Arginine vasotocin receptor in the vagina of the oviduct of the hen.
ABSTRACT The presence of receptor for arginine vasotocin (AVT) in the vagina of the oviduct of the hen was demonstrated by the use of radioligand binding assays on membrane fractions of the tissue. The binding to [125I]AVT was highly competitive with unlabeled AVT. Scatchard analysis revealed that the binding sites are of a single class. The equilibrium dissociation constant (Kd) was 0.48+/-0.05 nM (x+/-SEM; n = 6) in laying hens holding a hard-shelled egg in the uterus (shell gland) and 1.01+/-0.02 nM (n = 6) in nonlaying hens. The maximum binding capacity (Bmax) was 0.41+/-0.04 pmol/mg protein (n = 6) in laying hens and 0.81+/-0.01 pmol/mg protein (n = 6) in nonlaying hens. The Kd value of the laying hens varied from 0.39 to 1.20 nM during an oviposition cycle, showing an increase just prior to oviposition, and the Bmax value also varied from 0.30 to 0.66 pmol/mg protein, showing a gradual increase after 6 h prior to oviposition. In the nonlaying hen, both values were almost constant during a 24-h day. The changes in the binding affinity and capacity of AVT receptor of the vagina may be related to oviposition in the hen.
Received for publication January 9, 1998.
Accepted for publication June 24, 1998.
Abbreviation Key: AVT = arginine vasotocin; B:F = the ratio of
specific binding to free; Bmax= maximum binding capacity; c = chicken;
E2= estradiol-17b; ID50= half-maximal inhibition; Kd= equilibrium
dissociation constant; LHRH = luteinizing hormone-releasing hormone;
MT = mesotocin; P4= progesterone.
Arginine Vasotocin Receptor in the Vagina of the Oviduct of the Hen
TETSUYA TAKAHASHI,* MITSUO KAWASHIMA,*,1TADASHI YASUOKA,†and KATUHIDE TANAKA‡
*Department of Biological Diversity and Resources, Gifu University, Yanagido, Gifu 501-1193, Japan,
†Department of Oral and Maxillofacial Surgery, Gifu University School of Medicine, Tsukasamachi,
Gifu 500-8705, Japan, and,‡Department of Zootechnical Science, Tokyo University of Agriculture,
Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
vasotocin (AVT) in the vagina of the oviduct of the hen
was demonstrated by the use of radioligand binding
assays on membrane fractions of the tissue. The binding
to [125I]AVT was highly competitive with unlabeled
AVT. Scatchard analysis revealed that the binding sites
are of a single class. The equilibrium dissociation
constant (Kd) was 0.48 ± 0.05 nM (x ± SEM; n = 6) in
laying hens holding a hard-shelled egg in the uterus
(shell gland) and 1.01 ± 0.02 nM (n = 6) in nonlaying
hens. The maximum binding capacity (Bmax) was 0.41 ±
The presence of receptor for arginine
0.04 pmol/mg protein (n = 6) in laying hens and 0.81 ±
0.01 pmol/mg protein (n = 6) in nonlaying hens. The Kd
value of the laying hens varied from 0.39 to 1.20 nM
during an oviposition cycle, showing an increase just
prior to oviposition, and the Bmaxvalue also varied from
0.30 to 0.66 pmol/mg protein, showing a gradual
increase after 6 h prior to oviposition. In the nonlaying
hen, both values were almost constant during a
24-h day. The changes in the binding affinity and
capacity of AVT receptor of the vagina may be related to
oviposition in the hen.
