Intermediate filaments in Sertoli cells.

MICROSCOPY RESEARCH AND TECHNIQUE 2050-72 (1992)

Intermediate Filaments in Sertoli Cells G. AUMffLLER, C. SCHULZE, AND C. VIEBAHN Department of Anatomy and Cell Biology, Philipps-Uniuersitat, 0-3550 Marburg (G.A.); Department ofdnatomy, Universztat Hamburg, 0-2000 Hamburg (C.S.); Department ofdnatomy, Rheinische Friedrich-Wilhelms-UniversitatBonn, 0-5300 Bonn (C.V.), Federal Republic of Germany

KEY WORDS

Vimentin, Cytokeratin, Testis, Cytoskeleton

ABSTRACT Using immunohistochemical techniques both at light and electron microscopic levels, the arrangement and distribution of intermediate filaments in Sertoli cells of normal testis (in rat and human), during pre- and postnatal development (in rabbit, rat, and mouse) and under experimental and pathological conditions (human, rat), have been studied and related to the pertinent literature. Intermediate filaments are centered around the nucleus, where they apparently terminate in the nuclear envelope providing a perinuclear stable core area. From this area they radiate to the plasma membranes; apically often a close association with microtubules is seen. Basally, direct contacts of the filaments with focal adhesions occur, while the relationship to the different junctions of Sertoli cells is only incompletely elucidated. In the rat (not in human) a group of filaments is closely associated with the ectoplasmic specializations surrounding the head of elongating spermatids. Both in rat and human, changes in cell shape during the spermatogenic cycle are associated with a redistribution of intermediate filaments. As inferred from in vitro studies reported in the literature, these changes are at least partly hormone-dependent (vimentin phosphorylation subsequent to FSH stimulation) and influenced by local factors (basal lamina, germ cells). Intermediate filaments, therefore, are suggested to be involved in the hormone-dependent mechanical integration of exogenous and endogenous cell shaping forces. They permit a cycledependent compartmentation of the Sertoli cell into a perinuclear stable zone and a peripheral trafficking zone with fluctuating shape. The latter is important with respect to the germ cellsupporting surface of the cell which seems to limit the spermatogenetic potential of the male gonad. INTRODUCTION Cell Biology of Intermediate Filaments Intermediate filaments (IF) are fibrous elements of about 10 nm (range 7-11 nm) thickness which, along with microfilaments (diameter about 6 nm) and microtubules (diameter about 20-22 nm), constitute the elementary components of the cytoskeleton. They are present in most vertebrate cells (Franke et al., 1978; Bennett et al., 1978) and have also been found in invertebrate (Bartnik et al., 1985) and plant cells (Dawson et al., 1985). They are markedly insoluble over a wide range of pH and ionic strength. According to their physical, chemical, immunological, and histogenetic properties, IF form a large and diverse family of cytoplasmic proteins (Steinert et al., 1984) that can be subdivided into 6 major members: keratin(s), vimentin, desmin, glial fibrillary acidic protein, neurofilaments, and peripherin (see Altmannsberger and Osborn, 1989). The largest subgroup of IF proteins are the keratins representing about 20 different forms (Moll et al., 1982), identified by two-dimensional gel electrophoresis in various human epithelia. Additional 8 "hard" keratin polypeptides were found in cells from hair follicles (Lynch et al., 1986). A given epithelium usually expresses subsets of 2-10 keratin polypeptides depending on the state of cellular differentiation and the location of the cell. The use of monoclonal antibodies monospecific for a single keratin polypeptide has al-

0 1992 WlLEY-LISS, INC.

lowed a further subdivision of normal epithelia and their respective tumors by immunohistochemical methods (Nagle, 1988; Fischer et al., 1987; Gown and Vogel, 1984). Vimentin (55-58 kDa; Franke et al., 1978) filaments are characteristic of mesenchymal cell types and nonmesenchymal cell types (Franke et al., 1979). Under in vitro conditions, vimentin filaments are expressed in cultured cells of epithelial and myogenic origin. They can occur simultaneously with intermediate filaments of the prekeratin-or desmin-type, respectively (Franke et al., 1979). Assembly and disassembly of vimentin filaments have recently been reported to be controlled by phosphorylation-dephosphorylation events (Inagaki et al., 1987; Lamb et al., 1989). Reversible phosphorylation may therefore be a means involved in the regulation of vimentin pol merization. Smaller fragments may be formed by Ca'+ -activated proteinases (Traub et al., 1987). Desmin (55 kDa) is the intermediate filament protein characteristic of cardiac, skeletal, visceral, and many but not all vascular smooth muscle cells (Lazarides and Balzer, 1987). In the testis, it has been detected in peritubular myoid cells (Virtanen et al., 1986).

Received February 28, 1990; accepted in revised form May 29, 1990. Address reprint requests to Dr. G. Aumiiller, Department ofAnatomy and Cell Biology, Robert-Koch-Str. 6, D-3550 Marburg, Federal Republic of Germany.

INTERMEDIATE FILAMENTS IN SERTOLI CELLS

The neurofilament triplet (68 kDa, 160 kDa, 200 kDa), the newly described peripherin, and the glial fibrillary acidic protein (52 kDa) represent the IF group typical of the various components of the nervous system (see Altmannsberger and Osborn, 1989). Desmin and vimentin, together with glial fibrillary acidic protein (GFAP), form a subgroup of closely related non-epithelial (or type 111)IF proteins. Sequence studies, inaugurated with the desmin molecule (Geisler and Weber, 19821,showed that IF molecules contain a central a-helical rod domain, some 310 residues in length, which is highly conserved. In contrast, the terminal domains (the head and the tail) are variable both in sequence and length. These domains of the molecule account for the differences in function, stability, and epitope characteristics of the different IF proteins. A comparison of cDNA sequences coding for different IF proteins shows a more or less conserved number and position in the genes coding for the keratin groups 1-11 and group I11 (desmin, vimentin and GFAP), while neurofilament proteins seem to form a group of their own (group IV; Lewis and Cowan, 1986). cDNA sequences of hamster desmin and vimentin have been published by Bloemendal et al. (1985). IF are usually centered around the nucleus where they form a basket-like network. There are indications that IF (vimentin in avian erythrocytes: Georgatos and Blobel, 1987) bind to lamin, an IF-like protein of the nuclear lamina (Gerace et al., 1984). From the perinuclear region IF radiate towards the cell surface where they interact directly or by means of intermediate-filament-associated-proteins (IFAPs). In the case of the avian erythrocyte system ankyrin has been identified as the essential linker (Georgatos et al., 19851, while desmoplakins (Jones and Goldman, 1985) or associated cell adhesion molecules (Yang et al., 1985; Volk and Geiger, 1986; Tsukita et al., 1989; Gumbiner et al., 1988) present in adhesion zones have been envisaged as possible candidates. IF have been shown to interact with microtubules (MT) and microfilaments (MF). Especially MT determine the distribution of IF, the latter always running in parallel with MT. Following depolymerization of MT (prior to mitosis and in the presence of MT disrupting agents) IF collapse into a dense mass around the nucleus (Blose et al., 1984). While IF orientation is dependent on the course of MT, the reverse is not the case, Klymkowski (1981) reported that collapse of IF (induced by antibody treatment) occurs without obvious effects on cell shape or motility, that is, IF can be disrupted but MT (and MF) arrangement remains unaltered. Despite many recent new findings the functional significance of IF remains unclear. It has been suggested that IF may play a cytoskeletal role in being “mechanical integrators of cellular space” (Lazarides, 1980). ln conjunction with MT and MF they may be involved in intracellular transport. As they appear to be associated with the nuclear lamina on the one hand and the cell surface on the other hand, they may be instrumental in nucleus-cell-surface interactions (Goldman et al., 1986).Based on their findings that all non-epithelial IF subunit proteins interact with nucleic acids and his-

51

tones, Traub et al. (1987) defined an alternative concept and supposed that IF proteins might fulfil nuclear functions. As a unifying hypothesis, the idea has been proposed “that IF and their associated proteins may comprise the cell type specific molecular infrastructure that is involved in transmitting and distributing information amongst major cellular domains: the cell surface/extracellular matrix, the cytoplasm and the nuclear surface/nuclear matrix” (Goldman et al., 1986).

