Cellular Interrelationships in the Fetal Rabbit Testis
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B. GONDOSand CAROLYN J. CONNELL The types and patterns of arrangement of membrane interrelationships in the fetal rabbit testis were studied, using coordinated freeze-fracture, lanthanum tracer and standard transmission electron microscopic techniques. The testicular cords contain a mixture of large spherical germ cells and oval to elongated Sertoli cells. Desmosomes are present between germ cells and Sertoli cells and between adjacent Sertoli cells. Tight junctional specializations of the type found between Sertoli cells in the adult are not seen. In freeze-fracture replicas, short strands of particles of two different size populations are found on the surfaces of Sertoli cells. Similar short strands of particles are present on the membranes of peritubular myoid cells. In lanthanum preparations, the tracer passes freely between pentubular cells and through intercellular spaces within the seminiferous cords, indicating lack of a barrier to its passage throughout the testicular tissue. Prominent macular gap junctions similar to those found between adult Leydig cells are readily observed on the surfaces of fetal Leydig cells. The presence of gap junctions in the fetal testis may be important in coordinating the active testosterone production which occurs during this period of development. Key Words: fetal testis; freeze-fracture; Sertoli cells; peritubular cells; Leydig cells; gap junctions.
INTRODUCTION
Studies on the fine structure of the fetal rabbit testis have indicated a close interrelationship of germ cells and Sertoli cells within the developing seminiferous cords and close aggregation of groups of Leydig cells in the testicular interstitium [6,7,20]. However, the precise types of membrane interrelationships present have not been demonstrated. With the use of freeze-fracture and lanthanum tracer techniques, it is possible to analyze the special characteristics of intercellular relationships. These techniques have recently been applied in the study of the adult and prepubertal mammalian testis I S , 11,19,24,26], but no such study of the fetal testis has been reported. The present investigation was undertaken to determine the types and patterns of arrangement of intercellular relationships present in the fetal rabbit testis, using coordinated freezefracture, lanthanum tracer and standard transmission electron microscopic techniques. MATERIALS AND METHODS Testicular specimens were obtained from 26 New Zealand white rabbit fetuses at 18, 19, 22, 23, 25, 28 and 29 days gestation. Specimens were fixed either by immersion or by intracardiac Received July 1977. From the Department of Pathology and Reproductive Endocrinology Center, University of California. San Francisco. Reprint requests to Department of Pathology, University of California, San Francisco, CA 94143 (Bernard Gondos MD).
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perfusion 111. Tissues for standard electron microscopy were fixed in a mixture of 0.5% paraformaldehyde. 2.5% glutaraldehyde and picric acid at pH 7.4 for 30 minutes, then minced into small fragments and fixed for two hours, and transferred to 2% OsO, in 0.1M cacodylate buffer for two hours. Specimens were embedded in Araldite and sectioned with a Porter Blum MT2-B ultramicrotome. After staining with uranyl acetate and lead citrate, sections were examined with a Philips 300 electron microscope. For the lanthanum experiments, specimens were treated with 2% lanthanum hydroxide in 5% glutaraldehyde in 0.1M cacodylate buffer at pH 7.4 for 30 minutes. Sections were viewed stained and unstained. Tissues for freeze-fracturing were fixed in O.S% paraformaldehyde and 5% glutaraldehyde in 0. IM cacodylate buffer, then immersed in 20% glycerol for two to eight hours prior to freezing in Freon 22 and liquid nitrogen. The tissue was fractured in a Balzer's 360M unit at - 115°C. Carbon-platinum replicas were cleaned in Purex, rinsed in water and mounted on copper grids for viewing with a Philips 300 electron microscope.
