Localization of 5-hydroxytryptamine-like immunoreactive cells and nerve fibers in the rat female reproductive system.код для вставкиСкачать
THE ANATOMICAL RECORD 233:478-484 (1992) Localization of 5-H ydroxytryptamine-Like Immunoreactive CelIs and Nerve Fibers in the Rat Female Reproductive System F. AMENTA, J.A. VEGA, A. RICCI, AND W.L. COLLIER Lab, Neuromorfologia e Neuropatologia, Dipartimento di Sanita Pubblica e Biologia Cellulare, Universita “Tor Vergata,” 00173 Roma, Italy (F.A.);Departamento de Morfologia y Biologia Celular, Universidad de Oviedo, 33006 Oviedo, Spain (J.A.V.); Dipartimento di Scienze Cardiovascolari e Respiratorie, Universita “La Sapienza,” 00161 Roma, Italy (A.R.); Department of Science, Manatee Community College, Venice, Florida 34292 (W.L.C.) ABSTRACT The presence of 5-hydroxytryptamine (5-HTl-like immunoreactivity (IR) was studied in the rat female reproductive system usin; polyclonal antibodies directed against 5-HT. Moreover, 5-HT levels in the ovary, oviduct, uterus, and cervix were measured by high-pressure liquid chromatography with electrochemical detection. The highest 5-HT concentrations were found in the oviduct, followed in descending order by the cervix, the ovary, and the uterus. Most 5-HT-like IR was observed in the cytoplasm of mast cells. These cells were found in the connective tissue around the fimbria, in the oviduct, in the uterus, and in the ovary. Mast cells are clustered in the proximity of the parenchymal blood vessels. Moreover, a few 5-HT-like nerve fibers were found distributed mainly perivascularily in the uterine cervix and in the uterine horns as well as in the oviduct. IR nerve fibers were rarely seen within the ovary. The present data provide direct evidence that 5-HT in the female reproductive system not only is associated with mast cells but is located in nerve fibre-like structures as well. The functional significance of this probable 5-HT-ergic innervation of the female reproductive tract discovered in the present study should be clarified in future investigations. 0 1992 Wiley-Liss, Inc. INTRODUCTION 5-Hydroxytryptamine (5-HT) is probably involved in the control of the functional activity of the female reproductive system by regulating the blood flow in uterine and ovarian arteries (Dynarowicz et al., 1988) and ovarian function (Schmidt et al., 1988). Several studies have shown, using both direct and indirect evidence, that in the female reproductive tract 5-HT is localized mainly within mast cells (Levier and Spaziani, 1966; McKercher et al., 1966). Moreover, the presence of 5HT within mononuclear phagocytes of the rat endometrium has been reported (Gunin and Gordon, 1990). The occurrence of 5-HT-like immunoreactive (IR) nerve fibers has been demonstrated in the perivascular nerves of different vascular beds (Costa et al., 1982; Dahlstrom and Ahlman, 1983; Gale and Cowen, 1988; Gale et al., 1989) where they are probably colocalized with noradrenaline in sympathetic nerve fibers (Gale et al., 1989; Cowen et al., 1986, 1987; Owman et al., 1990). Moreover, the existence of a 5-HT-ergic innervation of the gastrointestinal tract has been described (Gershon et al., 1990). However, although the presence of a serotoninergic innervation of the female reproductive system has been reported in some invertebrates (Fairweather et al., 1987; Maule et al., 19901, no information is available concerning the existence of 5-HT containing nerve fibers in the mammalian female reproductive tract. The purpose of the present investigation was to study 0 1992 WILEY-LISS, INC. the concentrations of 5-HT and the distribution of 5HT-IR structures within the rat female reproductive system using high-pressure liquid chomatrography (HPLC) with electrochemical detection and immunohistochemical techniques, resepectively. The main goal of our work was to try to correlate the findings obtained using HPLC with electrochemical detection and immunohistochemistry and to analyze the localization of tissue stores of 5-HT in the rat femal reproductive system. MATERIALS AND METHODS Animals and Tissue Treatment Twelve female Wistar rats in estrus, weighing between 250 and 300 g, were used in our experiments. Animals were sacrificed by a n overdose of ether, and the abdominal cavity was opened and the ovaries, oviducts, uterine horns, and cervix were removed and cleaned of surrounding adipose tissue. Samples were washed with ice-cold 0.9% NaCl solution to remove blood and cell debris and were treated differently for 5-HT assay or immunohistochemistry. Received August 5, 1991; accepted December 30, 1991. Address reprint requests to Dr. F. Amenta, Dipartimento di Sanita e Biologia Cellulare, Via 0. Raimondo, 8-1 00173 Roma, Italy. 