Ultrastructural localization of alkaline and acid phosphatases in the human fallopian tube epithelium during the menstrual cycle.код для вставкиСкачать
THE ANATOMICAL RECORD 203:67-72 (1982) Ultrastructural Localization of Alkaline and Acid Phosphatases in the Human Fallopian Tube Epithelium During the Menstrual Cycle ELLA S. LINDENBAUM, DVORAH BEACH, AND BEZALEL A. PERETZ Department of Morphological Sciences and Department o f Obstetrics and Rambam Medical Center, Faculty o f Medicine, Technion, Gynecology “B’: Haifa, Israel ABSTRACT Ultrastructural and cytochemicalmethods were utilized to study the human Fallopian tube fimbrial epithelium during the different stages of the menstrual cycle. Alkaline phosphatase reaction product was located along the apical and lateral plasma membranes of the secretory cells only, regardless of the stage of the cycle. The ciliated cells were almost devoid of any reaction product at all stages of the cycle. Acid phosphatase reaction product depicted the lysosomes. These appeared as electron-dense bodies, of almost equal numbers in the ciliated and the secretory cells at all stages of the cycle. Thus the number of lysosomes did not vary appreciably during the different stages of the menstrual cycle. Many lipid droplets were found in both cells; these were rimmed by acid phosphatase reaction product, and some were partially enveloped by electron-densebodies containing acid phosphatase deposits. Acid phosphatase deposits were also found on the inner face of Golgi vesicles. Evidence of morphologic differences in the secretory and ciliated cells of the Fallopian tube epithelium is amply supplied in the literature (Rhea et al., 1974; Brinkworth, 1978). In addition, ultrastructural studies on the alterations associated with the different physiologic stages of the menstrual cycle by Patek et al. (1972), Hashimoto et al. (1964),and Verhage et al. (1979)confirm the reported ebbing and waning of this epithelium as a function of the fluctuations in serum hormonal levels. This fact was elucidated by histochemical studies on human tissue made at the light microscopic level by Fredricsson (1959). The function of the alkaline phosphatase in the cell membrane is not completely understood even though its capacity to participate in dephosphorylation and transphosphorylation reactions is well accepted. Therefore, their presence or absence may act as an indicator of the activity of the cell membrane. Acid phosphatase reaction product is present in the lysosomal system, indicating the presence of hydrolytic enzymes. Until now, alkaline and acid phosphatase reactions have not been demonstrated at the ultrastructural level in human Fallopian tubes, although simi- 0003-276X182/2031-0067$02.000 1982 Alan R. Liss, Inc. lar studies have been made on rat and cow (Bjorkman and Fredricsson, 1960; Ono, 1974). I t seems that there is a need to ascertain whether the enzymatic activity reported during the estrous cycle exists in the menstrual cycle as well. This study is novel in its combination of (a) usage of nonpathologic human materiai; (b) light and electron microscopic localization of alkaline and acid phosphatases; and (c)correlation of the enzyme presence or absence with the phases of the menstrual cycle. MATERIALS AND METHODS Microbiopsy of fifty-four Fallopian tubes fimbrial epithelium as well as endometrial samples were obtained from patients undergoing hysterosalpingectomy operations for surgical indications. All patients were of reproductive age, ranging from 29 to 49. All cases of tubal or endocrine pathologies were eliminated from this study. Dating of the menstrual cycle was based on (a) endometrial biopsy histological examination of paraffin sections stained with hematoxReceived May 12.1981; accepted November 13,1981 68 E.S. LINDENBAUM, D. BEACH. AND B.A. PERETZ ylene and eosin, which were dated according to Noyes et al. (1950);(b) last menstrual period; and (c) radioimmunoassay of estrogen and progesterone serum levels on the day of the operation. Criteria for dating were based on hormonal ranges reported by Moawad and Hafez (1980). For alkaline and acid phosphatases localization, the tissues were fixed in 1.5%gluteraldehyde for 45-60 minutes at room temperature, rinsed in cacodylate buffer 0.1M containing 7.5% sucrose a t p H 7.4 three times for ten minutes each, and washed overnight in cacodylate