ARTICLE

Distribution of NADPH-diaphorase and Nitric Oxide Synthase (NOS) in Different Regions of Porcine Oviduct During the Estrous Cycle

Correspondence to: Barbara Gawronska, Pennington Biomedical Research Center Louisiana State U., 6400 Perkins Road, Baton Rouge, LA 70808. E-mail: gawronB@pbrc.edu

	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 48:867–875, 2000)

Key Words: NADPH-diaphorase, nitric oxide synthase, oviduct, pig

	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; Knowles and Moncada 1994 ; Sessa 1994 ). Neuronal, constitutive, or brain nitric oxide synthase (nNOS, bNOS or NOS I) was found in brain and peripheral nervous system (Sessa 1994 , Norman and Cameron 1996 ). Endothelial constitutive nitric oxide synthase (eNOS or NOS III) exists in endothelia of vessels (Sessa 1994 ; Norman and Cameron 1996 ). These two isoforms require calcium calmodulin for activation. Activated macrophages, hepatocytes, and neutrophils synthesize NO by an inducible calcium-independent form of NOS (mac NOS, iNOS or NOS II; Knowles and Moncada 1994 ; Sessa 1994 ; Norman and Cameron 1996 ). The three isoforms of NOS are products of separate genes exhibiting different protein molecular weights: eNOS, 135 kD (Sessa et al. 1992 ); iNOS, 130–135 kD (Xie et al. 1992 ; Sessa 1994 ); and nNOS, 160 kD (Sessa 1994 ; Lopez-Figueroa et al. 1996 ). Generation of NO from L-arginine requires, in addition, NOS cofactors: NADPH, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD; Knowles and Moncada 1994 ). Because NADPH-diaphorase (NADPH-d) and NOS activities are caused by different properties of the same enzyme molecule, NADPH-d activity can be used as a marker for NOS (Gabbott and Bacon 1993 ; Loesch et al. 1993 ). NO is synthesized in the female reproductive tract (for review see Rosselli et al. 1998 ). NOS has been demonstrated in mouse (Moorhead et al. 1995 ), rat (Suburo et al. 1995 ), and human (Telfer et al. 1995 ) uterus using histochemical methods for NADPH-d staining. NOS-like immunoreactivity was also localized in human (Telfer et al. 1995 ) and rat (Suburo et al. 1995 ) uterus. Several studies have indicated that NO is responsible for inhibition of uterine contractions during pregnancy (Yallampalli et al. 1993 , Yallampalli et al. 1994 ; Buhimshi et al. 1995). NADPH-d activity has been determined in blood (Zezula-Szpyra et al. 1996a ) and lymph (Zezula-Szpyra et al. 1996b ) vessels in the broad ligament of the uterus of ovariectomized, estradiol-treated gilts. Recently, NOS has been identified in human (Rosselli et al. 1996 ; Ekerhovd et al. 1997 ), bovine (Rosselli et al. 1996 ), and rat (Bryant et al. 1995 ; Chatterjee et al. 1996 , Martinez et al. 1997 ) fallopian tubes. It has also been suggested that NO participates in the regulation of tubal function. The aim of our studies was to examine (a) the presence and potential changes in NADPH-d activity in the porcine oviduct at different stages of the estrous cycle, (b) the distribution of eNOS and iNOS isoforms, and (c) to assess the molecular weight of iNOS and eNOS protein in homogenates of porcine oviduct by Western blotting analysis.

	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 (Akins and Morrisette 1968 ; Leiser et al. 1988 ). These observations allowed the classification of the oviductal tissues into (a) early proestrus, Days 16–18; (b) late proestrus, Days 18–20; (c) estrus; (d) postovulation, Days 2–4; (e) early luteal stage, Days 6–8; (f) midcycle, Days 8–12; and (g) late luteal stage, Days 13–15. Oviducts were collected immediately after sacrifice. One fallopian tube was used for histo- and immunohistochemistry and the other one for Western blotting analysis.

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 37–38C 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 (Sigma–Aldrich; 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 1030–1209 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 961–1144 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 (Lowry et al. 1951 ). Equal amounts of protein (50–100 µg) were dissolved in SDS gel-loading buffer [50 mM Tris-HCl, pH 6.8, 2% SDS (BioRad, Hercules, CA), 10% glycerol (Sigma) 1% ß-mercaptoethanol (Sigma) and heated at 95C for 4 min. Protein fractions were loaded on 10% SDS-PAGE and electrophoretically separated for 2 hr at constant current (30 mA). Separated proteins were electrotransferred onto 0.45-µm nitrocellulose membrane for 2 hr at 250 mA in transfer buffer (25 mM Tris-Cl, pH 8.3; 192 mM glycine; 20% methanol A; 0.02% SDS). Then the membrane was washed in TBS-T; (25 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.05% Tween-20) and incubated in blocking buffer [1% BSA (Sigma) in TBS-T] for 1 hr at room temperature (RT). The presence of eNOS was confirmed by incubating the membrane in TBS containing eNOS antibody (a mouse monoclonal antibody directed against the amino acids 1030–1209 of human endothelial NO synthase; Transduction Laboratories) at a dilution of 1:2500 overnight at 4C. After incubation, the nitrocellulose membrane was washed three times for 10 min in TBS-T at RT. The washed blot was reincubated for 2 hr at RT with a 1:3000 dilution of biotinylated antimouse IgG (Vectastain ABC kit) washed three times in TBS for 10 min at RT, and incubated for 1 hr at RT with 1:3000 dilution of avdin–biotin–HRPO complex (Vectastain ABC kit). To develop color, TBS containing 0.01% H₂O₂ and 0.04% 3,3'-diaminobenzidine was used.

