Changes in the distribution of microtubules in rat Sertoli cells during spermatogenesis

I have studied the distribution of microtubules in rat Sertoli cells during spermatogenesis. The distribution of microtubules was determined by indirect immunofluorescence in fragments of seminiferous epithelium which were mechanically dissociated from perfusion-fixed testes. The presence of microtubules in regions indicated by immunofluorescence was confirmed with electron microscopy. Microtubules are not evident in Sertoli cell regions surrounding early spermatogenic cells, but are abundant in cytoplasm adjacent to apical crypts containing elongate spermatids. In regions surrounding crypts, microtubules are uniformly aligned parallel to the long axis of Sertoli cells. Microtubules in these more central, or columnar, regions of the cells extend from supranuclear levels to the apex of the epithelium. In apical processes surrounding late spermatids, microtubules conform to the contours of the hook-shaped spermatid heads. These microtubules appear continuous with those in the columnar regions of the Sertoli cells. Changes in microtubule distribution observed in the rat are generally similar to, but less elaborate than, those reported previously in the squirrel. These changes may be characteristic of mammalian spermatogenesis in general.     

Plectin is concentrated at intercellular junctions and at the nuclear surface in morphologically differentiated rat Sertoli cells

Intermediate filaments in Sertoli cells have a well-defined pattern of distribution. They form a basally situated perinuclear network from which filaments extend peripherally to adhesion plaques at the plasma membrane and to sites of codistribution with other major elements of the cytoskeleton, particularly with microtubules. Although the general pattern of intermediate filament distribution is known, the molecular components involved with linking the filaments to organelles and attachment plaques in these cells have not been identified. One candidate for such a linking element is plectin. In this study we test for the presence of, and determine the distribution of, plectin in Sertoli cells of the rat testis. Fixed frozen sections and fixed epithelial fragments of rat testis were probed for plectin and vimentin using antibodies. Tissue was evaluated using standard fluorescence microscopy and confocal microscopy. Plectin in Sertoli cells was concentrated in a narrow zone surrounding the nucleus, and at focal sites, presumably desmosome-like plaques, at interfaces with adjacent cells. Plectin was also concentrated at sites where intermediate filament bundles project into specialized actin-filament containing plaques at sites of attachment to elongate spermatids. Plectin in Sertoli cells is concentrated at the nuclear surface and in junction plaques associated with the plasma membrane. The pattern of distribution is consistent with plectin being involved with linking intermediate filaments centrally (basally) to the nucleus and peripherally to intercellular attachment sites.     

Distribution of actin isoforms within cells of the seminiferous epithelium of the rat testis: Evidence for a muscle form of actin in spermatids

Recently, a cDNA that coded for an enteric smooth muscle γ-actin (SMGA) that was expressed in post-meiotic mouse testicular cells was identified. To determine the cellular location(s) of the protein encoded by this cDNA, this SMGA was probed for by immunocytochemistry in the cells of the seminiferous epithelium with two different monoclonal antibodies (Mabs), B4 and HUC 1–1, known to be muscle actin selective. As a control, we also examined the immunoreactivity of a third Mab, C4, that reacts with all non-muscle and muscle vertebrate isoactins. Using light and electron microscopy, a progressive increase in immunolabeling was observed with the muscle selective HUC 1–1 Mab over a loose actin filamentous network distributed throughout the cytoplasm of steps 4–16 spermatids. Thereafter, the labeling decreased such that at step 17 spermatids, only cytoplasmic labeling in the tail of the spermatids was observed. No labeling of this network was noted with the C4 or B4 Mabs. However, myoid cells enveloping seminiferous tubules and smooth muscle cells of interstitial blood vessels demonstrated comparable intense labeling with each of the three Mabs. The C4 Mab intensely labeled actin filaments of the Sertoli-Sertoli and Sertoli-spermatid ectoplasmic specializations. Also well labeled were numerous actin filaments found in the apical Sertoli cell processes encapsulating the heads of late step 19 spermatids at stage VII of the cycle of the seminiferous epithelium. In addition, actin filamentous bundles enveloping tubulobulbar complexes of the late spermatids within the Sertoli cell apical processes were intensely labeled. The actin filaments in the Sertoli apical processes and surrounding the tubulobulbar complexes were also strongly immunolabeled with the HUC 1–1 Mab. The C4 Mab but not the B4 or HUC 1–1 Mabs, recognized actin in the subacrosomal space of steps 4–18 spermatids. This study suggests that there are muscle isoforms of actin within the cytoplasm of developing spermatids and within apical processes of Sertoli cells.     

