Cell Tiss. Res. 173, 325-334 (1976)
Cell and Tissue Research 9 by Springer-Verlag 1976
Effects of d-Propranolol and Estradiol on the Cervicovaginal Epithelium* Correlation with Adenylate Cyclase Activity Stener Kvinnsland Institute of Anatomy, University of Bergen, Bergen, Norway
Summary. The cervicovaginal epithelium of neonatal mice produces a material with specific antigenic properties (CVA) and this material is produced in increased amounts after estradiol treatment. Using a cytochemical method, estradiol treatment was shown to result in an increase of adenylate cyclase activity in the same epithelium. When d-propranolol is injected together with estradiol, the increase in CVA is inhibited, while the hormone-induced proliferation of epithelial cells is not influenced. When adenylate cyclase activity is studied under identical conditions, the estradiol-promoted increase in enzyme activity is largely counteracted by d-propranolol. These findings would suggest that Adenosine 3'5'-cyclic monophosphate (cAMP) has a role in some, but not all, estradiolmediated effects in the neonatal cervicovaginal epithelium.
Key words: Cervical epithelium - Mouse - d-propranolol - Estradiol Adenylate cyclase activity - Adenosine 3'5'-cyclic monophosphate. Introduction The role of adenosine 3'5'-cyclic monophosphate (cAMP) in the mechanism of action of estradiol is still uncertain. Conflicting evidence exists with regard to estradiol-promoted c A M P elevation in target tissues (Szego and Davis, 1967; Zor et al., 1973; Dupont-Mairesse et al., 1974; Kuehl et al., 1974). The matter is complex because the number of eosinophils and tissue levels of agents such as histamine, epinephrine, and serotonin are under estrogenic control (Spaziani and Szego, 1958; Tchernitchin, 1970). The interactions between these agents, cAMP, and estradiol in relation to target organ responses to steroid influences, are not fully understood. Dr. Stener Kvinnsland, Institute of Anatomy, Arstadvollen, Bergen, Norway * This work was supported by grants from the Norwegian Research Council for Science and the Humanities and from the Norwegian Cancer Society (Landsforeningenmot Kreft) Send offprint requests to :
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Propranolol, a beta-adrenergic blocking drug, has been shown to suppress the estradiol-induced elevation of cAMP (Szego and Davis, 1969; DupontMairesse et al., 1974). However, the estradiol-induced increase in activity of glycolytic enzymes in the uterus (Singhal et al., 1972), of increased RNA and protein synthesis (Dupont-Mairesse et al., 1974), and of increased synthesis of the Gorski protein (Notides and Gorski, 1966; Dupont-Mairesse and Galand, 1975), are not influenced by treatment with propranolol. In an earlier study undertaken in this laboratory, it was shown that in neonatal mice the amount of a material with specific antigenic properties, confined to the cervicovaginal epithelium (CVA), was increased after estradiol treatment. This increase was suppressed by treatment with d,l-propranolol (Kvinnsland, 1973), but proliferation of the vaginal epithelial cells was not affected. Cytochemical studies have demonstrated an increase in adenylate cyclase activity in the same epithelium after treatment with estradiol alone (Abro and Kvinnsland, 1974). Some results in the study by Kvinnsland (1973) indicated that the effect of d,l-propranolol was due to a membrane stabilizing influence. For this reason it was of interst to study d-propranolol which has almost no beta-blocking effect (Parmley and Braunwald, 1967) but possesses the same membrane effect as d,l-propranolol. Moreover, we wanted to investigate whether or not cytochemically demonstrable adenylate cyclase activity correlated with a decreased epithelial CVA content after d-propranolol treatment. Material and Methods Animals. All mice used belonged to a randomly bred N M R I strain. The animals were fed a standard pellet diet and given water ad libitum. Starting on day 3 after birth, one group of female animals (positive controls) received subcutaneous injections of 5 txg estradiol-17/3 (Sigma) in 0.025 ml olive oil for 3 days. The animals of the experimental group were given the same estradiol treatment and, in addition, subcutaneous injections of d-propranolol (ICI). A third group (negative controls) received the vehicles for estradiol-17/~ and d-propranolol according to the same time schedule as the animals of the experimental group. For concentrations used see Table 1. All animals were killed by decapitation on day 6 (24 h after the last estradiol injection).
Immunofluorescence Method for Demonstration of CVA. Antiserum was obtained from rabbits after immunization with vaginal epithelium from castrated adult, estradiol-treated mice. A vaginal epithelial homogenate was centrifuged for 1 h at 105,000g in a B-60 International Ultracentrifuge, the supernatant emulsified in Freund's complete adjuvant and injected into the rabbits' foot pads; for details see Forsberg and Kvinnsland (1972). The serum obtained was absorbed and treated as previously described but without further purification as the serum after centrifugation showed the same specificity as the crude lgG fraction used in earlier studies. The cervicovaginal canal from the 6-day animals was dissected out under a dissecting microscope, and frozen in a CO2-jet. Transverse sections (10 microns) were cut in a cryostat at - 2 0 ~ C. The indirect immunofluorescence method was used as described by Forsberg and Nord (1969). N o r m a l rabbit serum and phosphate buffered saline were used for the control sections. The sections were examined in a Zeiss photomicroscope equipped with an Osram High-Pressure mercury arc lamp, HBO 200W. Exciter filter: BG3 and BG12; barrier filters 44 and 50 (cut off at 500 mla). The photographs were taken on 35 m m K o d a k Tri-X Pan film 135-36. For an estimation of the a m o u n t of fluorescent material, the semiquantitative method previously described was used (Kvinnsland, 1973). Recently, Doskeland etal. (1976) have used a mixed haemagglutination technique for the demonstration of CVA. With this method which is far more sensitive, they have confirmed important
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Table 1 No. of preparation
Type of experiment
Fluorescence superficial zone
Epithelial proliferation basal zone
4
Estradiol-17fl
+ + +
Strongly increased
4
Olive oiP Parker 199
+
normal
4
Estradiol- 17 fl d-propranolol b
+
Strongly increased
" 5 gg 0.025 ml olive oil b Concentration used: 1 gg in 0.02 ml Parker 199 five times daily for 3 days, starting twelve hours before the first estradiol injection. Estradiol-17fl as in a results on CVA reported in earlier studies using the immunofluorescence technique, thereby supporting the method of semiquantitation used here.
