Zeitschrift fiir Zellforschung 78, 92--113 (1967)

THE POSTNATAL DEVELOPMENT OF THE SERTOLI CELLS OF THE MOUSE

CHARLESJ. ~LICKI:NGEI~* Department of Anatomy, Harvard Medical School, Boston, Massachusetts, U.S.A. Received October 27, 1966

Summary. The development of the Sertoli cells of the testis of the white mouse was studied with the electron microscope at weekly intervals from birth to sexual maturity, utilizing fixation in a glutaraldehyde-formaldehyde mixture followed by OsO4 . The Sertoli cells underwent ~ rapid structural differentiation between 1--2 and 4--5 weeks postnatally. Large increases were noted in cell size, in the extent of cell processes extending between germinal cells, and in the abundance of certain cytoplasmic organel]es, particularly the agranular endoplasmic reticulum and Golgi apparatus. The apparent sequences of development of the characteristic junctional complexes and tri-partite nucleoli of Scrtoli cells were observed. The development of structural specializations is discussed in terms of established and proposed functions of the Sertoli cells, and the demonstration of abundant agranular endoplasmic reticulum is considered in relation to the possibility of steroid hormone production by Sertoli cells. Introduction The seminfferous t u b u l e s of the m a m m a l i a n testis are composed of s p e r m a t o genic or g e r m i n a l cells a n d Sertoli cells. A l t h o u g h t h e t r a n s f o r m a t i o n s of t h e g e r m i n a l cells resulting in t h e p r o d u c t i o n of s p e r m h a v e become well known, tile role of the Sertoli or s u s t e n t a c u l a r cells in the n o r m a l functioning of t h e seminiferous t u b u l e s is n o t e n t i r e l y clear. On t h e basis of morphological studies, it is a p p a r e n t t h a t as a c o m p o n e n t of the seminiferous epithelium, the Sertoli cells p r o v i d e m e c h a n i c a l s u p p o r t for the s p e r m a t o g e n i c cells. The Sertoli cells also function in t h e p h a g o c y t o s i s of t h e residual bodies shed b y m a t u r i n g s p e r m a t i d s (SMITH a n d LACY, 1959; B~OKELMAN~, 1963; NICANDE~, 1963) a n d are capable of p h a g o c y t i z i n g i n j e c t e d p a r t i c u l a t e m a t t e r (CLEG~ a n d ~/[ACMILLA~,1965) a n d d e g e n e r a t i n g g e r m cells (LACY a n d LOFTS, 1965; VILA~, 1965; I-I~YGoN a n d Bo]cGE~S, 1966). B y virtue of their position, i n t e r p o s e d between capillaries a n d m o s t of t h e s p e r m a t o g e n i c cells, t h e y m a y p l a y a p a r t in t h e conveyance of n u t r i e n t s a n d m e t a b o l i t e s between the two (VILA~ et al., 1962). The e x a c t n a t u r e , however, of a n y such n u t r i t i v e or m e t a b o l i c c o n t r i b u t i o n is n o t known. I n addition, m a n y i n v e s t i g a t o r s h a v e suggested t h a t the Sertoli cells are the source of a non-androgenic t e s t i c u l a r h o r m o n e (see review b y ]~ISHO1% 1954; LACY, 1962). H o w e v e r , t h e r e is little a g r e e m e n t as to the n a t u r e of the h o r m o n e produced, or even if h o r m o n e p r o d u c t i o n is a n a c t i v i t y of Sertoli cells. I n v e s t i g a t o r s s t u d y i n g t h e p o s t n a t a l d e v e l o p m e n t of t h e testis b y light m i c r o s c o p y n o t e d t h a t m a t u r e Sertoli cells, as identified b y nuclear m o r p h o l o g y a n d cell shape, d i d n o t a p p e a r u n t i l p u b e r t y in t h e h u m a n (CHARNY et al., * He is indebted to Dr. I)ON W. FAWCETTand Dr. S~:suMv Iwo for their guidance during the course of the study and the preparation of the manuscript. - - This study was supported by training grant G-406 from the Institute of General Medical Sciences, National Institutes of Health.

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1952; S~IFF~N, 1952; MANCINI et al., 1960) o r u n t i l t h e t i m e of m a t u r a t i o n of t h e first crop of s p e r m a t i d s in r o d e n t s (HAI~GITT, 1926; BOOKHOUT, 1937; SAPSFORD, 1963; CLERMONT a n d P ~ l ~ r , 1957). D e s c r i p t i o n of earlier d e v e l o p m e n t a l changes in these cells was l i m i t e d b y t h e resolution of t h e light microscope. I n a d d i t i o n , m a n y a u t h o r s c o n c e n t r a t e d on t h e long s t a n d i n g c o n t r o v e r s y over t h e origin of t h e definitive g e r m cells. CL~I~ONT a n d PEREY (1957) h a v e r e v i e w e d t h e h i s t o r y of t h e d i s p u t e a n d p r e s e n t e d evidence for t h e now w i d e l y a c c e p t e d belief t h a t t h e r e are two i n d e p e n d e n t cell lines in m a m m a l i a n testis, t h e g e r m . cells a n d t h e s u s t e n t a c u l a r cells of Sertoli. The u l t r a s t r u c t u r e of t h e Serto]i cells of t h e a d u l t s of several m a m m a l i a n species has been d e s c r i b e d b y FAWCETT a n d Bu~Gos (1956), HO~STMANN (1961), VILA~ et al. (1962), B ~ O K ~ L M A ~ (1963), NICANDER (1963), BAWA (1963), GAl~DNER a n d HOLYOKE (1964), a n d NAGS.NO (1966); a n d NICANDER e t al. (1961) also s t u d i e d t h e Sertoli cells of calves. I n these studies t h e n o r m a l c y t o l o g y was described a n d t h e persisting c o n t r o v e r s y over w h e t h e r t h e Sertoli cells were s e p a r a t e units or a s y n c y t i u m was s e t t l e d in favor of t h e cellular units. The fine s t r u c t u r a l changes d u r i n g t h e d e v e l o p m e n t of t h e Sertoli cells h a v e n o t been r e p o r t e d . I t was h o p e d t h a t a s t u d y of t h e d e v e l o p m e n t of s t r u c t u r a l specializations of t h e Sertoli cells a n d t h e t i m e of their a p p e a r a n c e would help e l u c i d a t e t h e functional r e l a t i o n s h i p of Sertoli cells to g e r m i n a l cells. This e x p e c t a t i o n has n o t been fully realized, b u t t h e p r e s e n t s t u d y defines t h e fine s t r u c t u r e of t h e p r e s u m p t i v e Sertoli cells of t h e n e w b o r n mouse a n d traces t h e s t r u c t u r a l changes t h a t occur as t h e animals reach sexual m a t u r i t y .

