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Abstracts from VI International Symposium on Avian EndocrinologyMarch 31 - April 5, 1996 Chateau Lake Louise, Alberta Continued Distribution of GNRH-I Synthesizing Neurons in the Diencephalon of Cockerels: Effects of Age and PhotostimulationR.T. Gladwell, D. Finn, P.J. Sharp1, I. Dunn1, R. Talbot1, F.J. Cunningham School of Animal and Microbial Sciences, The University of
Reading, Reading RG6 7AJ England; 1Roslin Institute, Roslin,
Edinburgh EH25 9PS Scotland Neurons containing the mRNA encoding chicken
gonadotropin-releasing hormone-I (GnRH-I) were identified by in
situ hybridization with digoxigenin-labelled riboprobes. The
distribution and number of GnRH-I-synthesizing cells were
examined in juvenile and sexually mature cockerels maintained on
short (8h light:16h dark) or long (16L:8D) day photoschedules. In
17 week old cockerels photostimulated by transfer to long days
for one week 189.3 ± 32.9 (mean ± SEM, n=3) neurons containing
GnRH-I mRNA were found, a significant (P<0.05) 3-fold increase
in cell number over that in the brains of non-photostimulated
controls. Positive cells were mainly distributed in the preoptic
and anterior hypothalamic areas; medial and lateral septal
regions and caudal to the septal regions, in and ventral to the
corticohabenular-corticoseptal tracts. Positive cells were not
detected in the brains of short day birds at 9 weeks (n=4), but
low numbers were observed in the brains of some photostimulated
birds. In short day birds of 12, 17 and 24 weeks of age there
were no significant differences in the number of positive neurons
detected. The results suggest no increase in the number of
GnRH-I-positive cells at the onset of sexual maturation, but that
photostimulation significantly increased the number of neurons
actively synthesizing GnRH-I in birds approaching puberty. Morphological Relationships between VIP- and GnRH-like Immunoreactive Neurons in the Lateral Septum of the PigeonK. Kiyoshi, a. Adachi1, S. Ebihara1, K. Hirunagi Departments of Veterinary Anatomy and 1Animal Physiology,
School of Agricultural Sciences, Nagoya University,
Chikusa-ku,Nagoya 464-01, Japan In the lateral septum of the avian brain, the presence of vasoactive intestinal polypeptide (VIP)-and gonadotrophin releasing hormone(GnRH)-containing neurons has been reported. An accumulation of VIP-like immunoreactive(ir) neurons is found in the pars medialis of the lateral septal organ (LSOm), adjacent to the lateral ventricles. Many of these neurons display the characteristic features of CSF-contacting neurons. GnRH-ir neurons are located in the deeper layer of LSOm. In the present study, we used double-labelling techniques at the light (LM) and electron-microscopic (EM) levels to investigate whether VIP-and GnRH-ir neurons communicate in the lateral septum of the pigeon. At the LM levels, VIP-ir neurons and GnRH-ir neurons were seen in close association and some VIP-ir fibres ran near GnRH-ir cell bodies. Examination of these neurons at the EM level revealed that VIP-ir axon terminals had synaptoid contacts with perikarya and dendrites of GnRH-ir neurons. These results suggest that VIP neurons could communicate directly with GnRH neurons in the lateral septum of the pigeon and this area might play an important role for reproductive phenomena. Secretion of Luteinizing Hormone during a Forced Moult in Turkey HensW.L. Bacon, D.L. Long The Ohio State University, Ohio Agricultural Research and
Development Center, Department of Animal Sciences, Wooster, OH
44691, USA The secretory pattern of LH was determined in turkey hens during a forced molt induced by water (1 day) and feed (5 days) restriction and by switching the photoschedule from 14L:10D (light,L; dark,D;LD) to 6L:18D (SD) on the first day of feed and water restriction. Four sets of serial blood samples (every 10 minutes for 6 h and then every 4 minutes for 2 hr) were collected. Set 1 was collected one day before forced molting was initiated. Set 2 was collected seven days after the start of forced molting and SD lighting. Set 3 was collected after 7 wk and 3 days of SD lighting. Set 4 was collected 2 days after photostimulation by returning the hens to LD, after 8 wk of SD lighting. Egg production was stopped within 5 days of initiating the forced molt. Egg production-initiated by 34 days (range 22 to 34 days) after returning to LD lighting. Overall and baseline concentrations of LH were low at the end of