Subcellular distribution of the nonspecific esterase in the mouse epididymis with special reference to regional differences.код для вставкиСкачать
THE ANATOMICAL RECORD 214:148-153 (1986) Subcellular Distribution of the Nonspecific Esterase in the Mouse Epididymis With Special Reference to Regional Differences MARCELLE-ANNE FAIN-MAUREL AND AIDA ABOU-HAILA Laboratoire de Biologie cellulaire, Universitd Rend Descartes, 75270 Paris Cedex 06, France ABSTRACT The subcellular distribution of esterases was studied in mouse epididymis by using 5-bromo-indoxyl-acetate as a substrate. In all the cells of the duct, a low level of esterase activity was detected except in one of the five segments of the head-segment Tv; in one of the three types of apical cells-the “prominent cells”; and in the “clear cells” scattered in the middle and distal parts. In these cells, the intensity of the reaction was high. The reaction product was consistently found in the endoplasmic reticulum and was more abundant in cells showing a high level of activity than in others. In cells with low esterase activity, the reaction was mainly restricted to this organelle. In highly active cells, the spectrum of subcellular locations was selectively enlarged and esterase was demonstrated in almost all cell compartments, including the cell membrane, nuclear envelope, mitochondria, lytic structures, and, more rarely in the Golgi apparatus or microvilli. These locations were dependent on cell type. A weak enzyme activity also appeared on mature spermatozoa. Numerous studies have demonstrated the structural complexity of the epididymis in various mammals: the rat (Reid and Cleland, 1957),the rabbit (Nicander, 19571, man (Holstein, 1969), the macaque (Ramos and Dym, 1977), the guinea pig (Hoffer and Greenberg, 1978), the hamster (Flickinger et al., 19781, and the mouse (Soranzo et al., 1982). A previous study of postnatal differentiation of enzyme activities in mouse epididymis has shown that an esterase activity was present in the epithelium at birth, and persisted along the undifferentiated duct at the same intensity during the first 2 weeks and then varied, leading to the regional differentiation of the adult state (Abou-Ha‘ilaand Fain-Maurel, 1986). This suggests that these later variations could be related to a differentiated pattern of gene activation associated with the maturation of the different cell types and the functional zonation of the duct. Thus, esterase appears to be a valid marker enzyme for cellular activity. In the postpubertal mouse epididymis, esterase activity detected histochemically differed across cell types (Allen and Slater, 1958; Kirkeby and Blecher, 1981) and even in histologically identical cells (Abou-Ha‘ila and Fain-Maurel, 1984). These variations are probably the expression of different functional states. They may correspond to quantitative changes, or to a different subcellular distribution in cell compartments as shown in other organs (Deimling and Bocking, 1976). For example, in the interstitial cells or prepubertal mouse testis, esterase is exclusively associated with lipid droplets whereas in the Leydig cells of testosterone-producing adult testis, the esterase reaction is predominantly in mitochondria and at cellular membranes. 0 1986 ALAN R. LISS, INC. The aim of the present investigation was to determine the subcellular distribution of esterase along the mouse epididymis by comparing cells with a low or a high level of enzyme activity. This study may provide insight into regional and cellular differences in the function of the epididymis. Furthermore subcellular location of esterase has not previously been reported in this organ. MATERIALS AND METHODS This study was conducted on six male mice (Swiss OF 1) aged about 3 months. The basic procedure was repeated three times on two animals. The male mice were anesthetized with chloral hydrate and intracardially perfused with fixative (3%paraformaldehyde and 1.5% glutaraldehyde in 20 mM collidine-S buffer pH 7.4) for 5 minutes. For each animal, the two epididymides were removed, subdivided into ten parts (five for the head, three for the corpus, two for the cauda) under a binocular microscope while in the fixative solution, fragmented, fixed for 2 hours (4OC), and washed in several changes of collidine-S buffer. The samples, chopped into minced slices, were rinsed twice in 10 mM Tris-HC1 kuffer (pH 6.5) and incubated (37OC, 70 or 90 minutes) iil Holt’s esterase medium (1958) containing 5-bromoincloxyl-acetate (Sigma) as a substrate and buffered with Tris-HC1 to pH 6.5. Controls made on each of the ten parts of one animal, included incubation of tissues in medium lacking the substrate, and secondly in the complete medium after preincubation for 30 minutes in M eserine, a n inhibitor of cholinesterases. After incubaReceived April 2, 1985; accepted September 23, 1985. 