Ultrastructural studies on the boundary tissue of the seminiferous tubules of different mammals

The aims of our studies were to compare the ultrastructure of the boundary tissue of seminiferous tubules of various mammals (rat, mouse, hamster, guinea pig, rabbit, ram, bull and man). Visual analysis of electron micrographs revealed the similarity of structure of all layers at investigated animals. The boundary tissue consists of 4 layers: 1) amorphous inner lamina, 2) cellular inner lamina, 3) amorphous outer lamina, 4) cellular outer lamina. The outer lamina of boundary tissue of rat, mouse and hamster revealed in histochemical reactions meshes resembling honey-combs. The wall of seminiferous canalicules of bull and ram consists of more bigger and different structure than one at the other laboratory animals. The most different structure of boundary tissue in man was observed. The capillary vessels penetrate in the myofibroblastic layer, when comparted to that found in other mammals on the surface of the wall.     

The lamina propria of the bovine seminiferous tubule

Summary The boundary tissue of bovine testicular seminiferous tubules exhibits remarkable regional differences at the level of the seminiferous tubule proper, as compared with its terminal segment. The basal lamina of the seminiferous tubule proper is multilayered and possesses knob-like protrusions. At the level of the terminal segment the basal lamina is highly specialized; in the region of the terminal plug candelabrum-like projections of the tubular basal lamina invade the bases of the modified supporting cells up to a depth of 3.5 m. The adjoining surface of these supporting cells is densely studded with hemidesmosomes. The elongated peritubular cells are arranged in 3–5 concentric layers around the tubulus seminiferus proper but form a loose association at the level of the terminal segment. Where the terminal segment joins the testicular straight tubule, peritubular cells may assemble to constitute a contractile spiral. Elastic tissue is situated mainly subjacent to the tubular basal lamina and to a lesser degree between the peritubular cell layers. A peritubular space lined by endothelium-like cells may surround the seminiferous tubule proper and also the transitional zone of the terminal segment.Supported by a grant from the Deutsche Forschungsgemeinschaft     

Evidence for protein absorption from the lumen of the seminiferous tubule and rete of the rat testis

As luminal fluid moves from the seminiferous tubule and enters the rete testis, its protein concentration declines from approximately 6 mg/ml to 1 mg/ml. It was therefore suggested that protein is either 1) utilized by the spermatozoa, 2) transported across the epithelium of the terminal segment of the seminiferous tubule, the tubuli recti or rete testis, or 3) absorbed and degraded by the epithelium. Horseradish peroxidase (HRP), a protein marker, was microperfused into single seminiferous tubules or perfused directly into the rete. After fixation, the HRP was localized histochemically and the tissue observed under the light- and electron microscope. HRP was taken up via pinocytotic vesicles into the cytoplasm of the Sertoli cells and germ cells but did not permeate extracellularly beyond the tight junctions. Similar results were obtained in the cells lining the terminal segment and the tubuli recti. The rete epithelium showed uptake of HRP into coated and noncoated vesicles, while some cells additionally revealed diffuse cytoplasmic distribution of HRP. The terminal segment, tubuli recti, and rete testis may be important routes by which proteins may leave the testicular fluid either to be degraded or to enter the blood.     

Studies on the testis of the camel (Camelus dromedarius). III. Histochemical observations

Synopsis The histochemical localization of carbohydrates and lipids and some oxidative, hydrolytic and steroid-linked enzymes has been studied in the testis of the camel with particular reference to the effect of the season on the distribution of these substances. PAS-positive, but diastase-resistant, material was seen mainly in the wall of blood vessels and in the boundary tissues of the seminiferous tubuli recti and rete testis. Clear cyclical changes were seen for glycogen in the lining epithelium of the seminiferous tubules. Glycogen was most abundant in early stages and very scanty or absent in the late stages of the cycle of the seminiferous epithelium. Numerous small lipid droplets were seen in the interstitial cells and towards the lumen of the seminiferous tubules that contain elongate spermatids or spermatozoa. Large lipid droplets were also demonstrable in the basal layer of the seminiferous epithelium and in the cytoplasmic debri. Alkaline phosphatase was demonstrated in the boundary tissues of the seminiferous tubules, tubuli recti and reti testis and in the cells bordering the lumen of the seminiferous tubules. Succinate and lactic dehydrogenases showed similar patterns of distribution in the interstitial elements and intratubularly.⁵-3 hydroxysteroid dehydrogenase was exclusively demonstrated in the interstitial cells. 17-hydroxysteroid dehydrogenase could not be demonstrated. The season seems to have no effect on the distribution of all these substances. The possible significance of all these findings is discussed.     