Distribution of actin isoforms within cells of the seminiferous epithelium of the rat testis: evidence for a muscle form of actin in spermatids.

Recently, a cDNA that coded for an enteric smooth muscle gamma-actin (SMGA) that was expressed in post-meiotic mouse testicular cells was identified. To determine the cellular location(s) of the protein encoded by this cDNA, this SMGA was probed for by immunocytochemistry in the cells of the seminiferous epithelium with two different monoclonal antibodies (Mabs), B4 and HUC 1-1, known to be muscle actin selective. As a control, we also examined the immunoreactivity of a third Mab, C4, that reacts with all non-muscle and muscle vertebrate isoactins. Using light and electron microscopy, a progressive increase in immunolabeling was observed with the muscle selective HUC 1-1 Mab over a loose actin filamentous network distributed throughout the cytoplasm of steps 4-16 spermatids. Thereafter, the labeling decreased such that at step 17 spermatids, only cytoplasmic labeling in the tail of the spermatids was observed. No labeling of this network was noted with the C4 or B4 Mabs. However, myoid cells enveloping seminiferous tubules and smooth muscle cells of interstitial blood vessels demonstrated comparable intense labeling with each of the three Mabs. The C4 Mab intensely labeled actin filaments of the Sertoli-Sertoli and Sertoli-spermatid ectoplasmic specializations. Also well labeled were numerous actin filaments found in the apical Sertoli cell processes encapsulating the heads of late step 19 spermatids at stage VII of the cycle of the seminiferous epithelium. In addition, actin filamentous bundles enveloping tubulobulbar complexes of the late spermatids within the Sertoli cell apical processes were intensely labeled. The actin filaments in the Sertoli apical processes and surrounding the tubulobulbar complexes were also strongly immunolabeled with the HUC 1-1 Mab. The C4 Mab but not the B4 or HUC 1-1 Mabs, recognized actin in the subacrosomal space of steps 4-18 spermatids. This study suggests that there are muscle isoforms of actin within the cytoplasm of developing spermatids and within apical processes of Sertoli cells.     

Changes in the arrangement of actin filaments in myoid cells and sertoli cells of rat testes during postnatal development and after experimental cryptorchidism

Background: Abundant actin filaments are present in myoid cells and Sertoli cells in the testis. In the adult rat, the filaments form a lattice arrangement within the myoid cell, and show a hexagonal pattern in the basal junctional regions of Sertoli cells. Methods: Isolated seminiferous tubules and frozen sections were prepared from juvenile to adult Wistar rat testes, stained with FITC-conjugated phalloidin, and observed by confocal microscopy. Unilateral cryptorchidism was induced in adult rats, and seven days later, their testes were also examined. Results: In the myoid cell, parallel actin filaments running circularly around the seminiferous tubules were observed at 15 and 20 days of age. Then, at 30 days, actin filaments arranged longitudinally along the tubular long axis appeared in addition to the circular bundles. A lattice arrangement of actin-filament bundles in myoid cells became obvious at 40 days, when elongated spermatids are found in the tubule. Actin filaments in the basal junctional regions of Sertoli cells did not acquire the hexagonal pattern seen in the adult testis until 30 days of age. In the cryptorchid testes, the arrangement of actin filaments in the both cells showed a remarkable change compared to the control testis; the filaments became thinner and disrupted. Conclusions: A lattice arrangement of the actin filaments in the myoid cell appear at around 30 days, before the completion of spermatogenesis. A hexagonal pattern of the filaments in the junctional regions of Sertoli cells has already developed at this age. Cryptorchidism affects the actin filaments of the both cells. © 1995 Wiley-Liss, Inc.     

