Region-specific expression of nitric oxide synthases in the bovine oviduct during the oestrous cycle and in vitro

in Journal of Endocrinology

Nitric oxide synthases (NOS) account for the endogenous production of nitric oxide (NO), a small and permeable bioreactive molecule. NO is known to act as a paracrine mediator during various processes associated with female reproduction. In the present study, the mRNA expression of the endothelial (eNOS) and inducible (iNOS) NO synthases were examined in bovine oviduct epithelial cells (BOEC) during the oestrous cycle. In addition, eNOS and iNOS mRNA and protein were localised by in situ hybridisation and immunocytochemistry respectively. Furthermore, the effects of exogenously applied oestradiol-17β and progesterone on NOS mRNA regulation were studied in a suspension culture of BOEC. The eNOS mRNA abundance was low around ovulation (day 0) and increased significantly until pro-oestrus (day 18) in the ampulla. Immunoreactive protein of eNOS was detected predominantly in endothelial cells as well as in secretory oviduct epithelial cells at pro-oestrus. The iNOS mRNA concentration was significantly reduced in the isthmus at pro-oestrus (day 18) and oestrus (day 0) compared with persistently high levels in the ampulla. By in situ hybridisation, specific iNOS transcripts were additionally demonstrated in the oviduct epithelium. Immunoreactive iNOS protein was localised in secretory epithelial cells as well as in the lamina muscularis. The in vitro stimulation showed that both NOS were stimulated by progesterone, but not by oestradiol-17β. The region-specific modulated expression of eNOS and iNOS provides evidence for an involvement of endogenously produced NO in the regulation of oviductal functions.

Abstract

Nitric oxide synthases (NOS) account for the endogenous production of nitric oxide (NO), a small and permeable bioreactive molecule. NO is known to act as a paracrine mediator during various processes associated with female reproduction. In the present study, the mRNA expression of the endothelial (eNOS) and inducible (iNOS) NO synthases were examined in bovine oviduct epithelial cells (BOEC) during the oestrous cycle. In addition, eNOS and iNOS mRNA and protein were localised by in situ hybridisation and immunocytochemistry respectively. Furthermore, the effects of exogenously applied oestradiol-17β and progesterone on NOS mRNA regulation were studied in a suspension culture of BOEC. The eNOS mRNA abundance was low around ovulation (day 0) and increased significantly until pro-oestrus (day 18) in the ampulla. Immunoreactive protein of eNOS was detected predominantly in endothelial cells as well as in secretory oviduct epithelial cells at pro-oestrus. The iNOS mRNA concentration was significantly reduced in the isthmus at pro-oestrus (day 18) and oestrus (day 0) compared with persistently high levels in the ampulla. By in situ hybridisation, specific iNOS transcripts were additionally demonstrated in the oviduct epithelium. Immunoreactive iNOS protein was localised in secretory epithelial cells as well as in the lamina muscularis. The in vitro stimulation showed that both NOS were stimulated by progesterone, but not by oestradiol-17β. The region-specific modulated expression of eNOS and iNOS provides evidence for an involvement of endogenously produced NO in the regulation of oviductal functions.

Keywords:

Introduction

The oviduct is responsible for the accommodation of the gametes and the early embryo by providing an optimal environment for successful fertilisation. In addition, it accounts for the transport of the gametes and the embryo into the uterus. The anorganic free radical nitric oxide (NO) is a small, unstable and permeable molecule that can pass through membranes by diffusion. NO is an important intercellular regulatory molecule and a major paracrine mediator, functioning as a vascular, immunological and neuronal signalling molecule (Ignarro et al. 2001). NO can react with several targets by exerting unspecific immune responses (Guzik et al. 2003). By binding to guanylyl cyclase, cyclic guanosine monophosphate (cGMP)-specific proteinases (protein kinase G), Na+ ions and phosphodiesterases can be activated, leading to modulated gene expression (Nathan & Xie 1994). The effect of NO on vasodilatation has been demonstrated (Furchgott & Vanhoutte 1989). NO is assumed to have a broad range of functions in reproductive processes such as oocyte maturation, ovulation, implantation, pregnancy maintenance, labour and delivery (Shukovski & Tsafriri 1994, Jablonka-Shariff et al. 1999, Sengoku et al. 2001, Maul et al. 2003).

NO is produced by the conversion of l-arginine to l-citrulline by the enzyme NO synthase (NOS) with several co-factors in a number of different tissues and cell types. To date, three isoforms of NOS, products of separate genes with different molecular weight but apparently similar molecular structure, have been described: neuronal NOS (nNOS) in the brain and peripheral nervous system; endothelial NOS (eNOS) as a constitutive NOS mainly in the endothelium; and inducible NOS (iNOS), synthesised primarily by activated macrophages, hepatocytes and neutrophils in several tissue types and organs and upon inflammatory stimulation (Moncada et al. 1997).

