Molecular Human Reproduction, Vol. 6, No. 2, 185-190,
February 2000
© 2000 European Society of Human Reproduction and Embryology
Uterus and pregnancy |
Endothelial nitric oxide synthase is differently expressed in human endometrial vessels during the menstrual cycle
1 Department of Obstetrics and Gynecology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-0034, Japan, 2 Department of Anatomy and Reproductive Biology, RWTH Aachen, 52057 Aachen and 3 Research Laboratories, Schering AG, 13342 Berlin, Germany
Abstract
Endogenous nitric oxide (NO) can be synthesized by endothelial cells and can act as a potent vasodilator. We investigated endothelial nitric oxide synthase (eNOS), one of the three different enzymes responsible for the synthesis of NO by immunohistochemical methods throughout the menstrual cycle on 34 endometrial samples and compared its detection with the von Willebrand Factor (vWF) as a reliable marker molecule of the endothelium on serial sections. Immunoreactivity for eNOS was clearly localized in various types of arterial and venous endothelial cells as well as in capillaries. In addition, in some samples there was a positive staining in endometrial glandular epithelium. There was no staining in endometrial fibroblasts or in myometrial smooth muscle cells. Whereas the endothelium was constantly stained by the monoclonal antibody against vWF, eNOS was not always expressed in the endothelial lining of the vessels during the menstrual cycle. The number of vessels positively stained for eNOS increased gradually during the proliferation phase and most of the vessels were positive in the early secretory phase. These results suggest that its markedly increased expression during the early secretory phase of the menstrual cycle indicates a physiological significance.
endothelial nitric oxide synthase/human endometrium/immunohistochemistry/menstrual cycle
Introduction
Endogenous nitric oxide (NO) is synthesized by a number of different cells, mediates endothelium-dependent relaxation, neurotransmission and cell-mediated immune response and is involved in paracrine and autocrine regulation of neurotransmitter, polypeptide and ion secretion (Schmidt and Walter, 1994
). The endothelium-derived relaxing factor, which was first demonstrated by Furchgott and Zawadzki (1980), a locally produced substance that modulates the blood flow, was shown to be NO in subsequent studies (Palmer et al., 1987; Furchgott and Vanhoutte, 1989). Nitric oxide is produced by the nitric oxide synthase (NOS) family of enzymes which oxidizes the amino acid L-arginine to form L-citrulline and NO (Palmer et al., 1988).
NOS is known to exist in at least three isoforms: besides the endothelial NOS (eNOS, type III) there exist a neuronal constitutive form (nNOS, type I) and an inducible NOS (iNOS, type II), which was first purified from macrophages (Xie et al., 1992; Nathan and Xie, 1994; Förstermann and Kleinert, 1995). Types I and III are strongly calcium dependent whereas type II is independent of calcium. Neuronal NOS and endothelial NOS are regarded as constitutive and were initially identified in neurons or in endothelial cells, respectively, while iNOS is generally inducible in almost every cell.
eNOS is capable of synthesizing NO in vascular endothelial cells and plays an important role in the control of vasotension (Janssens et al., 1992). Modifications of uterine blood flow are implicated in many aspects of reproductive physiology and in several of their pathological disorders. These modifications are hormonally regulated but remain poorly understood, and various complex regulations have been proposed (White et al., 1995).
There have been several studies in rat (Schmidt et al., 1992; Biswas et al., 1998), mouse (Huang et al., 1995) and human endometrium (Tseng et al., 1996; Telfer et al., 1997) investigating NOS which have addressed whether the vasodilatory and anti-platelet action of NO may play a role in implantation (Cameron and Campbell, 1998).
We studied eNOS localization in human endometrium by immunohistochemical methods throughout the menstrual cycle to determine whether the expression is related to the stage of the menstrual cycle, which is known to be controlled by steroid hormones and paracrine factors.
