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Abstracts from VI International Symposium on Avian EndocrinologyMarch 31 - April 5, 1996 Chateau Lake Louise, Alberta Continued Neuroendocrine Control of Gonadotrophin SecretionP.J. Sharp, R.W. Lea1, R.T. Gladwell2, F.J. Cunningham2 Roslin Institute, Roslin, Midlothian EH25 9PS; 1University of
Central Lancashire, Preston PR1 2TQ; 2University of Reading,
Reading RG6 2AJ UK The tonic secretion of luteinizing hormone (LH) in the domestic chicken increases at the onset of puberty and decreases during the development of incubation behaviour (broodiness). It is maintained around a "set point" by the negative feedback effects of gonadal hormones, since gonadectomy of prepubertal and incubating chickens results in an increase in LH secretion. This "set point" is altered by changes in input to gonadotrophin releasing hormone-I (GnRH-I) neurones. Support for this view comes from the finding that hypothalamic GnRH-I mRNA content increases at the onset of puberty and decreases during incubation. Changes in hypothalamic "set point " can be seen as being due to changes in sensitivity to inhibitory steroid feedback or to a steroid-independent mechanism. The change in "set point" at the onset of puberty in the chicken is unlikely to be principally controlled by a change in sensitivity to negative steroid feedback since LH secretion increases steeply in birds castrated as juveniles, at the usual age for the onset of puberty. The reason for the decrease in hypothalamic GnRH-I "set point" in incubating birds is less certain, since there are no reports on the induction of incubation in gonadectomized birds without the use of steroids. An inhibitory role for prolactin is possible, but a direct inhibitory action on GnRH-I release has not been established. The maintenance of LH secretion around a "set point" by the inhibitory action of gonadal steroids operates both at the level of the anterior pituitary gland and of the hypothalamus. Treatment of anterior pituitary glands in vivo with oestrogen decreases the response to GnRH-I. Treatment of chronically castrated birds with oestrogen decreases the visible number of immunocytologically identifiable GnRH-I neurones and decreases hypothalamic GnRH-I mRNA. The inhibitory effects of steroids may be principally mediated by oestrogen: in males this is derived from androgens by aromatization within the hypothalamus. Inhibin from Embryo till Adult HenE. Decuypere, L. Rombauts, D. Vanmontfort, G. Verhoeven Leuven Poultry Research Group, Kard. Mercierlaan 92 (blok E),
3001 Heverlee, Belgium In non-mammalian vertebrates, information about the source, regulation and function of inhibin is rather scarce. In the chick embryo, inhibin is present in plasma, amniotic fluid and gonadal tissues at early stages of development (day 8). No obvious relationship between plasma inhibin and FSH is observed, suggesting a non-functional inhibin-FSH feedback loop during chick embryo development. On the other hand in vivo and in vitro data strongly support the hypothesis that the fetal adrenal is the major source of circulating inhibin in the chick embryo. Postnatally, the FSH-inhibin feedback loop becomes operational at the onset of sexual maturity in female hens. An inverse relationship between inhibin and FSH also occurs after short-term food deprivation and after ovariectomy. The adrenal is, however, an additional source of inhibin. Consequently, since plasma inhibin is predominantly derived from the chicken ovary during adult life, the role of inhibin may shift from the hypothalamo-pituitary-adrenal axis in the embryo to the hypothalamo-pituitary-gonadal axis in adult hens. Besides evidence for an endocrine role of inhibin in the fowl, the intragonadal roles of inhibin and related peptides were investigated in isolated and cultured embryonic gonadal cell populations and in granulosa cells of the follicular hierarchy of adult hens. These studies revealed a paracrine role for inhibin and activin as modulators of gonadal steroidogenesis. Follistatin antagonized the local effects of inhibin and activin, supporting previous evidence that follistatin is an activin-binding protein. Role of Estrogen in the Epididymal Region of the RoosterJ. Bahr, S. Kwon, R. Hess, D. Bunick Department of Animal Sciences and Physiology, University of
