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Faculty of Integrated Arts and Sciences (K.T., D.L., K.U.), Hiroshima University, Higashi-Hiroshima 739-8521, Japan; and the Department of Biology (M.K., S.I.), Waseda University, Nishi-Waseda 169–50, Japan
Address all correspondence and requests for reprints to: Dr. Kazuyoshi Tsutsui, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. E-mail: tsutsui{at}ue.ipc.hiroshima-u.ac.jp
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Abstract |
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Thus, ovarian sex steroids may contribute at least in part as hormonal factors to galanin receptor induction, which takes place in the uterine oviduct during development.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are both potent inducers of
oviposition in the domestic hen (3, 4, 5). PG receptors in the avian
uterus have also been demonstrated (7). In contrast to knowledge of hormonal control, little information is available on the neuronal mechanisms controlling oviposition. Some unidentified factors in the oviduct may play an important role in the evoking of oviposition as a neurotransmitter or a neuromodulator, as abundant nerves are terminated in the musculature in several oviduct regions such as the uterus and vagina. When studying the regulation of avian oviposition, poultry provides an excellent model, as oviposition occurs almost daily in the domestic quail and hen. In these birds, the functional oviduct consists of the infundibulum, magnum, isthmus, uterus, and vagina (8). Recently, we have isolated an oviposition-inducing peptide from the quail oviduct (9). This peptide is identical to avian galanin (9), which is a C-terminally amidated, 29-residue peptide. Galanin was originally isolated from porcine intestinal extracts (10), and avian and mammalian galanins differ at several positions in the C-terminal part (9, 10, 11, 12, 13, 14). Immunohistochemical analysis using the antigalanin serum showed that immunoreactive fibers were distributed in muscle layers of the quail uterus and vagina (9). In addition, in vitro and in vivo experiments (9) have revealed that the administration of avian galanin immediately evokes oviposition through the induction of uterine contractions. We have also demonstrated the presence of galanin receptors in the quail oviduct (15). Interestingly, our preliminary studies indicate that a large number of galanin receptors are restricted to the uterus in the quail oviduct (15). Taken together, these results suggest that avian galanin acts directly on the uterus to induce contraction. This mechanism may be essential for avian oviposition.
With these findings as a background, the following questions were addressed in this present study. Firstly, what change occurs in galanin receptors in the maturing and mature oviduct? Secondly, what is the factor(s) involved in the regulation of galanin receptors during oviduct maturation? As the binding capacity was greatest in the uterus compared with those in other oviduct regions (15), the investigations were performed using the uterine oviduct of the quail. In the present study, not only uterine galanin receptors but also circulating levels of ovarian sex steroids, i.e. 17ß-estradiol and progesterone, were measured to determine the effects of these steroids.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental schedules
When newly hatched females of the Japanese quail are exposed to
LD, they reach sexual maturity at around 3 months of age (16). In the
quail, sexually mature females usually lay an egg every day at the same
time (16). Ovulation occurs 6–8 h after the ovulatory surge of LH, and
the egg then spends about 24 h in the mature oviduct before it is
laid (16).
In the first series of experiments, female quails at 13 weeks of age
were killed by decapitation to confirm our preliminary findings showing
localization of galanin receptors in the mature oviduct. To determine
the normal change in uterine galanin receptors during oviduct
development, female quails at 4, 6, 10, and 13 weeks of age were killed
by decapitation in the second series of experiments. In the final
experiment, immature females at 4 weeks of age were treated with a
SILASTIC brand (Dow Corning, Midland, MI) plate containing
17ß-estradiol, progesterone, or 17ß-estradiol plus progesterone to
examine the effects of sex steroids on galanin receptors. A SILASTIC
plate (1.5 x 15 x 2 mm; 
10 mg crystal/plate) made of a
mixture of medical SILASTIC adhesive (silicone type, Dow Corning) and
crystalline sex steroid (Sigma, St. Louis, MO) was intraabdominally
implanted around the oviduct as described previously (17). After 1
week, sex steroid-implanted quails were killed by decapitation at 5
weeks of age along with control quails implanted with only SILASTIC
adhesive.