Materials and Methods Male Webster strain Swiss mice, newborn, 7, 14, 21, 28, and 35 days old were killed by cervical dislocation. The whole testes were fixed by immersion at room temperature in modified Karnovsky's fixative (KAR~OVSKY, 1965) containing 2.5 % glutaraldehyde, 2 % formaldehyde and 0.05% calcium chloride buffered to pH 7.3 with 0.1 M cacodylatebuffer. After one hour of fixation, the testes were diced into 1 ram. cubes and allowed to remain in the fixative for a total of two hours. The tissue was rinsed for 10 to 30 minutes in 0.05 M cacodylate buffer, and following one hour in ice cold 1% OsOa in 0.1 M cacodylate buffer, was rapidly dehydrated in ethanol or methanol and embedded in Araldite. Sections showing silver to pale gold interference colors were cut with glass knives on a Porter-Blum MT-1 microtome/picked up on uncoated copper grids, and stained with lead citrate (VENABLE and CO(~G~S~L, 1965). Electron micrographs were obtained with an RCA EMU-3E or 3F electron microscope. Sections 0.5 to 1.0 micron in thickness were prepared from the same material and stained with toluidine blue in sodium borate for observation with the light microscope.

Observations Newborn. The seminiferous t u b u l e s of t h e n e w b o r n mouse testis are r e p r e s e n t e d b y solid cords 40 t o 50 m i c r a in d i a m e t e r , c o m p o s e d of t w o m o r p h o l o g i c a l l y dist i n c t t y p e s of cells (Fig. 1). B y far t h e m o s t n u m e r o u s are p r e s u m p t i v e Sertoli cells (the " i n d i f f e r e n t cells" of some of t h e earlier authors). These cells (Fig. 4), resting on t h e b a s a l l a m i n a of t h e seminiferous tubule, are cuboidal or columnar, 5 to 8 b y 5 to 25 micra, a n d h a v e s m o o t h outlines, lacking i n t e r d i g i t a t i o n s w i t h a d j a c e n t cells. The nucleus, which is l o c a t e d in t h e center of t h e c u b o i d a l cells or in t h e b a s a l p o r t i o n of t h e c o l u m n a r cells, has a t y p i c a l n u c l e a r envelope conr a i n i n g pores. T h e r o u n d or oval n u c l e a r profile is o n l y slightly i r r e g u l a r in t h e m a j o r i t y of cells, b u t 10 to 20% m a y h a v e one or t w o deep i n d e n t a t i o n s or folds

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Fig. 1

Fig, 9_

Fig, 1. L i g h t micrograph of seminiferous cord from a newborn mouse. The large pale staining germinal cells (go) are easily distinguished from the more numerous smaller presumptive Sertoli cells (pS). Toluidine blue stain, x 810 Fig. 2. L~gh$ micrograph from a 1_week old animal. The nuclei of the prestlmlative Sertoli celts (pSI are located in the ba~al portions of the cells while their apical parts fill the center of the cot& ~c, germinal celt, Toluidine blue stain, 7, 810

of the nuclear envelope. The nucleus contains clumps of granular electron dense chromatin material and from one to four roughly circular areas of greater electron density composed of fine fibrillar material and ~ 150 A dense granules, identified as nucleotar material (Figs. 4, 17, 18). In some of these bodies the nucleolar material is organized into poorly defined anastomosing cords, suggestive of a nucleolonema (Fig. 18), whereas others appear to lack this reticular organization (Fig. 17). The majority of the nucleolar bodies are located at the periphery of the nucleus, adjacent to the nuclear envelope. The cytoplasm of the presumptive Sertoli ce~s (Figs. 4, 5) contains mitechondria which are spherical, 0.3 to 0.5 ~ in diameter, or have the shape of short rods, about 0.6 ~ long. Most of the mitochondrial cristae are oriented transversely and are embedded in a matrix of greater density than the surrounding cytoplasm. Intramitochondrial dense granules are not abundant. Tubular and cisternal profiles of granular endoplasmic reticulum are found in moderate abundance and scattered tubular or vesicular elements of agranular reticulum are present. The Golgi apparatus (Fig. 5), which occupies a supranuclear position, is composed of anastomosing tubules, small vesicles, and 4 - - 6 centrally flattened cisternae. The cytoplasm between the membranous organelles contains a large number of free ribosomes often arranged in clusters, and scattered ~ 200 A microtubules embedded in a finely granular cytoplasmic matrix (Figs. 4, 5, 6).

