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Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University (H.S., T.U., D.L., K.U., K.T.), Higashi-Hiroshima 739-8521, Japan; and Radioisotope Center, Hiroshima University (C.K.), Higashi-Hiroshima 739-8526, Japan
Address all correspondence and requests for reprints to: Dr. Kazuyoshi Tsutsui, Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. E-mail: tsutsui{at}hiroshima-u.ac.jp
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These results suggest that lumbosacral sympathetic ganglionic neurons innervating the uterine muscle produce avian galanin in mature birds. Because this peptide acts directly on the uterus to evoke oviposition through a mechanism of the induction of vigorous uterine contraction, galaninergic innervation of the uterine oviduct may be essential for avian oviposition.
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Introduction |
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Extensive knowledge of the hormonal regulation of oviposition in birds
as well as parturition in mammals has been accumulated. Avian
oviposition is considered to be regulated at least in part by a
neurohypophysial hormone, i.e. arginine vasotocin (AVT)
(2, 3), and PGs derived from the ovary
(4, 5, 6). It has been reported that AVT causes contractions
of the hen uterine muscle (2, 3). The presence of AVT
receptors in the uterine myometrium has been demonstrated in the hen
(7). Therefore, AVT may act directly on the musculature of
the hen uterus, causing oviposition. It has also been reported that
PGE2 and PGF2
are both
potent inducers of oviposition in the hen (4, 5, 6) and that
the avian uterus possesses PG receptors (8).
In contrast to extensive studies regarding the hormonal action, little information is available on the neuronal mechanism controlling oviposition. Because abundant nerves terminate in the uterine muscle, some functional substances that are secreted from the terminals may play an important role in the evoking of oviposition as a neurotransmitter or a neuromodulator. In fact, we recently isolated an oviposition-inducing peptide from the quail oviduct (9). This peptide, a 29-residue peptide, including an amidated threonine at the C-terminus, was identical with avian galanin (9), which has been previously isolated from the chicken intestine (10). Galanin was originally identified from porcine intestinal extracts (11), and avian and mammalian galanins differ at several positions in the C-terminal part (9, 10, 11, 12, 13, 14). Interestingly, in vitro and in vivo experiments (9) revealed that administration of avian galanin immediately evoked oviposition through the induction of vigorous contractions of the quail uterine muscles (9). Immunocytochemical analysis using the antigalanin serum further showed that immunoreactive fibers were distributed in muscle layers of the quail uterus (9). A large number of receptors for avian galanin was also restricted to uterine muscles in the quail and hen (15, 16). Taken together, these results suggest that avian galanin acts directly on uterine muscles, by its secretion from neuronal terminals projecting to the uterus, to induce contraction. This is the first role of galanin in controlling oviposition reported in a vertebrate.
The purpose of the present studies was to identify the neurons involved in avian oviposition using immunocytochemical, retrograde labeling and molecular biological (Northern blot analysis) methods. The reproductive system of mature birds (poultry) consists of a single left ovary and oviduct (17). Here we show that the lumbosacral sympathetic ganglionic neurons located in the same left side project to the uterine muscle and produce avian galanin. We recently cloned a complementary DNA (cDNA) for avian galanin from quail brain (18). In this study we therefore conducted Northern blot analysis to determine the expression of avian galanin messenger RNA (mRNA) in the identified neurons projecting to the uterine muscle. To investigate developmental changes in avian galanin in the neurons, we further measured the levels of avian galanin and its mRNA in immature and mature birds.
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Materials and Methods |
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When newly hatched females are exposed to a long day photoperiod, they reach sexual maturity at around 3 months of age (19). In sexually mature females 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 (19).
Retrograde labeling with neurobiotin
To identify the neurons projecting to uterine muscles,
retrograde tracing experiments were conducted using sexually mature
females (n = 15) according to the method previously reported
(20, 21, 22). In brief, a small incision was made in the
uterine muscle, and neurobiotin (Vector Laboratories, Inc., Burlingame, CA) was applied using a fine tungsten needle
with a small amount of its crystals on the tip. Five to 8 days after
application of neurobiotin to the uterine muscle each bird was deeply
anesthetized by an ip injection of sodium pentobarbital (Abbott Laboratories, North Chicago, IL) and then perfused with 200 ml
PBS, followed by 200 ml of a fixative solution [4% paraformaldehyde
in 0.1 M phosphate buffer (PB), pH 7.3]. The lumbosacral
part of the bird was immediately dissected and postfixed with the same
fresh fixative solution for 16–20 h at 4 C. After fixation, the
uterus, spinal cord, and sympathetic ganglia were cut out from each
bird, washed thoroughly in PBS containing 0.4% Triton X-100 (0%
solution for electron microscopic study), and embedded in 6% agar
(gelling temperature, 30–31 C) dissolved in distilled water. The
preparation was then sectioned with a microslicer (Dosaka EM, Kyoto,
Japan) in the transverse or horizontal plane at a thickness of 50 µm
in PBS. Sections were washed three times with PBS for 10 min each
time.
