The aim of the study was to investigate the presence, localisation and function
of brain nitric oxide synthase (bNOS) in the male genital accessory glands of rats
in the course of their postnatal development. Localisation of the bNOS was
immunocytochemically investigated in the epididymis, seminal vesicle and ventral
prostate of male Wistar strain rats at 1, 5, 10, 20, 28, 35, 45 and 59 days of
age. The method employed involved mouse monoclonal antibodies against rat
bNOS in combination with tyramide signal amplification (CSA). The intensity of
the reaction in the organs studied was determined using computer software to
demonstrate the optical density of the reaction product obtained. In the epididymis
a weak reaction was observed in the connective tissue/muscular sublayer
on the 28th and 45th days of life. In the seminal vesicle and ventral prostate
a positive reaction appeared in the epinuclear portions of glandular epithelial
cells on the 20th day of life, reaching a maximum intensity on the 28th day and
thus before the rats reached maturity. The results obtained allow the conclusion
to be drawn that nitric oxide resulting from bNOS-activity participates in the
processes of differentiation and of function in the epididymis, seminal vesicle
and ventral prostate

A, Konwerska

A, Limanowski

B, Miśkowiak

M, Partyka

English

Via Medica Journals

Folia Morphol. Vol. 63, No. 2, pp. 189–194Copyright © 2004 Via MedicaISSN 0015–5659www.fm.viamedica.pl O R I G I N A L  A R T I C L E189Immunohistochemical studies on brain nitricoxide synthase (bNOS) in the male genitalaccessory glands of the rat during postnataldevelopmentAndrzej Limanowski1, Bogdan Miśkowiak1, 2, Małgorzata Partyka1, Aneta Konwerska11Chair and Department of Histology and Embryology, University of Medical Sciences, Poznań, Poland2Chair of Optometry and Biology of the Visual System, University of Medical Sciences, Poznań, Poland[Received 3 October 2003; Revised 30 December 2003; Accepted 10 January 2004]The aim of the study was to investigate the presence, localisation and functionof brain nitric oxide synthase (bNOS) in the male genital accessory glands of ratsin the course of their postnatal development. Localisation of the bNOS wasimmunocytochemically investigated in the epididymis, seminal vesicle and ven-tral prostate of male Wistar strain rats at 1, 5, 10, 20, 28, 35, 45 and 59 days ofage. The method employed involved mouse monoclonal antibodies against ratbNOS in combination with tyramide signal amplification (CSA). The intensity ofthe reaction in the organs studied was determined using computer software todemonstrate the optical density of the reaction product obtained. In the epid-idymis a weak reaction was observed in the connective tissue/muscular sublayeron the 28th and 45th days of life. In the seminal vesicle and ventral prostatea positive reaction appeared in the epinuclear portions of glandular epithelialcells on the 20th day of life, reaching a maximum intensity on the 28th day andthus before the rats reached maturity. The results obtained allow the conclusionto be drawn that nitric oxide resulting from bNOS-activity participates in theprocesses of differentiation and of function in the epididymis, seminal vesicleand ventral prostate.Key words: accessory gland, epididymis, seminal vesicle, prostate,nitric oxide synthase, NOS, postnatal developmentAddress for correspondence: Prof. Bogdan Miśkowiak, Department of Histology and Embryology, University of Medical Sciences,ul. Święcickiego 6, 60–781 Poznań, Poland, tel: +48 61 869 91 81, w. 546, fax: +48 61 865 81 30, e-mail: bmiskow@usoms.poznan.plINTRODUCTIONDuring the last 20 years of the last century nu-merous studies appeared which demonstrated theimportant role of nitric oxide (NO) in the control ofseveral vital functions in the mammalian body.In the endocrine system, NO is supposed to beresponsible for the control of hormonal secretion by,for example, the adrenal cortex. Nitric oxide has beenestablished as an important messenger molecule invarious aspects of brain physiology, from develop-ment to synaptic plasticity, learning and memory [4].In the peripheral nervous system it acts as a neu-rotransmitter. However, NO has also been viewed asa major agent of neuropathology [4].Bredt and Snyder [2] are of the opinion that NOrepresents an unconventional new generation neu-rotransmitter. It is not present in synaptic vesicles,does not act through receptors