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Fig. 8. L i g h t micrograph of testis of a 14-day old animal, iUustrating the variable d e v e l o p m e n t of the lumens of the seminiferous tubules a t this age. A t the arrow, the center of a cord is still filled with the apical processes of Sertoli cells. A comparable area is illustrated in an electron micrograph in Fig. 11. x 280

Most of the cells contain a few small membrane-bounded homogeneous or granular electron dense bodies resembling lysosomes (Fig. 6). These are predominately in the basal region of the cells. Occasionally, a larger irregular homogeneous dense lipid droplet is seen (Fig. 4). The less frequent cells of the second type found in the newborn are identified as germinal cells. They are larger than the presumptive Sertoli cells, 20 to 25 micra in diameter, and are nearly round or regularly polygonal (Fig. 1; Fig. 8, 7 day old animal). They contain a round nucleus with evenly distributed fine chromatin and a prominent nucleolus organized in the form of a nncleolonema. Their cytoplasm contains spherical mitochondria, one to several Golgi bodies, free ribosomes, microtubules, vesicles and scattered elements of granular and agranular endoplasmic retienlum. In 1 micron sections stained with toluidine blue and observed with the light microscope (Fig. 1) these cells are easily distinguished from the young Sertoli cells b y their large size, round shape, large circular nucleus, distinct cell outline, and relatively pale-staining cytoplasm. They evidently correspond to the "gonocytes" of the earlier authors. The intercellular space between adjacent Sertoli cells is occasionally observed to narrow from the usual 200/~ to 70--90 A for distances of from 200 to 1000/~, and there is an associated accumulation of electron dense material in the adjacent cytoplasm (Fig. 4). Several "coated" vesicles, 800 to 900 A in diameter are often seen at the periphery of both the Sertoli and germinal cells (Fig. 6). These are sometimes open to the intercellular space, forming a small pit.

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Fig. 4. S u r v e y electron m i c r o g r a p h of a p r e s u m p t i v e Sertoli cell f r o m a newborn mouse. The area within the rectangle is shown a t higher magnification in the inset. Note the short areas of narrowing of the intercellular space a n d s u b p l a s m a l e m m a l dense material. N , nucleus; ch c h r o m a t i n ; n u nueleolar material; n e nuclear envelope; 1 lipid droplet; db dense body; m mitochondrion; m t microtubule; b basal lamina. • 17,000. I n s e t • 26,500

Development o[ ~ertoh Cells

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9"/

Fig, 6

Fig. 5. A poxtion el t h e supra~melear c y t o p l a s m oi a p r e s u m p t i v e Sertoli cell of a newborn animal, illustrating some of the cytoplasmic organelles, m mitochondrion; ~er granular endoplasmic retieulum; q Golgi. • ZS,000 Fig. 6. Basal cytoplasm of 7-day old !oresumptive Sertoli cell, Note m e m b r a n e bounded mierotubules (mr), a n d " c o a t e d " vesicles (co), • 28,000

dense bodies (db),

One Week Old. I n testes of 1 week old mice the Sertoli cells have increased in size, m a n y having become high columnar in shape, filling the center of the seminiferous cord with their apical portions, and sending short lateral processes between neighboring cells (Figs. 2, 8). The apical parts of the Sertoli ceils contain an increased amount of tubular and vesicular agranular endoplasmic reticnlum, as well as the organel[es already described for the newborn specimens (Fig. 9). The number el areas of cl(~se m e m b r a n e apposition and suhplasmalemma[ dense material are increased, and occasionally, a few short tubules, vesicles or small cisternae of the endoplasmic reticulum are present in the cytoplasm immediately subjacent to the dense material (Fig. 10). The mitochondria m a y display an elliptical dilatation of their intracrista] spaces which have a relatively electron lucent interior (Fig. 6). I n sections of favorabl~ orientation, the eisternae of the Golgi apparatus display m a n y circular fenestrations 250--300 A in diameter (Fig. 9, GI). Some nuclei of the developing Sertoli cells are observed to be elongated in the direction perpendicular to the basal lamina of the cord, especially where the Sertoli cell is closely applied to a germinal cell (Fig. 8). The presence of a nueleolonema in ~ome of the nucleo]ar bodies i~ more evidex~t than in the newborn (Fig. 8, arrow). The second type of nucleoiar body continues to lack a detectable nucleolonema, b u t some now display a light amorphous center (Fig. 8, double

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r i g . 7. Portions of several Sertoli ceils from a 14-day old mouse. The bracket encloses a n area of contact specialization of two Sertoli cells with narrowing of the intercellular space aaad, in both cells, the aecumuIation of dense material between the plasma m e m b r a n e a n d a i)arMlel cisterns of the endoplasmic reticulum. Ribosomes are present on the cytoplasmic side of the cisternae, a e r agranular endopIasmie retieulum. × 31,000

