Cell Tissue Res. 202, 357-377 (1979)

Cell and Tissue Research 9 by Springer-Verlag 1979

The Lamina Propria of the Bovine Seminiferous Tubule* Karl-Heinz Wrobel, Roswitha Mademann, and Fred Sinowatz* Institut fiir Anatomie der Universit~itRegensburg, Regensburg, BundesrepublikDeutschland

Summary.The boundary tissue of bovine testicular seminiferous tubules exhibits remarkable regional differences at the level of the seminiferous tubule proper, as compared with its terminal segment. The basal lamina of the seminiferous tubule proper is multilayered and possesses knob-like protrusions. At the level of the terminal segment the basal lamina is highly specialized; in the region of the terminal plug candelabrum-like projections of the tubular basal lamina invade the bases of the modified supporting cells up to a depth of 3.5 lam. The adjoining surface of these supporting cells is densely studded with hemidesmosomes. The elongated peritubular cells are arranged in 3-5 concentric layers around the tubulus seminiferus proper but form a loose association at the level of the terminal segment. Where the terminal segment joins the testicular straight tubule, peritubular cells may assemble to constitute a contractile spiral. Elastic tissue is situated mainly subjacent to the tubular basal lamina and to a lesser degree between the peritubular cell layers. A peritubular space lined by endothelium-like cells may surround the seminiferous tubule proper and also the transitional zone of the terminal segment. Key words: Seminiferous tubule (bull) - Regional differences - MyofibroblastsElastic tissue - Elastin.

Zusammenfassung. Die Membrana propria der Hodentubuli des Rindes weist im Bereich des eigentlichen Tubulus seminiferus contortus und seines Terminalsegmentes bemerkenswerte regionale Unterschiede auf. Die Basallamina des eigentlichen Tubulus seminiferus hat einen geschichteten Bau und besitzt knopff6rmige Verdickungen. Im Terminalsegment zeigt die Basallamina eine zunehmende Spezialisierung; in H6he des terminalen Pfropfes besitzt sie kandelaberartige Forts/itze, die bis zu einer Tiefe von 3,5 lam in die Basalabschnitte der hier gelegenen modifizierten Sttitzzellen v0rdringen. Die der Prof. Dr. Dr. K.-H. Wrobel, Institut fiir Anatomie, UniversitfitRegensburg, Universit~itsstraBe 31, D-8400 Regensburg, Federal Republic of Germany * Supported by a grant from the Deutsche Forschungsgemeinschaft

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K.-H. Wrobelet al. Basallamina anliegende Oberflfiche dieser St/.itzzellen bildet eine groge Anzahl von Halbdesmosomen aus. Die flachen peritubulgren Zellen umgeben mit 3-5 konzentrischen Lagen den eigentlichen Tubulus seminiferus und formieren sich im Terminalsegment zu einem stark aufgelockerten Verband. Am Ubergang vom Terminalsegment zum Tubulus rectus k6nnen sich peritubul~re Zellen zu einer kontraktilen Spirale zusammenlagern. Alle peritubulfiren Zellen des Rindes sind dem Typ nach als Myofibroblasten einzuordnen. Elastisches Gewebe ist vor allem unter der tubulfiren Basallamina und in geringerer Menge auch zwischen den peritubul~ren Zellagen festzustellen. Um den eigentlichen Tubulus seminiferus und um die fSbergangszone des Terminalsegments kann ein peritubul/irer Spaltraum ausgebildet sein, der allseits yon flachen endothelfihnlichen Zellen begrenzt wird.

In ruminants the time elapse between spermiation and appearance of spermatozoa in the head portion of the epididymis amounts to maximally 3 days (Esnault et al., 1974). Testicular spermatozoa, which still lack active motility, are transported passively through the seminiferous tubules, the testicular straight tubules, the rete testis and the efferent ductules in order to reach the epididymal canal. It is now rather well established that transport through the testicular excurrent duct system depends mainly upon two conditions: (i) a directed flow of tubular secretion originating from the Sertoli cells, and (ii) the presence of contractile cells in the periphery of the duct system and the tunica albuginea. Since 1951, when RoosenRunge first observed periodical peristaltic contraction waves running over seminiferous tubules in isolated testes from rats and dogs, a large number of investigations has dealt with the structure and function of the membrana propria surrounding the seminiferous tubules and its contractile peritubular cells (for references, see Ross, 1967; Dierichs and Wrobel, 1973; Bustos-Obreg6n and Holstein, 1973; Unsicker and Burnstock, 1975). By these investigations not only the role of the peritubular cells in intratubular sperm transport has been elucidated, but also their postnatal differentiation from fibroblast-like elements under hormonal influence (Ross, 1967; Bressler and Ross, 1969, 1972; Kormano and Hovatta, 1972: Dierichs and Wrobel, 1973; Bustos-Obreg6n and Courot, 1974) as well as their importance as part of the blood-testis barrier (Heidger, 1969; Dym and Fawcett, 1970; Fawcett et al., 1970; Willson et al., 1973). Although the membrana propria of the seminiferous tubules in adult reptiles, birds and mammals concordantly includes contractile peritubular cells and follows a general scheme of organization, there is, on the other hand, considerable variation in the morphological appearance and histochemistry of the tubular boundary region in the species so far studied. From the tubular epithelium to the periphery the membrana propria of all adult species can roughly be subdivided into four layers (Courot et al., 1970): (i) an internal noncellular layer, (ii) an internal cellular layer with the specialized peritubular cells, (iii) an external noncellular layer, and (iv) an external cellular layer blending with the intertubular tissue. According to the appearance of the internal noncellular layer and the arrangement of the peritubular cells, Burgos et al. (1970) differentiated between a membrana propria of type a, b and c. Morphological information on the structural