Distribution of actin in isolated seminiferous epithelia and denuded tubule walls of the rat

We have studied the distribution of actin, using NBD-phallacidin as a probe, in isolated sheets of seminiferous epithelia and denuded tubule walls of the rat. Sheets of intact seminiferous epithelia were separated from tubule walls using EDTA in PBS. The isolated epithelia and denuded tubule walls were fixed, mounted on slides, made permeable with cold acetone (-20 degrees C), and then treated with NBD-phallacidin. Actin was observed in myoid cells, in ectoplasmic specializations of Sertoli cells, and in Sertoli cell regions adjacent to tubulobulbar processes of late spermatids. In myoid cells, filament bundles course in circular and longitudinal directions relative to the tubule wall. In Sertoli cells viewed at an angle perpendicular to the epithelial base, actin filaments in ectoplasmic specializations adjacent to junctional complexes circumscribe the bases of the cells. Filament bundles in ectoplasmic specializations adjacent to germ cells closely follow the contour of and are arranged parallel to the long axis of the developing acrosome. Sertoli cell regions adjacent to tubulobulbar processes of late spermatids stain intensely with NBD-phallacidin. Isolated seminiferous epithelia, combined with NBD-phallacidin as a probe for actin, provide an ideal model system in which to study further the contractile properties of Sertoli cell ectoplasmic specializations and the possible involvement of these structures in events that occur during spermatogenesis.     

Role of the spermatogenic–Sertoli cell interaction through cell adhesion molecule-1 (CADM1) in spermatogenesis

Endocrine and local secretory factors have long been known to be required for spermatogenesis. Evidence has been accumulating in recent years indicating that direct contact between spermatogenic and Sertoli cells is also required for spermatogenesis. Cell adhesion molecules of various types have been found in the mammalian testis that are expressed in spermatogenic and/or Sertoli cells and involved in homophilic and/or heterophilic binding. We have cloned a novel cell adhesion molecule, cell adhesion molecule-1 (CADM1), also known as immunoglobulin superfamily 4A or spermatogenic immunoglobulin superfamily, from the mouse testis. CADM1 belongs to the immunoglobulin superfamily and is composed of three immunoglobulin-like domains, a transmembrane domain, and a short intracellular domain. In the seminiferous epithelium, CADM1 is expressed in intermediate spermatogonia through to early pachytene spermatocytes as well as in elongating spermatids—but not in round spermatids, mature spermatozoa, or Sertoli cells. One of the heterophilic binding partners of CADM1 has proven to be a poliovirus receptor, another member of the immunoglobulin superfamily that is expressed in Sertoli cells. Knockout mice for CADM1 develop male infertility due to defective spermatogenesis. These findings suggest that cell adhesion molecules between spermatogenic and Sertoli cells play essential roles in spermatogenesis.     

Intermediate filaments in the Sertoli cells of the ageing human testis

The presence and distribution of intermediate filaments (vimentin, keratin, desmin) was studied in the Sertoli cells of elderly men by means of quantitative immunohistochemical methods. Sertoli cells from young men showed moderate immunogold labelling to vimentin throughout the entire cytoplasm between the cell organelles in tubules showing complete spermatogenesis. Immunogold particles were more numerous in the perinuclear cytoplasm and beneath the plasma membrane in all its faces. The testes from elderly men showed different tubule types; some showed complete spermatogenesis and a normal lamina propria, while others had spermatogenic arrest at different levels (spermatids, spermatocytes, spermatogonia). The immunohistochemical reaction to vimentin in the Sertoli cells of tubules with complete spermatogenesis (type a) was similar to that in the cells of young men. In the Sertoli cells of severely damaged tubules (type b) the immunohistochemical reaction was more intense and immunogold particles extended in similar proportions throughout the whole cytoplasm. When immunolabelling intensity was compared between the three groups of tubules, by counting the number of immunogold particles per square micrometre of cytoplasm, it was found to be significantly higher (P≤0.05) in type b tubules of elderly men than either in tubules of young men or in type a tubules of elderly men. Since the average cell surface of Sertoli cells was similar in all tubule types, these data suggest that an actual vimentin increase occurs in Sertoli cells of germ-cell-depleted tubules. Sertoli cell immunogold labelling to keratin was found neither in young men nor in type a tubules of ageing men, whereas a positive immunohistochemical reaction was observed in the Sertoli cells of type b tubules of elderly men. Immunogold particles were localized mainly in the perinuclear cytoplasm, and beneath the lateral and basal cell surfaces. The observation of vimentin increase and keratin re-expression in ageing Sertoli cells only in germ-cell-depleted tubules suggests that the changes in intermediate filaments are related to the local factors associated with completion of spermatogenesis, causing functional changes in Sertoli cells.     