149 ESTERASE ACTIVITY IN MOUSE EPIDIDYMIS tion, the tissues were washed in Tris-HC1 buffer and then rinsed with several changes of collidine-S bufYer. The samples were postfixed in buffered osmium tetroxide (1hour a t 4OC), dehydrated, and embedded in Epon. For each animal, semithin sections (1 pm) were used to localize the different segments for electron microscopy. For each part, several ultrathin sections were examined in a Philips EM 300 without grid staining. RESULTS In the mouse, as in other mammals, the epididymis is made up of three portions: the proximal (caput), middle (corpus), and distal (cauda) parts. The proximal part was subdivided into five segments (1,-V) characterized by the cytological and histochemical features of the principal cells. In this part, scattered between the principal cells, which contained a basal nucleus, “cells with apical nuclei” were found (Abou-Hai’la and Fain-Maurel, 1984). In the middle and distal parts “clear cells” have been cytologically (Soranzo et al., 1982) and histochemically (Abou-Hai’laet al., 1985) described. The ultracytochemical study of esterase activity in the mouse epididymis led to the following results: 1) In all the principal cells of the epididymis, a low level of esterase activity was detected (Fig. l),except in segment IV of the proximal part, where the amount of reaction product was abundant (Fig. 2). However, in this segment, groups of cells having a stronger reaction than in certain neighbouring cells were seen. 2) In all the principal cells of the duct, a reaction product was found in the vesicular or saccular endoplasmic reticulum (ER). The reaction product was more abundant in cells showing a high level of activity than in others. In cells with low activity, the reaction was mainly restricted to this organelle (Fig. 1). In highly active cells, the spectrum of subcellular locations was found to be selectively enlarged and esterase was shown to be present in almost all the cell compartments: the nuclear envelope, mitochondria, Golgi apparatus, cellular membrane (Fig. a), cytoplasmic structures resembling lysosomes or residual bodies (Fig. 31, and microvilli (Fig. 4). The nuclear envelope and mitochondria presented low activity in segment 11, high activity in segment IV,and a variable pattern in the middle and distal parts. The reaction deposit was observed in the Golgi apparatus essentially in segment IV.It appeared at the cellular membrane and in the lytic inclusions in segment IV in the middle and distal parts. The microvilli showed a heterogeneously distributed reaction in the middle and distal parts. 3) Among the three types of “cells with apical nuclei” described in the five segments of the proximal part (narrow cells in segment I, prominent cells in segment 11, mitochondria-goblet cells in segments 111-V) (Abou-Haila and Fain-Maurel, 1984), only the “prominent cells” of segment I1 presented a high esterase activity in the ER, nuclear envelope, lytic inclusions, as well as in the mitochondria and basal membrane (Fig. 5a, b). In segment IV,the level of activity was lower in the “mitochondriagoblet cells” with apical nuclei than in the adjacent principal cells, but remained pronounced in the nuclear envelope and lytic inclusions (Fig. 6). The “clear cells,” scattered in the medial and distal parts, were characterized by their high pattern of enzyme activity, which was located in the ER, the nuclear envelope, and the mitochondria (Fig. 7). The lytic inclusions which were numerous in the perinuclear area of this cell type showed a marked activity on their membrane or in their contents (Figs. 7,8). The basal cells generally presented a n undetectable level of esterase activity. 4) In the lumen of the middle and distal parts, the spermatozoa showed a low pattern of enzyme activity on the mitochondria and the plasma membrane lining the acrosome and principal piece (Fig. 9). 5) In the case of incubation in the substrate-free medium, the deposition of reaction product did not occur in the cells, thus confirming the specificity of the reaction (Fig. 10). In the case of incubation in the standard medium containing eserine, the enzymatic activity of the epididymis remained unaffected (Fig. 11). DISCUSSION A preliminary electrophoretical study employing several inhibitors has shown that carboxylic ester hydrolases were the major group of esterases in the mouse epididymis (unpublished data). Thus the substrate chosen for the ultrastructural demonstration of this enzyme was 5-bromo-indoxyl-acetate,one of the more selective substrates used to study the specific hydrolysis patterns of carboxylic esters (Deimling and Bocking, 1976). Apparently incubation duration (70 or 90 minutes) did not influence the enzyme distribution in ultrathin sections. Fixation with aldehydes is the usual procedure of keeping soluble esterases in the sections. In some cases, the demonstration of the presence of esterases is feasible only after prior fixation (Niemi and Kormano, 1965). It is known that in sections fixed with glutaraldehyde, activity is weaker than after formaldehyde treatment, but the distribution of reaction product is comparable in the two cases (Bocking et al., 1973; Kirkeby and Moe, 1984). In the present study, the fixative used for ultracytochemistry contained paraformaldehyde (3%) and glutaraldehyde (1.5%)in a n attempt to obtain a satisfactory preservation of cell structures and soluble enzymes. The distribution and variations of esterases on ultrathin sections corresponded to the histochemical data obtained on cryostat section (Abou-Hai’laand Fain-Maurel, 1984). This study particularly demonstrated a high level of esterase activity in the principal cells of segment IV, which, however, showed the same ultrastructural features as the cells of segment V. It also showed some differences in the location of reaction deposit across the five segments of the proximal part. Thus, it can be postulated that each segment plays a specific role. The variations in the