OBSERVATIONS
The fetal testis of the rabbit consists primarily of seminiferous cords and interstitial tissue. The cords contain a mixture of large spherical germ cells and oval to elongated Sertoli cells, and are surrounded by a basal lamina and a layer of peritubular cells. The interstitial tissue includes blood and lymphatic vessels, mesenchymal cells and Leydig cells. Seminiferous Cords
In sections prepared for standard electron microscopy, a close association is evident between germ cells and Sertoli cells, and between adjacent Sertoli cells. Cell membranes of neighboring cells are closely apposed throughout the cords. In peripheral areas, the Sertoli cells are arranged in a columnar configuration with their long axis perpendicular to the basal lamina (Fig. 1). The germ cells are randomly distributed within the cords. The junctions between germ cells and Sertoli cells are characterized by the presence of multiple desmosome-like structures. Similar structures also occur between adjacent Sertoli cells (Fig. 2a). N o Sertoli-Sertoli tight junctional specializations of the type found in the adult are seen. In freeze-fracture replicas, short strands of particles are found on the surfaces of Sertoli cells (Fig. 2b). O n the P-face, the strands appear as ridges of membraneassociated particles of two different size populations measuring 8-9 and 13-15 nm in diameter (Fig. 3a). The strands occasionally show branching and some degree of organization (Fig. 3b), but extensive anastomosis is not seen. In general, the rows of particles extend for only short distances. On the E-face of Sertoli cells, arrays of indentation are seen corresponding in configuration and extent to the ridges on the Pface, presumably representing complementary areas of attachment. In lanthanum preparations. the tracer permeates freely into the testicular cords and through the intercellular spaces within the cords (Fig. 4), indicating the absence of a barrier to its passage throughout the testicular tissue. Peritubular Cells
Surrounding the seminiferous cords is a single layer of elongated peritubular cells wrapped around the basal lamina (Fig. 5 ) . The cytoplasm of some of these cells
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contains myofilaments, indicating that differentiation of peritubular myoid cells has already begun in the fetus. There is frequent overlapping of the cytoplasmic extensions of adjacent peritubular cells; in the areas of overlapping, desmosomes are frequently seen. In freeze-fracture replicas, the peritubular cell surface is characterized by the presence of numerous pinocytotic vesicles (Fig. 6). Strands of particles similar to those described for Sertoli cells are present in the membranes of peritubular cells. Grooves and ridges occur in irregular linear arrays and extend for relatively short distances without evidence of extensive anastomosis. In lanthanum preparations, the tracer passes freely through the intercellular spaces between peritubular cells. Interstitial Tissue
The interstitial tissue of the rabbit fetal testis contains numerous Leydig cells during the second half of gestation. These cells have the characteristic nuclear and cyto-
FIGURE 1. Transmission electron micrograph of periphery of seminiferous cord showing Sertoli cell (SC) and germ cell (GC). ( X 9,095).
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plasmic features of steroid-secreting cells. They are commonly arranged in groups with closely apposed cell membranes (Fig. 7). Pinocytotic vesicles are frequently observed as are areas of narrowing of the intercellular space characteristic of gap junctions (Fig. 8a). Desmosome-like structures are also seen. In freeze-fracture replicas, prominent macular gap junctions are readily observed on the surfaces of Leydig cells (Fig. 8b). They range in size from 0.4 to 1.2 p m . On the Pface, the junctions are round to oval, with sharp demarcation from surrounding areas where membrane-associated particles are scattered. The particles comprising junctions are about 8-9 nm in diameter with a center to center distance of 10 nm. A
FIGURE 2. Top, Junction between Serotoli cells. Arrow indicates desmosomal attachment. Bottom, Freeze-fracture replica showing Sertoli cell membrane with linear strands of particles (arrows). (Top. x 16,660; Bottom, x 13,600).
