J. Cell Sci. 59, 71-79 (1983) 71Printed in Great Britain © Company of Biologists Limited 1983

OSMIUM-IMPREGNATION PATTERNS OF THE

GOLGI COMPLEX IN THE EPIDIDYMAL EPITHELIAL

CELLS OF CASTRATED AND TESTOSTERONE-

INJECTED MICE

IKUO YAMAOKA, KOZO YAMAMOTO, NOBUKO URABE ANDYOSHIMI NAGATANIBiological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753, Japan

SUMMARY

After prolonged exposure of mouse epididymal epithelial cells to a solution of osmium tetroxide,reduced osmium compounds were detected in the Golgi cisternae and in the cytoplasm adjacent to theGolgi complex. Their appearance changed in time under certain conditions. Eight days after castra-tion each cisterna of the regularly arranged Golgi lamellae fragmented into small vesicles, in whichdeposits of reduced osmium compound were in reduced amounts or completely absent, but no no-table decrease of the fine reduced osmium particles in the cytoplasm adjacent to the Golgi complexoccurred. The amount of deposit in the Golgi cisternae in castrated mice recovered to the normal levelafter the subcutaneous injection of testosterone for two weeks. On the other hand, the osmium part-icles observed in the cytoplasm adjacent to the Golgi complex during the recovery process of thelamellar structure increased in amount. This study showed that the reduced osmium compoundswere of two kinds, and that the deposits contained in the Golgi cisternae were related to secretoryproducts, and might be controlled by hormonal factors, but the fine reduced osmium particles thatappeared in the cytoplasm adjacent to the Golgi complex might be regulated by other factors.

INTRODUCTION

Since the osmium-impregnation technique as a cytological marker for the Golgicomplex in electron microscopy was introduced by Dalton & Felix (1954, 1956) somefurther evidence related to the distribution of osmium deposits in the Golgi complexhas appeared; deposits of reduced osmium occurred primarily in one or two cisternaeof the outer face of the Golgi complex (Friend & Murray, 1965; Friend, 1969;Novikoff, Novikoff, Quintana & Hauw, 1971), but no evidence related to biologicalfunction has been produced. In a variety of animal tissues some workers have reportedthat osmium deposits are found also within the nuclear envelope, cisternae of theendoplasmic reticulum, mitochondria and the vesicles of multivesicular bodies(Malhotra & Meek, 1960; Friend & Brassil, 1970; Winborn & Seelig, 1974). Thispaper describes the development and decay of osmium deposits in the Golgi cisternaeand the cytoplasm surrounding them in mouse epididymal epithelial cells after castra-tion and treatment with androgen.

MATERIALS AND METHODS

Three-month-old mice (dd strain) were purchased from a dealer, and kept at 25 °C until required.The tissues of the epididymis were harvested after the following treatments. Mice were killed at 4,

72 /. Yamaoka, K. Yamamoto, N. Urabe and Y. Nagatani

8 and IS days after castration. Eight days after castration some individuals were subcutaneouslyinjected daily for one or two weeks with 0-2 ml of sesame oil containing 100 ng testosterone (Merck),and were then killed. Control experiments were designed as follows: unoperated mice were com-pared with the other experimental groups; castrated mice were compared with mice injected withsesame oil, containing or not containing testosterone after castration, and mice injected with sesameoil were compared with the hormone-injected individuals. Five mice were used in each experiment.

The middle segment of the anterior epididymis was dissected out and fixed at 4°C for 3 h inCaulfield's fixative (pH 7-4) with 4-5% added sucrose (Caulfield, 1957). After being dehydratedin a series of ethanol gradients and acetone, it was embedded in Epon 812. Some specimens weretreated by the following procedure before dehydration: the tissue was incubated after fixation in anunbuffered 2% OsO4 solution at 37 °C for 48 h.

Ultrathin sections were stained with aqueous uranyl acetate and lead citrate. The post-osmicatedsections were stained with lead citrate only. They were examined with a JEM-100 C electronmicroscope (Jeol, Ltd).

