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Mesotocin Receptor Binding in Oviduct Uterus of the Hen Before and After Oviposition
Mesotocin Receptor Binding in Oviduct Uterus of the Hen Before and After Oviposition T. Takahashi and M. Kawashima1 Department of Biological Diversity and Resources, Gifu University, Yanagido, Gifu 501-1193, Japan
Key words: hen oviduct uterus, mesotocin receptor, receptor binding affinity, receptor binding capacity, oviposition 2008 Poultry Science 87:546–550 doi:10.3382/ps.2007-00284
tocin receptor in uterine tissues and to elucidate whether the MT binding to the receptor changes relative to oviposition.
INTRODUCTION Neurohypophysial hormones in the chicken are arginine vasotocin (AVT; Munsick et al., 1960) and mesotocin (MT; Acher et al., 1970). The AVT causes the contractions of the smooth muscles of the uterus in the hen (Munsick et al., 1960) through an increased binding to its receptor existing in the tissue due to an increased affinity (decrease in Kd value) of the receptor just before oviposition (Takahashi et al., 1994). Mesotocin also causes contractions of the uterine smooth muscle in vitro (Rzasa and Ewy, 1982). The specific binding component to MT has been reported to exist in the uterus (Takahashi et al., 1993), but it is not elucidated whether the binding component has the binding specificity to MT. If the MT relates to oviposition in hens, it is expected that the binding of receptor for MT in laying hens differs from in nonlaying hens, and changes during oviposition cycle, as the findings in the uterine AVT receptor reported earlier (Takahashi et al., 1992, 1994). The present experiment was performed to elucidate the existence of meso-
MATERIALS AND METHODS Animals White Leghorn hens (20 mo of age; 1.8 to 2.2 kg of BW) laying 5 or 6 eggs in a sequence with a 1-d pause between sequences for more than 2 wk and those that had not laid an egg for at least 10-d prior to experiments were used as laying hens and nonlaying (molting) hens, respectively. The hens were kept under 14 h (0500 to 1900 h) light per day with feed (15% CP; 2,800 kcal of ME; Japan Feeding Standard for Poultry, 1992) and water provided for ad libitum consumption. The ovarian weight of nonlaying hens was less than 8.8 g, and whole oviduct weight was less than 11.2 g, and the serum concentration of ovarian steroid hormones measured by a routine radioimmunoassay (Shodono et al., 1975) were less than 323 pM (estradiol17β), 337 pM (progesterone), and 319 pM (testosterone), respectively. The first experiment was performed to examine the binding specificity, affinity, and capacity of MT binding component of the uterus. The laying hens (4 birds) holding a hard-shelled egg in the uterus and the nonlaying hens (4
©2008 Poultry Science Association Inc. Received July 24, 2007. Accepted October 26, 2007. 1 Corresponding author: [email protected]
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capacity (Bmax) was 0.28 ± 0.03 pmol/mg of protein (n = 4) in laying hens and 0.19 ± 0.01 pmol/mg of protein (n = 4) in nonlaying hens. The Kd value of the laying hens varied from 0.37 to 0.91 nM during an oviposition cycle showing a decrease 30 min before oviposition and an increase 4 h after oviposition. The Bmax value also varied from 0.15 to 0.38 pmol/mg of protein showing an increase 3 h before oviposition, a decrease 30 min before oviposition, and an increase 4 h after oviposition. In the nonlaying hen, both values were almost constant during a 24h day. The changes in the binding affinity and capacity of MT receptor of the uterus may be related to oviposition in the hen.
ABSTRACT [125I]mesotocin (MT) binding of membrane fractions of the oviduct uterus in laying hens and nonlaying hens was measured by the use of radioligand binding assay to elucidate the presence of MT receptor in the uterine tissue, and whether the binding to the receptor changes before and after oviposition. The uterine tissue of the hen was found to contain a specific [125I]MT binding component having properties of MT receptor (i.e., binding specificity, saturable binding, high affinity, and limited capacity). The equilibrium dissociation constant (Kd) was 0.46 ± 0.05 nM (x ± SEM; n = 4) in laying hens holding a hard-shelled egg in the uterus (shell gland) and 0.78 ± 0.02 nM (n = 4) in nonlaying hens. The maximum binding
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birds) were killed by decapitation at 0900 h of a day, and the uterus of the oviduct was excised. The weight of uterus of the laying hens was 13.38 ± 0.46 g (x ± SEM, n = 4) and was significantly higher (P < 0.001) than that of the nonlaying hens (4.89 ± 0.55 g, n = 4). A second experiment was performed to examine changes in the binding affinity and capacity before and after oviposition. The time before oviposition was estimated from the oviposition time observed before the experiment. The clock time of oviposition of the first egg of the laying sequence was 0654 h ± 4 min (x ± SEM, n = 50). The uterus was obtained from the laying hens at 5 different times (16, 11, 6, 3 h, and 30 min) before oviposition of the first egg of sequence and at other 5 different times (within
Figure 2. Time course of the specific [125I]mesotocin (MT) binding in the plasma membrane fraction of the oviduct uterus of laying hens. Samples (10 g of protein per tube) were incubated at 4 (䊊) or 30 (䊉)°C for various hours with 0.6 nM [125I]MT in the absence or presence of 1 M unlabeled MT, and the specific [125I]MT binding was measured. Each point represents the mean of 3 separate pools of samples, and the vertical bars represent SEM.