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hexagonal pattern of the particles can occasionally be seen (Fig. 8c). On the E-face, pits of comparable dimension and arrangement to those described for the P-face particles can be observed. In areas where well-formed junctions are not present, irregular aggregates of small numbers of particles of similar dimension may be found (Fig. 9). These possibly represent forming gap junctions. In lanthanum preparations, there is free passage of the tracer between adjacent Leydig cells, except for partial restriction in the regions of gap junctions. DISCUSSION In previous studies of cell association within the seminiferous tubules, occluding junctions, which exclude the passage of electron-dense tracers such as lanthanum, have been found between Sertoli cells in the adult [9, 10, 13-15, 19, 24, 25, 27, 301 but not in the early prepubertal testis [8, 17, 19, 25, 261. These junctions are generally considered to constitute the morphologic basis for the blood-testis barrier. The present observations indicate that occlusive junctions between Sertoli cells are absent in the rabbit fetal testis and that there is free passage of lanthanum through the fetal seminiferous cords. Linear strands of particles resembling incomplete tight junctions were observed on the surface of fetal Sertoli cells. Similar arrays have been described in the newborn mouse [25] and rat [18], where it has been suggested that they represent initial stages
FIGURE 3. Left, Sertoli cell membranes with short rows of particles arranged in ridges on Pface (above) and grooves on E-face (below). Right, Branching network of strands of particles, Sertoli cell membrane. ( x 30,000).
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in the formation of occlusive junctions. The structures found in the rerll .abbit testis probably do not represent forming occlusive junctions, since occluding junctions first become evident more than two months after birth in the rabbit (Connell and Gondos, unpublished observations, 1976). While a definite function for the incomplete junctions remains to be established, one possibility is that they are related to movement of cells within the cords. Similar intermittent beaded rows of particles have been observed during amphibian neurulation and were considered to repre: dissolving tight junctions associated with cellular reorganization [ 12, 16,281. The ,pment of the testicular cords of the rabbit fetus involves movement of germ ce, i e tubular .eriphery periphery and realignment of Sertoli cells in a columnar configuration [20]. These changes may require reorganization of membrane a t t a c h m .A* uf Sertoli cells and may involve periodic formation and dissolution of intercellular junctions. Although peritubular myoid cells are closely aligned and often overlapping, the boundary tissue in the fetal rabbit testis does not present a barrier to the passage of lanthanum. The peritubular cells do exhibit linear arrays of particles on their
FIGURE 4. Lanthanum preparation indicating free passage of tracer through interc spaces of seminiferous cord. ( x 4,620).
'?r
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Cellular Interrelationships in the Fetal Rabbit Testis
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FIGURE 5. Section through periphery of seminiferous cord and boundary tissue showing overlapping elongated peritubular cells. ( x 7,395).
FIGURE 6. Left, Freeze-fracture replica of peritubular cell membrane showing strands of particles and pinocytotic vesicles (pv) on external face. Right, Branching strands of particles, peritubular cell membrane. Numerous pinocytotic vesicles are present. (Left, X 38,400; Right, X 58,240).
B. Gondos and C. J. Connell
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FIGURE 7. Pair of Leydig cells with closely apposed cell membranes. Arrow indicates pinocytotic vesicle (shown at higher magnification in inset). ( x 8,700; Inset, X 24,000.)
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Cellular interrelationships in the Fetal Rabbit Testis
FIGURE 8. Top, Gap junction (long arrow) and desmosomal attachment (short arrow) between adjacent Leydig cells. ( X 38,400.) Lower left, Freeze-fracture replica of Leydig cell membrane showing sharply defined macular gap junction. Surrounding membrane includes scattered individual particles. ( X 72,800.) Lower right, Leydig cell gap junction with particles on cytoplasmic face (P) and pits on external face (E). Note hexagonal pattern on E-face at lower right. ( x 72,800.)
