Search

Changes in the morphology of viscacha Sertoli cells were studied during the annual reproductive cycle. Sertoli cells exhibited marked nuclear and cytoplasmic changes. Seasonal variation in nuclear size and shape, chromatin texture, and nucleolus characteristics was observed. The seasonal patterns of the volume densities of the endoplasmic reticulum (ER), mitochondria, Golgi complex, dense bodies and lipid inclusions were distinct. Morphometric analysis revealed that the Golgi complex is the organelle most sensitive to seasonal change. It declined drastically in the regressed testes and its recovery was slow. The ER and mitochondria exhibited seasonal variations in their pattern and content, that was minimal during winter. In contrast, an accumulation of lipid and dense bodies, such as primary and secondary lysosomes, accompanied the spermatogenic arrest. The volume densities of both organelles were maximum during the restoration of spermatogenesis. The length and organization of the inter-Sertoli junctions also changed with the reproductive cycle. The Sertoli cell number per tubular cross section decreased significantly during the testicular regression, coincident with the presence of Sertoli cells with marked signs of involution. The degree of regression and recovery exhibited by the viscacha Sertoli cells was closely related to that shown by the associated germ cells. Therefore, seasonal endocrine fluctuations and local factors could be involved in the regulation of the morphological and functional characteristics of the viscacha Sertoli cells. These hormonal fluctuations are synchronized by the photoperiod through the pineal gland and its hormone, melatonin., (Copyright 2001 Wiley-Liss, Inc.)

In the prespermatogenesis period, male germ cells (gonocytes) begin to reproliferate and move to the basement membrane of the seminiferous tubule. Although these two events-reproliferation and relocation-are important for establishment of spermatogenesis, they have not been greatly analyzed both in a mechanical and in an endocrine or paracrine aspect. In this study, the relationship between reproliferation and relocation of gonocytes was examined, using the thymidine analog bromodeoxyuridine (BrdU) labeling method and transmission electron microscopy (TEM). BrdU was injected into the fetuses [day 13.5 post coitus (dpc) to 18.5 dpc] and pups [day 0. 5 post partum (dpp) to 6.5 dpp] of C57BL/6J mice. Two hours later, BrdU positive gonocytes were examined immunohistochemically and these data were analyzed. TEM and LM observation was carried out as well. Gonocytes began to relocate on the basement membrane from 18.5 dpc (1.4%) while BrdU-labeled gonocytes were first detected on 1.5 dpp (13.6%). Relocated BrdU-negative gonocytes were recognized from 18.5 dpc (1.4%), and relocated BrdU-labeled gonocytes were recognized from 1.5 dpp (8.4%). On the other hand, non-relocated BrdU-labeled gonocytes were detected from 1.5 dpp (5.2%). Gonocyte relocation began 2 days earlier than reproliferation during the late fetal period. After birth, the two events occurred at random. These results indicate that the reproliferation of the gonocyte does not correlate with relocation. The two events may be regulated by different mechanisms., (Copyright 2000 Wiley-Liss, Inc.)

We report the ultrastructural changes in acrosome morphology during the final steps of rat spermiogenesis, focusing on the relationship between the acrosome morphogenesis and the tubulobulbar complexes (TBC) development. During steps 18-19, the electron-lucent area in the dorsal cortex of the anterior acrosome gradually diminished, and finally, the acrosome became condensed and reduced its volume. Simultaneously with this tightening up of the acrosome, TBC developed from the head portion of late spermatids, protruding into the surrounding Sertoli cells. To investigate the incorporation of acrosomal contents into TBC, step 19 spermatids were stained by periodic acid-Schiff (PAS) reaction and by using the anti-acrosomal monoclonal antibody mMN7. Both PAS-reactivity and the mMN7-immunoreactivity were found in the TBC, as well as in the acrosome. In addition, the acrosome projected into the TBC-like structure, and materials of a density similar to that of the acrosome were observed in the core of the TBC. These results suggest that the TBC eliminate excess acrosomal contents prior to spermiation., (Copyright 1999 Wiley-Liss, Inc.)

Background: Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development., Methods: Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development., Results: The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42., Conclusions: Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.

Background: The testes of rats treated neonatally with propylthiouracil (PTU) grow to almost twice their normal size. The cause of testicular enlargement has been suggested to be the result of delayed maturation of Sertoli cells, allowing Sertoli cell division to occur beyond the 15th postnatal day, the commonly recognized cutoff date for Sertoli cell divisions. It has been shown that an increased population of Sertoli cells in postnatal development supports increased numbers of germ cells in adult animals. After examining developing rats treated neonatally with PTU, we hypothesized that an approximate 10-day delay in maturation was occurring and proceeded to test this hypothesis experimentally. Thus the purpose of this report was to determine if a 10-day delay in maturation could explain the increased numbers of Sertoli cells and increased testis size in PTU-treated animals., Methods: Both control animals and animals treated neonatally with PTU N = 5/group were sacrificed at 15 and 25 days of age and prepared for electron microscopy., Results: Micrographs show and morphometric ultrastructural analysis of numerous parameters demonstrated at the 95% probability level that Sertoli cells from 25-day-old PTU animals are not different in size and most constituents (volume and surface area) from 15-day-old control animals and are less mature than 25-day-old control animals. Mitosis of Sertoli cells was observed in PTU-treated animals in 25-day-old animals but not in age-matched controls. The number of Sertoli cells in 25-day-old PTU-treated animals is significantly increased over age-matched controls. Micrographs show the presence of immature Sertoli cell nuclei in 25-day-old animals receiving PTU as well as increased germ cell degeneration in this group. Sertoli cell tight junction formation is also delayed in PTU-treated animals as compared with controls., Conclusions: Together, the data show that delayed maturation of Sertoli cells occurs in treated animals that corresponds to a minimum of 10 developmental days. In the immature state, Sertoli cells continue to divide. Data presented herein and published data related to PTU treatment indicate that delayed maturation of the Sertoli cell results in delayed maturation and proliferation of other testicular cell types. From this and from published data, the hypothesis is presented that the Sertoli cell is responsible for the overall control of testis development.