Cytochemical Demonstration ofAdenylate Cyclase Activity. Animals from the three groups described above were used. Following decapitation the intestines were removed and the uterovaginal region was prefixed in situ by pipetting directly onto it 1% glutaraldehyde (TAAB Laboratories) in 50 mM cacodylate buffer (pH 7.4) containing 4.5% glucose. The uterovaginal region was removed, immersed in the fixing fluid (as for prefixation, see above), and cut into transverse slices with a razor blade. The fixation time was 1 h. Slices through the uterine cervix and vaginal fornices were used for the experiments. The slices were washed in several changes of cacodylate-glucose buffer. The slices were then incubated for adenylate cyclase activity in a Tris-maleate buffer (pH 7.4) containing glucose (8%), MgSO4 (2 mM), Pb (NOa)2 (2 mM), theophylline (2 mM) and with 0.5 mM ATP as substrate. In order to inhibit ATPase activity, NaF (10 mM) and ouabain (0.5 mM, Sigma) were included in the incubation medium; for details see ,~bro and Kvinnsland (1974). The following control slices for demonstration of enzyme activity were always run together with the experimental slices: 1. Slices were heated for 3 min in a boiling water bath in order to heat-inactivate adenylate cyclase activity. 2. Slices were incubated in the absence of ATP. 3. Incubation was performed after alloxan (5 mM, Sigma), an inhibitor of adenylate cyclase activity, had been added to the medium. After incubation the tissue slices were washed briefly in Tris-maleate buffer (pH 7.4), and postfixed for 1 h in 1% OsO4 (Merck) in cacodylate-glucose buffer (pH 7.4). After rinsing in buffer the slices were dehydrated in a series of cold ethanol solutions cleared in propylene oxide, infiltrated with, and embedded in Epon 812. Ultrathin sections were cut on a LKB ultramicrotome, counterstained for 15 min with lead citrate, and examined in a Siemens electron microscope IA. Terminology. The vaginal anlage in neonatal mice consists of two different parts: an anterior part, the mtillerian vagina, and a posterior part, the sinus vagina. In newborn animals, the miillerian vagina, derived from the fused miillerian ducts, has a pseudostratified epithelium. This later transforms into an epithelium consisting of a basal and a superficial zone (Forsberg and ,~bro, 1971 ; Forsberg and Abro, 1973). The former is the origin of the basal epithelial layer in the adult vaginal epithelium.
Results
Immunofluorescence Studies. T h e r e s u l t s o f t h e i m m u n o f l u o r e s c e n c e s t u d i e s a r e s u m m a r i z e d in T a b l e 1. A l l s e c t i o n s w e r e f r o m t h e a n t e r i o r p a r t o f t h e m i i l l e r i a n
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vagina. In control mice treated with vehicles only (Fig. 1B), a distinct basal zone of cells was seen in the epithelium. The superficial zone of columnar cells contained a small amount of fluorescent material in the apical portions of the cells. After estradiol treatment for three days the basal zone was higher (Fig. 1A), and a large amount of fluorescent material was seen apically in the superficial zone cells. In sections from estradiol and d-propranolol treated mice, the high basal zone was still present, but there was a substantial reduction of fluorescent material in the superficial zone (Fig. 1C).
Electron Microscope Studies. In 6-day old mice, the vaginal epithelium has a superficial zone of high columnar cells. The cells of this zone have small irregular microvilli and some m e m b r a n e - b o u n d granules are present in the apical part of the cytoplasm. The luminal surface is covered with a thin layer of filamentous material. N a r r o w intercellular spaces are seen between adjacent epithelial cells. In the basal zone the cells are small and irregular. After estradiol treatment on days 3, 4 and 5, the cells of the superficial zone develop more pronounced, club-shaped microvilli. The filamentous surface material becomes much more abundant. Large intercellular spaces develop, with extensive apical interdigitation between the cells. In the apical part of the cytoplasm are large numbers of m e m b r a n e - b o u n d granules of different sizes and with a homogeneous content. Some of them appear to extrude their content by exocytosis. The cells of the basal zone lack" such granules. When estradiol and d-propranolol are given simultaneously, no specific changes in the m o r p h o l o g y of the epithelium are seen, especially with respect to microvilli and mucous granules.
Adenylate Cyclase Activity. F o r the specificity of the cytochemical reaction, see Abro and Kvinnsland (1974). Varying amounts of lead phosphate precipitate were taken to indicate varying degrees of enzyme activity. In the present study, the lead phosphate precipitates indicating enzyme activity were found on the cell membrane of both epithelial and connective tissue cells. It was difficult to determine the precise localization of the precipitate in relation to the plasma membrane, but in a recent study, using the same technique, F a r n h a m (1975) has shown that in the slime mould Dictyostelium discoideum it is localized on the cytoplasmic side of the membrane. The patchy precipitates were far more pronounced in the superficial zone than in the basal zone. The basal cells had distinct precipitates on the cell membrane bordering the basal lamina. In the superficial zone, the luminal part of the plasma membrane had the highest activity, while in the basal zone the membranes bordering the basal lamina had the most extensive precipitates. Variations within a single
Fig. 1. A Epithelium from estradiol treated animal. CVA is seen in the superficial zone. Beneath is the high basal zone. Magnification x 900 (reduced to 90%). B Epithelium from control animal. Little CVA is found in the superficial zone. The basal zone is narrow. Magnification x 900 (reduced to 90%). C Epithelium from estradiol and propranolol treated animal. A small quantity of CVA is seen in the superficial zone. Beneath is the high basal zone. Magnification x 900 (reduced to 90%)
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animal with respect to the lead phosphate precipitate, were low. A consistent finding, however, was that some cells in the superficial zone were almost free of precipitates, but otherwise seemed to be morphologically normal. The reason for this is not known. In mice injected with vehicles only, a low but distinct enzyme activity was seen, with regional differences in activity as described above (Fig. 2C). The basal activity in 6-day old normal mice was higher than in 3-day old mice. After estradiol treatment a very prominent increase in enzyme activity was found, with the same pattern of distribution (Fig. 2A). When d-propranolol and estradiol were injected together, most of the estradiol effect on the enzyme activity was counteracted, and a pattern of precipitates was found almost identical to that seen in control animals (Fig. 2B).