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membrane surfaces similar to those found in Sertoli cells. The function of peritubular cells in the developing testis remains to be determined. The presence of well-formed gap junctions between adjacent Leydig cells in the fetus is of particular interest. Although the precise function of gap junctions remains to be established, it is generally felt that they are involved in cellular communication and coordination, allowing passage of ions and metabolites between adjacent cells [ 18,22,29]. Such junctions have been especially noted in steroid hormone producing tissues in the adrenal 1161. ovary [2-4,231 and testis [11,24]. The presence of specialized junctions assumed to facilitate intercellular communication may provide a morphologic basis for the coordination of synthetic and secretory activity within cellular aggregates. Previous studies in the rabbit have shown that fetal Leydig cells have ultrastructural features characteristic of steroid-secreting cells [6,7]. Moreover, active testosterone synthesis occurs during this period [7,21,3 13. Our observations indicate that gap junctions between fetal Leydig cells resemble those in the adult testis. Also evident in the fetal testis are structures resembling forming gap junctions correlating with the period when Leydig cell aggregation takes place. The significance of such structures
FIGURE 9. Leydig cell membrane (P-face), showing several loose aggregates of particles. Boxed area is shown at higher magnification in inset. ( X 38,400; Inset, x 76,800.)
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remains to be determined. Further studies on early stages of gonadal differentiation may be useful in providing information on the assembly of gap junctions and their possible role in the initiation and regulation of steroidogenesis.
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The authors wish to thank Janet K. Boyles, Clifford E. Lai and May F. McKoon for their excellent technical assistance. The research was supported by U.S.P.H.S. grant HD-08940 and a grant from the Rockefeller Foundation.
REFERENCES
1 . Abrunhosa, R. (1972): Microperfusion fixation of embryos for ultrastructural studies. J Ultrastruct Res 41, 176-188. 2. Albertini, D. F. and Anderson, E. (1974): The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. J Cell Biol 63, 234-250. 3. Albertini, D. F. and Anderson, E. (1975): Structural modifications of lutein cell gap junctions during pregnancy in the rat and the mouse. Anat Rec 181, 171- 194. 4. Albertini, D. F., Fawcett, D. W. and Olds, P. J. (1975): Morphological variations in gap junctions of ovarian granulosa cells. Tiss Cell 7, 380-405. 5. Bigliardi, E. and Vegni Talluri, M. (1976): Ultrastructural details of Sertoli cell junctional complexes in vivo and their modifications in tissue culture. Cell Tim Res 172, 29-38. 6. Bjerregaard, P., Bro-Rasmussen, F. and Reumert, T. (1974): Ultrastructural development of fetal rabbit testis. Z Zellforsch 147, 401-413. 7. Catt, K. J., Dufau, M. L., Neaves, W. B., Walsh, P. C. and Wilson, J. D. (1975): LH-hCG receptors and testosterone content during differentiation of the testis in the rabbit embryo. Endocrinol 97, 11571165. 8. Connell, C. J. (1976): A freeze-fracture and lanthanum tracer study of the development of the junction between Sertoli cells of the prepubertal dog. J Cell Biol 70, 80a. 9. Connell, C. J. (1977): The effect of hCG on pinocytosis within the canine inter-
Sertoli cell tight junction. A preliminary report. Amer J Anat 148, 149-153. 10. Connell, C. J . (1977): A freeze-fracture and lanthanum tracer study of the junction between Sertoli cells of the canine testis. J Cell Biol in press. 1 1 . Connell, C. J. and Connell, G. M. (1977): The interstitial tissue. In: The Testis, A. D. Johnson and W. R. Gomes, eds. Academic Press, New York, Vol. IV, pp. 333-369. 12. Decker, R. S. and Friend, D. S. (1974): Assembly of gap junctions during amphibian neurulation. J Cell Biol 62, 32-47. 13. Dym, M. and Fawcett, D. W. (1970): The blood-testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol Reprod 3, 308-326. 14. Fawcett, D. W. (1973): Interrelations of cell types within the seminiferous epithelium and their implications for control of spermatogenesis. In: The Regulation of Mammalina Reproduction, S. J. Segal, R. Crozier, P. A. Corfman and P. G. Condliffe, eds. Charles C. Thomas, Springfield, Ill., pp. 116-135. 15. Fawcett, D. W., Leak, L. V. and Hediger, P. M. (1970): Electron microscopic observations on the structural components of the blood-testis barrier. J Reprod Fertil Suppl 10, 105- 122. 16. Friend, D. S. and Gilula, N. B. (1972): Variations in tight and gap junctions in mammalian tissues. J Cell B i d 53, 758776. 17. Gilula, N. B. (1973): Development of cell junctions. Amer Zoo1 13, 1109- 11 17. 18. Gilula, N. B. (1974): Junctions between cells. In: Cell Communication, R. P. Cox, ed. Wiley and Sons, New York, pp. 1-29.