and is secreted byneurons as if, on request, facilitating communica-tion between neurons in the central nervous system.As is generally recognised, NO is formed fromL-arginine as a result of involvement of the enzyme190Folia Morphol., 2004, Vol. 63, No. 2nitric oxide synthase (NOS) in the presence of NADHas a co-factor. Three isoforms have been distin-guished, depending on their origin: bNOS (nNOS,ncNOS, type I NOS), eNOS (ecNOS, type III NOS) andiNOS (mNOS, type II NOS). NOS isolated from thecerebellum, bNOS, and that from the vascular en-dothelium, eNOS, are considered constitutively ex-pressed proteins. Constitutive NOS is regulated byCa++ and releases NO within a short time. InducibleNOS is thought to be induced by cytokines, is inde-pendent of Ca++ and releases NO for prolonged pe-riods of time. Such an isoform of NOS may be in-duced in macrophages [2, 14].Nitric oxide has been proved to be one of the mostimportant non-adrenergic, non-cholinergic mediatorsin the control of human reproductive organs [16]. Inthe reproductive tract, NO originates from the endot-helium, glandular epithelium and from neurons. In thevascular system, nitric oxide acts on smooth musclesof the blood vessels and controls blood pressure andblood flow through the vessels. NO from nerve fibresinside the muscles and from neuronal subepithelial plex-uses probably participates in the contractility of thesmooth muscles in blood vessel walls. The subepi-thelial localisation of NOS is also supposed to affectsecretory processes in a neuronal way [3, 6]. In theepithelium of the epididymis, NO may control itssecretory activity and in this way affects sperm mat-uration and its displacement [18]. In the seminalvesicle, NO is known to cause smooth muscle relax-ation by activating guanylate cyclase and increasingcGMP levels in the smooth muscle cells [2]. NO ofthe prostate epithelium probably influences auto-crine or paracrine processes [6].NOS is also detected in the organs of the malesexual system in various species. Activity of the en-zyme was demonstrated in the rat epididymis [3, 7]and in the prostate of the rabbit [1], dog [5] andman [10]. Slight activity of the enzyme has also beendescribed in the human seminal vesicle in the courseof postnatal development [11].We have not encountered any data in the avail-able literature on alterations in bNOS activity in thegenital tract of the male rat in the course of postna-tal development. Clarification of this matter is theaim of the present study.MATERIAL AND METHODSThe studies were performed on male Wistar strainrats housed in stable conditions at a temperature of20 ± 2oC with a light cycle of 10 L – 14 D and allowedfree access to chow and water. On days 1, 5, 10, 20,28, 35, 45 or 59 of life the rats were sacrificed by ex-sanguination under ether anaesthesia. The epididymis,ventral prostate and seminal vesicle were weighed, thefragments fixed in Bouin’s solution and then embed-ded in paraffin. Each of the time points to be analysedin rat life was represented by 6 animals.Immunocytochemical tests were performed onparaffin sections for the presence of brain nitric ox-ide synthase (bNOS). The method employed involvedmouse monoclonal antibodies against rat bNOS(clone NOS-B1, Sigma-Aldrich), diluted 1:1000, incombination with tyramide signal amplification (CSA,Catalyzed Signal Amplification System, DAKO). A re-combined fragment of NOS (a.a. 1–181) originatingfrom a rat brain served as an immunogen to triggerproduction of the antibodies. In the protocol for bNOSthe producer of the antibody (Sigma-Aldrich) excludedinteraction of this antibody with NOS originating frommacrophages (mNOS) or with NOS present in endot-helial cells (eNOS). As a negative control for the immu-nocytochemical reactions performed, a procedure wasused in which monoclonal IgG protein included in theCSA kit was substituted for specific anti-bNOS antibod-ies. In each age group the best 1–2 preparations wereselected, on each of which 2 to 10 areas were marked,giving a total area of 0.096 to 0.48 mm2.The intensity of staining of bNOS-immunoposi-tive areas as a reflection of the brown stain in thecytoplasm per cell (= mean optical density) wasevaluated under ¥ 350 magnification. Computersoftware was employed for automatic image anal-ysis via a Image-Pro Plus and Nikon Eclipse E600microscope equipped with a chromatic TV CCDcamera. From the mean optical density of thebNOS-immunopositive areas was subtracted