Discussion
F o r a general discussion concerning the problems and pitfalls involved in the cytochemical demonstration of phosphatases, see Essnu (1973) and more specifically for adenylate cyclase activity, see Abro and Kvinnsland (1974). The method used has been questioned by Lemay and Jarrett (1975) who conclude that the method cannot be used for the demonstration of adenylate cyclase activity because the concentration of lead ions in the incubation medium would completely inhibit the enzyme. Furthermore, non-enzymatic hydrolysis of A T P giving rise to lead phosphate and cAMP was considered to be another serious drawback. However, prefixation, which has been reported to decrease the sensitivity of enzymes to lead ions (Novikoff et al., 1958), was not included in the procedure used by Lemay and Jarrett. After prefixation in 1.25% glutaraldehyde, and with 3 m M lead nitrate present during the incubation, Cutler (1975) has recently reported that 10-25% of the basal activity of adenylate cyclase is still present. Moreover, the buffers used by Lemay and Jarrett (1975) in their biochemical and cytochemical studies were not the same (Rosenthal et al., 1966). Results obtained with isolated rat cell membranes should not be compared to results from whole tissue blocks. When alloxan (adenylate cyclase inhibitor) was included in the incubation medium for our cytochemical studies (Abro and Kvinnsland, 1974), no precipitate was observed in the tissue sections. Heat-inactivation of the adenylate cyclase before incubation abolished the production of visible precipitates. Thus, in our hands non-enzymatic hydrolysis of ATP would not appear to invalidate the results. According to earlier studies on specificity (Forsberg and Kvinnsland, 1972), the fluorescent material studied represents a cell product with specific antigenic Fig. 2A-C. The luminal plasma membrane of cells in the superficial zone of the cervicovaginal epithelium. Magnification x 48,000 (reduced to 90%). A An estradiol treated animal. Heavy lead phosphate precipitates are seen in the epithelial cells. B Estradiol and propranolol treated animal. The precipitates are less marked than in the estradiol treated animal. C Control animal. Less heavy precipitates are seen
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properties characteristic for the cervicovaginal epithelium (CVA). The amount of CVA increases after estrogen stimulation; it is localized apically in the superficial columnar cells in the cervicovaginal anlage, and it is also present in the lumen. A close correlation exists regarding the localization of adenylate cyclase activity and the presence of CVA. The difference between the amount of lead phosphate precipitates in the superficial columnar cells and in the cells of the basal epithelial zone is striking, the latter having much less precipitate. This supports the view of Dupont-Mairesse et al. (1974) that a cAMP increase after estradiol treatment may be confined to a specific part of the target tissue. In biochemical studies a localized increase in cAMP level might be masked by the basal level of total cAMP content in the target organ. The experimental results point to a role of the adenylate cyclase-cAMP system in the cellular production of cytochemically demonstrable CVA (Kvinnsland, 1973). The nucleotide could be involved in synthesis, transport and secretion phenomena related to CVA (Wicks, 1974; Cerasi, 1975). The estradiolinduced increase in adenylate cyclase activity in the basal epithelial zone, which was undergoing a dramatic proliferative activity, was an unexpected finding. However, an increased adenylate cyclase activity does not necessarily mean an elevated cellular cAMP level, since this is influenced also by phosphodiesterase activity which was not studied in this investigation. The amount of guanosine -3'5'-cyclic-monophosphate increases under conditions of increased proliferative activity (Hadden et al., 1972) and the level of this nucleotide is influenced by estradiol (Kuehl et al., 1974). An inhibitory influence of cAMP as regards cell proliferation has been suggested (Anderson et al., 1973). As long as the roles of cAMP and cGMP during proliferative activity in the cervicovaginal epithelium are not known, the conclusion is that estradiol increases the histochemically demonstrable adenylate cyclase activity and that this increase is more pronounced in the superficial epithelial cells with high CVA content than in the basal epithelial zone containing proliferating cells. d,l-propranolol counteracts the estradiol-induced increase of CVA (Kvinnsland, 1973). This effect was not abolished by the simultaneous use of isoproterenol (a beta-adrenergic agonist). Since d,l-propranolol has membrane effects other than those related to the beta-adrenergic receptors (Parmley and Braunswald, 1967), the effect of d-propranolol was studied. The latter has membrane effects similar to those of d,l-propranolol but its beta-blocking effect is far less. In this study dpropranolol had the same effect as the racemic mixture which indicates that catecholamines are not involved in the estradiol-induced increase in adenylate cyclase activity. Instead this may be due to some membrane labilizing effect mediated by estradiol (Szego and Davis, 1969). In biochemical studies, Rosenfeld and O'Mallay (1970) were unable to demonstrate that d-propranolol could be used instead of d,l-propranolol to inhibit the estradiol-induced increase in cyclase activity. The role of epinephrine in the estradiol action mechanism is complex. Chew and Rinard (1974) reported an increase in adenylate cyclase activity in the rat uterus after injecting epinephrine into estradiol pretreated animals. The results of Denari et al. (1974) show a decreased uptake of estradiol in rat uterus
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w h e n the s t e r o i d a n d e p i n e p h r i n e were injected s i m u l t a n e o u s l y . A c c o r d i n g to the p r e s e n t a u t h o r e p i n e p h r i n e m a y be i m p o r t a n t f o r different a s p e c t s o f the e s t r a d i o l effect, b u t it is e v i d e n t t h a t a n increased a d e n y l a t e cyclase activity in some t a r g e t cells at least m a y be the result o f effects o f e s t r a d i o l o n the cell m e m b r a n e w h i c h a r e n o t r e l a t e d to the b e t a - a d r e n e r g i c receptors. In this s t u d y no u l t r a s t r u c t u r a l c h a n g e s were seen after d - p r o p r a n o l o l treatm e n t as c o m p a r e d w i t h e s t r a d i o l t r e a t m e n t alone. T h e e s t r a d i o l - i n d u c e d increase in the a m o u n t o f C V A was c o u n t e r a c t e d b y d - p r o p r a n o l o l . J u d g i n g f r o m the sections p r e p a r e d f o r i m m u n o f l u o r e s c e n c e , n o effects were seen o n the p r o l i f e r a tive activity in the b a s a l e p i t h e l i a l zone. T h e h e i g h t a n d cellular c o m p o s i t i o n o f this z o n e was the s a m e in a n i m a l s t r e a t e d with e s t r a d i o l a l o n e a n d in those injected with b o t h the steroid a n d d - p r o p r a n o l o l . S o m e increase in a d e n y l a t e cyclase activity o c c u r r e d in the b a s a l zone a f t e r e s t r a d i o l t r e a t m e n t , a n d this increase was i n h i b i t e d b y d - p r o p r a n o l o l . In o t e r studies, p r o p r a n o l o l c o u l d n o t be d e m o n s t r a t e d to h a v e effects on e s t r a d i o l - i n d u c e d R N A a n d p r o t e i n synthesis, synthesis o f the G o r s k i protein, synthesis o f glycolytic e n z y m e s o r cell p r o l i f e r a t i o n (Singhal et al., 1972; D u p o n t - M a i r e s s e et al., 1974; D u p o n t M a i r e s s e a n d G a l a n d , 1975). The e s t r a d i o l - i n d u c e d effects o n the cell m e m b r a n e w h i c h have been p o s t u l a t e d to result in increased a d e n y l a t e cyclase activity m a y be specific a n d c o n f i n e d o n l y to some o f the cells in a t a r g e t o r g a n . M o r e o v e r , the effects o f e s t r a d i o l o n the a d e n y l a t e cyclase system m a y be different a t different p e r i o d s o f life, a n d v a r y b e t w e e n n e o n a t a l a n d a d u l t animals.
