Adjacent Sertoli Cells Research Articles

Identification and developmental regulation of cadherin messenger ribonucleic acids in the rat testis.

Cellular interactions in the rat testis are suggested by the presence of gap junctions between developing germ cells and Sertoli cells as well as tight junctions between adjacent Sertoli cells. Cadherins are cell surface proteins that mediate calcium-dependent intercellular adhesion. In these experiments the presence and developmental regulation of three cadherins have been examined: epithelial cadherin (E-Cad), neural cadherin (N-Cad), and placental cadherin (P-Cad). Northern blot analysis of testicular RNA indicates the presence of N-Cad [4.3 and 3.5 kilobases (kb)] and P-Cad (3.5 kb) transcripts. No E-Cad message was detected. To determine whether mRNA concentrations for P-Cad and N-Cad are regulated during postnatal rat testicular development, testes from rats ranging in age from 7-91 days were subjected to Northern blot analysis. Relative P-Cad mRNA levels were highest at 7 days of age and decreased to almost half of these levels by day 14. P-Cad mRNA levels subsequently decreased to low levels and remained constant thereafter. This contrasted with the developmental pattern observed for the 4.3-kb N-Cad transcript, which was low early in testicular development but increased to peak levels on day 42, coincident with the shedding of the first sperm. N-Cad mRNA concentrations decreased from 42 to 56 days and then remained constant until 91 days. While mouse P-Cad antibody did not cross-react with rat P-Cad, immunoblots of testicular membrane protein preparations identified the presence of immunoreactive N-Cad protein in the testis. The presence of N-Cad protein confirms that N-Cad mRNA is translated in this tissue. The developmental patterns of P-Cad and N-Cad mRNA suggest a role for P-Cad early in testicular development, while N-Cad appears to play a role in later stages of spermatogenesis.

Junctional complexes between the Sertoli cells in the testis of the crayfish, Procambarus paeninsulanus

The testis of the crayfish, Procambarus paeninsulanus, was prepared for light and electron microscopic study. It is composed of tubules containing germ-spermatogenic and somatic-Sertoli cells. In sections of tubules lacking sperm, the Sertoli cells rest on the basement membrane. A desmosome-like junction is found near the luminal surface between two adjacent Sertoli cells. It is closely associated with a long, septate junction. Between Sertoli cells which have surrounded numerous spermatids, the undulating membranes exhibit profiles of pleated septate junctions in tangential sections. The morphology of the pleated septate junctions between adjacent Sertoli cells suggests a possible role as a permeability barrier.

Dual compartment (bicameral) culture: role of basement membrane in epithelial differentiation.

A number of years ago we reported that tight junctions between adjacent Sertoli cells subdivide the seminiferous epithelium into two compartments, basal and adluminal, thus forming the morphological basis of the blood-testis barrier. It is now generally believed that the special milieu created by the Sertoli cells in the adluminal compartment is essential for germ cell differentiation. In order to duplicate the compartmentalization that occurs in vivo, Sertoli cells were cultured in bicameral chambers on Millipore filters impregnated with a reconstituted basement membrane. Confluent monolayers of these cells were tall columnar (40-60 microns in height) and highly polarized. These Sertoli cell monolayers established electrical resistance that peaked when the Sertoli-Sertoli tight junctions developed in culture. In addition, the monolayers formed a permeability barrier to 3H-inulin and lanthanum nitrate. The bicameral chambers were utilized in a number of studies on protein secretion, and it was revealed that numerous proteins are secreted in a polarized manner. In another study, hormone-stimulated aromatase activity was measured in Sertoli cells grown on plastic culture dishes, plastic dishes coated with laminin or Matrigel, and in the bicameral chambers. Cell culture on basement membrane substrates decreased the FSH-dependent estrogen production. No estrogen production was observed when the Sertoli cells were cultured in the bicameral chambers. These results are in accord with the hypothesis that differentiated Sertoli cells lose their ability to metabolize androgen to estrogen in an hormone-dependent manner, whereas undifferentiated cells in culture, or in vivo, have a very active FSH-dependent aromatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Laminin ultrastructural immunolocalization in rat testis during ontogenesis