Recently, NOS have been identified in the human, bovine and rat oviduct (Bryant et al. 1995, Rosselli et al. 1996, Ekerhovd et al. 1997). Nevertheless, more comprehensive analyses could help to bridge lack of congruency for different species (Chatterjee et al. 1996, Gawronska et al. 2000) with respect to cellular localisation, regions of the oviduct and cycle-dependent regulation. Therefore, the present study investigated thoroughly the expression and localisation of iNOS and eNOS in the bovine oviduct of days 0, 3.5, 12 and 18 of the oestrous cycle. In addition, the in vivo data were compared with NOS expression in bovine oviduct epithelial cells (BOEC) in vitro. The obtained data can help to assign a specific regulatory role to NOS in the oviduct.

Materials and Methods

Synchronisation of the oestrous cycle and collection of oviduct samples

Twelve cyclic Simmental heifers 18–24 months old were cycle synchronised by injecting intramuscularly a single dose of 500 μg cloprostenol (Estrumate; Essex Tierarznei, Munich, Germany) at dioestrus. Animals were intensively observed for sexual behaviour (i.e. toleration, sweating, vaginal mucus) to determine standing heat, which occurred around 60 h after Estrumate injection.

Ovary function was monitored by ultrasonographic examination to locate the time frame of ovulation, and blood samples were taken to determine serum progesterone (P4) levels, which were executed in 6-h rhythms. Three animals were slaughtered the morning after standing heat occurred (day 0) and three animals each at days 3.5, 12 and 18 after oestrus respectively. Final blood samples were taken just before slaughter to determine serum P4 levels. Animals slaughtered at oestrus (day 0) displayed low serum P4 levels (<1.0 ng/ml) and animals slaughtered at dioestrus (day 12) had high serum P4 levels (>6 ng/ml). Oviducts were trimmed free of surrounding tissue, subdivided into ampulla and isthmus, and dissected in 1-cm-long sections. To isolate the epithelial cells, the sections were stripped with two tweezers, and the epithelia were transferred directly in cryotubes. These tubes were dropped immediately into dry ice, transported in liquid nitrogen and stored at −80 °C until further processing. All samples were taken and frozen within 20 min after death.

In vitro cell suspension culture

Four heifers were slaughtered on day 3.5 of the oestrous cycle, and BOEC were obtained as described previously (Rottmayer et al. 2005). Briefly, the oviducts were squeezed along the ampulla with forceps. The cell sheets were separated mechanically by repeated passages through syringes and pipetting, and recovered by sedimentation. Cells were cultured in 24-well plates with 800 μl TCM-199 supplemented with 2% serum obtained from heifers on day 3.5 of the oestrous cycle and 0.25 mg/ml gentamicin at a density of 106 cells per well at 38 °C in a humidified atmosphere of 5% CO2 in air. BOEC were stimulated with oestradiol-17β (10 pg/ml) or progesterone (10 ng/ml) (both purchased from Sigma) for 6 and 12 h respectively. Both experiments were carried out with appropriate negative controls diluting the carrier of the steroid (ethanol and water respectively) in the same way as the steroid itself. Two animals were also used for a short-time stimulation of 2 h. Cells were collected by centrifugation, washed in buffer solution, snap-frozen in liquid nitrogen and stored at −80 °C until further investigation.

Reverse transcription and real-time RT–PCR

Total RNA from BOEC in vivo and in vitro was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Two-step quantitative real-time RT–PCR with the LightCycler DNA Master SYBR Green I protocol (Roche) was done as described previously (Ulbrich et al. 2004). Briefly, 1 μg of each sample of RNA was reverse transcribed in a total volume of 60 μl: 5X Buffer (Promega), 10 mM dNTPs (Roche), 50 μM hexameres (Gibco BRL), 200 U Superscript RT enzyme (Promega). The conventional PCR was performed in a thermal cycler (Biometra, Göttingen, Germany) as previously described (Berisha et al. 2002). A 7-μl volume of each reaction was subsequently subjected to agarose gel electrophoresis followed by ethidium bromide staining. For each of the following real-time PCR reactions, 1 μl cDNA was used to amplify specific target genes. In each PCR reaction, 17 ng/μl cDNA were introduced and amplified in a 10 μl reaction mixture (3 mM MgCl2, 0.4 μM primer forward and reverse each, 1 Light Cycler DNA Master SYBR Green I; Roche) and compared with a standard curve based on a specific PCR product. Primers were adapted to amplify specific PCR products for 18S rRNA (forward: 5′-AAGTCTTTGGGTTCCGGG-3′; reverse: 5′-GGACATCTAAGGGCATCACA-3′ [365 bp]), eNOS (forward: 5′-AGGAGTGGAAGTGGTTCCG-3′; reverse: 5′-GCCCCGGTACTACTCTGTCA-3′ [126 bp]) and iNOS (forward: 5′-ACCTACCAGCTGACGGGA GAT-3′; reverse: 5′-TGGCAGGGTCCCCTGTGATG-3′ [316 bp]). The predicted size of each PCR product is assigned in brackets. The amplified PCR fragments were sequenced once (MWG, Ebersberg, Germany) to verify the resulting PCR product (Einspanier et al. 2002). Thereafter, the specific melting point (MP) of the amplified products served as verification of the product identity (18S (MP 88 °C, fluorescence acquisition at 80 °C), eNOS (MP 93 °C, fluorescence acquisition at 87 °C) and iNOS (MP 90 °C, fluorescence acquisition at 86 °C) (Ulbrich et al. 2004). The annealing temperature was 60 °C for 18S, 61 °C for iNOS and 66 °C for eNOS. As negative control, water instead of cDNA was used. The nucleotide sequence for the partial bovine iNOS cDNA was subsequently submitted to the EMBL database (accession no. AJ699400). The cycle number (CP) required to achieve a definite SYBR Green fluorescence signal was calculated by the second derivative maximum method (LightCycler software, Version 3.5.28). The CP was correlated inversely with the logarithm of the initial template concentration.