References Abro, A., Kvinnsland, S. : Adenylate cyclase in an estradiol sensitive tissue: A cytochemical study. Histochemistry 42, 333-344 (1974) Anderson, W.B., Russel, T.R., Carchman, R.A., Pastan, I. : Interrelationships between adenylate cyclase activity, adenosine 3':5' cyclic monophosphate phosphodiesterase activity, adenosine 3':5' cyclic monophosphate levels, and growth of cells in culture. Proc. nat. Acad. Sci. (Wash.) 70, 3802-3805 (1973) Cerasi, E. : Mechanisms of glucose stimulated insulin secretion in health and in diabetes: Some re-evaluations and proposals. Diabetologia 11, 1-13 (1975) Chew, C.S., Rinard, G.A.: Estrogenic regulation of uterine cyclic AMP metabolism. Biochim. biophys. Acta (Amst.) 362, 493-500 (1974) Cutler, L.S.: Cytochemical and biochemical studies on adenylate cyclase in the developing rat submandibular gland. J. Cell Biol. 67, 82a (1975) Denari, J.H., Neuspiller, N.R., Garcia, E.P., Rosner, J.M. : Effects of epinephrine on the estradiol action in the rat uterus. Steroids Lipids Res. 5, 118-122 (1974) D~skeland, S.O., Kalland, T., Forsberg, J.-G. : Studies on the differentiation pattern and hormonal sensitivity of an antigenic material specific for the cervicovaginal epithelium in fetal and neonatal mice. Develop. Biol., 48, 184-192 (1976) Dupont-Mairesse, N., Galand, P. : Oestradiol-induced synthesis of a specific uterine protein in propranolol-treated rats. J. Endocr. 65, 215-218 (1975) Dupont-Mairesse, N., van Sande, J., Rooryck, J., Fastrez-Boute, A., Galand, P. : Mechanism of estrogen action independence of several responses of the rat uterus from the early increase in adenosine 3',5'-cyclic monophosphate. J. Steroid Biochem. 5, 173-178 (1974) Essner, E. : Phosphatases: In electron microscopy. In: Principles and methods (M.A. Hayat, ed.), Vol. 1, pp. 44-76. New York: D. van Nostrand Company Inc. 1973 Farnham, C.J.M. : Cytochemical localization of adenylate cyclase and 3'. 5'-nucleotide phosphodiesterase in Dictyosteliurn discoideum. Exp. Cell Res. 91, 3646 (1975)
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Forsberg, J.-G., Abro, A. : Ultrastructural differences between the sinus and the Miillerian epithelium of the mouse vaginal anlage. Z. Anat. Entwickl.-Gesch. 135, 67-75 (1971) Forsberg, J.-G., Abro, A. : Ultrastructural studies on cell degeneration in the mouse uterovaginal anlage. Acta anat. (Basel) 85, 353-367 (1973) Forsberg, J.-G., Kvinnsland, S. : The appearance and distribution of vaginal antigen during the differentiation of the cervicovaginal epithelium in normal and estradiol-treated mice. J. exp. Zool. 180, 403-412 (1972) Forsberg, J.-G., Nord, K. : Immunofluorescence studies on the distribution of mouse uterine epithelial antigen in foetal, immature and adult mice. J. Embryol. exp. Morph. 21, 85-95 (1969) Hadden, J.W., Hadden, E.M., Haddox, M.K., Goldberg, N.D. : Guanosine 3'5'-cyclic monophosphate: A possible intracellular mediator of mitogenic influences in lymphocytes. Proc. nat. Acad. Sci. (Wash.) 69, 3024-3027 (1972) Kuehl, F.A., Jr., Ham, E.A., Zanetti, M.E., Sanford, C.E., Nicol, S.E., Golberg, N.D.: Estrogenrelated increases in uterine guanosine 3'5'-cyclic monophosphate levels. Proc. nat. Acad. Sci. (Wash.) 71, 1866-t870 (1974) Kvinnsland, S.: Estradiol-17fl cyclic AMP and prostaglandins: In vivo and in vitro studies on the cervicovaginal epithelium from neonatal mice. Life Sci. 12 (part 1), 373-384 (1973) Lemay, A., Jarrett, L. : Pitfalls in the use of lead nitrate for the histochemical demonstration of adenylate cyclase activity. J. Cell Biol. 65, 39-50 (1975) Notides, A., Gorski, J. : Estrogen-induced synthesis of a specific uterine protein. Proc. nat. Acad. Sci. (Wash.) 56, 230-235 (1966) Novikoff, A.B., Hausman, D.H., Podberg, E. : The localization of adenosine triphosphatase in liver: in situ staining and cell fractionation studies. J. Histochem. Cytochem. 6, 61-74 (1958) Parmley, W.W., Braunwald, E. : Comparative myocardial depressant and anti-arrhythmic properties of d-propranolol, dl-propranolol and quinidine. J. Pharmacol. exp. Ther. 158, 11-21 (1967) Rosenfeld, M.G., O'Malley, B.W. : Steroid hormones: Effects on adenyl cyclase activity and adenosine Y,5'-monophosphate in target tissues. Science 168, 253-255 (1970) Rosenthal, A.S., Moses, H.L., Beaver, D.L., Schuffman, S.S. : Lead ion and phosphatase histochemistry. 1. Nonenzymatic hydrolysis of nucleotide phosphates by lead ion. J. Histochem. Cytochem. 14, 698-701 (1966) Singhal, R.L., Thomas, J.A., Parulekar, M.R. : Failure of beta-adrenergic blocking agents to alter estrogenic induction of uterine enzymes. Life Sci. 11 (part 1), 255-261 (1972) Spaziani, E., Szego, C.M. : The influence of estradiol and cortisol on uterine histamine of the ovariectomized rat. Endocrinol. 63, 669~78 (1958) Szego, C.M., Davis, LS. : Adenosine Y,5'-monophosphate in rat uterus: Acute elevation by estrogen. Proc. nat. Acad. Sci. (Wash.) 58, 1711-1718 (1967) Szego, C.M., Davis, J.S. : Inhibition of estrogen-induced cyclic AMP elevation in rat uterus. 11. By glucocorticoids. Life Sci. 8 (part 1), 110%1116 (1969) Tchernitchin, A., Roorijck, J., Tchernitchin, X., van den Hende, J., Galand, P. : Dramatic early increase in uterine eosinophils after oestrogen administration. Nature (Lond.) 248, 142-143 (1974) Wicks, W.D.: Regulation of protein synthesis by cyclic AMP. In: Advances in cyclic nucleotide research (P. Greengard, G.A. Robison, eds.), Vol. 4, pp. 335-438 1974 Zor, U., Hoch, J., Lamprecht, S.A., Ausher, J., Lindner, H.R.: Mechanism of oestradiol action on the rat uterus: independence of cyclic AMP, prostaglandin E2 and beta-adrenergic mediation. J. Endocr. 58, 525-533 (1973)
Accepted February 16, 1976 / in final form May 15, 1976
Cell and Tissue Research 9 by Springer-Verlag 1976
Effects of d-Propranolol and Estradiol on the Cervicovaginal Epithelium* Correlation with Adenylate Cyclase Activity Stener Kvinnsland Institute of Anatomy, University of Bergen, Bergen, Norway
Summary. The cervicovaginal epithelium of neonatal mice produces a material with specific antigenic properties (CVA) and this material is produced in increased amounts after estradiol treatment. Using a cytochemical method, estradiol treatment was shown to result in an increase of adenylate cyclase activity in the same epithelium. When d-propranolol is injected together with estradiol, the increase in CVA is inhibited, while the hormone-induced proliferation of epithelial cells is not influenced. When adenylate cyclase activity is studied under identical conditions, the estradiol-promoted increase in enzyme activity is largely counteracted by d-propranolol. These findings would suggest that Adenosine 3'5'-cyclic monophosphate (cAMP) has a role in some, but not all, estradiolmediated effects in the neonatal cervicovaginal epithelium.