Illinois, Urbana, IL 61801, USA The epididymal region of the rooster has an important role in the production of fertile sperm by controlling the luminal microenvironment required for sperm maturation. We tested the hypothesis that estrogen (E) has a role in the regulation of the epididymis of the rooster. To test the hypothesis, we determined: 1) if germ cells and epididymal sperm are sources of E and 2) if the epididymal region is a target for E. Techniques used were immunocytochemistry, in situ hybridization, northern blot analysis and aromatase activity assay. We found that rooster testicular germ cells and epididymal sperm contain active P450 aromatase (arom) and E receptors (R) and ER mRNA are present in the epididymal region. These data demonstrate that rooster germ cells and epididymal sperm are sites of E synthesis and that the epididymal region is a target for E. (Supported in part by USDA-Ag-93-37203-9021) Evolution of Three Dimensional Conformation of Gonadotropin Molecules: Avian PeculiaritiesS. Ishii, H. Wako School of Education and School of Social Science, Waseda
University, Tokyo 16950, Japan. Miya and Ishii (1995) reported that chicken LH binds specifically to the rat FSH receptor with a high affinity and that amino acid sequences in two regions in the beta subunit molecule of chicken LH are identical to those of the corresponding regions in the FSH beta subunit molecules of other vertebrates. We concluded that these regions are responsible for hormone specificity. Wako and Ishii (1995) reported that the secondary structure is highly conserved through all members of the gonadotropin/thyrotropin family and through all vertebrate species studied. They constructed a three dimensional model of the betasubunit molecule common to all the members of the family. Three dimensional models of the human chorionic gonadotropin molecule based on the crystallographic analysis data were presented by two groups (Lapthorn et al., 1994; Wu et al., 1994). Incorporating all the available data, we also constructed a three dimensional model representing the molecule of the gonadotropin/thyrotropin family by using a computer program (MOLSCRIPT) and found that all the regions reported to play roles in the hormonereceptor interaction in the gonadotropin molecule (i.e. the determinant and large loops, necessary for the receptor interaction in the beta subunit, the specificity determining regions and a region reported to be important to the biological action in the alpha subunit) are located in cluster in a portion of the three dimensional model. Molecular Evolution of Avian Gonadotropin ReceptorsM.K. Park, Y. Akazome Graduate School of Science, The University of Tokyo, Tokyo
133, Japan Gonadotropins (GTHs) and TSH are the member of the pituitary glycoprotein hormone family and are thought to be derived from a common ancesterol molecule. The receptors for these hormones are also similar and are members of the (G-protein coupled) seven transmembrane receptor super family. These receptors are, nevertheless, unique in that they have a long extracellular domain that accounts for half of the receptor's amino acid sequences. Specific binding of the beta subunits of the glycoprotein hormones is probably to these long extracellular domains. Comparative binding studies with purified GTHs to the gonadal receptors of heterologous species suggests the presence of a complex evolutional interaction between hormone and receptor. To elucidate this interaction, we have focused on the ligandbinding domain of the receptor, using reversetranscription polymerase chain reaction to determine the sequence of the extracellular domain in different vertebrate species. Molecular characteristics of Nterminal domain sequences within quail, lizard and gecko GTH receptors will be discussed, together with implications for ligand-receptor interactions. A High Correlation between the Electric Activity of Neurones in Preoptic/Anterior Hypothalamic Areas and Lh Release from the Hypophysis in DucksJ.P. Peng, H.L. Qiao, C.R. Yang Department of Animal Physiology and Biochemistry, Beijing