reproduction (Set 1) and after forced molting (Set 2). Number and amplitude of LH pulses were also low in Set 1 and Set 2. Overall and baseline concentrations of LH also remained low in Set 3, after 7 wk and 3 days of SD lighting, with no change in number of LH pulses/8 hr. However, the amplitude of LH pulses was increased in Set 3 in comparison with Set 2. Increases in overall, baseline and number of LH pulses were detected in Set 4, 2 days after LD photostimulation. Peak amplitude was not increased from Set 3 to Set 4. It was concluded that in turkey hens, at the end of a relatively long reproductive period, baseline LH is low and does not decrease during a forced molt and subsequent reconditioning period. Relatively large increases in circulating LH are induced by re-exposing the reconditioned hens to a LD photoperiod of 14L:10D, and this increase is associated with an increase in overall and baseline LH concentrations, and in frequency of LH peaks. Effects of Estradiol-17b and Feeding Level on Ovarian Follicular Development and Lipid Allocation in 59 week Old Meat Type ChickensR.a. Renema, F.E. Robinson, M.T. Clandinin, W.T. Dixon Department of Agricultural, Food and Nutritional Science,
University of Alberta, Edmonton, AB T6G 2P5, Canada Female meat type chickens are routinely feed restricted, to limit excessive ovarian mass and carcass lipid content. As the number of developing follicles declines with age, stimulation of follicle development is of interest. To attempt to restore follicular numbers to pubertal levels, meat type hens (either full fed or standard restricted fed) were assigned to the following groups at 59 wk of age: FF (Continue full-feed access); RR (Continue restricted-feed access); and RF (Previously restricted fed birds now full fed). The 16 birds in each feeding group were given either a blank (B) or an estradiol-17b (E) silastic implant (7.2 mg/pellet estradiol-17b administering 120 pg/ml plasma) (8 birds/tmt). Hence, the experimental design was a 3 x 2 factorial with the resulting treatments being FF-B, FF-E, RR-B, RR-E, RF-B, and RF-E. Following a 24 h adjustment period, lipid allocation was traced with a 5 µCi dose of 14C-Palmitate. Liver, abdominal fatpad, and ovarian tissues were collected after 72 h to determine radiolabel distribution (data not shown). Birds were blood sampled prior to silastic pellet implantation and at the end of the experimental period for subsequent plasma lipid composition and estradiol-17b analysis. Estradiol-17b treatment had no effect on body weight or feed intake of the birds. RF treatment birds became 5.6% heavier (vs 0.1% in other feeding treatments) and consumed 64% more feed than their RR counterparts. These changes most strongly affected liver weight, which was 2.7% of total body weight in RF birds vs 1.8% in FF and RR birds. The overall mean initial plasma estradiol-17b concentration was 150 pg/ml. Estradiol-17b concentrations of RF-B birds increased by 43 pg/ml due to feed effects while FF-B and RR-B concentrations declined slightly. Estradiol treatment caused the mean plasma estradiol-17b concentrations to increase to 252 pg/ml. Estradiol treatment increased whole ovary weight by 9% (P= .57), with small increases in follicle weights in all size classes. Ovarian stroma weights in FF and RF birds were 20% heavier than that of the RR birds. Small follicle numbers and stroma weights of RF birds were similar to those of FF birds. While estradiol treatment may affect lipid allocation to the ovary, feeding level has a greater effect on ovary morphology. L-Thyroxine Regulates Pituitary LH Function in Castrated Subtropical Finch Estrilda amandavaA. Choudhury, C.M. Chaturvedi Department of Zoology, Banaras Hindu University, Varanasi -
221 005, India Red Avadavat (Lal munia) Estrilda amandava is a seasonally dimorphic finch having inverse thyroid-gonad relationships. Males acquire LH dependent brightly coloured plumage and a brick red bill during the breeding phase (Aug-Sept). Males castrated in April, were divided into three groups and administered daily with normal saline, or with 1.2 or 2.4 µg L-thyroxine over a period of one month, in August. Plumage pigmentation and bill colour was observed monthly until December. Results indicate that castration advanced the acquisition of the nuptial plumage and bright red bill. These secondary sex characters were also maintained outside the normal breeding phase, indicating that pituitary becomes acyclic in the absence of sex steroid feedback. Following L-thyroxine administration, plumage and bill colour were similar to that in nonbreeding birds. It is concluded that increased pituitary LH activity induced by the absence of gonadal hormones is inhibited completely by L-thyroxine. These findings suggest that thyroid hormones may be a substitute of gonadal steroids to induce feed back inhibition of LH activity in this subtropical species. PTH/PTHrP and their Common Receptor: State of the ArtM. Rosenblatt, a. Adams, V. Behar, a. Bisello, M. Chorev, M. Pines, S. Stueckle, L. Suva Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215,