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o r g a n i z a t i o n o f the b o v i n e m e m b r a n a p r o p r i a is, however, scarce. C o u r o t et al. (1970) m e n t i o n e d for the bull a characteristic l a m e l l a r a p p e a r a n c e o f the internal n o n c e l l u l a r layer. A s in m o s t m a m m a l s , the m e m b r a n a p r o p r i a o f the b o v i n e shows h i s t o c h e m i c a l l y a positive r e a c t i o n for p h o s p h a t a s e (alkaline p h o s p h a t a s e a n d a d e n o s i n e t r i p h o s p h a t a s e ) activity ( W r o b e l a n d E1 Etreby, 1971; W r o b e l et a1.,1978). Since the t u b u l a r m e m b r a n a p r o p r i a reacts c h a r a c t e r i s t i c a l l y to deleterious influences u p o n the testis a n d with respect to the e c o n o m i c i m p o r t a n c e o f all questions d e a l i n g with b o v i n e r e p r o d u c t i o n , the first a i m o f the p r e s e n t s t u d y is to give a d e t a i l e d a c c o u n t o f the u l t r a s t r u c t u r a l o r g a n i z a t i o n o f the m e m b r a n a p r o p r i a o f the n o r m a l b o v i n e testicular seminiferous tubule. F u r t h e r m o r e , a n effort was m a d e to elucidate the s t r u c t u r a l specializations o f the different c o m p o n e n t s o f the m e m b r a n a p r o p r i a s u r r o u n d i n g the t e r m i n a l segment o f the seminiferous tubule. H i s t o l o g i c a l studies ( W r o b e l et al., 1978) have shown t h a t in the bull this t e r m i n a l segment is c o m p o s e d o f three zones: (i) a t r a n s i t i o n a l zone b o r d e r i n g o n the n o r m a l seminiferous tubule, (ii) a m i d d l e p o r t i o n consisting m a i n l y o f m o d i f i e d s u p p o r t i n g cells, a n d (iii) the t e r m i n a l p l u g with an extremely n a r r o w l u m e n t h r o u g h which all t u b u l a r fluid a n d all s p e r m a t o z o a o f a given t u b u l e m u s t p a s s before entering the testicular straight tubule. The l u m i n a l d i a m e t e r in the t e r m i n a l segment can very likely be r e g u l a t e d b y s t r u c t u r a l a d a p t a t i o n s o f the p e r i t u b u l a r tissue. C a v i c c h i a a n d B u r g o s (1977) d e s c r i b e d a sphincter-like ring o f c o n t r a c t i l e cells o n the b o r d e r o f seminiferous a n d s t r a i g h t testicular tubules in the h a m s t e r . In the bull a w e l l - d e v e l o p e d v a s c u l a r plexus s u r r o u n d s the t e r m i n a l segment a n d m a y exert a r e g u l a t o r y f u n c t i o n on the t u b u l a r l u m e n in this species ( W r o b e l et al., 1978).

Materials and Methods For the present study, 27 testes of sexually mature bulls (Deutsches Fleckvieh) were fixed by vascular perfusion. Five to 10rain after slaughter the testes were removed from the animals by severing the spermatic cord proximal to the convoluted portion of the testicular artery. The cannula was inserted either into the straight funicular portion or into an intratunical portion of this artery. Whereas the insertion into the first position gave inconstant results, intratunical cannulation of the testicular artery resulted in a complete and reproducible perfusion of the testicular vascular bed. Perfusion was carried out with a rotor roller pump using a Masterfiex | pumphead (Cole-Parmer Instruments Company). Rinsing fluid was instilled at 24 ml/min, fixative at ! 8 ml/min. During perfusion via the straight funicular portion of the testicular artery, the periorchium was incised in order not to impede the volume increase of the testis.

Preparationfor Light Microscopy. Following the rinsing procedure (for composition of rinsing solution, see Wrobel et al., 1978), fixation was accomplished using either Bouin's fluid, Stieve's fluid or a mixture of methanol, chloroform and glacial acetic acid (Puchtler et al., 1976). Paraplast-embedded 5-7 ~tmthick sections were subjected to the following staining procedures: Masson-Goldner, van Gieson, van Giesonresorcin-fuchsin, resorcin-fuchsin dissolved in 70 % alcohol (RF-70 %), resorcin-fuchsin dissolved in 100 K alcohol (RF-100 %), tannic acid-phosphomolybdic acid-thiazine method (Puchtler et al., 1975), PAS-reaction, alcian blue at pH 0.5 (AB 0.5), alcian blue at pH 2.5 (AB 2.5).

Preparationfor Electron Microscopy. After rinsing (as above) perfusion fixation was performed either with solutions I and II of Forssmann et al. (1977) or with a 3 ~ solution of phosphate-buffered glutaraldehyde. Small pieces of the border region between the central mediastinum and the adjoining testicular parenchyma were separated and washed in 0.2 M phosphate buffer. After or without

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osmication (1% OsO4) the blocks were dehydrated in graded ethanols and embedded in either Epon, Araldite, ERL 4206 (Spurr, 1969), or an Epon-Araldite mixture. Ultrathin sections were mounted on uncoated copper grids and stained routinely with uranyl acetate and lead citrate (Reynolds, 1963). For the electron microscopic visualization of elastic tissue components, ultrathin sections were treated with orcein (Nakamura et al., 1977) or phosphotungstic acid-uranyl acetate (Wiedmer-Bridel et al., 1977). Examination of ultrathin sections was carried out with a Zeiss EM 10A electron microscope.

Results Light Microscopy

In r o u t i n e histological sections o f the seminiferous tubule p r o p e r (Fig. 1) the t u b u l a r e p i t h e l i u m is b o u n d e d by a lightly stained, strongly refringent, slightly u n d u l a t e d b a s a l l a m i n a . A n a r r o w s t r a t u m o f flattened p e r i t u b u l a r cells is l o c a t e d a d j a c e n t to the b a s a l lamina. A m u l t i l a y e r e d a r r a n g e m e n t o f these cells is s u b s t a n t i a t e d by the o b s e r v a t i o n t h a t occasionally two elongated, intensively stained nuclei lie in close parallel arrangement. Even in optimally fixed specimens, p e r i p h e r a l to the p e r i t u b u l a r cell layers, n a r r o w p e r i t u b u l a r spaces which m a y s u r r o u n d c o n s i d e r a b l e p o r t i o n s o f the t u b u l a r circumference can be found. The p e r i t u b u l a r spaces are lined o n either side b y flattened e n d o t h e l i u m - l i k e cells. The c o m p o s i t i o n o f the m e m b r a n a p r o p r i a in the t r a n s i t i o n a l zone o f the t e r m i n a l segment is basically identical to the situation described for the seminiferous t u b u l e p r o p e r . I n the s u b s e q u e n t t w o p o r t i o n s o f the t e r m i n a l segment (middle p o r t i o n a n d t e r m i n a l plug) which are s u r r o u n d e d b y a sleeve-like vascular plexus, b o t h the b a s a l l a m i n a a n d the s t r a t u m o f p e r i t u b u l a r cells are m a r k e d l y thickened. The l a t t e r is n o t exclusively due to a n u m e r i c a l increase o f p e r i t u b u l a r cells b u t r a t h e r to a b r o a d e n i n g o f the intercellular areas. Since a p e r i t u b u l a r space is lacking a r o u n d the distal two p o r t i o n s o f the bovine t e r m i n a l segment, there is no distinct d e m a r c a t i o n between the p e r i t u b u l a r cell layer a n d the i n t e r v a s c u l a r connective tissue. A t the distal b o r d e r o f the v a s c u l a r plexus, a p p r o x i m a t e l y at the level where the t e r m i n a l s e g m e n t o f the seminiferous t u b u l e changes into the straight testicular tubule, cushions o f s t a c k e d p e r i t u b u l a r cells can be o b s e r v e d in cross as well as in l o n g i t u d i n a l sections (Figs. 2, 3). In f o r t u i t o u s oblique sections these densely