Figure 3. Relationship of specific [125I]mesotocin (MT) binding to the protein concentration in the plasma membrane fraction of the oviduct uterus of laying hens. Samples (2.5 to 30 g of protein per tube) were incubated at 30°C for 5 h with 0.6 nM [125I]MT in the absence or presence of 1 M unlabeled MT, and the specific [125I]MT binding was measured. Each point represents the mean of 3 separate pools of samples, and the vertical bars represent SEM.
5 min, 2, 4, 5, and 7 h) after oviposition of the first egg of sequence (6 birds at each time), and also from the nonlaying hens at 8 different times corresponding to the time of sampling in the laying hens during a 24-h day (6 birds at each time). The myometrium of the uterus was used for the preparation of plasma membrane fractions.
Preparation of Plasma Membrane Fractions The method of preparation of membrane fractions for the uterine tissue was the same as reported earlier (Taka-
Figure 4. Competition for [125I]mesotocin (MT) binding in the plasma membrane fraction of the oviduct uterus of laying hens. Samples (10 g of protein per tube) were incubated at 30°C for 5 h with 0.6 nM [125I]MT in the absence (control) or presence of various fold M excess of unlabeled MT (䊉), arginine vasotocin (AVT; 䊊), chicken luteinizing hormone releasing hormone-1 (cLHRH-I; 䊏), cLHRH-II (䊐), or cAngiotensin-II (▼). The amount of the [125I]MT binding in control value was 0.38 pmol/mg of protein. Each point represents the mean of 2 separate pools of samples.
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Figure 1. Relationship of specific bindings for [125I]mesotocin (MT) to the concentration of EDTA, Mg2+, or Ca2+ in the plasma membrane fractions of the uterus of hens. Samples (10 g of protein per tube) were incubated in 50 mM Tris buffer (pH 7.4) containing various concentrations of EDTA (䊉), Mg2+ (䊐), or Ca2+ (䊏) at 30°C for 5 h with 0.6 nM [125I]MT in the absence or presence of 1 M unlabeled MT, and specific [125I]MT bindings were measured. Each point represents the mean ± SEM of 3 separate pools of samples from the control, and the vertical bars represent SEM. Points that have different letters **Significantly different (P < 0.01) by Newman-Keuls’ test.
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Binding Assay The labeling of MT with 125I was performed by the Iodogen (Sigma Chemical Co., St. Louis, MO) method (Takahashi et al., 1993). Specific activity of [125I]MT was 1,886 to 2,272 Ci/mmol determined by the method of Copeland et al. (1979). In the binding assay, polypropylene tubes used were pretreated overnight at 4°C with TE buffer containing 1% BSA. Aliquots of the membrane fraction (10 g of protein/200 L per tube) were incubated at 30°C for 5 h with [125I]MT (0.05 to 1.6 nM) in the presence (for nonspecific bindings) or absence (for total bindings) of 1 M of unlabeled MT in a total volume of 300 L. To examine the binding specificity, unlabeled MT (Bachem Inc., Torrance, CA), AVT (Bachem Inc.), chicken luteinizing hormonereleasing hormone-I [cLHRH-I: Gln8-GnRH (Peninsula Laboratories Inc., Belmont, CA)], chicken LHRH-II [His,8 Trp,7 Tyr,8-GnRH and chicken angiotensin-II [cAngiotensin-II: Val5-angiotensin-II (Bachem Inc.)] were used as competitors. Concentrations of unlabeled peptides used were 0.006 to 6 M in MT and AVT, and 0.06 to 6 M in cLHRH-I, cLHRH-II, and cAngiotensin-II. Bound and free ligands were separated by centrifugation (10,000 × g, 20 min, 4°C). The radioactivity of the precipitate (bound ligand) was measured by a gamma counter (Packard Cobra, Packard Instrument Co., Meriden, CT). The counting efficiency was 74.1 to 84.4%. Specific bindings were obtained by subtracting the nonspecific binding from the total bind-
Figure 5. Saturation curve and Scatchard plot of specific [125I]mesotocin (MT) binding in the plasma membrane fraction of the oviduct uterus of laying hens. Samples (10 g of protein per tube) were incubated at 30°C for 5 h with various concentrations of [125I]MT in the absence or presence of 1 M unlabeled MT, and the specific [125I]MT binding was measured. The value of Kd and Bmax (calculated by the use of Scatchard analysis), and correlation coefficient (γ) between the specific binding and the ratio of specific binding to free [125I]MT was 0.45 nM (Kd), 0.28 pmol/mg of protein (Bmax), and −0.99 (γ), respectively. Each point represents the mean of duplicate determinations from 1 pooled sample. (䊉) Specific binding, (䊊) Nonspecific binding. B = bound; F = free.
ing and expressed as moles per milligram of protein. The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) were determined by the method of Scatchard (1949).
Preliminary Experiments Relationships of specific [125I]MT binding to presence of cations (2 to 8 mM of Mg2+ or Ca2+) or a chelator (1 to 10 mM EDTA), incubation time (¹⁄₄ to 8 h) and temperature (4 and 30°C), and protein concentration (2.5 to 30 g per tube) were examined. The specific [125I]MT binding was not changed by the presence of Mg2+ and Ca2+ and increased by the presence of EDTA (1 to 10 mM; Figure 1). The binding at 30°C increased during the first 4 h of incubation, and then reached a plateau up to 8 h, but a remarkable increase was not observed at 4°C (Figure 2). A linear increase in the specific binding with the increase in the protein concentration from 2.5 to 30 g per tube was observed when incubated at 30°C for 5 h (Figure 3). Based on these findings, the following experimental conditions were used in following experiments: presence of 2 mM EDTA, 30°C for 5 h incubation, and 10 g of protein per tube.