Data analysis of real-time RT–PCR

The CP determined for the target genes were normalised against the housekeeping gene 18S. Resulting data are presented as means of CP (n=3) ± s.e.m. For statistical analysis, the SAS program package release 9.1.3 (2002, SAS Institute, Cary, NC, USA) was used. All data were analysed by one-way ANOVA (analysis of variance). In case of significant different groups, a multiple t-test analysis was done with the Bonferroni correction. Results were considered statistically significant at P<0.05.

In situ hybridisation

The detailed in situ hybridisation procedure has been described previously (Bauersachs et al. 2005). Briefly, formalin-fixed, paraffin wax-embedded samples were used. Sections were deparaffinised with xylene and immersed in isopropanol. Dried sections were submerged in 2 saline sodium citrate buffer and preheated at 80 °C. Slides were then washed in distilled water and TBS and permeabilised with 0.05% proteinase K (VWR, Ismaning, Germany) in Tris-buffered saline (TBS) at room temperature. Sections were relocated in TBS followed by distilled water and post-fixed for 10 min in 4% paraformaldehyde/PBS. After washing in PBS and distilled water, slides were dehydrated and air-dried. Hybridisation was carried out by overlaying the dried sections with the corresponding biotinylated oligonucleotide probe (100 pmol/μl), diluted 1:20 in in situ hybridisation solution (DAKO, Munich, Germany), and incubating them in a humidified chamber at 38 °C overnight. The sequence of the iNOS antisense oligonucleotides was 5′-TCCAGCATCTCCTCCCAGTA-3′. RNase-free hybridisation solution (DAKO, Munich, Germany) contained 60% formamide, 5 × SSC, hybridisation accelerator, RNase inhibitor and blocking reagents. Subsequently, slides were washed in 2 × SSC (2 h 15 min, preheated to 38 °C), distilled water (25 min) and TBS (25 min). Hybridised probes were detected with HRP-labelled ABC kit reagents developed by DAB (DAKO, Munich, Germany) according to the manufacturer’s instructions. Negative controls were done by exchanging the oligonucleotide probe with the corresponding sense oligonucleotide.

Immunohistochemistry

For the immunohistochemical demonstration of eNOS and iNOS, tissue samples were fixed in Bouin’s solution for 12 h, as described previously (Berisha et al. 2004). The specimens were dehydrated and embedded in paraffin wax. Serial sections (5 μm) were cut on a Leitz microtome and collected on gelatin/chrom alum-coated slides. To expose antigenic sites, dewaxed sections were heated four times to 95 °C in a 600 W microwave oven in citrate buffer for 5 min. Endogenous peroxidase activity was then eliminated by incubation with 0.5% (v/v) H2O2 solution in absolute methanol for 15 min at 20 °C. Non-specific protein binding was eliminated by incubation with 10% normal goat serum in PBS for 1 h at 20 °C. Sections were then incubated with either polyclonal rabbit antibody against iNOS (Upstate, Lake Placid, NY, USA) (Sherman et al. 1999), which is known to cross-react with bovine iNOS, or monoclonal rabbit antibody against eNOS (Alpha Diagnostic, San Antonio, TX, USA), from which the human peptide sequence is 100% conserved in the bovine (Marsden et al. 1992, Welter et al. 2004). Each was used at a dilution of 1:200. Incubation was performed at 18 h at 4 °C in a humidified chamber. This was followed by incubating the sections with biotinylated anti-rabbit IgG 1:400 (Amersham-Pharmacia) for 1 h. The sections were then incubated with ABC reagent from a commercial kit (Vector Laboratories, Burlingame, CA, USA). The bound complex was made visible by reaction with 0.05% 3,3-DAB and 0.0006% H2O2 in 0.1 × PBS. Sections were viewed unstained or counterstained in Mayer’s haematoxylin, dehydrated, cleared and mounted. Controls were performed by either replacing primary antibody with buffer or non-immune serum, or incubating with DAB reagent alone to exclude the possibility of non-suppressed endogenous peroxidase activity. Lack of detectable staining in the controls demonstrated the specificity of the reactions.

Results

A first screening of eNOS and iNOS mRNA in bovine oviduct total RNA by conventional RT–PCR led to different amplification signal intensities, as shown in Fig. 1. To further characterise these preliminary results, a real-time RT–PCR approach was applied to quantify the gene expression patterns in oviduct epithelial cells.

Transcripts of eNOS and iNOS were detected in BOEC throughout the oestrous cycle. A statistical analysis of eNOS/iNOS transcript concentrations from the ipsi-versus contralateral side revealed no significant differences at any time point (P=0.07) in either ampulla or isthmus. Therefore ipsi- and contralateral oviducts were grouped, so that each bar in Fig. 2 represents a data set of six individual oviducts from three different animals.