Fig. 1. Seminiferous tubules proper; van Gieson, • 560. General organization of tubular boundary region; arrow points to peritubular space Fig. 2. Cross section through the distal portion of the terminal segment of seminiferous tubule; van Gieson, • 560. Arrowhead indicates the small central lumen of the terminal plug; arrows delimit expansion of peritubular cell cushion Fig. 3. Longitudinal section through the border region between terminal segment of seminiferous tubule (right) and straight testicular tubule (left); van Gieson-resorcin-fuchsin, • 560. Arrow points to a thick strand of cross-sectioned peritubular cells Fig.4. Tangentially cut seminiferous tubule proper; RF-100%, x 560. Delicate, parallel orientated elastic fibrils (arrows) are situated immediately subjacent to the tubular epithelium (TE). A peripheral network of thicker, irregularly arranged fibrils, best seen in a horizontal section of the tubular surface (TS), corresponds to elastic tissue in the interstices between peritubular cell layers

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packed peritubular cells are often seen to surround nearly the entire tubular circumference. In sections stained according to Puchtler et al. (1975), a staining method for contractile proteins, the peritubular cells exhibit only a slight affinity for thiazine; in sections stained with van Giesoffs method the peritubular cells reveal an orange color. Thus, bovine peritubular cells differ from true smooth muscle cells in the same section by their tinctorial properties. Demonstration of elastic tissue with RF-70 ~o and RF-100 ~ results in a similar localization of positive material in the boundary tissue of the seminiferous tubule proper. After treatment with RF-100~o, however, the staining is significantly stronger and distinctly bound to fibrillary structures. The basal lamina is always negative. The bulk of positive material is situated in the narrow space between basal lamina and peritubular cells. Here, a rather homogeneous and continuous, thin RFpositive layer can be identified in longitudinal sections. In tangentially cut tubules, however, especially after staining with RF-100~o, a system of densely packed, parallel orientated, fine fibrils is recognized subjacent to the basal lamina. These fibrils surround the tubular circumference in a circular or helical manner (Fig. 4). Additional RF-positive material is found in the interstices between peritubular cell layers. Here again a fibrillary organization of this positive material becomes evident in tangentially cut tubules, especially with RF-100 ~ (Fig. 4). The fibrils between the peritubular cells do not run parallel but form a loose network and are less densely packed than the fibrils immediately subjacent to the basal lamina. At the level of the terminal segment differences between staining with RF-70 ~o and RF-100 ~ concerning localization and quantity of positive substances are more significant. After R F - 1 0 0 ~ up to seven partially interrupted, delicate positively staining lamellae are found between the peritubular cells or the elements of the sleeve-like vascular plexus (Fig. 5). After staining with RF-70~o the positive material is concentrated mainly subjacent to the basal lamina and forms a continuous layer with many short, tooth-like projections pointing to the tubular center (Fig. 6). That RF-70~o and RF-100~o differ in their affinities for various tissue components becomes quite evident by studying the tinctorial behavior of larger testicular arteries. With RF-100 ~ (Fig. 7) elastica interna, some delicate fibrils in the media and abundant separate fibrils in the adventitia are stained. On the other hand, components of the elastica interna and media have practically no affinity for RF-70 ~o, whereas the whole adventitia is intensively and nearly homogeneously stained (Fig. 8). With the PAS-reaction, a weak to moderate coloration of the basal lamina and the intercellular substances in close proximity to the peritubular cells is attained. The tubular basal lamina has no or only a very weak affinity forAB 0.5 andAB 2.5 (Fig. 9). The ground substance between the peritubular cells in the boundary region of the seminiferous tubule proper stains rather uniformly and moderately intensely with AB 2.5. With AB 0.5 the intensity of the stainin~ reaction shows greater variation. The intercellular ground substance at the interstitial side of the peritubular cell layer has a very weak affinity for AB at both pHs; the peritubular spaces are negative. At the level of the tubular terminal segment (Fig. 10) the AB reaction of the

Fig. 5. Longitudinal section through terminal segment of seminiferous tubule; RF-100~, • Delicate lamellae of positive material are situated in the peritubular region. TE tubular epithelium Fig. 6. Cross section through terminal plug; RF-70 ~, x 560. The dentate inner contour of the positive subepithelial layer is best seen in the upper sector between the two arrows. TE tubular epithelinm Fig. 7. Wall of testicular artery; RF-IO0%, x 140 Fig. 8. Wall of testicular artery; RF-70 ~, • 140 Fig. 9. Boundary region of seminiferous tubule proper; AB 2.5, x 560. The positive material in the tubular wall (between arrows) subjacent to the negative basal lamina corresponds to ground substance between peritubular cells. TE tubular epithelium Fig. 10. Longitudinal section through the border region between terminal segment and straight testicular tubule; AB 0.5, x 350. Small amount of positive material in the area of the perivascular plexus (left third); more positive material and stronger tinctorial reaction in the area of a peritubular cell cushion (arrow); TE unstained tubular epithelium

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intercellular ground substance between the peritubular cells and the cellular elements of the vascular plexus is generally weak. The reaction, however, is stronger in the narrow interstices between those densely packed peritubular cells that participate in the formation of the multilayered cushions.

