assays indicated that Pit1γ does not bind to the proximal Pit1-bindng site of the cGH promoter, suggesting a possible mechanism underlying its inactivity. We found a functional advantage for having multiple isoforms expressed. When Pit1β1 was coexpressed with Pit1α or Pit1β2, significantly greater activation of the cGH promoter occurred than with any one isoform alone, with synergistic activation occurring when Pit1α and Pit1β1 were coexpressed. Whether this increased activation required, or was facilitated by, heterodimerization of two isoforms is not known. Identification of isoforms with specific functions will facilitate identification of their respective interacting partners that are essential for GH gene expression.

Hyperprolactinemia is associated with incubation behavior and ovarian regression in birds. To investigate the association of prolactin (PRL), vasoactive intestinal peptide (VIP), and dopamine (DA) with the neuroendocrine regulation of incubation behavior, changes in the number of visible VIP-immunoreactive (VIP-ir) neurons in the nucleus inferioris hypothalami (IH) and nucleus infundibuli hypothalami (IN) and tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the nucleus intramedialis (nI) and nucleus mamillaris lateralis (ML) of incubating native Thai hens were compared with those of nest-deprived hens. TH was used as a marker for dopaminergic (DAergic) neurons. Blood samples were collected to determine PRL levels. The localization and the number of visible VIP-ir and TH-ir neurons were determined by immunohistochemistry. Disruption of incubation behavior was accompanied by a precipitous decline in plasma PRL levels. The number of visible VIP-ir neurons in the IH-IN and TH-ir neurons in the nI and ML were high during incubation and decreased when hens were deprived of their nests. This study indicated an association between VIP neurons in the IH-IN and DA neurons in the nI and ML with the degree of hyperprolactinemia, suggesting that the expression of incubation behavior in birds might be, in part, regulated by the DAergic input from the nI and ML to VIP neurons in the IH-IN and subsequent PRL release.

The anterior pituitary gland plays an important role in the regulation of many physiological processes. Formation of Rathke's pouch (RP), the precursor of the anterior pituitary, involves evagination of the oral ectoderm in a multi-step process regulated by cell interactions, signaling pathways, and transcription factors. Chickens are an excellent model to study development because of the availability of large sample sizes, accurate timing of development, and embryo accessibility. The aim of this study was to quantify mRNA expression patterns in the developing chicken anterior pituitary to evaluate the chicken embryo as a model for mammalian pituitary development. The expression profiles of 16 genes differentially expressed in RP and neuroectoderm were determined in this study. Among these, Pitx1, Pitx2, and Hesx1 mRNA levels were high on embryonic days (e) 2.5 to e3 in RP and decreased during development. Expression of Pit1 and Tbx19 mRNA in RP reached the highest levels by e7 and e6.5, respectively. Levels of glycoprotein subunit α mRNA increased beginning at e4. FGF8 mRNA showed the highest expression at e3 to e3.5 in neuroectoderm. BMP2 showed slight decreases in mRNA expression in both tissues during development, while Isl1 and Noggin mRNA expression increased in later development. Taken together, we present the first quantitative transcriptional profile of pituitary organogenesis. Our results will help further understanding of the functional development of this gland. Moreover, because of the high similarity in gene expression patterns observed between chicken and mouse, chickens could serve as an excellent model to study genetic and molecular mechanisms underlying pituitary development.

The neuroendocrine system consists of five major hypothalamic-pituitary hormone axes that regulate several important metabolic processes, and it develops in all vertebrates during embryogenesis. In order to define initiation and establishment of these five axes, mRNA expression profiles of hypothalamic releasing and release-inhibiting factors, their pituitary receptors, and pituitary hormones were characterized during the second half of embryogenesis and first week post-hatch in the chick. Axis initiation was defined as the age when pituitary hormone mRNA levels began to increase substantially, and establishment was defined as the age when mRNA for all components had reached maximum expression levels. The adrenocorticotropic axis appears established by e12, as there were no major increases in gene expression after that age. Hypothalamic thyrotropin-releasing hormone and pituitary thyroid-stimulating hormone β-subunit increased between e10 and e18, indicating establishment of the thyrotropic axis during this period. Pituitary growth hormone substantially increased on e16, and hypothalamic growth hormone-releasing hormone did not increase until e20, indicating that somatotropic axis activity is established late in embryonic development. Lactotropic axis initiation is evident just prior to hatch, as pituitary prolactin and vasoactive intestinal peptide receptor 1 did not increase until e18 and e20, respectively. Hypothalamic gonadotropin-releasing hormone 1 increased after hatch, and pituitary luteinizing hormone β-subunit expression remained low until d3, indicating the gonadotropic axis is not fully functional until after hatching. This study is the first to characterize major hypothalamic and pituitary components of all five neuroendocrine axes simultaneously and considerably increases our understanding of neuroendocrine system establishment during development.

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.