Background: There is little information about the distribution of cytoskeletal components in the testes of teleost fish. The aim of this paper was to know the distribution of some major cytoskeletal proteins (tubulin, actin, vimentin, desmin, and cytokeratins) in the Sertoli cells of Gambusia affinis holbrooki and in their efferent duct epithelial cells which are possibly originated from the Sertoli cells., Methods: Light and electron microscopic immunocytochemical studies and Western blotting analysis were performed in G. affinis testis., Results: Actin immunoreaction was observed in the Sertoli cells at all spermatogenic stages, although the intensity of this reaction varied from one stage to another. Sertoli cells that support spermatogonia or spermatocytes showed 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 stages of spermiogenesis and was mainly localized beneath the plasma membrane. This immunoreaction was more intense in the basal than in the apical cytoplasm of Sertoli cells. In a more advanced stage of spermiogenesis, actin immunoreaction become stronger in the apical cytoplasm where Sertoli cells displayed cytoplasmic projections around each spermatid. After sperm release, the apical Sertoli cell cytoplasm still showed an intense actin immunoreaction. Intense immunoreaction to actin was also observed in the epithelial cells lining the efferent ducts. Immunoreaction to tubulin was diffuse throughout the Sertoli cell cytoplasm. No immunoreaction to vimentin or desmin was observed in the Sertoli cells during the spermatogenic process. Immunoreaction to both vimentin and desmin was observed in the efferent duct cells. Desmin immunoreaction was also observed in the seminiferous tubule boundary cells, mainly in the sections showing germ cell cysts at the last stages of spermiogenesis and in the peritubular cells that surrounded the efferent duct epithelium. 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. affinis differs from that reported in mammalian Sertoli cells. These differences include the distribution of actin filaments and the absence of detectable vimentin immunoreaction in G. affinis Sertoli cells.

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.

Background: Paracrine effects of germ cells on Sertoli cell structure were examined in a mouse model with the W locus (dominant white spotting) mutation in which animals with the W/Wv genotype (referred to as mutants) lack virtually all germ cells., Results: Morphometric determination of Sertoli cell parameters in mutant and control (+/+) animals showed that although the testes of mutant animals were about eight times smaller than controls, the numbers of Sertoli cells in the two groups did not differ. Sertoli cell volume, Sertoli cell cytoplasmic and nuclear volumes, and Sertoli cell surface area in mutant animals were significantly smaller than in control animals. Organelle volumes and surface areas, expressed per cell, did not differ significantly in the two groups with one exception: the volume and surface area of smooth endoplasmic reticulum was significantly reduced in mutant animals. Plasma testosterone levels and tissue testosterone levels/testis were normal, indicating that the effects observed in the mutant animal were not a consequence of androgen insufficiency. Plasma FSH was elevated, probably as a consequence of germ cell depletion, and was thought not to affect Sertoli cell parameters observed., Conclusions: The data suggest that paracrine interactions with germ cells do affect Sertoli cells by modifying the amount of smooth endoplasmic reticulum. These data focus attention on the function of this abundant Sertoli cell organelle in promoting spermatogenesis.

Seminiferous tubules in mammals are composed of cell associations that show a cyclic pattern of renewal and development. The cyclic nature of germ cell development suggests that the cells supporting the spermatogenic process, the Sertoli cells, might also differ structurally during the spermatogenic cycle in terms of the quantity of their constituents. In the present study, cyclic differences in volumes, and surface areas were determined using a sampling technique at the electron microscope level that proportionally samples the Sertoli cell within the seminiferous tubule. Among the many parameters studied, only the surface area of the cell, the volume of lipid, and the volume and surface area of the rough endoplasm reticulum were shown by statistical analysis to vary cyclically. Regarding rough endoplasm reticulum, the volume and surface area of this organelle peaked at mid-cycle and its low was recorded near the end of the cycle, exhibiting an approximate 15-fold difference between extremes. The rough endoplasm reticulum parameters generally correlated with known patterns of protein secretion within the tubule and with the secretion of specific proteins as well as the factors important in controlling protein secretion. Many Sertoli cell structural parameters suggested to be influenced cyclically in the rat in other studies could not be confirmed by the present study. Methodological differences in the present study and past studies are discussed as potential sources of error for these discrepancies.

Adult male viscachas (Lagostomus maximus maximus) were gathered from their natural habitat during the period of complete spermatogenesis (June) and during the month of maximum testicular regression (August). The testes were processed by conventional electron microscopic technique using lanthanum nitrate (electron-dense intercellular tracer) to define the intercellular spaces below the inter-Sertoli tight junctions and by freeze-fracture techniques. During complete spermatogenesis the tracer surrounds spermatogonia, preleptotene, and leptotene spermatocytes and stops at the level of the inter-Sertoli tight junctions below all germ cells displaying synaptonemal complexes (zygotene-pachytene spermatocytes) and germ cells in more advanced stages of differentiation. Conversely, during testicular regression the tracer percolates all intercellular spaces between Sertoli cells and the remaining germ cells (spermatogonia and few preleptotene and leptotene spermatocytes). During complete spermatogenesis, freeze-fracture replicas exhibit numerous inter-Sertoli tight junction strands parallel to each other and to the basal lamina. During spermatogenesis decay, the inter-Sertoli tight junctions are found to be short, tortuous, frequently interrupted, and often associated with extented membranous areas of gap junctions.

Ectoplasmic specializations are complex actin-containing structures found at certain sites of intercellular attachment in Sertoli cells. Current evidence indicates that these structures are a form of actin-associated adhesion junction. In the turtle (Pseudemys scripta) and rooster (Gallus domesticus) ectoplasmic specializations are known to occur adjacent to sites of attachment to elongate spermatids and are characterized by a layer of "loosely" cross-linked actin filaments that lies next to the plasma membrane. In the turtle, a cistern of endoplasmic reticulum is associated with the cytoplasmic face of the filament layer. We have found that, in fixed frozen sections of turtle and rooster testes, immunological probes for myosin II react with epithelial regions that also stain with probes for filamentous actin and that are known to be sites at which ectoplasmic specializations occur. Furthermore, when exposed to standard contraction buffers, the diameters of glycerinated ectoplasmic specializations of the turtle are statistically smaller than those of the same structures exposed to control buffers. We interpret these smaller diameters as being produced by the contraction of actin bundles within the ectoplasmic specializations. Our results indicate that ectoplasmic specializations in the rooster and turtle, unlike those in mammals, possess contractile properties. We speculate that ectoplasmic specializations in eutherian mammals may have evolved from actin-associated adhesion junctions in which the actin bundles were initially contractile and from which myosin II was secondarily lost.