(Key words: hen oviduct vagina, arginine vasotocin receptor, receptor binding affinity,
receptor binding capacity, oviposition)
1998 Poultry Science 77:1699–1703
The process of oviposition (laying an egg outside of
body) in the hen involves the contraction of the uterus
(shell gland) of the oviduct, sending the egg to the
vagina, and successive peristaltic contractions of the
vagina, expelling the egg out through the cloaca (Sykes,
1953). The initial contraction of the uterus is caused by
(AVT), through an increased binding to its receptor
existing in the tissue due to an increased affinity
[decrease in the equilibrium dissociation constant (Kd)]
of the receptor just prior to oviposition (Takahashi et al.,
1994). Further contractions of the uterus may be caused
by an increased release of AVT from the neurohypophy-
sis together with an increased production of prostaglan-
din in the uterine tissue (Takahashi et al., 1994). The
peristaltic contractions of vagina are thought to be
induced by the entering of the egg into the vagina
(Sykes, 1953). In vitro studies have revealed that not only
uterine muscles but also vaginal muscles are contractile
by the action of AVT (Rzasa, 1972). The direct action of
AVT on the vagina suggests the presence of a receptor
in this tissue. The purpose of this study was to
determine whether AVT receptors were present in
vaginal tissue and, if so, to determine whether AVT
binding to the receptor changed relative to oviposition.
MATERIALS AND METHODS
Laying and nonlaying White Leghorn hens (20 mo of
age; 1.8 to 2.2 kg body weight) kept under 14 h light/d
(0500 to 1900 h) with feed (15% CP; 2,800 kcal ME; Japan
Feeding Standard for Poultry, 1992) and water provided
for ad libitum consumption were used. The laying hens
used were those laying five or six eggs in a sequence with
a 1-d pause between sequences and the nonlaying hens
used were those that had not laid an egg at least for 10 d
prior to experiments.
TAKAHASHI ET AL.
2Kishida Chemical Co., Ltd., Osaka 571-0063, Japan.
3Nacalai Tesque, Inc., Kyoto 604-0855, Japan.
5Hitachi Koki Co., Ltd., Hitachinaka 312-8502, Japan.
6Seikagaku Corp., Tokyo 103-0027, Japan.
7Sigma Chemical Co., St. Louis, MO 63178-9916.
8Bachem Inc., Torrance, CA 90505.
9Peninsula Laboratories, Inc., San Carlos, CA 94070.
10Packard Instrument Co., Meriden, CT 06450.
Experiment 1 was performed to examine the binding
specificity, affinity, and capacity of AVT binding compo-
nent of the vagina. The laying hens (35 birds) holding a
hard-shelled egg in the uterus (approximately 3 to 6 h
before expected oviposition) and the nonlaying hens (30
birds)werekilledat 0900honasingleday,and the vagina
of the oviduct was excised. Five tissues were pooled and
used as one sample. The weight of vagina of the laying
hens was 2.40 ± 0.10 g (x ± SEM, n = 35) and that of the
nonlaying hens was 0.99 ± 0.11 g (n = 30).
A second experiment was performed to examine
changes in the binding affinity and capacity before and
before oviposition of the first egg of sequence and at
another five different times (within 5 min, 2, 3, 5, and 7 h)
after oviposition (six birds at each time), and also from the
nonlaying hens at seven different times corresponding to
the time of sampling in the laying hens during a
24-h day (six birds at each time). The samples were
assayed individually rather than being pooled as in
The ovarian weight of nonlaying hens was less than 8.8
g and whole oviduct weight was less than 11.2 g. The
serum concentration of progesterone (P4) and estradiol-
17b (E2) measured by a routine radioimmunoassay
pM (n = 72), respectively.