Hypothalamic neuropeptides, neurotrophins, and systemic hormones modulate food intake and body composition. Although advances toward elucidating these interactions have been made, many aspects of the underlying mechanisms remain vague. Hypothalami from fat and lean chicken lines were assessed for differential expression of anabolic/orexigenic and catabolic/anorexigenic genes. Effects of triiodothyronine (T3), corticosterone (Cort), and brain-derived neurotrophic factor (BDNF) on expression of anabolic/orexigenic and catabolic/anorexigenic genes were tested in cultures of hypothalamic neurons. From this, we found that BDNF increased and T3decreased gene expression for BDNF, leptin receptor (LEPR), pro-opiomelanocortin (POMC), thyrotropin releasing hormone (TRH), and agouti-related protein (AGRP). Thyroid hormone levels were manipulated during development to show that T3inhibited BDNF, TRH, and BDNF receptor gene expression. Delivery of T3, Cort, T3plus Cort, or vehicle in vivo continuously for 72 h indicated that Cort and T3have overlapping roles in regulating TRH, LEPR, and POMC gene expression and that Cort and T3regulate BDNF, neuropeptide Y, and AGRP in opposite directions. Collectively, these findings suggest that interactions between the neuropeptide BDNF and the hormones T3and/or Cort may constitute a homeostatic mechanism that links hypothalamic energy regulation controlling body composition.

The expression profile of glucocorticoid-induced leucine zipper (GILZ) in the anterior pituitary during the second half of embryonic development in the chick is consistent with in vivo regulation by circulating corticosteroids. However, nothing else has been reported about the presence of GILZ in the neuroendocrine system. We sought to characterize expression and regulation of GILZ in the chicken embryonic pituitary gland and determine the effect of GILZ overexpression on anterior pituitary hormone levels. Pituitary GILZ mRNA levels increased during embryogenesis to a maximum on the day of hatch, and decreased through the first week after hatch. GILZ expression was rapidly upregulated by corticosterone in embryonic pituitary cells. To determine whether GILZ regulates hormone gene expression in the developing anterior pituitary, we overexpressed GILZ in embryonic pituitary cells and measured mRNA for the major pituitary hormones. Exogenous GILZ increased prolactin mRNA above basal levels, but not as high as that in corticosterone-treated cells, indicating that GILZ may play a small role in lactotroph differentiation. The largest effect we observed was a twofold increase in FSH β subunit in cells transfected with GILZ but not treated with corticosterone, suggesting that GILZ may positively regulate gonadotroph development in a manner not involving glucocorticoids. In conclusion, this is the first report to characterize avian GILZ and examine its regulation in the developing neuroendocrine system. We have shown that GILZ is upregulated by glucocorticoids in the embryonic pituitary gland and may regulate expression of several pituitary hormones.

Treatment of fetal rats and embryonic chickens with exogenous glucocorticoids induces premature GH cell differentiation. However, it is unknown whether the developing adrenal gland is capable of mounting this response autonomously. The present study determined whether stimulation of the adrenal gland in developing chicken embryos through administration of ACTH could induce a premature increase in GH cells. We found that plasma corticosterone and ACTH levels increased between embryonic day (e) 11 and e17, consistent with GH cell (somatotroph) ontogeny. Injection of ACTH into eggs on e9, e10, or e11 increased somatotrophs on e14. In contrast, thyroid-stimulating hormone, CRH, α-MSH, GHRH, and TRH were ineffective. Culture of e11 pituitary cells with ACTH failed to induce somatotrophs, suggesting an indirect action of ACTH on GH cells in vivo. Intravenous administration of ACTH dramatically increased plasma levels of corticosterone within 1 h and increased the percentage of pituitary somatotrophs within 24 h. Although ACTH administration increased the relative abundance of pituitary GH cells, there was no effect on plasma levels of GH, IGF-I, or IGF-II, or in hepatic expression of IGF-I or IGF-II mRNA. We conclude that ACTH administration can increase the population of GH cells in the embryonic pituitary. However, this treatment alone does not lead to downstream activation of hepatic IGF production. These findings indicate that the embryonic adrenal gland, and ultimately anterior pituitary corticotrophs, may function to regulate pituitary GH cell differentiation during embryonic development.

Glucocorticoid hormones are involved in functional differentiation of GH-producing somatotrophs. Glucocorticoid treatment prematurely induces GH expression in mammals and birds in a process requiring protein synthesis and Rat sarcoma (Ras) signaling. The objective of this study was to investigate mechanisms through which glucocorticoids initiate GH expression during embryogenesis, taking advantage of the unique properties of chicken embryos as a developmental model. We determined that stimulation of GH expression occurred through transcriptional activation of GH, rather than enhancement of mRNA stability, and this process requires histone deacetylase activity. Through pharmacological inhibition, we identified the ERK1/2 pathway as a likely downstream Ras effector necessary for glucocorticoid stimulation of GH. However, we also found that chronic activation of ERK1/2 activity with a constitutively active mutant or stimulatory ligand reduced initiation of GH expression by glucocorticoid treatment. Corticosterone treatment of cultured embryonic pituitary cells increased ERK1/2 activity in an apparent cyclical manner, with a rapid increase within 5 minutes, followed by a reduction to near-basal levels at 3 hours, and a subsequent increase again at 6 hours. Therefore, we conclude that ERK1/2 signaling must be strictly controlled for maximal glucocorticoid induction of GH to occur. These results are the first in any species to demonstrate that Ras- and ERK1/2-mediated transcriptional events requiring histone deacetylase activity are involved in glucocorticoid induction of pituitary GH during embryonic development. This report increases our understanding of the molecular mechanisms underlying glucocorticoid recruitment of somatotrophs during embryogenesis and should provide insight into glucocorticoid-induced developmental changes in other tissues and cell types.