Electron Microscopy The slightly undulated basal lamina of the seminiferous tubule proper generally possesses smooth contours but occasionally exhibits knob-like projections which invaginate into the basal portions of spermatogonia and Sertoli cells (Fig. 11). The thickness of the basal lamina measures between 0.4 and 0.8 gm and amounts up to 1.2 gm at the knob-like projections. With most of the techniques presently applied the basal lamina exhibits a multilayered appearance. The lamellar organization is best seen following fixation with the solutions I and II of Forssmann et al. (1977), embedding in Araldite and staining with phosphotungstic acid-uranyl acetate (Fig. 12). In the even or slightly undulated portions of the basal lamina 6-9 approximately parallel, electron-dense layers are recognized which are separated by less electron-dense or transparent interspaces. In the knob-like projections up to 15 electron-dense layers can be identified. Here, the dark layers do not always run parallel, are sometimes bifurcated and are often separated by broader transparent interspaces. In the transitional zone of the terminal segment the knob-like projections of the basal lamina increase in size and frequency (Fig. 13). A lamellar organization of the basal lamina is distinct in its inner half, however, less defined in its outer half. The thickness of the basal lamina at the level of the knobs amounts to 2 lam. In the middle portion of the terminal segment there is a further increase in thickness of the basal lamina (Fig. 14). In sections treated with methods revealing the lamellar organization, the dense layers do not run parallel but converge and diverge. Also the number of dense layers is diminished compared with the situation around the seminiferous tubule proper, resulting in development of rather large interlamellar areas containing some minute granular material and single collagen fibrils. Thick and thin irregular projections of the basal lamina, which are never lamellar in appearance, invaginate for a remarkable distance into the base of the modified supporting cells. A clear-cut external demarcation of the basal lamina is difficult to ascertain in this region, since an abundant amorphous ground substance of a similar morphological structure is situated immediately subjacent to it. In the third region of the terminal segment, where the basal portions of those supporting cells that form the terminal plug can be identified, the basal lamina is never lamellated, notwithstanding fixation, embedding and staining procedures applied (Fig. 15). However, the basal lamina comprises indistinct bordering light areas in a predominantly dense surrounding. The basal lamina is composed of a continuous 0.3-1.6 ~tm thick basal plate and slender, branched, candelabrum-like projections which invade the basal portions of the supporting cells up to a depth of 3.5 lam and deeper (Figs. 16, 17). The basal cytoplasm of the supporting cells in question contains bundles of approximately 11 nm ( = 110A) thick filaments. The cell surface adjacent to the basal lamina and its projections is densely studded with

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Fig. 11. Seminiferous tubule proper. Fixative according to Forssmann et al. [ref. Forssmann] (1977), Epon-Araldite, uranyl acetate - lead citrate, x 12,325. Basal lamina (BL) with knob-like projection (arrow); PC layer of peritubular ceils; TE tubular epithelium Fig. 12. Seminiferous tubule proper. Forssmann, Araldite, phosphotungstic acid-uranyl acetate, x 40,000. Multilayered appearance of basal lamina. In the space between basal lamina and peritubular cell (PC) collagen fibrils (CF), elastin (E), microfibrils (arrow) and ground substance are identified Fig. 13. Transitional zone of terminal segment. Forssmann, Epon-Araldite, uranyl acetate- lead citrate, x 10,075. Note increasing frequency of knob-like projections of the multilayered basal lamina and broadened sublaminal space compared with the seminiferous tubule proper (see Fig. 11). PC peritubular cell

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Fig. 14. Middle portion of the terminal segment. Forssmann, Epon-Araldite, uranyl acetate - lead citrate, x 12,325. The basal lamina (BL) reveals a reticular appearance and possesses irregular projections into the tubular epithelium (TE). The area surrounding this part of the terminal segment contains dark single collagen fibrils, bundles of weakly stained collagen fibrils (CF) and peritubular cells (PC) loosely interspersed within abundant intercellular ground substance. Peritubular cells of this region may possess at some sites a thin, separate basal lamina (arrows) Fig. 15. Distal portion of the terminal segment (area of terminal plug). Forssmann, Epon-Araldite, uranyl acetate - lead citrate, x 12,325. The basal lamina (BL) consists of a basal plate and long, slender, branched projections invaginating the tubular epithelium (TE)

Fig. 16. Candelabrum-like projection of basal lamina invaginating the base of a modified supporting cell from the terminal plug. Within the basal lamina dark collagen fibrils (arrows) are incorporated. Forssmann, Epon-Araldite, uranyl acetate - lead citrate, • 30,000 Fig. 17. Cross-sectioned projections of basal lamina (arrows) within the basal portion of a modified supporting cell. Forssmann, Epon-Araldite, uranyl acetate lead citrate, • 32,000

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hemidesmosomes (Fig. 18). Occasional collagen fibrils are located within the basal lamina including its candelabrum-like projections. At the level of the seminiferous tubule proper the narrow space between basal lamina and the layer of peritubular cells contains some collagen fibrils, a few microfibrils and patchy accumulations of a material very similar to the electrondense portions of the basal lamina (Fig. 11). In places where this material and the true tubular basal lamina are in close contact, the assumed borderline between both components is only vaguely discernible. At the periphery this basal lamina-like material may come in contact with the surface of the peritubular cells, which in the bovine do not possess a distinct separate basal lamina, as is the case in a number of other species. Following Epon-Araldite or ERL embedding and conventional staining, empty areas of irregular size are seen subjacent to the tubular basal lamina. Following orcein and lead citrate treatment, the entirety of the narrow space between tubular basal lamina and peritubular cells is stained in a specific manner (Fig. 19): Collagen fibrils have the strongest, the tubular basal lamina has the weakest affinity for this staining sequence; the remainder, composed of microfibrils and the empty areas of conventionally treated sections, has an affinity of medium strength. Especially following embedding in Araldite and staining with phosphotungstic acid-uranyl acetate, the "empty spots" of routinely treated sections contain elastin (Fig. 20). Elastin is also observed subjacent to the basal lamina of the terminal segment but in smaller quantities than at the level of the seminiferous tubule proper. The peritubular cell sheath of the seminiferous tubule proper has a total thickness of 1.5-2.5 pm and contains three to five layers of elongated, flattened cells (Fig. 11). Their diameters vary from approximately 1.6 ~tm in the perinuclear region to less than 0.1 ~tm in the attenuated ends. Adjacent layers of peritubular cells may be separated for long distances by relatively broad intercellular spaces. Occasionally, blunt lateral processes span these spaces and establish localized contacts between adjacent layers. The slender, sometimes bifurcated terminal processes of contiguous peritubular cells in the same layer generally overlap for a certain distance. Here, the opposing membranes are separated by intercellular gaps of approximately 20 nm; morphologically specialized attachment sites, however, are very rare in the zones of overlapping. The cytoplasmic matrix of peritubular cells appears appreciably electron-dense and contains a few mitochondria and some elongated profiles of rough endoplasm reticulum (Fig. 21). Especially in the terminal processes but also elsewhere in the peritubular cells, bundles of 7 nm ( = 70A) filaments are observed (Fig. 21), by preference following Araldite embedding. Electron-dense areas in the course of the filament bundles or anchoring of the filaments onto subplasmalemmal densities are occasionally identified. A peculiar and frequent feature of the cell periphery are round or pear-shaped micropinocytotic vesicles of 70-80 nm (Fig. 21) which may contain a fine granular material of varying distribution. In the areas of overlapping these vesicular inpocketings are virtually absent. The nuclei of peritubular cells are elongated, fusiform or rod-like and may attain a length of 15 ~tm and more. The broad spaces between different layers of peritubular cells comprise only a few collagen fibrils but abundant cell coat-like material. A continuous and distinct basal lamina, however, is absent from the surface of the peritubular cells.