USA Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) share limited structural homology, yet are both able to bind to a common receptor (Rc). The PTH/PTHrP Rc is expressed in kidney and bone. This seven-transmembrane guanyl nucleotide protein-coupled receptor is linked to two signal transduction and second messenger systems: cAMP and cytosolic calcium. Structure--activity studies of these hormones have generated analogs of enhanced activity, potent pure antagonists, and partial agonists. In order to elucidate the nature of the ligand--receptor bimolecular interface and delineate differences between agonist and antagonist interactions with Rc at the submolecular level, we have undertaken an effort to map the contact surface between ligand and receptor. Using photoaffinity crosslinking methodology and human cells engineered to stably express high levels of human PTH/PTHrP Rc, we have succeeded in crosslinking both agonists and an antagonist to Rc. These steps are prerequisite to microsequencing of ligand--receptor covalent conjugates in order to identify directly specific "contact points". The recent discovery of a new receptor in the family, the PTH-2 receptor, provides further impetus for applying this approach. Finally, these studies (which examine the first event in expression of hormone bioactivity) are complemented by a parallel effort to elucidate events downstream in the cascade of hormonal action. Our laboratory is engaged in an effort to identify genes up- or down-regulated by PTH or PTHrP using the differential mRNA display (DRD) technique. Genomic and Non-Genomic Effects of Vitamin D on Calcium TransportI. Nemere Department of Nutrition and Food Sciences and Biotechnology
Center, Utah State University, Logan, UT 84322-8700, USA The genomic effects of the hormonally active form of vitamin D, 1,25dihydroxyvitamin D3 [1,25(OH)2D3] in intestine are, for the most part, concerned with inducing the components of the calcium transport pathway. Once the components are present, calcium transport can be initiated by nongenomic means. The working model for the calcium transport pathway, as deduced by subcellular fractionation studies, is as follows: calcium is recognized at the brush border by a "receptor" or "carrier", resulting in internalization into endocytic vesicles. The vesicles, in turn, fuse with lysosomes (perhaps as a means of concentrating the cation), which move along microtubules to the basal lateral membrane (BLM) where exocytosis completes the transport process. The vitamin Dinduced calcium binding protein (calbindinD28k) has been localized in transport vesicles by both biochemical and microscopic means. The secosteroid hormone has also been found to alter the levels of a-tubulin mRNA and increase the levels of protein, as judged by isoelectric focusing. Additionally, immunofluorescent staining intensity of microtubules increases with vitamin Drepletion of deficient chicks. The nongenomic effects of 1,25(OH)2D3 (rapid stimulation of calcium and phosphate transport in perfused duodenal loops) are apparently mediated by a BLM receptor (BLMVDR), which is absent from brush borders. Binding of ligand to plamalemmal receptor activates a dihydropyridinesensitive calcium channel (also absent from the brush border); influx of cytosolic calcium promotes the exocytosis of vesicular transport calcium. The BLMVDR has an apparent mw of 66000 and a Kd of 0.7 nM. Analog studies have revealed a functional correlation between receptor binding and efficacy in stimulating the rapid transport of calcium across the intestine. Regulation of Plasma CalciumS. Hurwitz Institute of Animal Science, The Volcani Center, P.O. Box 6,