Agricultural University, Beijing 100094, China Unit discharges were recorded by a glass microelectrode from POA/AHA neurones in urethaneanaesthetized female ducks. Blood samples were withdrawn from the hypophyseal-portal veins at 5 to 10 min intervals during the recording in order to measure the changes in the serum LH concentration by in vitro bioassays, i.e. the response of granulosa cells of ovarian follicles to LH. Unit activity of 126 POA/AHA neurons was recorded. 65/126 were antidromically identified by stimulation of the median eminence (45 were spontaneously active and 20 were silent). The unidentified neurons (61/126 units) were determined to be spontaneously discharged (mean blood LH level 2.5±0.2ng/ml). The effects of norepinephrine (NE) and 5-HT on the electric activities of POA/AHA neurons were observed with multimicropipette and iontophoresis techniques: Discharge of 58/122 units was significantly increased by microntophoretically applied (MIA) 5-HT, but the response was blocked by MIA cyproheptadine. 33/122 units were inhibited, while 31/122 units were non-responsive. Serum LH concentrations increased from 2.5±0.2ng/ml to 4.1±0ng/ml (P<0.01) as a result of the stimulation of discharge frequency. Discharge of 86/138 units was also increased by MIA NE. The response resulted in a 2 ½ fold (6.3ng/ml) increase in serum LH levels (P<0.01): 58/86 units were reversed by MIA phenoxybenzamine. 23/138 units were inhibited, and 9/138 units were silent. In the adenohypophysis, MIA 5-HT and NE have little effect on LH levels. These experiments indicate: (1) there are GnRH neurons in the POA/AHA sending their axons into the external layer of the medial eminence; (2) discharge frequency of POA/AHA neurons is correlated closely to pulsatile GnRH/LH release; (3) electrical activity of POA/AHA neurons is modulated by either 5-HT or NE. Studies on Puberty of the Female Duck: Patterns of Pituitary and Circulating Lh Levels, and Pituitary LH Release in Response to GnRH ChallengeJ.Y.L. Yu, C.F. Weng, S.T. Shen, L.Y. Yang Endocrinology Laboratory, Institute of Zoology, Academia
Sinica, Taipei, Taiwan 115, ROC Pituitary and circulating luteinizing hormone (LH) levels and pituitary LH responses to GnRH challenge were investigated in female ducks (Tsaiya duck, Chinese common duck, Anas platyrhychos var. domestica), at intervals during 10-130 d of age. The first eggs were generally laid in 120-130 d of age, with oviduct weight of 40-50 g. In one expt, pituitary and serum LH were determined for untreated ducks; and in another expt, pituitary and serum LHs were measured after porcine GnRH challenge (20 µg/kg, sc). LH was measured by bioassay using rooster testicular testosterone formation in vitro. Pituitary LH remained extremely low during the first 60 d of age, rapidly increased during 60-100 d, and reached a plateau thereafter. Serum LH was rapidly increased during 60-100 d of age , but decreased during 100-130 d of age. Responses of pituitary LH following GnRH challenge were greater during the early phase of sexual maturation; the maximal response occurred in the ducks with oviduct weights of 1-3 g. Thus, rapid increases in both pituitary and circulating LH levels occur during the sexual maturation of the female ducks; however, the sensitivity of pituitary LH release and the circulating LH levels are greatest at the early phase of sexual maturation. (Supported by National Science Council and Academia Sinica, Taipei, Taiwan, R.O.C.) Phylogenic Immunology of Avian Gonadotropin: Crossreactivity of Duck Luteinizing Hormone AntibodyC.F. Weng, J.T. Chien, W.H. Yang, S.T. Shen, J.Y.L. Yu Endocrinology Laboratory, Institute of Zoology, Academia
Sinica, Taipei, Taiwan 115, ROC A Polyclonal antibody was prepared for duck luteinizing hormone( d-LH) by immunization of a rabbit with multiple injections. A radioimmunoassay (RIA) was established for d-LH, using 125I labelled d-LH and the antiserum. The phylogenic patterns and specificity of the d-LH antibody (d-LH Ab) with LHs and pituitary homogenates from various vertebrate classes were investigated by cross reactivity in this RIA system. The d-LH Ab crossreacted extensively with LHs from chicken, ostrich, turkey and goose, but did not crossreact with mammalian LHs and teleost gonadotropins (GTHs). We further observed the d-LH Ab crossreacted with pituitary homogenates from 20 species of 6 orders of birds, but did not crossreact with those from mammals (11 species of 5 orders) and teleosts (20 species of 10 orders). All of these pituitary homogenates contained bioactive LHs and GTHs, as demonstrated by in vitro bioassay. The present study demonstrates that the crossreactivity of d-LH Ab has a low degree of "Order Specificity", but a high degree of "Class Specificity" with respect to mammals and teleosts. Our findings support the proposal that the immunological binding sites of pituitary LHs are highly conserved within the Class Aves. The d-LH Ab may be useful for quantification of LHs from all or most avian species. (Supported by National Science Council and Academia Sinica, Taipei, Taiwan, R.O.C.) Effects of Food Restriction on cLHRH-I and cLHRH-II in Hypothalamic Nuclei of the ChickenE. D'Hondt, L.R. Berghman, E. Decuypere, F. Vandesande Laboratory for Neuroendocrinology and Immunological