The synthesis of one of the main glycoproteins of the basement membrane, the laminin, was demonstrated by ultrastructural immunolocalization during rat foetal (16th day to 20th day of gestation) and postnatal development of the testis. The lamina densa, part of seminiferous tubular basement membrane, is labeled uniformly at all studied stages. The lamina lucida is not well defined before the postnatal stages, at which times discrete immunostaining extends from the lamina densa to the adjacent seminiferous epithelial cells (spermatogonia and Sertoli cells). The extracellular matrix around the peritubular cells is not labeled before birth. Intracellular immunostaining was detected as early as the 16th day of gestation in both Sertoli cells and cells around the seminiferous tubules which will transform later into peritubular cells. It was located in rough endoplasmic reticulum (RER) cisternae and secretory vesicles. After 18-20 days of postnatal life, the immunostaining faints progressively. Some positive material is seen in the RER of the gonocytes at all studied stages. Sertoli cells and peritubular cells are the main producing cells of laminin after the 16th of gestation. The laminin secreted by gonocytes may play an important role in adhesion of gonocytes to the lamina densa and adjacent Sertoli cells before their transition from basal compartment to adluminal compartment.

Deep-etch visualization of the Sertoli cell (blood-testis) barrier in the boar

The Sertoli cell (blood-testis) barrier in the boar was visualized by the freeze-fracture, deep-etch, rotary-replication technique. Three kinds of cross-bridging structures were clearly recognized in the following three ectoplasmic specialization (ES) regions: (1) cross-bridges in the intercellular space between adjacent Sertoli cell membranes; (2) cross-bridges in the space between the Sertoli cell membrane and microrilament bundles; and (3) cross-bridges in the space between microfilament bundles and subsurface cisternae. Results from immunolocalization, vinculin and α-actinin were recognized in the Sertoli cell barrier. Our findings show that these structural elements of the Sertoli cell barrier are held together by these cross-bridging structures, and provide important morphological evidence that implicates the ES in the dynamic function of the microfilament bundles of the Sertoli cell barrier.

Functional Coupling of Neonatal Rat Sertoli Cells and Gonocytes in Coculture*

Interaction between Sertoli cells and germ cells is likely to be critical for normal development of the testis. We have established and characterized cocultures of neonatal Sertoli cells and gonocytes and have begun to study the physical and functional relationship between these cells in vitro. Cells were isolated from rat pups by sequential enzymatic treatment and cultured in serum-free medium. When plated on Matrigel, Sertoli cells rapidly attach, and gonocytes adhere to the underlying Sertoli cells shortly thereafter. We observed that some of these germ cells develop cytoplasmic processes and elongate during the first day of culture, essentially mimicking their behavior in vivo. Electron microscopic examination of typical cultures revealed the presence of desmosome-like adhesion sites and apparent gap junctions between Sertoli cells and gonocytes. To determine whether Sertoli cells and gonocytes are functionally coupled in the cocultures, we used the glass bead-loading technique of McNeil and Warder to introduce Lucifer yellow (LY), a gap junction-permeant probe, and Rhodamine-dextran (RD), a larger marker excluded by gap junctions, simultaneously into cultures 24 h after plating. Immediate fixation and viewing of cultures with fluorescence microscopy indicated that all bead-loaded cells received both probes. We studied other living cultures 10 min after bead-loading and located RD-negative (i.e. nonbead-loaded) gonocytes that were in obvious contact with RD- and LY-positive bead-loaded Sertoli cells; these gonocytes were scored for the presence or absence of cytoplasmic LY. This analysis revealed that many gonocytes were able to obtain LY from adjacent Sertoli cells, presumably via gap junctions maintained with these cells. In addition, we quantified the percentage of gonocytes that elongated with increasing time in vitro and correlated the morphology of these cells with their ability to acquire LY from adjacent Sertoli cells. Our findings indicate that although the absolute numbers of gonocytes present decreases, more of those remaining elongate as time in vitro increases. We can also conclude from our data that gonocytes with and without processes are equally likely to be coupled with Sertoli cells under these conditions. These observations provide the first demonstration of functional coupling between Sertoli cells and premeiotic germ cells. Together with our morphological observations, they suggest that gap junction-mediated communication between these cells may be involved in stimulating or regulating changes in the gonocyte population during postnatal development of the testis.

