FINE STRUCTURAL LOCALIZATION OF ADENOSINE TRI-PHOSPHATASE IN THE EPITHELIUM OF THE RABBIT OVIDUCT 1

FINE STRUCTURAL LOCALIZATION OF ADENOSINE TRI-PHOSPHATASE IN THE EPITHELIUM OF THE RABBIT OVIDUCT 1 Ramesh K. Nayak 2 and Arthur S. H. Wu Oregon State University, Corvallis 97331 SUMMARY The distribution of adenosine triphosphatase (ATP-ase) in prepubertal rabbit, intact and ovariectomized, oviductal epithelium under different hormonal influences was studied by examining the cytochemical products in the cell with an electron microscope. ATP-ase activity was found: (a) on the cell membrane, (b) on the membrane of cilia and microvilli, (c) in the Golgi cisternae and (d) in the basal cell membrane and basement membrane. Mitochondrial ATP-ase was also demonstrated by this procedure. The intensity of the enzyme reaction product at both the intracellular and extracellular sites was significantly reduced following oophorectomy and became distinctly more pronounced following the treatment with estrogen or progesterone. The effects of these two hormones appeared to be synergistic. Estrogen appeared to exert a significant effect on the ATP-ase activity of oviductal cilia. Acid phosphatase activity was also examined. It occurred within the Golgi cisternae, few secretory granules, cell membrane and microvilli of rabbit oviductal epithelium. The enzyme activity was also present within the large membrane-bound bodies similar to the dense bodies described in other cell types. (Key Words: Adenosine Tri-Phosphatase, Rabbit Oviduct; Oviduct Epithelium, Electron Microscopy, Acid Phosphatase.) INTRODUCTION The identification of enzymes at the subcellular level is one of the most recent and rapidly developing fields of cytochemistry. With the well-known metabolic role of the adenosine triphosphatase (ATP-ase) the subcellular local- 1Technical Paper No. 3231, Oregon Agricultural Experiment Station. 2Present Address: Dept. Anatomy, Coll. Med., Howard Univ., Wash., D. C. ization of this enzyme in oviductal epithelium and the possible changes of its activity under hormonal influence should contribute to a better understanding of the role of hormones in reproduction. Since the introduction by Wachstein and Meisel (1957) of a histochemical procedure for the localization of nucleoside phosphatases by heavy metal precipitation of inorganic phosphate, the technique has been widely accepted for the demonstration of tissue phosphatases. Many enzyme systems in the animal and human oviduct have been studied with the conventional biochemical and light microscopic histochemical methods (see review by Fredricsson, 1969) and several studies have been made on the fine structural localization of phosphatase activity in a variety of cell types (Wachstein and Besen, 1964; Bartoszewicz and Barrnett, 1964; Kaye and Pappas, 1965; Farquhar and Palade, 1966; Abel, 1969; lwayama, 1969; Smith and Farquhar, 1966). Bjorkman and Fredricsson (1961) studied the lipids, non-specific esterase, and acid phosphatase in the bovine oviduct epithelium. The present study was designed for fine structural localization of ATP-ase in rabbit oviduct under the influence of estrogen and progesterone. MATERIALS AND METHODS Immature female rabbits of the New Zealand White Strain were used in this study. The animals were in apparent good health and were under close supervision throughout the investigation. They were fed on Purina Lab Chow with water ad libitum. Two series of experiments were performed: Experiment I. Intact Rabbits: The animals were randomly assigned to four groups of four rabbits each. The following daily treatments were given to each animal for 7 days. Group 1. Control --.1 ml propylene glycol. Group 2. Estradiol benzoate -5/~g in.1 ml propylene glycol. 1077 JOURNAL OF ANIMAL SCIENCE, Vol. 41, No. 4, 1975