Histochemical and structural characteristics of the testis of the vampire bat (Desmodus rotundus rotundus, Geoffrey, 1810)

The testicular stroma of the vampire bat including the testicular capsula and the lamina propria of the seminiferous tubuli, was strongly PAS-positive. This observation was a possible indication of great amounts of structural glycogen and other glycoconjugates at the level of smooth muscle cells; elongated contractile cells and/or collagen frameworks of the tunica albuginea and tubular lamina propria. In the last the basement membranes of the seminiferous tubules were particularly strongly PAS positive, as an indication of their neutral mucosubstances structural composition, previously described (Malmi et al., 1987). The epithelium lining from the cavitary and surface rete testis complex showed low reactivities to mucosubstances; total proteins and lipids and oxidative enzymes studied. Although the apical granulation at the rete testis epithelium showed an intense PAS reactivity with hypothesis of glycoprotein secretion, through the rete. The PAS, Sudan Black B, NADH, MDH and LDH reactions of the testicular interstitium seem correlate to steroid metabolism (biosynthesis and secretion), at the Leydig cells level. The seminiferous epithelium generally had low reactions to all the histochemical studies realized. Particularly in the adbasal compartment the histochemical localizations of NADH diaphorase and LDH were possible related to glycolytic activities and general carbohydrates metabolism, both enzymes, and hydrogen transport, the NADH. The strong PAS, diastase and PAS, and alcian blue pH 2.5 and PAS reactions observed in the adluminal seminiferous epithelium compartment were directly related to the spermatids acrosomal glycoconjugates structuration. Also the SDH localization at this level seems to be related to the mitochondrial activities at the middle piece level in the late spermatids.     

Myoid cells in the peritubular tissue (lamina propria) of the reptilian testis

The arrangement and fine structure of peritubular myoid cells was studied in the testes of three species of reptiles (Lacerta dugesi, Testudo graeca and Natrix natrix) during two short periods of the seasonal cycle (European spring and autumn) and correlated with some ultrastructural properties of Leydig cells. The lamina propria consists of myoid cells, fibroblasts and non-cellular components comprising collageneous and non-striated microfibrils. Both components are arranged in alternating layers surrounding seminiferous tubules. In spring the lamina propria of lacertilian testis shows 1-5 layers of myoid cells which are rich in 50-70 A filaments and exhibit plasmalemmal and intracellular dense patches, smooth vesicles along the cell membrane and a concentration of organelles in a juxtanuclear position. Leydig cells are rich in smooth ER profiles and have few lipid droplets. In atumumn most myoid cells are replaced by fibroblast-like elements. Leydig cells display large numbers of lipid droplets and dense bodies, but only small amounts of agranular ER. Similar changes are noted in Leydig cells of Testudo and Natrix. However, in these species the boundary tissue of seminiferous tubules fails to show significant alterations comparing spring and autumn animals. In both species the lamina propria exhibits a few fibroblast-like cells interspersed among myoid cells.     

