Effect of castration and testosterone replacement on high glucose 6-phosphatase activity in principal cells of the mouse epididymis.код для вставкиСкачать
THE ANATOMICAL RECORD 207239-295 (1983) Effect of Castration and Testosterone Replacement on High Glucose 6-Phosphatase Activity in Principal Cells of the Mouse Epididymis KAZUO KANAI, SHINSUKE KANAMURA, J U N WATANABE, MAR1 ASADA-KUBOTA, AND MOTOKO YOSHIKAWA Department of Anatomy, Kansai Medical University, Fumizonecho I , Morigguchi, Osaka 570, Japan ABSTRACT Glucose 6-phosphatase activity is higher in the principal cell than in other cell types in the terminal segment and caudal half of the middle segment of the mouse epididymis. Effect of castration and testosterone replacement on the high enzyme activity in the principal cell was studied in the terminal segment and the caudal half of the middle segment (cytochemical study), and in the whole epididymis (biochemical study). Ten, 20, or 30 days after castration, the abundant amount of reaction product seen in principal cells from intact control animals decreased to the level in basal cells, halo cells, and smooth muscle cells. However, in animals treated with testosterone following castration, the reaction product in principal cells remained abundant. Changes in the biochemical activity after castration or testosterone administration following castration paralleled the cytochemical results. Thus, the high activity in the principal cell is under the control of testosterone. The ductus epididymidis is the site of maturation (initial and middle segments) and storage (terminal segment) of spermatozoa (Glover and Nicander, 1971; Hamilton, 1975; Brooks, 1981). The principal cell is the major constituent of the ductus epididymidis. Because of well developed rough endoplasmic reticulum and Golgi apparatus, although there are no secretory granules and no indication of exocytosis, the principal cell is believed to be a secretory cell (Hamilton, 1975; Bloom and Fawcett, 1975; Nicander and Malmqvist, 1977; Flickinger, 1979; Brooks, 1981). On the other hand, morphological evidence of absorption has been demonstrated in the principal cell (Nicander, 1965; Friend and Farquhar, 1967; Hoffer et al., 1973; Hamilton, 1975; Brooks, 1981). Thus, the functions of the principal cell are not completely explored and understood. Recently, we observed that in the terminal segment and caudal half of the middle segment of mouse epididymis, glucose 6-phosphatase (G6Pase) activity was apparently higher in the principal cell than in other cell types (Kanai et al., 1981). In the present study, we examined the effect of castration and testosterone administration following 0 1983 ALAN R. LISS, INC. castration on G6Pase activity in the cells of the terminal segment and caudal half of the middle segment (cytochemical study), and in the whole epididymis (biochemical study) in order to know whether the high G6Pase activity in the principal cell is androgen dependent. MATERIALS AND METHODS Male dd-Y mice, about 3 months old, were used. The animals had free access to food and water prior to the experiments. Animals were divided into three groups. Animals of one group were bilaterally castrated. Those of the second group were bilaterally castrated and immediately injected intraperitoneally (IP)with testosterone propionate in a dose of 1 mg per kg body weight once a day. Those of the third were intact. Castration was carried out under Nembutal anesthesia. Treated animals were killed by cervical dislocation 10,20, or 30 days after castration. Cytochemical Method Terminal segments and caudal halves of the middle segments were fixed by immerReceived October 4,1982; accepted June 6, 1983. 290 K. KANA1 ET AL. sion in 2% glutaraldehyde-0.1 M sodium cacodylate (pH 7.2) for 45 minutes at 4"C, and immersed in 0.1 M sodium cacodylate-8% sucrose for 1 hour at 4°C. The tissues were sectioned at 30 pm with a freezing microtome and incubated for 1 hour a t room temperature in a reaction medium (3.7 mM glucose 6-phosphate, 80 mM sodium cacodylate, 3.6 mM lead nitrate, 230 mM sucrose, pH 6.71, modified from the Wachstein and Meisel medium (1956). The sections were post-fixed in buffered osmium tetroxide, dehydrated and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate, and examined in a Hitachi 500 electron microscope. For light microscopic observations, fresh frozen sections of the epididymides were incubated in the reaction medium for 1hour a t room temperature, washed in distilled water, immersed briefly in ammonium sulfide, washed again in distilled water, and mounted in glycerine jelly. Biochemical Methods Whole epididymides were homogenized a t 4°C in 50 volumes of 0.25 M sucrose in a Potter-Elvehjem type homogenizer for 1.5 minutes a t 2000 rpm. The homogenates were centrifuged