Free floating transverse or horizontal sections were then incubated with avidin-biotin-peroxidase complex (ABC) in PBS for 2–8 h at 4 C or room temperature. Incubation periods shorter than 2 h usually yielded light staining, and periods longer than 8 h not only did not enhance neurobiotin labeling, but the tissue became deteriorated at the electron microscopic level (20). After washing in PBS three times for 10 min each time, the sections were reacted with 0.05% diaminobenzidine (DAB; Sigma, St. Louis, MO) and 0.03% H2O2 in 0.1 M Tris-HCl buffer (pH 7.4) for 30 min at room temperature. Stained sections were then rinsed twice, mounted on gelatin-coated slides, and examined under an Olympus Corp. BH-2 microscope (New Hyde Park, NY). Some stained sections were further subjected to electron microscopic studies as described below.
Electron microscopic analysis
To examine the ultrastructure of neurobiotin-labeled neurons,
tissue sections from six different birds were treated after the DAB
reaction with 1% OsO4 in 0.1 M PB
for 2 h at 4 C, dehydrated in graded ethanols, and embedded flat
in epoxy resin (Quetol-812, Nisshin EM, Tokyo, Japan) using a method
described previously (20). Serial semithin sections, 1–2
µm in thickness, were cut with a glass knife and stained with 1%
toluidine blue. These sections were preliminarily examined and
photographed with a light microscope. Ultrathin sections, 60 nm thick,
were then cut using a diamond knife (Diatome, Nisshin EM, Tokyo, Japan)
and double stained with uranyl acetate and lead citrate. Stained
sections were examined under an electron microscope (H-600A,
Hitachi, Tokyo, Japan).
Immunocytochemical analysis of galanin
Light microscopic level. In this immunocytochemical
experiment, sexually mature females at 3 months of age (n = 4)
were killed by decapitation, and lumbosacral sympathetic ganglia
(LS1–LS7, both left and right sides; Fig. 1
) were carefully dissected. Immature
females at 3 weeks of age (n = 4) were also used
(LS2–LS3, only the left side; Fig. 1). Tissues
of each bird were immediately immersion-fixed in 4% paraformaldehyde
in 0.1 M PB (pH 7.3) for 14–16 h at 4 C before
immunocytochemical procedures. Subsequently, tissues were dehydrated in
ethanol and xylene and embedded in paraffin wax. Serial sections of
each ganglion were cut horizontally on a microtome at 5-µm thickness.
Deparaffinized sections were processed according to the ABC
immunocytochemical technique described previously (9, 23, 24).
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Control procedures consisted of 1) preabsorbing the working dilution of the primary antiserum with a saturating concentration of the antigen (18 µg avian galanin/ml), and 2) substituting normal rabbit serum for the primary antiserum in a dilution of 1:500. The sections were also incubated with these control sera according to the same procedure as the antiserum. The localization of immunoreactive neurons was studied using an Olympus Corp. BH-2 microscope.
Electron microscopic level. Lumbosacral sympathetic postganglionic neurons projecting to the uterus were retrogradely labeled with neurobiotin as described above, and then processed for postembedding ultrastructural immunocytochemistry (25). For immunogold staining of the neurobiotin-labeled neurons, tissue sections from different birds (n = 5) were dehydrated after the DAB reaction with an ethanol series and embedded flat in acryl resin (LR White, London Resin, London, UK). The resin was then polymerized by heating at 60 C for 48 h. Ultrathin sections, 80–100 nm thick, were collected on nickel grids, rinsed with PBS for 5 min, and treated with 5% normal goat serum and 1% BSA in PBS for 30 min. The sections were then incubated with the primary antiserum raised against avian galanin for 5–10 h at 4 C. After washing with Tris-HCl-buffered saline (TBS; pH 8.2), the sections were incubated with a 1:50 dilution of goat antirabbit IgG-gold conjugate (10-nm particle diameter; British BioCell, Cardiff, UK) for 1–2 h at room temperature. The sections were rinsed in TBS and refixed in 2% glutaraldehyde in PB (pH 8.2) for 20 min at 4 C. They were then electron-stained with uranyl acetate and lead citrate. The stained sections were viewed and photographed using an electron microscope (H-600A, Hitachi, Tokyo, Japan). For controls, sections were incubated with preabsorbed primary antiserum.