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hashi et al., 1992). All steps were performed at 4°C. Uterine tissues were rinsed with ice-cold Tris-EDTA buffer [TE; 50 mM Tris (Kishida Chemical Co., Ltd., Osaka, Japan)HCl, 2 mM EDTA (Nacalai Tesque Inc., Kyoto, Japan), pH 7.4] containing 0.25 M sucrose. The tissues were blotted with a filter paper, weighed, and homogenized in the same buffer (5 vol/wt) using the Ultra-Turrax homogenizer (Type 18-10, Ika Labortechnik, Janke & Kunkel GmbH & Co KG, Staufen, Germany). The homogenate was filtered through gauze. The filtrate was centrifuged (1,000 × g, 10 min) and the supernatant was obtained. The precipitate was rehomogenized in the same buffer and recentrifuged. Two supernatants were combined and centrifuged at 30,000 × g for 30 min. The precipitate was suspended in the 0.25 M sucrose-TE buffer (5 vol/wt) with a PotterElvehjem type glass-Teflon homogenizer. The suspension was gently poured on equal volume of TE buffer containing 1.0 M sucrose and centrifuged at 90,000 × g for 90 min in a RPS-25 swinging rotor (Hitachi Koki Co., Ltd., Hitachinaka, Japan). The interface fraction of the 2 buffers was obtained and washed twice with TE buffer not containing sucrose by centrifugations (30,000 × g, 30 min). The final precipitate was suspended in TE buffer (0.5 vol/wt) and used as the plasma membrane fraction after determinations of the protein concentration by the method of Lowry et al. (1951) using BSA (Seikagaku Corp., Tokyo, Japan) as a standard. The amount of protein in the membrane fraction was 0.35 ± 0.02 (x ± SEM, n = 4) mg/g of tissue in laying hens and 0.36 ± 0.01 (n = 4) mg/g of tissue in nonlaying hens, respectively.
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Statistical Analyses A half-maximal inhibition (ID50) of the [125I]AVT binding was estimated by the use of a log-logit linear regression (Finney, 1964). The data were analyzed by 1-way ANOVA (Snedecor and Cochran, 1967). When significant effects were found at 5% level, Student’s t-test was used to assess statistical significance between 2 means and NewmanKeuls’ multiple range test (Snedecor and Cochran, 1967) was used to compare means of more than 2 groups.
RESULTS Binding Specificity The [125I]MT binding was markedly reduced by the presence of a 100- to 10,000-fold M excess of unlabeled MT
but was not reduced by the presence of an equivalent M excess of unlabeled cLHRH-I, cLHRH-II, and cAngiotensin-II (Figure 4). A 10,000-fold M excess of AVT reduced the binding to about 50% (Figure 4). The half-maximal inhibition (ID50) value calculated from the data of doseinhibition curve was 14 nM for MT and 8,458 nM for AVT.
Binding Affinity and Capacity The specific [125I]MT binding increased when increasing amounts of [125I]MT were added (i.e., when increasing amounts of free [125I]MT), and was saturable at about 1.0 nM (Figure 5). Scatchard analysis revealed a linear relationship between the amount of specific binding and the ratio of specific binding to free [125I]MT (Figure 5), indicating 1 single class of binding sites. The Kd and Bmax values ob-
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Figure 6. The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) of the specific binding component of [125I]mesotocin (MT) in the plasma membrane fraction of the oviduct uterus of laying hens at various times before and after oviposition (䊉) and of nonlaying hens at corresponding times (䊊). Samples (10 g of protein per tube) were incubated at 30°C for 5 h with [125I]MT (0.05 to 1.6 nM) in the presence and absence of 1 M unlabeled MT, and the specific [125I]MT binding was measured. The values of Kd and Bmax were obtained by Scatchard analysis. The amount of protein in membrane fractions, expressed as milligrams per gram of wet tissue weight, was 0.30 ± 0.03 (x ± SEM, n = 60) in laying hens and 0.27 ± 0.03 (n = 48) in nonlaying hens, respectively, and was not significantly different among the hens at different times. The wet weight of the uterus expressed as gram per hen was 13.07 ± 0.20 (n = 60) in laying hens and 4.56 ± 0.13 (n = 48) in nonlaying hens, respectively. Each point represents the mean of 6 birds, and the vertical bars represent SEM. *Significantly different (P ≤ 0.01) from the preceding value by Newman-Keuls’ test. +0 h: within 5 min after oviposition.
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Table 1. Equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) of mesotocin (MT) receptors in the plasma membrane fraction of the oviduct uterus (shell gland) of laying and nonlaying hens1 Hen Laying Nonlaying
Kd2 (nM)
Bmax2 (pmol/mg of protein)
0.46 ± 0.053** 0.78 ± 0.02
0.28 ± 0.03* 0.19 ± 0.01
1 Laying hens holding a hard-shelled egg in the uterus and nonlaying hens were killed at 0900 h. 2 Calculated by the use of Scatchard (1949) analysis. 3 Mean ± SEM of 4 birds. **Significantly different from the value of nonlaying hens by t-test (P < 0.01). *Significantly different from the value of nonlaying hens by t-test (P < 0.05).