Bet Dagan 50250 Israel Due to the importance of Ca2+ in regulation of vital cellular and tissue functions, the concentration of Ca2+ in body fluids is closely guarded by an efficient feedback control system. This system includes Ca2+ transporting subsystems bone, intestine and kidney, Ca2+ sensing (possibly by a calciumsensing receptor) and calcium regulating hormones parathyroid hormone (PTH), calcitonin (CT) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. In birds, amino acid sequences of both peptide hormones are known, but the importance of CT is questionable. The newly discovered Ca2+sensing receptor has been cloned for the chicken and is about 80% homologous with the bovine receptor. The control system has been formulated into a computer algorithm in which the behavior and importance of each of the subsystems can be evaluated. Acute Ca2+ perturbations are handled by modulation of kidney Ca2+ reabsorption, and by bone Ca2+ flow under PTH regulation. Chronic perturbations are handled by the more sluggish but economic genomic and nongenomic regulatory action of 1,25(OH)2D3 on intestinal calcium absorption. In the growing chicken, plasma calcium is kept nearly unchanged within a wide range of Ca intakes but changes rapidly once the capacity of the control system is overwhelmed. However, plasma Ca and its control systems exhibit diurnal oscillations. Hormonal and Ionic Control of Osteoclast FunctionC.V. Gay Departments of Poultry Science and Biochemistry and Molecular
Biology, The Pennsylvania State University, University Park, PA
16802, USA As occurs with most cells, osteoclasts respond to a multitude of factors. This presentation will focus mainly on parathyroid hormone (PTH) and calcium ions as modulators of osteoclast activity. In bone, the major effect of PTH is widely held to be stimulation of osteoblasts which, in turn, transmit signals that activate osteoclasts and cause bone resorption. We have shown that the osteoblast response to PTH is mediated, in part, by a surge in cytosolic Ca2+ which is rapidly restored to basal level by efflux of Ca2+ to the cell exterior (J. Bio. Chem. 270:22445, 1995). Zaidi and colleagues (J. Clin. Invest. 96:1582, 1995) have clearly shown that osteoclasts are inactivated by Ca2+ through a unique plasma membrane calcium receptor. It is possible that Ca2+ released by PTH-stimulated osteoblasts inhibit neighboring osteoclasts at a time when osteoclast stimulation is required. We hypothesize that osteoclasts override this effect by responding directly to PTH. This concept is supported by several reports which show specific binding of PTH to osteoclasts. Further, Minkin and colleagues (J. Bone. Miner. Res. 10:S322, 1995) have identified mRNA for PTH receptor in murine osteoclasts. We have shown that PTH activates acid formation by avian osteoclasts in a relatively pure cell culture system. The response is transmitted and modulated through Gs, Gi and possibly Go proteins. Using Western blot technology, the profile of G-proteins in purified osteoclast preparations appears markedly different from that of osteoblasts, a finding that lends further support that the osteoclast preparations being investigated are largely devoid of osteoblasts. In conclusion, a growing body of evidence indicates that osteoclasts are activated directly by PTH, a strategy which may have evolved to counteract osteoclast inactivation by local surges in ionic calcium. Involvement of Osteopontin in Epiphyseal Growth Plate Calcification and Egg Shell FormationM. Pines, V. Knopov, a. Bar Institute of Animal Science, Agricultural Research
Organization, The Volcani Center, Bet Dagan 50250, Israel Osteopontin (OPN), a phosphorylated growth-plate, OPN gene was expressed mainly at the lower hypertrophic zone in the region of cartilage to bone transition. In culture, non-differentiated chondrocytes exhibited lower levels of OPN mRNA than the differentiated cells. OPN gene expression was stimulated by FGF while its phosphorylation was regulated by EGF and parathyroid hormone. OPN synthesis and phosphorylation were found to be regulated by separate mechanisms and the response to the various controlling agents varied with the state of chondrocyte differentiation. In the oviduct of the laying hen, OPN gene expression was detected exclusively in the egg shell gland (ESG), where massive calcification occurs. OPN gene was expressed in a circadian fashion during the daily egg cycle only during the period of egg shell calcification. No OPN gene expression was detected in the ESG of a pre-laying hen before the onset of reproduction, or after forced removal of the egg close to its entrance into the ESG. OPN was found to be synthesized by the epithelial cells of the ESG lining the lumen. Upon synthesis, OPN is immediately secreted out of cells and accumulates in the egg shell. These findings demonstrate a temporal and spatial association of OPN with bone calcification and egg shell formation. OPN, which was found to be part of cartilage and egg shell organic matrix, may play an important role in these processes. Calcium Transport Mechanisms during Embryonic DevelopmentR.S. Tuan Departments of Orthopaedic Surgery, and Biochemistry and
