| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Physiology, Medical School, Institute for Medical Sciences, Jeonbug National University, Jeonju 560–180, Republic of Korea
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The reproductive organ is one of the important extraatrial sites that synthesize ANP. ANP and its binding sites were found in the corpus luteum (14), ovary (15, 16, 17), testis (18), and sperm (19). In luteal cells, ANP increases progesterone synthesis by increasing cGMP (16, 20). We have found that the ANP is synthesized in granulosa cells of ovary (21) and oocytes (22), and the content of ovarian ANP has a cyclic change during the estrous cycle of the rat (23). The physiological functions of ANP in the reproductive system have been known to increase steroidogenesis (20), to inhibit oocyte maturation (24), and to induce acrosomal exocytosis (25). However, no report on the presence and function of ANP in the oviduct exists. In the present study, we have defined for the first time the ANP system in the oviduct using RIA, HPLC, immunohistochemistry, reverse transcriptase-PCR (RT-PCR), in vitro autoradiography, and bioassay.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Tissue preparations were performed as described previously (15, 21, 26). Briefly, both oviducts and atria were boiled for 10 min, homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY), and centrifuged at 10,000 x g for 10 min at 4 C. The irANP in oviducts was extracted with Sep-Pak C18 cartridges (Waters Associates, Milford, MA), and the eluates were dried under vacuum using a Speed-Vac evaporator (Savant, Farmingdale, NY). The recovery rate of cold ANP was 70 ± 1.5% (n = 7).
Reverse phase HPLC
Oviducts from rats and rabbits were removed and immediately put
into liquid nitrogen. After boiling in 0.1 N acetic acid
containing protease inhibitors for 10 min, oviducts were homogenized,
centrifuged, and extracted using Sep-Pak C18 cartridge as
described above. The oviductal extracts were dried, reconstituted, and
subjected to reverse phase HPLC on a µBondapak column (Waters
Associates). Elution was performed with a linear gradient of 20–60%
acetonitrile in 0.1% trifluoroacetic acid for 40 min at a flow rate of
1 ml/min. The fractionated samples were dried and assayed. The column
was calibrated with synthetic ANP [atriopeptin III (APIII)] and with
pro-ANP isolated from rat atria (26).
ANP RIA
irANP in tissue homogenates and HPLC fractions was measured by
RIA as previously described (15, 21, 26). Anti-ANP antibody was made
against APIII (NovaBiochem, Laufelfingen, Switzerland), and APIII
(Peninsula Laboratories, Belmont, CA) was iodinated using the
chloramine-T method. Lyophilized samples were reconstituted and atrial
homogenates were diluted with Tris-acetate buffer (0.1 M;
pH 7.4) containing 0.2% neomycin, 10 mM EDTA, 50
benzoyl-L-arginine ethyl ester units/ml soybean trypsin
inhibitor, 200 kallikrein inhibitor units/ml aprotinin, 0.4 mg% PMSF,
0.02% sodium azide, and 1% BSA. After incubation with anti-ANP
antibody for 24 h at 4 C, [125I]APIII was added, and
incubation proceeded for another 24 h. The separation of the free
from the bound form was achieved by the addition of second
antibody.
Immunohistochemistry
Ovaries from adult female rats were obtained and fixed in 4%
paraformaldehyde for 4 h at 4 C. The tissues were then washed
overnight with 70% alcohol, dehydrated stepwise, and embedded in
paraffin. Immunostaining using the avidin-biotin complex method
(Vectastain ABC kit, Vector Laboratories, Burlingame, CA) was performed
as described previously (21, 22). After deparaffinization and
hydration, the sections were treated with H2O2
in methanol for 20 min, then washed in 0.02 M PBS (pH 7.4).
The sections were incubated for 30 min at room temperature with 10%
normal goat serum, incubated for 16 h at 4 C with the primary
antisera against APIII, rinsed in PBS for 1 h at room temperature,
and then incubated with biotinylated antirabbit IgG (Vector
Laboratories) for 30 min. After the sections were rinsed for 30 min in
PBS, they were treated with the avidin-biotin peroxidase complex for 30
min. The locations of the labeling peroxidase were visualized using
3,3'-diaminobenzidine (0.025% in PBS) in the presence of 0.003%
H2O2.
