Anatomy and Embryology

Anat Embryol (1986) 174:413-423

9 Springer-Verlag1986

Postnatal development of bovine Sertoli cells Fred Sinowatz and Werner Amselgruber Institut f/it Tieranatomie der Universit/it, Veterin/irstraBe 13, D-8000 Mfinchen 22, Federal Republic of Germany

Summary. The fine structure of bovine Sertoli cells was studied from the 4th to the 40th week post nature in order to correlate the progressive acquisition of normal adult morphology with functional development. The considerable increase in tubular size during the first 20 weeks is due to the proliferation of both presumptive Sertoli and germ cells. Aside from this, the presumptive Sertoli cells are seen to expand radially and lengthen considerably. From then on however, the observed increase in tubular diameter during the later period of postnatal development is solely due to the great increase in the number of germ cells. Presumptive Sertoli cells undergo morphological differentiation to mature Sertoli cells during the first 28 weeks of proliferative development. The maturation process includes distinct changes in cell shape, nucleus and cellular organelles, as well as an increase in and differentiation of Sertoli cell surface specializations. At 24 weeks the development of interSertoli cell junctions has reached a point of differentiation where, in our opinion, a functional blood-testis barrier can be expected. During the first 8 weeks an extensive development of rough endoplasmic reticulum and a well-developed Golgi apparatus can be observed, which suggests a high secretory activity in the presumptive Sertoli cells at this time. We speculate that these secretory activities may play a role in the formation of the basal lamina which is extremely well developed during early postnatal life. The subsequent reduction of the basal lamina correlates well with diminished secretory activity in the Sertoli cells. Key words: Sertoli cells - Postnatal development

Bull

paratively little information is available on the ultrastrucrural development and differentiation of this cell type, however (Black and Christensen 1969; Flickinger 1967; Ramos and Dym 1979; Hatier and Grignon 1980; Hadziselimovic' and Seguchi 1974; Schulze 1984). No detailed data are available on the postnatal development of bovine Sertoli cells. In the present investigation, the fine structure of bovine Sertoli cells was studied from 4 to 40 weeks of postnatal development in order to correlate the progressive acquisition of normal adult morphology with functional development.

Material and methods Testes of bull calves 4, 8, and 16 weeks of age were removed after slaughter and fixed by vascular perfusion via the testicular artery. The fixation was performed with solutions I and II according to Forssmann et al. (1977), or with formaldehyde-glutaraldehyde solution as described by Karnovsky (1965). Additional testicular biopsies were obtained from bull calves at the ages of 4, 8, 16, 20, 24, 30 and 40 weeks. The biopsied tissue was fixed by immersion for 2 h in 6% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). All specimens were postfixed in 1% cacodylate buffered osmium for 1 h. After dehydration in a graded series of ethanol and propylene oxide, the specimens were embedded in ERL 4206 (Spurt 1969). Semithin sections were cut with a Reichert O M U 3 ultramicrotome and stained with azure II methylene blue. Ultrathin sections were routinely contrasted with uranyl-acetate and lead citrate (Reynolds 1963) and examined with either a Zeiss EM 10 A or a Siemens 101 electron microscope.

Introduction Morphological, biochemical and physiological studies on the testis of many mammalian species indicate that Sertoli cells have a broad spectrum of functions which are of great importance in the physiology of spermatogenesis. The ultrastructure of mature Sertoli cells of several mammalian species (Nicander 1963; Dym 1974; Fawcett 1975; Br6kelmann 1963; Means et al. 1980)including man (Nagano 1966; Schulze 1974; Schulze 1984; Vorstenbosch et al. 1984; Zibrin 1972) has been studied extensively. CornOffprint requests to: F. Sinowatz, Institut f/it Tieranatomie der Universitfit, Veterin/irstral3e 13, D-8000 Mfinchen 22, Federal Republic of Germany

Results 4 weeks (Figs. 1, 2, 11, 12)

At 4 weeks post nature the tubuli seminiferi appear small and solid and are widely separated from each other by a well-developed interstitium. The average diameter of the solid cords is about 55 lain. Their epithelium (Fig. 1) consists of undifferentiated Sertoli cells and a few, usually more centrally located, prepuberal spermatogonia. The nuclei of presumptive Sertoli cells are round or oval. A small rim of heterochromatin runs along the inner nuclear membrane. Numerous flakes ofheterochromatin are dispersed through-