The eNOS mRNA expression in the ampulla was low at oestrus (day 0) and increased significantly, more than threefold, until reaching the highest levels at pro-oestrus (day 18) (70.7 and 245.4 fg mRNA/μg total RNA respectively) (Fig. 2A). The same tendency was observed in the isthmus, yet here the differences between days 0 and 18 were not significant. Immunoreactive protein was observed in endothelial cells of blood vessels (data not shown). Additionally, supranuclear staining in secretory epithelial cells of the oviduct epithelium was visible at day 18 (Fig. 3A). Nuclear and cytoplasmic staining of both smooth muscle and endothelial cells of small blood vessels could be detected also in the lamina muscularis (Fig. 3B).

The highest transcript amounts for iNOS were detected in the ampulla (3680 fg mRNA/μg total RNA at day 18), high levels being retained throughout the oestrous cycle (Fig. 2B). At oestrus (day 0), transcript levels in the isthmus were almost 10-fold lower than in the ampulla (333.8 and 2950 fg mRNA/μg total RNA respectively). At day 3.5 the iNOS expression in the isthmus was already again as high as in the ampulla (3246 and 2657 fg mRNA/μg total RNA respectively). It declined to an intermediate level at day 12, which was significantly lower than at day 3.5 (810.7 fg mRNA/μg total RNA in the isthmus). At pro-oestrus (day 18), there was a significantly lower iNOS mRNA concentration (threefold) in the isthmus than the ampulla (1222 and 3676 fg mRNA/μg total RNA respectively). In situ hybridisation revealed transcripts in the epithelial cells mainly in the oviductal ampulla at pro-oestrus and oestrus (Fig. 3 G and H), in accordance with the high mRNA transcript levels measured by real-time RT–PCR (Fig. 2). Immunohistochemical analysis revealed a conspicious supranuclear staining in secretory epithelial cells mainly toward the lumen (Fig. 3C). The staining appeared mostly at the apical, and not in the basal parts of the luminal branching folds (Fig. 3E). Additionally, pronounced nuclear staining of the lamina muscularis was observed (Fig. 3D and F). The stroma was consistently devoid of iNOS protein (Fig. 3C and E).

The stimulation of BOEC with oestradiol-17β had no effect on the transcript regulation of eNOS (Fig. 4A). However, progesterone significantly stimulated eNOS transcript levels more than threefold only 2 h after application (36.7 and 112 fg mRNA/μg total RNA respectively) (Fig. 4A).

Nor did oestradiol-17β have an effect on the transcript regulation of iNOS in cultured BOEC (Fig. 4B). But a significant twofold upregulation of iNOS transcript abundance was observed 6 and 12 h after progesterone stimulation (399 and 904 fg mRNA/μg total RNA, and 368 and 883 fg mRNA/μg total RNA respectively) (Fig. 4B).

Discussion

This study demonstrates the presence of the two iso-enzymes eNOS and iNOS in the bovine oviduct during the oestrous cycle. Moreover, immunoreactive protein of eNOS and iNOS could be located in distinct cell types of the oviduct.

In agreement with our mRNA data for iNOS, the lowest expression of NADPH diaphorase as a marker for NOS was found in the isthmus at oestrus (Gawronska et al. 2000). High expression was noticed during the luteal phase (Gawronska et al. 2000). The presence of NADPH diaphorase activity in the porcine oviduct has been demonstrated during the oestrous cycle. Bryant et al.(1995) showed reduced NO activity during late pro-oestrus by measuring the conversion of l-arginine to l-citrulline in the rat oviduct, and eNOS in the rat oviduct was most prevalent at pro-oestrus and oestrus (Chatterjee et al. 1996). The latter result may point to a direct oestradiol effect on eNOS in rats, but this was not confirmed in the present study for the cow. Instead, the rising levels of eNOS mRNA between oestrus (day 0), dioestrus (day 3.5 and 12) and pro-estrus (day 18) indicate the dependency of eNOS on progesterone. This assumption is supported by the stimulation of NOS mRNA expression by progesterone in cultured BOEC as well as the eNOS protein staining in vivo mainly at pro-oestrus.

Current data on the localisation of NOS in the oviduct are controversial. NADPH diaphorase was demontrated mainly in the oviduct epithelium of man, rat and pig (Bryant et al. 1995, Ekerhovd et al. 1999, Gawronska et al. 2000), and eNOS protein was consistently present in oviduct epithelial cells of different species (Bryant et al. 1995, Rosselli et al. 1996, Ekerhovd et al. 1997, Gawronska et al. 2000). In the present study, we clearly demonstrate the presence of both eNOS and iNOS in BOEC. Previously, iNOS protein was observed in the epithelium of the human and the rat oviduct (Bryant et al. 1995, Ekerhovd et al. 1997), but not in the pig (Gawronska et al. 2000). These peculiarities may indicate species differences. In the porcine oviduct, eNOS was found to be the predominant isoform (Gawronska et al. 2000). The present results reveal that the absolute expression of iNOS is much higher than eNOS, in addition to a more pronounced regulation during the oestrous cycle. Since the latter isoform produces lower quantities of NO (Ekerhovd et al. 1999), eNOS in the bovine oviduct might be the isoenzyme responsible for the rather constitutive presence of NO.

