Elastic tissue in the limiting membrane of the human seminiferous tubule

The structure and distribution of elastic tissue were studied in the limiting membrane of the seminiferous tubule from normal human testis. The elastic and elastic-related fibers (oxytalan and elaunin) were recognized by their tinctorial and ultrastructural characteristics. The connective structures of the limiting membrane, including the fibrotubules and the amorphous material of the elastic system, were studied after tannic acid-glutaraldehyde fixation. Fibrotubules (oxytalan fibers) were observed in almost all intercellular spaces of the limiting membrane, closely related to the contractile cells; the elaunin fibers (patches of amorphous material surrounded by bundles of fibrotubules) were evident in the outermost layers. The function of this system of elastic tissue and myoid cells is discussed, considering the permeability membrane and the role of the myoid cells in the elastogenesis and contractility of the seminiferous tubule.     

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

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

Isolation of cyclic protein-2 from rat seminiferous tubule fluid and Sertoli cell culture medium

Cyclic Protein-2 (CP-2), a stage-specific secretory product of the rat seminiferous epithelium, has been isolated from seminiferous tubule fluid (STF) and Sertoli cell culture medium. Isolation from STF was accomplished by mixing STF with radiolabeled proteins secreted by Stage VI-VII seminiferous tubules and sequential fractionation of these proteins by hydroxylapatite, DEAE-agarose, and quaternary amine ion-exchange chromatography. Radiolabeled proteins were used to identify the chromatographic fractions that contained CP-2. Through use of these procedures, a highly purified preparation of radioinert CP-2 was obtained from seminiferous tubule fluid. Cyclic Protein-2 was also isolated from Sertoli cell culture medium, indicating that the Sertoli cell is its most likely source. Preliminary characterization of CP-2 was conducted. First, CP-2 appeared to be highly enriched in methionine. Second, the molecular weight of CP-2 was found to be 20,000. Third, analysis by reverse-phase hydrophobic chromatography indicated that CP-2 was relatively hydrophobic. We conclude that CP-2 is a small hydrophobic glycoprotein secreted in vivo and in vitro in a stage-specific manner by Sertoli cells.     

Spent media from immature seminiferous tubules and Sertoli cells inhibit adult rat Leydig cell aromatase activity

Adult rat Leydig cell aromatase activity is stimulated 2.5 fold by LH or dbcAMP. Spent media prepared from seminiferous tubules or Sertoli cells of immature rats depress both the basal and the LH stimulated estradiol syntheses (25 and 20% decreases, respectively). These inhibitory effects are further enhanced when FSH is added to the culture medium of seminiferous tubules or Sertoli cells. Rat serum as well as culture media from other cell lines are ineffective while seminiferous tubule media from other immature animals (mouse, guinea-pig, calf) inhibit the aromatase activity. This Sertoli cell factor is a heat stable protein (molecular weight greater than 10 kDa), different from the LHRH-like Sertoli cell compound, which acts on the aromatase activity at a step beyond the adenylate cyclase.     

Ultrastructural study of crystalloids in Sertoli cells of the normal,intersex and experimental cryptorchid swine

Spindle- or needle-shaped crystalloids are observed in Sertoli cells of the intersex and experimental cryptorchid swine in the light and electron microscopes. Small crystalloids are also observed in Sertoli cells of the normal swine only by electron microscopy. These crystalloids consist of fine filaments. The filaments are about 5 nm in diameter and arranged parallel to the long axis of the drystalloid. In cross sections of the crystalloid, the close backing of the filaments shows hexagonal arrays. The interfilamentous distance is about 5 nm. In all animals, bundles of short filaments, which are 5nm in diameter, are observed in the basal part of the Sertoli cells. Ultrastructural similarities among the crystalloids, the bundles of fine filaments, and the filamentous layer in the junctional specialization of the Sertoli cell are shown. These morphological similarities suggest that the crystalloids are formed by the aggregation of the bundles in the Sertoli cells of azoospermic testes.     