Reproductively active hamsters were hypophysectomized and examined 6 or 20 days later in a combined morphometric and endocrine study of the Sertoli cell to determine 1) the morphological and endocrine effects of hypophysectomy of both short- and long-term duration, 2) if regression of Sertoli cells after hypophysectomy in a seasonal breeder resembles regression due to seasonal changes, and 3) if effects of hypophysectomy in a seasonal breeder are equivalent to the effects of hypophysectomy in a nonseasonal breeder. Six days after hypophysectomy, at a period when germ cell degeneration is first noted, there was a significant decrease in testis weight, interstitial space, tubule diameter and length, volume of seminiferous tubule, and tubular lumen. There were no significant changes in Sertoli cell nuclear and cytoplasmic volume although cell surface area was decreased significantly. Most organelles exhibited no significant change in volume or surface area except for secondary lysosomes which expectedly increased in volume as the result of phagocytosis of germinal cells. Thus at an early time period when functional changes in germ cells and Leydig cells are clearly evident (Russell et al. [1992] Endocrinology), the Sertoli cell shows minimal changes. Twenty days after hypophysectomy, the cell, nuclear and cytoplasmic volumes and surface area of the Sertoli cells, and volumes and surface areas of nearly all organelles were significantly decreased from values measured in normal and in short-term hypophysectomized hamsters. The exceptions were the total volumes of lipid which increased significantly and lysosomes which were similar to normal but significantly lower than short-term hypophysectomized animals. The long-term hypophysectomized hamster Sertoli cell, like that of the short-day hamster (Sinha Hikim et al. [1989b] Endocrinology, 125:1829-1843) is structurally regressed as a whole rather than exhibiting selective decreases in cellular and subcellular components. The size of the Sertoli cell in pituitary-intact, long- and short-term hypophysectomized animals showed positive and significant correlations with the volumes and surface areas of all its cytoplasmic organelles except the volume of lipid which showed a negative, significant correlation. Comparisons of long-term hypophysectomized hamsters (in long-day light exposure) and short-day exposed animals (Sinha Hikim et al. [1989b] (Endocrinology, 125:1829-1843) suggested that hypophysectomy, in general, resulted in similar, but slightly more severe regressive changes in the testis and germ cell population than those seen during seasonal regression.(ABSTRACT TRUNCATED AT 400 WORDS)

Movement of postnatal gonocytes to the periphery of the seminiferous cord, where they contact the basement membrane, and resumption of mitosis by these previously quiescent cells are likely to be critically important in establishing spermatogenesis in neonatal rats. We used several approaches both in vivo and in vitro to determine precisely when each of these two events begins, to study their temporal relationship to each other, to determine whether gonocyte division is a prerequisite for relocation or vice versa, and to probe the source of factors initiating and/or regulating these events. Both light and electron microscopy were used to determine that the first gonocytes make contact with the basement membrane on postnatal day 4, while quantitative autoradiography following 3H-thymidine administration in vivo indicated that the first gonocytes to re-initiate cell division do so one day earlier, on day 3, and that the percentage of gonocytes dividing remains at a stable level through day 5. Moreover, we organ-cultured neonatal testes from birth onwards in the presence of defined, serum- and hormone-free medium and determined that both proliferation and relocation of gonocytes begin and continue in vitro as in vivo. This observation argues against involvement of extratesticular factors in stimulating gonocyte relocation and division, and points to the testis itself as the most likely source of agent(s) regulating postnatal maturation of these cells. In other, similar incubations we included 3H-thymidine for varying periods of time to label either those gonocytes that are the first to divide or all gonocytes that divide during the first 48 hr of culture. From these studies, we confirmed that the first gonocytes to divide do so while separated from the basement membrane and found that, although some cells divide before moving peripherally, others do not.

Ectoplasmic specializations of Sertoli cells are actin containing structures found at sites of attachment to spermatids and to neighboring Sertoli cells. We suspect that these cytoskeletal structures are a form of actin-associated adhesion junction. If this is true, then molecular components, such as vinculin, that characterize actin-associated adhesion junctions in general should be present in ectoplasmic specializations. In this paper we have used two approaches to verify the prediction that vinculin is a component of ectoplasmic specializations. First, we have used fluorescence microscopy to probe immunologically for vinculin in ectoplasmic specializations associated with spermatids of the ground squirrel. Second, we have used immunogold techniques to probe for vinculin in ectoplasmic specializations of rat testis. Our results indicate that the immunological probe for vinculin was reactive with ectoplasmic specializations. In single label fluorescence experiments, linear patterns obtained with the vinculin probe were similar to those obtained with probes for filamentous actin. In double label experiments, the vinculin probe was co-distributed with the actin probes. In immunogold studies, specific labelling with the probe for vinculin occurred in ectoplasmic specializations both at sites of attachment to spermatids and adjacent to basal Sertoli cell junctions. Moreover, gold particles were concentrated adjacent to filament bundles within each ectoplasmic specialization. Our results support the conclusion that vinculin is present in ectoplasmic specializations. Further, they indicate that vinculin is co-distributed with actin bundles within each ectoplasmic specialization.

Pregnant rats (day 13) received 10 mg/kg of Busulfan i.p. The seminiferous tubules of their offspring from post-natal age 1 day up to day 35 were examined with TEM after fixation plus intercellular tracers, and with freeze-fracture techniques. During this period, the inter-Sertoli tight junctions of controls increase both in number and in length. Between days 10 and 13 the seminiferous cords have numerous preleptotene and leptotene spermatocytes surrounded by tracer. The inter-Sertoli junctions are tortuous and predominantly perpendicular to the basal lamina. Between ages 13 and 20 days the seminiferous epithelium reaches zygotene-pachytene stages. The tracer is stopped at the inter-Sertoli junctions at this stage, whereas it still permeates tubules displaying preleptotene and leptotene spermatocytes. Freeze-fracture shows that the orientation of inter-Sertoli junctions has changed to parallel, both to each other and to the basal lamina. In the Busulfan-treated rats, the tubules continue having, up to post-natal day 30, only Sertoli cells and scanty spermatogonia. In these, lanthanum penetration goes as far as the apical Sertoli cell region; the inter-Sertoli junctions still show tortuous strands, and most are oriented perpendicular to the basal lamina. This indicates that formation of the first competent inter-Sertoli junctions is temporo-spatially simultaneous with the appearance of zygotene-pachytene spermatocytes.