RT-PCR
Five hundred nanograms of total cellular RNA from atrium and 5
µg messenger RNA (mRNA) from oviduct of rats were suspended in 20
µl RT buffer containing 10 mM Tris (pH 8.3); 50
mM KCl; 5 mM MgCl2; 1
mM each of deoxy (d)-ATP, dCTP, dGTP, and dTTP; 20 U
ribonuclease inhibitor; 2.5 µM random hexamers; and 150 U
Moloney leukemia virus reverse transcriptase (Perkin Elmer, Branchburg,
NJ) and reverse transcribed at room temperature for 10 min and at 42 C
for 30 min. The reaction was stopped by heat inactivation for 5 min at
99 C and then chilled on ice. Complementary DNA products were amplified
by PCR with sense 5'-AGCATGGGCTTCTTCTCCATCACC (66–89) and antisense
5'-AGGGCCAGCGAGCAGAGCCCTCAGTTTGCT (402–431) primers (27). One hundred
microliters of PCR buffer contained 10 mM Tris (pH 8.3); 50
mM KCl; 2 mM MgCl2; 200
µM each of dATP, dCTP, dGTP, and dTTP; 2.5 U
Taq polymerase; and 100 pmol each of sense and antisense
primers. PCR was started at high temperature to increase the
specificity of amplification. The temperature profile of amplification
consisted of 30-sec denaturation at 95 C, 1-min 30-sec annealing at 54
C, and 2-min 30-sec extension at 72 C for 35 cycles. PCR products were
separated in 2% agarose gels, and bands were visualized by ethidium
bromide staining. Photographs of gels were taken with Polaroid 665 film
(Polaroid Corp., Cambridge, UK). PCR products were confirmed by
sequence analysis.
In vitro autoradiography
Oviducts in rats and rabbits were removed after death and
immediately snap-frozen by liquid nitrogen. Sections (20 µm) were cut
by a cryostat at -20 C, thaw-mounted onto gelatin-chrom-alum-coated
slides, and dried in a desiccator at 4 C overnight. The incubation
conditions of [125I]rANP were followed as previously
reported (28, 29). Briefly, the sections were washed with 0.1% acetic
acid for 10 min to remove the endogenous ANP and then preincubated with
30 mM sodium phosphate buffer (pH 7.2) containing 120
mM NaCl and 1 mM phenanthroline at room
temperature for 8 min. They were incubated with 250 pM
[125I]rANP in fresh preincubation buffer containing 40
µg/ml bacitracin, 100 µg/ml PMSF, 10 µg/ml leupeptin, and 0.5%
BSA at room temperature for 60 min. After incubation, the sections were
rinsed and washed with fresh preincubation buffer for 5 min at 4 C.
Subsequently, they were rinsed three times in cold distilled water at 4
C and immediately dried under a stream of cold air.
For analysis of the distribution of binding sites, competitive inhibition of the binding of [125I]rANP was examined on consecutive sections by coincubation with various concentrations (1 pM to 1 µM) of unlabeled rat ANP (rANP) or des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) (C-ANP) as a selective ligand of the clearance receptor. To test the specificity of [125I]rANP binding, the adjacent sections were incubated in the presence of unrelated peptides, angiotensin II, or arginine vasopressin (all 10 µM). Autoradiographic images were generated by exposing the slides to Hyperfilm-3H (Amersham International, Aylesbury, UK) in x-ray cassettes for 2–3 days. Autoradiograms were developed in Kodak D-19 (Eastman Kodak, Rochester, NY) at room temperature for 5 min. The slides were then counterstained with hematoxylin and eosin for tissue localization.
Autoradiographic images were viewed with a Leica Wild M420 Macroscope (Leica, Heerbrugg, Switzerland), and captured using a Sony video camera with CCD iris and a Hamamatsu AC adaptor connected to a Power Macintosh. Binding of [125I]rANP in the oviduct was analyzed for a mean grey scale value using the Prism image program (version 3.6–1, Improve Vision, Coventry, UK). An apparent dissociation constant (Kd) and maximal binding capacity (Bmax) were derived separately in each individual by Scatchard analysis using the Ligand iterative model-fitting computer program (30).
Tension study of oviduct
Oviducts were immediately removed after death, placed in
physiological salt solution (PSS), and gently trimmed to remove excess
fat and connective tissues. Oviducts were longitudinally cut about
25 x 2.5 mm, and the strips were suspended in a constant
temperature organ chamber containing 5 ml continuously oxygenated PSS.
The isometric tension was recorded by means of a force transducer
(Myograph F-60, Narco Bio-Systems, Houston, TX) with a multichannel
recording system (MK-IV, Narco Bio-Systems). The composition of PSS
was: NaCl, 119 mM; KCl, 5 mM;
MgCl2, 1 mM;
CaCl2, 2.5 mM;
KH2PO4, 0.5 mM;
glucose, 5.5 mM; HEPES, 20 mM; and 0.1%
BSA.