GHRH stimulates GH secretion in chickens as in mammals. However, nothing is known about the chicken GHRH receptor (GHRH-R). Here we report the cDNA sequence of chicken GHRH-R. Comparison of the cDNA sequence with the chicken genome localized the GHRH-R gene to chicken chromosome 2 and indicated that the chicken GHRH-R gene consists of 13 exons. Expression of all exons was confirmed by RT-PCR amplification of pituitary mRNA. The amino acid sequence predicted by the GHRH-R cDNA is homologous to that in other vertebrates and contains seven transmembrane domains and a conserved hormone-binding domain. The predicted size of the GHRH-R protein (48.9 kDa) was confirmed by binding of 125I-GHRH to chicken pituitary membranes and SDS-PAGE. GHRH-R mRNA was readily detected by RT-PCR in the pituitary but not in the hypothalamus, total brain, lung, adrenal, ovary, or pineal gland. Effects of corticosterone (CORT), GHRH, ghrelin, pituitary adenylate cyclase-activating peptide, somatostatin (SRIF), and TRH on GHRH-R and GH gene expression were determined in cultures of chicken anterior pituitary cells. GHRH-R and GH mRNA levels were determined by quantitative real-time RT-PCR. Whereas all treatments affected levels of GH mRNA, only CORT, GHRH, and SRIF significantly altered GHRH-R mRNA levels. GHRH-R gene expression was modestly increased by GHRH and suppressed by SRIF at 4 h, and CORT dramatically decreased levels of GHRH-R mRNA at 72 h. We conclude that adrenal glucocorticoids may substantially impact pituitary GH responses to GHRH in the chicken through modulation of GHRH-R gene expression.

Increased thyroid hormone production is essential for hatching of the chick and for the increased metabolism necessary for posthatch endothermic life. However, little is known about the ontogeny and distribution of pituitary thyrotrophs during this period or whether pituitary thyroid-stimulating hormone (TSH) production is regulated by endogenous thyroid hormones during chick embryonic development. This study assessed the abundance and location of pituitary thyrotrophs and the regulation of TSHβ peptide and mRNA levels by endogenous thyroid hormones prior to hatching. TSHβ-containing cells were first detected on embryonic day (e) 11, and the thyrotroph population increased to maximum levels on e17 and e19 and then decreased prior to hatching (d1). Thyrotroph distribution within the cephalic lobe of the anterior pituitary was determined on e19 by whole-mount immunocytochemistry for TSHβ peptide and by whole-mount in situ hybridization for TSHβ mRNA. Thyrotroph distribution within the cephalic lobe was heterogeneous among embryos, but most commonly extended from the ventral medial region to the dorsal lateral regions, along the boundary of the cephalic and caudal lobes. Inhibition of endogenous thyroid hormone production with methi-mazole (MMI) decreased plasma thyroxine (T4) levels and increased pituitary TSHβ mRNA levels on e19 and d1. However, control pituitaries contained significantly more TSHβ peptide than MMI-treated pituitaries on e17 and e19, suggesting higher TSH secretion into the blood in MMI-treated groups. We conclude that thyrotroph abundance and TSH production increase prior to hatching, that thyrotrophs are localized heterogenenously within the cephalic lobe of the anterior pituitary at that time, and that TSH gene expression and secretion are under negative feedback regulation from thyroid hormones during this critical period of development.

The embryo of the domestic fowl (Gallus domesticus) tenders one distinctive advantage over general mammalian models for investigating the development of the hypothalamo-pituitary-adrenocortical (HPA) axis. This is the relative simplicity with which the embryonic endocrine environment can be influenced without confounding maternal influences. The ease of direct manipulation of the embryonic endocrine system has facilitated analysis of the development and function of the HPA axis in the chick embryo. As the chick embryo develops, functional activation of the adrenal gland is regulated at three different levels: the adrenal gland itself, the anterior pituitary, and the hypothalamus. The adrenal gland appears capable of independent secretion of glucocorticoids from day 8 until shortly after day 14 of embryonic development, at which point the pituitary influences adrenocortical activity. Around the same age, the hypothalamic level of control also begins. The information covered in this review will describe the major steps in the development of the HPA axis in the chicken embryo and show that the chicken has an emblematic HPA neuroendocrine axis.

Thyrotropin or thyroid-stimulating hormone (TSH) secretion in the chicken is controlled by several hypothalamic hormones. It is stimulated by thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH), whereas somatostatin (SRIH) exerts an inhibitory effect. In order to determine the mechanism by which these hypothalamic hormones modulate chicken TSH release, we examined the cellular localization of TRH receptors (TRH-R), CRH receptors type 1 (CRH-R1) and somatostatin subtype 2 receptors (SSTR2) in the chicken pars distalis by in situ hybridization (ISH), combined with immunological staining of thyrotropes. We show that thyrotropes express TRH-Rs and SSTR2s, allowing a direct action of TRH and SRIH at the level of the thyrotropes. CRH-R1 expression is virtually confined to corticotropes, suggesting that CRH-induced adrenocorticotropin release is the result of a direct stimulation of corticotropes, whereas CRH-stimulated TSH release is not directly mediated by the known chicken CRH-R1. Possibly CRH-induced TSH secretion is mediated by a yet unknown type of CRH-R in the chicken. Alternatively, a pro-opiomelanocortin (POMC)-derived peptide, secreted by the corticotropes following CRH stimulation, could act as an activator of TSH secretion in a paracrine way.

