Identification of Ghrelin in Fertilized Eggs of Chicken

http://www.jstage.jst.go.jp/browse/jpsa doi:+*.,+.+/jpsa..0.,/1 Copyright ῌ ,**3, Japan Poultry Science Association. ῌResearch Note῍ Yukinori Yoshimura+, Chinatsu Tsuyuki+, Kalpana Subedi+, Hiroyuki Kaiya,, Toshihisa Sugino+ and Naoki Isobe+ + Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 1-3῎2/,2, Japan , National Cardiovascular Center Research Institute, Suita, Osaka /0/῎2/0/, Japan The aim of this study was to determine whether ghrelin was present in the internal contents of eggs. Ghrelin in the internal contents of fertilized eggs incubated for * to / days was measured by a time-resolved fluoro-immunoassay. Ghrelin was identified in both yolk and albumen of the fresh fertilized eggs before incubation with a higher concentration in the yolk than albumen. The concentration in the whole fertilized egg internal contents (mixture of yolk, albumen and embryo) did not show significant changes during / days of incubation. These results suggest that ghrelin is contained in the internal contents of fertilized eggs, which may a#ect the functions of embryonic cells during early stage of development in chickens. Key words: chicken, fertilized egg, ghrelin J. Poult. Sci., .0: ,/1῎,/3, ,**3 ῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍ Introduction The chicken ghrelin, a ,0-amino acid peptide, has a unique structure that the active form is acylated with n-octanoic acid at Ser- residue. It interacts with the growth hormone secretagogue receptor (GHS-R), which consists of two variants of GHS-R+a and GHS-R+aV, to exert multiple biological e#ects (Kaiya et al., ,**1). Ghrelin stimulates growth hormone release in both chickens and mammals (Ahmed and Harvey, ,**,; Kaiya et al., ,**,). Central ghrelin acts as an inhibitor for food intake in birds, whereas it acts as stimulator in mammals (Shousha et al., ,**/; Geelissen et al., ,**0; Saito et al., ,**/). Ghrelin is synthesized in the proventriculus, gastrointestinal tissues and brain in chicken (Chen et al., ,**1). Recently, it has been also reported that mRNAs of ghrelin are expressed in the chicken ovarian follicles (Sirotkin et al., ,**0), and that ghrelin may be involved in the local regulation of ovarian cell functions such as proliferation, apoptosis and progesterone secretion (Sirotkin et al., ,**1, ,**2). We found that the ghrelin mRNA and protein were expressed in the epithelial cells in the cephalic magnum of Japanese quail, and the ghrelin immunoreactivity in the epithelial cells was decreased after egg passing through the magnum (Yoshimura et al., ,**/). Although these reports suggest that ghrelin is synthesized in the ovary and oviduct, it remains unknown whether ghrelin is present in the egg internal contents. Received: August +*, ,**2, Accepted: February ,/, ,**3 Correspondence: Y. Yoshimura, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 1-3῎2/,2, Japan. (E-mail: yyosimu@hiroshma-u.ac.jp) Ghar et al. (,**.) reported that chicken embryos express GHS-R mRNA, which was low during embryonic day (ED) * to ED ., followed by an increase on ED/, and it remained constant to ED +1. In mammals, active-form of ghrelin was present in fetal and maternal circulation, which may regulate the energy supply and development in the utero (Yokota et al., ,**/). Nakahara et al. (,**0) reported that maternal ghrelin was transferred to fetal circulation and GHS-R was expressed in various fetal tissues such as skin, bone and intestine of rats. They also showed that chronic treatment of mothers with ghrelin resulted in a significant increase in birth weight, and suggested that maternal ghrelin may regulate fetal development during the stage of pregnancy in rats. Harison et al. (,**1) reported that both ghrelin and GHS-R+a were expressed in ovine placenta, and their expression pattern was in accordance with fetal development. This finding suggests that the ghrelin system may play a role in fetoplacental development. Furthermore, ghrelin enhanced blastocyst formation of porcine embryos (Zhang et al., ,**1). All of these findings suggest that maternal ghrelin may a#ect embryonic development in mammals. If ghrelin that was synthesized in the ovary and oviduct is transmitted to the fertilized eggs, the ghrelin may participate in the regulation of development of embryos in birds as suggested in mammals. The aim of this study was to determine the presence of ghrelin in the yolk and albumen of chicken fertilized eggs. Materials and Methods Experimental Animals White Leghorn hens regularly laying / or more eggs in 258 J. Poult. Sci., .0 (-) a sequence were kept in individual cages under a light regimen of +.-h light and +*-h dark, and provided with feed and water ad libitum. Ten birds were weekly inseminated with *.*/ mL of undiluted fresh semen to obtain fertilized eggs as described by Das et al. (,**1). The fertilized eggs were incubated at -1./῎ in a humidified air condition for / days using an incubator (model P-**2, Showa Furanki Ltd., Saitama, Japan). Time-resolved Fluoro-immunoassay (TR-FIA) of Ghrelin in Eggs The ghrelin concentrations in fertilized eggs before and after incubation for / days (from day * to day /) were measured by a competitive solid-phase immunoassay using Europium (Eu)-labeled synthetic rat ghrelin and polystyrene microtiter strips (Nunc-Immuno Modules, Nalge Nunc Int., Japan) coated with anti-rabbit g-globulin as described by Sugino et al. (,**,). Yolk and albumen of eggs before incubation were separately homogenized to measure the concentration of ghrelin in them, whereas all the internal contents (yolk, albumen and embryo) were homogenized together using a Polytron homogenizer (Kinematica AG, Switzerland). Five eggs were used for each measurement. Homogenized sample (+ mL) was treated with + mL of + M acetic acid (pH ,.