Molecular Biology, Thomas Jefferson University,
Philadelphia, PA 19107, USA The Ca requirement of a developing chicken embryo is derived sequentially from two sources, the egg yolk and the eggshell. Calcium translocation by the yolk sac endoderm provides the initial Ca supply and is a vitamin Ddependent process, involving the expression of the vitamindependent Cabinding protein, calbindinD28K. Eggshell Ca mobilization, on the other hand, is carried out by the ectodermal cells of the chorioallantoic membrane (CAM) and is turned on at ~day 12 of incubation. Several functional components have been identified with the CAM Ca transport function, including a membraneassociated Cabinding protein (transcalcin), a membrane CaATPase pump, and the cytosolic enzyme, carbonic anhydrase. Analysis of subcellular Ca traffic in isolated CAM ectodermal cells reveals that a vesicular, transcytotic mechanism, which maintains Ca in a compartmentalized state, is responsible for transcellular Ca transport. Recently, applying the technology of reverse transcription/polymerase chain reaction, we have identified and isolated the fulllength cDNA for annexin I (Anx I), a gene which is differentially expressed in the CAM ectoderm at the onset of Ca transport. Anx I binds phospholipids in a Cadependent manner and appears to be involved in active Ca uptake by cellfree CAM membrane vesicles. Current studies are directed towards elucidating the exact functional role of Anx I and the mechanisms regulating its tissue and developmentspecific expression. (Supported in part by NIH HD 158221, HD 29937, ES 07005, and DE 11327) The Effects of Growth Hormone on Avian Skeletal Muscle Satellite Cell Proliferation and DifferentiationO. Halevy, V. Hodik, a. Mett Department of Animal Science, Faculty of Agriculture, The
Hebrew University of Jerusalem, P.O.B. 12, Rehovot 76100, Israel Growth hormone receptor (GHR) mRNA was expressed in avian skeletal muscle tissue and satellite cells in culture, and was capable of binding cGH. In the satellite cells, GHR gene expression was regulated by chicken GH (cGH) in a biphasic manner which correlated with the GH effect on cell proliferation: 210 ng/ml of the hormone increased GHR mRNA and DNA synthesis, whereas higher concentrations attenuated these effects. GH induced insulinlike growth factor I (IGFI) mRNA, a potential factor for satellite cell proliferation and differentiation. However, GH inhibited the gene expression of myogenin and the expression of musclespecific proteins in a dosedependent manner. These results suggest a role of GH for inhibiting satellite cell differentiation in an IGFIindependent manner. During satellite cell differentiation, both GHR mRNA expression and cGH binding, peaked when cells were still proliferating and beginning to fuse, and then declined as cells fully differentiated. GHR mRNA expression in muscle tissue and the satellite cell fraction was evaluated during chicken growth. In both fractions, GHR mRNA peaked at 4 days of age and then declined in correlation with the reduction of muscle regulatory gene expression. Our results are in contrast with previous studies on rat muscle satellite cells, suggesting a difference in the mode of action of GH in these cells between mammalian and avian species. Our notion is that GH, via its own receptor, promotes more satellite cells to proliferate by inhibiting their differentiation, leading to the addition of more nuclei to the growing muscle. Growth Hormone Receptor and Epiphyseal Growth-Plate Chondrocyte DifferentiationE. Monsonego, a. Gertler1, S. Hurwitz, W.R. Baumbach2, M. Pines Institute of Animal Science, The Volcani Center, Bet Dagan
50250; 1Faculty of Agriculture, The Hebrew University of
Jerusalem, Rehovot 76100, Israel; 2American Cynamid Company,