Key words: Cervical epithelium - Mouse - d-propranolol - Estradiol Adenylate cyclase activity - Adenosine 3'5'-cyclic monophosphate. Introduction The role of adenosine 3'5'-cyclic monophosphate (cAMP) in the mechanism of action of estradiol is still uncertain. Conflicting evidence exists with regard to estradiol-promoted c A M P elevation in target tissues (Szego and Davis, 1967; Zor et al., 1973; Dupont-Mairesse et al., 1974; Kuehl et al., 1974). The matter is complex because the number of eosinophils and tissue levels of agents such as histamine, epinephrine, and serotonin are under estrogenic control (Spaziani and Szego, 1958; Tchernitchin, 1970). The interactions between these agents, cAMP, and estradiol in relation to target organ responses to steroid influences, are not fully understood. Dr. Stener Kvinnsland, Institute of Anatomy, Arstadvollen, Bergen, Norway * This work was supported by grants from the Norwegian Research Council for Science and the Humanities and from the Norwegian Cancer Society (Landsforeningenmot Kreft) Send offprint requests to :
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Propranolol, a beta-adrenergic blocking drug, has been shown to suppress the estradiol-induced elevation of cAMP (Szego and Davis, 1969; DupontMairesse et al., 1974). However, the estradiol-induced increase in activity of glycolytic enzymes in the uterus (Singhal et al., 1972), of increased RNA and protein synthesis (Dupont-Mairesse et al., 1974), and of increased synthesis of the Gorski protein (Notides and Gorski, 1966; Dupont-Mairesse and Galand, 1975), are not influenced by treatment with propranolol. In an earlier study undertaken in this laboratory, it was shown that in neonatal mice the amount of a material with specific antigenic properties, confined to the cervicovaginal epithelium (CVA), was increased after estradiol treatment. This increase was suppressed by treatment with d,l-propranolol (Kvinnsland, 1973), but proliferation of the vaginal epithelial cells was not affected. Cytochemical studies have demonstrated an increase in adenylate cyclase activity in the same epithelium after treatment with estradiol alone (Abro and Kvinnsland, 1974). Some results in the study by Kvinnsland (1973) indicated that the effect of d,l-propranolol was due to a membrane stabilizing influence. For this reason it was of interst to study d-propranolol which has almost no beta-blocking effect (Parmley and Braunwald, 1967) but possesses the same membrane effect as d,l-propranolol. Moreover, we wanted to investigate whether or not cytochemically demonstrable adenylate cyclase activity correlated with a decreased epithelial CVA content after d-propranolol treatment. Material and Methods Animals. All mice used belonged to a randomly bred N M R I strain. The animals were fed a standard pellet diet and given water ad libitum. Starting on day 3 after birth, one group of female animals (positive controls) received subcutaneous injections of 5 txg estradiol-17/3 (Sigma) in 0.025 ml olive oil for 3 days. The animals of the experimental group were given the same estradiol treatment and, in addition, subcutaneous injections of d-propranolol (ICI). A third group (negative controls) received the vehicles for estradiol-17/~ and d-propranolol according to the same time schedule as the animals of the experimental group. For concentrations used see Table 1. All animals were killed by decapitation on day 6 (24 h after the last estradiol injection).
Immunofluorescence Method for Demonstration of CVA. Antiserum was obtained from rabbits after immunization with vaginal epithelium from castrated adult, estradiol-treated mice. A vaginal epithelial homogenate was centrifuged for 1 h at 105,000g in a B-60 International Ultracentrifuge, the supernatant emulsified in Freund's complete adjuvant and injected into the rabbits' foot pads; for details see Forsberg and Kvinnsland (1972). The serum obtained was absorbed and treated as previously described but without further purification as the serum after centrifugation showed the same specificity as the crude lgG fraction used in earlier studies. The cervicovaginal canal from the 6-day animals was dissected out under a dissecting microscope, and frozen in a CO2-jet. Transverse sections (10 microns) were cut in a cryostat at - 2 0 ~ C. The indirect immunofluorescence method was used as described by Forsberg and Nord (1969). N o r m a l rabbit serum and phosphate buffered saline were used for the control sections. The sections were examined in a Zeiss photomicroscope equipped with an Osram High-Pressure mercury arc lamp, HBO 200W. Exciter filter: BG3 and BG12; barrier filters 44 and 50 (cut off at 500 mla). The photographs were taken on 35 m m K o d a k Tri-X Pan film 135-36. For an estimation of the a m o u n t of fluorescent material, the semiquantitative method previously described was used (Kvinnsland, 1973). Recently, Doskeland etal. (1976) have used a mixed haemagglutination technique for the demonstration of CVA. With this method which is far more sensitive, they have confirmed important
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Table 1 No. of preparation
Type of experiment
Fluorescence superficial zone
Epithelial proliferation basal zone
4
Estradiol-17fl
+ + +
Strongly increased
4
Olive oiP Parker 199
+
normal
4
Estradiol- 17 fl d-propranolol b
+
Strongly increased
" 5 gg 0.025 ml olive oil b Concentration used: 1 gg in 0.02 ml Parker 199 five times daily for 3 days, starting twelve hours before the first estradiol injection. Estradiol-17fl as in a results on CVA reported in earlier studies using the immunofluorescence technique, thereby supporting the method of semiquantitation used here.