at 2000 rpm for 15 minutes at 4"C, and assayed for G6Pase activity according to the method described by Leskes et al. (1971). Levamizole (10 mM) was added to the reaction mixture for inhibiting alkaline phosphatase activity (Van Belle, 1972). Incubation time was 30 minutes. The inorganic phosphorus released was determined by the method of Fiske and SubbaRow (1925). Protein was estimated according to the method of Lowry et al. (1951). Enzyme activity was expressed as nanograms of phosphorus liberated per minute per milligram of protein of ductus epididymidis. All of the data were subjected to statistical analysis (Student's t-test). RESULTS treated with testosterone following castration. In normal animals, the reaction product for G6Pase activity was observed in the endoplasmic reticulum and nuclear envelope of all cell types composing the terminal segment and caudal half of the middle segment. The amount of reaction product was apparently more abundant in principal cells than in basal cells, halo cells, and smooth muscle cells (Fig. 1).Ten, 20, or 30 days after castration, the abundant reaction product seen in principal cells from intact control animals decreased to the level of basal cells, halo cells, and smooth muscle cells and, therefore, the amount of reaction product appeared similar in the four cell types (Figs. 2, 3). In animals treated with testosterone following castration, however, the reaction product in principal cells remained abundant until 30 days after castration (Fig. 4). The deposition of final product was also observed in some lysosomes of principal cells and basal cells. Golgi apparatus, mitochondria, plasma membrane, and other organelles in the four cell types showed no reaction product. In order to ascertain whether the reaction product is due to G6Pase activity, control experiments were carried out as described previously at light and electron microscopic levels (Kanamura, 1975). Immersion of the glutaraldehyde-fixed sections in 0.1 M acetate buffer (pH 5.0) a t 37°C for 15 minutes before incubation in the reaction medium, and the use of P-glycerophosphate in place of glucose 6-phosphate in the reaction medium, resulted in complete absence of the reaction product except in lysosomes of principal cells and basal cells. Pre-incubation of the sections in 0.25 M sucrose containing 1mM Cu2+, 10 mM Zn2', 10 mM F- or 10 mM CN-, and then incubation in the reaction medium containing equal moles of these ions, caused a loss of the histochemical staining. These results indicate that the reaction product in the endoplasmic reticulum and nuclear envelope is due to G6Pase activity, but the deposition of final product in lysosomes of principal cells and basal cells is probably related to acid phosphatase activity. Cytochemical Results Castration caused regressive changes in the fine structure of principal cells, i.e., reduction in the height and number of apical miBiochemical Results crovilli, and in the amount of rough endoProtein (pg) per wet weight (mg) of plasmic reticulum. These regressive changes epididy mis appeared more pronounced with time after castration. However, no morphologic changes Ten days after castration, the value deoccurred in principal cells from animals creased (from 90.3 f 1.45 to 55.2 f 7.53, the GGPASEIN EPIDIDYMIS Figs. 1-4. Cytochemical demonstration of G6Pase activity in the cells of the mouse epididymis. Sections (30 fim) cut from glutaraldehyde-fixed tissues were incubated in a modified Wachstein and Meisel medium for 1 hour. Figures 2-4 appear on following pages. Fig. 1. Principal cells (PI, a basal cell (B), and smooth mean of 5 animals S.E.M., P < 0.01) and then did not change significantly. Testosterone administration after castration maintained the protein level of the intact control animals. G6Pase activity The value decreased to 35% (P < 0.001) of that of the intact animals 10 days after castration, and thereafter remained unaltered 291 muscle cells (S) of caudal half of the middle segment from a n intact control animal. The reaction product for GGPase activity is seen in the endoplasmic reticulum and nuclear envelope of these cells. Note that deposition of reaction product is apparently more abundant in principal cells than in basal cell and smooth muscle cells. x5,500. (Fig. 5). In animals treated with testosterone following castration, the value was unchanged except €or a decrease (P < 0.05) between 10 and 20 days. When the activity was expressed as nanograms of phosphorus per minute per wet weight of epididymis, the difference in the pattern of changes in the activity between castrated animals and testosterone-treated animals after castration became more distinct. This is due to the 292 K. KANA1 ET AL. GGPASE IN EPIDIDYMIS 293 Fig. 4. Principal cells (P), a basal cell (B), and smooth muscle cells (S) of caudal half of the middle segment from an animal treated with testosterone (1 mgkg, once a day) during 30 days after castration. The reaction product for the activity is apparently more abundant in principal cells than in basal cell and smooth muscle cells (cf. Fig. 1).