Methodological Considerations The most frequently used approaches to study the distribution and arrangement of IF are electron microscopy of thin sections and immunohistochemistry . The latter is more favorable, since the use of isolated cells or squash preparations (see Franke et al., 1979) allows a three-dimensional view of the arrangement of IF (Figs. 12-14). The results of immunohistochemical studies clearly depend on the quality of the antibody used. Highly selective monoclonal antibodies against vimentin have been developed (Virtanen et al., 1985; Kierszenbaum et al., 19861, which are superior to most commercial polyclonal antibodies. Often, the monoclonal antibodies operate only in a species specific manner. Using four different (commercially available) monoclonal antibodies against vimentin, we achieved a positive immunoreaction in human testis material, but not in rat testis. A polyclonal antibody against vimentin, kindly provided by Prof. D. Drenckhahn (Department of Anatomy and Cell Biology, Univ. Wiirzburg) on the other hand, worked not only in all species tested, but also in osmium-treated, epon-embedded material (Aumuller et al., 1988; for details of the antibody see Drenckhahn and Wagner, 1986). Western blotting analysis of the antibody employed should be used in any case to assess the specificity of the antibody (Amlani and Vogl, 1988; Alvarez-Buylla et al., 1987). Functional studies, such as phosphorylation experiments (Spruill et al., 1983a,b) are useful as indicators of regulatory effects, but they have to be supplemented by morphological (immunohistochemical) experiments. Very instructive preparations of intermediate filament distribution are the so-called IF-enriched cytoskeletsons (Starger and Goldman, 1977; Zackroff and Goldman, 1979)which allow the discrimination of perinuclear and subsurface portions of the intermediate filament network. They can be used either as whole mount preparations or stained with tannic acid, embedded, and thin-sectioned (Amlani and Vogel, 1988). Significance of Intermediate Filaments in Testis Biology The dual function of the testis as an endocrine and an exocrine gland requires a highly specialized cytology confined to the interstitial (endocrine) and tubular (exocrine) compartment. The different cells interact at different levels and over different distances (for review, see de Kretser and Kerr, 1988; Bardin et al., 1988). The cytoskeletal filament system as well as the extracellular matrix form a common superstructure within the elements of the different compartments. Three intermediate filament types are essential in the testis: vimentin, keratin, and desmin.

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Franke et al. (1979) have reported the presence of vimentin in vascular endothelium, Leydig cells, fibrocytes, lymphocytes, peritubular cells, and particularly in Sertoli cells (rat: Franke et al., 1978, 1979; Virtanen et al., 1986; Paranko et al., 1986; Kierszenbaum et al., 1986; Mali et al., 1987; Amlani and Vogl, 1988; ground squirrel: Vogl et al., 1983; pig and dog: van Vorstenbosch et al., 1984; human: Aumiiller and Peter, 1986; Aumiiller et al., 1988),but it has also been described in spermatids (Virtanen et al., 1986; Ochs et al., 1986). Keratin is present in the rete testis epithelium of the rat (Tung et al., 1987) and in Sertoli cell precursors (Paranko et al., 1986). Desmin has been described in rat peritubular cells by Virtanen et al, (1986), but has been missed in this location by van Vorstenbosch et al. (1984) and Kierszenbaum et al. (1986). In Sertoli cells which play a key role in spermatogenesis IF are essential for several reasons: 1. Information exchange. Cell-to-cell interaction in the testis occurs between Leydig and Sertoli cells (Bergh, 1983; Morrera et al., 1988; Saez et al., 1987; Perrard-Sapori et al., 1986) in a paracrine fashion and in a similar way between peritubular and Sertoli cells (Verhoeven and Cailleau, 1988; Ailenberg et al., 1988) as well as between Sertoli cells and germ cells (Means et al., 1976; Galdieri et al., 1983,1984; La Maguerresse et al., 1986; Kumari and Duraiswami, 1987). The possible role of intermediate filaments (e.g., in exposing receptors at the respective plasma membranes during volume/size changes in the contact area of the Sertoli cells) is conceivable. 2. Histogenesis and differentiation.In the male genital system the co-expression of vimentin with keratin has been reported (epididymis: Kasper and Stosiek, 1989; prostate: Wernert et al., 1987). The early findings of vimentin immunoreactivity in Sertoli cells by Franke et al. (19791, recently corroborated by Amlani and Vogl (19881, have prompted these authors to assume a mesenchymal origin of Sertoli cells. Contrary to that, Paranko et al. (1986) have pointed to the primary expression of keratin in Sertoli cell precursors. IF immunohistochemistry therefore has been used t o verify the dual origin (Wartenberg et al., 1989) of Sertoli cell precursors (kinetics: see Orth, 1982; Miething, 1989). The same approach has been used to identify local variants of Sertoli cells (e.g., in tubuli recti) and their relationship to rete testis cells (Tung et al., 1987). This is particularly interesting with regard to the histogenesis of gonadal tumors and the relationship of Sertoli and granulosa cell tumors (Gown and Vogel, 1984; Czernobilsky et al., 1985; Benjamin et al., 1987; Miettinen et al., 1985). 3. Parameters of Sertoli cell function. The morphological observation of distinct stages of spermatogenesis (Clermont, 1963, 1972) have made possible the detection of a cyclic variation of a number of biochemical and endocrine functions in the seminiferous epithelium (see Parvinen et al., 1986). One intracellular system which is thought to participate in many of these changes is the Sertoli cell cytoskeleton (Amlani and Vogl, 1988). Using vimentin as a marker, characteristic distribution patterns during the spermatogenic cy-

cle have recently been described in the rat by Mali et al. (1987) and in man by Aumuller et al. (1988). Parallel t o the dynamic changes in Sertoli cell outline (which is hormone-dependent; Spruill et al., 1983a,b), an interdependence of the intermediate filaments with intercellular junctions (Pelletier, 1988; Amlani and Vogl, 1988), with the extracellular matrix (Hadley et al., 1985; Ailenberg et al., 1988) and cell polarity (Neely and Boekelheide, 1988) has been considered. A mechanical significance of Sertoli cell intermediate filaments has to be envisaged with respect to intracellular transport (Janecki and Steinberger, 19871, membrane moulding (Hamasaki, 1987), and spermatid and nuclear positioning (Franke et al., 1979; Amlani and Vogl, 1988; Aumiiller et al., 1988). 4. Regulation of intermediate filament distribution. The dynamic aspects mentioned are germane to a highly regulated system of synthesis, arrangement, and distribution as well as degradation of IF in Sertoli cells during postnatal development, hormonal stimulation, Sertoli cell cycle, and pathological situations. Although a number of studies have been performed using in vitro system of Sertoli cells (De Philip and Kierszenbaum, 1982; Spruill et al., 1983a,b; Kierszenbaum et al., 1986; Monaco et al., 1988; Ireland et al., 19861, as yet only the hormonal effects of FSH and of second messengers on IF phosphorylation (Spruill et al., 1983a,b) have been well documented. However, little is known of synthetic and proteolytic events during IF remodeling or the degradation and turnover of Sertoli IF in normal and regressive situations. 5. Pathophysiology of IF in Sertoli cells. Significant alterations in the distribution of IF in Sertoli cells under various conditions such as androgen insensitivity (Aumiiller and Peter, 19861, estrogen challenge (Schulze, 1988) and other, more complex intra- and extratubular disturbances have been reported, but their subcellular pathogenetic mechanisms are only incompletely understood. The whole range of aspects mentioned above is necessary to get more insight into the complex regulation of Sertoli cell IF and their significance in testis biology. Furthermore it must be kept in mind that morphological analysis is only one approach and must be supplemented by biochemical and endocrine studies. For the sake of clarity three major situations of IF in Sertoli cells will be examined: pre- and postnatal development in laboratory animals, the in vivo situation during the spermatogenetic cycle and in vitro experiments, and pathological alterations.

INTERMEDIATE FILAMENTS IN DEVELOPING SERTOLI CELLS Vimentin During development, Sertoli cells express vimentin from early stages onwards. In the rat, vimentin is expressed in the precursor of the Sertoli cells, the so called pre-Sertoli cells in the 15-day-old embryo (Paranko et al., 1986). The level of vimentin expression changes very little, if at all, during further prenatal and postnatal development (Paranko et al., 1986), while Sertoli cells undergo dramatic biochemical and


Figs. 1-5. Cross-sections of immature seminiferous tubules of 27 dpc rabbit testis (Figs. 1-3) and 21 dpc mouse testis (Figs. 4,5). x 700. Fig. 1. Methylene blue-stained semithin section of a Karnovskyfixed Durcupan-embedded 27 dpc rabbit testis (specimen by courtesy of Dr. I. Kinsky, Bonn). Figs. 2,3. Methanol-(10 min at -20") and acetone- (1min at -20°C) fixed frozen sections of a freshly frozen (liquid nitrogen) 27 dpc rabbit testis, immunostained (indirect immunofluorescence using FITC- and TRITC-conjugated goat antimouse antibodies from NORDIC, Tilburg, Holland) with monoclonal antibodies against vimentin (Fig. 2, BV1118) and keratin 18 (Fig. 3, RCKlO6). The different vimentin

morphological changes (Magre and Jost, 1980; Russell et al., 1989) until the adult pattern of vimentin expression, as described by Franke et al. (1978)) is established. The intracellular arrangement of intermediate filament bundles is constantly reorganized according to the changes in cell shape occurring during this developmental period (i.e., from a nearly columnar cell shape to the highly irregular outlines known from the adult Sertoli cell). This developmental sequence of events in the rat can also be followed in the developing testis of the rabbit, a species in which vimentin expression has been found in developing gonads at even earlier stages (i.e., before sexual differentiation at 14 days post conceptionem [dpc], Wartenberg et al., 1989). In the 27-day-old fetal testis (Figs. 1-3) strong expression of vimentin is found in Sertoli cells (Fig. 2) as well as in the surrounding interstitial cells. During postnatal development, a t around the time when spermatogenesis begins (Gondos et al., 1973)-that is, in the 63-day-old testis (Figs. 6-8)-vimentin is still strongly expressed in Sertoli cells, and the slender morphology of the Sertoli cell can clearly be seen in the vimentin immuno-