NADPH diaphorase as well as eNOS was demonstrated in the myosalpinx of the rat and pig (Bryant et al. 1995), and iNOS was also found in smooth muscle of the human oviduct (Ekerhovd et al. 1997). The relaxing effect of NO on smooth muscle, possibly controlled by progesterone (Chwalisz 1994), is well known, particularly for uterine quiescence during pregnancy (Yallampalli et al. 1993). The motility patterns of the oviduct show rising frequency and amplitude of motility around oestrus (Bennett et al. 1988). Therefore, several mediators of contraction could be involved in this regulation, influencing or orchestrating the NOS expression in the oviduct, namely, oestradiol, oxytocin, prostaglandin (PG)F, PGE2 and endothelin-1 (Moore & Croxatto 1988, Gilbert et al. 1992, Salvemini et al. 1993, Rosselli et al. 1994, Perez et al. 1998). PGF can induce NO production by NOS in rat oviduct cells (Perez et al. 1998). The highest concentrations of PGF receptors are found around oestrus in the rat (Orlicky & Williams-Skipp 1993), and oestradiol is known to activate PG synthase. NO could therefore negatively modulate or antagonise the contractile response of PGF. Furthermore, NO participates in the release of PGE2 (Salvemini et al. 1993). PGE2 is increased at oestrus (Wijayagunawardane et al. 1998) and has been held to cause relaxation of the oviduct in the presence of progesterone (Gawronska et al. 2000). Subsequently, PGE2, together with progesterone, could regulate NOS expression (Milano et al. 1995), an effect that would agree with the in vitro findings of the present study. Beside these findings, endothelin-1 (ET-1) is high in the ipsilateral oviduct during the follicular and postovulation stage (Wijayagunawardane et al. 1998). ET-1 has been shown to stimulate NO in BOEC via endothelin receptor beta (Rosselli et al. 1994). NO reduces the contractile effects of ET-1, hence the interplay of ET-1 and NO might contribute to the physiological relaxation of the oviduct.

Using L-NAME (N-nitro-l-arginine methyl ester), a well-known inhibitor of NOS, Perez et al.(2000) found evidence of increased tubal motility that resulted in accelerated ovum transport into the uterus. Moreover, oestradiol treatment caused increasing contraction frequency of the smooth muscle of the isthmus (Moore & Croxatto 1988). This could be mediated by oestrogen receptor β, which is more abundant in the isthmus (Ulbrich et al. 2003). The endogenous local downregulation of iNOS in the isthmus could support similar effects. Therefore, our hypothesis is that the downregulation of iNOS at oestrus in the isthmus leads to an increase of oviduct motility by circular smooth muscle activity.

There is evidence that the accelerated movement of microspheres through the isthmus is due to peristaltic smooth muscle contractions, and to ciliary activity, and this is supported by the observation of a reduced number of ciliated cells in the isthmus (Perez et al. 1998). Moreover, the ciliated epithelial cells were mostly devoid of iNOS protein, whereas the lamina muscularis was clearly stained.

In secretory epithelial cells of the oviduct, the nucleus is shifted toward the apical side of the epithelium (unpublished data). We may deduce from this that both eNOS and iNOS proteins target mostly secretory epithelial cells, as has also been shown for eNOS in the human Fallopian tube (Ekerhovd et al. 1999). iNOS protein was found in secretory cells toward the oviduct lumen in the supra-nuclear region; therefore, it can be assumed that NO is released into the lumen. It might be of further interest to know whether the produced NO is also secreted toward the stroma and myosalpinx, or whether eNOS found in smooth muscle cells affects the contractibility of the oviduct. The contribution of NOS might be important for relaxation together with a local exertion during (pro-)oestrus, facilitating capture, retention and fertilisation of the released oocyte and the active transport of the conceptus (Chatterjee et al. 1996). Furthermore, through the ability of iNOS to produce cytotoxic levels of NO (Guo et al. 1995), the downregulation of iNOS in the isthmus at oestrus could be an implicit protective mechanism for advancing sperm and the developing embryo.

The present study does not take into account further regulation of NOS enzyme activity. In particular, the proposed differences in enzyme activity of both NOS isoforms (Presta et al. 1997) cannot be measured through expression analysis solely. Nonetheless, this investigation provides new data on a local NO-regulating mechanism in the bovine oviduct.

In summary, the present results provide evidence of the presence of NOS in the bovine oviduct. The region-specific modulated mRNA expression patterns of eNOS and iNOS during the oestrous cycle indicate a local regulatory system of NO in the bovine oviduct and suggest a different role of NO in the ampulla and the isthmus region. Although functional analyses still remain to be done, the conspicious downregulation of iNOS at oestrus in the isthmus requires further research. NO might represent another important local factor regulating oviducal functions with possible impact on contractility response. The present findings underline the physiological influence of both NOS in supporting a successful fertilisation by regulating the oviduct environment.

