J. Anat. (1978), 127, 3, pp. 603-613 603With 17 figuresPrinted in Great Britain

On the ultrastructure of modified Sertoli cells in the terminalsegment of seminiferous tubules in the boar

D. I. OSMAN*Department ofAnatomy and Histology, Faculty of Veterinary Medicine,

Swedish University of Agricultural Sciences, Uppsala, Sweden

(Accepted 18 January 1978)

INTRODUCTION

The terminal segment of a seminiferous tubule is that portion which connects it tothe tubulus rectus. Because of the difficulty of its identification it has received com-paratively little attention, and this has not been aided by the lack of consistency inthe terminology used for this part of the intratesticular tubular system. Thus, Martin-Padilla (1964) described it as a ruptured portion of the seminiferous tubule invagi-nated into a dilation of the tubulus rectus, while other authors have considered it asa part of the tubulus rectus (Roosen-Runge, 1961; Barack, 1968), an intermediateregion (Perey, Clermont & Leblond, 1961; Vitale-Calpe & Aoki, 1969), a tran-sitional zone (Dym, 1974) or (we believe correctly) a terminal segment of a semi-niferous tubule (Fawcett & Dym, 1974).

In the present study the term 'terminal segment' will be used. This region possessesfeatures which have suggested that it may act to prevent reflux of fluid from the retetestis into the seminiferous tubules (Perey et al. 1961; Roosen-Runge, 1961), althoughthis view has been challenged as a result of the physiological studies of Tuck et al.(1970) and Setchell & Waites (1971) which indicate that such reflux can occur.

MATERIALS AND METHODS

The testes of sexually mature boars used in this study were fixed by perfusionfor 30 minutes through the testicular artery with 3 % glutaraldehyde in 0-067 Mcacodylate buffer (pH 7-2) containing 2 % dextran. Usually fixation was precededby a brief rinse with Ringer's solution containing 2% dextran. Under the dissectingmicroscope small blocks of tissue were cut out which included the septulae testis andthe area at the junction of the mediastinum with the parenchyma of the testis. Thesesites were chosen after a study of paraffin sections stained with haematoxylin andeosin to locate the terminal segments. The small blocks were then treated with 2%osmium tetroxide in S-collidine for 3 hours, dehydrated in graded ethanols, clearedin propylene oxide and embedded in Epon.

Semithin sections were cut with glass knives on an LKB Ultratome and stainedwith buffered toluidine blue. It was found that the terminal segments could beidentified in unstained sections in the Ultratome by the reflexion of light on the edgeof the glass knife. This procedure made it easier to cut serial sections while searchingfor the desired segments in semithin sections. The terminal segments were thenmarked, the blocks trimmed, and thin sections with silver to gold interference colours

* Permanent address: Department of Anatomy and Histology, Faculty of Veterinary Sciences,University of Khartoum, Sudan.

were cut with glass or diamond knives. The thin sections were doubly stained withuranyl acetate and lead citrate and examined in a Philips EM 201 electronmicroscope.

RESULTS

The terminal segments were usually short, narrow tubules lined by a single layerof modified Sertoli cells. These tall columnar cells rested upon a basal lamina andhad long processes directed 'downstream' which projected into the lumen of thedilated proximal part of the tubulus rectus, the 'receptacle', for variable distances toform a plug-like structure (Fig. 1). The protruding portions of some cells were seento b- in contact with the surface of the lining epithelium of the 'receptacle'.Specialized cell contacts between the cellular elements of the 'receptacle' and the'plug' were not seen. On the basis of their electron density, the cells of the terminalsegments could be divided in two groups: pale cells, which constituted the majorcomponents of the region, and a few dark cells, mainly situated near the tip of the'plug'. There were no major morphological differences between these two types ofcell (Fig. 2).The cell nuclei in the terminal segment were sometimes situated near the basal

lamina but were mostly in the portions forming the 'plug'. When situated near thebasal lamina they were oriented with their long axis either perpendicular to it orinclined towards the 'receptacle'. They were highly irregular in shape, beinglobulated or indented (Fig. 4). The chromatin was evenly distributed as a rule andperipheral heterochromatin was seen only occasionally. Spherical structures oflower electron density and with cores of dark chromatin-like material were seen insome nuclei. A prominent nucleolus similar to that of more typical Sertoli cells wasusually observed.The cytoplasm showed an uneven distribution and a variable number of organelles