level of activity in the histologically identical principal cells of segment IV are probably the expression of different functional states, as shown in other tissues by biochemical and cytochemical determinations (Feustel et al., 1970; Bocking and Deimling, 1982). The main reaction, with the substrate used, is found in the ER in all cell types despite the regional differences described for the mouse epididymis (Soranzo et al., 1982; Abou-Hai’la and Fain-Maurel, 1984). This preferential location was found in many tissues and has been revealed with different substrates (cf. Deimling and Bocking, 1976). In the mouse epididymis, no difference was seen in staining patterns of the ER, which can 150 M.A. FAIN-MAUREL AND A. ABOU-HAILA Fig. 1. Ultracytochemical demonstration of the very low level of nonspecific esterase activity in the principal cells of segment I11 of the proximal part. ~ 6 , 9 0 0 . Fig. 2. Ultracytochemical demonstration of the high level of nonspecific esterase activity in the principal cells of segment Iv of the proxima1 part. Note the reaction deposits on cell membrane, ER, nuclear envelope, mitochondria (el, and Golgi apparatus (b).~ 6 , 9 0 0 . Fig. 3. Nonspecific esterase in lytic inclusions of the principal cells of the distal cauda. x 13,600. Fig. 4. Nonspecific esterase on microvilli of the principal cells of the distal part. x7,800. ESTERASE ACTIVITY IN MOUSE EPIDIDYMIS Fig. 5. Ultracytochemical demonstration of nonspecific esterase in segment I1 with a high level of activity in the apical (a) and basal (b) parts of “prominent cells.” Note the distribution of reaction product in the nuclear envelope, mitochondria, and cell membrane. a, X6,900; b, x9.200. Fig. 6. Demonstration of a higher esterase activity in a principal cell (m) than in a “mitochondria goblet-cell’’(b)in segment IV. X7,800. 151 Fig. 7. Demonstration of a higher level of activity i n the “clear cell” than in the two adjacent principal cells (proximal caudal. Note the location of reaction deposit around the lytic inclusions, on ER-rich cytoplasm, mitochondria, and plasma membrane. x 7,800. Fig. 8. High magnification of a “clear cell” basal part evidencing the location of reaction product in the RER saccular caveolae (a),perinuclear space (b),and on the membrane surrounding the lytic inclusions. ~15,000. 152 M.A. FAIN-MAUREL AND A. ABOU-HAILA Fig. 9. Location in the lumen of the epididymal medial part of the reaction product on the spermatozoa mitochondria1 sheath (*) and the plasma membrane of the principal piece. X7,800. Flg. 10. Principal cells in proximal corpus after incubation of tissue in medium lacking the substrate. ~ 7 , 8 0 0 . Fig. 11. The basal part of a “clear cell” in the distal corpus after treatmentwith 10-4Meserine.Notethedistributionofenzymaticactivity on the nuclear envelope, cytoplasm, mitochondria, residual bodies, and plasma membrane. ~ 6 . 9 0 0 . ESTERASE ACTIVITY IN MOUSE EPIDIDYMIS appear (Soranzo et al., 1982) either as vesicular, with rarely studded ribosomes in segments I and 11, or a s saccular, with numerous ribosomes in the other parts. On the contrary, the nuclear envelope which is a part of the ER presented a variable enzymatic reaction, depending on the regional differences of the canal and the various cell types. It will, therefore, be of interest to know whether these two subcellular components contain identical esterases. The location of esterase on the membrane or in the matricial substrate of the mitochondria and the lytic inclusions (lysosomes, residual bodies) could be correlated with their functional state. However, no activity was seen in the multivesicular bodies which now are considered as secondary lysosomes. Because of their esterase activities, as is the case for other enzymes (acid phosphatase, 6-glucuronidase) (unpublished data), the “prominent cells” seemed to play the same function as “clear cells,” which are implicated in reabsorption (Moore and Bedford, 1979). Even if their supranuclear cytoplasmic content was less rich in multivesicular bodies than “clear cells,” they showed a n active endocytosis process. This suggests that reabsorption occurs in the two cell types but that the ingested substances can differ. The presence of esterase activity, observed on cell microvilli and spermatozoa plasmalemma in the middle and distal parts of mouse epididymis, could reflect the selective adsorption on spermatozoa of a cell product released in the lumen. No reaction deposit was observed on acrosomes containing the “corona penetrating enzyme” (CPE), which nevertheless is believed to be a n esterase (Bradford et al., 1976a,b). Other inhibitors and methods are required to further specify individual esterases inside the cell. A preliminary study of electrophoretic banding patterns has already shown the presence of several specific esterases and isoenzymes, some of which were regulated by androgens, in tissues, luminal fluids, or spermatozoa of the different parts of the mouse epididymis (unpublished data). ACKNOWLEDGMENTS The authors wish to thank Mrs. Georgette Hedouin for her skillful technical assistance, Mrs. Myriam Largeau in typing the manuscript, and Constance Greenbaum for stylistic corrections. LITERATURE CITED Abou-Halla, A,, and M.A. Fain-Maurel (1984) Regional differences of the proximal part of mouse epididymis. Morphological and histochemical characterization. Anat. 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