Changes in Kd and Bmax Values The Kd value decreased 30 min before expected oviposition and increased 4 h after oviposition (Figure 6). The Bmax value increased 3 h before expected oviposition, decreased 30 min before oviposition and just after oviposition, and then increased 4 h after oviposition. In nonlaying hens, neither Kd nor Bmax changed during a 24-h day.
DISCUSSION Membrane fractions of oviduct uterus of the hen showed to contain a binding component having the properties of a receptor [i.e., binding specificity (Figure 4), saturable binding (Figure 5), as well as high affinity and limited capacity (Figure 5)]. The Kd expressed as moles per liter, which is an index of the binding affinity (the smaller the higher), was of the order of 10−10 and was similar to that reported for the MT receptor in the kidney (Takahashi et al., 1996) or the AVT receptors in the uterus (Takahashi et al., 1992) or the vagina (Takahashi et al., 1998) of the hen. The binding affinity and the binding capacity of the uterine MT receptor found in the present study were higher in laying hens than in nonlaying hens. The binding affinity and binding capacity of the uterine MT receptor in laying hens showed a change during a period before and after oviposition, but no change was found in nonlaying hens during a 24-h period (Figure 6). These results suggest that the MT may relate to the oviposition system in the uterus of hens. The Bmax value of the uterine MT receptor in laying hens increased 3 h before oviposition. A similar change in the Bmax value was also reported in the AVT receptor of the uterus in laying hens (Takahashi et al., 1994). In contrast within 30 min before oviposition, changes in the Kd and Bmax values of MT receptor differed from that reported on the uterine AVT receptor. The Kd and Bmax values of the MT receptor showed a decrease 30 min before oviposition, whereas that of the AVT receptor showed a decrease just before oviposition. (Takahashi et al., 1994). The decrease in the Kd value of the receptor indicates an increase in
REFERENCES Acher, R., J. Chauvet, and M.-T. Chauvet. 1970. Phylogeny of neurohypophysial hormones. The avian active peptides. Eur. J. Biochem. 17:509–513. Copeland, K. C., M. L. Aubert, J. Rivier, and P. C. Sizonenko. 1979. Luteinizing hormone-releasing hormone: Sequential versus conformational specificity of antiluteinizing hormone-releasing hormone sera. Endocrinology 104:1504–1512. Finney, D. J. 1964. Assays based on quantal responses. Pages 468–490 in Statistical Method in Biological Assay. 2nd ed. D. J. Finney, ed. Charles Griffin & Co. Ltd., London, UK. Japan Feeding Standard for Poultry. 1992. Pages 18–21 in Japan Feeding Standard. Agriculture, Forestry and Fisheries Research Council Secretariat, Ministry of Agriculture, Fisheries and Forestry, ed. Central Association of Livestock Industry, Tokyo, Japan. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275. Munsick, R. A., W. H. Sawyer, and H. B. van Dyke. 1960. Avian neurohypophysial hormones: Pharmacological properties and tentative identification. Endocrinology 66:860–871. Rzasa, J., and Z. Ewy. 1982. The effect of ovarian steroids on the response of the hen uterus to neurohypophysial hormones. Acta Physiol. Pol. 33:249–255. Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51:660–672. Shodono, M., T. Nakamura, Y. Tanabe, and K. Wakabayashi. 1975. Simultaneous determinations of estradiol-17β, progesterone and luteinizing hormone in the plasma during the ovulatory cycle of the hen. Acta Endocrinol. (Copenh.) 78:565–573. Snedecor, G. W., and W. G. Cochran. 1967. One-way classifications. Analysis of variance. Pages 258–298 in Statistical Methods. 6th ed. G. W. Snedecor and W. G. Cochran, ed. Iowa State Univ. Press, Ames, IA. Takahashi, T., M. Kawashima, M. Kamiyoshi, and K. Tanaka. 1992. Arginine vasotocin binding component in the uterus (shell gland) of the chicken. Acta Endocrinol. (Copenh.) 127:179–184. Takahashi, T., M. Kawashima, M. Kamiyoshi, and K. Tanaka. 1993. Mesotocin binding component in various tissues of the hen. Jpn. Poult. Sci. 30:108–113. Takahashi, T., M. Kawashima, M. Kamiyoshi, and K. Tanaka. 1994. Arginine vasotocin receptor binding in the hen uterus (shell gland) before and after oviposition. Eur. J. Endocrinol. 130:366–372. Takahashi, T., M. Kawashima, T. Yasuoka, M. Kamiyoshi, and K. Tanaka. 1996. Mesotocin binding to receptors in hen kidney plasma membranes. Poult. Sci. 75:910–914. Takahashi, T., M. Kawashima, T. Yasuoka, and K. Tanaka. 1998. Arginine vasotocin receptor in the vagina of the oviduct of the hen. Poult. Sci. 77:1699–1703.
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tained from each 4 birds in laying hens and nonlaying hens are shown in Table 1.
the binding affinity, which may cause an increase in the hormonal action (Takahashi et al., 1994). It seems likely that the MT action increases before the increase in that of AVT in the oviduct uterus within 30 min before oviposition. Therefore the role of MT may be different from that of the AVT in the uterus. The MT is reported to cause the contraction of the uterine smooth muscle in vitro (Rzasa and Ewy, 1982). If the uterine MT receptor binding is concerned in the contraction of the uterine muscles, the change in the binding affinity and the binding capacity found in the present study may be related to the process of oviposition in a different role from the AVT.