The purposes of this study were to develop a probe for the detection of thyroid-stimulating hormone (TSH) beta subunit mRNA, to validate the usefulness of that probe in measuring TSH, and to use it to investigate the effects of thyroid suppression on TSH and the reproductive axis in Japanese quail. The objectives of experiment 1 were to isolate and characterize a partial cDNA for quail TSH and validate a riboprobe transcribed from this cDNA. This riboprobe was then used to assess changes in TSHbeta mRNA levels in Japanese quail. We isolated a cDNA of 168 bp with 94% identity to the corresponding sequence in chicken TSHbeta. The transcribed riboprobe was shown to be pituitary gland specific, and differences in TSHbeta mRNA levels were detectable with 2.5 microg of total RNA in Northern blot analysis. In experiment 2, our objective was to determine if thyroid inhibition would result in a detectable change in TSHbeta mRNA and alterations in the pituitary luteinizing hormone (LH) or indices of gonadal function. We used adult, reproductively active, male Japanese quail on a long-day photoperiod. Treatment with a goitrogen, methimazole (MMI), increased (P < 0.05) thyroid gland and liver weights and decreased (P < 0.05) serum thyroxine (T4) concentrations compared to control birds. We detected increased TSHbeta mRNA in the pituitaries of MMI-treated birds compared to controls. There was no effect of MMI treatment on the reproductive variables measured, including LHbeta mRNA levels, serum androgen and estradiol concentrations, gonad weight, or cloacal gland area. Therefore, it appears that thyroid axis inhibition and the consequent increase in TSHbeta mRNA did not have direct effects on reproductive axis function in male Japanese quail.

Prior research indicates that growth hormone (GH) cell differentiation can be induced prematurely by treatment with glucocorticoids in vitro and in vivo. However, the nature of these responses has not been fully characterized. In this study, the time course of corticosterone induction of GH-secreting cells in cultures of chicken embryonic pituitary cells, responsiveness of differentiated somatotrophs to GH secretagogues, localization of somatotroph precursor cells within the pituitary gland, and the effect of corticosterone on GH gene expression were determined to better define the involvement of glucocorticoids in somatotroph recruitment during development. Anterior pituitary cells from embryonic day 12 chicken embryos were cultured in 10(-9) M corticosterone for 4 to 48 h and were then subjected to reverse haemolytic plaque assays (RHPAs) for GH. Corticosterone treatment for as short as 16 h increased the percentage of GH cells compared with the control. When corticosterone was removed after 48 h and cells were cultured for an additional 3 days in medium alone, the percentage of GH secretors decreased but remained greater than the proportion of somatotrophs among cells that were never treated with corticosterone. To determine if prematurely differentiated somatotrophs were responsive to GH secretagogues, cells were exposed to corticosterone for 48 h and then subjected to GH RHPAs in the presence or absence of GH-releasing hormone (GHRH) or thyrotropin-releasing hormone (TRH). Approximately half of the somatotrophs induced to differentiate with corticosterone subsequently released more GH in response to GHRH and TRH than in their absence. The somatotroph precursor cells were localized within the anterior pituitary by culturing cells from the caudal lobe and cephalic lobe of the anterior pituitary separately. Corticosterone induction of GH cells was substantially greater in cultures derived from the caudal lobe of the anterior pituitary, where somatotroph differentiation normally occurs. GH gene expression was evaluated by ribonuclease protection assay and by in situ hybridization. Corticosterone increased GH mRNA in cultured cells by greater than fourfold. Moreover, corticosterone-induced somatotroph differentiation involved GH gene expression in cells not expressing GH mRNA previously, and the extent of somatotroph differentiation was augmented by treatment with GHRH in combination with corticosterone. We conclude that corticosterone increases the number of GH-secreting cells within 16 h, increases GH gene expression in cells formerly not expressing this gene, confers somatotroph sensitivity to GHRH and TRH, and induces GH production in a precursor population found primarily in the caudal lobe of the anterior pituitary, a site consistent with GH localization in adults. These findings support the hypothesis that glucocorticoids function to induce the final stages in the differentiation of fully functional somatotrophs from cells previously committed to this lineage.

We reported that corticosterone administration into the albumen of fertile chicken eggs on embryonic day (e) 11 induces an increase in the population of GH-secreting cells. The present study evaluated the ontogeny, dose response, localization, and persistence of the glucocorticoid-induced increase in the somatotroph population during chicken embryonic development. Corticosterone (0, 0.02, 0.2, and 2 microg in 300 microl saline) was injected into separate eggs on e9, e10, e11, and e12, and the population of GH-secreting cells was assessed 2 days later using reverse hemolytic plaque assays. Corticosterone treatment on e9 or e10 was unable to increase the population of GH-secreting cells on e11 or e12. In contrast, 0.2 and 2 microg of corticosterone on e11 increased the population of GH-secreting cells on e13 (P < 0.05, n = 3 experiments) to 8.2 +/- 0.6 and 6.4 +/- 0.5% of all cells, respectively, relative to controls (2.4 +/- 0.2%). For e14 embryos treated on e12, only the 2 microg dose increased the proportion of GH-secreting cells (6.4 +/- 0.6%) relative to controls (3.6 +/- 0.4%). In a second experiment, 0, 0.02, 0.2, 2, and 20 microg of corticosterone were injected on e0, e8, e9, e10, e11, and e12, and the population of GH-secreting cells was assessed on e13 in all groups. No dose of corticosterone was effective when given on e0, e8, e9, or e10. The 0.2 microg and 2 microg doses increased the population of GH-secreting cells (7.6 +/- 0.9% and 6.7 +/- 0.8%, respectively) relative to controls (2.3 +/- 0.4%) when injected on e11 (P < 0.05, n = 4 experiments). The 2-microg dose also increased GH cell abundance when injected on e12 (5.6 +/- 0.4%), relative to controls (2.7 +/- 0.5%). Treatment with 20 microg on e11 and e12 induced the greatest responses (10.3 +/- 1.1% and 8.7 +/- 0.9%, respectively). However, in subsequent experiments, administration of 20 microg on e11 resulted in embryonic death by e18. In a third set of experiments, two groups of eggs were injected either with 2 microg of corticosterone in saline or saline alone on e11, and the number of GH-secreting cells was estimated on e13, e16, e19, and the day of hatch (d1). The population of GH-secreting cells in corticosterone treated embryos was significantly higher than in saline treated embryos only on e13 (7.1 +/- 0.8% and 2.7 +/- 0.3%, respectively). No significant differences were observed on e16 (12.4 +/- 1.5% and 13.6 +/- 1.2%), e19 (19.0 +/- 1.0% and 18.2 +/- 1.7%) and d1 (23.8 +/- 2.1% and 25.1 +/- 1.8%) between corticosterone treated and control embryos, respectively. In a fourth set of experiments, whole mount in situ hybridization indicated that injection of corticosterone on e11 induced GH messenger RNA expression in the caudal part of the pituitary gland on e13, where somatotrophs are located normally later in development. We conclude that corticosterone administration in ovo can increase the population of GH-secreting cells in the caudal anterior pituitary only during a small window of development between e11 and e13 and that this premature increase of GH-secreting cells does not affect the percentage of GH-secreting cells later in development.