Princton, NJ, USA. Longitudinal bone growth occurs as a consequence of proliferation and hypertrophy of growth plate chondrocytes. Avian chondrocytes in situ and in culture exhibit a single growth hormone receptor (GHR) transcript of ~4.2 kb, as demonstrated by Northern blot analysis. In culture, avian chondrocytes are in their proliferative state as indicated by the measurable proliferation and collagen type II gene expression. Chondrocyte differentiation, induced by ascorbic acid, is associated with attenuated collagen type II gene expression and an increase in the expression of osteopontin and collagen type X genes, and in alkaline phosphatase activity. No differences in the GHR gene expression are observed between nondifferentiated and the differentiated cells. However, GH binding to its receptor and down regulation of GHR gene expression by GH are observed exclusively in the undifferentiated cells. Chondrocyte differentiation is also accompanied by the appearance in the medium of GH binding protein (GHBP), previously suggested to result from cleavage of the extracellular domain of GHR. This was demonstrated by Western blot analysis using avian GHBP antibodies and by ligand blotting with iodinated chicken GH. These results suggest that the response of avian growth plate chondrocytes to GH is dependent on their state of differentiation. Furthermore, the loss of the extracellular domain of the GHR appears to be responsible for the loss of responsiveness to GH of chondrocytes during differentiation. The Effect of 1,25(OH)2D3 and PTH on the Movement of Calcium Across the Cam of the Chicken EmbryoM.J. Packard, N.B. Clark1, J.P. Erikson1 Biology, Colorado State University, Fort Collins, CO 80523,
USA; 1Physiology and Neurobiology, University of Connecticut,
Storrs, CT 06269 US. The hormonal form of vitamin D [1,25(OH)2D3 ], PTH, or appropriate vehicle were injected into the yolk sac of domestic fowl eggs on days 16 and 17 of incubation. The chorioallantoic membrane (CAM) and overlying inner shell membrane were subsequently removed from eggs on day 18 and mounted in a Ussing-type apparatus. Electrical characteristics (potential difference, resistance, and short-circuit current) and movement of calcium were assessed for 3-5 h. Electrical properties exhibited minor variation over time but were not affected in any consistent manner by hormone treatment. Movement of calcium from the chorionic aspect of the CAM to the allantoic aspect increased from around 3 ng/cm2/h during the first hour of measurement to around 20 ng/cm2/h during the fifth. "Back-flux" of calcium, i.e. movement of calcium from the allantoic aspect to the chorionic, was negligible at all sampling periods. Injection of eggs with PTH did not affect movement of calcium from allantois to chorion but reduced movement from chorion to allantois considerably. The underlying cause of this effect has not been identified. The vitamin D hormone did not affect movement of calcium in either direction. These data indicate that 1,25(OH)2D3 does not affect the transport of calcium across the CAM. We suggest that the role of this hormone during avian embryogenesis is to mediate mobilization of calcium from the eggshell and that effects of the hormone on calcium transport observed in eggs that are deficient in the hormone is a secondary effect attributable to the calcium deficient state that is induced by 1,25(OH)2D3 deficiency. The Effect of Acetazolamide and Benzolamide on Shell Calcium Mobilization in vitroM.J. Packard Biology, Colorado State University, Fort Collins, CO 80523,
USA The role of carbonic anhydrase in shell calcium mobilization was examined by exposing explants of eggshell to acetazolamide (AZ), benzolamide (BZ), or vehicle (DMSO). Explants of shell with (SHELL + CAM) or without (SHELL ONLY) the underlying chorioallantoic membrane were removed from fertile eggs of domestic fowl on day 16 of incubation and cultured in vitro using procedures similar to those used to examine bone resorption in vitro. Explants were cultured for 24 h at 37.5° C and 6% CO2 in air in DMEM with high glucose, glutamine, BSA, penicillin, and streptomycin. Explants were exposed to 10-4, 10-5, and 10-6 M AZ or BZ in DMSO. Control cultures were exposed to an equal volume of DMSO. Change in calcium content of culture medium between the onset and end of culture was used as an index of calcium release from the shell. Release of calcium by SHELL ONLY explants was used as an index for the non-specific release of calcium. SHELL + CAM explants released considerably more calcium during culture than SHELL ONLY