Figure 1
Figure 1

Messenger RNA transcripts of the housekeeping gene 18S and the target genes eNOS and iNOS in the bovine oviduct during the oestrus cycle, exemplified by conventional RT–PCR demonstrating differential expression between ampulla and isthmus as well as between the days of the oestrous cycle. One representative experiment is shown.

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06526

Figure 2
Figure 2

Messenger RNA expression (real-time RT–PCR) of (A) eNOS and (B) iNOS in bovine oviduct cells during the oestrous cycle in either ampulla (▪) or isthmus (□). eNOS mRNA was significantly upregulated in the ampulla between oestrus (day 0) and pro-oestrus (day 18). There was a remarkable downregulation of iNOS mRNA in the isthmus compared with the ampulla at oestrus (day 0) and dioestrus (day 18). Data are presented as means of mRNA/total RNA ± s.e.m. normalised by 18S (n=6). *Significant differences between ampulla and isthmus (P<0.05). Different superscript letters indicate significant differences between days of the oestrous cycle (P<0.05).

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06526

Figure 3
Figure 3

Immunohistochemical localisation of eNOS (A and B) and iNOS (C–F) in bovine oviducts. Black arrowheads (A–F) point at specific immunopositive secretory epithelial cells with supranuclear staining mainly toward the lumen. The staining appeared mostly at the apical, and not in the basal, parts of the luminal branching folds (E). Arrows (A–F) point at immunopositive cells for eNOS and iNOS in the lamina muscularis with pronounced nuclear staining. In situ hybridisation of iNOS (G and H) in bovine oviducts (brown staining) clearly showed positive iNOS mRNA transcripts specifically in epithelial cells of the ampulla (black spearheads). The black bar indicates 50 μm.

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06526

Figure 4
Figure 4

Messenger RNA expression (real-time RT–PCR) of (A) eNOS and (B) iNOS transcripts in a bovine oviduct epithelial cell suspension culture after stimulation with progesterone or oestradiol-17β Transcripts of both eNOS and iNOS were stimulated by progesterone, but not by oestradiol-17β. Data are presented as means of mRNA/total RNA ± s.e.m. normalised by 18S (n=4). *Significant differences between control and stimulation (P < 0.05).

Citation: Journal of Endocrinology 188, 2; 10.1677/joe.1.06526

This study was supported by Deutsche Forschungsgemeinschaft (DFG Ei 296/10–2) and Evangelisches Studienwerk e.V. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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  • MarsdenPA Schappert KT Chen HS Flowers M Sundell CL Wilcox JN Lamas S & Michel T 1992 Molecular cloning and characterisation of human endothelial nitric oxide synthase. FEBS Letters307287–293.

    • Search Google Scholar
    • Export Citation
  • MaulH Longo M Saade GR & Garfield RE 2003 Nitric oxide and its role during pregnancy: from ovulation to delivery. Current Pharmaceutical Design9359–380.

    • Search Google Scholar
    • Export Citation
  • MilanoS Arcoleo F Dieli M D’Agostino R D’Agostino P De Nucci G & Cillari E 1995 Prostaglandin E2 regulates inducible nitric oxide synthase in the murine macrophage cell line J774. Prostaglandins49105–115.

    • Search Google Scholar
    • Export Citation
  • MoncadaS Higgs A & Furchgott R 1997 International Union of Pharmacology nomenclature in nitric oxide research. Pharmacological Reviews49137–142.

    • Search Google Scholar
    • Export Citation
  • MooreGD & Croxatto HB 1988 Effects of delayed transfer and treatment with oestrogen on the transport of microspheres by the rat oviduct. Journal of Reproduction and Fertility83795–802.

    • Search Google Scholar
    • Export Citation
  • NathanC & Xie QW 1994 Nitric oxide synthases: roles tolls and controls. Cell78915–918.

  • OrlickyDJ & Williams-Skipp C 1993 Immunohistochemical localisation of PGF2 alpha receptor in the rat oviduct. Prostaglandins Leukotrienes and Essential Fatty Acids48185–192.

    • Search Google Scholar
    • Export Citation
  • PerezMS Franchi AM Viggiano JM Herrero MB & Gimeno M 1998 Effect of prostaglandin F2 alpha (PGF2 alpha) on oviductal nitric oxide synthase (NOS) activity: possible role of endogenous NO on PGF2 alpha-induced contractions in rat oviduct. Prostaglandins and Other Lipid Mediators56155–166.

    • Search Google Scholar
    • Export Citation
  • PerezMS Viggiano M Franchi AM Herrero MB Ortiz ME Gimeno MF & Villalon M 2000 Effect of nitric oxide synthase inhibitors on ovum transport and oviductal smooth muscle activity in the rat oviduct. Journal of Reproduction and Fertility118111–117.

    • Search Google Scholar
    • Export Citation
  • PrestaA Liu J Sessa WC & Stuehr DJ 1997 Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymatic activity. Nitric Oxide174–87.

    • Search Google Scholar
    • Export Citation
  • RosselliM Dubey RK Rosselli MA Macas E Fink D Lauper U Keller PJ & Imthurn B 1996 Identification of nitric oxide synthase in human and bovine oviduct. Molecular Human Reproduction2607–612.