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19. Cilula. N. B., Fawcett, D. W.. and Aoki. A. (1976): The Sertoli cell occluding junctions and gap junctions in mature and developing mammalian testis. DCIYI B i d 50, 142-168. 20. Condos. B. and Conner. L. A. (1973): Ultrastructure of developing germ cells in the fetal rabbit testis. Ainpr J Aiiat 136, 23-42. 21. Lipsett. M. B. and Tullner, W . W. (1965): Testosterone synthesis by the fetal rabbit gonad. Eiidoc riiiol 77, 273-277. 22. McNutt, N . S. and Weinstein. R. S. (1973): Membrane ultrastructure at mammalian intercellular junctions. Prog Biophqs Molrc Biol 26, 45- 101. 23. Merk. F. B. and McNutt, N . S. (1972): Nexus junctions between dividing and interphase cells of the rat ovary. J Ccll Biol
complexes of the mouse Sertoli cells as revealed by freeze-fracture. Anat R w 185, 403-418. 26. Nagano. T.. Suzuki. F., Kitarnura, Y., and Matsumoto, K . (1977): Sertoli cell junctions in the germ cell-free testis of the congenic mouse. Lab f n w s t 36, 8-17. 27. Neaves, W . B. (1973): Permeability of Sertoli cell tight junctions to lanthanum after ligation of ductus deferens and ductuli efferentes. J Ccll Biol 59, 559-572. 2 8 . Revel, J . P. and Brown, S. S. (1976): Cell junctions in development with particular reference to the neural tube. Cold Spring Harbor Symp Qiiarit Biol 40, 443455. 29. Staehelin, L. A. (1974): Structure and function of intercellular junctions. f n t Rciq C y t ~ 39, l 191-284. 30. Vitale. R., Fawcett. D. W., and Dym, M. 55, 51 1-515. (1973): The normal development of the 24. Nagano, T. and Suzuki. F. (1976): Freezeblood-testis barrier and the effects of fracture observations on the intercellular clomiphene and estrogen treatment. Anat junctions of Sertoli cells and Leydig cells 176, 333-344. in the human testis. C ' d l Tiss R1.s 166, 37- 31. Wilson, J . D. and Siiteri, P. K. (1973): 48. Developmental pattern of testosterone 25. Nagano. T. and Suzuki. F. (1976): The synthesis in the fetal gonad of the rabbit. postnatal development of the junctional Eizdoc~irtol92, 1182- 1 191.
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B. GONDOSand CAROLYN J. CONNELL The types and patterns of arrangement of membrane interrelationships in the fetal rabbit testis were studied, using coordinated freeze-fracture, lanthanum tracer and standard transmission electron microscopic techniques. The testicular cords contain a mixture of large spherical germ cells and oval to elongated Sertoli cells. Desmosomes are present between germ cells and Sertoli cells and between adjacent Sertoli cells. Tight junctional specializations of the type found between Sertoli cells in the adult are not seen. In freeze-fracture replicas, short strands of particles of two different size populations are found on the surfaces of Sertoli cells. Similar short strands of particles are present on the membranes of peritubular myoid cells. In lanthanum preparations, the tracer passes freely between pentubular cells and through intercellular spaces within the seminiferous cords, indicating lack of a barrier to its passage throughout the testicular tissue. Prominent macular gap junctions similar to those found between adult Leydig cells are readily observed on the surfaces of fetal Leydig cells. The presence of gap junctions in the fetal testis may be important in coordinating the active testosterone production which occurs during this period of development. Key Words: fetal testis; freeze-fracture; Sertoli cells; peritubular cells; Leydig cells; gap junctions.