explants, but exposure to AZ or BZ did not affect release of calcium by either preparation. These results indicate that carbonic anhydrase is unlikely to mediate shell calcium mobilization in vitro. However, levels of calcium mobilization in vitro are considerably lower than would be expected, based on rates of calcium accumulation in vivo. Thus, the in vitro process may not depend on the catalyzed rate of hydration of CO2. Indirect evidence implicates the vitamin D hormone as crucial for activity of carbonic anhydrase in vivo. Inadequate levels of this hormone during culture could underlie the low levels of calcium mobilization in vitro and the lack of effect of the carbonic anhydrase inhibitors. Relationship between Gene Expression of CaBP-D28K and Vitamin D3 Receptor and Calcium Deposition in the Shell Gland of Laying HensT. Ieda, N. Saito, T. Ono, K. Shimada Laboratory of Animal Physiology, School of Agricultural
Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan The intracellular intestinal calcium binding protein, calbindinD28K (CaBPD28K) is specifically regulated by 1a,25(OH)2D3. However, the role of 1_,25(OH)2D3 in the shell gland is not clear. In this study, the relationship between CaBPD28K and 1a,25(OH)2D3, levels and of CaBPD28K mRNA and 1a,25(OH)2D3 receptor (VDR) mRNA in the intestine and shell gland were measured in the laying hen during the ovulatory cycle. In addition, the effects of the presence of an egg and calcium deposition in the shell gland on the levels of both mRNAs were studied after premature or delayed oviposition. mRNA levels of CaBPD28K and of VDR remained relatively constant in the intestine during the ovulatory cycle. In contrast, both mRNA levels were low in the shell gland when there was no egg in the shell gland, but increased significantly during shell formation. Delayed oviposition by the i.m. injection of indomethacin 3 hours before expected ovulation affected neither shell thickness nor mRNA levels of CaBPD28K or VDR. On the other hand, premature oviposition reduced mRNA levels of both CaBPD28K and VDR in the shell gland. These results suggest that the presence of an egg and calcium deposition in the shell gland may be a stimulatory factor for synthesis and accumulation of CaBPD28K and VDR mRNA in association with calcification. Immunohistochemical Localization of Growth Hormone and Growth Hormone Receptor in the Early Chick EmbryoC.D.M. Johnson, M.a. Wride, K.L. Hull S. Harvey Department of Physiology, University of Alberta, Edmonton, AB
T6G 2H7 Canada Growth hormone (GH) is the primary hormone responsible for regulating postnatal growth, but its role in the development and growth of vertebrate embryos is uncertain. Indeed, chicken embryos continue to grow after decapitation and the removal of the pituitary gland although exogenous GH is able to potentiate embryonic development in decapitated embryos1. The possibility that embryonic growth may be independent of pituitary GH was therefore examined by determining the presence of GH immunoreactivity in the extrapituitary tissues of early embryonic chicks. GH immunoreactivity was observed during the 3rd, 4th, 6th and 8th days of embryonic development in the embryonic ectoderm, notochord, myotome, paravertebral ganglia, ventral nerve, and throughout the neural tube, specifically in the marginal zone and the neuroepithelium. The early ontogenic appearance of GH immunoreactivity in theses tissues therefore occurs before the appearance of GH immunoreactivity in Rathkes Pouch, the pituitary precursor, on day 4.5 of development2. The expression of the GH gene during early embryonic development is therefore probably independent of the pituitary specific factors that later restrict GH gene expression to pituitary somatotrophs. In the 8 day chick limb bud, GH staining was found in the
nuclei of the chondrogenic mesenchyme, particularly in the
nucleoli. Immunoreactivity for growth hormone receptor (GHR) was
also found in the cells of the chondrogenic mesenchyme of the 8
day chick embryo limb bud, with a similar distribution to that
observed for GH. These results could suggest a role for GH and
GHR in some aspects of early chick embryo development, perhaps in
the differentiation of nervous tissue and in chondrogenesis
during limb bud and sclerotome differentiation. These roles may
be mediated by paracrine or autocrine actions of GH produced
within these extrapituitary tissues of the early embryo. (Supported
by NSERC of Canada) |