Using the reverse hemolytic plaque assay described in the present investigation, a secretagogue activity of bursal antisteroidogenic peptide (BASP) for growth hormone (GH) or prolactin (PRL) secretion was observed in chicken Day 20e pituitary cell monolayers. Partially purified BASP (ppBASP), at all concentrations evaluated (0.25 BEQ/ml, 0.75 BEQ/ml, or 1.5 BEQ/ml), induced PRL secretion by isolated lactotrophs above (P < 0.05) basal levels during the 2- and 6-hr incubation. At the 18-hr time point, neither ppBASP nor vasoactive intestinal polypeptide (VIP) was efficacious (P < 0.05) in causing an elevation in PRL-secreting cells above basal levels. ppBASP, at all concentrations evaluated (0.25 BEQ/ml, 0.75 BEQ/ml, or 1.5 BEQ/ml), caused an increase in the percentage of GH-secreting cells above (P < 0.05) basal levels during the 18-hr incubation. When evaluating the 2-hr time point alone, ppBASP, at 0.75 or 1.5 BEQ/ml, significantly (P < 0.05) elevated the percentage of GH-secreting cells to above basal levels. After the 6-hr incubation, ppBASP at 0.25 or 0.75 BEQ/ml, was efficacious in causing elevated (P < 0.05) GH secretion above basal levels. The present study indicates a secretagogue activity of BASP on PRL or GH secretion by chicken embryonic anterior pituitary cells in vitro.

Somatotroph differentiation in the embryonic pituitary of avian and mammalian species can be stimulated by glucocorticoids in vitro, and this effect can be augmented by concomitant treatment with growth hormone-releasing hormone (GHRH). Owing to its isolation from maternal influences, the chick embryo is a useful model for studying humoral regulation of pituitary cell differentiation. Somatotroph differentiation in chickens occurs between embryonic day (e-) 14 and e-16, and treatment of e-12 pituitary cells with e-16 serum or corticosterone induces growth hormone (GH) cell differentiation within 2 d in culture. The objective of the present study was to determine whether direct administration of embryonic serum and corticosterone to developing chick embryos was effective in vivo in inducing somatotroph differentiation prematurely. The albumen of fertile eggs was injected on e-11 with 300 approximately microL of 0.9% saline or 150 microL of serum from e-12 or e-16 chick embryos diluted 1:1 with saline. The embryos were allowed to develop until e-14, when pituitaries were dispersed and the resulting pituitary cells were subjected to reverse hemolytic plaque assays (RHPA) and immunocytochemistry to detect GH-secreting and GH-containing cells, respectively. Injection of e-16 serum increased (p0.01) GH-secreting and GH-containing cells to 11.5 +/- 1.0% and 1 7.4 +/- 3.3% of all pituitary cells, compared to 5.0 +/- 0.3% and 5.5 +/- 0.9% for saline-injected controls, respectively. Day 12 serum increased GH-containing cells to 9.8 +/- 0.9%, without changing percentages of GH-secreting cells. In experiment 2, saline, e-16 serum, and corticosterone were injected on e-11, and pituitary cells were subjected to GH RHPA on e-14. GH secretors were increased by e-16 serum and corticosterone. In experiment 3, we tested whether GHRH would magnify the effect of corticosterone, as we had seen in extended 6-d cultures previously. Saline, corticosterone, and corticosterone plus GHRH were injected on e-11, and pituitary cells were subjected to GH RHPA on e-18. Treatment with corticosterone alone and combined with GHRH increased the percentage of GH-secreting cells. However, combined treatment with corticosterone and GHRH was not more effective than corticosterone alone. The present findings demonstrate that glucocorticoid administration can stimulate somatotroph differentiation in living vertebrate embryos isolated from maternal interactions.