*), and the protein fractions were precipitated by adding . mL acetone. After centrifugation, supernatant was evaporated and resuspended in an assay bu#er (/* mM Tris-HCl, +.* mM NaCl, *./ῌ BSA, *.*/ῌ g-globulins, *.***12ῌ DTPA, *.*/ῌ sodium azide, and *.*+ῌ Tween .*, pH 1.2) containing +* KIU/ mL aprotinin. Ghrelin (- mg/+** mL +* mM bicarbonate saline, pH 2./) was labeled with Eu according to the manufacturer’s instructions (PerkinElmer, Inc, USA). Each well was incubated overnight with a rabbit anti-ghrelin serum (([Cys+,]-rat ghrelin [+ῌ++]) polyclonal antiserum; Hosoda et al., ,***) diluted at +: ,,***,***. After washing the ghrelin antibody out, a serial diluted synthetic chicken ghrelin standard (*.*+ῌ+* ng/mL) or extracted samples dissolved in an assay bu#er (+** mL/well) was added to the wells and incubated overnight. Then, Eulabeled ghrelin (ca. /* pg/+** mL) was added to all wells, and incubated for , h. After washing six times, +** mL of the enhancement solution (PerkinElmer, Inc, USA) was added to each well and fluorescence was measured with a time-resolved fluorometer (Multilabel Counter, +.,* ALVO, PerkinElmer, Inc, USA). Ghrelin concentrations of all samples were determined in the same assay. The recovery rate was obtained using homogenized samples (+ mL) exogenously added with or without +* ng synthetic chicken ghrelin. Extraction and measurement of ghrelin amount was performed in a same manner as described above. The recovery value of added ghrelin was obtained from the di#erence between the values of samples added with and without ghrelin, and then the recovery rate was calculated against the exogenously added amount of ghrelin. The mean recovery of ghrelin from extracted sample was 22.-ῌ. The intra-assay of coe$cients of variation was ..0ῌ. Least detectable dose and IC/* in this assay system Fig. +. Ghrelin concentration in yolk and albumen of nonincubated fresh fertilized eggs. Values are meanῌSE (n ῍/). * Significantly di#erent between yolk and albumen. Changes in the ghrelin concentration in the internal contents of fertilized eggs (yolk, albumen and embryo) from day * (before incubation) to day /. Values are mean ῌSE (n῍/). Fig. ,. were *.*+, and *..0- ng/mL, respectively. Statistical Analysis Ghrelin concentrations were expressed as meanῌSE (n ῍/). Significance of di#erences between yolk and albumen was examined by Student’s t-test, whereas those among di#erent incubations days were examined using one-way ANOVA followed by Duncan’s multiple t-test. Di#erences were considered significant when P value was less than *.*/. Results and Discussion Ghrelin was identified in both yolk and albumen of the fresh fertilized eggs before incubation, and the concentration was significantly greater in the yolk than albumen (Fig. +). Changes in the ghrelin concentration in the internal contents of whole fertilized egg (mixture of yolk, albumen and embryo) from day * to day / of incubation are shown in Fig. ,. Ghrelin concentration did not change significantly during / days of incubation. This is the first report to show the presence of ghrelin in both yolk and albumen. Sirotkin et al. (,**0) reported the expression of genes encoding ghrelin in the ovarian follicular cells in laying hens. The surface epithelium of the infundibulum and cephalic magnum contained immunoreactive ghrelin in Japanese quail (Yoshimura et al., ,**/). Thus we assume that ghrelin in the eggs was synthesized in the ovary and oviduct and transmitted to the yolk and albumen. Yoshimura et al.: Ghrelin in Fertilized Egg Gahr et al. (,**.) reported that GHS-R expression was identified in the chicken embryo, although it was low on day * of incubation, and remained at the same level through day ., and increased about ,./-fold on day /. The ghrelin concentration in whole internal contents in fertilized eggs was unchanged for / days of incubation (Fig. ,). The ghrelin in the egg may interact with the GHS-R expressed in the embryonic cells. In mammals, maternal ghrelin may regulate the blastocyst formation (Zhang et al., ,**1), feto-placental development (Harrison et al., ,**1) and fetal development (Nakahara et al., ,**0). The development of embryonic cells may be a#ected by ghrelin also in birds as suggested in mammals. In conclusion, we have shown the presence of ghrelin in the egg internal contents. The endogenous maternal ghrelin may interact with their receptors of embryonic cells at early stage of development in chickens. Acknowledgments We wish to thank Dr. S. Heaphy, Department of Infection, Immunity and Inflammation, University of Leicester, UK, for critically reviewing this manuscript. This work was supported by Grant-in-Aid for Exploratory Research from Japan Society for the Promotion of Science. References Ahmed S and Harvey S. Ghrelin: a hypothalamic GH-releasing factor in domestic fowl (Gallus domesticus). Journal of Endocrinology, +1,: ++1ῌ+,/. ,**,. Chen LL, Jiang QY, Zhu XT, Shu G, Bin YF, Wang XQ, Gao P and Zhang YL. Ghrelin ligand-receptor mRNA expression in hypothalamus, proventriculus and liver of chicken (Gallus gallus domesticus): studies on ontogeny and feeding condition. 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