(Fig. 10). The mitochondria were moderate in number, small, slender and dark, andconcentrated in the vicinity of the nucleus where they were randomly scattered. Inthe cell processes they were few and oriented parallel to the long axes of theseprocesses. Some mitochondria showed tubular cristae and electron-dense matrixgranules.The Golgi apparatus was well developed. It consisted of many small stacks of

smooth cistemae situated at different levels in the cell (Fig. 3), i.e. in the vicinity ofthe nucleus, near the free border facing the epithelial surface of the 'receptacle', andadjacent to the large circumscribed spaces that bear microvilli. In some instances thecisternae at one face of the Golgi stack and/or the extremities of the cisternae of the

Fig. 1. A reconstructed low power micrograph from a serial thin section of a terminal segment.The terminal segment is short and narrow and the lining epithelium forms a plug-like structurein the dilated proximal part of the tubulus rectus. The cellular elements of the 'plug' have largespaces (s). The arrows show the beginning of the dilated proximal part of the tubulus rectus'receptacle'. TS, terminal segment; TR, tubulus rectus. x 400.Fig. 2. Adjacent cells of different electron densities. The dark cell shows three obliquely cutcrystalloids (C) and residual bodies (RB). x 4500.Fig. 3. Part of a cell near a dilated intercellular space (IS) showing a Golgi apparatus consistingof several stacks (arrows). An intracellular space (IC) contains flocculent material. x 15000.Fig. 4. The nuclei mostly have irregular outline and prominent nucleoli. The cells possess longcytoplasmic processes dominated by microtubules (MT), as seen at the lower part of the micro-graph. RB, residual bodies. x 2700.

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Modified Sertoli cells of the boarwhole stack appeared to be dilated (Fig. 5). Fuzzy-coated and bristle-coated vesicleswere usually encountered in the area occupied by the Golgi apparatus (Fig. 5).The rough endoplasmic reticulum was more profuse than in typical Sertoli cells.

It formed either long chains or concentric circles concentrated in one part of the cell(Fig. 6). The number of free ribosomes was variable: they were abundant in the cellbody and processes of some cells, but scanty in others (Fig. 10). Smooth endoplasmicreticulum was relatively rare, and when seen it formed short saccules and multiplelayers of concentric cisternae which often surrounded lipid droplets (Fig. 6). Largelipid droplets, characteristic of the more typical Sertoli cells in the boar were alsoseen in the cells of the terminal segment. Glycogen, mostly in the form of fl-granules,was distributed unevenly in the bodies and processes of the cells.A conspicuous feature of the cells in the terminal segment was the large number

and uneven distribution of microtubules and microfilaments. The microfilamentswere either concentrated in the cell body near the basal lamina (Figs. 7, 8), or aroundthe nucleus, or were packed to form crystalloids in different parts of the cell (Fig. 2).Microfilaments were also observed running individually between the microtubules.The microtubules were abundant in the cell processes (Figs. 4, 9), where they ranlongitudinally, but they were also found in the cell body (Fig. 7). In some sites themicrotubules and microfilaments were so abundant that other organelles were crowdedout (Fig. 7). Many crystalloids, resembling the Charcot-Bottcher crystalloids oftypical human Sertoli cells, were observed near the basal lamina and in the parts ofthe cells that formed the 'plug'. They were either surrounded by cisternae of roughendoplasmic reticulum or, when they were situated inside the nuclear indenta-tions, by nuclear membrane (Fig. 12). The crystalloids consisted of bundles of closelypacked microfilaments which ran longitudinally to form spindle-shaped structures.

Microvesicles and vacuoles were observed near the cell surfaces and towards thelarge spaces that showed microvilli. Centrioles (Fig. 13), multivesicular bodies, andstructures resembling lysosomes were seen. The microbodies in the cells of theterminal segment did not form basal aggregations at the sides of the nucleus as theydo in typical Sertoli cells. Instead, they were found away from the basal lamina,either aggregated between the concentric layers of the endoplasmic reticulum, orscattered at random, but still closely surrounded by cistemae of the endoplasmicreticulum (Fig. 16). In a few cases microbodies were observed in continuity with thesmooth part of the endoplasmic reticulum.