The blood-testis barrier and its breakdown following immunization to testis material in the Nile tilapia, Oreochromis niloticus

The blood-testis barrier and its changes following immunization to testis material, were investigated by light- and electron microscopy in a teleost fish, the Nile tilapia Oreochromis niloticus, using horseradish peroxidase and bovine serum albumin as tracers. In the normal testis, histochemistry using horseradish peroxidase revealed that a barrier composed of junctional complexes connecting adjacent Sertoli cells existed around the central lumina of the seminal lobules, and also around the germ-cell cysts containing spermatids at the middle or late phase of chromatin condensation. By contrast, bovine serum albumin was prevented from passing through the basement membrane and could not penetrate any of the spermatogenetic cysts, indicating that the basement membrane may be an ion-selective barrier. In tilapia immunized with allogeneic testis homogenate emulsified in Freund's complete adjuvant, bovine serum albumin could penetrate the spermatogenetic cysts, and horseradish peroxidase was able to pass through the intercellular spaces between Sertoli cells to the region nearer the seminal lobule lumen, due to the junctional complexes becoming loosened. The results suggest that the blood-testis barrier, both junctional complexes and the basement membrane, are broken down during immune responses.

Sertoli cell ectoplasmic specializations: a type of actin-associated adhesion junction?

In this paper we provide evidence that ectoplasmic specializations are a form of intercellular adhesion junction. Ectoplasmic specializations, found at basal junctions between adjacent Sertoli cells and at sites of adhesion between Sertoli cells and germ cells, consist of actin filament bundles sandwiched between the plasma membrane and a cistern of endoplasmic reticulum. The actin filaments in each bundle are unipolar and are hexagonally packed. The bundles are coupled to the adjacent membranes and to each other. Because ectoplasmic specializations are associated with junctional sites, they may play a role in intercellular adhesion. In this study, we report a procedure for obtaining samples enriched for ectoplasmic specializations and identify polypeptides that may be associated with ectoplasmic specializations. On SDS-polyacrylamide gels, an 83K (K = 10(3) Mr) polypeptide is specific to the ectoplasmic specialization-enriched sample, suggesting that it may be a component of ectoplasmic specializations. Other polypeptides at 38, 53, 56 and 69K also may be associated with ectoplasmic specializations. Immunoblots further indicate that fimbrin and vinculin are present in the ectoplasmic specialization-enriched fraction. In addition, immunofluorescence indicates that vinculin is associated with spermatid-Sertoli cell and Sertoli-Sertoli cell junctions. We suspect that fimbrin, an actin-bundling protein, may be involved in cross-linking the hexagonally packed actin filaments in ectoplasmic specializations while vinculin may be associated with actin-membrane linkages. If so, ectoplasmic specializations may be a new class of actin-associated junctional site. Moreover, the presence of vinculin in testicular fractions enriched for ectoplasmic specializations and at junctional sites supports the view that these structures may play a role in intercellular adhesion, possibly by stabilizing an adhesive membrane domain.

Pattern of compartmentation in human seminiferous tubules showing dislocation of spermatogonia.