RESULTS

General structure and osmium deposition

The epithelial cells of the middle segment tubule of the epididymis in normal micehad a well-developed Golgi complex lying in the supra-nuclear region (Fig. 1A). Itconsisted of ten or more parallel lamellae, arranged in a circular or semi-circular form.Discontinuities in the outer four or five lamellae were sometimes observed, andcisterna of the endoplasmic reticulum penetrated into the gaps (Fig. 1B) . On the innerconcave face of the Golgi complex many vesicles containing moderately electron-dense materials were observed.

After prolonged exposure of the tissue to a solution of osmium tetroxide, reducedosmium compounds appeared in the Golgi cisternae and the cytoplasm adjacent tothem. The pattern of deposition in the Golgi cisternae was classified as follows: either(1) all cisternae contained the reduced osmium compounds (Fig. 2A); or (2) one ortwo cisternae on both the outer convex face and the inner concave face of the Golgicomplex contained reduced osmium compounds (Fig. 2B). In both cases manyreduced osmium particles (about 50 nm in diameter) were seen in the cytoplasmadjacent to the Golgi complex (Fig. 2, arrows).

Effects of castration

Four days after castration, no significant change in the fine structure or number ofthe Golgi cisternae was observed, but the reduced osmium compounds in the Golgicisternae decreased in amount. The reduced osmium particles were observed in theouter convex cisternae of the Golgi complex (Fig. 3A). At the same time muchreduced osmium was observed in the endoplasmic reticulum in the basal region of thecell.

Eight days after castration the number of cisternae had decreased and many hadfragmented into small vesicules. The reduced osmium compounds of the Golgi cister-nae had disappeared (Fig. 3B), but the particulate deposits in the adjacent cytoplasmusually remained. In the most degenerated Golgi complex, the particular depositsadjacent to it could not be observed.

Osmium-impregnation patterns of the Golgi complex 73

Fig. 1. General structure of the epididymal epithelial cell in normal mouse. The epithelialcell is characterized by a well-developed Golgi complex at the supranuclear region (Fig.1 A) . B shows part of the Golgi complex. It is composed of about ten flattened cisternae withmany small vesicles adjacent to the convex face. Intrusions of endoplasmic reticulumcisternae into gaps in the Golgi lamellae are shown, n, nucleus; gc, Golgi complex; er,endoplasmic reticulum; t;, vesicles, A, X 4000; B, X 35 000.

CEL59

/. Yamaoka, K. Yamamoto, N. Urabe and Y. Nagatani

Fig. 2. Two types of distribution of reduced osmium compounds in the Golgi complex.Either all cisternae contain the reduced osmium compounds (A), or the central four or fivelayers of the cisternae lack osmium compounds (B). In both cases reduced osmium par-ticles (arrows) are shown adjacent to the outer face of the Golgi complex. X 15 000.

Osmium-impregnation patterns of the Golgi complex

3A

•?

•n

B * rFig. 3. Distribution of the reduced osmium compounds in the Golgi complex of castratedmouse epididymal epithelial cells. Four days after castration the reduced osmium com-pounds in the cisternae disappear, and many reduced osmium particles remain adjacentto the outer face of the Golgi complex (A). Eight days after castration some scatteredreduced osmium particles remain adjacent to the disintegrated Golgi complex (B). n,nucleus. X 12500.

76 /. Yamaoka, K. Yamamoto, N. Urabe and Y. Nagatani

Effects of injection of testosterone

When testosterone was subcutaneously injected daily for 1 week from 8 days aftercastration, the regularly arranged flattened cisternae recovered to the state seen innormal mice. This occurred before the recovery of reduced osmium deposits. Thenormal distribution of reduced osmium compounds in the Golgi cisternae was notseen until 2 weeks after treatment with androgen. Many deposits were observed in allcisternae of the Golgi lamella, and many reduced osmium particles also appeared inthe cytoplasm adjacent to the Golgi complex (Fig. 4). At the same time the depositionin the endoplasmic reticulum of the basal region of the cell disappeared. In the controlmice injected with sesame oil, the number and structure of the lamellae were similarto those of normal mice. Reduced osmium compounds were not observed in the Golgicisternae, but many reduced osmium particles were present in the cytoplasm adjacentto the Golgi complex (Fig. 5).