Fig. 5. A Golgi stack at a higher magnification to show dilated cisternae (DC), fuzzy-coated(FC) and bristle-coated (BC) vesicles. x 24000.Fig. 6. A lipid droplet (LD) surrounded by concentric layers of smooth endoplasmic reticulum(SER). Concentric layers of rough endoplasmic reticulum (RER) and a residual body (RB)are also shown. x 4000.Fig. 7. Basal parts of cells from a terminal segment. They have remarkably electron-dense basalplasma membranes (arrowheads) resting upon a tortuous basal lamina (BL). The cells containabundant microfilaments (MF) and microtubules (MT). The area inscribed in the lower part isshown at higher magnification in Fig. 8. x 5000.Fig. 8. A higher magnification from Fig. 7 to show that the basal parts of the cells are packedwith microfilaments running in different directions. x 23000.Fig. 9. Adjacent cytoplasmic processes showing that the microtubules are the main organellesand usually run parallel to the long axes of the cell processes. x 10000.

Fig. 10. The cells of the terminal segment show great variation in the type of organelles they con-tain, especially in the numbers of microfilaments (MF), microtubules (MT) and free ribosomes(FR). x 8000.

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Modified Sertoli cells of the boar 609One of the peculiar features of the cells in the terminal segment was their

possession of large circumscribed and membrane-bounded spaces which demon-strated well developed microvilli, and were mostly concentrated in the region of the'plug' (Fig. 1). In some cases these spaces appeared to be completely intracellular(Fig. 11), while in a few instances they were intercellular dilations. Both the intra-cellular and intercellular spaces were quite variable in size, but usually tended to becircular. Some of the spaces appeared to contain a lightly stained, flocculent material(Fig. 3).The borders of the cells were often difficult to follow for long distances, especially

in the 'plug'. This was largely because of the complicated arrangement of the cellborders. It was sometimes noticed that a narrow cytoplasmic process of one cell eithercompletely encircled another cell or protruded deeply into its cytoplasm. The freecell surfaces which were facing the epithelial lining of the 'receptacle' demonstrateda moderate number of short microvilli directed towards the rete channel. The lateralborders of the cell were attached to neighbouring cells by desmosome-like devices(Fig. 15) and tight junctions (Fig. 17) at different levels of the borders. The des-mosome-like devices resembled, in general, the type of junctions attaching neigh-bouring, typical Sertoli cells (Kaya & Harrison, 1976). Typical inter-Sertoli junctionsof the seminiferous epithelium were not observed. The basal plasma membrane wasremarkably electron-dense (Fig. 7) and closely followed the extraordinarily tortuousbasal lamina (Fig. 7).A patent central lumen was not clear, but long narrow clefts could be traced

between the cells for variable distances. These clefts also demonstrated welldeveloped microvilli and long cytoplasmic protrusions (Fig. 14).

Phagocytic activity of the cells of the terminal segment was suggested by thepresence intracellularly of sperm heads and segments of sperm tails at different stagesof degeneration (Fig. 14). No germ cells, other than a few spermatogonia anddegenerating spermatocytes, were found in the terminal segment. Dense bodies andresidual bodies (Figs. 6, 13, 16), similar to those observed in typical Sertoli cells, werealso seen in the cells of this region.

The boundary tissue. The cells of the terminal segment rested upon a typical basal

Fig. 11. Intracellular space demonstrating well developed microvilli. The intercellular dilations(IS) do not show microvilli. The arrowheads demarcate the cell boundary. x 1800.Fig. 12. An oblique section of a crystalloid (C) situated in a nuclear indentation. Lighter areasare seen between the bundles of microfilaments of the crystalloid. x 18000.Fig. 13. A centriole cut longitudinally and surrounded by microtubules. Dense bodies (DB)are also noticed. x 23000.Fig. 14. An apical part of a cell showing a phagocytized sperm head which seems to be normalapart from partial disappearance of the plasma membrane. The luminal border of the cell haslong cytoplasmic protrusions which come in contact with similar protrusions ofthe opposite side,separated only by a remnant of a central lumen. x 8000.Fig. 15. Two adjacent cells attached by desmosome-like devices. Note that the intercellular spaceis narrowed and contains electron-dense material. There are condensations of electron-densematerial and longitudinally oriented microfilaments on the cytoplasmic side of the plasmamembrane. x 55000.Fig. 16. A micrograph showing Golgi stacks (G) and microvesicles. In the Golgi region micro-bodies (arrowheads) and a residual body (RB) are found. x 10000.Fig. 17. Two adjacent cells joined together by a button-like tight junction (arrowhead). At thesite of the tight junction a condensation of material is seen. x 55000.