Key words: hen oviduct uterus, mesotocin receptor, receptor binding affinity, receptor binding capacity, oviposition 2008 Poultry Science 87:546–550 doi:10.3382/ps.2007-00284
tocin receptor in uterine tissues and to elucidate whether the MT binding to the receptor changes relative to oviposition.
INTRODUCTION Neurohypophysial hormones in the chicken are arginine vasotocin (AVT; Munsick et al., 1960) and mesotocin (MT; Acher et al., 1970). The AVT causes the contractions of the smooth muscles of the uterus in the hen (Munsick et al., 1960) through an increased binding to its receptor existing in the tissue due to an increased affinity (decrease in Kd value) of the receptor just before oviposition (Takahashi et al., 1994). Mesotocin also causes contractions of the uterine smooth muscle in vitro (Rzasa and Ewy, 1982). The specific binding component to MT has been reported to exist in the uterus (Takahashi et al., 1993), but it is not elucidated whether the binding component has the binding specificity to MT. If the MT relates to oviposition in hens, it is expected that the binding of receptor for MT in laying hens differs from in nonlaying hens, and changes during oviposition cycle, as the findings in the uterine AVT receptor reported earlier (Takahashi et al., 1992, 1994). The present experiment was performed to elucidate the existence of meso-
MATERIALS AND METHODS Animals White Leghorn hens (20 mo of age; 1.8 to 2.2 kg of BW) laying 5 or 6 eggs in a sequence with a 1-d pause between sequences for more than 2 wk and those that had not laid an egg for at least 10-d prior to experiments were used as laying hens and nonlaying (molting) hens, respectively. The hens were kept under 14 h (0500 to 1900 h) light per day with feed (15% CP; 2,800 kcal of ME; Japan Feeding Standard for Poultry, 1992) and water provided for ad libitum consumption. The ovarian weight of nonlaying hens was less than 8.8 g, and whole oviduct weight was less than 11.2 g, and the serum concentration of ovarian steroid hormones measured by a routine radioimmunoassay (Shodono et al., 1975) were less than 323 pM (estradiol17β), 337 pM (progesterone), and 319 pM (testosterone), respectively. The first experiment was performed to examine the binding specificity, affinity, and capacity of MT binding component of the uterus. The laying hens (4 birds) holding a hard-shelled egg in the uterus and the nonlaying hens (4
©2008 Poultry Science Association Inc. Received July 24, 2007. Accepted October 26, 2007. 1 Corresponding author: [email protected]
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capacity (Bmax) was 0.28 ± 0.03 pmol/mg of protein (n = 4) in laying hens and 0.19 ± 0.01 pmol/mg of protein (n = 4) in nonlaying hens. The Kd value of the laying hens varied from 0.37 to 0.91 nM during an oviposition cycle showing a decrease 30 min before oviposition and an increase 4 h after oviposition. The Bmax value also varied from 0.15 to 0.38 pmol/mg of protein showing an increase 3 h before oviposition, a decrease 30 min before oviposition, and an increase 4 h after oviposition. In the nonlaying hen, both values were almost constant during a 24h day. The changes in the binding affinity and capacity of MT receptor of the uterus may be related to oviposition in the hen.
ABSTRACT [125I]mesotocin (MT) binding of membrane fractions of the oviduct uterus in laying hens and nonlaying hens was measured by the use of radioligand binding assay to elucidate the presence of MT receptor in the uterine tissue, and whether the binding to the receptor changes before and after oviposition. The uterine tissue of the hen was found to contain a specific [125I]MT binding component having properties of MT receptor (i.e., binding specificity, saturable binding, high affinity, and limited capacity). The equilibrium dissociation constant (Kd) was 0.46 ± 0.05 nM (x ± SEM; n = 4) in laying hens holding a hard-shelled egg in the uterus (shell gland) and 0.78 ± 0.02 nM (n = 4) in nonlaying hens. The maximum binding
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birds) were killed by decapitation at 0900 h of a day, and the uterus of the oviduct was excised. The weight of uterus of the laying hens was 13.38 ± 0.46 g (x ± SEM, n = 4) and was significantly higher (P < 0.001) than that of the nonlaying hens (4.89 ± 0.55 g, n = 4). A second experiment was performed to examine changes in the binding affinity and capacity before and after oviposition. The time before oviposition was estimated from the oviposition time observed before the experiment. The clock time of oviposition of the first egg of the laying sequence was 0654 h ± 4 min (x ± SEM, n = 50). The uterus was obtained from the laying hens at 5 different times (16, 11, 6, 3 h, and 30 min) before oviposition of the first egg of sequence and at other 5 different times (within
Figure 2. Time course of the specific [125I]mesotocin (MT) binding in the plasma membrane fraction of the oviduct uterus of laying hens. Samples (10 g of protein per tube) were incubated at 4 (䊊) or 30 (䊉)°C for various hours with 0.6 nM [125I]MT in the absence or presence of 1 M unlabeled MT, and the specific [125I]MT binding was measured. Each point represents the mean of 3 separate pools of samples, and the vertical bars represent SEM.
