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Ultrastructural localization of alkaline and acid phosphatases in the human fallopian tube epithelium during the menstrual cycle.

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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.
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