shafted arrow). I n a few instances nueleolar bodies of both types are seen in apposition (Fig. 8, arrow). Two Weeks Old. By fourteen days, most of the cords have acquired a small lumen, but the extent of lumen development varies from one animal to the next (Fig. 3). The Sertoli ceils have increased greatly in extent, sending lateral cytoplasmic processes between neighboring spermatogenic cells and interdigitating with adjacent Sertoli ceils (Fig. 7). In the central areas of the cords which have not yet acquired a lumen (e.g. Fig. 3, arrow) there is extensive interdigitation of the apical parts of Sertoli cells (Fig. 11). Apical interdigitation to this degree is not present in those areas where a lumen has formed. There is a striking increase in the amount of agranular endoplasmie reticulum, especially in the apical portions of the Sertoli cells (Fig. If), and the number of Golgi bodies is greater than that seen one week earlier. At this age, more mitoehondria have dilated cristae that form elliptical or circular electron lucent areas (Figs. 7, ll). These are similar to the areas described by ANDI~£ (1962) in rat testis and termed the "pseudomatrix". Free ribosomes remain abundant, but the granular endoplasmic reticulum is seen less frequently. More of the nneleolar bodies t h a t lack a typical nueleolonema have b y this time developed light cores, and there is more frequent association of these nncleolar bodies with those exhibiting a nneleolonema (Fig. 19). The unusual contact specializations found between adult Sertoli cells (FLIC~INGE~ and FAWCE~T, 1967) are present by two weeks of age (Fig. 7, bracket). These structures display 1) narrowing of the intercellular space to about 70 to

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Fig. 8. Seven day old animal. A portion of the seminiferous cord adjacent to the basal lamina. Several presumptive Sertoli cells (S) are closely applied to a large germinal cell (gc). x 8100 9 0 / ~ f o r e x t e n d e d a r e a s u p t o 15 o r 2 0 m i c r a , 2) a f l a t c i s t e r n a o f e n d o p l a s m i e r e t i c u l u m p a r a l l e l t o b o t h S e r t o l i cell p l a s m a m e m b r a n e s at a distance of 400 to 7*

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Fig. 9

(legends see p. 101)

Fig. l0

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Fig, 11. F o u r t e e n d a y old mouse. T h e c e n t e r of a cord t h a t h a s n o t yet acquired a l u m e n is composed of the extensively intcrdigitated apical processes of Sertoli cells~ containing m u c h a g r a n u l a r endoplasmic r e t i c u l u m (aer). 15,500 •

Fig. 9. The supranuclear cytoplasm of 7-day old p r e s m n p t i v e Sertoli cells. The extensive Golgi a p p a r a t u s is sectioned parallel to a fenestrated cistcrna a t G~. The Golgi a p p a r a t u s of a n a d j a c e n t cell (G2) is sectioned perpendicular to the cistcrnae. Granular a n d a g r a n u l a r endoplasmic reticulum occupy m u c h of the cell, • 24,000 Fig. 10. Same specimen as Fig. 9. P a r t s of two p r e s u m p t i v e Sertoli cells are shown. There are several short areas of n a r r o w i n g of the intercellular space a n d associated s u b p l a s m a l e m m a l dense material. Some elements of the endoplasmic reticultun arc a r r a n g e d parallel to the p l a s m a m e m b r a n e s . • 34,000

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t~ig, 12. T w e n t y - o n e d a y old animal. Low power electron micrograph. Two germinal cells in mitosis (gcm) are shown. The processes of the Scrtoli cells (S) arc v e r y prominent, extending between the germinal cells for long distances, sc spermatoeyte. • 4300

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Fig. 13. Twenty-one day old mouse. The basal part of a Sertoli cell. h r nucleus; aer agranular endoplasmic reticulum; get granular endoplasmic reticuhlm; m mitochondrion. • 18,000

Fig. 14. Same specimen as Fig. 13. Part of a Sertoli cell process between germinal cells (gc) containing abundant agranular retieulum (aer) and several Golgi bodies (q). m mitochondrion. • 24,000

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Fig. 15. The portion of a m a t u r e Sertoli cell containing the nucleus (N) rests on the basal lamina of the seminiferous tubule. Several spermatids (sp) are surrounded by Sertoli cell processes, 35-day old animal, x 8200

600 A, bearing ribosomes on the c y t o p l a s m i c side, a n d 3) the a c c u m u l a t i o n of a continuous or discontinuous l a y e r of electron dense filamentous m a t e r i a l between

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Fig. 16. The apical part of a Sertoli cell surrounding a late spermatid (sp). The contact specialization (bracket) of the Sertoli cell consists of narrowing of the intercellular space, and accumulation of dense material between the cell membrane and a cisterna of the endoplasmic retieulum. Tubular agranular endoplasmie reticulum (aer) and microtubules (mr) arc prominent in the Sertoli cell cytoplasm. • 19,000

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Figs. 17--21. The proposed sequence of development of the nucleolus of the Sertoli cell