The pattern of compartmentation of the seminiferous epithelium was investigated, using a lanthanum tracer technique, in human testicular biopsies of adult infertile men (age 27 to 44 years), where dislocation of spermatogonia from the basal lamina occurred. Spermatogonia type A and B were found in a two- or three-layered arrangement, in aberrant locations throughout the seminiferous epithelium, and in intratubular positions associated with fragments of Sertoli cell cytoplasm. Tracer impregnation was found around spermatogonia in a multilayered arrangement, indicating the extension of the basal compartment in a luminal direction. Single spermatogonia within the second or third layer of the seminiferous epithelium were regularly found to be surrounded by tracer. The junctional complex between the lateral membranes of adjacent Sertoli cells was devoid of tight junctions. Tracer penetration around spermatogonia in a more luminal position was prevented by intact Sertoli cell junctional complexes; tracer was also absent from intraluminal located spermatogonia associated with cytoplasmic fragments of Sertoli cells. The luminal extension of the basal compartment associated with the dislocation of spermatogonia clearly differs from the pattern of compartmentation during the movement of primary spermatocytes within undisturbed epithelium. There is a strong incidence of elevated serum levels of follicle-stimulating hormone (greater than 7 U/l), indicating a suppression of Sertoli cell function; this may be the cause for the dislocation of spermatogonia and the changes of compartmentation.

Reproduction in the mexican leaf frog, Pachymedusa dacnicolor. IV. Spermatogenesis: A light and ultrasonic study.

Primary spermatogonia have highly lobate nuclei and can be distinguished as pale and dark types on the basis of nuclear and cytoplasmic features. Nuclei of secondary spermatogonia are also lobate. Primary spermatocytes have spherical nuclei and display synaptinemal complexes in late zygotene-pachytene. Spermatocytes are connected by intercellular bridges, which persist through spermiogenesis. During spermiogenesis no acrosomal granule is formed. The acrosomal vesicle is large and forms in the apical part of the cell. A helical system of perinuclear microtubules accompanies the phase of nuclear elongation. Microtubules disappear in late spermatids and there forms a compact bundle of filaments which extends into the subacrosomal area. These filaments probably derive from the breakdown of the microtubules. A mitochondrial sleeve is formed around the proximal portion of the tail and much of it is cast off in the mature spermatid. The tail is composed of a spirally coiled contractile element and a stiff fibrous axial rod connected together by an undulating membrane. The axial rod and the axoneme-associated rodlet derive from a dense, juxtacentriolar fibrous mass. Sertoli cells surrounding the spermatogonial and spermatocyte cysts are slender and have oblong nuclei. In contrast, those associated with spermatids are columnar and have deeply indented nuclei. They possess many Golgi complexes, elongated mitochondria, cisternae of smooth endoplasmic reticulum, lysosome-like bodies, masses of glycogen particles, few lipid droplets, and an array of microtubules running longitudinally around the elongating spermatid nuclei. Desmosomes are formed between adjacent Sertoli cells.

Effects of cytochalasin D on the integrity of the Sertoli cell (blood-testis) barrier.