Peptide extraction and enzyme-linked immunosorbent assay (ELISA) of
galanin
In this study avian galanin levels in the lumbosacral
sympathetic ganglionic neurons were quantified by a competitive ELISA
using the antiserum raised against avian galanin. Sexually immature
females (n = 56) at 3 weeks of age and mature females (n =
40) at 3 months of age were divided into eight groups each. We pooled
the tissue from seven immature and five mature birds for each of a
total of eight samples per group. All birds were killed by decapitation
between 1000–1300 h during the light cycle. Lumbosacral sympathetic
ganglia (LS1-LS7, left side; Fig. 1
) were
carefully removed using fine forceps under a dissecting microscope,
snap-frozen immediately in liquid nitrogen, and used for peptide
extraction.
Peptides were extracted according to our previous methods (9, 26). Frozen ganglia were boiled for 7 min and homogenized in 5% acetic acid using a homogenizer (Ultra-Turrax T8 IKA, Labortechnic, Germany). The homogenate was centrifuged at 16,000 x g for 30 min at 4 C. The supernatant was collected into a tube, and the resulting precipitate was again homogenized and centrifuged. The two supernatants were then pooled and forced through a disposable C18 cartridge (Sep-Pak Vac 1cc, Waters Corp., Milford, MA). The retained material was then eluted with 60% methanol. The eluate was concentrated in a vacuum centrifuge and subjected to competitive ELISA, as previously described (27). In brief, different concentrations of avian galanin (1–1,000 pmol/ml) and adjusted ganglionic extracts were added together with the antiserum against avian galanin (1:500 dilution) to each antigen-coated well of a 96-well microplate (ELISA plate, Corning, Inc., Corning, NY) and incubated for 1 h at 37 C. After the reaction with alkaline phosphatase-labeled goat antirabbit IgG, immunoreactive products were obtained in a substrate solution of p-nitrophenylphosphate, and the absorbance was measured at 415 nm on a microtiter plate reader (MTP-120, Corona Electric, Ibaraki, Japan). Protein contents in the ganglionic tissues used for ELISA were measured by the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL) with BSA as a standard. The concentration of avian galanin was calculated in terms of picomoles per µg protein.
If avian galanin is involved in quail oviposition, we expect that the peptide in the uterus would change under different physiological conditions. To test this hypothesis, uterine galanin levels were measured in different oviposition phases. Before this experiment, the oviposition time of laying quails was observed for 2 weeks. Birds were divided into two groups, oviposition phase (n = 4) and quiescent phase (4 h after oviposition; n = 4), and subjected to ELISA.
RNA isolation and Northern blot analysis of galanin
mRNA
To determine the expression of mRNA encoding for avian galanin
in the lumbosacral sympathetic ganglionic neurons, Northern blot
analysis was further conducted in this study. Lumbosacral sympathetic
ganglia (LS1–LS7, ipsilateral side of the
uterus; Fig. 1) of sexually immature females (3 weeks of age; n =
23) and mature females (3 months of age; n = 33) were carefully
removed, frozen immediately in liquid nitrogen, and used for RNA
isolation. In addition, entire brains, uterine oviducts, and livers of
sexually mature females (3 months of age; n = 4) were used for RNA
isolation.
Total cellular RNAs of lumbosacral sympathetic ganglia at each
age group and entire brains, uterine oviducts, and livers at the adult
were isolated by the guanidinium thiocyanate-phenol-chloroform
extraction method (28). Total RNA contains ribosomal RNA
and mRNA. Total cellular RNA (5 µg) isolated from each tissue was
denatured, separated on 1.2% agarose gel containing 1.2 M
formaldehyde, and transferred onto Hybond N+
nylon membrane. The membrane was prehybridized for 3 h at 42 C in
5 x SSC (0.75 M NaCl and 0.075 M sodium
citrate), 50 mM
NaH2PO4 (pH 7.0), 1%
blocking reagent (Roche Molecular Biochemicals, Tokyo,
Japan), 3.5% SDS, 0.1% N-lauroylsarcosine, and 50%
formamide and hybridized for 15 h at 42 C in the same buffer
containing a 32P-labeled probe. We have recently
cloned a cDNA for avian galanin from quail brain using degenerated
rapid amplification of cDNA end (3' and 5') techniques
(18). A cloned cDNA for avian galanin contained an open
reading frame consisting of 117 amino acids that had overall amino acid
homologies of 63%, 60%, 54%, 58%, and 62% with bovine, human,
mouse, porcine, and rat galanins, respectively (18).