    • Search Google Scholar
    • Export Citation
  • RottmayerR Ulbrich SE Kölle S Prelle K Meyer HH Sinowatz F Wolf E & Hiendleder S 2005 A novel suspension culture system for bovine oviduct epithelial cells. Reproduction Fertility and Development1722.

    • Search Google Scholar
    • Export Citation
  • SalveminiD Misko TP Masferrer JL Seibert K Currie MG & Needleman P 1993 Nitric oxide activates cyclooxygenase enzymes. PNAS907240–7244.

    • Search Google Scholar
    • Export Citation
  • SengokuK Takuma N Horikawa M Tsuchiya K Komori H Sharifa D Tamate K & Ishikawa M 2001 Requirement of nitric oxide for murine oocyte maturation embryo development and trophoblast outgrowth in vitro. Molecular Reproduction and Development58262–268.

    • Search Google Scholar
    • Export Citation
  • ShermanTS Chen Z Yuhanna IS Lau KS Margraf LR & Shaul PW 1999 Nitric oxide synthase isoform expression in the developing lung epithelium. American Journal of Physiology276L383–L390.

    • Search Google Scholar
    • Export Citation
  • ShukovskiL & Tsafriri A 1994 The involvement of nitric oxide in the ovulatory process in the rat. Endocrinology1352287–2290.

  • UlbrichSE Kettler A & Einspanier R 2003 Expression and localisation of estrogen receptor alpha estrogen receptor beta and progesterone receptor in the bovine oviduct in vivo and in vitro. Journal of Steroid Biochemistry and Molecular Biology84279–289.

    • Search Google Scholar
    • Export Citation
  • UlbrichSE Schoenfelder M Thoene S & Einspanier R 2004 Hyaluronan in the bovine oviduct – modulation of synthases and receptors during the estrous cycle. Molecular and Cellular Endocrinology2149–18.

    • Search Google Scholar
    • Export Citation
  • WelterH Bollwein H Weber F Rohr S & Einspanier R 2004 Expression of endothelial and inducible nitric oxide synthases is modulated in the endometrium of cyclic and early pregnant mares. Reproduction Fertility and Development16689–698.

    • Search Google Scholar
    • Export Citation
  • WijayagunawardaneMP Miyamoto A Cerbito WA Acosta TJ Takagi M & Sato K 1998 Local distributions of oviductal estradiol progesterone prostaglandins oxytocin and endothelin-1 in the cyclic cow. Theriogenology49607–618.

    • Search Google Scholar
    • Export Citation
  • YallampalliC Garfield RE & Byam-Smith M 1993 Nitric oxide inhibits uterine contractility during pregnancy but not during delivery. Endocrinology1331899–1902.

    • Search Google Scholar
    • Export Citation

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    Messenger RNA transcripts of the housekeeping gene 18S and the target genes eNOS and iNOS in the bovine oviduct during the oestrus cycle, exemplified by conventional RT–PCR demonstrating differential expression between ampulla and isthmus as well as between the days of the oestrous cycle. One representative experiment is shown.

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    Messenger RNA expression (real-time RT–PCR) of (A) eNOS and (B) iNOS in bovine oviduct cells during the oestrous cycle in either ampulla (▪) or isthmus (□). eNOS mRNA was significantly upregulated in the ampulla between oestrus (day 0) and pro-oestrus (day 18). There was a remarkable downregulation of iNOS mRNA in the isthmus compared with the ampulla at oestrus (day 0) and dioestrus (day 18). Data are presented as means of mRNA/total RNA ± s.e.m. normalised by 18S (n=6). *Significant differences between ampulla and isthmus (P<0.05). Different superscript letters indicate significant differences between days of the oestrous cycle (P<0.05).

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    Immunohistochemical localisation of eNOS (A and B) and iNOS (C–F) in bovine oviducts. Black arrowheads (A–F) point at specific immunopositive secretory epithelial cells with supranuclear staining mainly toward the lumen. The staining appeared mostly at the apical, and not in the basal, parts of the luminal branching folds (E). Arrows (A–F) point at immunopositive cells for eNOS and iNOS in the lamina muscularis with pronounced nuclear staining. In situ hybridisation of iNOS (G and H) in bovine oviducts (brown staining) clearly showed positive iNOS mRNA transcripts specifically in epithelial cells of the ampulla (black spearheads). The black bar indicates 50 μm.

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    Messenger RNA expression (real-time RT–PCR) of (A) eNOS and (B) iNOS transcripts in a bovine oviduct epithelial cell suspension culture after stimulation with progesterone or oestradiol-17β Transcripts of both eNOS and iNOS were stimulated by progesterone, but not by oestradiol-17β. Data are presented as means of mRNA/total RNA ± s.e.m. normalised by 18S (n=4). *Significant differences between control and stimulation (P < 0.05).

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  • MarsdenPA Schappert KT Chen HS Flowers M Sundell CL Wilcox JN Lamas S & Michel T 1992 Molecular cloning and characterisation of human endothelial nitric oxide synthase. FEBS Letters307287–293.