Fig. 18. A b u n d a n c e of 110A (11 nm) - filaments in the basal cytoplasm of a modified supporting cell from the area of the terminal plug. The basal lamina (BL) and its projections are almost continuously covered by hemidesmosomes. Forssmann, Epon-Araldite, uranyl acetate - lead citrate, • 32,000 Fig. 19. Seminiferous tubule proper. Glutaraldehyde, ERL 4206, orcein - lead citrate, • 64,000. Following this treatment basal lamina (BL), elastin (E) and collagen fibrils (arrows) are identified by their differing contrast. TE unstained tubular epithelium; PC peritubular cells Fig. 20. Seminiferous tubule proper. Glutaraldehyde - OsO4, Araldite, phosphotungstic acid-uranyl acetate, • 64,000. Elastin accumulations (E) subjacent to the basallamina (BL) a n d between peritubular cell layers (PC). Arrows indicate collagen fibrils. TiE tubular epithelium

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Furthermore, irregularly limited accumulations of elastin are identified in the intercellular spaces, especially following phosphotungstic acid-uranyl acetate staining (Fig. 20). However, the quantities of elastin between peritubular cell layers are lower than subjacent to the tubular basal lamina. The stratum of peritubular-cell layers is covered at the interstitial side by discontinuous accumulations of basal lamina-like material in which collagen fibrils - sometimes in thick bundles - are embedded. The organization of the peritubular-cell layers in the transitional zone of the terminal segment is very similar to the situation of the seminiferous tubule proper; the intralamellar spaces, however, are generally broader. In the middle portion of the terminal segment and in the region of the terminal plug, where the vascular plexus is situated, a regular stratification of the peritubular cells is no longer visible, except in the multilayered cushions. Generally, subjacent to the tubular basal lamina, capillaries, fibroblasts and the characteristic dark peritubular cells (sometimes with a true, distinct basal lamina) are found loosely interspersed within an intercellular ground substance containing a few microfibrils, small accumulations of elastin and collagen fibrils (Fig. 14). The latter form either thick bundles, often with a remarkably low affinity for the conventional stains, or are strongly stained and lie singly. The multilayered cushions of peritubular cells, that are best seen flanking the transition of the terminal segment to the straight testicular tubule (Fig. 22), are characterized by relatively narrow interlamellar spaces (0.1-0.3 gm) containing no collagen but some elastin and accumulations of basal lamina-like material. Overlapping of terminal processes with intercellular gaps measuring 20 nm and morphological attachment sites are again common features. At the level of the seminiferous tubule proper and the transitional region of the terminal segment, a peritubular space may be developed outside the peritubular cell layers (Fig. 23 a, b). These peritubular spaces are lined on either side by flattened endothelium-like cells. The cytoplasm of these cells generally contains many vesicles 70-80 nm in diameter (Fig. 23b). The terminal cell margins of adjoining endothelium-like cells overlap for a certain distance, but are nevertheless not in close contact with each other, and special attachment sites are not observed. No basal lamina can be attributed to the endothelium-like lining. Occasionally, migrating cells with many morphological features of lymphocytes are observed traversing the peritubular spaces (Fig. 23 a, b).

Fig. 21. Two peritubular cells from the boundary tissue of seminiferous tubule proper. Forssmann OSO4, Araldite, uranyl acetate - lead citrate, x 64,000. Note 70 A (7 nm) filaments, micropinocytotic vesicles and absence of a separate basal lamina. Following appropriate staining the empty regions in the investing ground substance contain elastin (compare with Figs. 19 and 20)

Fig. 22. Sector of multilayered peritubular cell cushion from border region between terminal segment and straight testicular tubule. Forssmann - OsO4, Epon-Araldite, uranyl acetate - lead citrate, x 32,000 Fig. 23a. Peritubular space (PS) lined by overlapping endothelium-like cells. L lymphocyte entering the peritubular space. Forssmann - OsO4, Araldite, uranyl acetate lead citrate, • 6,270. b Detail of Fig. 23 a, x 12,540. Note abundance of light vesicles (arrow) in the cytoplasm of endothelium-like cell and lack of a basal lamina subjacent to lining of peritubular space

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Discussion

The membrana propria or boundary tissue of the bovine seminiferous tubule shows some very specific features, although its overall organization follows the general pattern known from other ruminants, for example goat and sheep (Courot et al., 1970; Bustos-Obr6gon and Courot, 1974). Most remarkable in the bull are the structure and regional specialization of the tubular basal lamina. In the area of the seminiferous tubule proper the basal lamina is multilayered and exhibits knob-like projections into the bases of Sertoli cells and spermatogonia. A stratified organization of the tubular basal lamina is also reported for the ram, monkey and the human (Bustos-Obr6gon and Courot, 1974; Bustos-Obr6gon and Holstein, 1973; Seguchi and Hadziselimovic, 1974; Hermo et al., 1977; B6ck, 1978). While Seguchi and Hadziselimovic (1974), Hermo et al., (1977) and B6ck (1978) described the lamellar appearance for the seminiferous tubule under normal conditions, Bustos-Obr6gon and Holstein (1973) have seen this feature only in pathologically altered specimens. These discrepancies may be partly due to technical processing of the tissue. In our sections a stratification of the basal lamina was most clearly seen after fixation with the method of Forssmann et al., (1977), embedding in Araldite and staining with phosphotungstic acid-uranyl acetate; stratification was inconspicuous, however, after gtutaraldehyde fixation and embedding in ERL. In the transitional zone and middle portion of the terminal segment, the tubular basal lamina becomes thicker and the projections into the tubular cells become longer and more frequent. Furthermore, a small quantity of irregularly arranged collagen fibrils is incorporated within the basal lamina and its projections. At the level of the terminal plug the basal lamina is always unstratified, notwithstanding the techniques employed, and sends candelabrum-shaped processes with broad stalks and slender branches into the bases of the modified supporting cells. The tips of these branches are found at a depth of 3.5 Ixm and more. Stalks and branches may contain incorporated collagen fibrils. The invaginated basal surface of the supporting cells opposing the candelabrum-shaped processes of the basal lamina forms a large number of halfdesmosomes. As a consequence of this peculiar arrangement, a mechanically very tight connection between the extremely elongated modified supporting cells and the underlying basal lamina is established. Whether the increased surface area of the invading basal lamina also facilitates exchange of material between supporting cells and the extracellular compartment warrants further investigation. Since detailed studies on the composition of the tubular basal lamina in the area of the terminal segment are lacking for other mammalian species, it remains uncertain whether the situation seen in the bovine represents a common scheme. Deep protrusions of the basal lamina into the Sertoli cells of human seminiferous tubules of subjects suffering from tunica vaginal hydrocele (Chakraborty et al., 1976) differ from the bovine condition inasmuch as they are not reported for the terminal segment, do not contain collagen fibrils and are not covered by hemidesmosomes. In resorcin-fuchsin stained sections we observed positive material subjacent to the tubular basal lamina and between the layers of peritubular cells. At the level of the terminal segment the amount of resorcin-fuchsin positive structures is