buffer containing 7.5% sucrose. The tissues were then incubated with and without the respective substrates /3-glycerophosphate in tris buffer containing lead nitrate at pH 9.0 for alkaline phosphatase (Matsugawa and Anderson, 1971) and cytidine-5-monophosphoricacid in acetate buffer containing lead nitrate at pH 5.0 (Willingham and Yamada, 1978) for acid phosphatase. Incubation lasted one hour at 37°C; the tissues were then transferred to a 1% ammonium sulfide solution to stabilize the reaction and to observe the reaction product in the light microscope, for three minutes, rinsed and postfixed in osmium tetroxide solution, dehydrated, embedded in epon 812, sectioned on an LKB I11 Ultramicrotome, and observed and photographed on the JEOL EM 100B, operating a t 80 kV. Some sections were stained with uranyl acetate and lead citrate. Observations were made on unstained and stained sections. Semi-thin l y sections of all specimens were stained with toluidine blue and observed with the light microscope. Lysosomal counts were made with the light microscope. Lysosomes of ciliated and secretory cells of each specimen were counted, and averages and standard deviations were calculated. RESULTS Dating of the cycle Endometrial histology of the fifty-four cases confirmed the stage of the cycle reported. Measurements of serum estrogen and progesterone levels were correlated with the histological observations on the endometrial biopsies and with the last reported menstrual cycle. Only twelve valid determinations were obtained for E, and sixteen valid determinations were obtained for progesterone (see Table 1). Alkaline phosphatase Observations under the light microscope (Fig. 1)revealed alkaline phosphatase reaction product delineating the apical plasma membrane of the secretory cells only. No reaction product was found along the ciliated cell membranes. This observation was further confirmed by EM examination where the alkaline phosphatase reaction product appeared mostly along the apical cell membrane, while the ciliated cell membranes were almost devoid of any reaction product (Figs. 2, 3). These appeared as discrete deposits scattered along the apical membrane including the microvilli. In addition to the delineation of the luminal surfaces, and depending upon the degree of penetration of the reaction substrate into the tissue, the lateral membranes were also delineated by the alkaline phosphatase reaction product (Fig. 4). The junction points between a secretory and an adjacent ciliated cell exhibited an abrupt transition in which deposits of the reaction product were present on the secretory cell membrane and absent on the ciliated cell membrane (Fig. 3). The presence and location of the alkaline phosphatase reaction product were independent of the phase of the menstrual cycle. Acid phosphatase Acid phosphatase reaction product was observed a t the light and electron microscopic level within both the secretory and ciliated cell lysosomes. These included both demiluneshaped electron-dense bodies as well as round electron-dense bodies, which were located mostly in the apical part of the secretory and ciliated cells (Figs. 5a,b,c); their sizes varied. No morphologic difference was observed in the lysosomes of the secretory as compared with those of the ciliated cells. Some acid phosphatase reaction product was detected on the inner face of the Golgi vesicles (Fig. 6). Many electron-lucid lipid droplets were found in both types of cells, the peripheral circumference of which was rimmed by acid phosphatase reaction product. Some of the lipid droplets were partially enveloped by demiluneshaped bodies containing acid phosphatase (Figs.5b,c, and 6).The acid phosphatase deposits appeared as dense aggregates within a granular matrix (Fig. 5b). In some cases, electronlucid vesicles were surrounded by the reaction product (Fig. 5c). The impression that an almost equal number of lysosomes were found in ciliated and in secretory cells a t all phases of the cycle was confirmed by lysosomal counts. This observation was made on semi-thin sections of the same material which was examined with the electron microscope, with the light microscope (Table 2). The intensity of the reaction product dif- ULTRASTRUCTURAL LOCALIZATION OF PHOSPHATASES IN FALLOPIAN TUBE 69 Fig. 1. Photomicrograph of Fallopian tube fimbrial epithelium ohtained on the 11th day of