Temporal appearance and cyclic behavior of Sertoli cell-specific secretory proteins during the development of the rat seminiferous tubule

Electrophoretic and morphologic methods have been used to study the time course of [35S] methionine-labeled proteins accumulated in the incubation medium of rat fetal testes and seminiferous cords/tubules during their development. We have found that Sertoli cell-specific secretory proteins S70, S45 and S35 became progressively prominent as premeiotic, meiotic and postmeiotic spermatogenic events were established in the seminiferous tubules. In the sexually mature rat, S70, S45 and S35 were expressed in a spermatogenic stage-dependent manner. While S70, S45 and S35 were present in Stage VII-VIII, S45 and S35 were observed in Stages X and XIV. Neither S70, S45 nor S35 were detected in Stage IV. A relevant group of high molecular weight proteins, previously reported as characteristic products of cultured peritubular cells, accumulated in the incubation medium of seminiferous cords from postnatal Day 0-15 rats. This group of high molecular weight proteins appears when peritubular cells are proliferative and are engaged in the organization of the seminiferous tubular wall. A low molecular weight protein, designated T35, was also detected. T35 was prominent in the medium of incubated fetal testes and seminiferous cords of postnatal rats 0- to 5-days-old and disappeared gradually thereafter. A set of proteins (designated SP1 and SP2) previously ascribed to both cultured Sertoli and peritubular cells, were recognized during the early postnatal stages of seminiferous tubular development. SP1 and SP2 displayed age-dependent fluctuations in their [35S] methionine labeling. The timing of appearance of S70, S45, and S35 indicates both age- and spermatogenic stage-related activity that, in the future, may prove to be functionally significant in the spermatogenic process.     

The arrangement of germ cells in the rat seminiferous tubule: an electron-microscope study.

The spermatogonia and early spermatocytes of 13 samples of rat seminiferous epithelium (about 0-05 mm2 each) were mapped from electron micrographs of serial sections. Clones of cells, connected by cytoplasmic bridges (syncytia of 2-100 cells), in various stages of spermatogenic development were identified. Maps of 7 separate areas are illustrated. It is concluded that, contrary to the models of spermatogonial proliferation based on light-microscope observations, regions of seminiferous epithelium which are identical in terms of spermatid and spermatocyte criteria have, in fact, quantitative and qualitative differences in their spermatogonial population. The data are interpreted that for a given epithelial area there is a periodic build-up of spermatogonia which then produce several successive quanta of spermatocytes and when the spermatogonia are depleted the process repeats. That cell numbers less than double following a mitotic cycle has generally been attributed to systematic degeneration. Evidence from electron microscopy indicates, however, that at the mitotic peaks not all the syncytia undergo division but that some remain arrested. Similarly, within a dividing syncytium a few cells do not divide while they advance developmentally with the syncytium as a whole. The observed large size of spermatocyte syncytia further argues against systematic degeneration with its attendant fragmentation of syncytia.     

Fine structure of the transitional zone of the rat seminiferous tubule

An electron microscopic study was made on the structure of the testicular transitional zone (TZ) in the adult rat. The TZ proper consists of modified Sertoli cellss, with only a few spermatogonia and macrophages, surrounding distally a very narrow lumen. The TZ Sertoli cells have nuclei with a somewhat coarser matrix and more peripheral heterochromatin than Sertoli cell nuclei of the nearby seminiferous tubules, and the electron density of the cytoplasm varies from cell to cell. Smooth endoplasmic reticulum is abundant, but usually there are also scattered ribosomal rosettes and an occasional profile of rough endoplasmic reticulum. Microtubules are very numerous in the columnar portion of the cell, and laminar structures seemingly joining the cell surfaces are sometimes seen. Lipid droplets and lysosmal structures are frequent cellular components in proximal TZ Sertoli cells. Empty intracellular vacuoles are abundant, sometimes arranged around areas of smooth endoplasmic reticulum. Occasionally, membrane-limited fine granules and vacuoles are seen within Sertoli cells and also in the TZ lumen, suggesting a possible secretory activity by these cells. The apical processes of the Sertoli cells form large vacuolar structures, and in the basal parts of the epithelium vacuoles with capillary-like appearance are frequently seen. Phagocytosis of germinal cells by the Sertoli cells occurs in the proximal region of the TZ. Round waste bodies in contact with the Sertoli cell apices protruding into the tubulus rectus, are also common. The tunica propria of the TZ is thickened and somewhat wrinkled, and in the proximal region the myoid cell layer loses its continuity and is replaced by fibroblasts. The epithelium of the tubulus rectus adjacent to the TZ consists of several overlapping epithelial cells. The typical junctional complexes between TZ Sertoli cells appear to be impermeable to the lanthanum tracer.     