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diminished compared with the seminiferous tubule proper. Although Bodley and Wood (1972) could prove by enzymatic digestion experiments that the elastin matrix of elastic fibers is indeed responsible for a positive RF-staining, Puchtler et al., (1976) and Meloan and Puchtler (1979) deny any specificity of RF for elastin. According to Puchtler et al. (1976), RF dissolved in absolute alcohol stains certain precursors of collagen and pseudoelastica (which corresponds to type III collagen) but has no affinity for elastin; RF dissolved in 70 ~o alcohol is thought to stain elastin, pseudoelastica and certain types of collagen. This critical viewpoint taken into account, the early reports on the occurrence of elastic fibers in the membrana propria of testicular tubules (Nelson and Heller, 1945; Sniffen, 1950; Mancini et al., 1952; de la Baize et al., 1954) became again questionable, especially since Leeson (1966), Ross and Long (1966), de Kretser (1968), B6ck et al., (1972), BustosObr6gon and Hostein (1973), and de Kretser et al., (1975) were unable to prove the existence of elastic fibers in the tubular boundary tissue at the ultrastructural level. One possibility for these negative results is the fact that elastic fibers in the tubular membrana propria are not easily revealed by conventional electron microscopic techniques. Recently, both ultrastructural components of the elastic system, i.e., (i) fibrotubules approximately 10-15 nm in diameter (oxytalan fibers) staining with cationic stains, and (ii) amorphous material (elastin) staining with anionic stains, have been identified with appropriate techniques in the tubular membrana propria of mouse, monkey and man (Brissie et al., 1975; Hermo et al., 1977; Menezes, 1977; Wiedmer-Bridel et al., 1977). In electron micrographs of bovine seminiferous tubules the amorphous component (elastin) of the elastic system clearly dominates, while fibrotubules or microfibrils (oxytalan fibers) are rarely seen. Accumulations of elastin with irregular outlines are observed mainly in the space between tubular basal lamina and peritubular cells, especially after embedding in Araldite and staining with phosphotungstic acid-uranyl acetate. In the intercellular spaces between peritubular cell layers elastin is less well developed. In the region of the terminal segment the amount of elastin is lower than in the boundary tissue of the seminiferous tubule proper, but again is situated subjacent to the thick tubular basal lamina and in close proximity to the partly concentrated, partly scattered peritubular cells, indicating that these cells are responsible for elastin formation. The sites of elastin accumulations following Araldite embedding and phosphotungstic acid-uranyl acetate staining are represented in conventionally treated sections by empty spaces and correspond to spots with a medium strong affinity to orcein. The visual demarcation ofelastin complexes in orcein-stained sections from collagen fibrils and tubular basal lamina, however, is not so feasible as in Aralditeembedded and phosphotungstic acid-treated sections. When the ultrastructural localization of elastin is compared with the result of the RF-100~-staining, generally a rather good topographic conformity is observed. The row of elastin accumulations subjacent to the tubular basal lamina corresponds to cross and oblique sections of the parallel-arranged, fine RF-100 ~-positive fibrils. The outer loose network of coarser RF-100~-positive fibrils is represented at the ultrastructural level by elastin accumulations between the peritubular cell layers. Since also single collagen fibrils are found in the neighborhood of elastin, a synchronous reaction of these fibrils with RF-100 ~o cannot be excluded with

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certainty. After treatment with RF-70 ~o a simultaneous staining of elastic and specific collagen fibers representing pseudoelastica in the sense of Puchtler et al., (1976) is still more likely; in the region of the terminal segment, subjacent to the negative basal lamina, a continuous RF-70 ~-positive layer with tiny tooth-like processes extending inward toward the epithelium is observed. For this characteristically formed, relatively thick positive structure no equivalent amount of elastin is present in electron micrographs. The tubular basal lamina of the terminal segment and its candelabrum-shaped processes, however, contain a considerable quantity of incorporated collagen fibrils. Sublaminal elastin plus these incorporated collagen fibrils could well be responsible for the appearance seen with the light microscope following RF-70 ~o-treatment. A simultaneous reaction of elastic and collagen fibers after staining with RF-70 ~o is also quite evident in the adventitia of arteries within the mediastinum testis. The postnatal differentiation of vertebrate testicular peritubular cells from fibroblast-like elements to contractile cells, morphologically closely resembling smooth muscle cells, apparently may cease at different levels in different species. According to ultrastructural features as well as to alkaline phosphatase histochemistry, B6ck et al. (1972) and Breitenecker et a1.(1974) distinguished between two types of peritubular cells, i.e., (i) myoid cells (prototype: man) and (ii) myofibroblasts (prototype: rat). The further differentiated myoid cells possess a continuous basal lamina and tightly packed, parallel-orientated cytoplasmic filaments. They exhibit a positive reaction for alkaline phosphatase. Myofibroblasts are surrounded by a discontinuous basal lamina, possess bundles of crossing cytoplasmic filaments and lack alkaline phosphatase. Bovine peritubular cells do not represent myoid cells according to the definition ofB6ck et at. (1972). Instead, they resemble more closely myofibroblasts but exhibit also some particular features. Bovine peritubular cells contain single bundles of contractile filaments embedded in a quantitatively dominating dark cytoplasmic matrix. Consequently, dense subplasmalemmal attachment sites and localized densities in the course of the fiber bundles are less frequent as, for instance, in the typical myoid peritubular cells of the porcine testis (Dierichs and Wrobel, 1973). Bovine peritubular cells are not covered by a distinct basal lamina of their own. However, abundant cell coat-like material reacting positively with alcian blue fills the interstices between peritubular cells and the alcian blue-negative tubular basal lamina. Embedded in this cell coat-like material are collagen fibrils and elastic elements. The peritubular cells of the terminal segment, which in general do not form distinct layers but are found loosely interspersed between connective tissue elements and components of the peritubular vascular plexus, may exhibit at certain sites a true basal lamina. All bovine peritubular cells give a positive reaction for alkaline phosphatase, but the intensity is not uniform (Wrobel et al., 1978). According to their ultrastructure and histochemical behavior, bovine peritubular cells can be considered as myofibroblasts with a somewhat higher degree of differentiation than postpuberal human peritubular cells. For the hamster, Cavicchia and Burgos (1977) described a ring of contractile peritubular cells surrounding the border between the terminal segment of the seminiferous tubule and testicular straight tubule. In the bull we observed in this