Preparations of plasma and receptor samples
Trunk blood was collected into heparinized glass tubes and
centrifuged at 1,800 x g for 30 min at 4 C. Plasma was
stored at -20 C until assayed for 17ß-estradiol and progesterone.
Immediately after blood collection, oviducts of various ages were
removed and weighed. Several regions of each maturing and mature
oviduct from 5–13 weeks of age, i.e. the infundibulum,
magnum, isthmus, uterus, and vagina, were carefully dissected on ice.
The receptor samples at these ages were obtained from the uterine
oviduct, although the samples from 4 weeks of age consisted of the
whole oviduct because differentiation of the regions had not occurred
by this stage. Tissues were snap-frozen in liquid nitrogen and stored
at -80 C until the binding assay for avian galanin was performed. For
the receptor preparation, frozen samples were rapidly thawed and
homogenized with a glass homogenizer with a Teflon pestle in cold
Tris-HCl buffer (0.04 M; pH 7.4) containing 5
mM MgSO4, 0.1% BSA, and 0.1 mM
phenylmethylsulfonylfluoride, a protease inhibitor, as described
previously (15). The homogenates were centrifuged at 11,000 x
g for 20 min at 4 C. The resulting pellets were resuspended
in cold buffer and adjusted to contain 5 mgeq wet tissue/100 µl.
Peptide preparations
In our previous study (9), an isolated peptide from the quail
oviduct was identified as avian galanin with the following sequence:
Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-His-Ala-Val-Asp-Asn-His-Arg-Ser-Phe-Asn-Asp-Lys-His-Gly-Phe-Thr-NH2.
For the galanin binding assay, we synthesized a peptide with the
proposed sequence using a manual method followed by hydrogen
fluoride-anisole cleavage and purification by reverse phase
HPLC. It has been previously confirmed that the synthetic and native
peptides show identical retention times on the C18 reverse
phase column and the cation exchange column (9). The synthetic peptide
also evokes contractions of the uterus in a manner similar to that of
the native peptide (9).
Radioiodination of avian galanin
The synthetic avian galanin was radioiodinated with
125I (Na125I, Radiochemical Center, Amersham,
Aylesbury, UK) in the presence of lactoperoxidase and hydrogen
peroxidase using a method described previously (18). Labeled galanin
was separated from free 125I on a C18 reverse
phase column (15, 19). The specific activity of
[125I]iodoavian galanin was calculated from data obtained
in a peak with OD at 220 nm on the C18 reverse phase column
and was estimated to be 80 µCi/µg (15, 19).
Assay of avian galanin receptors
Binding experiments were performed as described previously (15, 19, 20, 21). In brief, 50 µl of the nonradioactive avian galanin in assay
buffer [0.04 M Tris-HCl buffer (pH 7.4) containing 5
mM MgSO4, 0.1% BSA, and 0.1 mM
phenylmethylsulfonylfluoride] or the assay buffer alone, 50 µl of
the [125I]iodoavian galanin, and 100 µl of the receptor
preparation were added to disposable plastic centrifuge tubes with a
capacity of 1.5 ml. All of the reaction tubes had previously been
coated with BSA to reduce the adsorption of peptides to the tube wall.
The tubes were placed in a water bath incubator with continuous shaking
at 20 C for 1 h. Previous studies (15, 19) demonstrated that the
specific binding of avian galanin to the quail oviduct is temperature
dependent and reaches a maximum level after 1 h at 20 C. At the
end of the incubation period, 1 ml cold assay buffer was added to each
tube, and the tubes were centrifuged at 11,000 x g for
3 min at 4 C. The pellets were washed twice with cold buffer, and the
radioactivity of the resulting pellets was counted in an autowell
-counter. The specific binding of [125I]iodoavian
galanin to the receptor preparations was calculated as the difference
between binding in the absence (total binding) and that in the presence
(nonspecific binding) of an excess of unlabeled avian galanin.