The effect of tunicamycin (TM) on testicular cord organization in the fetal mouse was examined in vitro at light and electron microscopic levels, with special reference to the glycoprotein functions during Sertoli cell differentiation. In testicular explants treated with TM, testicular cord organization was inhibited. TM treatment affected basal lamina formation by Sertoli cells, resulting in a discontinuous basal lamina or none at all in certain areas. The disorganized Sertoli cells were amorphous in shape, exhibited poor epithelial polarity, and were irregularly arranged in the testicular parenchyma. Extracellular matrix and collagen fibers were often observed in the intercellular spaces between the disorganized Sertoli cells. Lectin histochemical observation revealed that the number of wheat germ agglutinin binding sites on the plasma membrane and basal lamina of disorganized Sertoli cells was significantly decreased by TM treatment. However, junctions were normally observed in the plasma membrane between disorganized Sertoli cells. Leydig cells showed a normal differentiation in the testicular parenchyma in the presence of TM. These observations suggest that basal lamina formation of Sertoli cells and/or the expression of their cell surface glycoconjugates may be crucial for the establishment of Sertoli cell polarity and/or the Sertoli-Sertoli cell interactions required for proper testicular cord formation. Sertoli cell organization into testicular cords and Leydig cell differentiation may be controlled by different regulatory mechanisms.

The scanning and transmission electron microscopes were used to examine the processes of spermiation and sperm maturation in the marmoset. We observe that the heads of late spermatids are embedded in the apical aspect of the large sleeve-like columnar portion of Sertoli cells. As spermiogenesis progresses, spermatids become associated with numerous small apical Sertoli cell extensions. These finger-like processes undergo a sequence of changes during spermiation. Spermatozoa from the caput, corpus, and cauda epididymides were examined. In caput epididymis of marmoset, the apical segment of the spermatozoa extends well beyond the rostral edge of the nucleus and folds back on itself. In sagittal sections, the acrosome exhibits a distinct hook shape. In the corpus, the distinctive hook-shaped apical segment of the acrosome is observed in some spermatozoa, but the apical extension is significantly smaller or projects out only slightly beyond the nucleus. In cauda epididymis, the extension is absent. A similar acrosomal hook has been reported in the pigtailed monkey, which is an Old World species. We suggest that changes in acrosome structure during sperm maturation may be fairly widespread among primates.

The cells of the seminiferous epithelium of the rat testis are a rich source of microtubules and contain distinct microtubular structures such as the meiotic spindle and manchette. Microtubule diversity can be maintained by differential genetic expression of the multiple alpha- and beta-tubulin polypeptides or by tubulin monomer acetylation and detyrosination, post-translational modifications of alpha-tubulin. In the present analysis, antibodies that specifically recognize acetylated (antiacetylated), tyrosinated (anti-Tyr) and detyrosinated (anti-Glu) alpha-tubulins were employed to examine the distribution of post-translationally modified microtubules in the cells of the seminiferous epithelium. In the light microscope, a distinct pattern of staining for each antibody was detected using immunoperoxidase techniques on paraffin-embedded testicular sections. In the case of the anti-Glu antibody, a dense immunoperoxidase staining was detected in the cytoplasm of steps 4-7 spermatids. Thereafter, staining was noted over the area corresponding to the manchette of steps 8-15 spermatids, but not over their cytoplasm. The tails of spermatids were also reactive with this antibody. The anti-Tyr antibody was observed to be localized over the cytoplasm of Sertoli cells in their basal, supranuclear, and apical regions. A dense immunoperoxidase staining was also noted in the cytoplasm of pachytene spermatocytes, but it was negligible in the cytoplasm of spermatocytes undergoing their meiotic division; in these cells the centrioles and meiotic spindle were reactive. The spermatid's tails were also reactive. The antiacetylated antibody showed reactivity only over the tails of spermatids. With the electron microscope, a similar pattern of labeling was noted using immunogold labeling on Lowicryl K4M embedded testicular sections. The anti-Glu antibody heavily labeled microtubules of the manchette and the axoneme of tails of spermatids as well as microtubules of the proximal and distal centrioles and centriolar adjunct. The anti-Tyr antibody strongly labeled microtubules of Sertoli cells and the meiotic spindle and midbody of dividing spermatocytes. The anti-Tyr antibody also labeled the microtubules of the axoneme, centrioles, and centriolar adjunct of spermatids, but to a lesser degree than the anti-Glu antibodies; the manchette was faintly labeled. Of the three antibodies, the antiacetylated antibody showed the weakest labeling of microtubules of the centrioles, centriolar adjunct, and midbody, whereas those of the manchette and Sertoli cells were unreactive; the axoneme was moderately labeled.(ABSTRACT TRUNCATED AT 400 WORDS)

Nomarski optics were used to identify stages of the spermatogenic cycle of seminiferous tubules in sectioned tissue or in whole dispersed tubules and to characterize the equine spermatogenic wave. Embedded tissues were sectioned at 20 microns. Whole dispersed tubules were obtained by enzymatic digestion of thin slices of fresh testis. Dispersed tubules were fixed, dehydrated in graded levels of alcohol, infiltrated with Epon, and mounted in toto on glass slides. Stages of the spermatogenic cycle could be identified under Nomarski optics in both histologic sections and tubules mounted in toto. Stage dependent nuclear chromatic and cytoplasmic changes in spermatogonia, spermatocytes, and spermatids were evident. Spermatid development included chromatin condensation, nuclear elongation, acrosomal development from the Golgi and proacrosomic granules, migration of the annulus and mitochondrial alignment, and the transient appearance of the chromatoid body and manchette. Both nuclear and cytoplasmic details of Sertoli cells were revealed. In tubules mounted in toto, the spermatogenic wave along the length of the tubules occurred as a consecutive set of stages occupying small regions along the tubular length. The spermatogenic wave in the horse is more similar to that of humans than that of rats. The combination of enzymatic isolation of seminiferous tubules and identification of spermatogenic stages by Nomarski optics facilitates examination of the spermatogenic wave in species whose tubules are tightly bound and not easily teased apart.