It was previously reported that prolactin (PRL)-containing cells differentiate by days 15 to 19 of embryonic development and that vasoactive intestinal peptide (VIP) increased plasma PRL concentrations and pituitary PRL protein and mRNA levels in vivo. In the present study, anterior pituitaries derived from day 15, 16, 17, 18, and 19 embryos were subjected to reverse hemolytic plaque assays (RHPA) for chicken PRL to determine the ontogeny of lactotrophs. We found that PRL secreting cells were first consistently detected on day 17 of embryonic development, indicating that lactotroph differentiation during normal development occurs by this age. However, extended treatment in the RHPA with VIP exposed lactotrophs as early as day 15, suggesting that a lactotroph precursor population was present earlier. Next, primary cultures of embryonic anterior pituitary cells from day 12, 13, 14, 15, 16, and 17 embryos were incubated for 0, 2, or 4 days in serum-free medium or medium supplemented with 10 nM VIP. After the culture periods, cells were subjected to RHPAs for chicken PRL. PRL-secreting cells differentiated spontaneously by 2 days in culture for day 15 and 16 cells and by 4 days in culture for all embryonic ages tested, except day 17. Culturing with VIP for 2 days did not increase PRL-secreting cells at any embryonic age tested, whereas VIP treatment for 4 days increased lactotroph numbers at all ages except day 12. Cells from days 15, 16, and 17 were responsive to VIP in the RHPA after 2 days in untreated cultures, but after 4 days only day 17 cells continued to respond to VIP in the RHPA. Treatment with VIP in culture for 4 days maintained VIP responsiveness in the RHPA for cells derived from embryos as early as day 14. We conclude that lactotroph differentiation during normal chicken development occurs by embryonic day 17. Moreover, our results indicate that responsiveness to VIP is an early event in PRL cell development and that lactotroph differentiation may be stimulated in vitro by VIP as early as embryonic day 13.

This study was designed to test for differences in somatotroph ontogeny, abundance, growth hormone (GH) secretion rate and GH-releasing hormone (GHRH) responsiveness between pituitaries of slow and fast growing chicken strains during embryonic development. Day 10, 12, 14 and 16 embryonic anterior pituitary cells from slow and fast growing chickens were subjected to reverse hemolytic plaque assays (RHPA) for GH. No differences were found in the day on which GH-secreting cells were first detected; somatotrophs were first present on day 14 of embryonic development for both strains. Similarly, no differences were found in the proportions of pituitary cells that secreted GH between the two strains at any of the ages tested. In contrast, differences were observed in GH secretory characteristics of individual somatotrophs between slow and fast growing embryos on day 16 of development, when a substantial somatotroph cell population was first present. Somatotrophs of fast growing embryos released more GH per hour in the presence of GHRH and had a greater capacity for GH release than those of slow growing birds. Furthermore, the majority of GH-secreting cells of fast growing embryos were responsive to GHRH on embryonic day 16, while less than half of the somatotrophs found in slow growing embryos were GHRH responsive. In contrast to these enhanced GH secretory characteristics in the fast growing strain during embryonic growth, a greater percentage of GH-secreting cells was found in the slow growing strain 5 weeks after hatch. It is concluded that differences in GH secretion during embryonic development may contribute to the increased growth rate of chickens selected for greater body weight.

Somatotrophs become a significant population by day 16 of chicken embryonic development. We have previously demonstrated that an earlier induction of GH cell differentiation is possible with the addition of day 16 embryonic serum to cultures of day 12 pituitary cells, an age when somatotrophs are rare. The present study was designed to identify the blood-borne signal(s) responsible for the serum activity, using reverse hemolytic plaque assays to identify individual GH-secreting cells. The activity was found to be a heat-stable, ether-soluble compound(s) that is bound or inhibited by a trypsin-sensitive protein. The extent of GH cell differentiation was greater (P < 0.05; n = 3) in response to the ether phases of heated day 16 (14.1 +/- 0.4% of all cells) and day 12 sera (9.3 +/- 0.4%) than with untreated serum from days 16 and 12 (6.1 +/- 0.4% and 0.82 +/- 0.4%, respectively). Furthermore, ether-extracted day 16 serum was more effective than ether-extracted day 12 serum, which was also different from basal (0.85 +/- 0.4%; P < 0.05). Based on this biochemical profile, the abilities of various steroids to stimulate differentiation were tested. Three steroids were found to stimulate somatotroph differentiation in vitro: 17beta-estradiol, corticosterone, and progesterone. However, the estradiol receptor antagonist, tamoxifen, while abolishing the effect of estradiol, had no effect on the induction of differentiation by day 16 serum. In contrast, RU486, a specific glucocorticoid receptor antagonist in chickens, blocked the stimulatory effects of corticosterone, progesterone, and day 16 serum on somatotroph differentiation. We next tested whether the active compound in day 16 embryonic serum was corticosterone, the predominant glucocorticoid in chickens. Incubation of day 16 serum with corticosterone antiserum, but not control antiserum, suppressed day 16 serum-induced GH cell differentiation. Therefore, we conclude that corticosterone is the blood-borne signal capable of stimulating somatotroph differentiation in vitro. The present findings together with previous reports indicate that somatotroph differentiation during embryonic development may result from an increase in circulating glucocorticoid concentrations.