1078 NAYAK AND WU Group 3. Progesterone - 2 mg in.1 ml propylene glycol. Group 4. Estradiol benzoate (5/lg) and progesterone (2 mg) in.1 ml propylene glycol. Crystalline hormones were dissolved in propylene glycol and administered intramuscularly. Experiment 11. Ovariectomized Rabbits: Eight rabbits were ovariectomized at 8 weeks of age and randomly assigned to four groups of two animals each. Treatments exactly like those given to intact animals were initiated on the 40th day after surgery. The animals in both experiments were sacrificed 24 hr after the last injection. The reproductive tracts were removed immediately. The freshly excised oviductal tissues from fimbria, ampulla, isthmus, and uterotubal junction were cut into small blocks (approx. 1 mm 3) and fixed for 1 hr in cold 3% glutaraldehyde buffered to ph 7.4, with.1 M cacodylate buffer containing.22 M sucrose and the tissue was kept overnight in the buffer at 5 C. Tissue slices of 25 to 45 /1 thickness were also cut from strips of tissue with TC-2 Tissue Sectioner (Smith and Farquhar, 1966) and stored in cacodylate buffer for short periods prior to incubation. The tissue slices or blocks were incubated with the appropirate media and substrates for 1 hr at 37 C, washed twice in cold cacodylate buffer over a period of 30 rain and postfixed in buffered 1% oxmium tetroxide. They were then dehydrated in graded concentrations of ethanol and propylene oxide and embedded in Epon (Luft, 1961). Thin sections were made with a Porter Blum MT-2 Ultramicrotome and mounted on naked or formvarcoated grids. They were examined, either unstained or doubly stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963), in a Philips 300 or RCA--3G Electron Microscope at an operating voltage of 40 to 60 kilovolts. The activity of ATP-ase was demonstrated by the method of Wachstein and Meisel (1957). To assure the ATP-ase specificity, control experiments were conducted in which tissue slices or blocks were incubated in the same media without the substrate or with equimolar concentrations of sodium beta glycerphosphate instead of a nucleoside triphosphate as substrate before refixation in osmium tetroxide. Other control preparations were also made by inactivating enzymes through pretreatment of specimens for 1 hr in buffered 1% oxmium tetroxide prior to incubation with the substrate. R ESU LTS Experiment 1. In the epithelium of the oviduct of the immature rabbit, the final reaction product resulting from the hydrolysis of ATP was deposited as a fine granular electron-dense precipitate along the outer and lateral cell membranes and outer membrane of microvilli (figure 1). Such evidence of ATP-ase activity was also present on the basal bodies. Occasional deposits of the reaction product were seen in the Golgi apparatus and mitochondria (figures 2 and 3). In the mitochondria, the reaction product occurred as dense clumps of electron-dense precipitate usually extending from the inner membrane into the matrix. The tight junction, desmosome, nuclear envelope, basement membrane and the stromal cell were devoid of ATP-ase activity. As compared to the control group, ATP-ase activity was markedly pronounced on the cell membranes of the oviductal epithelium from estrogen treated rabbits. The ATP-ase activity also appeared to be much more pronounced on the membrances of cilia and microvilli, fibrils of cilia and basal bodies (figure 4). The ATP-ase activity in oviducts of progesterone treated rabbits appeared to be more pronounced on the microvilli and on the cell membrane than the control group (figure 5). However, the reaction product on the cell membrane was less intense when compared to the estrogen treated group. ATP-ase activity was also found on basal bodies and mitochondria, but only sporadically on the basement membrane (figure 6). Experiment 2. The ATP-ase activity of oviducts from oophorectomized rabbits was found only sporadically on the cell membrane and microvilli in the oviductal epithelium (figures 7 and 8). Evidence of enzymatic activity was absent on the mitochondria, Golgi apparatus and other intracellular organdies. After 7 days of estrogen treatment (figure 9), ATP-ase activity in the epithelial cells became distinctly more pronounced on the cell membrane, microvilli and cilia. It also appeared in the Golgi saccules, fibrils of cilia and basal bodies, mitochondria, and the basement membrane. The final reaction product resulting from the hydrolysis of ATP was found at the same structural localizations

LOCALIZATION OF RABBIT OVIDUCT ATP-ase 1079 PLATE I. Sections of oviduct from intact prepubertal rabbits. Figures 1--3: Control animals receiving the vehicle propylene glycol for 7 days. Figure 1. Note ATP-ase activity in the cell membrane and microvilli. X7750. Figure 2. Note ATP-ase activity in some mitochondria. X 25100. Figure 3. Note ATP-ase activity in the basal bodies. 25100. Figures 4--6: Animals receiving hormone treatments. Note the intense ATP-ase activity along cell membrane, microvilli and cilia. Figure 4. Animals receiving estrogen for 7 days. Figure 5. Animals receiving progesterone for 7 days. X17100. Figure 6. Animals receiving both estrogen and progesterone for 7 days. 19200.

1080 NAYAK AND WU PLATE II. Sections of oviduct from prepubertal rabbits 48 days following ovariectomy. Figures 7 and 8. ATP-ase activity is greatly reduced in the cell membrane and microvilli. Figure 9. Animal receiving estrogen (40 days following ovariectomy) for 7 days. Note the ATP-ase activity along the cell membrane, microvilli and cilia. 12300. Figure I0. Animals receiving both estrogen and progesterone (40 days following ovariectomy) for 7 days. Note the ATP-ase activity along the cell membrane and some reaction products are also seen in the Golgi complex.