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the plasma membrane and the associated cisterns of the endoplasmic reticulum. Occasionally, a series of short tubules or vesicles replaces the usual cisterna of this junctional complex. About 20 to 30% of the area of contact between Sertoli cells in their basal portions is specialized at this age. Only rarely are such modifications observed in the apical areas of contact, and they have not been observed adjacent to the lumen. By fourteen days, the germinal cells are represented by spermatogonia and m a n y primary spermatocytes in various stages of the first meiotic division. Multiplication of the spermatogonia b y mitosis (N~B~L et al., 1961) has led to an increase in the number of germinal cells so that, in contrast to newborn and one week old animals, the spermatogenic cells now equal or outnumber the Sertoli cells. Three Weeks Old. The seminiferous tubules have all acquired a lumen b y twenty-one days, and the Sertoli cells display increased development of m a n y of the features noted in the description of the two week old animals. Lateral processes have become very extensive (Fig. 12), surrounding the spermatogenic cells and making contact with other Sertoli cells. The granular endoplasmic reticulum is not prominent, while the agranular reticulum is highly developed into a system of tubules and vesicles in all parts of the cell (Figs. 13, 14). I t is particularly abundant in the processes which extend between spermatogenic cells and toward the lumen. The number of Golgi bodies is increased in number such t h a t four to eight are commonly located in the same plane of section of a portion of a single Sertoli cell process (Fig. 14). Therefore, hundreds or even thousands of Golgi bodies must be present in a single Sertoli cell. The perinuclear region, to a depth of several hundred A, contains randomly arranged fine filaments which appear to contact the outer membrane of the nuclear envelope (Figs. 13, 19). The cytoplasmic organelles appear to be absent from this region. The junctional complexes have increased in extent and now cover about 50% of the area of contact between Sertoli cells, predominately in their basal portions. Many primary spermatocytes are present and early (Golgi phase) spermatids as well. Late spermatids have not been observed at this age. I t is estimated t h a t 80 to 90% of the seminiferous epithelium is occupied b y germinal cells and the remainder b y Sertoli cells (Fig. 12). Four to S i x Weeks Old. By twenty-eight to thirty-five days the Sertoli cells appear to complete their morphologic differentiation and attain their adult structure. A "perikaryon" containing the nucleus rests on the basal lamina of the seminiferous tubule (Fig. 15) wt-dle numerous processes extend between the deveFig. 17. A nucleolar body lacking in nucleolonema is in contact with the inner mmlear membrane. :Newborn mouse. • 26,000 :Fig. 18. A nucleolar body with areas of increased density suggesting a developing nucleolonema. Newborn. • 26,000 :Fig. 19. A nucleolar body composed of nucleolonema is associated with a nucleolar body lacking nucieolonema, A second body of the latter type lies a short distance away. • 24,000 :Fig. 20. A n electron-lucent core is present in a nucleolax body w i t h o u t nucleolonema. A second body of the same type has a smaller central area of decreased electron density. The first is associated with a nucleolar body composed of nucleolonema. • 18.500 Fig. 21. The nucleolus of a m a t u r e Sertoli cell has a central portion composed of nucleolonema a n d two peripheral spheres with less dense centers. Areas of fine fibrillar material and a b o u t 150 A dense granules are present i n b o t h components. • 15,000

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loping spermatogenic cells and to the lumen. Contact specializations cover as much as 70 to 80% of the area of apposition of Sertoli cells. The cytoplasmic organelles (Figs. 15, 16) present in the "perikaryon" and in the processes, consist of abundant tubular and vesicular agranular endoplasmic reticulum, sparse granular reticulum, many Golgi bodies, round or rod shaped mitochondria with dilated cristae, microtubules, free ribosomes, and membrane-bounded dense bodies interpreted as lysosomes. Lipid droplets are seen more frequently in Sertoli cells of animals more than 4 weeks old. The nuclei are irregularly shaped, frequently having several deep folds of the nuclear envelope (Fig. 15). The two types of nucleolar bodies are now fre quently in close association, and in sections favorably oriented, the characteristic tripartite organization (FAWCETT, 1966) of the adult Sertoli cell nucleolus may be seen (Fig. 21). There is a central round area of nucleolonema, flanked by two smaller round bodies with less dense cores. Fine fibrillar material and dense granules ~ 150 A may be found in both central and lateral components while the cores of the lateral bodies are composed of amorphous material of low electron density. Cap phase spermatids have appeared by twenty-eight days, and acrosome and maturation phase spermatids by thirty-five days. Surface specializations of Sertoli cells at points of contact with the acrosome region of late (acrosome and maturation phases) spermatids are observed for the first time at thirty-five days (Fig. 16, bracket). These contact modifications resemble the Sertoli-Sertoli specializations described previously. That is, there is a narrowing of the intercellular space to 70 to 90 A and an accumulation of dense material between the Sertoli cell plasma membrane and a parallel cisterna of endoplasmic rcticulum. There is no complementary specialization of the spermatid membrane.

Discussion By three to five days, the germinal cells have begun to multiply by mitosis (NEBEL et al., 1961), and as early as seven days some maturational changes have been observed in the Sertoli cells, The most rapid differentiation of the Sertoli cells, however, is between seven and fourteen days. Likewise, this is the time of first appearance of early meiotic stages in the germ cell population. This coincidence of maturation in both cell lines suggests that they may be simultaneously influenced by some control mechanism, e.e., pituitary gonadotropin or interstitial cell hormone. It is possible, however, that changes nnobservable in the present study take place earlier in one of the cell lines which subsequently play a role in the initiation of maturation in the other cell type. Some of the structural features developed by the Sertoli cells appear to be related to the function of providing mechanical support for the germinal cells. The extensive processes of the Sertoli cells may be visualized as forming a series of compartments and shelves in the interstices of which the spcrmatogenic cells rest. The large number of free ribosomes present in the cytoplasm may be necessary to maintain such a large volume of cytoplasm, and the microtubules may function in imparting some rigidity to the processes and maintaining cell shape, as suggested by CItl:~ISTENSI~N(1965) for the microtubules in guinea pig Sertoli cells.