Materials and methods
Human endometrial samples
Endometrial tissue was obtained from 34 patients undergoing hysterectomy due to benign uterine diseases. Specimens were excluded when any bleeding disorders appeared in the patient's records or the actual diagnosis. The mean age of the women was 43 (range 30–54) years. Seventeen specimens were obtained from the proliferative phase and 17 from the secretory phase. The menstrual cycle was divided into the early to mid proliferative phase (days 3–9), the late proliferative phase (days 10–14), the early secretory phase (days 15–19) and the mid–late secretory phase (days 20–28). The cycle date of each specimen was confirmed by menstrual history, histological examination (Noyes et al., 1950) and assessment for 17ß-oestradiol, progesterone and luteinizing hormone (LH) on the day of hysterectomy by routine laboratory diagnosis. All patients were normally cycling women and none had received medication or hormones for at least 6 months before surgery. The tissue samples were quickly frozen in liquid nitrogen and stored at –20°C until processed.
Immunohistochemistry on cryostat sections
Tissue blocks were embedded in Tissue-Tek O.C.T. (Miles, USA). Cryostat sections (4–7 µm) were cut on a cryostat Reichert Jung 2800 Frigocut E, mounted on aminopropyltriethoxysilane-coated slides and fixed for 10 min in acetone at 4°C. Immunohistochemical staining was performed by a streptavidin–biotin–peroxidase method (Histostain-SP Kit; Zymed Laboratories Inc., CA, USA) at room temperature. The sections were rehydrated in PBS and blocked against endogenous peroxidase by incubating with 3% H2O2/methanol for 10 min. After blocking with 5% swine serum (Dako, Denmark), sections were incubated for 1 h with a specific polyclonal rabbit antibody against human eNOS (Berlex, Biosciences, Richmond, CA, USA) which was diluted 1:120 with phosphate-buffered saline (PBS)/1.5% bovine serum albumin (BSA). Lack of cross-reactivity of this antibody with human iNOS and nNOS was confirmed by solid-phase enzyme-linked immunosorbent assay and Western blots (P.Medberry and F.Parkinson, unpublished data). The second antibody, a multi-link biotinylated swine anti-goat, -mouse, -rabbit immunoglobulin (Dako, Denmark) diluted 1:50 with PBS/1.5% BSA, was added for 1 h. From serial sections, immunostaining for von Willebrand Factor (vWF; also called factor VIII-associated antigen) as a reliable marker of human endothelium was carried out simultaneously. After blocking with 10% non-immune goat serum, the cryosections were incubated for 1 h with a monoclonal primary antibody against vWF (Dako) at a concentration of 1:50. The second antibody, a biotinylated goat anti-mouse immunoglobulin, was incubated for 30 min. After reaction of streptavidin–peroxidase for 10 min, the antigenic sites were visualized by the chromogen aminoethylcarbazole. The immuno histochemical procedure was controlled by replacing the primary antibodies with non-immune serum or with IgG of the appropriate non-immunized species. None of the controls revealed a positive staining (see Figure 12). The reproducibility of the results was confirmed by repeating immunohistochemistry for each specimen. In addition, the specificity of localization was investigated between fundus, corpus and isthmus uteri of the same specimen. However, no difference could be found.
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Histochemistry
Enzyme histochemistry of NADPH diaphorase was performed to detect all sites of putative NOS activity using a published protocol (Vincent et al., 1983
). The cryostat sections were fixed in acetone at 4°C for 10 min and rinsed in PBS three times. The medium for NADPH diaphorase contained 1.2 mmol/l ß-NADPH and 0.25 mmol/l tetranitrozolium blue chloride (Sigma, Deisenhofen, Germany) in PBS, pH 7.4. Sections were incubated for 30 min at room temperature. The specificity was controlled omitting the ß-NADPH in the incubation mixture.
Evaluation of results
More than five fields per section and more than five sections per specimen of each patient were assessed and scored separately by three individual investigators (M.T., I.C.L., J.A.). The scores per field, per specimen and per patient were added and the accumulated final scores divided by three. The quantification of coincidental positive immunostaining of vWF and eNOS was calculated on the basis of these scores per patient (see Figure 13). All three investigators were blinded to the final menstrual cycle dating and the hormonal assessment of the patient.