39-2

lamina that consisted of two zones differentiated according to their electron density:an inner lighter, and an outer darker zone. The basal lamina, which was extremelyinfolded (Fig. 7), was usually bounded by a layer of collagen fibres. The cells of theboundary tissue were arranged in more than one overlapping layer. Two types ofcell could be identified. The cells nearer to the basal lamina had a filamentouscytoplasm and numerous vesicular inpocketings of the plasma membrane, and theirnuclei were either elongated and smooth or deeply indented. They were thus similarto the contractile myoid cells around the seminiferous tubules. The outer cells hadfewer microfilaments, but abundant rough endoplasmic reticulum, thus resemblingfibroblasts.

DISCUSSION

The cells of the terminal segment are considered as modified Sertoli cells becauseof their general similarity to typical Sertoli cells of the seminiferous tubules. Thesesimilarities include their columnar shape with long cytoplasmic processes, and thecharacteristically indented nuclei with prominent nucleoli.The absence of germ cells in the terminal segment allows an opportunity of

visualizing the complicated arrangement, columnar shape and long cytoplasmicprocesses of these modified Sertoli cells. The two types of cells seen in this region,and distinguished according to their electron density, may represent the same cell intwo different metabolic states.The virtual absence of typical inter-Sertoli cell junctions in the modified Sertoli

cells perhaps explains the absence of post-mitotic germ cells in this region.The dominant feature of the modified Sertoli cells in the boar, as in the monkey

(Dym, 1974), is the enormous amount of microtubules and microfilaments. In factthese form the major constituents of some cell processes, other organelles being veryscanty. Microfilaments are found in various types of cell in the body includingsmooth muscle cells, typical Sertoli cells and myoid cells surrounding the semi-niferous tubules (cf. Ross, 1967; Bloom & Fawcett, 1975; Toyama, 1977). Toyama(1976, 1977) demonstrated actin-like filaments in the typical Sertoli cell junctionalspecializations, and in the myoid cells of the seminiferous tubules. These obser-vations may lead to the speculation that microfilaments in modified Sertoli cells havea contractile function in addition to acting as a cytoskeleton. It is suggested thatmicrotubules in typical Sertoli cells are involved in the internal movements of thecytoplasm, changing the cell shape in conformity with movements of the developinggerm cells (Fawcett, 1975). However, since germ cells are absent in the terminalsegment, movement of the modified Sertoli cells requires another explanation, e.g. thatthese cells play an active role in a valve-like device. But such a role was excluded byDym (1974) on the basis of the distribution and size of the microfilaments.

In all sections examined the cellular elements of the terminal segment were directedtowards the tubuli recti, and loosely telescoped into the 'receptacle'. The configurationof the epithelium of the terminal segment, and the direction of microvilli in theterminal segment, and in the 'receptacle' seem to indicate a unidirectional flow offluid from the seminiferous tubules to the tubuli recti. Nevertheless, the plug-likestructure of the terminal segment was seen before the formation of the lumen of theseminiferous tubules in juvenile testes, and hence before fluid secretion starts (Osman,unpublished observations). This observation excluded the possibility that the flowof fluid is the factor responsible for the configuration of the epithelium of theterminal segment. Thus the present observations neither support nor contradict the

610 D. I. OSMAN

Modified Sertoli cells of the boar 611physiological conclusions of Tuck et al. (1970) and Setchell & Waites (1971) thatreflux of fluid occurs into the seminiferous tubules.One observation which is also attributable to the absence of germ cells is the

presence of many crystalloids in the modified Sertoli cells. Such crystalloids arerarely seen in the typical Sertoli cells of normal boars (Toyama, 1975; Osman &Ploen, 1978 a). The best explanation of this phenomenon may be obtained from theresults of the study of typical Sertoli cells in intersex 'boars' and those with crypt-orchid testes (Toyama, 1975). The assumption that crystalloids in typical Sertoli cellsrepresent aggregations of randomly distributed microfilaments has already beenmade by Bawa (1963).

It could be suggested that modification of the seminiferous tubule fluid begins inthe terminal segment, on the ground of the differences between the modified andtypical Sertoli cells mentioned above.