Biotechnology, Zoological Institute, Katholieke Universiteit,
Leuven, Naamsestraat 59, 3000 Leuven, Belgium Intensive selection for increased muscle growth in meattype chickens has led to a decrease in reproductive performances of the parent stocks. To improve the efficiency of reproduction, these animals are submitted to an early quantitative food restriction. In order to determine whether this procedure alters the concentration of cLHRHI and cLHRHII, ad libitum fed and food restricted female broiler chickens were killed at 4, 10, 16 and 22 weeks of age. A quantitative determination of both peptides was performed in different hypothalamic nuclei. Therefore the brains were sectioned and the different areas were punched out. After an acidic extraction of these micropunches cLHRHI and cLHRHII were measured by specific radioimmunoassays. In most hypothalamic nuclei, no significant concentration differences were found. However, food restriction did affect the concentration of both cLHRHI and cLHRHII in the median eminence (ME) of the chicken. The amount of cLHRHI is significantly higher in ad libitum fed broilers after 10 weeks of age. On the contrary, food restriction seems to cause an increase of cLHRHII in the ME . These changes seem to begin from the 16th week but in some cases they appear around 22 weeks. The precise function of these changes remains to be elucidated, but these quantitative data provide an excellent basis for further research on the altered feeding and reproductive behaviour in the meattype chicken. Expression of Central Progesterone Receptors during the Breeding Cycle of the Ring DoveG.C. Georgiou, J.a. Askew, P.J. Sharp, R.W. Lea Department of Applied Biology, University of Central
Lancashire, Preston, PR1 2HE UK Both sexes of the ring dove participate in the incubation of eggs and subsequent care of the young. Previous studies have shown exogenous progesterone can induce incubation behaviour in both the male and female. In this study, an immunocytochemical method was employed in order to localise and quantify changes in progesterone receptor (PR) number in non-breeding, incubating, brooding and oestrogen-treated non-breeding male and female doves. Progesterone receptors were localised in similar regions of the brain in both sexes, in particular the nucleus hypothalami lateralis posterioris (PLH) and the nucleus preopticus anterior (POA), however only one brain region in the male dove exhibited a significant increase in PR at the onset of incubation, the nucleus tuberalis (NT). Treatment with exogenous oestrogen increased PR in this region in non-breeding male doves. In brooding birds, PR number diminished in the NT of both sexes, this was particularly evident in the ventrolateral region, but remained unchanged or even elevated in the POA. Double-immunolabelling for VIP (vasoactive intestinal polypeptide) and PR was carried out at the onset of incubation, mid-incubation and during the brooding period. The distribution of VIP-immunoreactive cells in the NT were consistent with an earlier study (Cloues et al., 1990). A small number (10-20%) of neurones co-expressing VIP and PR were found to exist in the dorsolateral region of the NT during the period of mid-incubation. However, during the brooding period, dense labelling of VIP was seen in the ventrolateral region of the NT, a region which had become devoid of PR during this period. These results suggest that PR in discrete brain regions differ in their sensitivity to oestrogen and PR may be involved in both the behavioral and neuroendocrine changes that occur during the breeding cycle. (Supported by the BBSRC and The Wellcome Trust) Increased Vasoactive Intestinal Polypeptide mRNA in the Hypothalami of Incubating