Actin Disruption Results in Altered Morphology of Basal Tubulobulbar Complexes in Rat Seminiferous Epithelium

Basal tubulobulbar complexes (TBCs) that occur at attachment sites between neighboring Sertoli cells are subcellular machines that internalize intercellular junctions during movement of spermatocytes from basal to adluminal compartments of the seminiferous epithelium. Each complex consists of an elongate tubular extension of two attached plasma membranes, and is capped at its distal end by a clathrin‐coated pit. The tubular region is surrounded by a cuff of actin arranged in a dendritic network. Near the end of the complex, a bulbous region forms that lacks the actin cuff but is closely associated with cisternae of endoplasmic reticulum. The bulb eventually buds from the complex and enters endocytic compartments of the Sertoli cell. Previous research has shown that when the actin network is perturbed using the actin filament–disruptor, cytochalasin D, apical tubulobulbar complexes that are associated with spermatids were associated with lower levels of actin, patchy actin networks and swollen tubular regions. Here we explored the effects of actin network perturbation on the morphology of basal tubulobulbar complexes in stage V seminiferous tubules. Isolated rat testes were perfused ex vivo for one hour with oxygenated Krebs‐Henseleit buffer (with BSA) containing either 40 μM cytochalasin D or control solution containing DMSO and perfusion‐fixed for electron microscopy. Compared to control, actin cuffs in drug‐treated TBCs appeared less uniform and patchy. In addition, the tubular regions of the complexes appeared swollen. Our results are consistent with the conclusion that intact networks of actin filaments are required for maintaining the structural integrity of basal TBCs. Anat Rec, 299:1449–1455, 2016. © 2016 Wiley Periodicals, Inc.     

Ultrastructure of sertoli-cell penetrating processes found in germ cells of the golden-mantled ground squirrel (Spermophilus lateralis)

We have studied the ultrastructure of Sertoli-cell processes that extend into developing germ cells of the ground squirrel (Spermophilus lateralis). In other mammals it is speculated that these processes anchor germ cells to the seminiferous epithelium and transfer materials between Sertoli and germ cells. In the ground squirrel, Sertoli-cell projections first appear in round spermatids and consist of regions containing numerous mitochondria and intermediate filaments together with areas composed mainly of a fine filamentous matrix. Also present are what may be desmosomelike junctions with adjacent germ cells. During spermatogenesis, numerous changes in the penetrating processes and their internal composition occur. Especially significant are those occurring during the movement of residual cytoplasm basaly over spermatid heads: some Sertoli-cell processes contain microtubules, mitochondria and vesicular elements, but also present are regions that lack organelles and appear simply as thin lamellae of cytoplasm that line cavernous invaginations of the germ cell. Coated vesicles and pits are present in processes and adjacent germ-cell regions at all stages of spermatogenesis. Our observations are consistent with the suggestions that Sertoli-cell processes have an attachment function and that they also may facilitate the movement of residual cytoplasm into the epithelium. Further, they indicate that these structures might be involved with receptor-mediated edocytosis.     

Cytoskeleton in sertoli cells of the mosquito fish (Gambusia affinis holbrooki)