Throught stage VII and early stage VIII of the cycle of the seminiferous epithelium, the heads of the late spermatids, located in a juxtaluminal position, are embedded in apical processes of Sertoli cells. These processes contain cisternae of endoplasmic reticulum (ER) of two main types, i.e., flattened and tubular, which communicate with each other to form a continuous system. Throughout the long stage VII of the cycle, these two types of cisternae undergo marked changes. In early stage VII, the flattened cisternae, developing from the subsurface cisternae which compose the "junctional specialization," form concentric sheets at the periphery and in the middle of each apical process. The less conspicuous tubular cisternae form a continuous network which is present in the bridge connecting the Sertoli cell body to the apical process, and extends along the dorsal and ventral aspects of the spermatid's head to end up as cup-shaped flattened cisternae capping the bulbs of the tubulobulbar complexes described by Russell and Clermont ('76). In mid stage VII, the flattened cisternae start to regress, while the tubular cisternae become more abundant. In late stage VII, only fragments of the flattened cisternae are present, while the tubular cisternae form a profuse and elaborate network throughout the apical process. In the following stage VIII, the tubular cisternae disperse and only remnants of ER are present at the time of the release of the spermatid into the tubular lumen. These transformations of ER cisternae suggest a complex alteration in the relationship between Sertoli cells and late spermatids prior to their release as spermatozoa.

The mitochondrial population of sustentacular cells in the testis of the amphibian, Xenopus laevis, has been examined by electron microscopy. Three distinct types of mitochondria have been observed. The first and most common mitochondrial type is a "typical" organelle with a rod-like profile containing tubular to plate-like cristae. The second mitochondrial form is characterized externally by irregular bulbous protrusions and internally by increased numbers of tubular cristae. The third mitochondrial type, which is usually ovoid in profile, has the most unusual internal membrane configuration, consisting of pleated folds pierced by regular rows of fenestrations. Mitochondrial type one is found in all sustentacular stages, mitochondrial type two first appears in sustentacular cells surrounding early spermatids, and mitochondrial type three is observed only in sustentacular cells surrounding spermatozoa. These observations indicate that discrete subclasses of mitochondria are present in sustentacular cells of Xenopus.

The permeability of Sertoli tight junctions to lanthanum administrated during fixation is demonstrated in biopsies of patients with partial germinal aplasia. In freeze-fracture replicas the number of fibrils is not significantly different from the data obtained in normal testis. Thus, in these pathological conditions junctional permeability is not related solely to the complexity of the network revealed by freeze-fracture.

The present work reports novel findings in the toad testis using freeze-fracture techniques. Tight and gap junctions are observed in the nonfenestrated endothelial cells. Numerous gap junctions are present between interstitial cells. Sertoli-Sertoli junctional specializations in the toad are similar to those described in mammals although they appear to be less elaborate. The appearance of nuclear pores, in the seminiferous epithelium, ranges from a uniformly and apparently random distribution in Sertoli cells to large aggregations of closely spaced pores separated by free areas in the nuclear envelope of spermatocytes. Inasmuch as these features have also been described in mammals, the fact that they are present in amphibia might indicate that they represent a widespread pattern. Concomitantly with increasing chromatin condensation, nuclear pores diminish in round spermatids and seem to disappear in the more mature germ cells. The en face freeze-fracture views of annulate lamellae in the postnuclear cytoplasm and membranous features of the acrosome formation, flagellum, and undulating membrane of elongating spermatids are also described.

In adult Sertoli cells of most strains of mice, all the centromeric heterochromatin is condensed in two chromocenters, one on each side of a single, large nucleolus. In a random-bred Swiss OF-1 strain, however, the nucleus has a different structural organization. Part of the heterochromatin Is seen as chromocenters in contact with the nucleolus; the rest of it is dispersed in granules of unequal size in the nucleoplasm. Such an unusual spatial arrangement of heterochromatin in interphase nucleus cannot be explained either by a difference in the nucleolar organizing regions or by a polymorphism of the C-banding of metaphase chromosomes.

Biochemical analyses of Squalus testis indicates that key enzymes involved with androgen production increase progressively from immature regions containing spermatogonia to mature regions in the late spermatid stage of maturation (Canick et al., 1983). In an effort to identify cells possessing the cytological characteristics of steroid production and to determine the structural correlates of the observed functional changes, we have carried out an electron microscopic study of Squalus testis. This report demonstrates that Sertoli cells contain a well-developed agranular reticulum, mitochondria with tubulovesicular cristae, and numerous lipid droplets. Moreover, as germ cells mature, there is an increase in abundance of agranular reticulum in the adjacent Sertoli cells. By the time of spermatid elongation, this has reached dramatic proportions and fills the Sertoli cell as a mass of tubules. These results lead us to conclude that the Sertoli cell is responsible for secretion of the increasing amounts of androgen during the spermatogenetic cycle in Squalus.

This paper provides a description of the intercellular junctions in the rat testis, as observed using the freeze fracture technique. These intercellular junctions were categorized into four general types: Sertoli cell tight junctions, myoid cell tight junctions, gap junctions, and "heterogeneous junctions." The Sertoli cell tight junctions had a mean depth of 3.1 micron in the basal to apical direction, and contained 25 to 50 (average: 36) parallel rows of particulate sealing elements. The myoid cell tight junction was neither continuous nor extensive, but focal in nature. Interestingly the sealing elements of this junction, like those of the Sertoli cell tight junction, were quite particulate in nature. Typical gap junctions were observed between Sertoli cells where they were intercalated between the parallel rows of the Sertoli cell tight junction. The most interesting observation was the identification of gap junction-like structures, in various stages of formation, on germ cell membrane fracture faces, both in the basal and adluminal compartments. Lastly, the unusual "heterogeneous junction" was observed on large membrane fracture faces in close proximity to cells in the adluminal compartment, presumably between Sertoli cells. These junctions appeared to consist of both tight and gap junction elements.