Preparation of Plasma
The method of preparation was the same as reported
earlier for the uterus of the hen (Takahashiet al., 1992). All
steps were performed at 4 C. Vagina tissues were rinsed
with ice-cold Tris-EDTA buffer (TE; 50 mM Tris2-HCl, 2
mM EDTA,3pH 7.4) containing 0.25 M sucrose, blotted
with a filter paper, and weighed. The tissues were then
homogenized in the same buffer (5 vol/wt), using the
Ultra-Turrax homogenizer (Type 18-10).4
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. The 1,000 × g supernatant was
combined and centrifuged (30,000× g, 30 min). The 30,000
× g precipitate was suspended in the 0.25 M sucrose-TE
buffer (5 vol/wt) with a Potter-Elvehjem glass-teflon
homogenizer. The suspension was gently pored on an
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.5The interface fraction of the two buffers was
obtained and washed twice with TE buffer not containing
sucrose by centrifugation (30,000 × g, 30 min). The final
precipitate was suspended in TE buffer (0.5 vol/wt) and
used as the plasma membrane fraction. The protein
concentration was determined by the method of Lowryet
al. (1951) using BSA6as a standard. The amount of protein
in the membrane fraction was 0.22±0.03 ( x ±SEM, n = 7)
mg/g tissue in laying hens and 0.23 ± 0.02 (n = 6) mg/g
tissue in nonlaying hens, respectively.
The AVT was labeled with125I using the Iodogen7
method (Fraker and Speck, 1978; Salacinski et al., 1981).
The specific activity of radioiodinated AVT was 2,117 to
2,284 Ci/mmol as determined by the method of Copeland
et al. (1979). Binding assays of the membrane fractions
were performed by the use of the same method as
reported earlier (Takahashi et al., 1992; 1993). The
membranefraction(10mg protein per tube) was incubated
at 30 C for 16 h with [125I]AVT (0.1 to 1.4 nM) in the
presence (for nonspecific binding) or absence (for total
mL. For experiments on the competitive binding, 8 to 8,000
nM of unlabeled AVT (Arg8-vasotocin)8and mesotocin
(MT: Ile8-oxytocin),8and 80 to 8,000 nM of unlabeled
chicken luteinizing hormone-releasing hormone-I
(His5,Typ7,Tyr8-GnRH),8 and chicken angiotensin-II
(cAngiotensin-II: Val5-angiotensin-II)8were used. After
the incubation, the tubes were placed immediately in an
ice-bathandthen centrifuged (10,000× g, 20 min, 4 C). The
precipitatewas washed with 500mL TE buffer by the same
centrifugation. The radioactivity was measured by a
gamma counter (Packard Cobra).10The counting effi-
ciency for125I was 74.0 to 84.6%. Specific binding was
obtained by subtracting the nonspecific binding from the
total binding and expressed as moles per milligram
protein. The equilibrium dissociation constant (Kd) and
the maximum binding capacity (Bmax) were determined
by the method of Scatchard (1949).
Relationships of specific [125I]AVT binding to incuba-
tion time (0.25 to 17 h) and temperature (4 and 30 C), and
to protein concentration (1.25 to 15 mg per tube) were
examined. The specific [125I]AVT binding at 30 C of
incubation temperature was greater than at 4 C, increased
during the first 8 h of incubation, and then reached a
plateau (Figure 1). The specific binding increased linearly
with the increase in the protein concentration from 1.25 to
15 mg per tube (Figure 2). Based on these findings, the
experiments: 30 C, 16 h incubation, and 10 mg protein per
ARGININE VASOTOCIN RECEPTOR IN HEN VAGINA
FIGURE1.Timecourseof thespecific[125I] argininevasotocin (AVT)
bindinginthe plasma membrane fraction of the oviduct vagina of laying
hens.Samples (10mg protein per tube) were incubated at 4 (o) or 30 (ÿ) C
for various hours with 0.8 nM [125I]AVT in the absence or presence of 1
mM unlabeled AVT, and the specific [125I]AVT binding was measured.
Each point represents the mean of three separate pools of samples, and
the vertical bars represent SEM.
FIGURE 2. Relationship of specific [125I]arginine vasotocin (AVT)
binding to the protein concentration in the plasma membrane fraction of
the oviduct vagina of laying hens. Samples (1.25 to 15 mg protein per
or presence of 1 mM unlabeled AVT, and the specific [125I]AVT binding
wasmeasured.Each pointrepresentsthe mean of three separate pools of
samples, and the vertical bars represent SEM.