53

antibodies used (see Table 1)react principally in the same manner. Also, the antibodies specific for keratin 8 (see Table 1)give principally the same reactions as the antibodies against keratin 18 (see Table 1). Figs. 4, 5. Frozen sections of 21 dpc mouse testis immunostained with BV1118 (Fig. 4) and LE61 (Fig. 5). Methods as for Figs. 2, 3. In the mouse Sertoli cell, too, the different monoclonal antibodies against vimentin and keratins 8 and 18 give principally the same immunoreactions, respectively, except that antibodies LE41 and CAM5.2 do not react in-even unfixed-frozen sections of mouse Sertoli cells. Note different intracellular staining pattern between keratin staining in rabbit (Fig. 3) and mouse (Fig. 5).

staining (Fig. 7). When a t 84 days post partum (dpp) spermatogenesis is firmly established (Gondos et al., 1973)) vimentin expression has changed very little compared to previous juvenile stages, except that it seems to be expressed even more strongly in the cells of the interstitial compartment (Fig. 10). Thus, during development, the expression level for vimentin remains seemingly the same while Sertoli cells gradually acquire their adult highly irregular shape.

Keratins Of the family of keratins it is the simple keratins (e.g., 8, 18, and 19) which have been detected in developing Sertoli cells so far. This has been shown by Paranko et al. (1986) for the rat fetus during early stages following sexual differentiation. This keratin expression is transient and terminates between 10 and 14 dpp, a t which time spermatogenesis begins in this species (Clermont and Perey, 1957). In the rabbit, too, antibodies to keratin 18 have been found to stain differentiating Sertoli cells during fetal development and even the precursor cells of expression of Sertoli cells

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G. AUMULLER ET AL.

Figs. 6-11. Cross-sections of immature seminiferous tubules of 63 dpp (Figs. 6-81 and 84 dpp (Figs. 9-11) rabbit testes. Figs. 6, 9: Methylene blue-stained semithin sections. Figs. 7, 10: Anti-vimentinstained (monoclonal antibody BV1118) frozen sections. Figs. 8, 11: Anti-keratin-stained (monoclonal antibody RCK106) frozen sections.

Note germ cells at 63 dpp (Fig. 6 ) in pre-leptotene, whereas at 84 dpp (Fig. 91 most germ cells have proceded to pachytene stage. In Fig. 11 note seminiferous tubule with positively stained rete testis cells (R, left third of picture) next to negatively stained Sertoli cells (S). x 700.


55

TABLE 1 . Mouse monoclonal antibodies used for analysis of uimentin and keratin expression in the postnatal rabbit testis’ ~

Antibody LE 41 RCK 102 CAM5.2 RCKlO6 RGE53 CK18-2 LE61 RV202 v9 BV1118

IF-protein keratin 8 keratin 5,s keratin 8,18 keratin 18 keratin 18 keratin 18 keratin 18 vimentin vimentin vimentin

IgG-subclass IgGl IgGl IgG2a IgGl IgGl IgGl IgGl IgGl IgGl ItzM

Dilution undiluted undiluted undiluted undiluted undiluted undiluted undiluted undiluted 1:lO undiluted

~

_

_

_

Reference Lane (1982) Broers et al. (1986) Makin et al. (1984) Ramaekers et al. (1987) Ramaekers et al. (1985, 1987) Broers et al. (1986) Lane (1982) Ramaekers et al. (1987) Osborn et al. (1984) Viebahn et al. (1991b)

‘Numbers of keratins refer to human keratins catalogued by Moll et al. (1982).

during development, with vimentin expression at the same stages. The expression of keratins 8 and 18, seen at 27 dpc (Fig. 3), is retained until postnatal stages (63 dpp; Fig. 8). Keratin expression is strongest in the basal compartment of Sertoli cells, but extends also with thin filament bundles up to the luminal tip of the Sertoli cells. During the next 2 weeks of development, dramatic changes can be observed in the expression pattern of keratins. At first, in 70-day-old testis, some seminiferous tubules contain Sertoli cells almost keratin-negative, while other tubules contain Sertoli cells which still strongly express keratins 8 and 18. One week later, only a few Sertoli cells express keratins, the staining signal being concentrated near the base of the Sertoli cell. Finally, at 84 days, only rete cells can be stained with keratin antibodies (Fig. 11)which represent the pattern seen in the adult testis. In the rabbit, the developmental period between 63 and 84 dpp also covers the onset of spermatogenesis (Gondos et al., 1973) (see Table 2) so that, like in the rat, the disappearance of keratins in developing Sertoli cells seems to be correlated with the onset of spermatogenesis (see also below). However, in the rat, this correlation is based mainly on comparing immunohistochemical data of intermediate filament protein expression in one study with conventional morphological data of spermatogenic stages in another study. Therefore, the fine tuning of these two testicular maturational events was established by a combined immunohistochemical and electron microscopical study (Viebahn and Miething, 1991a):separate histological blocks were obtained from one and the same testis specimen and were fixed using different methods, because sensitive immunohistochemical detection of intermediate filament proteins cannot be obtained on optimally (glutaraldehyde-) fixed specimens. Figures 6-11 illustrate the results of this investigation: frozen sections prepared for immunofluorescent analysis shown in Figures 7, 8, 10, and 11 were obtained from the same testis as the semithin sections shown in Figures 6 and 9, respectively. The semithin sections (Figs. 6 and 9,63 and 84 dpp, respectively) show the changing nuclear morphology (mainly smooth contours a t 63 dpp versus ample indentations a t 84 dpp), the progression of spermatogenesis (preleptotene at 63 dpp versus pachytene stages at 84 dpp), and, in general, the establishment of a tubular lumen during this developmental period. At the ultrastruc-

tural level (data not shown), these changes are accompanied by the appearance of typical Sertoli cell junctions which is in agreement with the timetable established by Sun and Gondos (1986). Species Differences There seem to be only little species differences in the IF expression in developing Sertoli cells. These results mentioned above, together with results obtained on immature Sertoli cells from mouse, chick, and golden hamster, suggest that, as far as vimentin is concerned, there is a cross-speciesconsensus that vimentin belongs to the standard repertoire of the developing Sertoli cell cytoskeleton as it does in the adult Sertoli cell (see lntroduction). The expression of simple keratins also has a remarkable similarity between a t least two species (rat and rabbit) with its close correlation to the onset of spermatogenesis. However, the intracellular staining patterns as compared between species (e.g., rat and rabbit) seem to be different: while in the rat, keratin expression is only found in the basal aspects of developing Sertoli cells (see Fig. 5f in Paranko et al., 19861, in the rabbit, keratins are regularly expressed also in the apical compartments. In the immature Sertoli cells of mouse (Figs. 4,5) and golden hamster, keratin expression is similar to the pattern found in the rat. Thus, in all species studied so far vimentin is the regular intermediate filament protein in developing Sertoli cells, while keratins are expressed only transiently. Regulatory Aspects When vimentin expression was first discovered in Sertoli cells (Franke et al., 1979) it was regarded as an unusual occurrence because vimentin was considered specific for mesenchymal cells (Franke et al., 1978). One of the first conclusions as to vimentin’s regulatory meaning was that it might tip the balance in the dispute over the still elusive origin of Sertoli cells towards a likely mesenchymal origin. Consequently, the recent finding of keratin expression in immature Sertoli cells (Paranko et al., 1986) had to be considered as an indication for an epithelial origin of Sertoli cells. However, available data on changes of one intermediate filament isotype to another during development, such as during the development of the nervous system (Stagaard and Mgllghrd 19891, rather suggest that intermediate filament isotypes are changed with changing functional needs of the cells concerned and, therefore, are unreli-

G. AUMULLER ET AL.

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TABLE 2 . Postnatal maturation of testicular tubules in the rabbit Days post partum

Tubular lumen Sertoli cells mitosis DNA synthesis nuclear indentations vimentin expression keratin expression tight junctions blood-testis barrier Germ cells meiosislspermatogenesis

56

70

84

-

-I+

+

+ + -1 + ++

-

-

Sun and Gondos (1981) Sun and Gondos (1981)

+ ++ + i+ +

++

+ +

Sun and Gondos (1986) Sun and Gondos (1986)

leptotene

pachytene

Gondos (1980) Sun and Gondos (1986)