    • Search Google Scholar
    • Export Citation
  • MaulH Longo M Saade GR & Garfield RE 2003 Nitric oxide and its role during pregnancy: from ovulation to delivery. Current Pharmaceutical Design9359–380.

    • Search Google Scholar
    • Export Citation
  • MilanoS Arcoleo F Dieli M D’Agostino R D’Agostino P De Nucci G & Cillari E 1995 Prostaglandin E2 regulates inducible nitric oxide synthase in the murine macrophage cell line J774. Prostaglandins49105–115.

    • Search Google Scholar
    • Export Citation
  • MoncadaS Higgs A & Furchgott R 1997 International Union of Pharmacology nomenclature in nitric oxide research. Pharmacological Reviews49137–142.

    • Search Google Scholar
    • Export Citation
  • MooreGD & Croxatto HB 1988 Effects of delayed transfer and treatment with oestrogen on the transport of microspheres by the rat oviduct. Journal of Reproduction and Fertility83795–802.

    • Search Google Scholar
    • Export Citation
  • NathanC & Xie QW 1994 Nitric oxide synthases: roles tolls and controls. Cell78915–918.

  • OrlickyDJ & Williams-Skipp C 1993 Immunohistochemical localisation of PGF2 alpha receptor in the rat oviduct. Prostaglandins Leukotrienes and Essential Fatty Acids48185–192.

    • Search Google Scholar
    • Export Citation
  • PerezMS Franchi AM Viggiano JM Herrero MB & Gimeno M 1998 Effect of prostaglandin F2 alpha (PGF2 alpha) on oviductal nitric oxide synthase (NOS) activity: possible role of endogenous NO on PGF2 alpha-induced contractions in rat oviduct. Prostaglandins and Other Lipid Mediators56155–166.

    • Search Google Scholar
    • Export Citation
  • PerezMS Viggiano M Franchi AM Herrero MB Ortiz ME Gimeno MF & Villalon M 2000 Effect of nitric oxide synthase inhibitors on ovum transport and oviductal smooth muscle activity in the rat oviduct. Journal of Reproduction and Fertility118111–117.

    • Search Google Scholar
    • Export Citation
  • PrestaA Liu J Sessa WC & Stuehr DJ 1997 Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymatic activity. Nitric Oxide174–87.

    • Search Google Scholar
    • Export Citation
  • RosselliM Dubey RK Rosselli MA Macas E Fink D Lauper U Keller PJ & Imthurn B 1996 Identification of nitric oxide synthase in human and bovine oviduct. Molecular Human Reproduction2607–612.

    • Search Google Scholar
    • Export Citation
  • RottmayerR Ulbrich SE Kölle S Prelle K Meyer HH Sinowatz F Wolf E & Hiendleder S 2005 A novel suspension culture system for bovine oviduct epithelial cells. Reproduction Fertility and Development1722.

    • Search Google Scholar
    • Export Citation
  • SalveminiD Misko TP Masferrer JL Seibert K Currie MG & Needleman P 1993 Nitric oxide activates cyclooxygenase enzymes. PNAS907240–7244.

    • Search Google Scholar
    • Export Citation
  • SengokuK Takuma N Horikawa M Tsuchiya K Komori H Sharifa D Tamate K & Ishikawa M 2001 Requirement of nitric oxide for murine oocyte maturation embryo development and trophoblast outgrowth in vitro. Molecular Reproduction and Development58262–268.

    • Search Google Scholar
    • Export Citation
  • ShermanTS Chen Z Yuhanna IS Lau KS Margraf LR & Shaul PW 1999 Nitric oxide synthase isoform expression in the developing lung epithelium. American Journal of Physiology276L383–L390.

    • Search Google Scholar
    • Export Citation
  • ShukovskiL & Tsafriri A 1994 The involvement of nitric oxide in the ovulatory process in the rat. Endocrinology1352287–2290.

  • UlbrichSE Kettler A & Einspanier R 2003 Expression and localisation of estrogen receptor alpha estrogen receptor beta and progesterone receptor in the bovine oviduct in vivo and in vitro. Journal of Steroid Biochemistry and Molecular Biology84279–289.

    • Search Google Scholar
    • Export Citation
  • UlbrichSE Schoenfelder M Thoene S & Einspanier R 2004 Hyaluronan in the bovine oviduct – modulation of synthases and receptors during the estrous cycle. Molecular and Cellular Endocrinology2149–18.

    • Search Google Scholar
    • Export Citation
  • WelterH Bollwein H Weber F Rohr S & Einspanier R 2004 Expression of endothelial and inducible nitric oxide synthases is modulated in the endometrium of cyclic and early pregnant mares. Reproduction Fertility and Development16689–698.

    • Search Google Scholar
    • Export Citation
  • WijayagunawardaneMP Miyamoto A Cerbito WA Acosta TJ Takagi M & Sato K 1998 Local distributions of oviductal estradiol progesterone prostaglandins oxytocin and endothelin-1 in the cyclic cow. Theriogenology49607–618.

    • Search Google Scholar
    • Export Citation
  • YallampalliC Garfield RE & Byam-Smith M 1993 Nitric oxide inhibits uterine contractility during pregnancy but not during delivery. Endocrinology1331899–1902.

    • Search Google Scholar
    • Export Citation