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Fig. 1. At 4 weeks post nature the epithelium of the seminiferous cords consists of presumptive Sertoli cells (pSc) and usually more centrally located prepubertal spermatogonia (psg). x 3,300 Fig. 2. Seminiferous cords, 4 weeks post natum. The epithelium rests on an extremely well developed multi-layered basal lamina (BL); rER, rough endoplasmic reticulum; FR, 15broblast-like cells, which are precursors of contractile peritubular cells, x 10,000 out the nucleoplasm. One or two larger aggregates of heterochromatin are often seen in the center of the nucleus, and are sometimes associated with a small nucleolar-like structure. The nuclei of prepuberal spermatogonia are dis-

tinctly different as evidenced by a consistently larger average diameter (12 gm), relative paleness and the presence of a well-developed nucleolus. Lateral cell membranes of neighboring presumptive Sertoli cells run comparatively

415

Fig. 3. Seminiferous cord, 8 weeks post natum; pSc, presumptive Sertoli cell; BL, basal lamina. • 3,300 straight in the basal aspect of the epithelium and contain few interdigitations and no special junctional complexes. A small, circumscribed thickening of opposing cell membranes can sometimes be seen. The cytoplasm of presumptive Sertoli cells is characterized by large amounts of rough endoplasmic reticulum arranged in parallel, often in anastomosing arrays or concentric whorls. The absolute quantity

of rough endoplasmic reticulum varies between neighboring cells but is extremely well developed in many cases. Many of the cisternae are dilated and contain a moderately electron dense material. A pronounced Golgi apparatus, consisting of stacks of cisternae with many associated vesicles, can generally be observed in a supranuclear position. Small mitochondria of the crista-type are scattered abundantly

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Fig. 4. Bovine seminiferous cord, 16 weeks post nature. Note the more elongated and irregular form of the nuclei of presumptive Sertoli cells (pSc). • 3,300 Fig. 5. Bovine seminiferous cord, 16 weeks post natum, pSc, presumptive Sertoli cells; many of the prepubertal spermatogonia (pspg) are now displaced to the basal aspect of the seminiferous cord. x 5,200 throughout the cytoplasm. M a n y of the presumptive Sertoli ceils contain a few, electron-dense, m e m b r a n e b o u n d bodies. Lysosomes were also identifiable on the basis of their morphology. The epithelium of the cords rests on an extremely well-developed multi-layered basal lamina (Fig. 2),

which is about 1.9 gm thick, The frequent appearances of small vesicles which are fused with the basal cell membrane seems to indicate the emptying of their contents to the basal lamina. A few collagen fibers and one or two layers of fibroblast-like cells are located peripherally to the basal la-

417

Fig. 6. Bovine seminiferous tubule, 16 weeks postnatal. Junctional complexes (arrows) between neighboring Sertoli cells have increased in extent within the basal portions of the tubuli. x 4,100 mina and represent precursors of contractile peritubular cells. 8 weeks (Figs. 3, 11, 12)

By 8 weeks the tubular diameter has increased to ca. 70 gm, due mainly to the mitotic activity of presumptive Sertoli cells, which now exhibit a more centrally-located nucleus (central supporting cells). Their cytoplasm does not extend to the basal lamina but the overall ultrastructure is identical to that of those presumptive Sertoli cells which rest on the basal lamina. The outstanding feature of both cell types is the enormous amount of rough endoplasmic reticulum, which frequently sandwiches the small mitochondria between adjacent cisternae. The well-developed Golgi apparatus seen in many of the presumptive Sertoli cells is indicative of a high secretory activity. Replication of prepubertal spermatogonia via mitosis has lead to a significant increase in the number of germ cells in the epithelium of the still solid seminiferous tubules. 16 weeks (Figs. 4, 5, 11, 12)