+-

preleptotene

-

References

+-+

ular stages of the cycle. Cyclic changes of a number of biochemical and endocrine functions such as FSH-dependent CAMPproduction (Parvinen et al., 1986) have been related to the morphological stages (Parvinen, 1982; Parvinen et al., 1986). Interaction and cell recognition between Sertoli cells and germ cells have been postulated to be required for the differentiation of germ cells and their transport to the adluminal compartment of the seminiferous tubule (Russell, 1977). In terms of cell-to-cell signalling and regulation of Sertoli cell function, changes in Sertoli cell shape were considered to operate also as a regulatory factor (Aumuller et al., 1988). Different phenotypic configurations of Sertoli cells have been described by Clermont et al. (1959) and reconstructed by Wong and Russell (1983). Weber et al. (19831, and Russell et al. (1983, 1986). As compared t o the two cell configurations of Sertoli cells in rat and monkey, Schulze et al. (1976) described four to five different phenotypes of Sertoli cells in the human testis which, however, have not been correlated with specific stages of spermatogenesis. Rat. Cyclic changes. In order to investigate if there are cyclic changes in the distribution of vimentin in Sertoli cells of the rat, vimentin immunocytochemistry and Sertoli cell shape were monitored at different stages of the seminiferous epithelium by Mali et al. (1987) and by Amlani and Vogl (1988). Both groups agree with the observation of two basic configurations of vimentin distribution during the cycle: 1)a reaction in the perinuclear region and the presence of apical extensions projecting towards the developing spermatid bundles during stages XII-V of the cycle, and 2) a perinuclear concentration of vimentin immunoreactivDISTRIBUTION AND ARRANGEMENT ity and only a few small and narrow apical extensions OF INTERMEDIATE FILAMENTS IN during stages VI-XI (Mali et al., 1987). Particularly SERTOLI CELLS DURING during stages 11-IV immunoreactive material surNORMAL SPERMATOGENESIS rounding the crypts or recesses within the Sertoli cells Species Differences-Immunohistochemistry which engulf elongating spermatid heads is prominent. General. With the formation of junctions (Dym and As the spermatids develop and become situated more Fawcett, 1970) and the onset of cyclic changes in the apically, immunofluorescence in the area of the reseminiferous epithelium (see Clermont, 1972) Sertoli cesses diminishes (stages v-VI). A t stages VII-VIII, cells undergo extensive morphological and functional when spermatids have reached the apex of the Sertoli changes such as redistribution of intercellular junc- cells, fluorescence is evident only a t the base of the tions, formation of cellular extensions, and invagina- cells. With the appearance of a new generation of elontions in association with sDermatog.enic cells . ~ at . .~ oartic_ .. . ~ .gate spermatids at stage XIV, linear tracts of im-

able candidates a t least for “long range” cell lineage markers (discussed by Viebahn et al., 1987). Transient keratin expression in Sertoli cells might be correlated with the transient appearance of desmosome-like junctions between immature Sertoli cells such as the ones described in the dog (Connell, 1980). However, such junctions, which would precede the development of typical Sertoli cell tight junctions, have not been described as yet in the 2 species most thoroughly investigated for the transient keratin expression in immature Sertoli cells, the rat and the rabbit. At the same time, the close correlation between keratin expression and the presence of desmosomes is a wellestablished fact (Arnn and Staehelin, 1981). Another regulatory aspect concerns the possible function of the vimentin intermediate filament isotype in Sertoli cells during development, which might also be applicable to the adult Sertoli cell involved in the spermatogenetic cycle (see below). As epithelial cells, Sertoli cells are unusual in that they constantly change their cellular outlines. At very early stages, germ cells invade the gonadal anlage and are received into the primitive sex cords by the pre-Sertoli cells. Subsequently, they are displaced from their central position to a more basal position in the immature seminiferous cord (Gondos and Conner, 19731, again with the aid of the Sertoli cells. Both these morphogenetic events require a highly flexible cytoplasmic shape of the Sertoli cells involved. This is reminiscent of the fact that vimentin is frequently associated with highly dynamic cell shapes, albeit that the cells are organized in epithelial compartments such as in the embryonic neural tube (Viebahn et al., 1990).

~


Figs. 12-18. Immunohistochemical visualization of IF in Sertoli cells. The antiserum against vimentin was kindly provided by Prof. D. Drenckhahn, Wiirzburg. Figs. 12-14 demonstrate changes in the arrangement of IF during Sertoli cell isolation from rat seminiferous tubules (Fig. 12: mechanical dissociation; Fig. 13: enzymatic digestion) and in tissue culture (Fig. 14). Figs. 15-18 are semithin and paraffin (Fig. 16) sections of human testis biopsies. Fig. 18a,b is a

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double staining showing Sertoli cells (S) with vimentin immunoreactivity (a)detected by a TRITC-labeled secondary antibody and tubulin immunoreactivity (b: monoclonal antibody) in spermatids, detected by a FITC-labeled secondary antibody. Fig. 12: Bar = 10 pm; X 635. Fig. 13: Bar = 10 pm; x 665. Fig. 14: Bar = 25 pm; x 305. Fig. 15: Bar = 25 pm; x 175. Fig. 16: Bar = 25 pm; x 280. Fig. 17: Bar = 10 pm; x 1,185. Fig. 18: Bar = 0.25 pm; x 450.

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munoreactive material reappear apical to the Sertoli cell nucleus (Amlani and Vogl, 1988). Mali and collaborators (1987) found residual bodies in stages IX-XI within Sertoli cells to be surrounded by vimentin positive filaments. Using a monoclonal antibody, they observed a weak granular vimentin immunoreaction in the mid piece area of the flagella of steps 16-19 spermatids. These filaments are thought to be related to the positioning of mitochondria. There is some discrepancy between the two reports with regard to the exact location of Sertoli cell intermediate filaments neighbouring the elongate spermatid head. Mali et al. (1987)found them in the proximity of the concave surface of spermatid heads during stages 111-V, while Amlani and Vogl (1988) found them concentrated in those Sertoli cell regions immediately adjacent to the convex or dorsal aspects of the sickleshaped spermatid head. This finding was corroborated by electron microscopic observations. On the whole, there is a core of vimentin filaments surrounding the Sertoli cell nucleus, from where, in a cycle-dependent manner, apical extensions develop which surround the elongating spermatid recesses. The distribution of vimentin filaments in Sertoli cells in the straight tubules is unknown. Human. Regional differences. Both within the contorted and the straight portions of the seminiferous tubule, the local variants of the Sertoli cells are invariably immunoreactive for vimentin (Figs. 15-18). The epithelium lining the human rete testis is vimentin immunoreactive. In our specimens, no cytokeratin immunoreaction of the rete testis epithelium was observed. This, however, is the case in the testis of the ram (Tung et al., 1987). Cyclic changes. Using a computer-assisted reconstruction of semithin sections of human testicular tissue processed with the PAP method for vimentin immunoreactivity (Aumuller et al., 19881, we have observed two basic configurations of the Sertoli cells, named AS (ante spermiationedbefore spermiation, stages V, VI, I, and 11, classification of Clermont, 1963) and PS (post spermiationem/after spermiation, stages I11 and IV), respectively. The method used apparently depicts the complete shape of Sertoli cells including their finest ramifications, mostly missed on cryostat sections processed for immunof luorescence. This finding is in accordance with that of Russell et al. (1983, 19861, Wong and Russell (19831, and Weber et al. (1983) who distinguished an A type and a B type configuration of the Sertoli cell. The breaking point between these two configurations seems to be spermiation. The PS configuration observed during stage I11 shows an ample contact area of the Sertoli cell base with the basement membrane. Just above the region of the Sertoli cell junctions, deep invaginations into the Sertoli cell cytoplasm are present, destined for primary spermatocytes. The indentations surrounding spermatids are only shallow. At stage V of the cycle the AS configuration results in increased extension of the cells from the basement membrane into the adluminal compartment, where thin apical sheets surround major portions of the spermatid heads. The basal cytoplasm accumulates underneath the nucleus, which, therefore,