Phagocytic ability of typical Sertoli cells has been reported for several species(Oettle & Harrison, 1952; Clegg & Macmillan, 1965; Niemi & Kormano, 1965;Nicander, 1967; Carr, Clegg & Meek, 1968; Dym, 1973). Similar activity has beenobserved in the modified Sertoli cells of the boar and monkey (Dym, 1974). Themodified Sertoli cells may act as a second 'line of filtration' for unwanted sperma-tozoa (after the typical Sertoli cells). Such a function would be facilitated by thenarrow lumen of this segment and the loose arrangement of the lining epithelium,which forces close contact between passing spermatozoa and modified Sertoli cells.A puzzling feature of the modified Sertoli cells is their possession of large circum-

scribed intracellular spaces, and extremely dilated intercellular spaces having welldeveloped microvilli. These intracellular spaces and intercellular dilations are moreconcentrated at the tip of 'plug', and only in a few thick sections were somespermatozoa seen in them, The vacuolated cytoplasm of the modified Sertoli cellswas observed in some mammals with the aid of the light microscope (Roosen-Runge,1961; Barack, 1968), when some modified Sertoli cells appeared very pale, as ifdehydrated or vacuolated; but with the electron microscope these clear areas wereseen to be occupied by microfilaments (Dym, 1974). This was not the case in thepresent material, as the modified Sertoli cells were vacuolated when examined bothby light and electron microscopy.The large intracellular spaces that bear microvilli might be interpreted as cross

sections of infoldings of the cell surfaces, infoldings which increase the surface areaof the cells available for fluid exchange. This suggestion is based on the fact that theterminal segment in the boar is short, whereas the terminal segments of the bull,ram, goat, rabbit and rat are relatively long, and the spaces and free surfaces havefew or no microvilli (Osman, unpublished observations). On the other hand, theconsiderably dilated intercellular spaces may be interpreted as sites where germ cellswould normally be found and become visible only when the germ cells are depletedin this segment. The experimental study of Gravis, Chen & Yates (1977) in epi-nephrine-induced testicular degeneration supports this assumption.

There has been much controversy over the question of a central lumen in theterminal segment. Furthermore, Martin-Padilla (1964) claimed that the connexionbetween the seminiferous tubules and the tubuli recti is not established until the timeof sexual maturity. This seems not to be true, at least not in the immature goat,because a connexion has been observed between the seminiferous tubules and thetubuli recti (Osman, unpublished observations). Although no conspicuous lumen wasseen even in serial sections of the 'plug' in the rat (Roosen-Runge, 1961), it was

reported that the apical borders of the cells in the terminal segment were never seenin direct contact in well oriented sections in the guinea-pig (Vitale-Calpe & Aoki,1969). In the present study only traces of a central lumen could be seen. It can beconcluded that the loosely arranged long cell processes in the 'plug' can easilycollapse, but, owing to the pressure flow of contained fluid, these processes may besqueezed against the epithelial surface of the 'receptacle', thus maintaining a per-manent communication between the seminiferous tubule and the tubulus rectus. Inthe rabbit and rat the lumen was clearly seen under conditions of increased intra-testicular pressure following ligation of the ductuli efferentes (Osman& Ploen 1978 b).Lymphocytes observed in the epithelial lining of the terminal segment of the

monkey (Dym, 1974) were taken as evidence of a leaky blood-testis barrier. In thepresent study lymphocytes were not seen in the terminal segment, which mayindicate a species variation. Using lanthanum as a tracer substance, researches onthe efficiency of the blood-testis barrier in the terminal segment of the rat are nowbeing made.

SUMMARYThe seminiferous tubules are linked to the tubuli recti by a short terminal segment

which is lined by a single layer of modified Sertoli cells whose long cytoplasmicprocesses occlude the lumen and form a plug-like structure in the 'receptacle'. Themain features of the modified Sertoli cells are: enormous numbers ofmicrotubules andmicrofilaments, many crystalloids, abundance of rough endoplasmic reticulum, andpaucity of smooth endoplasmic reticulum. The intercellular spaces are considerablydilated, and the cells possess intracellular spaces having well developed microvilli.Degenerated spermatozoa were seen engulfed by the modified Sertoli cells. Desmo-some-like devices and tight junctions were observed joining adjacent cells. Theconfiguration of the epithelium and the fine structure of the cells of the terminalsegment are discussed in relationship to their possible roles as modifiers of theseminiferous tubule fluid and as regulators of fluid reflux from the rete testis into theseminiferous tubules.

The author wishes to express his gratitude to Professor L. Nicander and Dr L.Ploen for many valuable suggestions, and for critical reading and improvements ofthe manuscript. This study was supported by a research grant from "Anslaget forframjande av ograduerade forskares vetenskapliga verksamhet vid Veterinar-hogskolan".

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