Ectoplasmic specializations (ES) containing packed actin microfilaments are associated with the numerous parallel rows of occluding junctions which form the Sertoli cell (blood-testis) barrier. To determine if ES regulate the structure of the occluding junctions and/or barrier permeability, we experimentally disrupted ES microfilaments in vivo with intratesticularly injected cytochalasin D (CD). Electron microscopic observations of seminiferous tubules from CD-treated (150-500 microM CD; 0.5-12 hr) animals indicated that ES was absent from regions where the Sertoli cell barrier is located. Seminiferous epithelial sheets from uninjected or vehicle-injected animals (1 DMSO: 1 saline) stained with NBD-phallacidin demonstrated the presence of patterned ES actin surrounding the basolateral regions of adjacent Sertoli cells. After exposure to CD, epithelial sheets exhibited increasingly patchy fluorescence indicating progressive F-actin disruption. Freeze-fracture replicas of CD-injected testes revealed numerous focal alterations in the region of occluding junctions which included disorganization of the parallel arrangement of junctional rows, the presence of free-ending rows, clustering of intramembranous particles (IMPs) between rows, reduction in the number of rows, and loss of IMPs on both the P-face and E-face. Tracer experiments, following CD exposure, were conducted to test the integrity of occluding junctions: lanthanum hydroxide, dextrose, or filipin was added, in separate experiments, to the fixative during perfusion-fixation. In another study, serum containing an antibody against adluminal germ cells was injected intratesticularly, and frozen sections were processed for immunofluorescence study. A final study consisted of simultaneous intratesticular infusions of CD and radiolabelled inulin with subsequent intraluminal and peritubular fluid sampling. In animals which were injected with CD, lanthanum was found to enter the adluminal compartment; fixative made hypertonic by addition of dextrose caused germ cells within the adluminal compartment to shrink and produce exaggerated intercellular spaces; filipin-cholesterol perturbations were present between some Sertoli cell junctional rows and on spermatid plasma membranes; and IgG was detected within the adluminal compartment of many seminiferous tubules. None of these adluminal manifestations was noted in control animals or those which received vehicle. Quantitatively, in the in vivo micropuncture experiments, significantly more radiolabelled inulin entered the lumen of seminiferous tubules from CD-treated animals than from those exposed to vehicle.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of mono-(2-ethylhexyl) phthalate and mono- n-pentyl phthalate on the ultrastructural morphology of rat Sertoli cells in Sertoli/germ cell co-cultures: Correlation with the in vivo effects of di- n-pentyl phthalate

The effects of mono-(2-ethylhexyl) phthalate (MEHP) and mono- n-pentyl phthalate (MPP) on the ultrastructure of Sertoli cells in primary co-cultures of rat Sertoli and germ cells has been examined and compared with the in vivo effects of di- n-pentyl phthalate (DPP) on Sertoli cells in the rat. MEHP and MPP produced changes in the configuration of the plasma membrane, resulting in long thin cytoplasmic processes or filopodia. This was accompanied by changes in microfilament distribution and an increased density of ribosomes, smooth endoplasmic reticulum and the Golgi complex. Changes were qualitatively similar for both monoesters, but MEHP was effective at lower concentrations than MPP. In vivo, DPP produced extensive convolutions in the basal plasma membranes separating adjacent Sertoli cells and retraction of the lateral processes surrounding germ cells. The similarity of the membrane changes in vivo and in vitro suggests a comparable response by the Sertoli cell in the two systems. Furthermore, the similarity of the in vitro response to the two monoesters suggests a shared mechanism of toxicity.

The ultrastructural effects of di- n-pentyl phthalate on the testis of the mature rat

A sequential morphological study has been carried out to examine the ultrastructural effects of di- n-pentyl phthalate (DPP) on the mature rat testis. A single oral dose of 2.2 g DPP/kg body wt was administered, and testes, perfuse-fixed 3–48 hr after dosing, were examined by transmission electron microscopy. By 3 hr, rarefaction of the basal Sertoli cell cytoplasm was seen and the basal plasma membranes separating adjacent Sertoli cells were thrown into a series of convoluted profiles with the appearance of interdigitating cell processes. The subjacent ectoplasmic specializations that normally face these membranes were disrupted, and by 12 hr the inter-Sertoli junctions showed numerous membrane discontinuities. The lateral processes of Sertoli cell cytoplasm, which separate germ cells, showed retraction and fragmentation, resulting in direct contact between adjacent germ cells or the isolation of germ cells unapposed by Sertoli cell plasma membrane. In addition, the ectoplasmic specializations associated with Sertoli-spermatid and Sertoli-pachytene spermatocyte junctions were often disrupted or absent. The mitochondria in the Sertoli cells were enlarged and, in some tubules, increased in number. The changes seen were restricted to tubules in the successive stages XI–XIV, I, and II of the spermatogenic cycle. Elongating spermatids (steps 12–15) showed cytoplasmic condensation and vacuolation by 12 hr and were necrotic by 24 hr. A small proportion of zygotene and early pachytene spermatocytes showed necrosis by 24 hr after dosing. By 48 hr, the cytoplasmic rarefaction and convoluted plasma membranes had regressed and ectroplasmic specializations had reformed along Sertoli-Sertoli junctions.