~ 8 , 0 0 0 . marked decrease in protein concentration in the epididymis after castration. tains the abundant amount of reaction product in principal cells, if animals are castrated. Further, the biochemical activity of whole epididymis parallels generally these cytochemical results. Therefore, the high G6Pase activity in principal cells of the mouse epididymis is androgen dependent. A marked regressive change occurred in the endoplasmic reticulum of principal cells after castration. Therefore, the reduction of G6Pase activity in principal cells after castration may be related to the regressive change in the endoplasmic reticulum. Brooks (1976a,b, 1978, 1979, 1981) showed that castration results in a decline in the activity of a number of enzymes associated with glycolysis, tricarboxylic acid cycle, lipid oxidation, and others in the epididymis. Alkaline phosphatase (Allen and Slater, 1957; Ono, 1973; Sreenivasulusetty et al., 1977), aliesterase DISCUSSION As revealed in the present cytochemical results, castration results in an apparent reduction of the amount of reaction product for G6Pase in principal cells to the level in basal cells, halo cells, and smooth muscle cells. However, testosterone administration main- Figs. 2, 3 . Principal cells (P) and basal cells (B) of caudal half of the middle segment from animals 10 days (Fig. 2) and 30 days (Fig. 3 ) after castration. Note a reduction in the amount of reaction product for the activity in principal cells compared to that in principal cells in Figure 1. The amount of reaction product appears similar in principal cells and basal cells. ~ 5 , 5 0 0 . 294 K. KANA1 ET AL. I 0 10 20 30 Days a f t e r castration Fig. 5. Effect of castration and testosterone administration following castration on G6Pase activity. Testosterone was injected in a dose of 1 mgkg once a day. Each point represents the mean of 5 animals and the stan- dard error is shown as a vertical line. * P < 0.05, ** P i0.01, *** P < 0,001: significantly different from value of animals castrated and injected with testosterone. (Allen and Slater, 1957), acid phosphatase (Allen and Slater, 1958; Sreenivasulusetty et al., 1977) and hyaluronidase (Sreenivasulusetty et al., 1977) activities in the epididymis or principal cell also decrease after castration. Brooks (1981)considered that the reduction in enzyme activity reflects a decrease in the number of enzyme molecules rather than in the catalytic activity of each enzyme molecule, because the reduction in activity of the affected enzymes is relatively slow (about 12 days) after castration. The reduction in enzyme molecules is effected when their rate of degradation is more than their rate of synthesis in the endoplasmic reticulum. In the regressed endoplasmic reticulum after castration, the decline in the enzyme activities occurs probably by such a mechanism. This situation is probably applicable to G6Pase. G6Pase activity has been detected in a variety of cell types of various organs. The activity is high in hepatocytes (Tice and Barrnett, 1962; Kanamura, 1971a; Leskes et al., 19711,proximal convoluted tubule cells of the kidney (Kanamura, 1971b1, and jejunal epithelial cells (Hugon et al., 1970, 1971). The activity of hepatocytes and proximal convoluted tubule cells is concerned with supplying glucose to the blood from the liver and kidney (Krebs, 1963; Nordlie, 1969), and that of jejunal epithelial cells might relate to the absorption of nutrient. However, the function of this enzyme is unknown in other various cell types containing low activity. In the terminal segment and caudal half of the middle segment, the activity is apparently higher in the principal cells than in basal cells, halo cells, and smooth muscle cells. Therefore, it is supposed that the enzyme in principal cells performs a specific function like the enzyme in hepatocytes and proximal convoluted tubule cells. As revealed in the present results, the high G6Pase activity in principal cells appears to be under the control of testosterone. Therefore, it is suspected that the enzyme plays some role in the maturation and storage of the spermatozoa in the ductus epididymidis. The spermatozoa use fructose in the semen as an energy source for their movement (Mann, 1964).However, the spermatozoa may need glucose during their maturation and storage in the ductus epididymidis. The appearance of glucose has been reported in the G6PASE IN EPIDIDYMIS epididymal fluid (Cooper, 1982). 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