LOCALIZATION OF RABBIT oviduct ATP-ase 1081 from the fimbria, ampulla, isthmus and uterotubal junction of the fallopian tube. Intense activity was present in these regions after estrogen treatment. The lateral surfaces of the epithelial cells which tortuously interdigitate with those of similar adjoining cells also showed heavy deposits of the final reaction product. Cell membranes of some stromal cells of the area close to the superficial epithelium were also reactive after estrogenic influence. In the ovariectomized rabbits receiving progesterone treatment, the distribution of the reaction product in the fimbriae and ampulla epithelium was similar to that observed in the estrogen treated group and the enzymatic activity was more intense as compared to the ovariectomized controls. A sparse deposition of final product occurred on the basal cell membrance, basement membrane and stromal cell membrane. In some instances, the deposition of the reaction produce was in a discontinuous beaded fashion at the luminal surface. In the ovariectomized rabbits receiving both estrogen and progesterone (figure 10), ATP-ase activity in the superficial cell layer was highly pronounced on the cilia, microvilli, Golgi lamellae, along the entire length of cell membrances, and the basement membrane of the columnar layer. The distribution and intensity of the reaction product in the epithelium from tubal junction appeared to be the same as in the epithelium from other regions of the fallopian tube. Furthermore, the effects of estrogen and progesterone appeared to be synergistic on the ATP-ase activity of the cell membrane of the epithelial cells. A comparative small number of oviductal tissues were studied for the ultrastructural localization of acid phosphatase (figure 11-13), according to the method of Barka and Anderson (1962). The presence of acid phosphatase in the dense bodies suggested the close resemblance of these dense bodies to the lysosomes described in other cell types. Acid phosphatase activity also occurred on the luminal cell membrane and microvilli of oviductal epithelial cells from the fimbria and ampulla of immature rabbits. Lead phosphate reaction product of ATP-ase was also intermittently deposited in the Golgi cisternae, secretory granules and secretory material in the process of extrusion. Discussion Many investigators have discussed the advantages and disadvantages of using blocks of tissue in electron histochemistry (Holt and Hicks, 1961; Tice and Barrnett, 1963 ; Goldfischer et al., 1964; Sabatini et al., 1964). Holt and Hicks (1962) have shown that, for rat liver fixed in formaldehyde, penetration difficulties arise when frozen sections exceed 50/a inthickness. Enzyme diffusion is a significant objection to the use of blocks of tissue in electron microscopy. In the present study, best localization of ATP-ase reaction product was obtained when tissue has been sectioned at 25-45 ~ with a TC-2 Tissue Sectioner (Smith and Farquhar, 1965) prior to incubation with the substrate. Our findings are similar to those of Smith and Farquhar (1966) who have reported sharper and better localization of nucelosidephosphatases in the mammotrophs of rat pituitary gland. Specific activators and inhibitors (other than Mg ++) have not been used in this study but experimental techniques were used to assure the specificity of the enzymes reported here. Ahmed and King (1960) found that.001 M arsenate strongly inhibits placental alkaline phosphatase and the inhibition could not be reversed by Mg ++. Since the tissues in the present study were washed in.1 M dimethyl arsenic acid (sodium cacodylate) prior to incubation, the hydrolysis of ATP is not due to the nonspecific alkaline phosphatase. Furthermore, the lack of the reaction product when the tissues were incubated either in the absence of ATP or with a lead phosphate precipitate (cloudy control) and in equimolar concentrations of sodium beta glycerophosphate indicated that the final reaction product observed in this study is not due to a nonspecific binding of lead with protein but represents the genuine activity of the enzyme ATP-ase. The hydrolysis of ATP by some glutaraldehyde resistant ATPase-like enzymes was unlikely. The precise intramitochondrial localization of ATP-ase at the electron microscope level is controversial. In our study, larger clumps of the reaction product occurred in the matrix and usually extended from the cristal membrane. Localization of the reaction product for ATP- Figures 11, 12 and 13. Animals receiving estrogen (40 days following ovariectomy) for 7 days; demonstrating the acid phosphatase activity. Figure 11. Acid phosphatase activity is discernable in cell membrane and microvilli. X23700. Figure 12. Intense acid phosphatase activity in the lysosome Figure 13. Acid phosphatase activity is visible in the Golgi complex. 15300.