The average diameter of the tubuli seminiferi has increased to 82 gm. This is primarily a result of mitosis on the part of the presumptive Sertoli cells, but may also be attributed to the replication of prepubertal spermatogonia. The inter-

tubular spaces are relatively reduced and many tubules appear to be in close contact. Tubuli with lumen are still not found at this stage. Presumptive Sertoli cells have sent out lateral processes containing rough endoplasmic reticulum between neighboring germ cells. Many of the prepubertal spermatogonia have been displaced to the basal aspect of the tubuli (Fig. 5) and now contact the basal lamina. Above the spermatogonia (i.e. in the direction of the future lumen), opposing cell membranes of adjacent presumptive Sertoli cells start the development of extended junctional complexes (Fig. 4). Marked changes have occurred in the morphology of the nuclei of presumptive Sertoli cells as well. The nuclear form is more elongated and irregular. The amount of heterochromatin is distinctly reduced, and in many of the presumptive Sertoli cells the development of the vacuolar nucleolus characteristic of the mature cell is in progress. The quantity of the rough endoplasmic reticulum appears considerably reduced and most of the cisternae are narrow and contain only a small amount of moderately electron-dense material. In the more apical regions of the epithelium a parallel orientation of these narrow cisternae along the spermatocytes is seen. By 16 weeks the secretory activity of presumptive Sertoli cells has diminished greatly, whereas the number of microfilaments and microtubuli has considerably increased. Microfilaments are especially prominent in the basal area of the cell where they concentrate along the basal cell membrane. The thickness of the multilayered basal lamina is reduced to 1.5 lam.

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Fig. 7. Bovine seminiferous tubule; 30th postnatal week. SC, Sertoli cells; SCN, Sertoli cell nuclei, x 5,200 20 weeks (Figs. 6, 11, 12) The increase in the extent of tubuli seminiferi (average diameter: 95 pm) is reflected by the decrease in relative vol-

ume of the intertubular space. Some o f the seminiferous tubules now show a small lumen. In the central area of those which have not yet acquired a lumen, extensive interdigitation of the cell apices and vacuolation is seen. In the

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Fig. 8. Bovine seminiferous tubule, 40th postnatal week. Spd, spermatid, cap phase; SC, Sertoli cells. Large gaps (x) often occur within the germinal epithelium, probably due to the degeneration of germ cells, x 3,900

basal portion of the tubuli, junctional complexes between neighboring Sertoli cells have increased in extent. The nuclei of the supporting cells are more elongated compared to previous stages and usually show a comparatively irregular outline. In the perinuclear region many randomly arranged microfilaments can be observed which appear to be in contact with the nuclear envelope and may play a role in nuclear reshaping. Most presumptive Sertoli cells reveal the development of the typical Sertoli cell nucleolus. The clumps of heterochromatin within the nucleus seen in earlier stages disappear and seem to be transformed into reticular and vacuolar structures. A small rim of heterochromatin can

be seen only at the periphery of the nuclei. The mitochondria have increased considerably in length and are orientated along the long axis of the extending presumptive Sertoli cells. The thickness of the basal lamina is further decreased (1.2 ~tm).

24 weeks (Figs. 11, 12) The number of tubuli with a lumen has increased significantly. The central area of those devoid of lumen contains many vacuoles of varying diameter which will merge and

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Fig. 9. Bovine seminiferous tubules; 40th postnatal week. Spd, elongated spermatids; L, tubular lumen, x 5,200 Fig. 10. Bovine seminiferous tubule; 40 weeks post nature. SCN, Sertoli cell nuclei; arrows, blood-testis barrier, x 6,450 eventually give rise to the c o m m o n central lumen. The number of germ cells is also significantly higher. M a n y primary spermatocytes in different stages of meiosis can be observed. The prevalence of junctional specialization be-

tween adjacent presumptive Sertoli cells is much greater and divides the tubular epithelium into a basal compartment containing spermatogonia and an adluminal compartment characterized by the later stages of spermatogenesis.

421 Number of Sertoli cells/cross section

Diameter of seminiferous tubules in gm 2OO 180

50

160

40

140 120

30

100 80 601

20

10'.