FIGURE 3. Competition for [125I] arginine vasotocin (AVT) binding
in the plasma membrane fraction of the oviduct vagina of laying hens.
Samples (10mg protein per tube) were incubated at 30 C for 16 h with 0.8
nM [125I]AVT in the absence (control) or presence of various fold molar
excess of unlabeled AVT (ÿ), mesotocin (MT; o), chicken luteinizing
hormone-releasing hormone-I (cLHRH-I; Gln8-GnRH; ⁄), chicken lu-
teinizing hormone-releasing hormone-II (cLHRH-II; His5, Trp7, Tyr8-
GnRH; ◊), or chicken Angiotensin-II (cAngiotensin-II; Val5-angiotensin-
II; …). The amount of the [125I]AVT binding in control value was 0.38
pmol/mg protein. Each point represents the mean of two separate pools
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 one-
way ANOVA (Snedecor and Cochran, 1967). When
significant effects were found at the 5% level, Tukey’s
multiple range test (Tukey, 1953) was used to compare
means of more than two groups.
The [125I]AVT binding was markedly reduced by the
presence of a 100- to 10,000-fold molar excess of unlabeled
10,000-fold molar excesses was used. However, the
binding was not reduced by the presence of a
100- to 10,000-fold molar excess of unlabeled cLHRH-I,
cLHRH-II, and cAngiotensin-II (Figure 3). The ID50value
calculated from the data of the dose-inhibition curve was
30 nM for AVT and 3,613 nM for MT.
Binding Affinity and Capacity
The specific [125I]AVT binding increased with the
increase in the amount of [125I]AVT, and was saturable at
about 0.7 nM (Figure 4). Scatchard analysis of the data
revealed a linear relationship between the amount of
specific binding and the ratio (B:F) of specific binding to
free [125I]AVT (Figure 4), indicating one single class of
binding sites. The Kdvalue was 0.48 ±0.05 nM (x ±SEM; n
= 6) in laying hens and 1.01 ±0.02 nM (n = 6) in nonlaying
hens. The Bmaxvalue was 0.41 ± 0.04 pmol/mg protein (n
= 6)inlayinghensand0.81±0.01pmol/mgprotein(n= 6)
in nonlaying hens. In the nonlaying hens used in this
experiment, the serum concentrations of P4and E2were
387±13pM(n= 30)and 316±12 pM (n = 30), respectively.
Changes in Kd and Bmax Values
The Kdvalue decreased 11 h before oviposition, and
increased just prior to oviposition (Figure 5). The Bmax
value decreased 6 h before oviposition, and then gradu-
ally increased up to oviposition. In nonlaying hens,
neither Kdnor Bmaxchanged during a 24-h day. In the
nonlaying hens used in this experiment, the serum
TAKAHASHI ET AL.
FIGURE 4. Saturation curve and Scatchard plot of specific [125I]
arginine vasotocin (AVT) binding in the plasma membrane fraction of
the oviduct vagina of laying hens. Samples (10mg protein per tube) were
incubatedat30C for 16h with various concentrations of [125I]AVT in the
absence or presence of 1 mM unlabeled AVT, and the specific [125I]AVT
Scatchard analysis), and correlation coefficient (g) between B:F and
specific binding was 0.40 nM (Kd), 0.36 pmol/mg protein (Bmax) and
–0.97 (g), respectively. Each point represents the mean of duplicate
determinations from one pooled sample. (ÿ) Specific binding, (o)
Nonspecific binding. B = bound; F = free.
FIGURE 5. The equilibrium dissociation constant (Kd) and the
maximum binding capacity (Bmax) of the specific binding component of
[125I]arginine vasotocin (AVT) in the plasma membrane fraction of the
oviduct vagina of laying hens at various times before and after
oviposition (ÿ) and of nonlaying hens at corresponding times (o).