We have previously demonstrated that chicken embryonic somatotropes are responsive to GHRH shortly after they differentiate. In contrast, relatively little is known about the regulation of GH secretion by somatostatin (SRIF) and IGF-I during chicken embryonic development. In the present study, anterior pituitary cells were isolated from day 16, 18 and 20 embryos and subjected to reverse hemolytic plaque assays (RHPAs) for chicken GH to assess the effect of SRIF and IGF-I on basal GH release and SRIF on GHRH-stimulated GH secretion. We found that all three ages responded to SRIF, under both basal and stimulated conditions. SRIF inhibition of basal GH release was evident for day 18 and 20 cells by 9 h, while 18 h were required for day 16 cells. After 18 h, 10−11 m SRIF depressed basal GH secretion by day 16 and 18 cells, while 10−9 m SRIF was required to depress GH plaque percentages by day 20 cells. GHRH stimulated GH release from all ages tested. After 2 h, SRIF partially suppressed GHRH-stimulated GH release by day 20 cells. After 6 h, day 18 cells responded to SRIF, reducing the percentage of plaque-forming cells under GHRH stimulated conditions. After 18 h, the percentage of day 16 cells forming GH plaques was reduced for cells treated with GHRH and SRIF, compared with cells treated with GHRH alone. All ages examined also responded to IGF-I. After 36 h, GH release by day 16 and 18 cells was decreased when exposed to IGF-I. While IGF-I decreased the relative amount of GH secreted per somatotrope on day 20, a paradoxical increase in the percentage of cells secreting GH was noted. These results indicate that anterior pituitary somatotropes are responsive to SRIF and IGF-I during late embryonic development of chickens. Journal of Endocrinology (1997) 154, 303–310

Exogenous gonadotrophins administered before AI can adversely alter endocrine dynamics and inhibit embryo development in felids. In the present study, we tested the hypothesis that priming the domestic cat ovary with progestin mitigates the negative influence of gonadotrophin therapy by normalising early embryogenesis and luteal function. Queens were given either: (1) progestin pretreatment plus chorionic gonadotrophins (n = 8; primed); or (2) gonadotrophins only (n = 8; unprimed). Ovulatory response was assessed laparoscopically, and cats with fresh corpora lutea (CL) were inseminated in utero. Ovariohysterectomy was performed 3 days later to recover intra-oviductal embryos for in vitro culture; one ovary was prepared for histology, and CL from the remaining ovary were excised and assessed for progesterone content and targeted gene expression. Of the six primed and seven unprimed queens inseminated, embryo(s) were recovered from five individuals per group. Embryos from progestin-primed donors more closely simulated normal stage in vivo development (P 0.05) 5–16-cell embryos progressed to morulae or blastocysts by Day 4 of culture. Although histological characteristics were unaffected by progestin priming (P > 0.05), luteal progesterone was unusually high (P

In the present study, a reverse hemolytic plaque assay (RHPA) for chicken GH was established and used to study the ontogeny of somatotroph differentiation and functional responsiveness to GH-releasing hormone (GHRH) during chicken embryonic development. Anterior pituitaries from embryos on days 10, 12, 14, and 16 of incubation were isolated and dissociated into single cells with trypsin. The resulting cells were then subjected to the GH plaque assay under basal and GHRH-stimulated conditions. No GH-releasing cells were detected on day 10 or 12 of embryonic development. In contrast, a few somatotrophs (< 2% of all cells) were consistently found on day 14, and a statistically significant population existed on day 16, when 6.3 +/- 1.4% of all anterior pituitary cells secreted GH. Thus, GH-secreting cells differentiated by embryonic day 16. Treatment of pituitary cells from day 16 embryos with GHRH was found to increase the proportion of GH plaque-forming cells during a shortened assay interval from 1.8 +/- 0.3% under basal conditions to 6.7 +/- 1.2% in the presence of GHRH. This nearly 4-fold increase in the proportion of plaque-forming cells indicates that at least 70% of the initial somatotrophs present on day 16 were responsive to the stimulatory effects of GHRH. To test whether the absence of GH cells on day 12 of embryonic development was due to the presence of cells that produced but did not release GH, pituitary cells from day 12 and day 16 embryos were subjected to immunocytochemistry for GH and to the GH RHPA in parallel. No significant differences were found in the percentage of cells that either contained or released GH on the two embryonic ages tested. On day 12, 1.1 +/- 0.8% of all cells contained GH, as determined by immunocytochemistry, whereas 0.5 +/- 0.5% released GH as determined by RHPA. By day 16, the proportions of cells that contained and released GH had increased to 9.5 +/- 0.6 and 11.2 +/- 2.5%, respectively. Taken together, these results indicate that GH-secreting cells differentiate by day 16 of chicken embryonic development and that these initial somatotrophs are responsive to GHRH. Given that growth and metabolism are regulated in part by GH in chick embryos, these findings suggest that these processes may be under hypothalamic control during late embryonic development in the chicken.

Vasoactive intestinal peptide (VIP) is the avian prolactin releasing factor and changes in the concentrations of plasma prolactin (PRL) are found during the avian reproductive cycle. This study investigated the changes in the VIP/PRL system of native Thai hens rearing their young as compared to hens deprived of rearing their chicks. The number of VIP-immunoreactive (VIP-ir) neurons in the Nucleus inferioris hypothalami (IH) and Nucleus infundibuli hypothalami (IN) of hens rearing chicks (R) were compared with those of non-rearing chicks (NR). Plasma PRL levels were determined by enzyme-linked immunosorbent assay. The localization and number of VIP-ir neurons were determined by immunohistochemistry. The numbers of VIP-ir neurons in the IH–IN areas were high in the R hens, whereas the number of VIP-ir neurons decreased in the NR hens as compared to their respective R hens. During the rearing period, changes in the VIP-ir neurons within the IH–IN were correlated with plasma PRL levels. The results of the present study indicate for the first time that the VIP/PRL system plays a role in neuroendocrine reorganization to establish maternal behavior in native Thai chickens. The VIP/PRL system functions not only as a well established key regulator of incubation behavior, but is also involved in the regulation of rearing behavior. It is possible that VIP and the decline in the number of VIP-ir neurons and in turn VIPergic activity and the decrease in PRL levels are related to their contribution to rearing behavior of this non-seasonal breeding, equatorial precocial species.