Background: There is little information about the distrib ution of cytoskeletal components in the testes of teleost fish. The aim of this (tublin, actin, vimentin, desmin, and cytokeratins) in the sertoli cells of Gambusia affinis holbrooki and in their efferent duct epithelial cells which are possibly orginated from the Sertoli cells Methods: Light and electron microscopic immunocytochemical studies and Western blotting analysis were performed in G. affinis testis. Results: Actin immuncor eaction was observed in the Sertoli cells at all spermatogenic stages, although the intensity of the reaction varied from one state to another. Sertoli cells that support supermatogonia or supermatocytes showd a weak immunoreaction which was uniformly distributed throughout the cytoplasm and somewhat more concentrated at the level of the inter-Sertoli specialized junctions, Immunoreaction to actin increased during the first states of supermiogenesis and was manly localized beneath the plasma membrane. This immunoreacction was more intense in the basal than in the aical ctoplasm of Sertoli cells. In a more advanced stage of supermiogenesis, actin immunoreaction become stroger in the apical cytoplasm where Sertoli cells displayed cytoplasmic projections around each supermatid. After sperm release, the apical Sertoli cell cytoplasm still showed an intense actin immunoreaction. Intense immuncreation to actin was also observed in the epithelial cells lining the efferent ducts. Immunoreaction to tubulin was diffuse throughout the Sertoli cell cytoplasm. No immunocreation to vimentin or desmn was observed in the Sertoli cells during the spermatogenic process. Immunoreation to both vimentin and desmin was observed in the efferent ducts cells. Desmin immunoreaction was also observed in the seminiferous tubule boundary cells, mainl in the sections showing germ cell cysts at the last stages of spermiogenesis and in the peritubular cells that surrounded the efferent duct epitheium. Immunoreaction to cytokeratins was found in the endothelium of testicular blood vessels but not in the Sertoli cells or in the efferent duct epithelium. Conclusions: Immunoreaction pattern to cytoskeletal proteins in the Sertoli cells of G. afinis: differs from that reported in mammalian Sertoli cells. These differences include the distribution of action filaments and the absence of dectectable vimentin immunoreaction in G. affinis: Sertoli cells. © 1995 Wiley-Liss, Inc.     

The distribution pattern of cytokeratin and vimentin immunoreactivity in testicular biopsies of infertile men

Testicular biopsies of infertile patients are often characterized by a mixed atrophy, in which different types of spermatogenic lesions are found in adjacent tubules. In order to evaluate a possible involvement of the state of differentiation of the Sertoli cells, the distribution pattern of cytokeratin and vimentin intermediate filaments within the seminiferous epithelium of 228 biopsy specimens with normal spermatogenesis (n=10), mixed atrophy (n= 206) or Sertoli Cell Only Syndrome (n=12) were investigated by means of immunohistochemical techniques. Sertoli cells were regularly found to show vimentin expression in tubules with normal spermatogenesis as well as in tubules with any kind of spermatogenic impairment including SCO. Cytokeratin expression as a marker showing lack of differentiation was common in Sertoli cells of tubules with arrest of spermatogenesis at the level of spermatogonia, and was occasionally associated with arrest at the level of primary spermatocytes or with SCO. Ultrastructural examination of tubules with spermatogonial arrest revealed Sertoli cells with features of typical fetal or prepubertal Sertoli cells, such as round to ovoid nuclei without indentations, stacks of rough ER and spot desmosomes. These data suggest that spermatogenic arrest at the level of spermatogonia might be due to functional impairment of the associated Sertoli cells, which have maintained or regained an undifferentiated state and are not able to initiate or trigger the process of spermatogonial differentiation.     

Testis of the lizard Mabuya carinata: a light microscopic and ultrastructural seasonal study

Histomorphology and ultrastructure of the testis during breeding and nonbreeding phases of the reproductive cycle of the lizard Mabuya carinata are studied. Observations of the ultrastructural features of the testis during breeding and nonbreeding phases of the reproductive cycle reveal a prenuptial type of spermatogenesis and a clearcut discontinuous spermatogenic cycle. Seminiferous tubules are enlarged and there is active spermatogenesis as shown by the presence of all the stages of spermatogenesis (spermatogonia to spermatids) and spermatozoa during the breeding phase (November). During the nonbreeding phase (April) only spermatogonia and Sertoli cells are seen in the shrunken seminiferous tubules. Leydig cells and Sertoli cells show distinct changes in the morphological appearance with hypertrophy of the cells in breeding phase and atrophy of the cells in the nonbreeding phase of the reproductive cycle. The present study suggests that Sertoli cells and Leydig cells functions are synchronous in the lizard M. carinata.     