Near the end of spermiogensis, the late spermatids remain attached to the superficial layer of the seminiferous epithelium for an appreciable period of time (i.e., 3 to 4 days). Ths sickle-shaped heads of the spermatids are embedded in an apical process of Sertoli cell cytoplasm which is connected to the rest of the cell by a narrow stalk. In the concavity of the head several long (2-3 mum) and very narrow (50 nm) tubular projections of the spermatid's plasma membrane invaginate the Sertoli cell cytoplasm. These tubular processes terminate by a bulbous swelling which may measure up to 1 mum in diameter. Along the process the plasma membrane of the Sertoli cell is closely apposed to the spermatid's membrane, the intracellular space being only 6-8 nm wide. In the Sertoli cytoplasm immediately surrounding the tubular portion of the structure there is an accumulation of filamentous material, while next to the bulbous extremity there are, at a shrot distance, smooth surfaced cisternae of endoplasmic reticulum. The whole structure was referred to as a tubulobulbar complex. These complexes, of which there are up to 24 per spermatid, appear as these cells complete their migration toward the apex of the Sertoli cells. They disappear just before the release of the spermatids in the lumen of the seminiferous tubule as a result of the fragmentation of the spermatid's plasma membrane followed by a resorption of the Sertoli plasma membrane. Morphological evidence suggests that the Tubulobulbar complexes serve as anchoring devices that retain the spermatids at the surface of the seminiferous epithelium while their dissolution contributes in part to the process of spermiation. Similar tubulobulbar complexes were also formed by the plasma membranes of two adjacent Sertoli cells close to the Sertoli-Sertoli tight junctions near the tubular limiting membrane.

Sertoli cell processes invaginating the cytoplasm of rat spermatids were investigated in thin or thick sections stained respectively with ferrocyanide-reduced osmium (Karnovsky, 1971) and the Ur-Pb-Cu metallic impregnation method (Thiéry and Rambourg, 1976). Two types of Sertoli cell processes were observed: Some, seen in steps 11-15 of spermiogenesis, were associated with a tight network of ER cisternae located within the spermatid cytoplasm subjacent to the plasma membrane facing the Seertoli cell process; others, seen from step 11 until the end of spermiogenesis, were not associated with such a spermatid subsurface ER network. Following their formation, the ER-associated Sertoli cell processes were cleaved into double-walled vesicles. Concomitantly, the enclosed Sertoli cell cytoplasm underwent autolysis followed by the dissolution of the Sertoli cell plasma membrane leaving within the spermatid cytoplasm many empty-looking vacuoles delimited only by the plasma membrane of the spermatid. Thereafter, i.e., in steps 15 and 16 of spermiogenesis, these vacuoles clustered, and with the collapse of the vacuoles they gave rise to a single compact mass of interlaced ER cisternae and small vacuoles. With the dissolution of ER cisternae, in step 18, this mass was finally dispersed. These observations suggested that the ER-associated Sertoli cell processes may constitute a pathway for the delivery of material from the Sertoli cells into spermatids. In contrast, the Sertoli cell processes not associated with a network of ER cisternae did not show cleavage or autolysis and appear to be involved mainly in holding the cytoplasm of the spermatid in place.

Normal swine testes, congenital cryptorchid swine testes, and normal human tests were exposed to HMM (heavy meromyosin) after either glycerination or saponin treatment in order to determine whether the fine filaments composing the crystalloids in the Sertoli cells of the cryptorchid swine testes bind HMM to form arrowhead complexes. Short bundles of microfilaments observed in the basal part of the Sertoli cells in both normal and cryptorchid testes also bind HMM. Similar bundles of HMM-bound filaments are observed in the vicinity of spermatocytes. The periodicity of the arrowhead complexes is about 35 nm, and all arrowheads on a given filament point in the same direction. In addition, the polarity of the HMM-bound filaments in a given crystalloid or bundle is uni-directional. A mechanism for the formation of the swine crystalloids has been strongly support this hypothesis. Fine filaments of Charcot-Boettcher's crystalloid in human Sertoli cells did not bind HMM. Therefore the fine filaments of the human crystalloid are not actin-like in nature.

A "ball and socket-like" junction between branches of the Sertoli cells and the developing spermatids is described. A cytoplasmic extension of the Sertoli cell fits into a pocket in the spermatid and has a constricted neck region. Frequently there are large multivesiculate bodies in the Sertoli cell extension and small vesicles frequently appear in the spermatid cytoplasm, in the area of the "ball and socket-like" junction. This suggests the possibility that there may be communication of materials between the two cells. The possible function of the junctions is discussed and it is concluded that they more likely have a role in nutrition than in coordination.

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.

Transmission electron microscopy of Thai leaf frog testis revealed a unique pattern of spermatid nuclear morphogenesis. Chromatin condenses into a continuous cylindrical coil within a roughly spherical nucleus. Later the nuclear membrane conforms to the contours of the uncoiling nuclear contents. In the mature sperm, the long, tapering nucleus is helically shaped. This developmental sequence occurs in the absence of a microtubular manchette, raising questions about the role of this structure in nuclear shaping in spermatozoa of other species.

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.

In order to gain insight into the earliest pathological changes underlying the development of autoimmune aspermatogenic orchitis (AIAO) the blood-testis barrier was studied by light and electron microscopy, freeze-etching, and cytochemical techniques early (from 1 to 8 days after adjuvant treatment of isoimmunization). At later times (16 to 21 days) the study was carried out by light microscopy only. Adult male guinea pigs were used either as controls or immunized with Freund's complete adjuvant alone or together with pertussis vaccine. An additional group comprised animals immunized with a suspension of isologous spermatozoa emulsified in Freund's complete adjuvant and with pertussis vaccine. Ultrastructural studies of the testes of experimental animals showed, at earlier periods, apparently normal Sertoli junctions. However, in the adluminal compartment, distended gaps were seen between the facing membranes of adjacent Sertoli cells. At later periods, a massive destruction of the germinal cells were observed. In freeze-fracture replicas, the Sertoli junctions of testes belonging to all the experimental groups were characterized by an irregular network of occasionally interrupted strands of particles associated with the P face (PF). Large concavities determined distensions between interconnecting ridges. The gap junctions were increased in number and in surface. Tracer studies using horseradish peroxidase showed that the marker permeated the myoid cells of a greater proportion of tubules than in control animals. Within the seminiferous epithelium there was only a limited passage of the marker towards the lumina of the tubules. Yet the tracer was always excluded from the adluminal compartment by the Sertoli tight junctions. Our observations suggest the possibility that the FCA causes a loosening of the Sertoli junctions. This condition could enhance exchanges between two antigenically different cellular compartments and, thus, favor occurrence of an autoimmune reaction when cytotoxic factors are experimentally induced, as in iso- or autoimmunization.