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Results
Immunoreactivity for eNOS was clearly localized in various types of arterial and venous endothelial cells, i.e. spiral arteries but also small capillaries, which were also stained by the antibody against vWF. In addition, in some samples with strong eNOS-staining in all vessels, glandular epithelial cells were stained as well, but reactions were in general rather weak compared to endothelium.
Whereas the vWF could be detected in all endothelial cells in various types of vessels and did not differ in intensity and degree of staining throughout the endometrial cycle, the staining for eNOS was different (see Figure 1–6). The eNOS expression was very low or negative in the early to mid proliferative phase (day 9, Figure 1) but strongly expressed in the late proliferative phase (day 14, Figure 5) and early (day 18, Figure 11
) to mid secretory phase (day 24, Figure 7).
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There was no staining in endometrial stromal cells or smooth muscle cells (Figures 7, 8
). In the spiral arteries of the secretory phase, the endothelium was clearly stained for eNOS, but the smooth muscle cells surrounding the endothelium were negative (Figure 9). The enzyme histochemical staining for NADPH diaphorase which detects all the three different NOS was similar to the immunoreactivity of eNOS, but in addition there was always a positive staining for glandular epithelium (Figure 10).
The immunostaining for eNOS was compared with the staining for vWF and the percentage of vessels which were stained by eNOS as well as by vWF was evaluated (Figure 13). In contrast to the immunoreactivity for vWF, which was uniformly positive for endothelial cells at all stages of endometrial development, eNOS immunoreactivity was found to change during the cycle. As shown in Figure 13, the immunostaining of eNOS was found to increase from the late proliferative phase to the early and mid secretory phase, based on the morphometric evaluation of the percentage of vessels stained positively by both eNOS and vWF.
Discussion
In addition to its role as a potent vasodilator, NO serves several other functions including activity as an anti-coagulating effector (Schmidt and Walter, 1994), in neurotransmission (Garthwaite et al., 1988) and in cell-mediated immune responses.
In this investigation, we have focused on eNOS immunoreactivity which was clearly located in the endothelium of spiral arteries, capillaries and veins, and to a weaker extent in glandular epithelium in accordance with earlier results (Telfer et al., 1997).
Studies on placenta, with the same polyclonal antibody we used (Nanaev et al., 1995), revealed positive staining for eNOS in the uteroplacental vessels and trophoblast cells. Endothelial NOS has been detected immunohistochemically in human endometrium by several groups (Telfer et al., 1995, 1997; Ota et al., 1998; Tschugguel et al., 1998) with different antibodies against eNOS but with contrasting results.
Whereas one study (Telfer et al. 1997), using a monoclonal antibody against human eNOS (from Affiniti, Nottingham, UK), detected eNOS in endothelial as well as epithelial endometrial cells, another study (Tschugguel et al. 1998), using a different monoclonal antibody (from Transduction Lab., Lexington, KY, USA), localized eNOS on cryostat sections exclusively in endothelial cells. Ota et al. (1998) on the other hand, with the same antibody as Tschugguel and co-workers but using paraffin sections, detected eNOS immunoreactivity on surface and glandular epithelium in addition to a weak staining in the endothelial cells.
Telfer et al. (1997) and Tschugguel et al. (1998) could not find any obvious correlation with the stage of the menstrual cycle, whereas Ota et al. (1998) assessed a similar phase-dependent change in the expression of eNOS as we have shown in our study. Although Telfer et al. (1997) noticed that eNOS staining was not present in all endothelial cells, they could not relate the observed variations to the stage of the menstrual cycle or to the degree of menstrual blood loss. On the other hand, further support for a cyclic expression of eNOS mRNA in the epithelial glands of human endometrium has been provided by Tseng et al. (1996).