Figure 3. Relationship of specific [125I]mesotocin (MT) binding to the protein concentration in the plasma membrane fraction of the oviduct uterus of laying hens. Samples (2.5 to 30 g of protein per tube) were incubated at 30°C for 5 h with 0.6 nM [125I]MT in the absence or presence of 1 M unlabeled MT, and the specific [125I]MT binding was measured. Each point represents the mean of 3 separate pools of samples, and the vertical bars represent SEM.
5 min, 2, 4, 5, and 7 h) after oviposition of the first egg of sequence (6 birds at each time), and also from the nonlaying hens at 8 different times corresponding to the time of sampling in the laying hens during a 24-h day (6 birds at each time). The myometrium of the uterus was used for the preparation of plasma membrane fractions.
Preparation of Plasma Membrane Fractions The method of preparation of membrane fractions for the uterine tissue was the same as reported earlier (Taka-
Figure 4. Competition for [125I]mesotocin (MT) binding in the plasma membrane fraction of the oviduct uterus of laying hens. Samples (10 g of protein per tube) were incubated at 30°C for 5 h with 0.6 nM [125I]MT in the absence (control) or presence of various fold M excess of unlabeled MT (䊉), arginine vasotocin (AVT; 䊊), chicken luteinizing hormone releasing hormone-1 (cLHRH-I; 䊏), cLHRH-II (䊐), or cAngiotensin-II (▼). The amount of the [125I]MT binding in control value was 0.38 pmol/mg of protein. Each point represents the mean of 2 separate pools of samples.
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Figure 1. Relationship of specific bindings for [125I]mesotocin (MT) to the concentration of EDTA, Mg2+, or Ca2+ in the plasma membrane fractions of the uterus of hens. Samples (10 g of protein per tube) were incubated in 50 mM Tris buffer (pH 7.4) containing various concentrations of EDTA (䊉), Mg2+ (䊐), or Ca2+ (䊏) at 30°C for 5 h with 0.6 nM [125I]MT in the absence or presence of 1 M unlabeled MT, and specific [125I]MT bindings were measured. Each point represents the mean ± SEM of 3 separate pools of samples from the control, and the vertical bars represent SEM. Points that have different letters **Significantly different (P < 0.01) by Newman-Keuls’ test.
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Binding Assay The labeling of MT with 125I was performed by the Iodogen (Sigma Chemical Co., St. Louis, MO) method (Takahashi et al., 1993). Specific activity of [125I]MT was 1,886 to 2,272 Ci/mmol determined by the method of Copeland et al. (1979). In the binding assay, polypropylene tubes used were pretreated overnight at 4°C with TE buffer containing 1% BSA. Aliquots of the membrane fraction (10 g of protein/200 L per tube) were incubated at 30°C for 5 h with [125I]MT (0.05 to 1.6 nM) in the presence (for nonspecific bindings) or absence (for total bindings) of 1 M of unlabeled MT in a total volume of 300 L. To examine the binding specificity, unlabeled MT (Bachem Inc., Torrance, CA), AVT (Bachem Inc.), chicken luteinizing hormonereleasing hormone-I [cLHRH-I: Gln8-GnRH (Peninsula Laboratories Inc., Belmont, CA)], chicken LHRH-II [His,8 Trp,7 Tyr,8-GnRH and chicken angiotensin-II [cAngiotensin-II: Val5-angiotensin-II (Bachem Inc.)] were used as competitors. Concentrations of unlabeled peptides used were 0.006 to 6 M in MT and AVT, and 0.06 to 6 M in cLHRH-I, cLHRH-II, and cAngiotensin-II. Bound and free ligands were separated by centrifugation (10,000 × g, 20 min, 4°C). The radioactivity of the precipitate (bound ligand) was measured by a gamma counter (Packard Cobra, Packard Instrument Co., Meriden, CT). The counting efficiency was 74.1 to 84.4%. Specific bindings were obtained by subtracting the nonspecific binding from the total bind-
Figure 5. Saturation curve and Scatchard plot of specific [125I]mesotocin (MT) binding in the plasma membrane fraction of the oviduct uterus of laying hens. Samples (10 g of protein per tube) were incubated at 30°C for 5 h with various concentrations of [125I]MT in the absence or presence of 1 M unlabeled MT, and the specific [125I]MT binding was measured. The value of Kd and Bmax (calculated by the use of Scatchard analysis), and correlation coefficient (γ) between the specific binding and the ratio of specific binding to free [125I]MT was 0.45 nM (Kd), 0.28 pmol/mg of protein (Bmax), and −0.99 (γ), respectively. Each point represents the mean of duplicate determinations from 1 pooled sample. (䊉) Specific binding, (䊊) Nonspecific binding. B = bound; F = free.
ing and expressed as moles per milligram of protein. The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) were determined by the method of Scatchard (1949).
Preliminary Experiments Relationships of specific [125I]MT binding to presence of cations (2 to 8 mM of Mg2+ or Ca2+) or a chelator (1 to 10 mM EDTA), incubation time (¹⁄₄ to 8 h) and temperature (4 and 30°C), and protein concentration (2.5 to 30 g per tube) were examined. The specific [125I]MT binding was not changed by the presence of Mg2+ and Ca2+ and increased by the presence of EDTA (1 to 10 mM; Figure 1). The binding at 30°C increased during the first 4 h of incubation, and then reached a plateau up to 8 h, but a remarkable increase was not observed at 4°C (Figure 2). A linear increase in the specific binding with the increase in the protein concentration from 2.5 to 30 g per tube was observed when incubated at 30°C for 5 h (Figure 3). Based on these findings, the following experimental conditions were used in following experiments: presence of 2 mM EDTA, 30°C for 5 h incubation, and 10 g of protein per tube.