Morphometric study of the prepubertal rabbit testis: germ cell numbers and seminiferous tubule dimensions

Testes from rabbits aged 1-9 weeks were examined by light microscopy. Changes in seminiferous tubule dimensions, testicular volume, and volume fraction of tubules were assessed. Germ cells and Sertoli cells were counted in round tubular cross sections and total germ cell number in each testis was estimated. Mitotic, meiotic, and degenerative activities of germ cells as well as their basal or central positions within tubules were quantified. A marked, steady increase in testis volume and in tubular length and volume occurred over the prepubertal period; but diameter underwent no significant increase and in fact decreased until week 4. Overall, tubules lengthened 40-fold and testis volume increased 25-fold; the percentage volume of the testis occupied by tubules rose from one-third neonatally to three-fifths at the onset of spermatogenesis. The ratio of germ cells to total tubular (germ and Sertoli) cells was lowest at 3 weeks. However, the total number of germ cells increased little until 3 weeks, after which it rose at a sharp rate commensurate with testis volume. Percentage of germ cells in mitosis peaked sharply at 3 weeks, dropped in subsequent weeks, and then rose at 7 weeks at the initiation of spermatogenesis. Importantly, the surge in mitosis at 3 weeks was followed by a redistribution of germ cells to a predominantly basal location from 3 to 7 weeks. Meiotic activity was sparse at 7 weeks and became abundant by 9 weeks. Germ cell degeneration remained relatively constant during weeks 1 through 6, with an increase at 7 weeks.     

Non-classic androgen actions in Sertoli cell membrane in whole seminiferous tubules: effects of nandrolone decanoate and catechin

Studies show a mechanism of action of testosterone, nandrolone and catechin as agonists of the membrane androgen receptor. The aim of this work is to investigate the non-classical effect of androgens and catechin in Sertoli cells from immature rats. The membrane potential of Sertoli cells in whole seminiferous tubules was recorded using a standard single microelectrode technique. It was performed a topical application of testosterone (1 μM), nandrolone (0.1, 0.5 and 1 μM) and the flavonoid catechin (0.1, 0.5 and 1 μM) alone and also after infusion with flutamide (1 μM), diazoxide (100 μM) or U73122 (1 μM). The immature testes were incubated for 5 min in KRb with (45)Ca(2+), with or without nandrolone (1 μM). The results were given as mean±SEM. The data were analyzed using ANOVA for repeated measures with Bonferroni post-test. Testosterone produces a depolarization in the membrane potential at 120 s after application. Catechin (1 μM) and nandrolone (1 μM) have shown a similar response to testosterone: depolarization at 120 s after the application. The same response of catechin and nandrolone was observed at different doses. The effects of testosterone, catechin and nandrolone were not affected after perfusion with flutamide. Perfusion with diazoxide and U73122 nullified the effect of nandrolone (1 μM) and catechin (1 μM). Nandrolone and testosterone increased (45)Ca(2+) uptake with or without flutamide within 5min. These results indicate that nandrolone and catechin act through a receptor on the plasmatic membrane, as well as testosterone, showing a non-classical pathway in Sertoli cells from immature rat testes.     

Content of K+ and Na+ in seminiferous tubule and rete testis fluids from Sertoli cell-enriched testes

Seminiferous tubules of rats exposed to x-irradiation before birth were subjected to micropuncture in situ at 50 days of age to obtain samples of fluid 4 h after ligation of efferent ducts. The concentrations of cations in this fluid were: potassium, 39.7 +/- 1.2 mM, and sodium, 136.3 +/- 1.2 mM (means and standard errors, n = 5). Histologic examination revealed that germ cells constitute less than 1% of the cell population within the seminiferous tubules of these rats; the remaining cells were all Sertoli cells. Sertoli cells showed efflux of 86Rb+ with t1/2 of approximately 11 min and an active ATPase in plasma membranes. These activities were similar to those of Sertoli cells from normal rats. Germ cells from normal rats showed less rapid efflux of 86Rb+ (t1/2 greater than 60 min) and less active Na+/K+ ATPase in plasma membranes. It is concluded that Sertoli cells are responsible for the high concentration of potassium in seminiferous tubule fluid and that plasma membranes of these cells contain an active K+ pump that is not inhibited by ouabain (1 mM).     

Estradiol and testosterone inhibit rat seminiferous tubule development in a hormone-specific way