In order to elucidate the percentage of vessels positively stained for eNOS in human endometrium, we employed antibodies to vWF which delineates endothelium in human tissue (Mukai et al., 1980). Coincidence between positively stained vessels increased gradually during the proliferative phase. Most vessels were positive in the late proliferative and early to mid secretory phase. During this period of time a complex subepithelial capillary plexus develops (Sheppard and Bonnar, 1980) and a significant dilatation of these vessels during the post-ovulatory phase has been described (Sheppard and Bonnar, 1980; Peek et al., 1992).
The up-regulation of eNOS might be correlated with the 17ß-oestradiol level throughout the menstrual period, particularly the pre-ovulatory oestradiol peak. As we have measured the serum 17ß-oestradiol concentration on the day of hysterectomy, we have tried to correlate this value with the coincidence between eNOS and vWF staining. The mean value of the 17ß-oestradiol concentration of all serum samples with 100% or >50% coincidence between eNOS and vWF (19 samples) was 101 pg /ml, compared to only 65 pg/ ml in the samples with <50% or no coincidence. This assessment reveals supporting evidence for a 17ß-oestradiol involvement in eNOS expression; nevertheless there was only one determination of oestradiol for each patient and there was considerable inter-individual variation in steroid concentrations.
However, there are several papers in which the up-regulation of eNOS by oestrogens is described. An increase in eNOS mRNA in the aorta of pregnant rats and after oestrogen treatment has been reported (Goetz et al., 1994). An increase of eNOS in the uterine artery of guinea-pigs in pregnancy as well as after treatment with oestradiol has also been shown (Weiner et al., 1994). Hayashi et al. reported that in-vitro preincubation with a physiological concentration of 17ß-oestradiol (10–12 to 10–8 M) over 8 h significantly enhanced the activity of eNOS in endothelial cells of cultured human umbilical vein and of bovine aorta (Hayashi et al., 1995), and Hishikawa et al. reported that treatment with 17ß-oestradiol enhanced calcium-dependent NO production and eNOS protein production in human aortic endothelium analysed by Western blotting (Hishikawa et al., 1995).
Nevertheless the up-regulation of eNOS by oestradiol is still a matter of debate. For example it has been shown (Figueroa and Massmann, 1995) that oestrogen increases NOS activity only in the myometrium, but not in the endometrium of non-pregnant castrated sheep, and Arnal et al. have revealed that oestradiol does not increase eNOS protein in a bovine aortic endothelial cell line (Arnal et al., 1996). Which factors actually may regulate the eNOS concentration in the vasculature during the cycle is not yet clear. Besides the ovarian steroid hormones, there are several paracrine factors, but also shear stress, pathological disorders or blood flow itself which could influence eNOS.
Nitric oxide synthesized by eNOS has a direct vasodilatory impact on vascular smooth muscles, probably ensuring an optimal blood supply which might be beneficial to the endometrium during the phase of attachment and implantation.
For a successful implantation, synchronization between embryo and endometrium is necessary. Various growth factors, cytokines and their receptor molecules are present in the endometrium during the peri-implantation period, and it has been suggested that their role is to provide a suitable microenvironment for the blastocyst expansion and embryonic development and to facilitate the implantation process (Beier et al., 1994; Chwalisz et al., 1996; Beier, 1997; Beier and Beier-Hellwig, 1998; Classen-Linke et al., 1998). NOS is present at the time of implantation to induce NO, and this may have a role not only in increasing blood supply by vasodilatation but also in mediating immunological effects.
In conclusion, we have demonstrated the expression of eNOS in human endometrium with a specific polyclonal rabbit antibody against human eNOS. We found a heterogeneity in the expression patterns of this molecule in the human endometrial vasculature. Increased reactivity was observed in the late proliferative and early to mid secretory phase. The importance of these findings must be further elucidated and assessment of the possible role of eNOS in the regulation of blood flow via NO synthesis in the endometrium, in turn facilitating endometrial receptivity and implantation, needs additional research efforts.