Samples (10 mg protein per tube) were incubated at 30 C for 16 h with
[125I]AVT (0.1 to 1.4 nM) in the presence and absence of 1 mM unlabeled
AVT,andthespecific[125I]AVTbinding was measured. The values of Kd
and Bmaxwere obtained by Scatchard analysis. The amount of protein in
membranefractions, expressedasmilligram per gram wet tissue weight,
was 0.22 ±0.02 (x ±SEM, n = 60) in laying hens and 0.20 ±0.01 (n = 42) in
nonlaying hens, respectively, and was not significantly different among
the hens at different times. The wet weight of the vagina expressed as
in non-laying hens, respectively. Each point represents the mean of six
birds, and the vertical bars represent SEM. *Significantly different (P ≤
0.01) from the preceding value by Tukey’s test. –0 h: within 5 min before
oviposition; 0 h: within 5 min after oviposition.
concentrations of P4and E2were 371 ± 9 pM (n = 42) and
333 ± 15 pM (n = 42), respectively.
The binding component to [125I]AVT in the mem-
brane fractions of vagina of the hen showed binding
specificity to AVT and saturable binding. The Kdvalue
(moles per litter) obtained by Scatchard analysis was of
the order of 10–10 and the Bmax value (moles per
milligram of protein) was of the order of 10–13. The
binding specificity, the high affinity (small Kdvalue),
and the minute capacity (Bmax) serve as the requisites of
a receptor, and therefore, the binding component in the
membrane fractions of the vagina is likely to be a
receptor for AVT. The specific binding component to
MT, another neurohypophysial hormone in chickens
(Acher et al., 1970), has been reported to exist in the
vagina (Takahashi et al., 1993), but it is unknown
whether the binding component has binding specificity
to MT. In the uterus, MT causes the contraction of
muscle strips in vitro (Rzasa and Ewy, 1982). However,
the effect of MT on the vagina or on oviposition is
obscure. The binding affinity of the vaginal AVT
receptor found in the present study was higher (Kdwas
less) in laying hens than in nonlaying hens. Although
the binding capacity (Bmax) per milligram of the protein
was less in laying hens, the Bmaxwhen expressed as
moles per vagina would be substantially more in laying
hens (0.25 ± 0.02 pmol per vagina; x ± SEM; n = 6) than
in nonlaying hens (0.16 ± 0.01 pmol per vagina; n = 6).
The Kdvalue of the AVT receptor of the vagina was
almost equal to that of the AVT receptor existing in the
uterus of the oviduct of the hen, although the Bmax
value (0.41 pmol/mg protein) was less (0.89 pmol/mg
protein in the uterus) (Takahashi et al., 1992). Both Kd
and Bmax values of the vaginal receptor showed a
change during a period before and after oviposition,
which differed from that reported on the uterine
receptor (Takahashi et al., 1994). The Kdvalue of the
vaginal receptor showed an increase just prior to
oviposition whereas that of the uterine receptor showed
a decrease. The Bmax value of the vaginal receptor
showed a gradual increase before oviposition whereas
that of the uterine receptor showed a decrease just prior
to oviposition. Takahashi et al. (1994) has described the
following hypothesis concerning mechanisms regulating
oviposition based on the study for changes in the
binding of uterine AVT receptor before and after
oviposition. The binding affinity of the AVT receptor in
the uterus increases just prior to oviposition, which
initiates contraction of the uterine musculature. A
message of the occurrence of the contraction is transmit-
ted to the central nervous system via a neural pathway.
It causes an abrupt release of AVT from the neuro-
hypophysis and an increase in the AVT level of blood
just prior to oviposition, as reported earlier (Sturkie and
Lin, 1966; Nouwen et al., 1984; Tanaka et al., 1984; Rice et
al., 1985; Shimada et al., 1986; 1987; Koike et al., 1988).