DISCUSSION

Since Dalton & Felix (1954, 1956) reported that in the mouse epididymal epithelialcell all three elements of the Golgi complex (lamella, vacuoles and vesicles) werestained by a post-osmication technique, the technique has been widely used as amarker for the Golgi complex in electron microscopy. In various types of cell thedistribution of reduced osmium compounds was usually limited to a few outer convexcisternae of the Golgi complex (Friend & Murray, 1965; Friend, 1969; Novikoff etal. 1971; Hand, 1980).

Kurosumi (1970) found that all cisternae and vesicles of the Golgi complex in themouse epididymal epithelium were blackened by the osmication technique in a fewcases. Although we also obtained similar staining results our observation suggests thatthe stainability depends on the specific nature of the epididymal epithelial cell. Twokinds of reduced osmium compounds were detected in and around the Golgi complex:one detected in the Golgi cisternae themselves, the other located adjacent to the Golgicomplex. In the early period after castration the former deposits disappeared from theGolgi cisternae, but the latter deposits persisted until the Golgi lamella completelydisintegrated.

Castration and hypophysectomy of the male mouse resulted in contraction of theaccessary sex organ (Allen & Slater, 1957; Honjin, Uozu& Nakanishi, 1960). Honjinet al. (1960) reported that the Golgi lamella of the epididymal epithelial cell contrac-ted after castration. Our results showed that segmentation of the Golgi lamellaoccurred 8 days after castration, and at about the same time the reduced osmiumcompounds in the Golgi cisternae disappeared.

Friend (1969) reported that in the epididymal epithelial cell the reduced osmiumcompounds were detected in the vesicles within multivesicular bodies and adjacent tothe outer face of the Golgi complex, and concluded that the multivesicular bodieswere formed by Golgi vesicles. Although our results do not agree with his description,several changes in the distribution of the reduced osmium compounds were also

Osmium-impregnation patterns of the Golgi complex 77

*

* - i « ^ ^

Fig. 4. Effects of testosterone injection on the reappearance of reduced osmium com-pounds. All cisternae contain the reduced osmium compounds, and many reduced os-mium particles are present adjacent to the Golgi complex. X 12500.

Fig. 5. Recovery of the lamellar structure and many reduced osmium particles followinginjection of sesame oil. Reduced osmium compounds in the cisternae are not present.X 12500.

78 /. Yamaoka, K. Yamamoto, N. Urabe and Y. Nagatani

observed in the cytoplasm. In normal mice many vesicles containing reduced osmiumwere observed at the cell apex, but in the castrated mice many deposits appeared inthe endoplasmic reticulum of the basal region of the cell. On the other hand testos-terone injection produced the same results as in normal mice. These results show thatthe changes in reduced osmium deposition in the Golgi cisternae and endoplasmicreticulum relate to the secretory activity in the epididymal epithelial cell.

The observation that there are many reduced osmium particles in close contact withthe Golgi complex is a new rinding. The particles were most readily detectable duringthe recovery process of the Golgi lamellar structure after testosterone injection.Similar particles had been observed in the acinar cell of Brunner's gland of the mouse(Friend & Murray, 1965; Nagatani & Yamaoka, 1967). Although he did not mentionit, similar particles are also visible in fig. 71 of Fawcett (1967). Hand & Oliver (1971)have reported diagramatically some cytochemical features of the Golgi complex, andhave shown that one of the reduced osmium compounds existed within some smallvesicles of the outer Golgi saccules. Although no cytochemical tests were carried outin the present work, some speculations can be presented here. In a previous paper,we reported that many deposits adjacent to the Golgi complex were detectable by asilver-impregnation technique (Nagatani & Yamaoka, 1963). Their distribution issimilar to that of the fine reduced osmium particles obtained by the osmium-impregnation technique. It is suggested that the reduced osmium particles may bechemically similar to the deposits seen by the silver-impregnation technique.

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{Received 25 February 1982)