INTRODUCTION
Studies on the fine structure of the fetal rabbit testis have indicated a close interrelationship of germ cells and Sertoli cells within the developing seminiferous cords and close aggregation of groups of Leydig cells in the testicular interstitium [6,7,20]. However, the precise types of membrane interrelationships present have not been demonstrated. With the use of freeze-fracture and lanthanum tracer techniques, it is possible to analyze the special characteristics of intercellular relationships. These techniques have recently been applied in the study of the adult and prepubertal mammalian testis I S , 11,19,24,26], but no such study of the fetal testis has been reported. The present investigation was undertaken to determine the types and patterns of arrangement of intercellular relationships present in the fetal rabbit testis, using coordinated freezefracture, lanthanum tracer and standard transmission electron microscopic techniques. MATERIALS AND METHODS Testicular specimens were obtained from 26 New Zealand white rabbit fetuses at 18, 19, 22, 23, 25, 28 and 29 days gestation. Specimens were fixed either by immersion or by intracardiac Received July 1977. From the Department of Pathology and Reproductive Endocrinology Center, University of California. San Francisco. Reprint requests to Department of Pathology, University of California, San Francisco, CA 94143 (Bernard Gondos MD).
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perfusion 111. Tissues for standard electron microscopy were fixed in a mixture of 0.5% paraformaldehyde. 2.5% glutaraldehyde and picric acid at pH 7.4 for 30 minutes, then minced into small fragments and fixed for two hours, and transferred to 2% OsO, in 0.1M cacodylate buffer for two hours. Specimens were embedded in Araldite and sectioned with a Porter Blum MT2-B ultramicrotome. After staining with uranyl acetate and lead citrate, sections were examined with a Philips 300 electron microscope. For the lanthanum experiments, specimens were treated with 2% lanthanum hydroxide in 5% glutaraldehyde in 0.1M cacodylate buffer at pH 7.4 for 30 minutes. Sections were viewed stained and unstained. Tissues for freeze-fracturing were fixed in O.S% paraformaldehyde and 5% glutaraldehyde in 0. IM cacodylate buffer, then immersed in 20% glycerol for two to eight hours prior to freezing in Freon 22 and liquid nitrogen. The tissue was fractured in a Balzer's 360M unit at - 115°C. Carbon-platinum replicas were cleaned in Purex, rinsed in water and mounted on copper grids for viewing with a Philips 300 electron microscope.
OBSERVATIONS
The fetal testis of the rabbit consists primarily of seminiferous cords and interstitial tissue. The cords contain a mixture of large spherical germ cells and oval to elongated Sertoli cells, and are surrounded by a basal lamina and a layer of peritubular cells. The interstitial tissue includes blood and lymphatic vessels, mesenchymal cells and Leydig cells. Seminiferous Cords
In sections prepared for standard electron microscopy, a close association is evident between germ cells and Sertoli cells, and between adjacent Sertoli cells. Cell membranes of neighboring cells are closely apposed throughout the cords. In peripheral areas, the Sertoli cells are arranged in a columnar configuration with their long axis perpendicular to the basal lamina (Fig. 1). The germ cells are randomly distributed within the cords. The junctions between germ cells and Sertoli cells are characterized by the presence of multiple desmosome-like structures. Similar structures also occur between adjacent Sertoli cells (Fig. 2a). N o Sertoli-Sertoli tight junctional specializations of the type found in the adult are seen. In freeze-fracture replicas, short strands of particles are found on the surfaces of Sertoli cells (Fig. 2b). O n the P-face, the strands appear as ridges of membraneassociated particles of two different size populations measuring 8-9 and 13-15 nm in diameter (Fig. 3a). The strands occasionally show branching and some degree of organization (Fig. 3b), but extensive anastomosis is not seen. In general, the rows of particles extend for only short distances. On the E-face of Sertoli cells, arrays of indentation are seen corresponding in configuration and extent to the ridges on the Pface, presumably representing complementary areas of attachment. In lanthanum preparations. the tracer permeates freely into the testicular cords and through the intercellular spaces within the cords (Fig. 4), indicating the absence of a barrier to its passage throughout the testicular tissue. Peritubular Cells