1082 NAYAK AND WU ase on the cristae and the membrances of mitochondria is in accord with the data obtained by ultracentrifugation of cell homogenates (Siekevitz et al., 1958). Recently, Ogawa and Mayahara (1969) reported the precise intramitochondrial localization of ATP-ase activity with special reference to the inner membrane particles in the normal rodent cardiac mitochondria. The outer membranes and spaces were devoid of enzymatic activity. Cheetham et al. (1970) have recently demonstrated the presence of the nucleoside phosphatases activity in the Golgi apparatus fraction isolated from rat liver. Novikoff et al. (1962) and Goldfischer et al. (1964) have discussed the existence of nucleoside phosphatases in the Golgi apparatus. The enzyme activities are revealed in the saccules and vesicles but it has not been determined whether the corresponding enzymes are situated in the membrane or inside the cavities of the saccules or vesicles. An enzymatic heterogeneity was evident among the individual elements of the Golgi apparatus. The significance of this has not yet been explained. Tice and Barrnett (1963) found both di- and triphosphatases activities in the Golgi apparatus in rat testes. Localization of the reaction product for ATP-ase in the Golgi saccules is also in accord with the demonstration of the enzyme in Golgi fractions obtained by ultracentrifugation of cell homogenates (Cheetham and his associates, 1970). Nelson (1958) found that, in the rat epididymal sperm, ATP-ase activity was localized in the nine outer fibers of the axial filaments. Nagano (1965) however, reported that, in the rat sperm tail, the ATP-ase reaction product was present both in the axial filament complex and on the surface membrane of the mitochondrial helix of the middle piece. Lansing and Lamy (1961) found ATP-ase activity in the peripheral filaments of the rotifer cilia and no activity in the region near the base of the cilium. In the present study, ATP-ase activity was demonstrated under the influence of estrogen, on the ciliary membrane, the outer fibrils of cilia and basal bodies of the ciliated cells. The enzyme ATP-ase, localized in cilia, is probably involved in energy-forming reactions related to contractile mechanisms. The demonstration of ATP-ase in the basal bodies by electron microscopy has not been accomplished until the recent work of Abel (1969). Since basal bodies participate in the formation and function of cilia, one might expect that these structures contain enzymes for hydrolyzing high energy bonds. Our findings agree with the biochemical and cytochemical localization of ATP-ase in the base and shaft of cilia (Gibbons, 1967; Gibbons and Rowe, 1965; Lansing and Lamy, 1961) and in the ciliary rootlet and basal bodies of retinal rods (Matsusaka, 1967). Early studies of acid phosphatase in bovine oviduct epithelium did not correlate the enzyme with the ultrastructural organization (Bjorkman and Fredricsson, 1961). Abdalla (1968), however, using light microscopic histochemical techniques, found acid phosphatase activity in the apical parts of the nonciliated cells and their cytoplasmic projections. In the present study, the dense bodies found in the rabbit oviductal epithelium are identified as lysosomes on the basis of their cytochemically demonstrable acid phosphatase activity and by such fine structural features as their delimitation by a unit membrane and their electrondense matrix. Acid phosphatase activity was also found in the Golgi saccules, microvilli, cell membrane and secretory granules. This is in accord with the recent findings in other cell types (Smith and Farquhar, 1966; Hopkins and Baker, 1968). These findings seem to support the suggestion that acid phosphatase is probably associated with the formation of secretory products. LITERATURE CITED Abdalla, O. 1968. Observations on the morphology and histochemistry of the oviducts of sheep. J. Anat. 102:333-344. Abel, J. H., Jr. 1969. Electron microscopic demonstration of adenosine triphosphate phosphohydrolase activity in herring gull salt glands. J. Histochem. Cytochem. 17:570-584. Ahmed, Z. and E. J. King. 1960. Kinetics of placental alkaline phosphatase. Biochim. Biophys. Acta 45:581--592. Barka, T. and P. J. Anderson. 1962. Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. Histochem. Cytochem, 10:741-753. Bartoszewicz, W. and R. J. Barrnett. 1964. Fine structural localization of nucleoside phosphatase activity in the urinary bladder of the toad. J. Ultrastr. Res. 10: 599-609. Bjorkman, N. and B. Fredricsson. 1961. The bovine oviduct epithelium and its secretory product as studied with the electron microscope and histochemical tests. Zeit. Zellforsch. Mikrosk. Anat. 55:500--506. Cheetham, R. D., J. D. Moore and N. W. Yunghans. 1970. Isolation of a Golgi apparatus-rich fraction from rat liver. 1I. Enzymatic characterization and comparison with other cell fractions. J. Cell. Biol. 44:492--500.

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