A tension of 100–200 mg was applied to each strip, and bathing fluid was changed every 20 min. The strips were allowed to equilibrate for 1 h. To determine whether ANP has an effect on the basal motility of the oviduct, the oviduct tension was observed for 10 min as a control, and then various doses (10-7, 3 x 10-7, and 10-6 M) of APIII were added at 20-min intervals.
Statistical analysis
Results are expressed as the mean ± SEM.
Comparisons of the means were performed using ANOVA with Duncan’s
multiple range test for significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
ANP immunoreactivity in oviductal extracts of rats and rabbits was
characterized by reverse phase HPLC, as shown in Fig. 2, A and B. Two immunoreactive peaks were observed: the elution time of
the major peak corresponded to that of the rat pro-ANP, whereas the
minor one corresponded to that of the synthetic APIII. The presence of
ANP prohormone suggests that ANP is synthesized in situ in
oviducts.
|
|
To determine whether there is a physiological role for ANP in the
oviduct, we measured changes in oviductal irANP contents throughout the
estrous cycle of the rat. The concentration of oviductal irANP at the
proestrous stage of estrous cycle (14.59 ± 3.24 pg/mg tissue wet
wt; n = 12) was significantly lower than that at the metestrous
(27.07 ± 4.41 pg/mg tissue wet wt) and diestrous (31.07 ±
4.87 pg/mg tissue wet wt) stages (Fig. 3). On estrus,
the level of irANP in oviducts was 21.18 ± 2.76 pg/mg tissue wet
wt (n = 16), which was lower than that at the diestrous stage. No
changes in plasma (112.5 ± 11.5 pg/ml; n = 28) and atrial
contents of irANP (2.19 ± 0.25 µg/left atrium; 4.77 ±
0.34 µg/right atrium; n = 28) were observed during the estrous
cycle.
|
). Immunostaining was
blocked by preadsorption of the anti-ANP antibody with APIII (Fig. 4B).
Figure 5 shows the agarose gel analysis after
electrophoresis of RT-PCR products from rat atrial and oviductal RNA. A
band of DNA is present in both the atrial and oviductal lanes
corresponding to the predicted size from the base pair ladders.
|
|
, the
comparison of auto-radiograms with their corresponding
hematoxylin-eosin-stained sections revealed specifically reversible
binding of [125I]rANP to the mucosal layer of the
oviducts of rats and rabbits. Specific bindings to the mucosal layer of
the rabbit oviducts were 81.73 ± 3.32% of total binding and were
not altered by the unrelated peptides, angiotensin II and vasopressin
(all 10 µM; data not shown). Analysis of competitive
inhibition of binding of [125I]rANP by unlabeled rANP on
the mucosal layer of rabbit oviducts was consistent with reversible
binding sites for rANP of uniform affinity (Fig. 7). The
mean Kd and Bmax of these sites were
18.69 ± 5.55 nM and 14.85 ± 6.19
fmol/mm2, respectively. However, C-ANP, a selective ligand
for the clearance receptors, had little or no inhibitory effect on the
specific binding of [125I]rANP to this structure.
|
|
shows basal motility of the oviduct in
rabbits. The frequency and amplitude of basal motility were
3.6 ± 0.6 beats/min and 56.0 ± 7.4 mg (n = 11),
respectively. After the addition of APIII to the organ bath, basal
motility was inhibited gradually, and its inhibitory effect reached a
nadir at 2–3 min. APIII (10-7, 3 x
10-7, and 10-6 M) dose
dependently caused decreases in the frequency of contraction by
86.6 ± 6.3%, 62.0 ± 6.45%, and 43.9 ± 11.8% of
control values (Fig. 8B). In addition, APIII (10-7, 3
x 10-7, and 10-6 M) caused
decreases in the amplitude of contraction by 66.8 ± 7.5%,
65.7 ± 6.43%, and 40.0 ± 11.4% of control values. In rat
oviduct, ANP also had an inhibitory effect on basal motility.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The total content of ANP was 10-fold higher in the rat oviduct (1.19 ± 0.19 ng/oviduct at metestrus) than in the rabbit (0.15 ± 0.01 ng/oviduct). The ANP contents in rat oviduct were lower than those found in the ovary (2.94 ± 0.40 ng/ovary; 43.48 pg/mg tissue wet wt at metestrus). The amount of locally synthesized ANP is very low (1/3,000- to 1/10,000-fold) compared to that of atrial ANP. However, the ANP secreted from extraatrial tissues to surrounding tissues may not be too diluted with blood and may not be degraded rapidly by serum protease. Therefore, local ANP may act in an autocrine or paracrine fashion despite its low amount. ANP in the reproductive system has been known to stimulate progesterone and testosterone production (31), inhibit oocyte maturation (24), and induce an acrosomal reaction (25).
