ARTICLE |
Correspondence to:
Barbara Gawronska, Pennington Biomedical Research Center Louisiana State U., 6400 Perkins Road, Baton Rouge, LA 70808. E-mail:
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Summary |
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Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs, including those of the reproductive tract. We investigated potential changes in NADPH-diaphorase (NADPH-d) activity (marker for NOS activity) and the presence and distribution of NOS in the porcine oviduct. Tissues were obtained from gilts (n=16) on different days of the estrous cycle. One fallopian tube was used for histo- and immunohistochemistry and the other for Western blotting analysis. NADPH-d activity was much higher in the epithelium of the mucosa than in the myosalpinx. The highest activity of NADPH-d was always found in the epithelium of the isthmus. The intensity of the reaction (arbitrary units ± SEM) in isthmus epithelium increased from the postovulatory period until early proestrus (96.2 ± 11.2) and then gradually decreased. The lowest intensity of NADPH-d reaction in the epithelium of the isthmus was seen at estrus (58.4 ± 7.7). The most intense NADPH-d activity in myosalpinx of all parts of the oviduct was observed at the postovulatory stage of the estrous cycle (isthmus 38.3 ± 2.5; ampulla 35.6 ± 4.2; infundibulum 24.7 ± 0.8) and then decreased during the remaining stages of the estrous cycle (p< 0.001). The presence of endothelial NOS (eNOS) was detected in epithelial cells of mucosa and in endothelium of vascular tissues and myosalpinx during all studied days of the estrous cycle. The positive reaction for inducible NOS (iNOS) was restricted only to the endothelium of lymph vessels and some blood vessels. Because our Western blotting analysis revealed that porcine oviduct contains eNOS but not iNOS, we suggest that eNOS is the main isoform of NOS expressed in the porcine oviduct. We concluded that the different activity of NADPH-d in the various regions of the oviduct, accompanied by changes in its activity during the course of the estrous cycle, could indicate an important role of NO in regulation of tubal function. (J Histochem Cytochem 48:867875, 2000)
Key Words: NADPH-diaphorase, nitric oxide synthase, oviduct, pig
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Introduction |
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Nitric oxide (NO) plays a crucial role in many physiological events, including vasodilatation, neurotransmission, platelet aggregation, and immune activation. NO is an inorganic free radical gas which is generated from L-arginine by the group of enzymes called nitric oxide synthase (NOS;
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Materials and Methods |
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Studies were carried out on 16 sexually mature, crossbred gilts that had exhibited at least two estrous cycles. The animals were kept in separate pens and checked daily for heat in the presence of a boar. The first day of behavioral estrus was established as Day 0 of the estrous cycle. Day of the estrous cycle was additionally confirmed by the morphological appearance of the ovaries after sacrifice (
NADPH-d Staining and Immunocytochemistry for eNOS and iNOS Protein
Three portions of each oviduct (isthmus, ampulla, and infundibulum) were fixed for 6 hr in 4% paraformaldehyde (Fluka Chemie; Buchs, Switzerland) in 0.1 M phosphate buffer (PB). Fixed tissues were stored in 18% sucrose (POCH; Giliwice, Poland) in PB with 0.01% sodium azide (POCH). Cryostat sections (8 µm) were used for immunocytochemistry and histochemistry. To demonstrate NADPH-d activity, cryostat sections of fallopian tube were incubated at 3738C in a freshly prepared solution of ß-NADPH (5 mg/ml; Sigma, St Louis, MO), nitroblue tetrazolium (0.5 mg/ml; Sigma) and Triton X-100 (5 µl/ml; Merck, Darmstadt, Germany) in 0.1 M PB, pH 7.4, for 1 hr. Control sections were exposed to the staining solution without NADPH. All oviduct samples were assayed in parallel and at least three complete sets of samples were examined.