Acknowledgments
We thank Dr J.E.Parkinson from Berlex Laboratories Inc. (Richmond, CA, USA) for providing the eNOS antibody and Dr M.Kusche at Marienhospital, Aachen for the cooperation of this investigation. We gratefully acknowledge the excellent technical skills of Mrs D.Seelis and S.Hennes-Mades during the course of these experiments. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (grant Cl 88/3-2).
Notes
4 To whom correspondence should be addressed
References
Arnal, J.F., Clamens, S., Pechet, C. et al. (1996) Ethinyloestradiol does not enhance the expression of nitric oxide synthase in bovine endothelial cells but increases the release of bioactive nitric oxide by inhibiting superoxide anion production. Proc. Natl. Acad. Sci. USA, 93, 4108–4113.
Beier, H.M. (1997) Cell biological aspects of the implantation window. In The Endometrium as a Target for Contraception. Ernst Schering Research Foundation Workshop 18. Springer, Berlin, pp. 51–90.
Beier, H.M. and Beier-Hellwig, K (1998) Molecular and cellular biological aspects of endometrial receptivity. Hum. Reprod. Update, 4, 448–458.
Beier, H.M., Hegele-Hartung, C., Mootz, U. and Beier-Hellwig, K. (1994) Modification of endometrial cell biology using progesterone antagonists to manipulate the implantation window. Hum. Reprod., 9 (Suppl. 1), 98–116.
Biswas, S., Kabir, S.N. and Pal, A.K. (1998) The role of nitric oxide in the process of implantation in rats. J. Reprod. Fertil., 114, 157–161.
Cameron, I.T. and Campbell, S. (1998) Nitric oxide in the endometrium. Hum. Reprod. Update, 4, 565–569.
Chwalisz, K., Buhimschi, I. and Garfield, R.E. (1996) Role of nitric oxide. Obstet. Prenat. Neonat. Med., 1, 292–328.
Classen-Linke, I., Alfer, J., Hey, S. et al. (1998) Marker molecules of human endometrial differentiation can be hormonally regulated under in-vitro conditions as in-vivo. Hum. Reprod. Update, 4, 539–549.
Figueroa, J.P. and Massmann, G.A. (1995) Oestrogen increases nitric oxide synthase activity in the uterus of nonpregnant sheep. Am. J. Obstet. Gynecol., 73, 1539–1545.
Förstermann, U. and Kleinert, H. (1995) Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn-Schmiedebergs Arch. Pharmacol., 352, 351–364.[Web of Science][Medline]
Furchgott, R.F. and Zawadzki, J.V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288, 373–376.[Medline]
Furchgott, R.F. and Vanhoutte, P.M. (1989) Endothelium-derived relaxing and contracting factors. FASEB J., 3, 2007–2018.[Abstract]
Garthwaite, J., Charles, S.L. and Chess-Williams, R. (1988) Endothelium-derived relaxing factor release on activation of NMDA receptors suggests a role as intracellular messenger in the brain. Nature, 336, 385–388.[Medline]
Goetz, R.M., Morano, I., Calovini, T. et al. (1994) Increased expression of endothelial constitutive nitric oxide synthase in rat aorta during pregnancy. Biochem. Biophys. Res. Commun., 205, 905–910.[Web of Science][Medline]
Hayashi, T., Yamada, K., Ezaki, T. et al. (1995) Oestrogen increases endothelial nitric oxide by a receptor-mediated system. Biochem. Biophys. Res. Commun., 214, 847–855,[Web of Science][Medline]
Hishikawa, K., Nakai, T., Marumo, T. et al. (1995) Up-regulation of nitric oxide synthase by oestradiol in human aortic endothelial cells. FEBS Lett., 360, 291–293.[Web of Science][Medline]
Huang, J., Roby, K.F., Pace, J.L. et al. (1995) Cellular localization and hormonal regulation of inducible nitric oxide synthase in cycling mouse uterus. J. Leukocyte Biol., 57, 27–35.[Abstract]
Janssens, S.P., Shimouchi, A., Quertermous, T. et al. (1992) Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J. Biol. Chem., 267, 14519–14522.