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location relatively frequently multilayered cushions of densely packed peritubular cells in longitudinal as well as in cross sections. In fortuitous oblique sections these cell arrangements may surround nearly the entire tubular circumference, thus indicating that the cushions seen in longitudinal and cross sections are very likely part of a spiral strand of contractile cells. Such a contractile spiral together with the vascular plexus investing the terminal segment in a sleeve-like fashion (Wrobel et al., 1978) may well act as a regulatory device at this position of the testicular excurrent duct system. Considerable portions of the tubular circumference in the bovine testis are surrounded by a peritubular fluid space which is lined on both sides by a thin layer of flat endothelium-like cells. The cytoplasm of these cells contains many light vesicles 70-80 nm in diameter. The terminal cell margins of adjoining endotheliumlike cells regularly overlap for a certain distance; special attachment sites were not observed. Similar peritubular spaces with a corresponding lining, but with occasional junctional complexes at the level of the overlapping area, were observed in rodent testicles (Fawcett et al., 1969; Dym and Fawcett, 1970; Fawcett et al., 1973; Clark, 1976; Hermo and Clermont, 1976), where, in contrast to the bovine, the spaces surround the complete tubular circumference. Fawcett et al. (1969), Fawcett et al. (1970), Dym and Fawcett (1970), Fawcett et al. (1973) and Clark (1976) consider these spaces as lymphatic channels; Hermo and Clermont (1976) described them as interstitial spaces lined by adventitial cells. Hundeiker (1969) demonstrated the bovine intratesticular lymph system by injection of colored gelatin and was able to show the existence of a delicate intertubular lymphatic network; however, the gelatin did not penetrate from the small lymphatics into tissue spaces. Thus, the topographic relationship between the true lymphatic vessels and the peritubular spaces observed by us in the bovine testicle requires further close examination. Regarding the functional significance, a dual role may be ascribed to the peritubular spaces: (i) they facilitate individual tubular movements by the contractile peritubular cells, and (ii) they favor an even distribution of substances gaining access from the intertubular tissue to the tubular wall. Occasionally, migrating cells with morphological features of lymphocytes were observed during their passage between membrana propria and peritubular space. In the rat, migrating cells have been found to be a regular component of the membrana propria of testicular tubules. Hoffer et al. (1973) identified them as agranular leucocytes; Hermo and Clermont (1976) observed occasional mitoses of these cells and therefore gave them the indifferent designation of "light cells". The migrating cells of human testicular tubular boundary tissue were recognized as mast cells and as monocytes (Hermo and Lalli, 1978). The view is presented that the migrating cells within the testicular tubular wall might react to antigens emanating from the seminiferous epithelium (Hoffer et al., 1973; Dym and Romrell, 1975; Hermo and Clermont, 1976). This opinion, however, warrants further investigation using comparative morphological and experimental techniques. References

Btck, P.: Histochemicaldemonstrationof disulfide-groupsin the lamina propria of human seminiferous tubules. Anat. Embryol. (Berl.) 153, 157-166 (1978) Btck, P., Breitenecker,G., Lunglmayr,G.: Kontraktile Fibroblasten(Myofibroblasteu)in der Lamina propria der Hodenkan~ilehenvom Menschen. Z. Zellforsch. 133, 519-527 (1972)

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Bodley, D.H., Wood, R.L.: Ultrastructural studies on elastic fibers using enzymatic digestion of thin sections. Anat. Rec. 172, 71-88 (1972) Breitenecker, G., B6ck, P., Lunglmayr, G.: Histochemical localization of phosphohydrolases in testes of man and rat. Z. Anat. Entwickl.-Gesch. 143, 301-313 (1974) Bressler, R.G., Ross, M.H.: Pituitary involvement in testicular peritubular cell maturation. Anat. Rec. 163, 158 159 (1969) Bressler, R.G., Ross, M.H.: Differentiation of peritubular myoid cells of the testis: effects of intratesticular implantation of newborn mouse testis into normal and hypophysectomized adults. Biol. Reprod. 6, 148-159 (1972) Brissie, R.M., Spicer, S.S., Thompson, N.T.: The variable fine structure of elastin visualized with Verhoeffs iron hematoxylin. Anat. Rec. 181, 83-94 (1975) Burgos, M.H., Vitale-Calpe, R., Aoki, A.: Fine structure of the testis and its functional significance. In: The testis, pp. 451 to 649 (A.D. Johnson, W.R. Gomes, N.L. Vandemark, eds.). New York and London: Academic Press 1970 Bustos-Obreg6n, E., Courot, M.: Ultrastructure of the lamina propria in the ovine seminiferous tubule. Cell Tissue Res. 150, 481-492 (1974) Bustos-Obreg6n, E., Holstein, A.F.: On structural patterns of the lamina propria of human seminiferous tubules. Z. Zellforsch. 141, 413-425 (1973) Cavicchia, J.C., Burgos, M.H.: Tridimensional reconstruction and histology of the intratesticular seminal pathway in the hamster. Anat. Rec. 187, 1-10 (1977) Chakraborty, J., Nelson, L., Ihunjhunwala, J., Young, M., Kropp, K.: Basal lamina of human seminiferous tubule - its role in material transport. I. In presence of tunica vaginal hydrocele. Cell Tissue Res. 174, 261-271 (1976) Clark, R.V.: Three-dimensional organization of testicular interstitial tissue and lymphatic space in the rat. Anat. Rec. 184, 203-226 (1976) Courot, M., Hochereau-de Reviers, M.T., Ortavant, R.: Spermatogenesis. In: The testis, pp. 339-432 (A.D. Johnson, W.R. Gomes, N.L. Vandemark, eds.). New York and London: Academic Press 1970 De la Baize, F.A., Bur, G.E., Scarpa-Smith, F., Irazu, J.: Elastic fibers in the tunica propria of normal and pathologic human testis. J. Clin. Endocrinol. Metab. 14, 626-639 (1954) De Kretser, D.M.: The fine structure of the immature human testis in hypogonadotrophic hypogonadism. Virchows Arch. [Cell Pathol.] 1, 283-296 (1968) De Kretser, D.M., Kerr, J.B., Paulsen, C.A.: The peritubular tissue in the normal and pathological human testis. An ultrastructural study. Biol. Reprod. 12, 317-324 (1975) Dierichs, R., Wrobel, K.-H.: Licht- und elektronenmikroskopische Untersuchungen an den peritubul~iren Zellen des Schweinehodens w/ihrend der postnatalen Entwicklung. Z. Anat. Entwickl.-Gesch. 143, 49-64 (1973) Dym, M., Fawcett, D.W.: The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod. 3, 308-326 (1970) Dym, M., Romrell, L.J.: Intraepithelial lymphocytes in the male reproductive tract of rats and rhesus monkeys. J. Reprod. Fertil. 42, 1-7 (1975) Esnault, C., Courot, M., Ortavant, R.: Transport et maturation des spermatozoides +pididymaires chez les animaux domestiques. En: Transport, survie et pouvoir f6condant des spermatozoides chez les vert+br+s, pp. 98-114 (E.S.E. Hafez, C.G. Thibault, Publ.). Paris: Editions INSERM 1974 Fawcett, D.W., Heidger, P.M., Leak, L.V.: Lymph vascular system of the interstitial tissue of the testis as revealed by electron microscopy. J. Reprod. Fertil 19, 109-119 (1969) Fawcett, D.W., Leak, L.V., Heidger, P.M.: Electron microscopic observation on the structural components of the blood-testis barrier. J. Reprod. Fertil 10, 105 122 (1970) Fawcett, D.W., Neaves, W.B., Flores, M.N. : Comparative observations on intertubular lymphatics and the organization of the interstitial tissue of the mammalian testis. Biol. Reprod. 9, 500-532 (1973) Forssmann, W.G., Ito, S., Weihe, E., Aoki, A., Dym, M., Fawcett, D.W.: An improved perfusion fixation method for the testis. Anat. Rec. 188, 307-314 (1977) Heidger, P.M.jr.: An electron microscopic study of a barrier to the entrance of horseradish peroxidase into the seminiferous tubule. (Abstract) Anat. Rec. 163, 198 (1969). Hermo, L., Clermont, Y.: Light cells within the limiting membrane of rat seminiferous tubules. Am. J. Anat. 145, 467-484 (1976) Hermo, L., Lalli, M.: Monocytes and mast cells in the limiting membrane of human seminiferous tubules. Biol. Reprod. 19, 92-100 (1978)