Chickens mimic an insulin-resistance state by exhibiting several peculiarities with regard to plasma glucose level and its control by insulin. To gain insight into the role of insulin in the control of chicken transcriptome, liver and leg muscle transcriptomes were compared in fed controls and “diabetic” chickens, at 5 h after insulin immuno-neutralization, using 20.7K-chicken oligo-microarrays. At a level of false discovery rate

We have reported previously that differentiation of PRL-secreting cells in rats is regulated by a maternal peptide transferred to the neonatal circulation after ingestion of mothers' milk. Inasmuch as milk contains numerous hormones and biologically active peptides, the present study was designed to test the capacity of various growth factors and hypothalamic peptides at inducing the differentiation of PRL cells in vitro. Anterior pituitary cells from 1-day-old rat pups were cultured in a serum-free system for 6 days with a wide concentration range of each test peptide. After this culture period, lactotrope differentiation was assessed by subjecting the anterior pituitary cells to reverse hemolytic plaque assays for PRL. Our efforts were focused on those growth factors and hypophysiotropic peptides found in milk and/or known to regulate pituitary function. Included among these were TRH, GH-releasing factor, somatostatin, vasoactive intestinal peptide, angiotensin-II, insulin-like growth factor-I and -II, LH-releasing hormone, arginine vasopressin, and acidic and basic fibroblast growth factors (aFGF and bFGF, respectively). Of these peptides, only aFGF and bFGF were capable of stimulating lactotrope differentiation. Specifically, we found that maximally effective concentrations of aFGF and bFGF increased the percentage of PRL-releasing cells by almost 8-fold, from about 0.5% to over 4% of all pituitary cells. In addition, bFGF was found to be about 10-fold more potent than aFGF at inducing the differentiation of PRL secretors, with minimum effective doses approaching 10(-11) and 10(-10) M for bFGF and aFGF, respectively. These results suggest that bFGF is a strong candidate to subserve a role in regulating the differentiation of lactotropes in vivo.

We have previously reported that hypophysial neurointermediate lobe peptides, di-acetylated α-melanocyte-stimulating hormone (di-ac-α-MSH) and N-acetylated β-endorphin (N-ac-β-END), can acutely increase the relative number of prolactin-secreting cells in anterior pituitary cell cultures from ovariectomized rats. Inasmuch as the des-acetylated forms of these peptides (des-ac-α-MSH and β-END) were not effective in this regard, we concluded that acetylation was an absolute requirement for manifestation of the recruitment response. The aim of the present study was to determine whether these des-acetylated variants could antagonize the mammotrope-recruiting activity of their acetylated congeners. Treatment of anterior pituitary cell cultures with di-ac-α-MSH and N-ac-β-END increased the relative amount of prolactin secretors above control values. Interestingly, des-acetylated variants of α-MSH and β-END blocked the mammotrope-recruitment activity of their respective acetylated forms. In addition, β-END antagonized the mammotrope-recruitment activity of di-ac-α-MSH while des-ac-α-MSH did not attenuate the stimulatory effect of N-ac-β-END. Given that mammotropes maintained in vivo are exposed to all these peptides, it is possible that these acetylated and non-acetylated congeners may act in an opposing manner to regulate dynamic prolactin release. Journal of Endocrinology (1993) 139, 295–300

We have previously reported that normal differentiation of PRL-secreting cells in neonatal rats requires a maternal signal specific to the first few days of lactation. Subsequently, we found that milk proteins from this period had a greater capacity than those from later in lactation to stimulate lactotrope differentiation in vitro, suggesting that the maternal signal was a milk-borne peptide. We reasoned in the present study that if this were the case, the signal should be present in the serum of suckling pups, and its level should reflect that in mothers' milk. To test this hypothesis, we first compared serum from 3-day-old pups with that from newborn pups that had not yet suckled. Anterior pituitary cells from 1-day-old pups were cultured for 6 days in the presence of various serum samples (0.01-1.0% by volume). Lactotrope differentiation was assessed using a reverse hemolytic plaque assay for PRL. Serum from 3-day-old pups was more effective (P0.01; n = 4) than that from unsuckled newborns at stimulating lactotrope differentiation in vitro (to 3.5 +/- 0.5% and 1.5 +/- 0.5% of all anterior pituitary cells, respectively). Next, we tested whether this increase in serum bioactivity in 3-day-old pups required maternal influences restricted to the first few days of lactation. Newborn pups were placed with foster mothers that had been lactating for 0 or 7 days. When the pups were 3 days old, their serum was tested for lactotrope-differentiating activity. Serum from pups placed with day 0 foster mothers (control) increased the abundance of PRL cells to 2.4 +/- 0.3% of all cells (P0.01; n = 3), whereas serum from pups placed with day 7 foster mothers failed to stimulate lactotrope differentiation. In similar experiments, litters placed the day after birth with day 7 foster mothers were separated on day 3 postpartum for 3 h, after which littermates were allowed to suckle for 90 min on different foster mothers on day 3 or 9 of lactation. This brief suckling bout increased (P0.01; n = 3) the capacity of serum from pups on day 3 mothers to stimulate PRL cell differentiation (to 3.9 +/- 0.4%) above that of serum from littermates placed with day 9 foster mothers (to 1.8 +/- 0.2%). Finally, the differentiating activities in pup serum and mothers' milk eluted from gel filtration chromatography in identical fractions (4-8 kilodaltons).(ABSTRACT TRUNCATED AT 400 WORDS)