Distribution of actin in spermatids and adjacent Sertoli cell regions of the rat.

In this study, we describe the distribution of actin filaments in and around spermatids that are mechanically dissociated, in the presence and absence of exogenous trypsin, from the seminiferous epithelium of the rat. NBD-phallacidin and subfragment 1 of the myosin molecule (S-1) are used as probes for filamentous actin at the light and electron microscopic levels, respectively. The fluorescence associated with spermatids mechanically dissociated in the absence of trypsin is due to actin both in the spermatogenic cells themselves and in attached Sertoli cell ectoplasmic specializations. Fluorescence generated by labelled actin in ectoplasmic specializations occurs in linear tracts that follow the outer contour of spermatid heads. The residual fluorescence seen when trypsin is used to detach Sertoli cell fragments is diffuse and due to f-actin in the subacrosomal space. Electron microscopic data agree with the fluorescence results. This study conclusively demonstrates that Sertoli cell ectoplasmic specializations remain attached to spermatids mechanically dissociated from the seminiferous epithelium. This observation may be useful when attempting to isolate ectoplasmic specializations for biochemical analyses.

Three-dimensional structure of the Sertoli cell in the shiba goat.

The three-dimensional structure of the Sertoli cell in the miniature "shiba" goat was examined using scanning electron microscopy. In the basal portion of the seminiferous epithelium, spermatogonia and/or spermatocytes were located in compartments enclosed by adjacent Sertoli cells. From the basal aspect, they were situated in successive recesses. In the middle portion, early round spermatids halfway embedded in the Sertoli cell were recognized. The exposed surfaces of these spermatids were wrapped with ramifying processes which were derived from the Sertoli cell. In the apical portion, only the heads of the maturing spermatids invaded the Sertoli cell. As the spermatid matured, the apical Sertoli process varied in range to finally release the spermatid head. It is probably that the maturing spermatids gradually leave the apical Sertoli process and ultimately segregate themselves from the seminiferous epithelium.

Distribution of actin in Sertoli cell ectoplasmic specializations and associated spermatids in the ground squirrel testis.

We have investigated the possibility that the complex patterns of fluorescence associated with spermatids of the ground squirrel labeled with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) are due to the presence of filamentous actin within the spermatids themselves rather than to actin in attached Sertoli cell ectoplasmic specializations, as previously reported (J. Cell Biol., 100:814-825). Enzymatic treatments (trypsin, DNAase 1) freed Sertoli cell ectoplasmic specializations from spermatids and resulted in a loss, from the spermatids, of the complex fluorescence patterns, suggesting that the latter were generated by labeled actin in ectoplasmic specializations. Moreover, ectoplasmic specializations that were detached enzymatically from spermatids demonstrated the same fluorescence patterns as those emitted from spermatids in the intact or mechanically fragmented seminiferous epithelium. Most spermatids, however, do display a weak and diffuse pattern of fluorescence that changes during spermatogenesis and that is localized between the acrosomal cap and nucleus. S-1 decoration confirmed this subacrosomal localization and further demonstrated that the actin in adjacent Sertoli cell ectoplasmic specializations is arranged in a unipolar fashion. We conclude that the complex patterns of actin fluorescence associated with mechanically isolated spermatids are a superimposition of both Sertoli cell and germ cell actin; however, the latter is either poorly detected or not detected at all when Sertoli cell ectoplasmic specializations overlie the germ cells.

Sertoli cell junctional complexes in gossypol-treated neonatal and adult guinea pigs.