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hashi et al., 1992). All steps were performed at 4°C. Uterine tissues were rinsed with ice-cold Tris-EDTA buffer [TE; 50 mM Tris (Kishida Chemical Co., Ltd., Osaka, Japan)HCl, 2 mM EDTA (Nacalai Tesque Inc., Kyoto, Japan), pH 7.4] containing 0.25 M sucrose. The tissues were blotted with a filter paper, weighed, and homogenized in the same buffer (5 vol/wt) using the Ultra-Turrax homogenizer (Type 18-10, Ika Labortechnik, Janke & Kunkel GmbH & Co KG, Staufen, Germany). The homogenate was filtered through gauze. The filtrate was centrifuged (1,000 × g, 10 min) and the supernatant was obtained. The precipitate was rehomogenized in the same buffer and recentrifuged. Two supernatants were combined and centrifuged at 30,000 × g for 30 min. The precipitate was suspended in the 0.25 M sucrose-TE buffer (5 vol/wt) with a PotterElvehjem type glass-Teflon homogenizer. The suspension was gently poured on equal volume of TE buffer containing 1.0 M sucrose and centrifuged at 90,000 × g for 90 min in a RPS-25 swinging rotor (Hitachi Koki Co., Ltd., Hitachinaka, Japan). The interface fraction of the 2 buffers was obtained and washed twice with TE buffer not containing sucrose by centrifugations (30,000 × g, 30 min). The final precipitate was suspended in TE buffer (0.5 vol/wt) and used as the plasma membrane fraction after determinations of the protein concentration by the method of Lowry et al. (1951) using BSA (Seikagaku Corp., Tokyo, Japan) as a standard. The amount of protein in the membrane fraction was 0.35 ± 0.02 (x ± SEM, n = 4) mg/g of tissue in laying hens and 0.36 ± 0.01 (n = 4) mg/g of tissue in nonlaying hens, respectively.
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Statistical Analyses A half-maximal inhibition (ID50) of the [125I]AVT binding was estimated by the use of a log-logit linear regression (Finney, 1964). The data were analyzed by 1-way ANOVA (Snedecor and Cochran, 1967). When significant effects were found at 5% level, Student’s t-test was used to assess statistical significance between 2 means and NewmanKeuls’ multiple range test (Snedecor and Cochran, 1967) was used to compare means of more than 2 groups.
RESULTS Binding Specificity The [125I]MT binding was markedly reduced by the presence of a 100- to 10,000-fold M excess of unlabeled MT
but was not reduced by the presence of an equivalent M excess of unlabeled cLHRH-I, cLHRH-II, and cAngiotensin-II (Figure 4). A 10,000-fold M excess of AVT reduced the binding to about 50% (Figure 4). The half-maximal inhibition (ID50) value calculated from the data of doseinhibition curve was 14 nM for MT and 8,458 nM for AVT.
Binding Affinity and Capacity The specific [125I]MT binding increased when increasing amounts of [125I]MT were added (i.e., when increasing amounts of free [125I]MT), and was saturable at about 1.0 nM (Figure 5). Scatchard analysis revealed a linear relationship between the amount of specific binding and the ratio of specific binding to free [125I]MT (Figure 5), indicating 1 single class of binding sites. The Kd and Bmax values ob-
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Figure 6. The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) of the specific binding component of [125I]mesotocin (MT) in the plasma membrane fraction of the oviduct uterus of laying hens at various times before and after oviposition (䊉) and of nonlaying hens at corresponding times (䊊). Samples (10 g of protein per tube) were incubated at 30°C for 5 h with [125I]MT (0.05 to 1.6 nM) in the presence and absence of 1 M unlabeled MT, and the specific [125I]MT binding was measured. The values of Kd and Bmax were obtained by Scatchard analysis. The amount of protein in membrane fractions, expressed as milligrams per gram of wet tissue weight, was 0.30 ± 0.03 (x ± SEM, n = 60) in laying hens and 0.27 ± 0.03 (n = 48) in nonlaying hens, respectively, and was not significantly different among the hens at different times. The wet weight of the uterus expressed as gram per hen was 13.07 ± 0.20 (n = 60) in laying hens and 4.56 ± 0.13 (n = 48) in nonlaying hens, respectively. Each point represents the mean of 6 birds, and the vertical bars represent SEM. *Significantly different (P ≤ 0.01) from the preceding value by Newman-Keuls’ test. +0 h: within 5 min after oviposition.
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Table 1. Equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) of mesotocin (MT) receptors in the plasma membrane fraction of the oviduct uterus (shell gland) of laying and nonlaying hens1 Hen Laying Nonlaying
Kd2 (nM)
Bmax2 (pmol/mg of protein)
0.46 ± 0.053** 0.78 ± 0.02
0.28 ± 0.03* 0.19 ± 0.01
1 Laying hens holding a hard-shelled egg in the uterus and nonlaying hens were killed at 0900 h. 2 Calculated by the use of Scatchard (1949) analysis. 3 Mean ± SEM of 4 birds. **Significantly different from the value of nonlaying hens by t-test (P < 0.01). *Significantly different from the value of nonlaying hens by t-test (P < 0.05).