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Hermo, L., Lalli, M., Clermont, Y.: Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey. Am. J. Anat. 148, 433-446 (1977) Hoffer, A.P., Hamilton, D.W., Fawcett, D.W.: The ultrastructure of the principal cells and intraepithelial leucocytes in the initial segment of the rat epididymis. Anat. Rec. 175, 169-202 (1973) Hundeiker, M.: Untersuchungen zur Darstellung der Lymphgef'~i6e im Hodenparenchym beim Stier mit der Injectionsmethode. Andrologia 1, 113-117 (1969) Kormano, M., Hovatta, O.: Contractility and histochemistry of the myoid cell layer of the rat seminiferous tubules during postnatal development. Z. Anat. Entwickl.-Gesch. 137, 239-248 (1972) Leeson, C.R.: An electron microscopic study of cryptorchid and scrotal human testis, with special reference to pubertal maturation. Invest. Urol. 3, 498-511 (1966) Mancini, R.E., Nolazco, J., de la Baize, F.A.: Histocbemical study of normal adult human testis. Anat. Rec. 114, 127-147 (1952) Meloan, S.N., Puchtler, H.: A re-investigation of early elastica stains. Anat. Rec. 193, 170-171 (1979) Menezes, A.P.de: Elastic tissue in the limiting membrane of the human seminiferous tubule. Am. J. Anat. 150, 349-374 (1977) Nakamura, H., Kanai, C., Mizuhira, V. : An electron stain for elastic fibers using orcein. J. Histochem. Cytochem. 25, 306-308 (1977) Nelson, W.O., Heller, C.G.: Hyalinization of the seminiferous tubules associated with normal or failing Leydig cell function. Microscopic picture in the testis and associated changes in the breast. J. Clin. Endocrinol. Metab. 5, 13-26 (1945) Puchtler, H., Waldrop, F.S., Meloan, S.N., Branch, W.B.: Myoid fibrils in epithelial cells: studies of intestine, biliary and pancreatic pathways, trachea, bronchi, and testis. Histochemistry 44, 105-118 (1975) Puchtler, H., Meloan, S.N., Pollard, G.R.: Light microscopic distinction between elastin, pseudoelastica (type III-collagen?) and interstitial collagen. Histochemistry 49, 1-14 (1976) Reynolds, E.G.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Roosen-Runge, E.C. : Motions of the seminiferous tubules of rat and dog. (Abstract)Anat. Rec. 109,153 (1951) Ross, M.: The fine structure and development of the peritubular contractile cell component in the seminiferous tubules of the mouse. Am. J. Anat. 121, 523-558 (1967) Ross, M.H., Long, I.R. : Contractile cells in human seminiferous tubules. Science 153, 1271-1273 (1966) Seguchi, H., Hadziselimovic, F.: Morphologie der peritubul~iren Strukturen des Tubulus seminiferus bei Kindern. Eine elektronenmikroskopische Studie. Z. Mikrosk. Anat. Forsch. (Leipzig) 88, 1149-1160 (1974) Sniffen, R.C.: The testis. I. The normal testis. Arch. Pathol. Lab. Med. 50, 259-284 (1950) Spurr, A.R.: A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 3143 (1969) Unsicker, K., Burnstock, G.: Myoid cells in the peritubular tissue (lamina propria) of the reptilian testis. Cell Tissue Res. 163, 545-560 (1975) Wiedmer-Bridel, J.T., Vogel, A., Hedinger, C.: Das Verhalten elastischer Fasern in der Tunica propria der Hodenkan~ilchen. Licht- und elektroneumikroskopische Untersuchungen. Acta Anat. (Basel) 99, 346 (1977) abstr. Willson, J.T., Jones, N.A., Katsh. S., Smith, S.W.: Penetration of the testicular-tubular barrier by horeseradish peroxidase induced by adjuvant. Anat. Rec. 176, 85-100 (1973) Wrobel, K.-H., el Etreby, M.F.: Enzymhistotopochemie an der miinnlichen Keimdrtise des Rindes w~hrend ihrer fetalen und postnatalen Entwicklung. Histochemie 26, 160-179 (1971) Wrobel, K.-H., Sinowatz, F., Kugler, P.: Zur funktionellen Morphologie des Rete testis, der Tubuli recti und der Terminalsegmente der Tubuli seminiferi des geschlechtsreifen Rindes. Zentralbl. Veterinaermed. [C] Anat. Histol. Embryol. 7, 320-335 (1978)

Accepted September 2, 1979