5-HT-LIKE IR IN RAT-FEMALE REPRODUCTIVE SYSTEM 5-HT Assay The ovaries, oviducts, uterine horns, and cervix of six rats were weighed and homogeneized in 1 ml of ice-cold perchloric acid (0.4N). Homogenates were centrifuged for 15 min at 4”C, and the supernatant obtained was used for 5-HT assay. 5-HT was assayed by HPLC with electrochemical detection after separation of the monoamine by ion-exchange chromatography according to the procedure described by Niwa and coworkers (1984). The significance of differences in 5-HT content among the female reproductive tract portions examined was evaluated by analysis of variance (ANOVA). Analysis was performed with programs 7D and 1V of the BMDP (using a mainframe NAS 60 with rel. 5.0 of VM/SP). Power function for ANOVA was analyzed according to Pearson and Hartley (1951). 479 6-HT (ng/g tlseue) 2600 [ OU4RY 2000 1600 1000 600 lrnrnunohistochernistry The ovaries, oviducts, uterine horns, and cervix of the remaining six rats were incubated for 3 h r at 37°C in a Krebs solution containing lop4M tryptophan and 5 x lop5 M pargyline. During incubation, the Krebs solution was bubbled with 95% oxygen and 5% carbon dioxide. After incubation, the tissues were cut in small pieces and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer solution (PBS) for 1 h r a t room temperature. The fixed tissues were then washed with PBS and immersed in a 20% sucrose solution in PBS overnight. Ten-micrometer-thick sections were cut in a microtome cryostat and processed for indirect immunohistochemistry according to the method of Coons (1958). The sections were exposed to a rabbit anti-5-HT polyclonal antiserum (RIA, U.K.) diluted 1:300 in a humid chamber overnight at room temperature. After washing with PBS containing 0.1% Triton X-100 (Sigma, St. Louis, MO), sections were exposed for 1 hr at room temperature to a fluoresceine isothiocyanate-conjugated antirabbit IgG diluted 1:50. Sections were then washed, mounted in PBS-glycerol, and observed under a Zeiss fluorescence photomicroscope equipped with epiillumination. After immunofluorescence, some sections had their coverslips removed and were stained with Astrablue (Bloom and Kelly, 1960) to establish the nature of cells displaying 5-H1’-like IR. Control sections were processed in the same way using a preabsorbed antiserum (anti-5-HT antiserum M for 12 hr, prior to preabsorbed with serotonin being used) instead of the primary antibody. Upon examination, the control sections did not show any immunof luorescence. The density of 5-HT-like IR cells was evaluated by three researchers independently in a blind study. Sections were examined using a x 16 Neofluar objective and a x 1.25 optovar to obtain a final magnification of x 80. We counted the number of IR cells present in five consecutive sections of each female reproductive tract portion investigated per animal (n = 6). A structure with more than 20 5-HT-like IR cells per microscopic field was considered “very rich” ( + + + ) in IR cells. If 5-HT-like cells were <20 but >10 per field, the structure examined was considered “rich” ( + + ) in IR cells. Structures with <10 5-HT-like IR cells per field was considered “poorly” ( + ) provided with 5-HT-like IR cells. 0 Fig. 1. Levels of 5-HT in the reproductive organs of diestrous female rat determined by HPLC with electrochemical detection. Values represent the mean -t S.E.M. of two independent determinations from different animals (n = 6). a, P < 0.001 vs. oviduct; b, P < 0.001 vs. oviduct, P < 0.01 vs. cervix, P < 0.05 vs. ovary. RESULTS 5-HT Assay The 5-HT levels assayed in the four female reproductive tract portions investigated are shown in Figure 1. As can be seen, the highest 5-HT concentrations were found in the oviduct, followed in descending order by the cervix ( P < 0.001 vs. oviduct), the ovary (P < 0.001 vs. oviduct), and the uterine horn ( P < 0.001 vs. oviduct, P < 0.01 vs. cervix, and P < 0.05 vs. ovary). lrnrnunohistochernistry No positive IR was observed in the control sections (data not shown). In all examined portions of the female reproductive system, a wide distribution of 5-HTlike IR was observed in the cytoplasm of cellular elements and within nerve fiber-like structures. Cells displaying 5-HT-like IR cells could be identified as mast cells, because of their metachromatic staining with Astrablue in control sections (Fig. 2). In the ovary, the 5-HT-like IR cells were found primarily in the medulla, close to blood vessels (Fig. 3). In