Surrounding the seminiferous cords is a single layer of elongated peritubular cells wrapped around the basal lamina (Fig. 5 ) . The cytoplasm of some of these cells
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contains myofilaments, indicating that differentiation of peritubular myoid cells has already begun in the fetus. There is frequent overlapping of the cytoplasmic extensions of adjacent peritubular cells; in the areas of overlapping, desmosomes are frequently seen. In freeze-fracture replicas, the peritubular cell surface is characterized by the presence of numerous pinocytotic vesicles (Fig. 6). Strands of particles similar to those described for Sertoli cells are present in the membranes of peritubular cells. Grooves and ridges occur in irregular linear arrays and extend for relatively short distances without evidence of extensive anastomosis. In lanthanum preparations, the tracer passes freely through the intercellular spaces between peritubular cells. Interstitial Tissue
The interstitial tissue of the rabbit fetal testis contains numerous Leydig cells during the second half of gestation. These cells have the characteristic nuclear and cyto-
FIGURE 1. Transmission electron micrograph of periphery of seminiferous cord showing Sertoli cell (SC) and germ cell (GC). ( X 9,095).
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plasmic features of steroid-secreting cells. They are commonly arranged in groups with closely apposed cell membranes (Fig. 7). Pinocytotic vesicles are frequently observed as are areas of narrowing of the intercellular space characteristic of gap junctions (Fig. 8a). Desmosome-like structures are also seen. In freeze-fracture replicas, prominent macular gap junctions are readily observed on the surfaces of Leydig cells (Fig. 8b). They range in size from 0.4 to 1.2 p m . On the Pface, the junctions are round to oval, with sharp demarcation from surrounding areas where membrane-associated particles are scattered. The particles comprising junctions are about 8-9 nm in diameter with a center to center distance of 10 nm. A
FIGURE 2. Top, Junction between Serotoli cells. Arrow indicates desmosomal attachment. Bottom, Freeze-fracture replica showing Sertoli cell membrane with linear strands of particles (arrows). (Top. x 16,660; Bottom, x 13,600).
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hexagonal pattern of the particles can occasionally be seen (Fig. 8c). On the E-face, pits of comparable dimension and arrangement to those described for the P-face particles can be observed. In areas where well-formed junctions are not present, irregular aggregates of small numbers of particles of similar dimension may be found (Fig. 9). These possibly represent forming gap junctions. In lanthanum preparations, there is free passage of the tracer between adjacent Leydig cells, except for partial restriction in the regions of gap junctions. DISCUSSION In previous studies of cell association within the seminiferous tubules, occluding junctions, which exclude the passage of electron-dense tracers such as lanthanum, have been found between Sertoli cells in the adult [9, 10, 13-15, 19, 24, 25, 27, 301 but not in the early prepubertal testis [8, 17, 19, 25, 261. These junctions are generally considered to constitute the morphologic basis for the blood-testis barrier. The present observations indicate that occlusive junctions between Sertoli cells are absent in the rabbit fetal testis and that there is free passage of lanthanum through the fetal seminiferous cords. Linear strands of particles resembling incomplete tight junctions were observed on the surface of fetal Sertoli cells. Similar arrays have been described in the newborn mouse [25] and rat [18], where it has been suggested that they represent initial stages
FIGURE 3. Left, Sertoli cell membranes with short rows of particles arranged in ridges on Pface (above) and grooves on E-face (below). Right, Branching network of strands of particles, Sertoli cell membrane. ( x 30,000).