Cytochemical Demonstration ofAdenylate Cyclase Activity. Animals from the three groups described above were used. Following decapitation the intestines were removed and the uterovaginal region was prefixed in situ by pipetting directly onto it 1% glutaraldehyde (TAAB Laboratories) in 50 mM cacodylate buffer (pH 7.4) containing 4.5% glucose. The uterovaginal region was removed, immersed in the fixing fluid (as for prefixation, see above), and cut into transverse slices with a razor blade. The fixation time was 1 h. Slices through the uterine cervix and vaginal fornices were used for the experiments. The slices were washed in several changes of cacodylate-glucose buffer. The slices were then incubated for adenylate cyclase activity in a Tris-maleate buffer (pH 7.4) containing glucose (8%), MgSO4 (2 mM), Pb (NOa)2 (2 mM), theophylline (2 mM) and with 0.5 mM ATP as substrate. In order to inhibit ATPase activity, NaF (10 mM) and ouabain (0.5 mM, Sigma) were included in the incubation medium; for details see ,~bro and Kvinnsland (1974). The following control slices for demonstration of enzyme activity were always run together with the experimental slices: 1. Slices were heated for 3 min in a boiling water bath in order to heat-inactivate adenylate cyclase activity. 2. Slices were incubated in the absence of ATP. 3. Incubation was performed after alloxan (5 mM, Sigma), an inhibitor of adenylate cyclase activity, had been added to the medium. After incubation the tissue slices were washed briefly in Tris-maleate buffer (pH 7.4), and postfixed for 1 h in 1% OsO4 (Merck) in cacodylate-glucose buffer (pH 7.4). After rinsing in buffer the slices were dehydrated in a series of cold ethanol solutions cleared in propylene oxide, infiltrated with, and embedded in Epon 812. Ultrathin sections were cut on a LKB ultramicrotome, counterstained for 15 min with lead citrate, and examined in a Siemens electron microscope IA. Terminology. The vaginal anlage in neonatal mice consists of two different parts: an anterior part, the mtillerian vagina, and a posterior part, the sinus vagina. In newborn animals, the miillerian vagina, derived from the fused miillerian ducts, has a pseudostratified epithelium. This later transforms into an epithelium consisting of a basal and a superficial zone (Forsberg and ,~bro, 1971 ; Forsberg and Abro, 1973). The former is the origin of the basal epithelial layer in the adult vaginal epithelium.
Results
Immunofluorescence Studies. T h e r e s u l t s o f t h e i m m u n o f l u o r e s c e n c e s t u d i e s a r e s u m m a r i z e d in T a b l e 1. A l l s e c t i o n s w e r e f r o m t h e a n t e r i o r p a r t o f t h e m i i l l e r i a n
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vagina. In control mice treated with vehicles only (Fig. 1B), a distinct basal zone of cells was seen in the epithelium. The superficial zone of columnar cells contained a small amount of fluorescent material in the apical portions of the cells. After estradiol treatment for three days the basal zone was higher (Fig. 1A), and a large amount of fluorescent material was seen apically in the superficial zone cells. In sections from estradiol and d-propranolol treated mice, the high basal zone was still present, but there was a substantial reduction of fluorescent material in the superficial zone (Fig. 1C).
Electron Microscope Studies. In 6-day old mice, the vaginal epithelium has a superficial zone of high columnar cells. The cells of this zone have small irregular microvilli and some m e m b r a n e - b o u n d granules are present in the apical part of the cytoplasm. The luminal surface is covered with a thin layer of filamentous material. N a r r o w intercellular spaces are seen between adjacent epithelial cells. In the basal zone the cells are small and irregular. After estradiol treatment on days 3, 4 and 5, the cells of the superficial zone develop more pronounced, club-shaped microvilli. The filamentous surface material becomes much more abundant. Large intercellular spaces develop, with extensive apical interdigitation between the cells. In the apical part of the cytoplasm are large numbers of m e m b r a n e - b o u n d granules of different sizes and with a homogeneous content. Some of them appear to extrude their content by exocytosis. The cells of the basal zone lack" such granules. When estradiol and d-propranolol are given simultaneously, no specific changes in the m o r p h o l o g y of the epithelium are seen, especially with respect to microvilli and mucous granules.
Adenylate Cyclase Activity. F o r the specificity of the cytochemical reaction, see Abro and Kvinnsland (1974). Varying amounts of lead phosphate precipitate were taken to indicate varying degrees of enzyme activity. In the present study, the lead phosphate precipitates indicating enzyme activity were found on the cell membrane of both epithelial and connective tissue cells. It was difficult to determine the precise localization of the precipitate in relation to the plasma membrane, but in a recent study, using the same technique, F a r n h a m (1975) has shown that in the slime mould Dictyostelium discoideum it is localized on the cytoplasmic side of the membrane. The patchy precipitates were far more pronounced in the superficial zone than in the basal zone. The basal cells had distinct precipitates on the cell membrane bordering the basal lamina. In the superficial zone, the luminal part of the plasma membrane had the highest activity, while in the basal zone the membranes bordering the basal lamina had the most extensive precipitates. Variations within a single
Fig. 1. A Epithelium from estradiol treated animal. CVA is seen in the superficial zone. Beneath is the high basal zone. Magnification x 900 (reduced to 90%). B Epithelium from control animal. Little CVA is found in the superficial zone. The basal zone is narrow. Magnification x 900 (reduced to 90%). C Epithelium from estradiol and propranolol treated animal. A small quantity of CVA is seen in the superficial zone. Beneath is the high basal zone. Magnification x 900 (reduced to 90%)
Role of c A M P in a Specific Effect of Estradiol on Cervicovaginal Epithelium
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Role of cAMP in a SpecificEffect of Estradiol on CervicovaginalEpithelium
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animal with respect to the lead phosphate precipitate, were low. A consistent finding, however, was that some cells in the superficial zone were almost free of precipitates, but otherwise seemed to be morphologically normal. The reason for this is not known. In mice injected with vehicles only, a low but distinct enzyme activity was seen, with regional differences in activity as described above (Fig. 2C). The basal activity in 6-day old normal mice was higher than in 3-day old mice. After estradiol treatment a very prominent increase in enzyme activity was found, with the same pattern of distribution (Fig. 2A). When d-propranolol and estradiol were injected together, most of the estradiol effect on the enzyme activity was counteracted, and a pattern of precipitates was found almost identical to that seen in control animals (Fig. 2B).