the different parts of the oviduct, clustered or isolated mast cells displaying 5-HT-like IR were found closely related to the blood vessels a s well as within the adventitia or the muscular layer (Fig. 3). A similar pattern of distribution was observed within the uterine horn and the cervix (Fig. 3). The relative density of mast cells in the different portions of the female reproductive tract investigated is summarized in Table 1. As can be seen, the highest number of mast cells was observed in the oviducts; a similar density was found in the cervix and in the ovaries, and the lowest density occurred in the uterine horns. In addition to the mast cells, 5-HT-like IR nerve fiber-like structures were found in the female reproduc- 480 F. AMENTA ET AL. Fig. 2. Section of rat oviduct processed for 5-HT-like immunohistochemistry (A) and subsequently stained with Astrablue (B). The mast cell nature of cellular elements displaying 5-HT-like IR (arrows) is confirmed by their positive Astrablue staining (arrows). Bar = 25 km. cells in the female reproductive system has been documented (Levier and Spaziani, 1966; McKercher et al., renroductive tract' 1973; Juorio et al., 1989). In the present study, we obRelative density of IR cells served that mast cells with a strong 5-HT-like IR in Orpan their cytoplasm are present in the connective tissue + Ovary Cortex around the female reproductive organs and within the + Medulla ovary, oviduct, and uterus. These cells, which are pri+ + + Oviduct Muscle marily associated with blood vessels, were usually clus++ Adventitia tered together in small groups. These findings, which + Uterine horns Muscle are among the first to characterize the 5-HT content of ++ Adventitia mast cells using immunohistochemistry (for refer++ Cervix Muscle ++ Adventitia ences, see Juorio et al., 19891, are in good agreement with former investigations carried out using monoam'Values represent the relative density of IR mast cells within the ine fluorescence techniques (Levier and Spaziani, different portions of the female reproductive tract and were obtained 1966; McKercher et al., 1973). Moreover, the high senas described in Materials and Methods. sitivity of immunocytochemical techniques for the detection of 5-HT IR (for references, see Costa e t al., 1982; Owman e t al., 1990), in comparison with histochemical tive system portions examined. In the ovaries, IR nerve techniques formerly available, could be considered as fiber-like profiles were observed only rarely, in associ- indicative of the presence of 5-HT in mast cells located ation with medullary vessels but not within the cortex within the rat female reproductive tract. Analysis of (Fig. 4). In the oviducts, the highest density of 5-HT- the density of 5-HT-IR mast cells in the different porlike IR nerve fiber profiles was observed. These nerve tions of the female reproductive tract showed that the fiber-like profiles were found perivascularly, in the organs displaying the highest 5-HT content revealed muscular layer (Fig. 4),and rarely at the level of the by HPLC with electrochemical detection were those mucosal layer (Fig. 4).In the uterine horns and in the with the highest density of mast cells. These findings cervix, a few 5-HT-like IR nerve fiber profiles were support the suggestion that the majority of 5-HT observed perivascularly (Fig. 4)or within the myome- present in the rat female reproductive tract is associtrial tissue, without any obvious perivascular localiza- ated with mast cells (Juorio et al., 1989). Moreover, tion (Fig. 4). these data indicate the sensitivity and reliability of 5-HT immunocytochemistry for the assessment of the DISCUSSION density of 5-HT stores in peripheral tissues. The observation that the majority of 5-HT-like IR Our results provide direct evidence that in the rat female reproductive system 5-HT IR has a double lo- mast cells are located in relationship with blood vescalization within mast cells a s well a s within nerve sels suggests that 5-HT released from mast cells may fiber-like structures. As far a s we know, this is the first be involved in the regulation of local blood flow, probreport describing a n innervation of the mammalian fe- ably in particular conditions such as postpartum or postabortion, when hemostasis is of primary impormale reproductive tract by 5-HT-IR nerve fibers. The existence of a pool of 5-HT originating from mast tance. In fact, it is known that 5-HT causes either vaTABLE 1. Density and distribution of 5-HT-like immunoreactive (IR) cells in the rat female 5-HT-LIKE IR I N RAT-FEMALE REPRODUCTIVE SYSTEM Fig. 3. 