The activity of NADPH-d in porcine oviduct was measured as distribution of formazan deposits (endproduct of the histochemical reaction). The intensity of histochemical reaction in oviduct tissues was estimated by measuring the optical density using the PC-IMAGE system (Foster Findlay Associates; Oxford, UK). All values are reported as mean ±SEM. The data were subjected to one-way analysis of variance (ANOVA; InStat GraphPAD, San Diego, CA).
For immunocytochemistry, consecutive sections were rinsed in 0.05 mol/liter Tris-hydroxylmethyl aminomethane (TBS; Sigma), then placed in ethanol in ascending concentration series (50%, 70%, 96%, absolute alcohol). The sections were treated with 1% H2O2 in methanol (SigmaAldrich; Steinhem, Germany) for 30 min to block endogenous peroxidases and then in 0.75% glycine (Sigma) in TBS for 30 min to block free aldehyde groups. After rinsing in TBS, the sections were incubated overnight with primary antibody against eNOS (diluted 1:50; a mouse monoclonal antibody directed against the amino acids 10301209 of human endothelial NO synthase; Transduction Laboratories, Lexington, KY). The consecutive sections were incubated with primary antibody against iNOS (diluted 1:50; a mouse monoclonal antibody directed against the amino acids 9611144 of mouse macrophage inducible NO synthase; Transduction Laboratories). Antibody binding was detected with the ABC complex (Vectastain ABC kit from Vector Laboratories; Burlingame, CA). Peroxidase activity was revealed using 3,3'-diaminobenzidine (Sigma) as a substrate. Two types of controls were performed: (a) the primary antibody was omitted during the immunostaining procedure; (b) the primary antibody was substituted with nonspecific immunoglobulin G (IgG) during the immunostaining procedure. The observations and photographs were made using a light microscope (Olympus IMT-2; Tokyo, Japan).
Western Blotting Analysis
Tissue samples were placed in ice-cold homogenization buffer [50 mM Tris-HCl, pH 7.4; 10 mM EDTA (Sigma) 150 mM NaCl, 1% Triton X-100 with 1 µM pepstatin A (Sigma) 5 µg/ml leupeptin (Sigma) 5 µg ml aprotinin (Sigma), 100 mM PMSF (Sigma) freshly made] and homogenized on ice. The homogenate was centrifuged at 5000 x g for 5 min at 4C and the pellet was discarded. The supernatant was then centrifuged at 29,000 x g for 30 min at 4C and the sediment suspended in PBS with proteinase inhibitors (concentration as above). The protein content of the resulting suspension was determined (
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Results |
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NADPH-d Histochemistry
Porcine fallopian tube showed NADPH-d activity in mucosal epithelium, myosalpinx, nerve fibers, and endothelium of blood and lymph vessels in all regions of oviduct during the entire estrous cycle (Fig 1 and Fig 2). However, the intensity of the histochemical reaction was different among regions of oviduct and stages of the estrous cycle. In general, NADPH-d activity was much higher in mucosal epithelium than in myosalpinx (Fig 1 Fig 2 Fig 3 Fig 4). The highest activity of NADPH-d was consistently found in the epithelium of the isthmus (Fig 1a and Fig 3). The intensity of reaction (arbitrary units ± SEM) in this part of oviduct increased from postovulation (62.6 ± 5.8) till early proestrus (96.2 ± 11.2; Fig 1a and Fig 3; p = 0.06) and then gradually decreased. The lowest intensity of reaction in the epithelium of the isthmus was seen at estrus (58.4 ± 7.7; Fig 1b and Fig 3). Epithelium lining the ampulla and infundibulum exhibited the most intense activity of NADPH-d at midcycle (Fig 1c and Fig 1e, respectively; Fig 3) and the lowest at estrus (p<0.01, p<0.05; Fig 1d and Fig 1f, respectively; Fig 3).