HensR.T. Talbot, P.W. Wilson, P.J. Sharp Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS UK Incubation behaviour in hens is associated with an increase in the concentration of plasma prolactin. This increase in prolactin is due to the prolactin releasing factor, vasoactive intestinal polypeptide (VIP) secreted from the basal hypothalamus. During incubation the number of immunocytochemically identified VIP neurones and the amount of VIP in the basal hypothalamus increases. The aim of this study was to establish whether the increase in the VIP content of the basal hypothalamus in incubating hens is associated with increased VIP mRNA per neurone or to the appearance of more neurones containing similar amounts of VIP mRNA. In situ hybridisation was used to quantify hypothalamic VIP mRNA on frozen sections using a 35S-labelled 45 base antisense oligonucleotide to the VIP peptide coding sequence. Control sections were hybridised with labelled oligonucleotide complementary to the antisense probe. The amount of radioactivity hybridised to each section was measured as the integrated optical density of silver grains. Sections through each basal hypothalamus were analyzed for the number of VIP mRNA neurones and their integrated optical density. Sections from incubating and laying hens were processed in pairs (n=3). There was an even distribution of VIP mRNA positive cells throughout the basal hypothalamus. The number of observed cells containing VIP mRNA was greater (P<0.01) in incubating than laying hens (107.7 ± 6.6 v s 41.3 ± 4.8; mean ± s.e.m.). The integrated optical density expressed in arbitrary units was significantly greater (P<0.01) in basal hypothalami of incubating than in laying hens, indicating a greater amount of VIP mRNA. The ratio of the integrated optical density of each section divided by the number of cells in that section was used to estimate the amount of VIP mRNA present in each cell. The amount of VIP mRNA per cell in the hypothalami from laying and incubating hens was not significantly different. In conclusion, the increase in VIP in the basal hypothalamus of incubating hens is associated with an increase in the number of visible VIP neurones each expressing similar amounts of VIP mRNA. cGnRH-I mRNA Levels are Increased by Transfer from Short to Long Days in Peripubertal Cockerels and Photosensitive Mature HensI.C. Dunn Division of Development and Reproduction, Roslin Institute
(Edinburgh), Roslin, Midlothian, EH25 9PS, UK Long daylengths advance the age of sexual maturity and stimulate the release of gonadotrophins. It is assumed that stimulation of the pituitary-gonadal axis is a result of activation of the GnRH neurones in the hypothalamus, but a change in the function of the GnRH neurone after photostimulation has not been observed. To demonstrate that GnRH neurones are stimulated by long daylengths a competitive PCR assay was used to estimate the amount of cGnRH-I mRNA in the hypothalamus, measured as the amount of reversed transcribed cGnRH-I cDNA. Two experiments were carried out to measure hypothalamic cGnRH-I mRNA after photostimulation; 1) prepubertal and peripubertal (8 and 16 week-old) cockerels were photo-stimulated by increasing the daylength from 8 to 16 hours, control cockerels remained on an 8 hour daylength, 2) hens at the end of a laying year were transferred from long days to short days (3L:21D) for 6 weeks after which half were transferred to 16L:8D. Control hens remained on short days. All birds were killed 1 week after transfer from short to long days. In both peripubertal cockerels and hens transfer from short to long days was associated with a significantly larger hypothalamic content of cGnRH-I mRNA (peripubertal cockerels, 3.02±0.67 vs 1.22±0.36 x 10-17 moles cDNA; photosensitive mature hens 4.58±1.27 vs 1.80±0.35 x 10-17 moles cDNA). In contrast, there was no difference in the hypothalamic content of cGnRH-I mRNA after photostimulation in 8 week-old cockerels (prepubertal cockerels, 1.69±0.21 vs 1.72±0.14 x 10-17 moles cDNA). It is concluded that cGnRH-I mRNA transcription or mRNA stability is increased in cGnRH-I neurones after transfer from short to long days. The absence of an increase in mRNA in 8 week old cockerels suggests that a maturational event may have to take place before photostimulation can cause an increase in the hypothalamic content of cGnRH-I mRNA. (Supported by MAFF) |