Arrangement and possible function of actin filament bundles in ectoplasmic specializations of ground squirrel Sertoli cells

We have investigated the arrangement and function of actin filament bundles in Sertoli cell ectoplasmic specializations found adjacent to junctional networks and in areas of adhesion to spermatogenic cells. Tissue was collected, from ground squirrel (Spermophilus spp.) testes, in three ways: seminiferous tubules were fragmented mechanically; segments of intact epithelium and denuded tubule walls were isolated by using EDTA in a phosphate-buffered salt solution; and isolated epithelia and denuded tubule walls were extracted in glycerol. To determine the arrangement of actin bundles, the tissue was fixed, mounted on slides, treated with cold acetone (-20 degrees C), and then exposed to nitrobenzoxadiazole-phallacidin. Myosin was localized using immunofluorescence. To investigate the hypothesis that ectoplasmic specializations are contractile, glycerinated models were exposed to exogenous ATP and Ca++; then contraction was assessed qualitatively by using nitrobenzoxadiazole-phallacidin as a marker. Actin bundles in ectoplasmic specializations adjacent to junctional networks circumscribe the bases of Sertoli cells. When intact epithelia are viewed from an angle perpendicular to the epithelial base, honeycomb staining patterns are observed. Filament bundles in Sertoli cell regions adjacent to spermatogenic cells dramatically change organization during spermatogenesis. Initially, the bundles circle the region of contact between the developing acrosome and nucleus. They then expand to cover the entire head. As the spermatid flattens, filaments on one side of the now saucer-shaped head orient themselves parallel to the germ cell axis while those on the other align perpendicularly to it. Before sperm release, all filaments course parallel to the rim of the head. Contrary to the results we obtained with myoid cells, we could not convincingly demonstrate myosin in ectoplasmic specializations or induce contraction of glycerinated models. Our data are consistent with the hypothesis that actin in ectoplasmic specializations of Sertoli cells may be more skeletal than contractile.     

Non-muscle cofilin is a component of tubulobulbar complexes in the testis

Tubulobulbar complexes are finger-like structures that form at the interface between maturing spermatids and Sertoli cells prior to sperm release and at the interface between two Sertoli cells near the base of the seminiferous epithelium. They originate in areas previously occupied by actin filament-associated intercellular adhesion plaques known as ectoplasmic specializations. Actin filaments also are associated with tubulobulbar complexes where they appear to form a network, rather than the tightly packed bundles found in ectoplasmic specializations. Cofilin, a calcium-independent actin-depolymerizing protein, previously has been identified in the testis, but has not been localized to specific structures in the seminiferous epithelium. To determine if cofilin is found in Sertoli cells and is concentrated at actin-rich structures, we reacted fixed frozen sections of rat testis, fixed fragmented tissue, and blots of seminiferous epithelium with pan-specific and non-muscle cofilin antibodies. In addition, GeneChip microarrays (Affymetrix, Santa Clara, CA) were utilized to determine the abundance of mRNA for all cofilin isoforms in Sertoli cells. Using the monoclonal pan-specific cofilin antibody, we found specific labeling exclusively at tubulobulbar complexes and not at ectoplasmic specializations. On one-dimensional (1D) Western blots this antibody reacted monospecifically with one band, and on 2D blots reacted with two dots, which we interpret as phosphorylated and nonphosphorylated forms of a single cofilin isotype. Messenger RNA for non-muscle cofilin in Sertoli cells is about 8.5-fold higher than for muscle-type cofilin. To confirm that the non-muscle isoform of cofilin is present at tubulobulbar complexes, we used antibodies specific to non-muscle cofilin for immunofluorescent localization. As with the pan-specific antibody, we found that the non-muscle cofilin antibody exclusively labeled tubulobulbar complexes. Results presented here indicate that non-muscle cofilin is concentrated at tubulobulbar complexes. Our results also indicate that cofilin is not concentrated at ectoplasmic specializations.     

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

Using commercial monoclonal antibodies against actin and tubulin (alpha and beta), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and - most intensely - in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail. Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.     