Distribution of vimentin-type intermediate filaments in Sertoli cells of the human testis, normal and pathologic

The presence, distribution and spatial arrangement of vimentin-type intermediate filaments in Sertoli cells from human testis biopsies, were studied in semithin and ultrathin sections using a polyclonal rabbit antiserum. At the ultrastructural level, vimentin immunoreactivity was seen concentrated around the nuclei, along fibrillary material within the cytoplasm and at the ectoplasmic specializations of the Sertoli cell junctions, as well as throughout the periphery of the Sertoli cell processes. It is therefore well suited as a marker for Sertoli cell configuration. In computer-aided 3D reconstructions of 20 serial sections, Sertoli cells displayed particular configurations of intermediate filaments in the different stages of spermatogenesis. Two basic configurations, named AS (before spermiation, stages V, VI, I and II), and PS (after spermiation, stages III and IV) respectively, could be differentiated. In addition to the reconstruction and morphological analysis of vimentin filaments in Sertoli cells from patients with unaltered spermatogenesis (obstructive azoospermia), pathological specimens (spermatogenetic arrest, Sertoli cells only-syndrome) were studied with respect to vimentin immunohistochemistry. The results indicate that vimentin filaments play an important role in the adaptation of Sertoli cells to the varying configurations of neighbouring cells during spermatogenesis as well as under pathological conditions.     

Compensatory upregulation of myelin protein zero-like 2 expression in spermatogenic cells in cell adhesion molecule-1-deficient mice

The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expressed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells.     

E-MAP-115, encoding a microtubule-associated protein, is a retinoic acid-inducible gene required for spermatogenesis

Cell type-specific microtubules, such as the Sertoli cell microtubules and the manchette and flagellum microtubules of the spermatids, play essential roles in spermatogenesis. We identified the gene encoding E-MAP-115 (epithelial microtubule-associated protein of 115 kD) as a retinoic acid-inducible gene using gene trap mutagenesis in mouse embryonic stem cells. The gene trap insertion led to a null allele of the E-MAP-115 gene and, in agreement with its high expression in the testis, male mice homozygous for the mutation were sterile because of deformation of spermatid nuclei and subsequent gradual loss of germ cells. Consistent with a possible role for E-MAP-115 in stabilizing microtubules, microtubule associations in the mutant were morphologically abnormal in the manchette of spermatids and in Sertoli cells. We hypothesize that the abnormal microtubules in these two cell types are responsible for deformation of spermatid nuclei and germ cell loss, respectively, and indicate an essential role for E-MAP-115 in microtubule functions required for spermatogenesis.     

Testicular damage caused by inhalation of ethylene oxide in rats: light and electron microscopic studies.

Although testicular damage caused by ethylene oxide vapor (EtO) has been previously reported, the morphological changes occurring in seminiferous tubules remain unclear. We examined the time course of the testicular lesion induced by EtO in order to clarify its morphogenesis. Wistar rats were exposed to 500 ppm EtO for 6 hr per day, 3 times per week for 2, 4, 6, or 13 weeks through inhalation. In the 2-week exposure group, Sertoli cells often showed condensation and retraction of the cytoplasm, and dilatation of the endoplasmic reticulum (ER). In apical Sertoli cells, processes which encapsulated the heads of elongate spermatids, ectoplasmic specializations, and tubulobulbar complexes were often deformed and many elongate spermatids were degenerated. In the 4- and 6-week exposure groups, many degenerated Sertoli cells were present, and deformed germ cells, sometimes with multinucleation, appeared to make direct contact with each other without interlocation of Sertoli cell lateral processes. A few scattered immature Sertoli cells were evident in the 6-week exposure group. In the 13-week exposure group, seminiferous tubules containing almost all types of germ cells reappeared, mixed with atrophic tubules containing Sertoli cells only. In the former tubules, Sertoli cells often possessed regularly regenerated lateral processes, which were interposed between germ cells. These results indicate that the germ cell damage may be associated with damage to Sertoli cells. In spite of the intermittent exposure, focal regeneration of Sertoli cells appeared after 6 weeks of exposure to EtO and preceded patchy recovery of germ cells. Therefore, the data suggest that Sertoli cell regeneration may permit regeneration of germ cells.     