The effect of gossypol, an experimental male contraceptive agent, on the development and maintenance of the blood-testis barrier was determined by feeding gossypol daily to prepubertal and adult guinea pigs, and then examining their testes by electron microscopy of thin sections and freeze-fracture replicas. In guinea pigs of 10 to 30 and 10 to 40 days of age that were fed gossypol, impermeable continuous junctional zones did not develop between adjacent Sertoli cells. Compartmentalization of germ cells in the seminiferous epithelium, therefore, was nonexistent. These findings were obtained by use of the sterol-binding polyene, filipin, used as a low molecular weight tracer in combination with freeze-fracture. In general, the seminiferous tubules lacked lumina and spermatogenesis did not progress beyond the pachytene spermatocyte stage. In adult guinea pigs fed gossypol daily for five weeks, continuous zonules at the base of the seminiferous epithelium appeared intact and were impermeable to filipin. Discontinuous zonules found higher in the epithelium showed distensions between interrupted junctional strands and were permeated by filipin. In addition, vacuolated spaces between Sertoli cells and clumps of heterochromatin were conspicuous in some of the Sertoli cell nuclei. Spermatogenesis was disturbed in about 10% of the seminiferous tubules examined. These perturbations included exfoliation of round and elongated spermatids with concomitant formation of multi-nucleated giant cells. Spermatozoa from these adult male guinea pigs were immotile. These findings suggest that, in neonatal animals, gossypol appears to prevent the maturation of Sertoli cells and this effect is expressed as the failure of focal Sertoli cell tight junctional strands to assemble into continuous zonules. In adult animals, gossypol appears to have no effect on the maintenance of the blood-testis barrier.

Formation of the blood-testis barrier in the rabbit

The time of establishment of the blood-testis barrier in the rabbit was studied by electron microscopy using lanthanum nitrate. This electron-dense tracer was present in the intercellular spaces in all regions of the seminiferous cords in 7- to 9-week-old animals. In 10- and 11-week-old rabbits, the penetration of lanthanum nitrate was restricted to the basal region of the seminiferous cords. Closer examination revealed the presence of numerous tight junctions between adjacent Sertoli cells. The morphological appearance of these junctions was similar to those described previously in other mammals. Entry of the tracer substance was restricted at these junctions. Pachytene germ cells, which reside beyond the junctions, were never surrounded by the tracer. Based on our observations it was concluded that the blood-testis barrier in the rabbit is formed between the 9th and 10th postnatal week, and that it is functionally effective by the 10th week.

Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation, and germ cell development in vitro.

Sertoli cell preparations isolated from 10-day-old rats were cultured on three different substrates: plastic, a matrix deposited by co-culture of Sertoli and peritubular myoid cells, and a reconstituted basement membrane gel from the EHS tumor. When grown on plastic, Sertoli cells formed a squamous monolayer that did not retain contaminating germ cells. Grown on the matrix deposited by Sertoli-myoid cell co-cultures, Sertoli cells were more cuboidal and supported some germ cells but did not allow them to differentiate. After 3 wk however, the Sertoli cells flattened to resemble those grown on plastic. In contrast, the Sertoli cells grown on top of the reconstituted basement membrane formed polarized monolayers virtually identical to Sertoli cells in vivo. They were columnar with an elaborate cytoskeleton. In addition, they had characteristic basally located tight junctions and maintained germ cells for at least 5 wk in the basal aspect of the monolayer. However, germ cells did not differentiate. Total protein, androgen binding protein, transferrin, and type I collagen secretion were markedly greater when Sertoli cells were grown on the extracellular matrices than when they were grown on plastic. When Sertoli cells were cultured within rather than on top of reconstituted basement membrane gels they reorganized into cords. After one week, tight junctional complexes formed between adjacent Sertoli cells, functionally compartmentalizing the cords into central (adluminal) and peripheral (basal) compartments. Germ cells within the cords continued to differentiate. Thus, Sertoli cells cultured on top of extracellular matrix components assume a phenotype and morphology more characteristic of the in vivo, differentiated cells. Growing Sertoli cells within reconstituted basement membrane gels induces a morphogenesis of the cells into cords, which closely resemble the organ from which the cells were dissociated and which provide an environment permissive for germ cell differentiation.

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components.

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.