Rai14 (Retinoic Acid Induced Protein 14) Is Involved in Regulating F-Actin Dynamics at the Ectoplasmic Specialization in the Rat Testis*

Rai14 (retinoic acid induced protein 14) is an actin binding protein first identified in the liver, highly expressed in the placenta, the testis, and the eye. In the course of studying actin binding proteins that regulate the organization of actin filament bundles in the ectoplasmic specialization (ES), a testis-specific actin-rich adherens junction (AJ) type, Rai14 was shown to be one of the regulatory proteins at the ES. In the rat testis, Rai14 was found to be expressed by Sertoli and germ cells, structurally associated with actin and an actin cross-linking protein palladin. Its expression was the highest at the ES in the seminiferous epithelium of adult rat testes, most notably at the apical ES at the Sertoli-spermatid interface, and expressed stage-specifically during the epithelial cycle in stage VII-VIII tubules. However, Rai14 was also found at the basal ES near the basement membrane, associated with the blood-testis barrier (BTB) in stage VIII-IX tubules. A knockdown of Rai14 in Sertoli cells cultured in vitro by RNAi was found to perturb the Sertoli cell tight junction-permeability function in vitro, mediated by a disruption of F-actin, which in turn led to protein mis-localization at the Sertoli cell BTB. When Rai14 in the testis in vivo was knockdown by RNAi, defects in spermatid polarity and adhesion, as well as spermatid transport were noted mediated via changes in F-actin organization and mis-localization of proteins at the apical ES. In short, Rai14 is involved in the re-organization of actin filaments in Sertoli cells during the epithelial cycle, participating in conferring spermatid polarity and cell adhesion in the testis.     

Hereditary defects in both germ cells and the blood-testis barrier system in as-mutant rats: evidence from spermatogonial transplantation and tracer-permeability analysis

The rat mutant allele as is located on chromosome 12. Homozygous (as/as) males show arrested spermatogenesis, mainly at the pachytene spermatocyte stage. It is not clear whether this defective spermatogenesis is caused by a failure in a somatic cell component that supports spermatogenesis or in the germ cell itself. Spermatogonial transplantation was performed to identify the genetically defective site in the as/as testis. In experiment 1, germ cells collected from as/as testes were transplanted into the testes of immunodeficient mice and normal rats. In experiment 2, normal rat germ cells were transplanted into as/as testes. The results of experiment 1 showed arrest of spermatogenesis at the pachytene spermatocyte stage, accompanied by a characteristic morphological feature, i.e., the formation of inclusion-like bodies in the cytoplasm, in both rat and mouse recipients. These results revealed the intrinsic effect of the mutant gene(s) on germ cells. In experiment 2, no restoration of spermatogenesis was detected in the recipient testes despite thorough histological examination. These results suggest that defects in a somatic cell component in as/as testes prevent the donor germ cells from colonizing and regaining their spermatogenetic ability. When the seminiferous epithelium of the as/as testis was examined by electron microscopy, no morphological abnormalities, including the formation of ectoplasmic specializations between adjacent Sertoli cells, were observed in the somatic cell components. However, when cytochrome c was applied as a tracer material, it penetrated the tight junctions between the Sertoli cells, indicating dysfunction of the blood-testis barrier in the as/as testis. The lack of restoration of spermatogenesis in the as/as testis after transplantation of normal germ cells may have been caused by the unfavorable environment in the seminiferous epithelium resulting from the incomplete barrier system between adjoining Sertoli cells. The gene(s) at the as locus may have a role in both germ cell differentiation and the establishment of the blood-testis barrier.     

THE INTER-SERTOLI CELL TIGHT JUNCTIONS IN GERM CELL-FREE SEMINIFEROUS TUBULES FROM PRENATALLY IRRADIATED RATS: A FREEZE-FRACTURE STUDY

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood—testis barrier in the normal testis.     

Structural abnormalities in spermatids together with reduced sperm counts and motility underlie the reproductive defect in HIP1-/- mice

Huntingtin interacting protein 1 (HIP1) is an endocytic adaptor protein with clathrin assembly activity that binds to cytoplasmic proteins, such as F-actin, tubulin, and huntingtin (htt). To gain insight into diverse functions of HIP1, we characterized the male reproductive defect of HIP1(-/-) mice from 7 to 30 weeks of age. High levels of HIP1 protein were expressed in the testis of wild-type mice as seen by Western blots and as a reaction over Sertoli cells and elongating spermatids as visualized by immunocytochemistry. Accordingly, major structural abnormalities were evident in HIP1(-/-) mice with vacuolation of seminiferous tubules caused by an apparent loss of postmeiotic spermatids and a significant reduction in mean profile area. Remaining spermatids revealed deformations of their heads, flagella, and/or acrosomes. In some Sertoli cells, ectoplasmic specializations (ES) were absent or altered in appearance accounting for the presence of spherical germ cells in the epididymal lumen. Quantitative analyses of sperm counts from the cauda epididymidis demonstrated a significant decrease in HIP1(-/-) mice compared to wild-type littermates. In addition, computer-assisted sperm analyses indicated that velocities, amplitude of lateral head displacements (ALH), and numbers and percentages of sperm in the motile, rapid, and progressive categories were all significantly reduced in HIP1(-/-) mice, while the numbers and percentages of sperm in the static category were greatly increased. Taken together, these various abnormalities corroborate reduced fertility levels in HIP1(-/-) mice and suggest a role for HIP1 in stabilizing actin and microtubules, which are important cytoskeletal elements enabling normal spermatid and Sertoli cell morphology and function.     

Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat

The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.     

Observations on rat sertoli ectoplasmic (‘junctional’) specializations in their association with germ cells of the rat testis

The ectoplasmic (‘junctional’) specialization, a subsurface modification of the Sertoli cell that is often seen facing germ cells, was studied in relation to the development and maturation of these germ cells. This structure is composed of sub-surface bundles of filaments and more deeply placed endoplasmic reticulum. The data indicate that these subsurface modifications of Sertoli cells are reutilized in a cyclic fashion, being transferred from their position facing late spermatids to one opposing less mature germ cells. Ectoplasmic specializations appeared to function mechanically in grasping the heads of the spermatids which are undergoing the elongation and maturation phases of spermiogenesis rather than in actually attaching Sertoli cells to these germ cells. It is postulated that the ectoplasmic specialization imparts rigidity to that area of the Sertoli cell that surrounds the head region of the germ cell, forming a recess and a mantle by which the germ cell may be moved toward the base or toward the surface of the seminiferous epithelium. The observed linkage of microtubules to the cisternae of the complex provided a morphological basis for the changes in the cytoarchitecture of the Sertoli cell, which must accompany these movements.     

An ultrastructural study of the effect of a diabetogenic dose of alloxan on the secretory ameloblasts of the rat incisor

Sertoli cells of the ground squirrel (Spermophilus lateralis), a seasonal breeder, were examined by light and electron microscopy and their structure, particularly the organization of the cytoskeleton, was related to events that occur in the seminiferous epithelium during spermatogenesis. Among the events considered and described are the apical movement of elongate spermatids, withdrawal of residual cytoplasm from germ cells, transport of smooth endoplasmic reticulum (SER) between the base and apex of the Sertoli cells, and sperm release. These events are dramatically evident in this species because the seminiferous epithelium is thin, i.e., there are few germ cells, and both the germ cells and Sertoli cells are large. Sertoli cells of the ground squirrel have a remarkably well developed cytoskeleton. Microfilaments occur throughout the cell but are most evident in ectoplasmic specializations associated with junctions. Intermediate filaments occur around the nucleus, as a layer at the base of the cell, and adjacent to desmosome-like junctions with germ cells. Intermediate filaments, together with microtubules, are also abundant in regions of the cell involved with the transport of SER, in cytoplasm associated with elongate spermatids, and in processes that extend into the residual cytoplasm of germ cells. Our observations of ultrastructure are consistent with the hypothesis that Sertoli cell microtubules are involved with the movement of germ cells within the seminiferous epithelium, and further implicate these structures as possibly playing a role in the retraction of residual cytoplasm from germ cells and the intracellular transport of SER. The abundance and organization of intermediate filaments suggest that these cytoskeletal elements may also be involved with events that occur during spermatogenesis.     

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the "blood-testis" barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated protein ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days, ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions, however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.     

Ultrastructure of sustentacular (Sertoli) cells in the bovine testis

In the present communication, ultrastructural and cytochemical aspects of mature bovine Sertoli cells and their relationship to the different stages of germ cell development are described. As in other mammalian species, different types of junctional specializations exist between Sertoli and germ cells in the bovine seminiferous epithelium, including desmosome-like junctions, Sertoli cell ectoplasmic specializations and tubulobulbar complexes. The functional significance of the morphological results and the interactions of Sertoli and germ cells during spermatogenesis are discussed.     

Immunocytochemical localization of actin and tubulin in rat testis and spermatozoa

Summary Using commercial monoclonal antibodies against actin and tubulin ( and ), the respective antigens were localized on semithin and ultrathin sections of the rat testis. Tubulin immunofluorescence was found in the socalled manchette surrounding the heads of the maturating spermatids as well as the sperm tail. The distribution pattern varied with sperm development. Modified Sertoli cells found at the transition between the seminiferous tubules and the rete testis displayed much filamentous tubulin-reactive material. The immunofluorescence findings could be confirmed at the ultrastructural level using the indirect immunogold method. Actin immunofluorescence was demonstrated in vascular smooth muscle cells, interstitial macrophages and — most intensely — in peritubular cells. Inside the seminiferous tubules the Sertoli cell junctions and the ectoplasmic specializations of the Sertoli cells that follow the outer contour of spermatid heads displayed distinct actin immunofluorescence. In addition to the locations mentioned, actin-like immunoreactivity was visualized at the ultrastructural level in the chromatoid body and the subacrosomal space of spermatids as well as on the outer dense fibers of the sperm tail.Immunoblotting experiments with actin antibodies showed that in extracts from testicular spermatozoa, intact or fragmented into heads and tails, from isolated Sertoli cells grown in vitro, and from testis tissue in addition to authentic actin a protein was present in sperm tail extracts that strongly bound the actin antibody. This protein may be an actin-related protein and may be responsible for the actin-like immunoreactivity of the outer dense fibers of the sperm tail.     