increases its distance from the cell base (Aumuller et al., 1988; Fig. 15). One interesting but not unequivocally discernible observation was the arrangement of the different germ cells surrounding the trunk of the Sertoli cells: they appear to follow a spiral route. The obvious differences in the appearance of rat and human Sertoli cells at different stages of the spermatogenic cycle are difficult to reconcile. Since only one computer reconstruction was performed per cell and stage, we may have missed other configurations, although this seems rather unlikely with regard to the meticulous studies of semithin sections. Subcellular Organization and Distribution of Intermediate Filaments in Sertoli Cells General Morphology of the Sertoli Cell Cytoskeleton. Sertoli cells can be subdivided into three major regions (Franke et al., 1979): a basal portion bordering the basal lamina and neighboring the spermatogonia, respectively; an intermediate portion containing the highly invaginated nucleus and complex junctional specializations with adjacent Sertoli cells; and an apical region with numerous invaginations (crypts) or recesses of the plasma membrane which are occupied by spermatocytes and particularly by spermatids (for reviews, see Dym and Fawcett, 1970; Fawcett, 1975; Schulze et al., 1976; Russell and Peterson, 1985; Tindall et al., 1985; de Kretser and Kerr, 1988). In view of the elaborate shape and both functional and morphological complexity of this cell type which undergoes extensive changes in external and internal organization during spermatogenesis (Ross, 1976; Clermont et al., 1980; Morales and Clermont, 1982; Russell, 1980, 1984) the structure and arrangement of the cytoskeleton has attracted much attention (Franke et al., 1978; Vogl et al., 1983a,b, 1985; Vogl and Soucy, 1985; Amlani and Vogl, 1988; Russell et al., 1989). Changes in the actin filament pattern in Sertoli cells during spermatogenesis have been correlated with changes in the germ cell population (Camatini et al., 1987; Vogl et al., 1985; Vogl and Soucy, 1985). Microtubule distribution has been studied by Christensen (19651, Fawcett (19751, Wolosewick and De Mey (19821, Vogl (19881, and Aumuller and Seitz (1988). Microtubules are concentrated in Sertoli cell cytoplasm apical to the nucleus, where they are oriented parallel to the apico-basal alignment of the cell (Neely and Boekelheide, 1988). They are thought to maintain cell shape (Russell et al., 1981; Amlani and Vogl, 19881, to facilitate both the movement of intracellular organelles and the translocation and modelling of spermatogenic cells (Fawcett, 1975) and other events associated with sperm maturation (Vogl et al., 1983b). The arrangement of intermediate filaments within Sertoli cells displays some peculiar features which are described below. Rat. Franke et al. (1979)have precisely documented the subcellular distribution of intermediate filaments in rat Sertoli cells. Their findings have recently been confirmed by Amlani and Vogl (1988). The filaments are concentrated around the nucleus and are also present in the nuclear invaginations. At tangentially sectioned nuclear pores they appear to approach the


nuclear surface a t a sharp angle and to merge into the nuclear envelope (Figs. 19-22). As previously shown by Jones et al. (1982) in keratinocytes the filaments frequently appear to insert directly into the nuclear envelope. A direct attachment of intermediate filaments at the nuclear envelope to the nuclear lamins has been reported by A. Goldman et al. (1986) and by Georgatos and Blobel (1987) in erythrocytes. In Sertoli cells, however, the transition of perinuclear fibers into the outer nuclear membrane is difficult to ascertain. Some fuzzy material obscures the direct contact zone. The often multilayered perinuclear zone, where the filaments form a dense feltwork without preferential direction, measures about 0.3-0.7 km. At more peripheral sites, where intermingled with different cytoplasmic elements, the packing density of the fibers is less prominent and the arrangement pattern is rather variable (Figs. 23,241. Mostly, a non-ordered meshwork of individual filament bundles can be found. These bundles are always loosely packed and never form the thick arrays, typical of tonofibrillar bundles of cytokeratin filaments-for example, in squamous epithelium (Franke et al., 1979). In areas of bundled intermediate filaments, Yang et al. (1985, in baby hamster kidney cells) have found a 300 kDa protein. It is unknown if this protein is present in Sertoli cells. There is no peculiar relationship between cytoplasmic organelles and intermediate filaments. Close to mitochondria, they appear to be arranged in parallel with the long axis of the mitochondria (pig: see van Vorstenbosch et al., 1984). Around stacks and vesicles of the Golgi apparatus only few and loosely arranged filaments are seen. In the apical region of Sertoli cells, where invaginations containing elongating spermatid heads are present, the feltwork of filaments gives rise to individual bundles that run in parallel with the microtubules, the latter being more obvious than the former. According to Goldman et al. (1984) such microtubule-intermediate filament complexes are involved in intracellular organelle movement as well as in cell shape formation and maintenance. A preferential concentration of intermediate filaments around lipofuscin granules, or residual bodies, as observed by immunofluorescence by Mali et al. (19871, is not prominent a t the ultrastructural level. Special regard has been given to the relationship and association of intermediate filaments with the plasma membrane and their junctional and contact devices. The latter have been reviewed by Russell and Peterson (1985) and discussed in functional terms by Pelletier (1988). A brief survey of the salient features of this complex system is presented here. Junctions of Sertoli cells are found at the basal plasma membrane and at the lateral plasma membrane where contacts between Sertoli cells and germ cells (spermatogonia, spermatocytes, spermatids) and neighbouring Sertoli cells are observed. Morphologically, focal contacts, adherens (or intermediate) type junctions, gap junctions, tight junctions, and ectoplasmic specializations (at tight or gap junctions and surrounding spermatid heads) as well as tubulobulbar complexes have been distinguished (see Russell and Peterson, 1985, for a review).

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The basal plasma membrane of Sertoli cells, where receptors, pumps, and transport and exchange mechanisms are thought to reside, is linked in a stage-dependent manner (Hamasaki, 1987) to the extracellular matrix (basal lamina) by focal adhesions (see Burridge et al., 1988). These have formerly been misinterpreted as hemidesmosomes (Russell, 1977; see Franke et al., 1979). Desmosomal proteins and plakoglobin are lacking a t these sites. Yet Ziparo et al. (1986) recently documented the presence of spectrin and fodrin in Sertoli cells grown in vitro and suggested that there might be an association between spectrin and vimentin in Sertoli cells. Although rather dense bundles of intermediate filaments are seen adjacent to the basal membrane the attachment of these filaments to the basal plasma membrane is difficult to ascertain. Amlani and Vogl (1988) recently studied the relationships of intermediate filaments in Sertoli cells with maturating germ cells (Figs. 25-30). Using detergenttreated preparations stained with tannic acid they found “the intermediate filaments extending from the nucleus to desmosome-likejunctions with spermatogenic cells” (Amlani and Vogl 1988). Another consistent finding was the presence of a bundle of 8 to 12 filaments in Sertoli cell cytoplasm adjacent to the convex aspect of the spermatid head in early stages of the spermatogenic cycle. According to Amlani and Vogl(1988) these filaments are closely related to the ectoplasmic specializations lining the apical crypts within the Sertoli cells. Grove and Vogl (1989) have recently discussed the function of these ectoplasmic specializations as a type of actin-associated adhesion junction. The presence of a-actinin (Franke et al., 19781, vinculin, and fimbrin (Grove and Vogl, 1989) in this area has been described. However, the site of anchorage of intermediate filaments to the ectoplasmic specialization is not fully elucidated (Figs. 32-34). Amlani and Vogl(1988) found a close association of these filaments with the actin filaments: IF traverse through gaps in the cisterns of the endoplasmic reticulum covering the actin bundles. This finding was difficult to verify in unextracted sections. In our own specimens termination of intermediate filaments on the cytoplasmic face of the cistern seems to be at least as frequent as the contact with actin bundles (Figs. 31-38). In enriched extracts of ectoplasmic specializations, Grove and Vogl (1989) described a 83 kD protein that might be related to filament anchorage. We have recently purified a transglutaminase from rat Sertoli cells with a molecular weight slightly above 80 kDa which could be involved in fixation of intermediate filaments in the region of the ectoplasmic specializations (unpublished observations). The intimate association of IF with ectoplasmic specializations on the one hand and the perinuclear network of filaments on the other hand is interpreted as indicative “that these apical bundles of intermediate filaments may anchor Sertoli cell crypts, and therefore the attached spermatids, in a supranuclear position during the early stages of the spermatogenic cycle” (Amlani and Vogl, 1988). Ectoplasmic specializations between Sertoli cells are usually associated with gap and/or tight junctions. In

Figs. 19-27. Distribution of intermediate filaments in Sertoli cells from experimental rats (three weeks unilateral cryptorchidism: Figs. 19,20,26,27;7 days thioglucose treatment Figs. 21,24,25 and after isolation from seminiferous tubules: Figs. 22, 23). The arrangement of IF (empty arrows) surrounding the nuclei (N) is shown in Figs. 19-23. In tangentially sectioned nuclear pores (Figs. 19-21) the close association with IF is seen. In Fig. 21 (thin arrow) IF terminate at a nuclear pore. After isolation of the Sertoli cells, a rearrangement of IF occurs, resulting in a dense accumulation of IF in the cell periphery (arrows in Fig. 231, while microtubules (thick arrow) are in-

terspersed between perinuclear IF (empty arrow in Fig. 22). Within the cytoplasm thin strands of IF (empty arrow) are found adjacent to cytoplasmic organelles (Fig. 24). Only few intermediate filaments are found in the vicinity of adherens type (Figs. 25,26) and tight junctions (Fig. 27). Fig. 19: Bar = 1 pm; x 32,100. Fig. 20: Bar = 0.25 pm; x 44,000. Fig. 21: Bar = 0.25 pm; x 55,000. Fig. 22: Bar = 0.25 pm; X 47,000; Fig. 23: Bar = 1 pm; x 21,800. Fig. 24: Bar = 1 pm; x 29,200. Fig. 25: Bar = 0.25 pm; x 47,000. Fig. 26: Bar = 0.25 pm; x 41,500. Fig. 27: Bar = 0.25 pm; x 82,000.