themean optical density of the negative control, ob-tained with the use of IgG on the same microscopeslide on which the immunocytochemical test forbNOS was performed. After the mean optical densityof bNOS-immunoreactive areas had been measured,the slides with bNOS sections were stained with hae-matoxylin to demonstrate the cell nuclei, thereby fa-cilitating analysis of the histological patterns.The results obtained were statistically analysedusing Student’s t test.RESULTSThe immunocytochemical studies of bNOS in theepididymis revealed a weak reaction, which appearedonly on the 28th and 45th days of life in the nervefibres of the connective tissue/smooth muscle sub-layer (Fig. 1). The optical density values of bNOS were191Andrzej Limanowski et al., bNOS in male rat genital accessory glandslow and were noted in the same compartment inthe two groups of animals.In the seminal vesicle a weak reaction developedin the epinuclear zone of cells of the glandular epithe-lium on the 20th day of life. Its mean optical densityreached peak values on day 28 and remained almostunchanged until day 35. The minimal value of thisparameter occurred in the 45-day-old animals. The levelof mean optical density of bNOS-immunopositive ar-eas reached its higher value later, on day 59 (Fig. 2). In the prostate bNOS was observed in the epi-nuclear zone of the cells of the glandular epitheli-um beginning on the 20th day of life and the opticaldensity was lowest at that time. The mean opticaldensity in the prostate reached its maximum inten-sity on the 28th day. Later, the mean optical densityof the immunopositive reaction for bNOS variedwithin a narrow range but always remained higherthan that found on the 20th day of life (Fig. 3).Changes in the mean optical density of the prod-uct of the immunocytochemical reaction for bNOSactivity in the genital accessory glands of male ratsduring their postnatal development are illustratedin Figure 4.DISCUSSIONIn these studies on the immunocytochemical lo-calisation of bNOS in the course of postnatal devel-opment, a weak reaction for the enzyme was observedin the epididymis. In 28- and 45-day-old rats the re-action was localised to the nerve fibres of the con-nective tissue/smooth muscle sublayer. No bNOS ac-tivity could be found in cells of the glandular epithe-lium. This specific localisation is consistent with thefindings of other authors, who have observed NOS-activity in noradrenergic nerve fibres in the sublayerof the epididymis of rats [7] and humans [10]. Theseauthors concluded that NOS and its product, nitricoxide, are responsible for the control of smooth mus-cle function in the epididymis of both species.Wiszniewska et al. [18] used immunocytochemicaltechniques to demonstrate iNOS activity in epinucle-ar portions of cells in the glandular epithelium in cul-ture or in organ fragments. It has been suggestedthat the nitric oxide produced affects the sublayer ofthe rat epididymis in the course of sperm passageand provides nutrients for sperm. Ventura and Burn-stock [17], using polyclonal antibody, observed en-hanced expression of bNOS in the course of post-natal development in the rat epididymis. In ratsboth of 28–35 and of 150–180 days of age all sec-tions of the cauda part of this organ showed virtu-ally no NOS-immunoreactive nerve fibres in thesmooth muscle layers. Only sections of the epididy-mis cauda taken from 24-month-old rats showed sig-nificantly increased bNOS-immunoreactivity. Burnettet al. [3] used rabbit antibodies to the whole rat cer-ebellar NOS protein to demonstrate nitric oxide syn-thase in nerve fibres of the sublayer and, in parallel,in endothelial cells of the epididymis as well as in theapical and perinuclear portion of the canalicular epi-thelium. NOS-activity in the rat epididymis was 7 timeshigher in the rat epididymis as compared to the semi-nal vesicle, while in humans the reverse was found,namely that NOS activity could be termed as negligi-ble in the epididymis, while in the seminal vesicle itwas high [3, 8].Ventura and Burnstock [17] and Burnnett et al.