40 ; 200 4

i 8

i 16

[

20

i 24

i 30

i 40

0

i 8

i 16

i 20

i 24

~ 0

i 40

Age in weeks

Age in weeks

Fig. 11. Diameter of seminiferous tubules during postnatal development

Fig. 12. Number of Sertoli cells/cross-section during postnatal development

30 weeks (Figs. 7, 11, 12)

Mitochondria scattered in the basal region of the Sertoli cells are usually small and round or oval, whereas those of the supranuclear cell area are elongated and run along the major cell axis. The Golgi apparatus consists of several dictyosomes and is usually situated close to the nucleus, frequently in a supranuclear position. Both rough and smooth endoplasmic reticulum are present, the latter occurring predominately in the basal cytoplasm, usually in form of narrow, anastomosing cisternae. Conspicous aggregations of the well-developed smooth endoplasmic reticulum are found in the basal lateral processes of the Sertoli cells, as mentioned previously. A considerable amount of smooth endoplasmic reticulure is also localized in that portion of the Sertoli cells immediately adjacent to developing spermatids in the capor acrosome-phase. It shows its greatest development just before the onset of nuclear condensation in the spermatids and appears as a circumscribed crescentic or conical mass of smooth tubuli. These accumulations of smooth endoplasmic reticulum disperse during the later stages of spermatid development subsequent to which only one or a few cisternae follow the indentated Sertoli cell membrane. These subsurface cisternae are separated from the Sertoli cell plasma membrane by a layer of cytoplasm which is rich in bundles of filaments. The complex probably constitutes a specialization for maintaining cohesion between Sertoli cells and developing spermatids. Numerous microtubules run parallel to the longitudinal axis of the Sertoli cells throughout the cytoplasm and form a well-developed cytoskeleton (Fig. 9). A varying number of membrane-bound dense bodies can be seen in their preferential location around the nuclei.

Most of the tubuli have now acquired a lumen. Round and elongated spermatids can be observed. The average diameter of the tubuli is about 170 gm. Many of the nuclei of presumptive Sertoli cells are elongated with deep indentations and possess a vacuolar nucleolus. Bundles of filaments surround the nuclei, but these areas are devoid of other cell organelles. The cytoplasmic organelles show a preferential orientation along the long axis of the cell. Many elongated mitochondria of the crista-type, a great number of microtubuli, and profiles of rough and smooth endoplasmic reticulum lie perpendicular to the basal lamina. The Golgi apparatus consists of several stacks of cisternae and associated vesicles and is generally found to occupy a supranuclear position. Many free ribosomes and some dense bodies occur regularly in the cytoplasm. Numerous lateral processes containing a large amount of endoplasmic reticulum extend between the developing germ cells. 40 weeks (Figs. 8, 9, 10, 11, 12)

The average diameter of the tubuli seminiferi is now about 190 gin. They are hollow and the tubular epithelium demonstrates all stages of spermatogenesis up to that of elongated spermatids (Figs. 8, 9). Comparatively large gaps often occur within the germinal epithelium, probably due to the degeneration of germinal cells. Most Sertoli cells have completed their morphological differentiation and now attain the adult structure: An irregularly-shaped nucleus containing a typical vacuolated nucleolus is situated in the basal part of the cell near the basal lamina. Bulky processes with whorls of smooth endoplasmic reticulum extend laterally into the basal region of the epithelium. The corresponding Sertoli cell processes in the more central portion of the epithelium separating the germ cells usually appear much thinner. Junctional complexes (Fig. 10) between the interdigitating lateral cell membranes of adjacent Sertoli cells are extremely well developed in the basal part of the tubular epithelium. They consist of many serially arranged points or lines of fusion involving neighboring Sertoli cell membranes, associated filaments and a varying amount of smooth endoplasmic reticulum situated close to the contact zone. In toto they are an important part of the so-called blood-testis barrier, which divides the tubular epithelium into a basal compartment containing the spermatogonia and preleptotene spermatocytes, and an adluminal compartment with the more advanced stages of spermatogenesis.

Discussion

Formation of the mammalian testis during prenatal development relies on the precisely-timed proliferation and, to a certain extent, differentiation of both germinal and somatic cells (Bellve' and Feig 1984). In many species, presumptive Sertoli cells also continue to proliferate rapidly during early postnatal life (Orth 1982), thereby enabling the presumptive Sertoli cells to expand radially and to lengthen considerably. However, following birth the proliferative activity of presumptive Sertoli cells diminishes quickly in many species. By day 16 neither DNA-synthesis nor mitotic divisions of presumptive Sertoli cells are evident in the rat (Orth 1982). Mitotic figures of presumptive Sertoli cells in humans are likewise seen only in the testes of the