To examine galanin binding levels during development and after steroid treatment, 0.365 ng [125I]iodoavian galanin was incubated with or without an excess of cold avian galanin (200 ng). According to a previous study (15), 200 ng cold avian galanin are sufficient for complete inhibition of specific binding of 0.365 ng [125I]iodoavian galanin. In the saturation binding experiment, different amounts of [125I]iodoavian galanin (0.038–1.22 ng) were incubated with or without an excess of cold avian galanin (0.032–1.00 µg). Scatchard plots were constructed from the data obtained from the saturation binding experiment. The dissociation constant (Kd) and the number of binding sites for avian galanin were then determined with Scatchard plots.
Assays of progesterone and 17ß-estradiol
Extraction of progesterone was performed according to a previous
method (22). In brief, plasma (200 µl) was diluted with 5 ml cold
PBS, and progesterone was extracted with 5 ml ethyl acetate three
times. To measure the concentrations of progesterone, aliquots of
organic extract were assayed in a specific RIA (23) that used an
antiserum to progesterone (Scantibodies Laboratories, Santee, CA) and
[1,2,6,7-3H]progesterone (SA, 115 Ci/mmol; New England
Nuclear, Boston, MA). The antiserum used in the present experiment
cross-reacted with deoxycorticosterone at 3.3%, with
17-hydroxyprogesterone at 0.6%, and with aldosterone and
17ß-estradiol less than 0.02%. According to a method described
previously (21, 22), the least detectable amount was 0.1 ng/ml, and
intraassay variation was less than 7%.
17ß-Estradiol in plasma samples was determined by an automatic enzyme immunoassay system (AIA-600, Tosoh Corp., Tokyo, Japan) using a kit (AIA-PACK E2, Tosoh Corp.) consisting of 17ß-estradiol conjugated with bovine alkaline phosphatase and magnetic beads coated with an antibody against 17ß-estradiol. The antiserum used in the present experiment cross-reacted with estrone at 8.8%, with estriol at 1.1%, and with progesterone and testosterone less than 0.001%. The least detectable level was 20 pg/ml according to the manufacturer’s data.
Statistical analyses
Statistics for linearity and parallelism of the Scatchard plots
and for 95% confidence intervals for the Kd and number of
binding sites were computed according to the method of Bliss (24).
Results for galanin binding levels and steroid levels during
development and after steroid treatment were expressed as the mean
± SEM and were analyzed for significance of difference by
Duncan’s multiple range test or Kruskal-Wallis test followed by
Mann-Whitney’s U test after verification of equality or inequality,
respectively, of variances among the groups compared (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
A high level of specific binding was also observed in the vagina (Table 1). Specific binding in the isthmus was lower than those in the uterus
(P < 0.01) and vagina (P < 0.05,
Table 1). As the uterus was much larger than the vagina and isthmus,
the total binding capacity in each region would be much greater in the
uterus than in other oviduct regions.
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). Specific galanin binding per 5 mg
tissue equivalent increased (P < 0.05) from 4–6 weeks
of age and was maintained at a higher level from 6–13 weeks of age
(Fig. 1b). In contrast to the marked change in specific binding,
nonspecific binding on a unit weight basis was low and almost constant
regardless of age (Fig. 1b). Specific galanin binding per uterus
increased continuously from 4–13 weeks of age (P <
0.05, 4 vs. 6 weeks; P < 0.01, 10
vs. 13 weeks; Fig. 1c).
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). Scatchard plots showed
significantly (P < 0.05) straight lines in the two
groups, suggesting the presence of a single class of galanin-binding
sites (Fig. 3). The equilibrium
Kd values calculated from the fitted lines of the plots
were 0.30 (95% confidence interval, 0.25–0.38) nM at 4
weeks of age and 0.34 (0.28–0.40) nM at 6 weeks of age.
Thus, the Kd values of these two groups indicated no
significant change in the affinity of galanin binding. The mean numbers
of galanin-binding sites (capacity) in the uterus at 4 and 6 weeks of
age were 0.232 (95% confidence interval, 0.210–0.262), and 0.827
(0.776–0.893) fmol/mg tissue, respectively. There was a significant
difference in the number of binding sites per mg tissue equivalent
between these two groups (P < 0.05). The total numbers
of galanin-binding sites per uterus at 4 and 6 weeks of age were 5.6
and 237 fmol, respectively. These data show that the change in galanin
binding during development was due to a change in the number of binding
sites.