The ANP profile of oviductal extracts on reverse phase HPLC showed two major peaks, corresponding to APIII and rat pro-ANP, respectively. The presence of pro-ANP in the oviduct strongly suggests its local synthesis. This suggestion was further confirmed by the results of both immunohistochemistry and RT-PCR. A distinct and strong ANP immunoreactivity was found in the mucosal layer of the oviduct, and ANP mRNA was also detected. Therefore, these data strongly suggest that ANP is synthesized in the mucosal layer of the oviduct.
The presence of ANP in the oviduct raised a question of its physiological role as a local modulator. In the present study, we found that oviductal ANP content showed a cyclic change characterized by the lowest level at proestrus. However, no significant changes in plasma or atrial ANP were observed. This later finding is consistent with previous reports (23, 32). Many reports exist about the cyclic change in ovarian hormones such as renin (33), inhibin (34), and GnRH (35). Ovarian ANP content has been shown to have a cyclic change, with the highest content at proestrus and the lowest content at diestrus (23). Therefore, it is clear from previous data (23) that the ovarian ANP content increases, but the oviductal ANP content decreases at proestrus. This means that both ovarian and oviductal ANP may be involved in the processes of ovulation and oocyte transportation. We hypothesized that oviductal ANP may be related to the control of oviductal motility.
As for the functional significance of the presence of ANP in the oviduct, we have characterized ANP receptors using in vitro autoradiography. The predominant subtype of ANP receptors in the mucosal layer of the oviduct was found to be biological receptors, with a single class of high affinity. The dense localization of ANP and its receptors in the inner layer of the oviduct suggests that the ANP system may be involved in oviductal function, possibly related to ovum transportation, and to influence ciliary movement, mucus secretion, or oviductal motility. It has been reported that ANP inhibits the ciliary movement of the trachea (36) and causes the relaxation of smooth muscle (1). If ANP inhibits either the motility or ciliary movement of the oviduct, it is understandable that the oviductal ANP content decreases during the ovulation period (late proestrus). We observed an inhibitory effect of ANP on oviductal motility in rats and rabbits. From the results showing the synthesis of and specific binding sites for ANP and its function, we suggest that the oviductal ANP system has an important role in maintaining a cyclic change in its motility at a time crucial to successful transportation of the ovum.
In summary, these data suggest that the oviduct has its own ANP system, and that the system is involved in the functional change during an estrous cycle.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
1 This work was supported by Korean Ministry of Education through the
Research Fund. 
Received December 18, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  M. Zhang, H. Hong, B. Zhou, S. Jin, C. Wang, M. Fu, S. Wang, and G. Xia The expression of atrial natriuretic peptide in the oviduct and its functions in pig spermatozoa. J. Endocrinol., June 1, 2006; 189(3): 493 - 507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Zhang, Y Tao, B Zhou, H Xie, F Wang, L Lei, L Huo, Q Sun, and G Xia Atrial natriuretic peptide inhibits the actions of FSH and forskolin in meiotic maturation of pig oocytes via different signalling pathways J. Mol. Endocrinol., April 1, 2005; 34(2): 459 - 472. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Kim, J. H. Han, C. Cao, S. Z. Kim, and K. W. Cho Postnatal changes in inhibitory effect of C-type natriuretic peptide on secretion of ANP Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2002; 282(6): R1672 - R1679. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P.B. Wijayagunawardane, A. Miyamoto, Y. Taquahashi, T. J. Acosta, M. Nishimura, and K. Sato Angiotensin II and Atrial Natriuretic Peptide in the Cow Oviductal Contraction In Vitro: Direct Effect and Local Secretion of Prostaglandins, Endothelin-1, and Angiotensin II Biol Reprod, September 1, 2001; 65(3): 799 - 804. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Lee, S. Z. Kim, X. Cui, S. H. Kim, K. S. Lee, Y. J. Chung, and K. W. Cho C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling Am J Physiol Heart Circ Physiol, January 1, 2000; 278(1): H208 - H221. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
) and rabbits (
).