the menstrual cycle. Note the selective delineation of the secretory cell apical membrane by the alkaline phosphatase reaction product. Complete absence of the reaction product on the ciliated cell membrane and cilia is apparent. x 1,033. Fig. 3. Electron micrograph of ciliated and secretory cells of Fallopian tube fimbrial epithelium obtained on the fifth day of the menstrual cycle. Alkaline phosphatase reaction product appears as discrete deposits scattered along the apical membrane of the secretory cells. The ciliated cell reveals glycogen rosettes (gl).x 23.500. Fig. 2. Electron micrograph of Fallopian tube fimbrial epithelium obtained on the fifth day of the menstrual cycle. Alkaline phosphatase reaction product delineates the apicaI cell membranes and their microvilli in the bulging secretory cell (S)only. Adjacent ciliated cell (C)membranes are devoid of any reaction product. x 7.500. Fig. 4. Electron micrograph of secretory cells of the Fallopian tube fimbrial epithelium obtained on the 27th day of the menstrual cycle. Alkaline phosphatase reaction product delineates the apical and lateral plasma membranes of thecells. x 8.400. 70 E.S. LINDENBAUM, D. BEACH, AND B.A. PERETZ Fig. 5. Electron micrographs of secretory cells of Fallopian tube fimhrial epithelium. (a) Day 26 of the menstrual cycle. Note the lysosomes (L)in which acid phosphatase reaction product appears condensed a s aggregates. x 56,000. (h) Day 10 of the menstrual cycle. Note acid phosphatase reaction product aggregates within an electron-lucid matrix of the lysosomes enveloping lipid droplets (LD)scattered in the apical cytoplasm. Note small, round vesicles surrounded by the reaction product (arrow).x 70,000. (c)Day 13of the menstrual cycle. Acid phosphatase reaction products appear as discrete aggregates within the matrix of lysosomes enveloping lipid droplets (LD) and aggregating in small, round vesicles (arrow). x 42,000. Fig. 6. Electron micrograph of secretory and ciliatedcells of Fallopian tube fimhrial epithelium on the 13th day of the menstrual cycle. Acid phosphatase reaction product can he detected along the inner face of the Golgi (G)vesicles of the secretory cell, in lysosomes, some of which envelop lipid droplets. x 16,800. ULTRASTRUCTURAL LOCALIZATION OF PHOSPHATASES IN FALLOPIAN TUBE 71 T A B L E 1. Correlation of day of cycle with serum E I (pglrnl)and progesterone (ng/ml)levels and with endometrial dating. Day of cycle: Estrogen: Progesterone: Endometrial examination: 7 8 9 12 13 17 19 20 13 14 20 22 27 28 33 28.0 88.0 195.0 81.0 186.0 - 257.0 21.0 37.0 64.0 42.0 52.4 71.0 0.70 0.38 0.74 0.38 0.25 0.80 0.52 0.31 0.78 4.90 1.56 18.20 2.30 2.90 2.40 2.80 2 - Proliferative phase -Luteal phase T A B L E 2. Average lysosome count per cell. Cycle Phase Ciliary cells Proliferative phase Luteal phase 17.56 + 3.24 16.05 f 4.07 fered from that of the osmiophic substances as compared with those incubed without the substrate, and therefore the two could be readily distinguished. DISCUSSION Our results demonstrate the expected correlation between the three criteria used for dating of the menstrual cycle. Both the histochemical examination of the endometrium and the radioimmunoassay levels of estrogen and progesterone confirmed the date of the last menstrual period. The morphologic observations made at the light microscopic level were further elucidated at the electron microscopic level. The localization of the enzymes was thus pinpointed within specific organelles of the epithelial cells. In spite of the short fixation time and the low concentration of gluteraldehyde (1.5%)in the fixative solutions which were dictated by the technique for optimal enzyme preservation, the cell organelles were sufficiently preserved to illustrate the presence of enzymes in them. Alkaline phosphatase Processes associated with cytoplasmic membrane alkaline phosphatase are mostly transphosphorylating and dephosphorylating reactions, and may be concerned with membrane transport systems and metabolic maintenance of the membrane structure. The alkaline phosphatase reaction product was found along the apical and lateral membranes of the secretory cells during all phases of the cycle. This fact agrees with reports on the localization of alkaline phosphatase during the rat estrous cycle (Ono, 1974).The stage of the estrous cycle had no effect on the enzyme. Fredricsson (1959)showed the same results in a histochemical study made on human tissues at the light microscopic level. In a biochemical Secretory cells 14.35 14.98 f f 3.45 3.03 and cytochemical study on the localization of ATPases (Rhea et al., 1974; Nayak and Wu, 1975)it was demonstrated that the luminal surface of the secretory cell was the prime source of these enzymes, and their presence was estrogen- and progesterone-dependent. Since in our results the localization of alkaline phosphatase did not show variation during the menstrual cycle, we may be observing a different group of enzymes. Our study finds the alkaline phosphatase in the exact same location as t h a t of the ATPases. The fact that in some cases the lateral membranes did not exhibit alkaline phosphatase presence is probably due to variability in penetration of the substrate. Acid phosphatase The lysosomes were observed within both the secretory and the ciliated cells. Lysosomes contain acid hydrolases such as acid phosphatases, P-glucuronidase, nuclease, and cathepsin. According to Clyman (1966),an increase in lysosome number indicates that an autolytic process related to cellular turnover is occurring. Weissman (1964)reported that pyrogenic steroids such as progesterone activate the lysosomal system. Therefore it seemed plausible to do lysosomal counts and correlate them with progesterone elevation. However, our results do not show significant differences in the lysosomal counts related to hormonal levels. The above observations demonstrate that (a) alkaline phosphatase activity is present in the secretory cell apical and lateral membranes throughout the menstrual cycle;(b)the number of lysosomes containing acid phosphatase were found not to be related to t h e stage of the menstrual cycle; and (c) the number of lysosomes containing acid phosphatases in the ciliated cells was almost equal to those in the secretory cells. 72 E.S. LINDENBAUM, D. BEACH, AND B.A. PERETZ traception. 2nd Edition. E.S.E. Hafez, ed. Harper and Row. New York, pp. 201-220. Nayak, R.K. and S.H. Wu (1975)Fine structural localization of adenosine tri-phosphatase in epithelium of the rabbit oviduct. J. Anim. Sci. 4(4):1077. LITERATURE CITED Noyes, R.W., A.T. Hertig, and J. Rock (1950) Dating the endometrial biopsy. Fertil. Steril. 1(1):3. Bjorkman, N. and B. Fredricsson (1960)The ultrastructural organization and alkaline phosphatase activity of the epi- Ono, K. (1974) Fine structural localization of alkaline phosphatase activity of the female genital canal surface during thelial surface of the bovine Fallopian tube. 2. Zellforsch. the estrous cycle and after castration in rats. Acta Anat. 5:589. 89:577-590. Brinkworth. R.I. (1978) On the localization of LDH in the Patek, E.. L. Nilssonand E. Johannison(l972)Scanningelecovaries and reproductive tractsof the rats and mice. Mech. tron microscopic study of the human Fallopian tube. Ageing Dev. 8(5):299. Report I. The proliferative and secretory stages. Fertil. Clyman, M.J. (1966) Electron microscope studies of human Steril. 23r459. Fallopian tube. Fertil. Steril. 17r281. Rhea, R.P., B. Anderson, N.B. Kim, and M.D. Rosenberg Fredricsson. B. (1959) Histochemical observations on the (1974) Biochemical, electron microscopic and cytochemiepithelium of human Fallopian tubes. Acta Obstet. Gynec. cal studies of ATPase localization in avian, murene and huScand 38:109. man oviducts. Fertil. Steril. 25(9): 788. Hashimoto. M., T. Shimoyama, M. Kosaka. A. Komori, T. Verhage, H.G., M.L. Bareither. R.C. Jaffe, and M. Akbar Hirasawa. Y. Yokoyama, N. Kawase, and T. Nakamura (1979)Cyclic changes in ciliation secretion and cell height (1964)Electron microscopic studies on the epithelial cells of the oviductal epithelium in women. Am. J. Anat. of the human Fallopian tube. J. Jpn. Obstet. Gynec. SOC. 156:505-522. 11.92. Matsugawa, T. and C. Anderson (1971)Phosphatases of epi- Weissman, G. (1964) Labilization and stabilization of lysosomes. Fed. Proc. 23:1009. physeal cartilage studies by electron microscopic cytoWillingham. M.C. and S.S. Yamada (1978)A mechanism for chemical methods. J. Histochem. Cytochem. 19:801. the destruction of pinosomes in cultured fibroblasts. J. Moawad, A.H. and E.S.E. Hafez (1980)Theoviduct and egg Cell Biol. 78:480. transport. In: Human Reproduction, Conception and ConACKNOWLEDGMENT This research was supported by Technion grant 180-326.