Morphometric and ultrastructural analysis of stage-specific effects of Sertoli and spermatogenic cells seen after short-term testosterone treatment in young adult rat testes.

The effects of testosterone treatment on spermatogenesis in the rat have been investigated by morphometric and structural analysis at the ultrastructural level in stages VII-IX. The aim has been to characterize the changes in Sertoli and spermatogenic cells to elucidate the mechanism of testosterone effects on spermatogenesis and to test the possibilities of developing male contraceptives. In stage VII, the morphometric parameters of volume and surface area in Sertoli cells (see abbreviations below): and the morphometric parameter of volume in the spermatogenic cells such as V(VPG,T), V(VPC,T), V(VrPT,T) and V(VelPT,T) decreased. In stage VIII, the respective values of Sertoli cells, VSN, and VSN/VSC decreased while SSJ increased, and the respective morphometric parameters in the spermatogenic cells, V(VPG,T), V(VPC,T), and V(VrPT,T) increased. In stage IX, in Sertoli cells VSC, VSN, VSN/VSC, and SSJ remained unchanged. In spermatogenic cells V(VPG,T), V(VPC,T), and V(VrPT,T) increased. Further, in all stages, a close apposition of mitochondria and rough endoplasmic reticulum in basolateral cytoplasm of Sertoli cells suggested active protein synthesis. In elongated spermatids in stage IX the microtubular manchette became disorganized. This disorganization and the unexpected shift after testosterone treatment from decrease in several morphometric parameters in stage VIII to increases in stage IX cannot be explained by alterations in testosterone (T), LH, FSH, and their respective receptors. Therefore, still unknown regulatory factors in spermatogenesis are apparently involved in the developmental interactions between Sertoli and spermatogenic cells.     

Vimentin expression during altered spermatogenesis in rats

The collapse of vimentin caused by some xenobiotics correlates with the loss of structural integrity of the seminiferous epithelium. In this study, we investigated the effect of busulphan (an anticancer drug with toxic effects on dividing germ cells) on vimentin filament distribution in rat seminiferous epithelium and compared it with changes found in testes of unilaterally cryptorchid rats. In the seminiferous epithelium, the vimentin labelling was observed only in the Sertoli cells, showing a stage-specific arrangement of the filaments. Both busulphan treatment and cryptorchism caused altered distribution of vimentin filaments in the Sertoli cells. In both models, the apical vimentin filaments collapsed towards the nuclei and were disorganized in the basal region of the Sertoli cells while the germ cells were diminished in the epithelium. After the busulphan effect subsided (4 weeks after administration), spermatogenesis began to restore and vimentin filaments began to organize in basal and perinuclear regions of Sertoli cells among the spermatogonia and spermatocytes. Vimentin labelling of the sloughed material in the lumen of cryptorchid testes (but not in busulphan treated animals) was observed. We conclude that the Sertoli cell vimentin filaments play an important role in the maintenance of spermatogenesis, their damage is associated with the seminiferous epithelium disintegration and their restoration with a recovery of spermatogenesis after the unfavourable conditions subside.     

Misregulation of histone acetylation in Sertoli cell-only syndrome and testicular cancer

In many species, including humans, chromatin remodelling during spermiogenesis is initiated with a marked increase in histone acetylation in elongating spermatids. We have investigated whether this process is disturbed when spermatogenesis is defective or in human testicular tumours. For this purpose, the presence of highly acetylated histone H4 was detected on testicular sections from men with a severe impairment of spermatogenesis of several origins, as well as in different types of testicular tumours. In most tubules devoid of germinal cells (including SCO, Sertoli cell only syndromes) or lacking spermatocytes and spermatids, the Sertoli cells' nuclei showed a global increase in histone H4 acetylation. A similar observation was made in the peritumoral seminiferous tubules of testicular tumour tissues, whenever they were lacking germinal cells, with carcinoma in situ (CIS) cells being hypoacetylated. The global hyperacetylation of elongating spermatids during spermatogenesis could be part of an intercellular signalling pathway involving Sertoli cells and germinal cells, which could be disturbed in cases of severe spermatogenesis impairment, as well as in tubes surrounding germ cells in testicular tumours.