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in the formation of occlusive junctions. The structures found in the rerll .abbit testis probably do not represent forming occlusive junctions, since occluding junctions first become evident more than two months after birth in the rabbit (Connell and Gondos, unpublished observations, 1976). While a definite function for the incomplete junctions remains to be established, one possibility is that they are related to movement of cells within the cords. Similar intermittent beaded rows of particles have been observed during amphibian neurulation and were considered to repre: dissolving tight junctions associated with cellular reorganization [ 12, 16,281. The ,pment of the testicular cords of the rabbit fetus involves movement of germ ce, i e tubular .eriphery periphery and realignment of Sertoli cells in a columnar configuration [20]. These changes may require reorganization of membrane a t t a c h m .A* uf Sertoli cells and may involve periodic formation and dissolution of intercellular junctions. Although peritubular myoid cells are closely aligned and often overlapping, the boundary tissue in the fetal rabbit testis does not present a barrier to the passage of lanthanum. The peritubular cells do exhibit linear arrays of particles on their
FIGURE 4. Lanthanum preparation indicating free passage of tracer through interc spaces of seminiferous cord. ( x 4,620).
'?r
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FIGURE 5. Section through periphery of seminiferous cord and boundary tissue showing overlapping elongated peritubular cells. ( x 7,395).
FIGURE 6. Left, Freeze-fracture replica of peritubular cell membrane showing strands of particles and pinocytotic vesicles (pv) on external face. Right, Branching strands of particles, peritubular cell membrane. Numerous pinocytotic vesicles are present. (Left, X 38,400; Right, X 58,240).
B. Gondos and C. J. Connell
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FIGURE 7. Pair of Leydig cells with closely apposed cell membranes. Arrow indicates pinocytotic vesicle (shown at higher magnification in inset). ( x 8,700; Inset, X 24,000.)
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Cellular interrelationships in the Fetal Rabbit Testis
FIGURE 8. Top, Gap junction (long arrow) and desmosomal attachment (short arrow) between adjacent Leydig cells. ( X 38,400.) Lower left, Freeze-fracture replica of Leydig cell membrane showing sharply defined macular gap junction. Surrounding membrane includes scattered individual particles. ( X 72,800.) Lower right, Leydig cell gap junction with particles on cytoplasmic face (P) and pits on external face (E). Note hexagonal pattern on E-face at lower right. ( x 72,800.)
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6. Gondos and C. J. Connell
membrane surfaces similar to those found in Sertoli cells. The function of peritubular cells in the developing testis remains to be determined. The presence of well-formed gap junctions between adjacent Leydig cells in the fetus is of particular interest. Although the precise function of gap junctions remains to be established, it is generally felt that they are involved in cellular communication and coordination, allowing passage of ions and metabolites between adjacent cells [ 18,22,29]. Such junctions have been especially noted in steroid hormone producing tissues in the adrenal 1161. ovary [2-4,231 and testis [11,24]. The presence of specialized junctions assumed to facilitate intercellular communication may provide a morphologic basis for the coordination of synthetic and secretory activity within cellular aggregates. Previous studies in the rabbit have shown that fetal Leydig cells have ultrastructural features characteristic of steroid-secreting cells [6,7]. Moreover, active testosterone synthesis occurs during this period [7,21,3 13. Our observations indicate that gap junctions between fetal Leydig cells resemble those in the adult testis. Also evident in the fetal testis are structures resembling forming gap junctions correlating with the period when Leydig cell aggregation takes place. The significance of such structures
FIGURE 9. Leydig cell membrane (P-face), showing several loose aggregates of particles. Boxed area is shown at higher magnification in inset. ( X 38,400; Inset, x 76,800.)
Cellular Interrelationships in the Fetal Rabbit Testis
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remains to be determined. Further studies on early stages of gonadal differentiation may be useful in providing information on the assembly of gap junctions and their possible role in the initiation and regulation of steroidogenesis.
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The authors wish to thank Janet K. Boyles, Clifford E. Lai and May F. McKoon for their excellent technical assistance. The research was supported by U.S.P.H.S. grant HD-08940 and a grant from the Rockefeller Foundation.
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