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Several features of the development of the Sertoli cells, b y virtue of their extent and coincidence with germinal cell differentiation, are worthy of more comment, even though their precise relationship to the economy of the seminiferous tubule remains to be determined. The extensive elaboration of tubular and vesicular membranes of the agranular reticulum is of note in relation to the claims for hormone production by Sertoli cells. I t is beyond the scope of this paper to consider all of the conflicting evidence for and against this hypothesis. (See BIsHoP, 1954, for a review of the literature up to t h a t time, and LACY, 1962, for a summary of some of the more recent work.) I t is of interest, however, to indicate briefly a few of the lines of evidence implicating Sertoli cells in hormone production. Patients with a disease t h a t apparently affects only the seminiferous tubules and not the interstitial cells m a y show an increased excretion of F S H (HowARD et al., 1950), and in primary testicular failure, more testosterone m a y be required to supress F S H than is necessary to restore secondary sex characteristics (HOWARD et al., 1950). Estrogen has been extracted from normal testes of several species (ZONDEK, 1934; BEALL, 1940; CU~INGHAM et al., 1942; BURROWS and HO~NING, 1952) and from Sertoli cell tumors of dog and man (HuGGINS and MOULD]~Ir 1945; BERTI-~O~IG et al., 1949; TEILUM, 1949). I n addition, lipid accumulating in Sertoli cells under certain conditions (radiation or estrogen treatment) m a y become histochemically positive for cholesterol (LACY, 1962; LACY and LOFTS, 1965). Extracts of such tissue have been reported to contain progestin (LACY, 1962) or estrogen (LACY et al., 1965). Some invgators, however, who agree t h a t the testis produces a non-androgenic hormone, believe t h a t it is produced b y the interstitial cells (M~DI)OCKet al., 1952) or the late spermatogenic cells (McCuLLAGHand SCHAFFE~BVRG, 1952; JOHNSEN, 1964) rather than b y the Sertoli cells. CHI~ISTENSEN and FAWCETT (1961) have advanced the idea that the site of hormone production in steroid producing cells is in the agranular endoplasmic reticulum on the basis of its abundance in known steroid producing cells and the biochemical localization of enzymes active in the synthesis of steroid hormones in the microsomal fraction of such cells. I f it could be assumed t h a t the enzyme systems associated with the smooth endoplasmic reticulum are similar in different cell types, the abundant agranular reticulum of the Sertoli cells would indicate t h a t they are adequately equipped for the manufacture of steroid hormones. However, in other cell types the agranular retieulum appears to be related to quite different functions; for example, the transport of recently absorbed lipid (PALAu and KARLIN,1959), salt transport (PnTLPOTT and COPELAND, 1963), acid secretion (ITo, 1961), and drug metabolism (JONES and FAWCETT, 1966). The agranular endoplasmic reticulum of the Sertoli cell, therefore, although morphologically consistent with the production of steroid hormones m a y perform some other function such as providing a route for transport of nutrients and metabolites to and from the germinal cells as suggested byYAsuzuMI et al. (1960), for the 750 A tubules observed in the nutritive cells of a snail testis. The structure of the characteristic contact specializations of Sertoli cells has been considered in detail elsewhere (FLIcKINGERand FAWCETT, 1967). Of note in the present context is the appearance and great increase in number and extent of these surface specializations between seven and fourteen days. I t is possible

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t h a t they develop by aggregation or extension of the small areas of close membrane apposition and cytoplasmic dense material seen in newborn and one week old animals (Figs. 4, 10). Favorable electron micrographs (Fig. 10) suggest that short tubular or vesicular elements of the smooth endoplasmic reticulum m a y line up on the cytoplasmic side of the dense material and then by progressive coalescence and acquisition of ribosomes on the cytoplasmic side produce the cisterna seen in the fully developed junctional complex. At birth the Sertoli cell nucleus contains two types of dense body identified as nucleolar material. One shows cordlike areas of greater density, suggesting a nucleolonema. This organization becomes progressively more evident with age and appears to give rise to the central component of the adult tripartite nucleoli. On the other hand, at birth some of the nuclear dense bodies are relatively uniform in density and remain so throughout subsequent development until they acquire a less dense core and apparently form the lateral components of the adult nucleolus. Association of the two elements into pairs and their apparent migration from the periphery into the interior of the nucleus was observed in seven to fourteen day old mice, but typical tripartite adult nucleoli were rarely observed prior to twenty-one days. According to SArSFORD (1963) the lateral elements ("perinucleolar spheres") are Feulgen positive while the central portion is negative. The possibility, therefore, must be considered that the lateral spheres are composed of nucleolus-associated heterochromatin which m a y include the nucleo]ar organizing region of the genome. In the younger animals, however, the dense areas lacking a nucleolonema are not associated with those displaying a nucleolonema as would be expected if the former contained a nucleolar organizer, but are located up to several micra distant. Furthermore, both the central and lateral components appear to have a similar fibrillo-granular substructure, although the lateral spheres are more dense. At present, then, it seems appropriate to consider the lateral spheres part of the nucleolus, pending chemical definition of their composition. Perhaps it is the light cores which are composed of the Feulgen positive material. If this unusual nucleolar morphology is related to some peculiar synthetic activity of the Sertoli cells coordinated with the beginning of spermiogenesis, its nature is completely unknown at present. Dense bodies resembling lysosomes are present in the Sertoli cells at all the stages studied. There is abundant evidence that Sertoli cells phagocytize the residual bodies shed from spermatids, and m a y generally clear the tubules of debris (SMITH and LACY, 1959; B]~OX~LMAN~, 1963; NICA~DE~, 1963; CLEGG and MACMILLAN, 1965; LACY and LOFTS, 1965; VILAR, 1965). These lysosomes are presumably used in the intracellular digestion of phagocytized material. There are a few lipid droplets present in the Sertoli cells of the newborn mouse, but the number and size of these droplets become greater at about twenty-eight days of age, when late spermatids appear for the first time. This is consistent with the contention that much of the Sertoli cell lipid is derived from the residual bodies, as suggested by several authors who correlated variations in adult Sertoli cell lipid content during the spermatogenic cycle with the release of residual bodies (LACY, 1962; SMIT~ and LACY, 1959; LOFTS, 1962; BROXELMANN, 1963; NIEMI and KORMANO, 1965). LACY (1962) has made the interesting suggestion t h a t the phagocytosis of residual body lipid m a y stimulate the Sertoli cell to