	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).

View larger version (102K): [in this window] [in a new window]   Figure 1. Histochemical reaction for NADPH-d in mucosal epithelium of porcine oviduct. (a,b) Isthmus; (c,d) ampulla; (e,f) infundibulum. (a) Early proestrus; (c,e) midcycle; (b,d,f) estrus. Bars: b,d,f = 50 µm; a,c,e = 25 µm.

View larger version (136K): [in this window] [in a new window]   Figure 2. NADPH-d staining of porcine myosalpinx. (a,c,d) Isthmus; (b) ampulla. (a,b) Postovulation stage of estrous cycle; (c) late luteal stage. Bars = 25 µm. Arrows, endothelium of vessels; arrowheads, nerve fibers. No staining was seen in control sections incubated without NADPH (d). Bar = 100 µm.

View larger version (33K): [in this window] [in a new window]   Figure 3. Quantitative analysis of NADPH-d activity in the mucosal epithelium of porcine oviduct. Columns represent the mean ± SEM of samples from separate experiments (n = 4) expressed as the relative optical density.

View larger version (27K): [in this window] [in a new window]   Figure 4. Quantitative analysis of NADPH-d activity in the myosalpinx of porcine oviduct. Columns represent the mean ± SEM of samples from separate experiments (n = 4) expressed as the relative optical density.

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 5a–5c). 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).

View larger version (92K): [in this window] [in a new window]   Figure 5. Immunohistochemical staining for NOS in porcine oviduct. Positive reaction for eNOS is shown in a–c and for iNOS in (d) meso–salpinx and (e) myosalpinx. (f) Control section in which nonspecific IgG was substituted for primary antibody. M, myosalpinx; large arrows, epithelium of mucous membrane; arrowheads, endothelium of blood vessels; small arrows, endothelium of lymph vessels. Bars: a,b,d,e = 50 µm; c = 25 µm; f = 100 µm.

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).

View larger version (50K): [in this window] [in a new window]   Figure 6. Representative Western blot of eNOS protein in porcine oviduct (Lane 3). Lane 1, marker of protein molecular weight; Lane 2, positive control in human endothelial cell lysate.

	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 (Tracey et al. 1993 ; Sessa 1994 ; Bryant et al. 1995 ; Rosselli et al. 1996 ; Tschugguel et al. 1998 ). However, Tracey et al. 1993 suggested that NOS represents only a fraction of total cellular NADPH-d activity and that these activities are not always co-localized. Non-NOS NADPH-d activity can be removed by formaldehyde fixation because the process does not affect the NOS-related NADPH-d activity (Nakos and Gossrau 1994 ; Moorhead et al. 1995 ). Therefore, in our study we used paraformaldehyde-fixed tissues to ensure that NADPH-d activity coincided with NOS activity.

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 (Bryant et al. 1995 ). NADPH-d staining was less dense in fallopian tube obtained from rats in late estrus and estrus than in samples taken at other stages of the estrous cycle. On the other hand, Tschugguel et al. 1998 observed only weak staining in the epithelial lining of the human oviduct. Rosselli et al. 1996 demonstrated that cultured bovine and human oviduct epithelial cells possessed areas of intense staining for NADPH-d and that this staining was not homogeneous. They suggested that NOS enzyme activity may be dependent on the differentiation of the cell.

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. Bryant et al. 1995 , examining rat oviduct, observed only light NADPH-d staining in the smooth muscle. It is interesting that porcine myosalpinx showed NADPH-d activity only at the postovulatory stage of the estrous cycle. Salvemini et al. 1993 showed that NO plays a critical role in the release of PGE₂ by direct activation of cyclo-oxygenase. Prostaglandin E₂ is a well-known factor that becomes increased at the postovulatory stage of the estrous cycle (Wijayagunawardane et al. 1998 ) and is responsible for relaxation of the oviduct in the presence of progesterone (Hunter 1988 ). Because progesterone can be locally concentrated in blood, reaching the oviduct immediately after ovulation (Pharazyn et al. 1991 ), it is possible that NO (apart from its own direct influence on oviduct; Ekerhovd et al. 1997 ) activates cyclo-oxygenase enzymes to increase the production of PGE₂. The increase of NADPH-d activity in myosalpinx exclusively at the postovulatory stage of the estrous cycle can be connected with relaxation of the oviduct for passage of embryos to the uterus after fertilization.

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. Chatterjee et al. 1996 observed immunolabeling for eNOS in mucosal epithelium and muscule wall of rat oviduct. They suggested that maximal immunostaining for eNOS during proestrus and estrus may indicate the role of NO in quiescence for reception, retention, and fertilization of ovulated oocytes. In contrast, Bryant et al. 1995 did not observe apparent differences in the density of NOS labeling in rat oviduct at different stages of the estrous cycle. Rosselli et al. 1996 demonstrated positive staining for eNOS in human and bovine cultured epithelial cells.

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 (Sessa 1994 ).

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. Ekerhovd et al. 1997 observed positive staining for iNOS in smooth muscle, epithelium, vascular endothelium, and connective tissues of human oviduct. Labeling for iNOS in rat oviduct was restricted to the epithelial cell lining (Bryant et al. 1995 ; Martinez et al. 1997 ). The differences between those data and our results may result from species differences or may be attributable to low antigenicity of the primary antibody, which could not detect low concentration of iNOS in porcine oviduct.

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 (Majewski et al. 1995 ) it appears likely that observed NADPH-d activity coincides with eNOS activity. eNOS is commonly described as a constitutive isoform of NOS. Our results demonstrate that, in porcine oviduct, changes in NADPH-d/eNOS activity can be associated with hormonal changes during the estrous cycle.

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.

	Footnotes

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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