Figs. 28-38. Distribution of intermediate filaments in the vicinity of Sertoli cell junctions from normal rats (Figs. 28, 29, 31) and humans (Figs. 32-34) and experimentally treated rats (Fig. 30: 7 days thioglucose; Figs. 35-38 6 weeks unilateral cryptorchidism). In Fig. 28 tubulobulbar complexes are outlined by lanthanum precipitates. Only a few IF (empty arrows in Figs. 28-30) are found in the vicinity of tubulobulbar complexes and adherens type junctions (Fig. 30) (thick arrow: microtubule). Figs. 31-38 demonstrate the relationships between actin filaments (arrowheads), endoplasmic reticulum (small asterisks), microtubules (thick arrow), and IF (empty arrows) under normal (Fig. 31) and pathological conditions (Figs. 35-38). No direct

continuation of IF into the zone of actin filaments is seen. Fig. 32 shows a post-embedding double-immunoreaction of action 10 nm gold particles and vimentin (5 nm gold particles) a t ectoplasmic specializations. Co-localization is rare; vimentin-immunoreactivity is prevalent in the cytoplasmic portion of the ectoplasmic specialization. Fig. 28: Bar = 1 pm; x 22,750. Fig. 29: Bar = 1 pm; x 22,500. Fig. 30: Bar = 0.1 pm; x 158,000. Fig. 31: Bar = 0.25 pm; x 103,600. Fig. 32: Bar = 1 pm; x 10,700. Fig. 33: Bar = 0.25 p m ; x 49,000. Fig. 34: Bar = 1 pm; x 23,400. Fig. 35: Bar = 0.25 pm; x 95,000. Fig. 36: Bar = 0.5 pm; x 88,000. Fig. 37: Bar = 0.25 pm; x 68,000. Fig. 38: Bar = 0.25 pm; x 91,000.

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such ar'eas particular proteins have been described serving different functions (L-CAM: Gumbiner et al., 1988; A-CAM: Volk and Geiger, 1986; ZO-1: Stevenson et al., 1986; Byers et al., 1988; spectrin: Ziparo et al., 1986; radixin: Tsukita et al., 1989) which are thought to act together in the highly complex cell junction-cell junction interactions (see Pelletier, 1988). These interactions are obviously necessary to allow the passage of spermatogenic cells from the basal into the adluminal compartment of the seminiferous tubule. This migration of germ cells requires a highly regulated process of opening, closing, formation, and disposal of junctions accompanied by changes in membrane rigidity and cell-to-cell interaction. Yet little is known as to what degree intermediate filaments that are the spacers between the stable perinuclear core zone and the remodelling peripheral zone of the Sertoli cell are involved in this process. Adherens type junctions are prominent in Sertoli cell membranes facing spermatocytes. Here modification of attachment structures is required because of the continuous production and migration of germ cells. This type of junction is likewise present between adjacent Sertoli cells. In the ovarian counterpart of Sertoli cells, the granulosa cells, the presence of adherens junctions and the co-expression of keratin, vimentin, and desmoplakin have been shown by Czernobilsky et al. (1985). In Sertoli cells, however, adherens type junctions have been described by Franke et al. (1981, 1982) to lack desmoplakin and other desmosomal proteins. Cowin et al. (1986) have indicated the presence of plakoglobin, a 83 kDa palypeptide in the vinculin-actin associated intercellular junctions. These junctions of Sertoli cells therefore represent typical adherens junctions. In the rat, there are only few intermediate filaments associated with Sertoli adherens junctions and their direct contact with the plaque material was difficult to establish (Figs. 25,26,29,30).Amlani and Vogl(1988) have pointed to the fact that adherens type junctions are transient (see Pelletier, 1988).Therefore the association of intermediate filaments with these junctions must change during spermatogenesis. Moreover, the distribution of intermediate filaments a t these junctions is asymmetrical, since they are lacking at the spermatogonial cell portion of the junction. So-called tubulobulbar complexes present between adjacent Sertoli cells and Sertoli and germ cells have been viewed as anchoring devices between germ and Sertoli cells in the rat (Russell and Clermont, 1976). Pelletier (1988) has suggested that the formation of these complexes, particularly in the basal regions, may be associated with pleating of a junctional membrane segment, formation of a tubulobulbar complex, removal from the original site, and internalization as a junctional vesicle. In our specimens basal tubulobulbar complexes were only infrequently associated with condensations of intermediate filament bundles inside the Sertoli cell portion (Fig. 28). Only when associated with an adherens type junction were increased numbers of filaments observed in Sertoli cell processes protruding into a neighbouring cell (Fig. 29). In addition to the normal situation in the rat described above, we studied the

distribution of intermediate filaments in experimental material such as unilateral cryptorchidism (Aumuller et al., 1980; Figs. 19, 20, 26, 27, 36-38) hypophysectomy and testosterone treatment (Aumuller and Schiller, 1978), thioglucose treatment (Figs. 21,24,25, 30), and in isolated Sertoli cells cultured in vitro (Figs. 12-14, 22, 23). The salient feature in all these cases was the sometimes considerable increase in the amount of filaments. In cryptorchidism, this was most obvious in the perinuclear region, where dense fascicles of filaments radiated into the periphery. After thioglucose treatment for 3 weeks, interlacing strands of filaments were seen interspersed between the cytoplasmic organelles. No significant changes were observed in the relationship of intermediate filaments with the different types of junctions. Human. Since essential deviations from the situations in the rat are not observed in the distribution of intermediate filaments in normal human Sertoli cells, only a brief description is presented here. More interesting are the changes of the system in pathological specimens which are described below. The subcellular distribution of intermediate filaments in human Sertoli cells (Figs. 39-56) was studied by Aumuller et al. (1988) who observed a similar pattern as in the rat (Franke et al., 1979), pig, and dog (van Vorstenbosch et al., 1984). Immunoelectron microscopically vimentin was identified subjacent to the plasma membrane at the level of the junctional complexes, where intermediate filaments are suggested to intermingle with actin filaments (Fig. 32-34). As previously described by Nagano (19661, intermediate filaments are found closely associated with the so-called Charcot-Bottcher crystalloids. In favorable sections filaments seemed to merge into the crystalloids (Figs. 44-46). The diameter of the filamentous structures inside the crystalloids (- 15 nm) was slightly larger than that of the filaments. As soon as the latter reached the crystals they appeared to be immersed with some electron dense material which seems to glue the individual filaments together. The immunolabeling of the Charcot-Bottcher crystalloids was only slightly elevated against the background labeling. The cytoplasm surrounding the nuclei was densely immunolabeled, well in line with ordinary thin sections of adequately fixed, epon-embedded testis biopsies. The relationship between individual cytoplasmic organelles (e.g., mitochondria) and microtubules with intermediate filaments was the same as in the rat (Figs. 43, 47, 48). We have scrutinized the association of elongating spermatids deeply inserted in Sertoli cell cytoplasm and surrounded by ectoplasmic specializations with intermediate filaments. In no case was a bundle found approaching the spermatid, as observed in the rat. Rather, most of the intermediate filaments are in contact with the cytoplasmic face of the cistern participating in the ectoplasmic specialization (Figs. 54-56). Regulatory Aspects. The active regulatory role of intermediate filaments within the Sertoli cells as well as their passive control is briefly considered in this section. In view of the current literature (see, e.g., Laza-


Figs. 39-48. Perinuclear and cytoplasmic distribution of IF in human Sertoli cells. The arrangement of IF (empty arrows) surrounding the nuclei (N) and their relationship to microtubules (thick arrows) is shown in Figs. 39-42. In the cell periphery (Figs. 43-48) IF are either arranged in parallel with microtubules (thick arrows in Fig. 43) or are randomly distributed. The relationship of IF with Charcot-Bottcher crystalloids is demonstrated in Figs. 44-46. The direct continuation of IF into longitudinally (Fig. 44) or transversely (Fig. 45) sectioned

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Charcot-Bottcher crystalloids is indicated by a curved arrow. The immunolabeling with vimentin is confined to the periphery of the crystalloid (Fig. 46). Fig. 39: Bar = 0.25 pm; x 107,000. Fig. 40: Bar = 0.25 pm; x 41,000. Fig. 41: Bar = 0.25 pm; X 61,000. Fig. 42: Bar = 0.25 pm. x 95,000. Fig. 43: Bar = 0.25 pm; x 43,000. Fig. 44: Bar = 0.25 pm; x 112,000. Fig. 45: Bar = 0.25 km; x 75,000. Fig. 46: Bar = 0.25 pm; x 102,000. Fig. 47: Bar = 0.25 km; x 40,000. Fig. 48: Bar = 0.25 Fm; x 38,750.