Changes in Kd and Bmax Values The Kd value decreased 30 min before expected oviposition and increased 4 h after oviposition (Figure 6). The Bmax value increased 3 h before expected oviposition, decreased 30 min before oviposition and just after oviposition, and then increased 4 h after oviposition. In nonlaying hens, neither Kd nor Bmax changed during a 24-h day.
DISCUSSION Membrane fractions of oviduct uterus of the hen showed to contain a binding component having the properties of a receptor [i.e., binding specificity (Figure 4), saturable binding (Figure 5), as well as high affinity and limited capacity (Figure 5)]. The Kd expressed as moles per liter, which is an index of the binding affinity (the smaller the higher), was of the order of 10−10 and was similar to that reported for the MT receptor in the kidney (Takahashi et al., 1996) or the AVT receptors in the uterus (Takahashi et al., 1992) or the vagina (Takahashi et al., 1998) of the hen. The binding affinity and the binding capacity of the uterine MT receptor found in the present study were higher in laying hens than in nonlaying hens. The binding affinity and binding capacity of the uterine MT receptor in laying hens showed a change during a period before and after oviposition, but no change was found in nonlaying hens during a 24-h period (Figure 6). These results suggest that the MT may relate to the oviposition system in the uterus of hens. The Bmax value of the uterine MT receptor in laying hens increased 3 h before oviposition. A similar change in the Bmax value was also reported in the AVT receptor of the uterus in laying hens (Takahashi et al., 1994). In contrast within 30 min before oviposition, changes in the Kd and Bmax values of MT receptor differed from that reported on the uterine AVT receptor. The Kd and Bmax values of the MT receptor showed a decrease 30 min before oviposition, whereas that of the AVT receptor showed a decrease just before oviposition. (Takahashi et al., 1994). The decrease in the Kd value of the receptor indicates an increase in
REFERENCES Acher, R., J. Chauvet, and M.-T. Chauvet. 1970. Phylogeny of neurohypophysial hormones. The avian active peptides. Eur. J. Biochem. 17:509–513. Copeland, K. C., M. L. Aubert, J. Rivier, and P. C. Sizonenko. 1979. Luteinizing hormone-releasing hormone: Sequential versus conformational specificity of antiluteinizing hormone-releasing hormone sera. Endocrinology 104:1504–1512. Finney, D. J. 1964. Assays based on quantal responses. Pages 468–490 in Statistical Method in Biological Assay. 2nd ed. D. J. Finney, ed. Charles Griffin & Co. Ltd., London, UK. Japan Feeding Standard for Poultry. 1992. Pages 18–21 in Japan Feeding Standard. Agriculture, Forestry and Fisheries Research Council Secretariat, Ministry of Agriculture, Fisheries and Forestry, ed. Central Association of Livestock Industry, Tokyo, Japan. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275. Munsick, R. A., W. H. Sawyer, and H. B. van Dyke. 1960. Avian neurohypophysial hormones: Pharmacological properties and tentative identification. Endocrinology 66:860–871. Rzasa, J., and Z. Ewy. 1982. The effect of ovarian steroids on the response of the hen uterus to neurohypophysial hormones. Acta Physiol. Pol. 33:249–255. Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51:660–672. Shodono, M., T. Nakamura, Y. Tanabe, and K. Wakabayashi. 1975. Simultaneous determinations of estradiol-17β, progesterone and luteinizing hormone in the plasma during the ovulatory cycle of the hen. Acta Endocrinol. (Copenh.) 78:565–573. Snedecor, G. W., and W. G. Cochran. 1967. One-way classifications. Analysis of variance. Pages 258–298 in Statistical Methods. 6th ed. G. W. Snedecor and W. G. Cochran, ed. Iowa State Univ. Press, Ames, IA. Takahashi, T., M. Kawashima, M. Kamiyoshi, and K. Tanaka. 1992. Arginine vasotocin binding component in the uterus (shell gland) of the chicken. Acta Endocrinol. (Copenh.) 127:179–184. Takahashi, T., M. Kawashima, M. Kamiyoshi, and K. Tanaka. 1993. Mesotocin binding component in various tissues of the hen. Jpn. Poult. Sci. 30:108–113. Takahashi, T., M. Kawashima, M. Kamiyoshi, and K. Tanaka. 1994. Arginine vasotocin receptor binding in the hen uterus (shell gland) before and after oviposition. Eur. J. Endocrinol. 130:366–372. Takahashi, T., M. Kawashima, T. Yasuoka, M. Kamiyoshi, and K. Tanaka. 1996. Mesotocin binding to receptors in hen kidney plasma membranes. Poult. Sci. 75:910–914. Takahashi, T., M. Kawashima, T. Yasuoka, and K. Tanaka. 1998. Arginine vasotocin receptor in the vagina of the oviduct of the hen. Poult. Sci. 77:1699–1703.
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tained from each 4 birds in laying hens and nonlaying hens are shown in Table 1.
the binding affinity, which may cause an increase in the hormonal action (Takahashi et al., 1994). It seems likely that the MT action increases before the increase in that of AVT in the oviduct uterus within 30 min before oviposition. Therefore the role of MT may be different from that of the AVT in the uterus. The MT is reported to cause the contraction of the uterine smooth muscle in vitro (Rzasa and Ewy, 1982). If the uterine MT receptor binding is concerned in the contraction of the uterine muscles, the change in the binding affinity and the binding capacity found in the present study may be related to the process of oviposition in a different role from the AVT.