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, the plasma
progesterone concentration in females significantly increased
(P < 0.05) from 6–10 weeks of age, with no
significant change thereafter. Plasma 17ß-estradiol levels in females
also tended to increase between 6–13 weeks of age, but the alteration
was not significant due to a large variance (Fig. 4b).
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).
Combined administration of 17ß-estradiol and progesterone also
significantly increased (P < 0.01) uterine weight
(Fig. 5a); however, this combined administration failed to induce a
more noticeable increase than when 17ß-estradiol alone was given
(Fig. 5a).
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). This combined effect of
17ß-estradiol and progesterone was not additive, but appeared
synergistic. Specific galanin binding per uterus was also increased
(P < 0.05) by administrations of both 17ß-estradiol
and progesterone alone (Fig. 5c). A synergistic action of
17ß-estradiol and progesterone was also observed on specific galanin
binding per uterus (Fig. 5c).
Saturation binding experiments were performed on uterine preparations
from control immature females and immature females treated with
17ß-estradiol and/or progesterone to determine whether the
steroid-induced increase in the binding of labeled galanin to the
uterus was due to an increase in the number of binding sites (capacity)
or an increase in the affinity of binding. Scatchard plots showed
significantly (P < 0.05) straight lines in all groups,
suggesting the presence of a single class of galanin-binding sites
(Fig. 6). The Kd
values calculated from the fitted lines of the plots were 0.20 (95%
confidence interval, 0.16–0.29) nM in the control group,
0.33 (0.27–0.42) nM in the 17ß-estradiol group, 0.39
(0.27–0.63) nM in the progesterone group, and 0.32
(0.27–0.38) nM in the 17ß-estradiol plus progesterone
group. Thus, no significant difference was detectable in the affinity
of galanin binding among these groups. In contrast, the mean numbers of
galanin-binding sites (capacity) in the groups of control,
17ß-estradiol, progesterone, and 17ß-estradiol plus progesterone
were 0.189 (95% confidence interval, 0.173–0.218), 0.480
(0.437–0.545), 1.08 (0.792–1.31), and 1.22 (1.12–1.36) fmol/mg
uterine tissue, respectively. There was a significant difference in the
number of binding sites per mg tissue equivalent among these four
groups. Thus, the increase in galanin binding to the uterus after
steroid treatment was related to an increase in the number of
galanin-binding sites. The calculated total numbers of galanin-binding
sites per uterus in the groups of control, 17ß-estradiol,
progesterone, and 17ß-estradiol plus progesterone were 85, 752, 472,
and 1665 fmol, respectively.
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).
17ß-Estradiol administration also induced a significant increase
(P < 0.05) in the circulating 17ß-estradiol level in
the immature birds (Fig. 7b). Such a steroid level occurring in
immature females treated with progesterone or 17ß-estradiol was
relatively similar to the maximal level observed in females exposed to
LD (Fig. 4, a and b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results provide a detailed profile of developmental changes in galanin binding to the quail uterus. At 4 weeks of age, when differentiation of the uterus has not occurred, specific galanin binding was detectable in the oviduct preparation, but the total binding capacity was extremely low. However, immature female quails responded to LD by showing not only an increase in uterine weight but also an increase in specific galanin binding to the uterus. Scatchard plot analyses of the binding suggested that an increase in galanin binding to the uterus during development was due to an increase in the number of binding sites and not to an increase in the affinity of binding. Thus, galanin receptors may appear in the oviduct before the differentiation of the uterus. Subsequently, the number of galanin receptors in the uterine oviduct may increase concurrent with development.