Figs. 49-56. Relationship of IF with cell junctions in human Sertoli cells. In Fig. 49 adjacent Sertoli cells bordering on ectoplasmic specialization (arrows) are shown displaying the distribution of IF between nucleus (N) and plasma membrane. Figs. 50 and 51 display ectoplasmic specializations in Sertoli cells prepared for conventional electron microscopy (Fig. 50) or for immunocytochemistry (Fig. 51: low concentration of glutaraldehyde, no osmium treatment, LR-White embedding). Vimentin immunoreactivity (Fig. 51) is seen on the cytoplasmic face of the cisterna of endoplasmic reticulum, correspond-

ing with empty arrow in Fig. 50. In the vicinity of adherens type junctions (Fig. 52) and focal adhesions (Fig. 53) condensations of IF (empty arrow) are seen. Cisternae of endoplasmic reticulum (small asterisks) that are part of ectoplasmic specializations surrounding spermatids (A = acrosome) separate I F from the contact zone. Fig. 49: Bar = 0.25 pm; x 85,000. Fig. 50: Bar = 0.1 pm; x 99,000, Fig. 51: Bar = 0.1 Fm; x 103,000.Fig. 52: Bar = 0.1 km; x 118,000.Fig. 53: Bar = 1.0 pm; x 41,000. Fig. 54: Bar = 1.0 bm; x 10,200. Fig. 55: Bar = 1.0 pm; x 40,000. Fig. 56: Bar = 0.1 pm; x 104,000.


rides, 19801, a unifying hypothesis of their function in Sertoli cells is presented. It is suggested that they may play an important role in the hormone-dependent mechanical integration of exogenous and endogenous cell shaping forces which permits 1)a relatively stable central positioning of the nucleus with its unique internal organization and the coordinated exposition of nuclear pores, 2) the highly variable peripheral configuration of the apico-lateral plasma membrane associated with variable cell-to-cell contacts during germ cell transportation, and 3) the fluctuating fixation of the basal plasma membrane resulting in quantitative changes of functional proteins residing in this area. Previous studies (e.g., by Wrobel and Schimmel, 1989) have pointed to the volumetric changes (ranging between 27% and 38% in bovine Sertoli cells: Wrobel and Schimmel, 1989) of Sertoli cells during the spermatogenic cycle. Spatial changes have to be buffered unless considerable dislocations of the nucleus are to occur. Spacing function is likely to be related to intermediate filaments. A rather similar situation occurs in certain glia form (see Alvarez-Buylla et al., 1987; Spruill et al., 1983b) where a comparable function of vimentin filaments is suggested. The phosphorylation studies performed by Kierszenbaum and his group (see Kierszenbaum et al., 1985; Spruill, 1983a,b) have shown that the conformational changes of the intermediate filaments secondary to hormonal stimulation result in considerable shape changes of the cells. According to Ireland et al. (1986) these changes occur very rapidly (within 5 min). “Multisite phosphorylation could provide a regulatory mechanism allowing cellular responses to diverse physiological stimuli to take place according to different control pathways” (Spruill et al., 1983a). Another aspect is the association of Sertoli cells with the extracellular matrix as well as peritubular cells which both influence Sertoli cell function (see Ailenberg et al., 1988). Kierszenbaum et al. (1986) have demonstrated a differential reaction of vimentin in Sertoli cells from that of peritubular cells, allowing a different reaction of both cells to identical stimuli. In addition, co-cultures of peritubular and Sertoli cells (Ailenberg et al., 1988), and cultures of Sertoli cells on (or in) extracellular matrix (Hadley et al., 1985), result in a polarized orientation of the Sertoli cells as well as the development of a functioning Sertoli cell barrier. It is premature, however, to define the functional role of intermediate filaments in these events and their significance of differential Sertoli cell functions, such as germ cell positioning, spermiation, phagocytosis, or bipolar secretion. The control of intermediate filament biosynthesis, the switch of intermediate filaments from keratin type to vimentin type, the coordination of their biosynthesis with that of membrane and junctional as well as nuclear envelope proteins, their disposal within the cell during cell shape changes, and their degradation are far from being elucidated. In their classic paper Kierszenbaum et al. (1986) related the presence of degradation forms of vimentin in cultured Sertoli cells under certain conditions to the activity of Ca2+-activatedproteases. Nevertheless, close to nothing is known of the

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influence of steroids (estradiol, testosterone), peritubular proteins (P-Mod-S), and germ cell factors on the regulation of Sertoli cell intermediate filaments. Experimental results from in vitro studies are only of limited value with regard to these questions, since often the in vitro effects cannot be observed in intact seminiferous tubules (Kierszenbaum et al., 1985).

INTERMEDIATE FILAMENTS IN SERTOLI CELLS DURING IMPAIRED SPERMATOGENESIS Sertoli cells display pathological alterations of the intermediate filament cytoskeleton in a diversity of spermatogenic disorders (Chemes et al., 1977; Schulze, 1984; Aumiiller and Peter, 1986; Aumuller et al., 1988). They are associated with either a reduction or an increase in the amount of intermediate filaments. Distribution of IF in Hypoplastic Zones, After Estrogen Treatment and in Androgen Insensitivity Sertoli cells in androgen insensitivity syndrome, in so-called hypoplastic zones, and after long-term estrogen treatment display features of immature cells and resemble precursors prior to puberty. Consequently, IF distribution follows a more primitive pattern. In androgen insensitivity syndrome, vimentin immunoreactive filaments were concentrated primarily in the basal cytoplasm (Figs. 57-61), while only a few apical processes were positive. Compared to mature cells, the perinuclear network often appeared less prominent (Aumuller and Peter, 1986). The same applies to the IF distribution in Sertoli cells that occupy the seminiferous cords of hypoplastic zones and those seen after estrogen treatment. Electron microscopic studies show that IF form a distinct network between nucleus and basal cell membrane, but only a thin perinuclear layer. They are only rarely found in the apical cytoplasm. Occasionally, microtubules are seen in parallel with IF, radiating from the apical portion of a nucleus to the adjacent plasma membrane. In all cases studied, predominantly adherens type junctions have been found, ranging from well-defined to more primitive structures. These junctions mainly occur between apical portions of cells. Only rarely could an association of individual IF with the junctions be verified. Distribution of IF in Sertoli-Cell-Only Syndrome, Cryptorchidism, Antiandrogen Treatment, and in the Vicinity of Testicular Tumors In idiopathic Sertoli-cell-only syndrome and postpubertal cryptorchidism, after administration of the antiandrogen cyproterone acetate, and in the vicinity of testicular tumors, IF are considerably increased in number, predominantly in the basal cytoplasm and adjacent to the nucleus. They form loosely arranged bundles which intermingle with cell organelles in the same region (Fig. 63) or (as is illustrated in Fig. 62) occupy extensive areas, thereby displacing the organelles to the cell periphery. These focal accumulations consist of randomly oriented, densely packed IF bundles. Occasionally, alternating layers of longitudinal and crosssectional profiles reflect a more regular arrangement

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Figs. 57-61. Distribution of IF (empty arrow) in Sertoli cells of a case of androgen insensitivity syndrome. Close to the ectoplasmic specializations (Fig. 57) bundles are seen that are dispersed in the perinuclear (N = nucleus) zone (Figs. 58-60). IF (empty arrow) merge

basally into a focal adhesion. Fig. 57: Bar = 0.25 pm; x 68,000. Fig. 58: Bar = 0.25 pm; x 40,800. Fig. 59: Bar = 0.25 pm; x 81,600. Fig. 60: Bar = 0.25 pm; x 24,400. Fig. 61: Bar = 0.25 km; x 40,800.