Discussion
F o r a general discussion concerning the problems and pitfalls involved in the cytochemical demonstration of phosphatases, see Essnu (1973) and more specifically for adenylate cyclase activity, see Abro and Kvinnsland (1974). The method used has been questioned by Lemay and Jarrett (1975) who conclude that the method cannot be used for the demonstration of adenylate cyclase activity because the concentration of lead ions in the incubation medium would completely inhibit the enzyme. Furthermore, non-enzymatic hydrolysis of A T P giving rise to lead phosphate and cAMP was considered to be another serious drawback. However, prefixation, which has been reported to decrease the sensitivity of enzymes to lead ions (Novikoff et al., 1958), was not included in the procedure used by Lemay and Jarrett. After prefixation in 1.25% glutaraldehyde, and with 3 m M lead nitrate present during the incubation, Cutler (1975) has recently reported that 10-25% of the basal activity of adenylate cyclase is still present. Moreover, the buffers used by Lemay and Jarrett (1975) in their biochemical and cytochemical studies were not the same (Rosenthal et al., 1966). Results obtained with isolated rat cell membranes should not be compared to results from whole tissue blocks. When alloxan (adenylate cyclase inhibitor) was included in the incubation medium for our cytochemical studies (Abro and Kvinnsland, 1974), no precipitate was observed in the tissue sections. Heat-inactivation of the adenylate cyclase before incubation abolished the production of visible precipitates. Thus, in our hands non-enzymatic hydrolysis of ATP would not appear to invalidate the results. According to earlier studies on specificity (Forsberg and Kvinnsland, 1972), the fluorescent material studied represents a cell product with specific antigenic Fig. 2A-C. The luminal plasma membrane of cells in the superficial zone of the cervicovaginal epithelium. Magnification x 48,000 (reduced to 90%). A An estradiol treated animal. Heavy lead phosphate precipitates are seen in the epithelial cells. B Estradiol and propranolol treated animal. The precipitates are less marked than in the estradiol treated animal. C Control animal. Less heavy precipitates are seen
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properties characteristic for the cervicovaginal epithelium (CVA). The amount of CVA increases after estrogen stimulation; it is localized apically in the superficial columnar cells in the cervicovaginal anlage, and it is also present in the lumen. A close correlation exists regarding the localization of adenylate cyclase activity and the presence of CVA. The difference between the amount of lead phosphate precipitates in the superficial columnar cells and in the cells of the basal epithelial zone is striking, the latter having much less precipitate. This supports the view of Dupont-Mairesse et al. (1974) that a cAMP increase after estradiol treatment may be confined to a specific part of the target tissue. In biochemical studies a localized increase in cAMP level might be masked by the basal level of total cAMP content in the target organ. The experimental results point to a role of the adenylate cyclase-cAMP system in the cellular production of cytochemically demonstrable CVA (Kvinnsland, 1973). The nucleotide could be involved in synthesis, transport and secretion phenomena related to CVA (Wicks, 1974; Cerasi, 1975). The estradiolinduced increase in adenylate cyclase activity in the basal epithelial zone, which was undergoing a dramatic proliferative activity, was an unexpected finding. However, an increased adenylate cyclase activity does not necessarily mean an elevated cellular cAMP level, since this is influenced also by phosphodiesterase activity which was not studied in this investigation. The amount of guanosine -3'5'-cyclic-monophosphate increases under conditions of increased proliferative activity (Hadden et al., 1972) and the level of this nucleotide is influenced by estradiol (Kuehl et al., 1974). An inhibitory influence of cAMP as regards cell proliferation has been suggested (Anderson et al., 1973). As long as the roles of cAMP and cGMP during proliferative activity in the cervicovaginal epithelium are not known, the conclusion is that estradiol increases the histochemically demonstrable adenylate cyclase activity and that this increase is more pronounced in the superficial epithelial cells with high CVA content than in the basal epithelial zone containing proliferating cells. d,l-propranolol counteracts the estradiol-induced increase of CVA (Kvinnsland, 1973). This effect was not abolished by the simultaneous use of isoproterenol (a beta-adrenergic agonist). Since d,l-propranolol has membrane effects other than those related to the beta-adrenergic receptors (Parmley and Braunswald, 1967), the effect of d-propranolol was studied. The latter has membrane effects similar to those of d,l-propranolol but its beta-blocking effect is far less. In this study dpropranolol had the same effect as the racemic mixture which indicates that catecholamines are not involved in the estradiol-induced increase in adenylate cyclase activity. Instead this may be due to some membrane labilizing effect mediated by estradiol (Szego and Davis, 1969). In biochemical studies, Rosenfeld and O'Mallay (1970) were unable to demonstrate that d-propranolol could be used instead of d,l-propranolol to inhibit the estradiol-induced increase in cyclase activity. The role of epinephrine in the estradiol action mechanism is complex. Chew and Rinard (1974) reported an increase in adenylate cyclase activity in the rat uterus after injecting epinephrine into estradiol pretreated animals. The results of Denari et al. (1974) show a decreased uptake of estradiol in rat uterus
Role of cAMP in a Specific Effect of Estradiol on Cervicovaginal Epithelium
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w h e n the s t e r o i d a n d e p i n e p h r i n e were injected s i m u l t a n e o u s l y . A c c o r d i n g to the p r e s e n t a u t h o r e p i n e p h r i n e m a y be i m p o r t a n t f o r different a s p e c t s o f the e s t r a d i o l effect, b u t it is e v i d e n t t h a t a n increased a d e n y l a t e cyclase activity in some t a r g e t cells at least m a y be the result o f effects o f e s t r a d i o l o n the cell m e m b r a n e w h i c h a r e n o t r e l a t e d to the b e t a - a d r e n e r g i c receptors. In this s t u d y no u l t r a s t r u c t u r a l c h a n g e s were seen after d - p r o p r a n o l o l treatm e n t as c o m p a r e d w i t h e s t r a d i o l t r e a t m e n t alone. T h e e s t r a d i o l - i n d u c e d increase in the a m o u n t o f C V A was c o u n t e r a c t e d b y d - p r o p r a n o l o l . J u d g i n g f r o m the sections p r e p a r e d f o r i m m u n o f l u o r e s c e n c e , n o effects were seen o n the p r o l i f e r a tive activity in the b a s a l e p i t h e l i a l zone. T h e h e i g h t a n d cellular c o m p o s i t i o n o f this z o n e was the s a m e in a n i m a l s t r e a t e d with e s t r a d i o l a l o n e a n d in those injected with b o t h the steroid a n d d - p r o p r a n o l o l . S o m e increase in a d e n y l a t e cyclase activity o c c u r r e d in the b a s a l zone a f t e r e s t r a d i o l t r e a t m e n t , a n d this increase was i n h i b i t e d b y d - p r o p r a n o l o l . In o t e r studies, p r o p r a n o l o l c o u l d n o t be d e m o n s t r a t e d to h a v e effects on e s t r a d i o l - i n d u c e d R N A a n d p r o t e i n synthesis, synthesis o f the G o r s k i protein, synthesis o f glycolytic e n z y m e s o r cell p r o l i f e r a t i o n (Singhal et al., 1972; D u p o n t - M a i r e s s e et al., 1974; D u p o n t M a i r e s s e a n d G a l a n d , 1975). The e s t r a d i o l - i n d u c e d effects o n the cell m e m b r a n e w h i c h have been p o s t u l a t e d to result in increased a d e n y l a t e cyclase activity m a y be specific a n d c o n f i n e d o n l y to some o f the cells in a t a r g e t o r g a n . M o r e o v e r , the effects o f e s t r a d i o l o n the a d e n y l a t e cyclase system m a y be different a t different p e r i o d s o f life, a n d v a r y b e t w e e n n e o n a t a l a n d a d u l t animals.