Adjudin-mediated Sertoli–germ cell junction disassembly affects Sertoli cell barrier function in vitro and in vivo

Adjudin, an analogue of lonidamine, affects adhesion between Sertoli and most germ cells, resulting in reversible infertility in rats, rabbits and dogs. Previous studies have described the apical ectoplasmic specialization, a hybrid-type of Sertoli cell–elongating/elongated spermatid adhesive junction, as a key target of adjudin. In this study, we ask if the function of the blood–testis barrier which is constituted by co-existing tight junctions, desmosome-gap junctions and basal ectoplasmic specializations can be maintained when the seminiferous epithelium is under assault by adjudin. We report herein that administration of a single oral dose of adjudin to adult rats increased the levels of several tight junction and basal ectoplasmic specialization proteins during germ cell loss from the seminiferous epithelium. These findings were corroborated by a functional in vitro experiment when Sertoli cells were cultured on Matrigel?-coated bicameral units in the presence of adjudin and transepithelial electrical resistance was quantified across the epithelium. Indeed, the Sertoli cell permeability barrier was shown to become tighter after adjudin treatment as evidenced by an increase in transepithelial electrical resistance. Equally important, the blood–testis barrier in adjudin-treated rats was shown to be intact 2 weeks post-treatment when its integrity was monitored following vascular administration of inulin-fluorescein isothiocyanate which failed to permeate past the barrier and enter into the adluminal compartment. These results illustrate that a unique mechanism exists to maintain blood–testis barrier integrity at all costs, irrespective of the presence of germ cells in the seminiferous epithelium of the testis.     

Expression of claudin-1 and -11 in immature and mature pheasant (Phasianus colchicus) testes

The expression of claudin-1 and -11, tight junctions (TJs) proteins was examined in immature and adult pheasant (Phasianus colchicus) testes. Claudin-1 and -11 cDNA were highly similar to those of human, mice, and chicken. Claudin-1 mRNA and protein (21 kDa) levels in immature testes were higher than those of adult testis. In immature testes until 6 weeks of age, Claudin-1 was found at contacts between adjacent Sertoli cells and between Sertoli cells and germ cells. In adult testis, Claudin-1 was found in early spermatocytes migrating the blood testis barrier (BTB). Blood vessels were positive for claudin-1. Claudin-11 mRNA and protein (21 kDa) increased during adulthood development of testis. In immature testis, Claudin-11 was found in apicolateral contacts between adjacent Sertoli cells, indicating its involvement in cell adhesion in immature testis. In adult testis, strong wavy Claudin-11 immunoreactivity was parallel to basal lamina at the basal part of seminiferous epithelium, indicating that Claudin-11 at the inter-Sertoli TJs may act as a structural element of the BTB. Weak Claudin-1 and -11 immunoreactivity at contacts between Sertoli cells to elongating/elongated spermatids, meiotic germ cells, and basal lamina suggests that they also participate in the cell-cell and cell-extracellular matrix adhesion in pheasant testis. Testosterone increased claudin-11 mRNA in testis organ culture and Sertoli cell primary culture, suggesting positive regulation of claudin-11 gene by androgen in Sertoli cells of pheasant testis. This is the first report on the claudins expression at BTB in avian testis.     