The increased AVT causes further contractions of the
uterus to move the egg to the vagina. If the vaginal AVT
receptor binding is concerned in the contraction of the
vaginal muscles, the change in the Kdand Bmaxvalues
found in the present study may be related to the process
ARGININE VASOTOCIN RECEPTOR IN HEN VAGINA
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
hormone-releasing hormone sera. Endocrinology 104:
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 and Co. Ltd., London, UK.
Fraker, P. J., and J. C. Speck, Jr., 1978. Protein and cell
membrane iodinations with a sparingly soluble chloroa-
mide, 1,3,4,6-tetrachloro-3a, 6a-diphenylglycoluril. Bio-
chem. Biophys. Res. Commun. 80:849–857.
Japan Feeding Standard for Poultry, 1992. Pages 18–21 in:
Japan Feeding Standard. Agriculture, Forestry and Fisher-
ies Research Council Secretariat, Ministry of Agriculture,
Livestock Industry, Tokyo, Japan.
Koike, T. I., K. Shimada, and L. E. Cornett, 1988. Plasma levels
of immunoreactive mesotocin and vasotocin during ovipo-
sition in chickens: Relationship to oxytocic action of the
peptides in vitro and peptide interaction with myometrial
membrane binding sites. Gen. Comp. Endocrinol. 70:
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.
Nouwen, E. J., E. Decuypere, E. R. Ku ¨hn, H. Michels, T. R.
Hall, and A. Chadwich, 1984. Effect of dehydration,
haemorrhage and oviposition on serum concentrations of
vasotocin, mesotocin and prolactin in the chicken. J.
Rice, G. E., S. S. A´rnason, Z. Arad, and E. Skadhauge, 1985.
Plasma concentrations of arginine vasotocin, prolactin,
aldosterone and corticosterone in relation to oviposition
and dietary NaCl in the domestic fowl. Comp. Biochem.
Rzasa, J., 1972. Effect of vasotocin and oxytocin on contractility
of the hen oviduct in vitro. Acta Physiol. Pol. 23:735–745.
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.
Salacinski, P.R.P., C. McLean, J.E.C. Sykes, V. V. Clement-
Jones, and P. J. Lowry, 1981. Iodination of proteins,
glycoproteins, and peptides using a solid-phase oxidizing
agent, 1,3,4,6-tetrachloro-3a,6a-diphenyl glycoluril (Iodo-
gen). Anal. Biochem. 117:136–146.
Scatchard, G., 1949. The attractions of proteins for small
molecules and ions. Ann. N.Y. Acad. Sci. 51:660–672.
Shimada, K., H. L. Neldon, and T. I. Koike, 1986. Arginine
vasotocin (AVT) release in relation to uterine contractility
in the hen. Gen. Comp. Endocrinol. 64:362–367.
Shimada, K., N. Saito, K. Itogawa, and T. I. Koike, 1987.
Changes in plasma concentrations of arginine vasotocin
after intrauterine injections of prostaglandin F-2a and
acetylcholine at various times during the oviposition cycle
of the domestic hen (Gallus domesticus). J. Reprod. Fertil.
Shodono, M., T. Nakamura, Y. Tanabe, and K. Wakabayashi,
progesterone and luteinizing hormone in the plasma
during the ovulatory cycle of the hen. Acta Endocrinol.
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. The Iowa State University Press, Ames, IA.
Sturkie, P. D., and Y.-C. Lin, 1966. Release of vasotocin and
oviposition in the hen. J. Endocrinol. 35:325–326.
Sykes, A. H., 1953. Some observations on oviposition in the
fowl. Q. J. Exp. Physiol. 38:61–68.
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.)
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.
Tanaka, K., K. Goto, T. Yoshioka, T. Terao, and O. Koga, 1984.
Changes in the plasma concentration of immunoreactive
arginine vasotocin during oviposition in the domestic
fowl. Br. Poult. Sci. 25:589–595.
Tukey, J. W., 1953. Some selected quick and easy methods of
statistical analysis. Trans. NY Acad. Sci. Ser. II 16:88–97.