Follicle stimulating hormone (FSH), testosterone (T) and estradiol (E₂) are known to regulate testis maturation, and changes in FSH secretion induced by sex steroid treatment may mediate the effects of sex hormones. The aim of this study was to compare the effects of T and E₂ on the pre-meiotic steps of first spermatogenesis and FSH level in rats. Male rat pups were injected daily with 17β-estradiol benzoate (EB; 12.5 μg) or testosterone propionate (TP; 2.5 mg) with the use of one of the two administration modes: 1/transient mode; hormone injections on postnatal days (PND) 1–5 followed by daily vehicle injections until PND 15 (t-EB and t-TP, respectively) or 2/continuous mode; hormone injections on PND 1–15 (c-EB and c-TP, respectively). The control group was injected with vehicle alone. On PND 16, blood was taken for serum hormone measurement and testes were collected for analysis of seminiferous tubule morphometry as well as cell number, proliferation and apoptosis. Testis weight, tubule length, Sertoli and germ cell numbers were reduced, and cell apoptosis in seminiferous epithelium was increased after transient EB and TP treatments. Despite normal or increased FSH secretion, the c-EB treatment inhibited pre-meiotic germ cell development and augmented cell apoptosis, whereas the c-TP treatment reduced the spermatocyte number and inhibited the formation of seminiferous tubule lumen. In conclusion, transient administration of EB or TP during PND 1–5 inhibited testis growth, whereas continuous administration (PND 1–15) impaired pre-meiotic germ cell development in a hormone-specific way.     

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.     

Apoptosis and cell removal in the cryptorchid rat testis

In order to determine that apoptosis is responsible for large-scale germ cell elimination, we analyzed cells from cryptorchid testes both in histological sections and among those isolated in vitro. Apoptotic testicular cells during 3 to 7 days were only 8 to 30%, reaching a maximum of 80% by the end of 15 days of cryptorchidism. A similar trend was also observed with the number of dead cells. The process of large-scale germ cell removal in the initial stages was facilitated by the formation of multinucleated giant cells, which stained negative for apoptosis. Increase in oxidative stress and decrease in intratesticular testosterone was also observed. The above findings indicate that large-scale germ cell removal, at least during initial stages of cryptorchidism is not solely as a result of apoptosis. Declined intra testicular testosterone, elevated temperature and high oxidative stress following cryptorchidism probably affect cell viability and trigger a fast pace cell removal through giant cell formation.     

Paracrine effect of seminiferous tubule factors on rat Leydig cell testosterone production: Role of cytoskeleton

In Percoll purified Leydig cells from mature rat we have demonstrated that the basal testosterone production (9.5 ng/10⁶ Leydig cells/24 h) is increased 10-fold in presence of a saturating amount of hCG (1 IU/mL) and diminished in a dose-related manner when larger concentrations of gonadotropin are used to reach 14 ng/10⁶ Leydig cells for 50 IU of hCG. If 40% (v/v) seminiferous tubule medium (STM) is added together with hCG (1 IU/mL) to the incubation medium, a further increase (62%) of testosterone output is noticed. Obviously, when the testosterone production is low as a consequence of a higher dose of hCG (50 IU/mL), the STM (80%) improves the steroid synthesis five-fold (67.4 ng). Concerning the cytoskeletal components (microtubules, intermediate filaments and microfilaments) which have been examined in presence or absence of hCG and STM, we have found a rearrangement of cytoskeletal elements as well as cell-shape changes in relation with hormonal activity of the cells. The most prominent alterations of cytoskeletal elements have been observed after 24 h of incubation with 1 IU/mL of hCG added together with 80% of STM. The obtained results suggest that paracrine factor(s) presents in STM and acting in synergy with LH/hCG generate(s) the rearrangement of cytoskeletal structures which, in turn, facilitates the availability of cholesterol for the mitochondria and finally enhances the testosterone production in the rat Leydig cells.     

Morphometrical analysis of Sertoli cell ultrastructure during the seminiferous epithelial cycle in rats

Stage-dependent variations of Sertoli cell organelles during the seminiferous epithelial cycle were analyzed morphometrically in rats by use of a point-counting method. Cyclic changes in volume and surface area of various organelles were observed. Mitochondria and rough endoplasmic reticulum increased at stages VII-VIII and stage VII, respectively. Lipid droplets accumulated markedly after spermiation, decreased rapidly after meiotic division, and remained at low levels for stages IV-VIII. The most prominent change was a topographic alteration of the Golgi apparatus. It was usually located exclusively in the basal cytoplasm, but shifted upwards to the mid and apical cytoplasm at stages VII-VIII. This shift may be implicated in an increase of plasma membrane and lysosomes in these regions. Consecutive increases of primary and secondary lysosomes were observed twice in the basal and mid cytoplasm. The first peak of the primary lysosomes at stage IV was followed by the first peak of the secondary lysosomes at stage VI; and the second peak of the primary lysosomes at stages VII-IX was followed by the second peak of the secondary lysosomes at stage IX. These consecutive increases may indicate that Sertoli cells anticipate the increase of structures to be removed and accordingly produce primary lysosomes before their appearance.