Northern blot analysis previously revealed that avian galanin mRNA was
expressed in the quail brain (18). So, the entire brain of
the mature bird was used as a positive control tissue. In this study,
labeling of the galanin cDNA fragment was also performed by the random
priming method using [-32P]deoxy-CTP
(ICN Biomedicals, Inc., Costa Mesa, CA), as
previously described (18). After hybridization, the
membrane was washed with SSC (2x, 0.5x, and 0.1x) containing 0.5%
SDS at 50 C. Hybridization signals on the membrane were analyzed on
BAS-2000 film (Fuji Photo Film Co., Ltd., Tokyo,
Japan).
Statistical analyses
The numbers of the galanin-immunoreactive neurons in lumbosacral
sympathetic ganglia (LS1–LS7; Fig. 1) in both
the left side (uterine side) and the right side (contralateral side of
the uterus) were computed, expressed as the mean ±
SEM, and analyzed for significance of difference by two-way
ANOVA. If significant in ANOVA, these analyses were followed by
Duncan’s multiple range test (29).
Differences for galanin concentrations in the ganglia between different ages (3 weeks and 3 months) or in the uterus between different phases (oviposition phase and quiescent phase) were analyzed by Bartlett’s test, followed by Student’s t test or Mann-Whitney’s U test (29).
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). As
shown in Fig. 2, retrograde transport of
neurobiotin (see arrows) was observed in somata of the
lumbosacral sympathetic ganglionic neurons (LS2; see
arrows) located in the uterine side (left side), suggesting
that these labeled ganglionic neurons project their axons to the
uterus. Although the accumulation of neurobiotin in neuronal somata was
observed in almost all of the lumbosacral sympathetic ganglia
(LS1–LS7) located in the uterine side (left
side), a large number of labeled neurons was detected in the
LS2–LS5 ganglia. Unlike these ganglia, the
LS1, LS6, and LS7 ganglia showed a
few labeled neurons. No neurons were labeled in the LS8
ganglion or the thoracic sympathetic ganglia. In addition, we could not
detect the accumulation of neurobiotin in the uterus and lumbosacral
spinal cord. In this study, retrograde tracing experiments were
repeated independently in 15 females 5–8 days after application of
neurobiotin and indicated the same results. No significant difference
in neuronal labeling was found among different survival periods
(5–8 days) after application of neurobiotin.
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). Many high electron-dense particles
showing neurobiotin accumulation were scattered in the cytoplasm of the
neurobiotin-labeled neurons at a higher magnification (Fig. 3b, see
arrowheads), and hence the labeled neurons were darker
compared with the nonlabeled cells under light and electron microscopic
levels (Figs. 2 and 3). The labeled neurons possessed well developed
rough endoplasmic reticula, extensive Golgi apparatuses, and abundant
elementary granules in the cytoplasm (Fig. 3). Abundant granules in the
neurobiotin-labeled neurons were 300–400 nm in diameter, homogeneous,
and of higher density (Fig. 3), suggesting that these neurons may
contain some neuropeptide(s) and function as a neurosecretory cell.
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, a and b, an intense
immunoreaction for galanin was observed in the relatively large cell
bodies, which were localized in the sympathetic ganglia
(LS2) as well as other ganglia (LS1,
LS3–LS7). These immunoreactive cell bodies
were distributed throughout each whole sympathetic ganglion.
Preabsorbing the antiserum (1:500 dilution) with an excess of avian
galanin at a concentration of 18 µg/ml resulted in a complete absence
of galanin-like immunoreactivity in all of the positively stained cells
in the sympathetic ganglia (Fig. 4, c and d). Controls in which normal
rabbit serum was substituted for the antigalanin serum also showed no
immunoreactivity in the sympathetic ganglia at the same concentration
(1:500 dilution; data not shown). Similar results were obtained by
repeated experiments using different four birds.
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summarizes the number of
galanin-immunoreactive cells per section in each sympathetic ganglion.