[3] showed that NADPH-d localisation, except forsmall staining differences, yielded similar results toNOS-immunohistochemistry in the nerve fibres andepithelium of the epididymis. Uckert et al. [16] re-ported a poor correlation between NADPH-d andimmunocytochemical staining of nNOS in the hu-man seminal vesicle.In our investigations on the rat seminal vesiclea positive reaction for bNOS appeared in the epinu-clear zone of the glandular epithelium cells in the20th day of life, while maximum optical density ofthe reaction was noted in the 28th and 35th days oflife. In the adult rat Burnett et al. [3] detected onlya weak reaction for NOS and did not indicate its lo-calisation. Immunocytochemical studies on NOS inthe seminal vesicle of 2-month-old to 3-year-old boysobtained at post-mortem examination detected weakbNOS-activity in the subepithelial nerve fibres andin noradrenergic nerve fibres linked to smooth mus-cles [11]. The data seem to suggest that nitric oxideparticipates in the autonomic control of organ func-tion. Moreover, immunocytochemical studies on NOSin nerve fibres of the seminal vesicle in human foet-uses detected an immunopositive reaction beginningat the 23rd week of pregnancy and the reaction wassignificant in the postnatal period [6].In the prostate gland bNOS-activity appeared onthe 20th day of life, while its localisation and day ofmaximal intensity resembled those observed in theseminal vesicle. According to Aikawa et al. [1], nitricoxide in the rabbit prostate was thought to inducerelaxation of the smooth muscles.Changes in NOS in the genito-urinary system havealso been examined in human foetuses between the13th and 30th weeks of intrauterine life [6]. A positivereaction in the prostate was detected as early as the192Folia Morphol., 2004, Vol. 63, No. 2Figure 1. Epididymis of a 28-day-old rat. Weak reaction for brain nitric oxide synthase in the nerve fibres of the connective tissue/smoothmuscle sublayer. Scale bar 24 mm.Figure 3. Rat prostate: A. 20-day-old rat. Weak reaction for brain NOS-immunoactivity in the glandular epithelium; B. 28-day-old rat;C. 35-day-old rat. Evident increase in bNOS-immunoactivity in the glandular epithelium. Scale bar 24 mm.Figure 2. Rat seminal vesicle: 28-day-old rat (A) and 35-day-old rat (B). Strong reaction for bNOS-immunoactivity in the glandular epithe-lium; C. 45-day-old rat. Weak reaction for brain nitric oxide synthase in the glandular epithelium. Scale bar 24 mm.193Andrzej Limanowski et al., bNOS in male rat genital accessory glands13th week of intrauterine life, while such a reactiononly developed between the 23rd and 30th weeks ofintrauterine life in the seminal vesicle and epididy-mis. The reaction was localised to nerve fibres sup-plying the smooth muscles and was also demonstrat-ed in cells of the human glandular epithelium [6].Constitutive NOSs (nNOS and eNOS) are expressedin both normal and hyperplastic human prostate andboth are expressed in epithelial cells [9].The appearance of a bNOS-specific reaction inthe seminal vesicle and prostate in the 20th day oflife and the development of maximum optical den-sity of the reaction on day 28 found in the presentstudy deserve attention. The reaction in the epididy-mis occurs slightly later, appearing in the 28th day oflife. The maximum intensity of the reaction for bNOSactivity markedly precedes the time when the ratsreach sexual maturity.In our previous morphometric studies on the ratmale reproductive system during postnatal develop-ment we have shown that in their histological pat-terns and in morphometric indices rats reach a dif-ferentiation status similar to that observed in adultanimals before they attain sexual maturity [15].Manifestation of  bNOS-activity in epinuclear partsof the glandular epithelium cells of the seminal ves-icle and prostate corresponds to the localisation ofcholecystokinin (CCK-8) and of bombesin observedin our earlier immunocytochemical studies [12, 13].To sum up, the localisation and activity of bNOSprior to the rats reaching sexual maturity may suggestthat synthesised NO affects not only the function ofaccessory genital glands and particularly their smoothmuscles, but also the growth and differentiation ofthe epididymis, seminal vesicle and ventral prostate.ACKNOWLEDGEMENTSWe sincerely thank Prof. Geoffrey Shaw for hisrevision of the English version of the manuscript.REFERENCES1. 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Immunohistochemical studies on brain nitric oxide synthase (bNOS) in the male genital accessory glands of the rat during postnatal development

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Immunohistochemical studies on brain nitric oxide synthase (bNOS) in the male genital accessory glands of the rat during postnatal development

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