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synthesize a s u b s t a n c e capable of influencing spermatogenesis, t h u s p l a y i n g a p a r t i n the r e g u l a t i o n of cyclical a c t i v i t y i n the seminiferons tubule. A~DR~ (1962) has described i n detail the d e v e l o p m e n t of areas of "pseudom a t r i x " b y d i l a t i o n of the cristae of the m i t o c h o n d r i a of r a t spermatogenic cells. I n the mouse the m i t o c h o n d r i a of the Sertoli cells u n d e r g o this change as well as those of the g e r m i n a l cells, suggesting t h a t some factor which acts througho u t the tissue m a y be responsible. The cause a n d significance of these mitochondrial alterations, however, r e m a i n obscure. Because of the avascular n a t u r e of the seminiferous t u b u l e , it appears t h a t n u t r i e n t s a n d metabolites of the m a j o r i t y of the germinal cells (excepting those t o u c h i n g the basal l a m i n a ) m u s t pass either t h r o u g h the Sertoli ceils or the intercellular space. Areas of m e m b r a n e fusion do n o t seem to be a feature of the Sertoli cells' c o n t a c t specialization, b u t the possibility exists t h a t these specializations m a y effectively limit the passage of materials t h r o u g h the intercellular space. If s u b s t a n c e s were t r a n s p o r t e d t h r o u g h the Sertoli cells, chemical modification of the n u t r i e n t s a n d metabolites m i g h t be a n a d d i t i o n a l f u n c t i o n of the Sertoli cell. The presence of a large n u m b e r of Golgi bodies w i t h i n one Sertoli cell is a t least suggestive of i n t e n s e secretory activity, b u t the chemical n a t u r e of the substances i n v o l v e d is u n k n o w n , a n d such a n a c t i v i t y on the p a r t of the Sertoli ceils m u s t r e m a i n a speculation for the present. References ANDR$, J. : Contribution ~ la connaisance du chondriome, l~tude de ses modifications ultrastructurales pendant la spermatog6nbse. J. Ultrastruct. Res. 6, Suppl. 3, 1--185 (1962). BAWA, S. R. : Fine structure of the Sertoli cell of the human testis. J. Ultrastruct. Res. 9, 459--474 (1963). BEALL, D . : The isolation of alpha-oestradiol and oestrone from horse testis. Bioehem. J. 34, 1293--1298 (1940). BEaTrmONG, M., W.E. GOOI)WIN, and W. W. SCOTT: Estrogen production by the testis. J. din. Endocr. 9, 579--592 (1949). Bishop, P. M. F. : Recent advances in endocrinology, p. 222--229. London: Churchill 1954. BOOX~OVT, C. G. : The germ cell cycle in the guinea pig. II. The postnatal development of the testis. Z. Zellforsch. 25, 749--763 (1937). BRSXrLMAN~, J. : Fine structure of germ cells and Sertoli cells during the cycle of the seminiferous epithelium in the rat. Z. Zellforsch. 59, 820--850 (1963). BVRROWS,H., and E. H O R ~ G : Oestrogens and neoplasia. Oxford: Blackwell Sci. Publ. 1952. C~AX~Y, C.W., A. S. CONSTON, and D. R. MERA~Zr: Testicular developmental histology. Ann. N. Y. Acad. Sci. 55, 597--608 (1952). Cn~ISTENSEN,A. K.: Mierotubules in the Sertoli cells of the guinea pig testis. Anat. Rec. 151, 335 (1965). --, and D. W. FAWCETT: The normal fine structure of opossum testicular interstitial ceils. J. biophys, biochem. Cytol. 9, 653--670 (1961). CLEGa, E. J., and E. W. MACMILLAi'~:The uptake of vital dyes and particulate matter by the Sertoli cells of the rat testis. J. Anat. (Lond.) 99, 219--229 (1965). CLERMONT,Y., and B. P~.REu Quantitative study of the cell population of the seminiferous tubules in immature rats. Amer. J. Anat. 100, 241--268 (1957). CUNNING~IA~,B., J. MAY, and S. GORDAN:The presence of estrogenic hormones in testicular material. Proe. Soc. exp. Biol. (N. Y.) 49, 130--132 (1942). FAWCETT,D. W. : The cell: Its organelles and inclusions, p. 30--31. Philadelphia and London: W. B. Saunders Co. 1966. --, and M. H. BURGOS: The fine structure of Sertoli cells in the human testis. Anat. Rec. 124, 401 (1956).