Mukai, K., Rosai, J. and Burgdorf, W.H.C. (1980) Localization of factor 8-related antigen in vascular endothelial cells using an immunoperoxidase method. Am. J. Surg. Pathol., 4, 273–276.[Web of Science][Medline]
Nanaev, A., Chwalisz, K., Frank, H.G. et al. (1995) Physiological dilatation of uteroplacental arteries in the guinea pig depends on nitric oxide synthase activity of extravillous trophoblast. Cell Tiss. Res., 282, 407–421.[Web of Science][Medline]
Nathan, C. and Xie, Q. (1994) Nitric oxide synthases: roles, tolls, and controls. Cell, 78, 915–918.[Web of Science][Medline]
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.
Ota, H., Igarashi, S., Hatazawa, J. and Tanaka, T. (1998) Endothelial nitric oxide synthase in the endometrium during the menstrual cycle in patients with endometriosis and adenomyosis. Fertil. Steril., 69, 303–308.[Web of Science][Medline]
Palmer, R.M., Ferrige, A.G. and Moncada, S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature, 327, 524–526.[Medline]
Palmer, R.M.J., Ashton, D.S. and Moncada, S. (1988) Vascular endothelial cells synthesise nitric oxide from L-arginine. Nature, 333, 664–666.[Medline]
Peek, M., Landgren, B.M. and Johannisson, E. (1992) The endometrial capillaries during the normal menstrual cycle: a morphometric study. Hum. Reprod., 7, 906–911.
Schmidt, H.H., Gagne, G.D., Nakane, M. et al. (1992) Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction. J. Histochem. Cytochem., 40, 1439–1456.[Abstract]
Schmidt, H.W. and Walter, U. (1994) NO at work. Cell, 78, 919–925.[Web of Science][Medline]
Sheppard, B.L. and Bonnar, J. (1980) The development of vessels of the endometrium during the menstrual cycle. In Diczfalusy, E. Fraser, I.S. and Webb, F.T.G. (eds), Endometrial Bleeding and Steroidal Contraception. Pitman Press, Bath, pp. 65–77.
Telfer, J.F., Lyall, F., Norman, J.E. and Cameron, I.T. (1995) Identification of nitric oxide synthase in human uterus. Hum. Reprod., 10, 19–23.
Telfer, J.F., Irvine, G.A., Kohnen, G. et al. (1997) Expression of endothelial and inducible nitric oxide synthase in non-pregnant and decidualized human endometrium. Mol. Hum. Reprod., 3, 69–75
Tschugguel, W., Schneeberger, C., Unfried, G. et al. (1998) Induction of inducible nitric oxide synthase expression in human secretory endometrium. Hum. Reprod., 13, 436–444.
Tseng, L., Zhang, J., Peresleni, T.Y. and Goligorsky, M.S. (1996) Cyclic expression of endothelial nitric oxide synthase mRNA in the epithelial glands of human endometrium. J. Soc. Gynecol. Invest., 3, 33–38.[Web of Science][Medline]
Vincent, S.R., Staines, W.A. and Fibiger, H.C. (1983) Histochemical demonstration of separate population of somatostatin and cholinergic neurons in the rat striatum. Neurosci. Lett., 35, 111–114.[Web of Science][Medline]
Weiner, C.P., Lizasoain, I., Baylis, S.A. et al. (1994) Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc. Natl. Acad. Sci. USA, 91, 5212–5216.
White, M.M., Zamudio, S., Stevens, T. et al. (1995) Oestrogen, progesterone, and vascular reactivity: potential cellular mechanisms. Endocr. Rev., 16, 739–751.
Xie, Q., Cho, H.J., Calaycay, J. et al. (1992) Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science, 256, 225–228.
Submitted on May 13, 1999; accepted on October 28, 1999.
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