In the LS2, LS3, and LS5 ganglia,
the number of galanin-immunoreactive cells in the uterine side (left
side) was significantly (P < 0.01 or P
< 0.05) greater than that in the contralateral side of the uterus
(right side; Table 1). Thus, the cells containing avian galanin were
more numerous in the sympathetic ganglia located in the uterine side
(left side). In addition, the number of galanin-immunoreactive cells
was maximal in the LS2 and LS3 ganglia and
minimal in the LS7 ganglia among the observed ganglia
(LS1–LS7; Table 1). Such a region-specific
distribution of the cells containing avian galanin was in agreement
with the distribution of retrograde labeling with neurobiotin. These
results taken together suggest that the LS2 and
LS3 ganglionic neurons located in the uterine side (left
side) project mainly to the uterine muscle and greatly possess avian
galanin.
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), intracellular localization of the galanin
immunoreactivity was examined by the method using immunolabeled gold.
In this analysis, neurobiotin-labeled neurons located in the
sympathetic ganglia of LS2 and LS3 in the
uterine side (left side) were used for electron microscopic
immunocytochemistry. The membrane structure in the immunostained
sections was not clearly visible due to postfixation without
OsO4, which prevents the reduction of
antigenicity of avian galanin. However, neurobiotin-labeled sympathetic
ganglionic neurons were darkly stained as well as conventional
ultrathin sections (Fig. 3a) and were also identifiable at a low
magnification at the electron microscopic level (Fig. 5a).
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, a–c), whereas other intracellular organs, such as mitochondrion, cell
nucleus, etc., and extracellular spaces possessed few gold
spheres as background labeling. Each immunopositive granule was
300–400 nm in diameter, which corresponded to the elementary granules
observed by the conventional electron microscopic analysis (Fig. 3).
Although many elementary granules in the sympathetic ganglionic neurons
showed the galanin immunoreactivity, some elementary granules did not
show immunoreaction with avian galanin. In this immunogold method,
immunostaining was not observed in any control grids (Fig. 5c). Similar
results were obtained by immunolabeling experiments using five
different birds.
Comparison of the expressions of avian galanin and its mRNA in
sympathetic ganglionic neurons between immature and mature birds
Avian galanin was measured in the sympathetic ganglia
(LS1–LS7, uterine side) of immature and mature
quails by a competitive ELISA using the antiserum against avian
galanin. As shown in Fig. 6, the
concentration of avian galanin on a unit protein basis of ganglia in
mature birds was significantly greater (P < 0.05;
6.0 times) than in immature birds.
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). The
initial amount of RNA used in Northern blot analysis was adjusted
spectrophotometrically. Northern blot analysis for ß-actin was
performed as the internal control (Fig. 7c). As shown in Fig. 7, a
(shorter exposure) and b (longer exposure), a single band corresponding
to avian galanin mRNA was detected in the sympathetic ganglia as well
as the brain. Interestingly, the expression of avian galanin mRNA was
much greater in the ganglia than in the entire brain (Fig. 7b).
Dilution of galanin-producing brain regions with brain regions that
contain relatively few galaninergic cells may be the reason for such a
difference. The expression in the ganglia in mature birds was also
greater than that in immature birds (Fig. 7, a and b), suggesting an
age-dependent change in the mRNA encoding avian galanin. In contrast,
no band corresponding to avian galanin mRNA was detected in the uterine
oviduct and liver under the same experimental conditions (data not
shown). Northern blot analyses were repeated four times using
independently extracted RNA samples from different animals and produced
similar results.