(Schulze, 1984). A comparable marked increase in the number of IF have been reported by Chemes et al. (1977) in men with germinal aplasia or severe germ cell depletion. The apical portions of these cells appear unaltered: a few IF can be seen coursing parallel t o microtubules aligned with the long axis of the cells. However, IF also concentrate in other than basal portions in Sertoli cells that have lost their normal apico-basal differentiation (e.g., in atrophic cellular cords neighbouring testicular tumors). All kinds of intercellular junctions are present between Sertoli cells in Sertoli-cell-only syndrome and cryptorchidism and after cyproterone acetate administration. In these pathological disorders Sertoli cells are the only or the predominating cell types in seminiferous tubules. Ectoplasmic specializations in Sertoli-cellonly syndrome and in cryptorchidism are unusually extensive (Chemes et al., 1977; Schulze, 1984); a direct contact of IF with any of these junctions, including adherens type junctions, was difficult t o establish. Yet conspicuously well-defined adherens type junctions with prominent dense plaques seen between Sertoli cells in the vicinity of testicular tumors show attachment of loosely arranged IF easily recognizable on electron micrographs (Fig. 64). Except for the dense midline structure these junctions morphologically resemble true desmosomes. Such well-developed desmosome-like junctions have never been observed between Sertoli cells associated with normal spermatogenesis. They are the only type of junction found in these cellular cords; ectoplasmic specializations do not occur. Apart from the conspicuous adherens type junctions and the most dense IF accumulations of the material studied, the nuclei of these Sertoli cells that directly abut upon seminoma cells contain unusual nuclear bodies. For all these special features, Sertoli cells from such cases may be considered a unique cell variant. This notion has lately been underlined by Altmannsberger, who observed that Sertoli cells in the vicinity of tumors display a strong cytokeratin immunoreaction (personal communication). The observation may indicate that Sertoli cells in these pathological conditions deviate not only morphologically but also chemically from normal adult cells. Relating data from animal experiments (see preceding section) to the findings attained in man, we assume that by expressing cytokeratin filaments Sertoli cells have possibly regained undifferentiated features. By modulating their IF proteins they may be able to meet the requirements of an altered milieu. An indication that such a functional variant expressing cytokeratin may also occur in normal testicular tissue of adult men has been pointed out by Miettinen et al. (1985). In an immunohistochemical study they found cytokeratin-positive cells in atrophic and occasionally also in normal-appearing seminiferous tubules with active spermatogenesis. However, it could not be ascertained whether the cells in question originated from Sertoli cells or from abnormally differentiated germ cells. Cytokeratin- in addition to vimentin-positive tumor cells, a t any rate, have recently been docu-

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mented in a malignant Sertoli cell tumor (Nielsen and Jacobsen, 1988). In the preceding section a close relationship between the formation of IF and the characteristic CharcotBottcher crystalloids has tentatively been suggested. Evidence in favor of this possibility has been provided by the observation that the increase in IF in Sertolicell-only syndrome, in cryptorchidism, and after administration of cyproterone acetate is paralleled by a marked increase in crystalloids (Schulze, 1984). These crystalloids are mostly larger than under normal conditions and are invariably found among loosely arranged bundles of IF. Their association with IF resembles that described above.

Pathophysiological Aspects Focal aggregates of IF are also observed in cells other than Sertoli cells. A disturbed organization of all classes of IF has been documented in a diversity of pathological conditions both in man and experimental animals. Either normal or morphologically and/or chemically altered IF have been described in this context. It is noteworthy that increased amounts of IF occur due to the influence of various toxins-for example, as Mallory bodies consisting of an altered cytokeratin skeleton in alcoholic hepatitis of man and in griseofulvin-induced liver damage of mouse (Denk and Krepler, 1982). Similarly, cultured hepatoma cells derived from the liver of diethyl-nitrosamine-treated rats show large aggregates of cytokeratin and vimentin filaments that closely resemble those described in Sertoli cells (Borenfreund et al., 1980). The same applies to accumulations of IF in human leucemic cells. These filamentous aggregates can be correlated to a distinct shape of the cells and therefore may be of functional significance (Felix and Strauli, 1978). Pathological accumulations have also been reported of neurofilaments, glial filaments, and desmin filaments. However, further details concerning the respective diseases are beyond the scope of this paper (for further information and references, see Denk and Krepler, 1982). The fact that a variety of lesions and drug-induced disturbances all result in an increase in IF suggests that this increase is merely a nonspecific reaction. The masses of IF in Sertoli cells under pathological conditions may be interpreted in the same way. In view of our incomplete knowledge of the biological functions of IF to date, one can only speculate upon the functional significance of the abnormal IF accumulations in Sertoli cells. As mentioned above, IF are so densely packed that they exclude other organelles from large areas of the cytoplasm. It may be simply inferred from this observation that important organelle-dependent functions may be reduced too. Moreover, it has been suggested that by connecting the nucleus with the cell membrane vimentin may contribute to signal transduction and transport processes between cell surface and nucleus (for references, see Introduction). It is conceivable, therefore, that surplus IF in the basal cytoplasm of Sertoli cells may interrupt such a communication and strongly interfere with the normal cell functions. Further investigations of the IF proteins in

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Figs. 62-64.

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Sertoli cells in the vicinity of a testicular tumor.

Fig. 62. The basal cytoplasm is filled with densely packed IF bundles. Bar = 0.25 pm; x 31,400. Fig. 63. Loosely arranged IF bundles intermingle with cell organelles. IF radiate from the nucleus and are seen to merge in

the focal adhesions of the basal cell membrane. Bar = 0.25 pm; x 31,400. Fig. 64. Adherens type junctions between adjacent Sertoli cells that show attachment of loosely arranged IF. Bar = 0.25 pm; x 28,000.


Sertoli cells under pathological conditions and of the molecular mechanisms involved in IF accumulations are necessary to support this idea. They may be useful in elucidating in more detail the physiological significance of IF in Sertoli cells associated with normal spermatogenesis.

CONCLUSIONS In immature testes intermediate filaments in Sertoli cells are of both the keratin and vimentin type, the latter becoming most prevalent during testicular maturation. With regard to the histogenesis of the cells, the immunocytochemical character of the filaments is not discriminatory. In normal adults only vimentin is expressed in Sertoli cells. Under pathological conditions keratin expression may be resumed in Sertoli cells. Depending on the stage of the spermatogenic cycle, the distribution of intermediate filaments and the shape of the Sertoli cells vary considerably. While in human material a clumsy postspermiation configuration (PS, stages 111-IV) has been distinguished from a ramified prespermiation configuration (AS, stages V-10, in the rat elongate apical vimentin extensions occur in stages XII-V, but are rare in stages VII and VIII. Focal accumulations of IF in Sertoli cells occur in a diversity of spermatogenic disorders. This disturbed organization is suggested to be the result of a non-specific reaction. In view of current hypotheses of their functions it is argued that IF may be involved in the hormone-dependent mechanical integration of exogenous and endogenous cell shaping forces. They permit the positioning of cytoplasmic organelles and the anchoring of the basolateral cell portion, thereby forming a central perinuclear stable compartment and a peripheral mobile zone adapted to cell-to-cell contacts. The intermediate filaments constitute a mechanical lattice which is fixed basally to the plasma membrane and laterally to the belt-like ectoplasmic specializations associated with the intersertoli cell tight junctions. Changes in membrane rigidity, junctional morphology, and membrane disposal typical of the apico-lateral plasma membrane seem to elicit an internal rearrangement of the intermediate filaments. Only limited data, resulting from in vitro studies, are available on the hormonal and local factors responsible for the biosynthesis, disposal, and degradation of intermediate filaments and their control in Sertoli cells. ACKNOWLEDGMENTS The authors gratefully acknowledge the engaged secretarial work of Mrs. K. Gerbig, who typed the manuscript. They are also grateful t o Mrs. I. Dammshauser, Mrs. M. Boge, and Mr. S. Schillemeit for technical and photographic work and to PD Dr. J. Seitz and Dr. M. Aumiiller for their help during the preparation of the manuscript. REFERENCES Ailenberg, M., Tung, P.S., Pelletier, M., and Fritz, LB. (1988) Modulation of Sertoli cell functions in the two-chamber assembly by peritubular cells and extracellular matrix. Endocrinology, 122:26042612.

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Intermediate-sized filaments of human endothelial cells.

Intermediate filaments in nervous tissues.

Molecular interactions in intermediate filaments.

Characterization of Sertoli cell perinuclear filaments.

HeLa cells contain intermediate-sized filaments of the prekeratin type.

Intermediate filaments in the human extraocular muscles.

Role of Intermediate Filaments in Vesicular Traffic.

Mechanosensing through focal adhesion-anchored intermediate filaments.

Intermediate filaments. Getting under the skin.

Epithelial Intermediate Filaments: Guardians against Microbial Infection?

Analyzing dynamic properties of intermediate filaments.

Immuno-histochemistry and three-dimensional architecture of the intermediate filaments in Purkinje cells in mammalian hearts.

Intermediate filaments in rat ovarian surface epithelial cells: changes with neoplastic progression in culture.

Heavy meromyosin labeling of intermediate filaments in cultured connective tissue cells.

Attachment of mitochondria to intermediate filaments in adrenal cells: relevance to the regulation of steroid synthesis.

Intermediate filaments and steroidogenesis in adrenal Y-1 cells: acrylamide stimulation of steroid production.

Transient storage of a nuclear matrix protein along intermediate-type filaments during mitosis: a novel function of cytoplasmic intermediate filaments.

Microfilaments, microtubules, and intermediate filaments in cultured corneal fibroblasts.

In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells.

Organization of intermediate filaments and their association with collagen-containing phagosomes in mouse decidual cells.

Novel origin of lamin-derived cytoplasmic intermediate filaments in tardigrades.

Quantum mechanism of light transmission by the intermediate filaments in some specialized optically transparent cells.

Errata: Quantum mechanism of light transmission by the intermediate filaments in some specialized optically transparent cells.

Dynamic properties of intermediate filaments: disassembly and reassembly during mitosis in baby hamster kidney cells.