In the present study, hormonal administration to immature birds was then performed to identify the hormonal factor(s) inducing the increase in galanin receptors during development. 17ß-Estradiol and progesterone individually induced an increase in specific galanin binding to the uterus. In addition, the binding further increased in a synergistically manner when both sex steroids were administered at the same time. These steroid treatments increased the number of galanin-binding sites but did not influence the affinity of binding judging from the Scatchard plot analysis. Oviduct development depends on ovarian function. It is well established that in birds these ovarian sex steroids are required in oviduct development (25, 26) and can act directly on oviduct tissue (for review, see Ref. 27). In domestic birds, the uterine oviduct possesses receptors for both 17ß-estradiol and progesterone (28, 29, 30, 31). Accordingly, it is probable that these two steroids exert their actions on galanin receptor induction as well as oviduct development after binding to specific steroid receptors localized in the uterine oviduct. Such a steroid action on galanin receptors might be induced through the activation of gene transcription for galanin receptors.
The hypothesis postulated here that ovarian sex steroids are hormonal factors for galanin receptor induction in the developing uterus may be partly supported by the results of circulating levels of these steroids during development. Circulating progesterone significantly increased during 6–10 weeks of age, with no significant change thereafter. An increase in circulating 17ß-estradiol also observed during 6–13 weeks of age, although the alteration was not significant due to a large variance. In addition, circulating 17ß-estradiol or progesterone levels in immature steroid-treated females were in the proximity of the maximal levels observed in females exposed to LD, suggesting that the levels may be within the physiological range. Therefore, the increase in these circulating sex steroids may be a possible cause of the induction of uterine galanin receptors.
There was a clear difference between the two steroids in the stimulatory effect on the uterus. Unlike 17ß-estradiol, progesterone increased only the number of galanin-binding sites without influencing uterine weight. Thus, it is considered that the effect of progesterone on the two observed parameters is specific for the induction of galanin receptors. In addition, we found that 17ß-estradiol and progesterone can act synergistically to increase the number of galanin-binding sites. There is evidence indicating that estrogen induces progesterone receptors in the hen uterus (31). If this also occurs in the quail uterus, the combined effect of 17ß-estradiol and progesterone on galanin receptors should be not additive but, rather, synergistic. Such a synergism between 17ß-estradiol and progesterone probably enables the marked increase in galanin receptors in the uterus occurring during development under normal physiological conditions. Interestingly, a synergism between these two steroids in the induction of galanin messenger RNA in the brain has recently been reported in the rat (32). According to Rossmanith et al. (32), 17ß-estradiol was the primary ovarian signal inducing galanin messenger RNA expression in GnRH neurons. In addition, progesterone facilitated the action of 17ß-estradiol on galanin gene expression in GnRH neurons (32). It may be that these two sex steroids act as important factors to induce an increase in both galanin and its receptors.
In the present study, however, the number of uterine galanin receptors did not correlate with the circulating sex steroid levels during early development. The initial rise in galanin receptors that occurred between 4–6 weeks of age was not accompanied by an increase in circulating steroid levels. Therefore, sex steroids may not be the major factors for galanin receptor induction at the initial phase of uterine growth. Although no report is available on galanin receptors, hormone-dependent and -independent induction has been documented in the developing testis for the regulation of peptide hormone receptors (18, 19, 20, 21, 33). FSH and testosterone act as hormonal factors to induce an increase in testicular FSH receptors (18, 19, 20, 21, 33). According to Tsutsui (21), other factors that are independent of pituitary and gonadal hormones may also contribute to FSH receptor induction, as the absence of a pituitary was followed by an increase in FSH receptors only during early development. The present and previous data suggest that some other factor(s) that is not associated with sex steroids may induce the initial rise in galanin receptors in the uterus. Another possibility is that a kind of self-induction mechanism of galanin receptors, such as spontaneous gene expression of receptors, may be present during the initial phase of development. Further study is required to draw a firm conclusion.
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Acknowledgments |
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Footnotes |
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Received January 21, 1998.
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References |
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in relation to
oviposition in the domestic hen (Glallus domesticus). Biol
Reprod 24:496–504[Abstract]
to membranes of shell gland muscle of
laying hens: correlations with contractile activity. Biol Reprod 20:390–398[Abstract]
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