5-HT-like IR cells (arrows) were found primarily perivascularly in the ovarian medulla (A), in the oviduct (C), in the uterine horn (E), and in the cervix (F).Moreover, IR cells were found within the adventitial and muscular layers of both oviduct and uterus (B-F). A few nerve fiber-like profiles (arrowheads) were also observed. a, adventitia; m, muscular layer. Bar = 50 pm. 481 482 F. AMENTA ET AL. Fig. 4. In the ovary, nerve fiber-like profiles showing 5-HT IR are associated with medullary blood vessels (A; arrowheads), whereas no immunoreactive nerve fibers were seen in the cortex or in the Graafian follicles (B). In the oviduct, the IR nerve fiber-like profiles are localized perivascularly (C, D arrowheads) within the muscle layers (C, D; arrowheads), and beneath the mucosa (C; arrowheads). In the uterine horns (E,F)and in the cerivx (G, H), a few 5-HT-like IR nerve fiber-like profiles are localized among the coats of the myometrium (E; arrowheads) and perivascularly (F; arrowheads). The same is true for the cervix (G, H), where 5-HT-like IR was found among smooth muscle (G; arrowheads) and around blood vessels. f, Graafian follicle; m, mucosa; mc, mast cells; v, blood vessels. Bar = 50 pm. 5-HT-LIKE IR IN RAT-FEMALE REPRODUCTIVE SYSTEM sodilation or vasoconstriction (for references, see Janssens and Van Neuten, 1990) and that it is involved in the regulation of blood flow through the ovarian and uterine arteries (Schmidt et al., 1988). On the other hand, it has been suggested that 5-HT participates in several reproductive activities, such as regulation of ovarian function (Dynarowicz et al., 1988). Moreover, 5-HT probably has a role during ovulation by causing follicular wall contraction, which leads to the expulsion of the oocyte from the ovary (for references, see Espey et al., 1989). In the oviduct, 5-HT is probably involved in the propulsion of the oocyte toward the uterus. In the uterus, 5-HT causes specific contractile responses or spasm (Robson et al., 1954; Holliningsworth et al., 1988, 19891, which is estrogen dependent (Campos-Lara et al., 1990). These responses are inhibited by 5-HT antagonists (Ichida et al., 1983) and are probably mediated by 5-HT2 receptors (Cohen et al., 1985). Finally, it has been hypothesized that basal levels of 5-HT in early gestational periods are necessary for conception (Acharya et al., 1990). It is probable, due to the widespread localization of 5-HTlike IR mast cells in the female reproductive tract, that 5-HT released from mast cells reaches 5-HT receptors located in the different structures of the female reproductive tract. Besides the mast cell pool, 5-HT-like IR has been found in nerve fiber-like structures. Although the existence of perivascular 5-HT-containing nerve fibers has been demonstrated in the cerebral, hindlimb, and mesenteric vascular beds (for references, see Griffith, 1988; Gale and Cowen, 1988; Gale et al., 19891, and a 5-HT-ergicinnervation of the gut has been documented (Gershon et al., 19901, the occurrence of 5-HT-like IR nerve fibers in the female reproductive tract of mammals has not been described previously. This is the first observation of a probable innervation of the female reproductive organs by 5-HT-like IR-containing nerve fibers. The localization of 5-HT-like IR nerve fiber profiles both perivascularly and between smooth muscle in the oviduct and in the uterus suggests their probable involvement in the regulation of blood flow in the female reproductive tract and in the activity of oviductal and uterine smooth muscle. Further studies are in progress to assess the nature, the source, and the occurrence in other mammals, and the functional significance of the 5-HT-like IR nerve fibers discovered in the present study. ACKNOWLEDGMENTS This study was supported in part by a grant from the Italian National Research Council (C.N.R., 90.03272 and 91.00687.CT 04) to F.A. J.A.V. was supported by a travel grant from the Italian C.N.R. LITERATURE CITED Acharya, S.B., N.G. Goswami, and P.K. Debnath 1990 Uterine and placental 5-HT profile in different gestational period of albino rats. Indian J. Exp. Biol., 27505-509. Bloom, G., and J.W. Kelly 1960 The copper phtalocyanin dye “Astrablue” and its staining properties, specially the staining of mast cells. Histochemistry, 2r48-57. Campos-Lara, G., F. Caracheo, A. Valencia-Sanchez, and H. PonceMonter 1990 The sensitivity of rat uterus to serotonin in vitro is a late estrogen response. Arch. Invest. Med. Mex., 22:71-74. Cohen, M.L., K.W. Schenck, W. Colbert, and L. 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