References Abro, A., Kvinnsland, S. : Adenylate cyclase in an estradiol sensitive tissue: A cytochemical study. Histochemistry 42, 333-344 (1974) Anderson, W.B., Russel, T.R., Carchman, R.A., Pastan, I. : Interrelationships between adenylate cyclase activity, adenosine 3':5' cyclic monophosphate phosphodiesterase activity, adenosine 3':5' cyclic monophosphate levels, and growth of cells in culture. Proc. nat. Acad. Sci. (Wash.) 70, 3802-3805 (1973) Cerasi, E. : Mechanisms of glucose stimulated insulin secretion in health and in diabetes: Some re-evaluations and proposals. Diabetologia 11, 1-13 (1975) Chew, C.S., Rinard, G.A.: Estrogenic regulation of uterine cyclic AMP metabolism. Biochim. biophys. Acta (Amst.) 362, 493-500 (1974) Cutler, L.S.: Cytochemical and biochemical studies on adenylate cyclase in the developing rat submandibular gland. J. Cell Biol. 67, 82a (1975) Denari, J.H., Neuspiller, N.R., Garcia, E.P., Rosner, J.M. : Effects of epinephrine on the estradiol action in the rat uterus. Steroids Lipids Res. 5, 118-122 (1974) D~skeland, S.O., Kalland, T., Forsberg, J.-G. : Studies on the differentiation pattern and hormonal sensitivity of an antigenic material specific for the cervicovaginal epithelium in fetal and neonatal mice. Develop. Biol., 48, 184-192 (1976) Dupont-Mairesse, N., Galand, P. : Oestradiol-induced synthesis of a specific uterine protein in propranolol-treated rats. J. Endocr. 65, 215-218 (1975) Dupont-Mairesse, N., van Sande, J., Rooryck, J., Fastrez-Boute, A., Galand, P. : Mechanism of estrogen action independence of several responses of the rat uterus from the early increase in adenosine 3',5'-cyclic monophosphate. J. Steroid Biochem. 5, 173-178 (1974) Essner, E. : Phosphatases: In electron microscopy. In: Principles and methods (M.A. Hayat, ed.), Vol. 1, pp. 44-76. New York: D. van Nostrand Company Inc. 1973 Farnham, C.J.M. : Cytochemical localization of adenylate cyclase and 3'. 5'-nucleotide phosphodiesterase in Dictyosteliurn discoideum. Exp. Cell Res. 91, 3646 (1975)
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Forsberg, J.-G., Abro, A. : Ultrastructural differences between the sinus and the Miillerian epithelium of the mouse vaginal anlage. Z. Anat. Entwickl.-Gesch. 135, 67-75 (1971) Forsberg, J.-G., Abro, A. : Ultrastructural studies on cell degeneration in the mouse uterovaginal anlage. Acta anat. (Basel) 85, 353-367 (1973) Forsberg, J.-G., Kvinnsland, S. : The appearance and distribution of vaginal antigen during the differentiation of the cervicovaginal epithelium in normal and estradiol-treated mice. J. exp. Zool. 180, 403-412 (1972) Forsberg, J.-G., Nord, K. : Immunofluorescence studies on the distribution of mouse uterine epithelial antigen in foetal, immature and adult mice. J. Embryol. exp. Morph. 21, 85-95 (1969) Hadden, J.W., Hadden, E.M., Haddox, M.K., Goldberg, N.D. : Guanosine 3'5'-cyclic monophosphate: A possible intracellular mediator of mitogenic influences in lymphocytes. Proc. nat. Acad. Sci. (Wash.) 69, 3024-3027 (1972) Kuehl, F.A., Jr., Ham, E.A., Zanetti, M.E., Sanford, C.E., Nicol, S.E., Golberg, N.D.: Estrogenrelated increases in uterine guanosine 3'5'-cyclic monophosphate levels. Proc. nat. Acad. Sci. (Wash.) 71, 1866-t870 (1974) Kvinnsland, S.: Estradiol-17fl cyclic AMP and prostaglandins: In vivo and in vitro studies on the cervicovaginal epithelium from neonatal mice. Life Sci. 12 (part 1), 373-384 (1973) Lemay, A., Jarrett, L. : Pitfalls in the use of lead nitrate for the histochemical demonstration of adenylate cyclase activity. J. Cell Biol. 65, 39-50 (1975) Notides, A., Gorski, J. : Estrogen-induced synthesis of a specific uterine protein. Proc. nat. Acad. Sci. (Wash.) 56, 230-235 (1966) Novikoff, A.B., Hausman, D.H., Podberg, E. : The localization of adenosine triphosphatase in liver: in situ staining and cell fractionation studies. J. Histochem. Cytochem. 6, 61-74 (1958) Parmley, W.W., Braunwald, E. : Comparative myocardial depressant and anti-arrhythmic properties of d-propranolol, dl-propranolol and quinidine. J. Pharmacol. exp. Ther. 158, 11-21 (1967) Rosenfeld, M.G., O'Malley, B.W. : Steroid hormones: Effects on adenyl cyclase activity and adenosine Y,5'-monophosphate in target tissues. Science 168, 253-255 (1970) Rosenthal, A.S., Moses, H.L., Beaver, D.L., Schuffman, S.S. : Lead ion and phosphatase histochemistry. 1. Nonenzymatic hydrolysis of nucleotide phosphates by lead ion. J. Histochem. Cytochem. 14, 698-701 (1966) Singhal, R.L., Thomas, J.A., Parulekar, M.R. : Failure of beta-adrenergic blocking agents to alter estrogenic induction of uterine enzymes. Life Sci. 11 (part 1), 255-261 (1972) Spaziani, E., Szego, C.M. : The influence of estradiol and cortisol on uterine histamine of the ovariectomized rat. Endocrinol. 63, 669~78 (1958) Szego, C.M., Davis, LS. : Adenosine Y,5'-monophosphate in rat uterus: Acute elevation by estrogen. Proc. nat. Acad. Sci. (Wash.) 58, 1711-1718 (1967) Szego, C.M., Davis, J.S. : Inhibition of estrogen-induced cyclic AMP elevation in rat uterus. 11. By glucocorticoids. Life Sci. 8 (part 1), 110%1116 (1969) Tchernitchin, A., Roorijck, J., Tchernitchin, X., van den Hende, J., Galand, P. : Dramatic early increase in uterine eosinophils after oestrogen administration. Nature (Lond.) 248, 142-143 (1974) Wicks, W.D.: Regulation of protein synthesis by cyclic AMP. In: Advances in cyclic nucleotide research (P. Greengard, G.A. Robison, eds.), Vol. 4, pp. 335-438 1974 Zor, U., Hoch, J., Lamprecht, S.A., Ausher, J., Lindner, H.R.: Mechanism of oestradiol action on the rat uterus: independence of cyclic AMP, prostaglandin E2 and beta-adrenergic mediation. J. Endocr. 58, 525-533 (1973)
Accepted February 16, 1976 / in final form May 15, 1976