Developmental regulation of calmodulin, actin, and tubulin RNAs during rat testis differentiation

Postnatal testis differentiation involves transition through neonatal, pre-meiotic, meiotic, haploid, and mature stages. We have examined the qualitative and quantitative changes in rat testis RNAs that specifically hybridize to cDNAs encoding the cytoskeletal proteins, calmodulin, beta-actin, alpha- and beta-tubulin at ages corresponding to each of these developmental periods. We compared the species and relative levels of specific RNAs from testes of animals engaged in normal spermatogenesis with RNA from germ cell-depleted, Sertoli cell-enriched (SCE) testis. Distinct developmental patterns of expression of the specific RNAs were found with each of the cDNAs in the two animal models. A 2.2 kb (kilobase) actin RNA and a 2.7 kb beta-tubulin RNA are maximal at 5-10 days of age, suggesting these RNAs are required by somatic and germ cells in the postnatal phase prior to puberty. Between 19 and 29 days, when pachytene spermatocytes appear in significant numbers, there is a slight increase in the 2.2-kb actin RNA, but a 4- to 10-fold increase in RNAs hybridizing to cDNAs for calmodulin, alpha- and beta-tubulin. These changes are much less pronounced in the SCE testis than in the normal testis, indicating increases in these RNAs are related to germinal cell maturation. The germ cell-related increase in 1.8-kb beta-tubulin RNA appears to reflect a developmental "switch" in the gene from which the RNA is derived. This hypothesis is based on the observation that the ratio of hybridization of a chicken brain beta-tubulin cDNA versus a rat spleen beta-tubulin cDNA to the 1.8-kb RNA band increases more than 40-fold between 5 and 29 days of age in normal testis, but is constant in SCE testis. These data suggest that a specific beta-tubulin gene is activated in maturing germ cells. Analogously, a 2.1-kb alpha-tubulin RNA is found only in maturing normal testis and increases as spermatids are produced. A 2.0-kb beta-tubulin RNA, not found in normal testes, is maximal in maturing SCE testes, suggesting this RNA is of somatic cell origin. All of the RNA species studied, except the 2.0-kb beta-tubulin RNA, decrease between 5 and 19 days in SCE testes, as Sertoli cell mitotic activity wanes, indicating that their levels may be regulated by the developmental signals that influence mitosis.     

Sertoli-germ cells interactions in the human testis.

In previous histoimmunochemical studies we reported that transferrin (TF) and insulin-like growth factor I (IGF-I) are present in the cytoplasm of the Sertoli cells of the adult human testis. Receptors for TF were found mainly in adluminal germ cells and type I receptors for IGF-I both in Sertoli and germ cells. Using electron microscopy, evidence of transfer of both TF and IGF-I from the Sertoli to the germ cells through a receptor-mediated endocytosis mechanism was also found. In this paper we report the results of the histoimmunochemical localization of alpha inhibin in the human fetal, prepubertal and adult testis. In 8- to 14-week-old fetal testes a positive immunostaining was found mainly in the interstitial cells, whereas no staining was found in the germ cords. In the prepubertal testis the immunostaining was present in the Sertoli cells but not in the interstitial cells. In the adult human testis the immunostaining was present not only in the Sertoli cells but also in the spermatocytes and in several Leydig cells. Using electron microscopy and immunogold labeling the presence of alpha inhibin immunoreactivity was found in the rough endoplasmic reticulum and in the Golgi cisternae of both Sertoli and Leydig cells. Moreover we found evidence of transfer of alpha inhibin from the Sertoli to the germ cells through receptor-mediated endocytosis.     

Intermediate filaments in the testis of the teleost mosquito fish Gambusia affinis holbrooki: a light and electron microscope immunocytochemical study and Western blotting analysis

Summary A light and electron microscope immunocytochemical study and Western blotting analysis has been performed on intermediate filaments (vimentin, desmin and cytokeratins) in the testis of the teleost fish Gambusia affinis holbrooki. An immunoreaction to vimentin was observed in the epithelium of the efferent ducts, testicular canal and their surrounding peritubular cells. Positive vimentin immunostaining was also observed in the cells located around seminiferous tubules (boundary cells), Leydig cells, interstitial fibroblasts, chromatophores, and blood vessel endothelial cells. In contrast to mammals, no vimentin immunoreactivity was found in the Sertoli cells. Immunoreactivity to desmin was weak in the epithelial cells of the efferent ducts and testicular canal and intense in the peritubular cells that surrounded these ducts. Desmin immunoreactivity was also observed in the seminiferous tubule boundary cells. The immunoreactivity was weak in the boundary cells that surrounded germ cell cysts containing spermatogonia or spermatocytes and intense in the boundary cells around cysts with elongated or mature spermatids. Immunoreactivity towards cytokeratins was observed only in testicular blood vessels. Cytokeratin immunolabelling was intense in the endothelium and weak in the vascular smooth muscle cells. No cytokeratin immunoreactivity was found in the Sertoli cells, germ cells, interstitial cells or in the efferent duct epithelium. The absence of intermediate filaments in the Sertoli cells, the absence of cytokeratins in the epithelium of the sperm excretory ducts, and the presence of desmin filaments in these epithelial cells are the most important differences with regards to the intermediate filament phenotype in mammalian testes.