Short pieces of seminiferous tubules from juvenile rats were grown in tissue culture and studied by phase contrast light microscopy while living and by transmission and scanning electron microscopy after fixation and appropriate processing. The pieces of tubules remodeled in vitro, with the original explant becoming surrounded closely by a sheet of epithelioid cells and more peripherally by elongate cells. The epithelioid cells were identifiable as Sertoli cells because of the presence of characteristic Sertoli-Sertoli cell junctions near their upper surface. The elongate cells were derived from peritubular tissues, but could not be specifically identified as to cell type. Clusters of stellate cells and of round cells were present on the upper surface of the Sertoli cell sheets, but not on the elongate cells or the bare floor of the culture dish. The stellate cells were spermatogonia and the round cells were spermatocytes, as identified by fine structural features. Intercellular bridges were maintained between germ cells in culture without being surrounded by processes of Sertoli cells. Rudimentary junctions were present between germ cells and Sertoli cells in culture. The shape of germ cells in vitro was the same as the shape in situ, indicating that shape is an inherent feature of germ cells and is not determined by surrounding Sertoli cells.

Mouse testes of newborn to adult were examined by freeze-fracture. Between the newborn Sertoli cells, gap junctions consisting of aggregations of the intramembranous particles (about 8 nm in diameter) are frequently found. Some of the junctions are about 1 mum in diameter and show particle-free regions in the aggregation. Linear arrangements of a few particles, which appear to be the initial formation of the occluding junctions, are seen in the newborn sertoli cells. The occluding junctions are arranged in a meshwork, in which the gap junctions are situated between the stages of newborn to six days of age. The particles of the occluding junctions are predominantly located on the B face in the center of the groove instead of the A face of the ridge. The occluding junctions do not appear to surround the entire circumference of the Sertoli cell of the 6-day-old mouse. The gap junctions decrease in size. In later stages, many parallel occluding junctions (up to forty in number) are found over one Sertoli cell surface and are distributed circumferentially around the entire cell surface, indicating establishment of the blood-testis barrier. The occluding junctions dominate and the gap junctions diminish in number as development proceeds.

We have studied the distribution of microtubules and intermediate filaments in mammalian Sertoli cells during spermatogenesis. The arrangement of microtubules was determined, by indirect immunofluorescence, in ground squirrel testes that were 1) fixed, mechanically fragmented, and attached to polylysine-coated slides, and 2) fixed, embedded in polyethylene glycol, and sectioned. Intermediate filament patterns were determined, also by indirect immunofluorescence, in sections of unfixed rat testis. Results from these studies were confirmed and extended using electron microscopy. Microtubules first become evident in lateral processes that embrace round spermatids. When spermatids elongate and become situated in apical crypts of Sertoli cells, the microtubules become oriented parallel to the long axis of Sertoli cells and surround the crypts. As spermatids mature and acquire a saucer shape, apical microtubules progressively concentrate in Sertoli cell regions adjacent to the acrosome and eventually form discrete C-shaped structures that disappear during spermiation. Intermediate filaments in rat Sertoli cells are centered around the nucleus. From perinuclear regions, filaments extend toward desmosome-like junctions with early spermatogenic cells and into the apical cytoplasm where they have a transient association with crypts containing elongate spermatids. Filaments amongst crypts are most evident in early stages of the spermatogenic cycle when apical crypts are situated deep within the epithelium. They become less evident and eventually disappear as spermatids assume a more apical position. Our fluorescence studies and ultrastructural analyses indicate that the association of intermediate filaments with crypts is specific to regions adjacent to the dorsal or convex aspect of spermatid heads. In these regions, approximately 8 to 12 uniformly aligned filaments are intimately associated with actin filaments in ectoplasmic specializations surrounding the crypts. We conclude that, like actin, the distribution of microtubules and intermediate filaments changes in Sertoli cells during spermatogenesis. The distribution of microtubules correlates with the irregular columnar shape of Sertoli cells. We suspect that the apically situated intermediate filaments may play a role in anchoring or positioning Sertoli cell crypts deep within the epithelium during the early stages of the spermatogenic cycle.

Random and serial thin sections of the nucleus in adult rat Sertoli cells were examined by electron microscopy. Besides the previously reported nucleoli and heterochromatin masses, the nuclei contain a coiled body and a new structure, the "mykaryon." The coiled body is 835 nm in maximum diameter. It is composed of distinct elements referred to as "coils." They are 32 nm wide on average and resemble the nucleolar pars fibrosa in their intense staining with heavy metal salts and their composition of narrow filaments. The coiled body is often close to a nucleolus, though no direct contact is established, and it sometimes exists at a distance from the nucleolus. The mykaryon is spherical, 460 nm in maximum diameter, and composed of a tridimensional network of 7-26 nm-wide electron-opaque "cords" separated by slightly thinner spaces. It has not been observed in the vicinity of a nucleolus. A literature survey showed no previous mention of a structure similar to the mykaryon. The coiled body and the mykaryon are interpreted as normal constituents of the Sertoli nucleus in the adult rat.

To further characterize Sertoli cell-germ cell junctional specializations seminiferous tubules from sexually mature Sprague-Dawley rats were dissociated by enzymatic and mechanical methods. Ultrastructural analysis of cell suspensions prepared by incubation in collagenase alone or by mechanical methods revealed that spermatids remained attached to Sertoli cells or Sertoli cell fragments. Such cellular associations were found only between Sertoli cell fragments and spematids in which the developing acrosome had made contact with the plasma membrane (step 8 and subsequent steps of spermiogenesis). Furthermore, the fragments were confined to that region of the plasma membrane over the acrosome. The Sertoli cell half of this adhesive site displayed the typical elements of Sertoli cell junctions, filamentous bundles and associated cisterna of endoplasmic reticulum, in apposition to the spermatids. The spermatids demonstrated no surface specializations at the attachment sites. In contrast, in cell suspensions prepared with trypsin, spermatids were free of attachments to Sertoli cells or their fragments. These results demonstrate that: (1) the junctions act to bind cells together, (2) adhesive type contact is established between Sertoli cells and spermatids at step 8 and subsequent steps of spermiogenesis, (3) contact is restricted to the spermatid plasma membrane over the acrosome, and (4) spermatids can be freed from the junctional specializations by treatment with trypsin.