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The most intense NADPH-d activity in myosalpinx of all parts of the oviduct was observed at the postovulatory stage of the estrous cycle (isthmus, 38.3 ± 2.5; ampulla, 35.6 ± 4.2; infundibulum, 24.7 ± 0.8; Fig 2a, Fig 2b and Fig 4) compared to estrus (isthmus, 12.4 ± 1.8; p<0.001; ampulla, 17.5 ± 1.7, p<0.001; infundibulum, 15.5 ± 1.3, p<0.01). The lower NADPH-d activity was also maintained in myosalpinx of isthmus and ampulla during the remaining stages of the estrous cycle (p<0.001; Fig 4). The myosalpinx was almost clear at midcycle, late luteal, early proestrus, late proestrus and estrus, except for some NADPH-d activity in the endothelial cells of vessels and in NADPH-d positive nerve fibers (Fig 2c).
The control specimens, in which ß-NADPH was omitted during the histochemical procedure, showed no NADPH-d activity (Fig 2d).
NOS Immunohistochemistry
Fig 5 shows that the gilt oviduct was immunostained for the NOS. eNOS-like immunoreactivity was identified in epithelial cells of mucosa, endothelium of vascular tissues, and myosalpinx at all stages of the cycle (Fig 5a5c). The positive reaction for the inducible calcium-independent isoform of NOS (iNOS) was restricted to the endothelium of lymph vessels and some blood vessels in both the mesosalpinx and the oviduct (Fig 5d and Fig 5e, respectively).
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This immunostaining for eNOS and iNOS (data not shown) was absent when the receptor antibody was omitted or substituted with nonspecific immunoglobulin G (IgG) during the immunostaining procedure (Fig 5f).
Western Blotting Analysis
Fig 6 is a representative Western blot showing the presence of an eNOS-specific band in porcine oviduct. This band, with a molecular weight of about 135 kD, corresponds to endothelial NOS from human endothelial cell lysate (Fig 6, lane 2). In parallel immunoblots, we probed with a specific iNOS monoclonal antibody and iNOS was not detected in porcine fallopian tubes (not shown).
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Discussion |
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This study demonstrates both the presence of NADPH-d activity in porcine oviduct and its fluctuation during the course of the estrous cycle. Furthermore, we documented the expression of NOS and assessed the molecular weight of eNOS in porcine fallopian tube.
NADPH-d co-localizes with all known NOS isoforms (
We observed the NADPH-d activity in porcine oviduct epithelium of mucous, myosalpinx, nerve fibers, and endothelium of blood and lymph vessels. The most intense staining was observed in the epithelium lining the porcine oviduct. The intensity of staining was variable, with the lowest intensity of reaction seen at estrus. This finding is in agreement with results obtained in rat oviduct (
The results of our study indicate that the muscle layer of the porcine oviduct was almost clear of NADPH-d staining but that samples taken at the postovulatory stage of the estrous cycle showed marked blue formazan reaction product, mainly in the isthmic region.
Our immocytochemical study revealed that eNOS is the main isoform of NOS expressed in porcine oviduct. Immunolabeling for eNOS was found in epithelial cells of mucosa, endothelium of vascular tissues, and myosalpinx.
The Western blotting analysis confirmed our immunocytochemical results. In homogenates of porcine oviduct, we detected a protein of approximately 135 kD which corresponded to the expected molecular weight of eNOS (
In contrast to eNOS, iNOS immunoreactivity was confined to endothelium of lymphatics and some blood vessels. These data are at variance with the results of others.
Taking into account the method of fixation, the lack of the expected band using Western blotting analysis for iNOS, and single nNOS-immunoreactive nerve fibers running among smooth myocytes of the porcine oviductal isthmus (
In summary, we conclude that the different activity of NADPH-d in various regions of the oviduct, accompanied by changes of its activity during the course of the estrous cycle, can indicate a considerable role of NO in regulation of tubal function. The increase in NOS activity in myosalpinx of the isthmus up to Day 4 of the estrous cycle can inhibit motility of this part of oviduct, facilitating passage of embryos into the uterus after fertilization.
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Footnotes |
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Reprints requests to: Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland. E-mail: ziecik{at}irzbz.pan.olsztyn.pl
Received for publication February 9, 2000; accepted February 9, 2000.
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