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). In contrast, ganglionic
immunoreactive cell bodies in the immature birds were smaller and fewer
than those in mature birds (Fig. 8, a and c). Preabsorbing the
antiserum with an excess of avian galanin (18 µg/ml) resulted in a
complete absence of galanin-like immunoreactivity in all of the
positively stained cells at both ages (Fig. 8, b and d). Similar
results were obtained by repeated experiments using different four
birds at each age. Such an immunocytochemical change with age (Fig. 8, a and c) was in agreement with the changes in avian galanin (Fig. 6)
and its mRNA (Fig. 7, a and b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on electron microscopic analysis, the presence of abundant elementary granules in the lumbosacral sympathetic ganglionic neurons retrogradely labeled with neurobiotin suggests the possibility that the labeled neurons produce some neuropeptide(s), such as avian galanin, and function as neurosecretory cells. This hypothesis is supported by the results of the present immunocytochemical analysis. An intense immunoreaction with the antiserum against avian galanin was detected in cell bodies of the lumbosacral sympathetic ganglia. In addition, abundant immunoreactive cells were localized in the LS2 and LS3 ganglia located in the uterine side (left side). Thus, the localization of neurobiotin-labeled cells is in agreement with the immunocytochemical finding showing the distribution of galanin-like immunoreactive cells. Colocalization of neurobiotin with galanin-like substance was also confirmed by the ultrastructural analysis. It is therefore possible that the sympathetic ganglionic neurons produce avian galanin and project to uterine muscles. Employing Northern blot analysis, the expression of galanin mRNA was also detected in these neurons. We have previously demonstrated in the same avian species that avian galanin causes, by acting directly on the uterus, contractions of uterine musculature and consequently induces oviposition (9, 15, 16). In addition, avian galanin in the uterus increased in the oviposition phase in this study, suggesting that avian galanin may act on the uterus to induce avian oviposition. Therefore, the identified efferent innervation of the sympathetic ganglionic neurons to uterine muscles may contribute to an essential role in avian oviposition. This is the first neuronal mechanism controlling oviposition reported in a vertebrate. To draw a firm conclusion, however, we need precise functional experiments that measure galanin release from terminals of the sympathetic ganglionic neurons during oviposition and examine the occurrence of oviposition after axotomy of the efferent fibers.
In birds, expression of the galanin-like substance in the brain has previously been verified by immunocytochemical analysis (23, 31), and brain galanin mRNA has been determined by RT-PCR analysis together with Southern hybridization (18). In this study, the expression of galanin mRNA, when compared by Northern blot analysis on a unit RNA basis, was clearly greater in the ganglionic tissue than in the brain tissue. Therefore, it is considered that the ganglionic neurons may actively produce avian galanin. On the other hand, we previously reported no expression of galanin transcripts in the quail uterus by Northern blot analysis (18). The present study obtained the same result. In contrast, there is a report showing the expression of galanin mRNA in the rat uterus (32). Although the cause of this discrepancy is not known, the present and previous results using the avian species support the hypothesis that sympathetic ganglionic galanin neurons project to the uterine oviduct.
The present study further investigated what change occurs in avian galanin in the sympathetic ganglia of immature and mature birds. As measured by ELISA, avian galanin was detectable in the sympathetic ganglia at 3 weeks of age, but the level was extremely low. In contrast, the concentration of avian galanin was greater in the ganglia at 3 months of age, indicating a marked increase in the concentration of avian galanin during development. Such an increase in avian galanin was in agreement with the finding of Northern blot analysis, showing a greater expression of galanin mRNA in the mature ganglia. Because galanin-like immunoreactivity was restricted to few ganglionic cells at 3 weeks of age, as ascertained by immunocytochemical staining, galanin neurons may increase in the sympathetic ganglia during development. The question that immediately follows the present finding is what mechanism is operating for the induction of avian galanin in the sympathetic ganglia during development. Ovarian sex steroids, such as 17ß-estradiol and progesterone, might contribute to galanin induction, as these hormones act on the rat brain to induce the galanin mRNA expression in GnRH neurons (33, 34). 17ß-Estradiol also induced galanin expression in the mouse pituitary (35). On the other hand, there is evidence indicating that 17ß-estradiol and progesterone contribute as hormonal factors to galanin receptor induction in the quail uterus (16). To identify the factors involved in the induction of avian galanin in the lumbosacral sympathetic ganglia during development, experiments that analyze a detailed profile of developmental changes in avian galanin in the ganglionic neurons and effects of sex steroids on galanin induction are now in progress.
From a series of our studies with quails, sympathetic ganglionic galanin neurons may evoke avian oviposition by innervation to the uterine oviduct. On the other hand, it is known that avian oviposition is hormonally regulated at least partly by PGs (4, 5, 6, 8) and AVT (2, 3, 7). On the basis of previous findings in birds, a hypothetical scheme for oviposition mechanisms is considered as follows. 1) PGs stimulate contractions of the uterus. 2) AVT has an additive action on the PG effect and further stimulates uterine contractions. Therefore, the following question arises. What relative importance does each of these hormonal and neuronal mechanisms assume? If these two mechanisms function in birds, it might be considered that PGs and/or AVT trigger galanin secretion from the sympathetic ganglionic neurons. We need to examine the presence of receptors for PGs and/or AVT in the sympathetic ganglionic galanin neurons.
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Acknowledgments |
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Footnotes |
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Received July 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in
relation to oviposition in the domestic hen (Gallus
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]
-dependent
manner. Endocrinology 140:2628–2631This article has been cited by other articles:
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