The appearance of seminiferous tubules and interstitial cells of children, aged 2.5 to 13 years, affected by acute lymphoblastic leukemia was analyzed in sections. The testicular biopsies were performed at the end of therapy (vincristine, prednisone, L-asparaginase, 6-mercaptopurine, intrathecal methotrexate), which was affected for the same period and at the same doses. Three age groups were considered (I, 2.5 to 5 years; II, 6 to 9 years; III, 12 to 13 years). Age groups I and II presented damage of some tubules (25-35%) and areas of degeneration. Histometric analysis performed for A type spermatogonial population gave a mean value corresponding to controls in age group I and a mean value significantly lower with respect to controls in age group II. Moreover, age group II presented a lack of increase in tubular cross section. These results suggest that there is a vulnerability both of whole tubules and of some areas of Sertoli cells and germ cels to cytotoxic-induced damage. Leydig cells appear to be the cells least sensitive to drugs, and hormonal data indicate that the hypothalamic pituitary function appears to be intact, despite chemotherapy. Long-term prospective studies of reproductive function in children receiving cancer chemotherapy are needed to determine the magnitude and duration of damage resulting from therapeutic treatment.

The inter-Sertoli junctions of children aged between 5 and 12 years, affected by acute lymphoblastic leukaemia, were analyzed in sections and freeze-fracture replicas. The testicular biopsies were performed at the end of therapy, when patients were in continuous remission for over 30 months. Chemotherapy does not seem to affect the development of junctions that were studied in sections and freeze fracture. Two age groups were considered (I, 5 to 8 years; II, 9 to 12 years). In age group I, oval Sertoli cells were connected by occasionally focal points of fusion, which in replicas appeared as scattered, interrupted ridges on the P face and grooves on the corresponding E face. In age group II Sertoli cells presented cytoplasmic extensions and interdigitations. Tight junctions appeared close to one another in conventional sections. Freeze fracture evidenced extensive although isolated areas formed by intervining strands. Lanthanum penetrated freely the intercellular spaces and gap junctions were observed in both age groups. The results suggest that tight junctions formation is initiated long before puberty; a progression in the complexity of the strand organization is present as the tubules mature; the strands reorganize in parallel and continuous rows only at puberty.

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.

Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding 125I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived.(ABSTRACT TRUNCATED AT 250 WORDS)

We report observations on the behavior of Sertoli cells in sparse culture during the period from the time of plating to the time of initial confluence (the transitional remodeling phase). Changes in shape, structure, and polarity of cells, as well as changes in migration patterns and cell-cell association patterns, have been followed during the transitional remodeling phase with the aid of topographical markers. These markers are based upon differences between ultrastructural features of the basolateral and apicolateral surfaces. The basolateral surface is characterized by plasmalemmal blebs, whereas the apicolateral surface is characterized by filopodial extensions. Structural differences observed in situ remain evident in Sertoli cells isolated by sequential enzymatic treatments that are described. Another marker is provided by laminin-binding sites, which are detected exclusively on the blebbed, basolateral surfaces of freshly prepared Sertoli cell aggregates. The orientation described is sustained during the initial radial migration of Sertoli cells explanted on uncoated glass coverslips. Under these conditions, blebs are detected only on the dorsal surfaces, and filopodial extensions are evident only on the ventral surfaces. In contrast, Sertoli cells sparsely plated on a reconstituted basement membrane (air-dried Matrigel) migrate rapidly, display an extraordinary capacity to form elaborate cytoplasmic extensions for cell-cell and cell-substratum contacts, and readily retract blebs and filopodial extensions. These cells do not form mosaic borders, whereas cells plated on uncoated glass do form a monolayer with mosaic-like borders. Cells sparsely seeded on gelated Matrigel migrate preferentially at gaps between adjacent cell explants, and develop a compact cell-cell association pattern. These cells display few, if any, cytoplasmic extensions. We compare the behavior of Sertoli cells sparsely plated on Matrigel with the behavior of Sertoli cells in situ during different stages of development.

Immunohistological studies on the localization of inhibin in human testes were carried out to investigate the cell types involved in inhibin synthesis/storage. The paraffin embedded testicular tissues were stained by an indirect immunoperoxidase technique. Specific antiserum to human testicular inhibin generated against the homogenous preparation of human testicular inhibin at our laboratory was used as the first antibody. Inhibin was found to be localized in Sertoli cells, spermatogonia, and primary spermatocytes. A weak positive reaction was also observed in spermatids. Interestingly, Leydig cells showed positive reaction indicating presence of inhibin in these cells too.

The combination of a progestin and androgen has received attention as a possible male contraceptive. The progestin is thought to reduce gonadotropin release and suppress spermatogenesis, while the sex accessory organs and male characteristics are maintained by the simultaneous administration of testosterone. In the present study, the histology and ultrastructure of parts of the male reproductive tract of rats treated with medroxyprogesterone (Provera, Upjohn) (1 mg/100 g body weight/day) alone and combined with testosterone (15, 30, or 100 mug/100 g/day) were studied following treatment for up to 16 weeks. The testes and epididymides of rats administered Provera alone or Provera and testosterone weighed less than those of control rats. The weights of the accessory glands of rats treated with Provera were greatly reduced; it was possible to maintain them at approximately control levels by simultaneously administering sufficient testosterone (100 mug/100 g body weight/day). The fertility of some of the animals was tested by caging them with female rats, and none of the treated rats tested in this way was fertile. Similar microscopic alterations were present in the testes of animals administered Provera alone or Provera and different levels of testosterone. Spermatogonia, spermatocytes, and early spermatids were abundant in treated rats and did not show ultrastructural changes. However, many degenerating or necrotic spermatids of the cap phase (approximately stages 6-7) and later were present. Late spermatids of the acrosome and maturation phases were rare. Some necrotic spermatids were surrounded by Sertoli cells, and parts of spermatids lay within lysosome-lyke structures in the cytoplasm of Sertoli cells. Many large lipid droplets were also present in Sertoli cells of treated rats. Leydig cells were smaller in treated animals than in control rats. The results suggest that germ cells can develop up to cap phase spermatids but then undergo degeneration. These alterations in spermatogenesis may be responsible in large part for the antifertility effect of the progestin and androgen combination. Some rats were permitted to recover following the end of treatment. The microscopic appearance of the testis returned to normal within three to six weeks, although epididymal alterations persisted in some animals six weeks after the end of treatment. By 9 to 12 weeks after the end of treatment the reproductive organs had a normal microscopic appearance in all the rats studied.