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FLICKINGER, C. J., and D. W. FAWCETT: The junctional specializations of Sertoli cells in the seminiferous epithelium. Anat. Rec. (in preparation) (1967). GARDNER, P. J., a n d E. A. HOLYOKE: Fine structure of the seminiferous tubules of the Swiss mouse. I. The limiting membrane, Sertoli cells, spermatogonia, and spermatocytes. Anat. Rec. 150, 3 9 1 ~ 4 0 4 (1964). HARGITT, G. T.: The formation of the sex glands and germ cells of mammals. II. The history of the male germ cells in the albino rat. J. Morph. 42, 253--294 (1926). HORSTMANN, E.: Elektronenmicroskopische Untersuchungen zur Spermiohistogenese beim Menschen. Z. Zellforsch. 54, 68--89 (1961). HOWARD, R. P., R.C. SNIFFEN, F . A . SIMMONS, a n d F. ALBRIGHT: Testicular deficiency: A clinical and pathological study. J. clin. Endocr. 10, 121--186 (1950). HUGGINS, C., and P. V. MOULDER: Estrogen production by Sertoli cell tumors of the testis. Cancer Res. 5, 510--514 (1945). HUGON, J., and M. BOROERS: Ultrastructural and cytochemical changes in spermatogonia and Sertoli cells of whole body irradiated mice. Anat. Rec. 15~, 15--19 (1966). ITO, S. : The endoplasmic reticulum of gastric parietal cells. J. biophys, biochem. Cytol. l l , 333--347 (1961). JOItNSEN, S. G.: Studies on the testicular-hypophyseal feedback mechanism in man. Acta endocr. (Kbh.), Suppl. 90, 9 9 - - t 2 4 (1964). JoNEs, A. L., and D. W. FAWCETT: H y p e r t r o p h y of the agranular endoplasmic reticulum in hamster liver induced b y phenobarbital. J. Histochem. Cytochem. 14, 215--232 (1966). KARNOVSKY, M. J. : A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A (1965). LACY, D.: Certain aspects of testis structure and function. Brit. reed. Bull. ]8, 205--208 (1962). , and B. LOFTS: Studies on the structure and function of the mammalian testis. I. Cytological a n d histochemical observations after continuous t r e a t m e n t with oestrogenic hormone a n d the effects of F S H and LH. Proc. roy. Soc. B 162, 188--197 (1965). - - - - G. KrNSON, D. HOPKINS, and H. DOTT: Sertoli cells and steroid synthesis. Gen. comp. Endocr. 5, 693 (1965). LOFTS, B.: Role of the Sertoli cell in spermatogenesis. Exerpta Medica, Int. Congr. Series No 51 (1962). MADDOCK, W. O., M. El'STEIN, a n d W. O. NELSON: The assay of urinary estrogens as a test of h u m a n Leydig cell function. Ann. N. Y. Acad. Sci. 55, 657--673 (1952). MANCINI, R. E., R. NARBA][TZ,and J. C. LAVIEm: Origin and development of the germinative epithelium and Sertoli cells in the h u m a n testis: cytological, cytochemical, and quantitative study. Anat. Rec. 136, 4 7 7 - 4 8 9 (1960). MCCULLAGIt, E. P., and C. SCHAFFENBURO: The role of the seminiferous tubules in the production of hormones. Ann. N. Y. Acad. Sci. ~5, 674--684 (1952). NAGANO, T. : Some observations on the fine structure of the Sertoli cell in the h u m a n testis. Z. Zellforsch. 73, 89--106 (1966). NEBEL, B. R., A. P. AMAROSE, and E. M. HACKETT: Calendar of gametogenie development in the pre-pubertal male mouse. Science 134, 832--833 (1961). NICANDER, L.: Some ultrastructural features of mammalian Sertoli cells. J. Ultrastruct. Res. 8, 190 (1963). - - N. ABDEL-RAOUF, and B. CRABO: On the ultrastructure of the seminiferous tubules in bull calves. Acta morph, neerl.-scand. 4, 127--135 (1961). NIEMI, M., a n d M. KORMANO: Cyclical changes in and significance of lipids and acid phosphataKe activity in the seminiferous tubules of the r a t testis. Anat. Rec. 151,159--170 (1965). PALAY, S. L., and M. J. KARLIN: An electron microscope study of the intestinal villus. II. The p a t h w a y of fat absorption. J. biophys, biochem. Cytol. ~, 373--384 (1959). PHILPOTT,C. W., a n d D. E. COPELAND: Fine structure of chloride cells from three species of Fundulus. J. Cell Biol. 18, 3 8 9 - 4 0 4 (1963). SAFSFORD, C. S. : The development of the Sertoli cell of the rat and nmuse; its existence as a mononucleate unit. J. Anat. (Lond.) 97, 225--238 (1963). SMITH, B. V., a n d D. LACY: Residual bodies of seminiferous tubules of the rat. Nature (Lond.) 184, 249--251 (1959).

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S~FFEN, R. C. : Histology of the normal and abnormal testis at puberty. Ann. N. Y. Aead. Sci. 55, 609--618 (1952). TEILU~I, G.: Estrogen producing Sertoli cell tumors (androblastoma tubulare lipoides) of the human testis and ovary. Homologous ovarian testicular tumors. III. J. clin. Endocr. 9, 301--318 (1949). V~CABLE, J. H., and R. COOOES~ALL:A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25, 407--408 {1965). VILAR, O.: Electron microscopical study of the phagoeytosis of germ cells by Sertoli cells in tissue cultures. Anat. Rec. 151, 428 (1965). - - M. I. PEREZ DEL CERRO, and R. I. M~ANCINI"The Sertoli cell as a "bridge cell" between the basal membrane and the germinal cells. Exp. Cell Res. 27, 158--161 (1962). Y A s v z v ~ , G., H. TAN~KA, and O. TEZUKA: Spermatogenesis in animals as revealed by electron microscopy. VIII. Relations between the nutritive cells and the developing spermatids in a pond snail. J. biophys, biochem. Cytol. 7, 4 9 9 ~ 0 4 (1960). ZOlffD:EK,B.: Mass excretion of oestrogenic hormone in the urine of the stallion. Nature (Lond.) 188, 209---210 (1934). Dr. CHARLES J. FLICKINGER Institute for Developmental Biology P S R B NO 1 Room 129 University of Colorado Boulder, Colorado 80302 USA

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