422 newborn (Nistal et al. 1982). Compared with the situation in rats and humans the mitotic activity in the bovine testes appears to continue over a comparatively long postnatal period. The number of presumptive Sertoli cells increases significantly from the 4th week of age (19_+ 1 presumptive Sertoli cells per tubular cross section) to the 20th week (41 + 1 cells per cross section). During that time the tubular diameter increases from 58 _+4 to 98 +_2. Similar results were also obtained by Curtis and Amann (1981). From the 20th week on, the number of Sertoli cells per cross-section appears to decrease (Curtis and Amann 1981), which is probably due to a considerable loss of presumptive Sertoli cells during the process of lumen formation in the tubuli seminiferi. The considerable increase in tubular diameter during later postnatal development is due to the massive proliferation of germ cells. An important event during postnatal tubular development is the formation of a lumen in the previously solid tubules. Tubules with a lumen were not found before 20 weeks in this study. At 20 weeks distinct signs of a progressing lumen formation could be observed. Convergence of differently sized vesicles in the central parts of presumptive Sertoli ceils produces large vacuoles which eventually fuse with the apical cell membranes to form a common central lumen within the tubular epithelium. Appearance of tubular lumina may be linked with an increase in fluid secretion by the presumptive Sertoli cells (Setchell 1980). Additional degeneration of germ cells may also contribute to lumen development. Several authors describe different types of Sertoli cells or Sertoli cell precursors. The distinction between clear and dark Sertoli cells (Vilar 1970) was not observed in this study. It is possible that dark cells are the consequence of imperfect fixation of testicular tissue (Chemes et al. 1977; Nistal et al. 1982). The claim has been made (Christensen 1970) that dark cells do not appear in perfusion-fixed testes. The present material consisted of perfusion-fixed tissue as well as immersion-fixed testicular biopsies. Dark cells never occurred following vascular perfusion but were sometimes seen after immersion fixation. Presumptive Sertoli cells have also been classified into basal indifferent supporting cells and "central indifferent supporting cells", using light microscopy (Abdel Raouf 1960). Our ultrastructural studies show that both cell types possess the same morphological features. The only major difference is a more central position of the nucleus in the "central indifferent supporting cells". In serial sections both cell types appear to be in contact with the basal lamina. On the basis of ultrastructural criteria there is no need to maintain this artificial distinction between two separate types of presumptive Sertoli cells. Presumptive Sertoli cells undergo their morphological differentiation to adult Sertoli cells during the first 28 weeks of postnatal development. The maturation process includes distinct morphological changes in the cell shape, nucleus and cellular organelles as well as an increase in surface specialization and subsequent interaction with other Sertoli and germ cells. After birth the presumptive Sertoli cells are polyhedral in form and display comparatively broad extensions which surround the centrally located germ cells. The lateral cell membranes of adjoining presumptive Sertoli cells are comparatively straight in the basal parts of the cells. Only in the central contact area of the cells are the Sertolian cell processes deeply enmeshed. The following months see the continued elongation of the presumptive

Sertoli cells. Due to the peripheral translocation of germ cells which eventually come into contact with the basal lamina, the basal contact zone between neighboring Sertoli cells also becomes increasingly complicated. Contact specialization between neighboring presumptive Sertoli cells is already evident at 4 weeks of age. Although restricted to small areas characterized by a narrowing of the intercellular space with accumulation of filamentous material in the underlying cytoplasm during the first 12 weeks, these contact structures are markedly increased by the end of the 4th month. In those regions of the tubuli where prepubertal spermatogonia now contact the basal lamina, an increasing number of tight junctions are evident between neighboring presumptive Sertoli cells just above the germ cells. The further development of these specialized contacts continues through the 24th week, at which time the development has reached a point where in our opinion a functional blood-testis barrier has been established. Since the initial formation of the blood-testis barrier coincides with the migration of germ cells to the basal region of the tubuli, it appears as though the latter may be a strong inductive stimulus for the development of specialized contact zones between presumptive Sertoli cells. This is in agreement with the results of Curtis and Amann (1981) who observed that the blood-testis barrier is functional by 24 to 28 weeks of age and coincides with the appearance of pachytene spermatocytes. Our observations also lend some support to the suggestions made by Bergmann and Dierichs (1983) who found that postnatal formation of the blood-testis barrier in rats is correlated with the onset of meiosis. Using tracer experiments they were able to demonstrate that tight junctions between neighboring Sertoli cells are first formed in those parts of the seminiferous cords where pachytene spermatocytes are located. It is generally agreed that mature Sertoli cells modulate germ cell development by secreting particular components such as androgen binding protein (Fritz 1978), plasminogen activator (LaCroix et al. 1977), inositol or transferrin (Skinner and Griswold 1980). Fetal Sertoli cells on the other hand are known to produce relatively high concentrations of Anti-Mullerian hormone (AMH), a glycoprotein important in male sexual differentiation (Tran etal. 1977). Shortly after birth the concentration of A M H drops sharply, but then rises again (Tran et al. 1977). A M H in bovine Sertoli cells was found to be localized in the cisternae of the rough endoplasmic reticulum (Tran and Josso 1982). Our results show an extensive development of rough endoplasmic reticulum and a well-developed Golgi complex at 4 and 8 weeks post nature with a high secretory activity of presumptive Sertoli cells during this period. No firm conclusions can be made on the data without accompanying biochemical studies of the secreted material; however, secretion of A M H as observed by Tran and Josso (1982) might account at least in part for the observed secretory activity at this stage. One might also suspect that the secretory activity of presumptive Sertoli cells plays an important role in the formation and maintenance of the basal lamina, which is extremely well-developed during fetal and early postnatal life. With the reduction of the amount of rough endoplasmic reticulum and the size of the Golgi complex in subsequent weeks, a concomitant and continuous reduction in the thickness of the basal lamina is observed. At 20 weeks its thickness is reduced to 1.2 pro, which is comparable to the adult situation.

423

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high osmolality for use in electron microscopy. J Cell Biol 27: 137A-138A LaCroix M, Smith FE, Fritz IB (1977) Secretion of plasminogen activator by Sertoli cell enriched cultures. Mol Cell Endocrinol 9 : 227-236 Means AR, Dedman JR, Tash JS, Tindall D J, Sickle van M, Welsh MJ (1980) Regulation of the testis Sertoli cell by FSH. Annu Rev Physiol 42 : 59-70 Nagano T (1966) Some observations on the fine structure of the Sertoli cell in the human testis. Z Zellforsch 73 : 89-106 Nicander L (1963) Some ultrastructural features of mammalian Sertoli cells. J Ultrastruct Res 8 : 190-191 Nistal M, Abaurrea MA, Paniagna R (1982) Morphological and histometric study on the human Sertoli cell from birth to the onset of puberty. J Anat 14:351-363 Orth JM (1982) Proliferation of Sertoli cells in fetal and postnatal rats: a quantitative autoradiographic study. Anat Rec 203:485492 Ramos ASI Dym M (1979) Ultrastructural differentiation of rat Sertoli cells. Biol Reprod 21 : 909-922 Reynolds EG (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17 : 208-212 Schulze C (1974) On the morphology of the human Sertoli cell. Cell Tissue Res 153 : 339-355 Schulze C (1984) Sertoli cells and Leydig cells in man. Adv Anat Embryol 88 : 1-104 Setchell (1980) The functional significance of the blood-testis barrier. J Androl 1:3-10 Skinner MK, Griswold MD (1980) Sertoli cells synthesize and secrete transferrin-like protein. J Biol Chem 255:9523-9525 Spurr A R (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26 : 31-43 Tran D, Josso N (1982) Localization of anti-Mullerian hormone in the rough endoplasmic reticulum of the developing bovine Sertoli cell using immunocytochemistry with a monoclonal antibody. Endocrinology 111:1562-1567 Tran D, Meusy-Desolle N, Josso N (1977) Anti-Mullerian hormone is a functional marker of fetal Sertoli cells. Nature 269:411 412 Vilar (1970) Histology of the human testis from neonatal period to adolescence. In: Rosenberg E, Paulsen CA (eds) The human testis. Plenum, New York, pp 95-101 Vorstenbosch CJAHV, Spek E, Colenbrander B, Wensing CJG (1984) Sertoli cell development of pig testes in the fetal and neonatal period. Biol Reprod 31:565-577 Zibrin M (1972) Multivesicular nucleus body with nucleolar activity in Sertoli cells of bulls. An ultrastructural study. Z Zellforsch 135:155-164 Accepted May 13, 1986