Morphologic study of the testes from spontaneous unilateral and bilateral abdominal cryptorchid boars

Macroscopical and histological characteristics were examined in both testes from three healthy boars, three boars with unilateral abdominal cryptorchidism on the right side, and three boars with bilateral abdominal cryptorchidism. Abdominal cryptorchidism, unilateral and bilateral, provoked a significant decrease of the weight and volume of the ectopic testes. The scrotal testis of the unilateral cryptorchid boars showed an increase in its volume and weight. Cryptorchidism also induced abnormalities in the histological structure of seminiferous tubules, lamina propria, and interstitial tissue of the abdominal testes. The number of seminiferous tubules decreased; the seminiferous epithelium was constituted by few spermatogonia with an atypical pattern and by abnormal Sertoli cells. The lamina propria showed a variable degree of thickening and collagenization. The interstitial tissue was very developed but displayed a decrease in the Leydig cell population. These abnormalities were more critical in bilateral cryptorchidism than in unilateral cryptorchidism. The scrotal testis of the unilateral cryptorchid boars showed normal appearance, but a decrease of the number of seminiferous tubules was observed. Moreover, the seminiferous tubules showed impaired spermatid maturation. The alterations observed in the abdominal testes of the unilateral and bilateral cryptorchid boars were attributed to defective proliferation and differentiation of Sertoli cells and Leydig cells. The anomalies in the scrotal testis of the unilateral cryptorchid boars were due to disturbances in the Sertoli cell activity.     

Quantitation and tissue localization of the cellular retinoic acid-binding protein

The distribution of the cellular retinoic acid-binding protein (CRABP) in some rat tissues has been determined, and the protein has been localized by immunocytochemical techniques in sections from rat testis. In the testis CRABP was found in the seminiferous tubuli with Sertoli cells and the spermatogonia most intensely stained. All other cells of the germinal epithelium appeared largely devoid of CRABP. By use of an enzyme-linked immunosorbent assay CRABP was quantitatively estimated in several tissues and the highest levels were found in testis and eye. Comparisons of the tissue levels of CRABP and of the cellular retinol-binding protein (CRBP) did not reveal any apparent correlation.     

Changes in the Peritubular Tissue of Rat Testis after Cadmium Treatment

The present study demonstrates histological and immunohistochemical changes in the peritubular testicular tissue of rat testis after application of cadmium chloride. After 5-day cadmium exposure, advanced deterioration of the boundary testicular tissue, mainly oedema, disarrangement of collagen fibres and peritubular cells, dilatation and thrombosis of blood vessels were observed. Changes in the boundary tissue were accompanied with desquamation of the germinal epithelium. Immunohistochemically, positive reaction for α-smooth muscle actin and desmin in tunica media of large testicular blood vessels basically was not affected. No reaction for vimentin was seen in endothelial cells of blood capillaries, whereas positive reaction presented only these cells in large blood vessels. The myofibroblasts positively reacting for desmin and α-smooth muscle actin form a single incomplete layer in the lamina propria of seminiferous tubules. Vimentin reactivity in the myofibroblasts and in the supporting Sertoli cells as well as Leydig cells in damaged testicular tissue was not observed. An increase in fibroblasts and free inflammatory cells positive for vimentin in the peritubular space on the peripheric area of the testis was observed.     

Intraepithelial lymphocytes in the male reproductive tract of rats and rhesus monkeys.

Although lymphocytes are never present in 'normal ' seminiferous epithelium, they are found in the terminal portions of the seminiferous tubles near their junctions with the tubuli recti. Intraepithelia lymphocytes are also found in the tubuli recti testis, ductuli efferentes, epididymis and ductus deferens. The ultrastructural morphology of these cells closely resembles that of the intraepithelial lymphocytes in the intestinal mucosa and those obtained from the lymph nodes, spleen blood and thoracic duct. The mucleus is spherical and is characterized by clumps of chromatin near the nuclear membrane. A thin rim of cytoplasm is usually found, and is remarkably free of most cell organelles except for free ribosomes. Frequently, a blunt cytoplasmic process can be seen extending from one end of the cell. Membrane-bounded granules and other dense bodies are occasionally encountered in the cytoplasm. The possible functional significance of intraepithelial lymphocytes in the male reproductive tract is discussed.     

Involution of seminiferous tubules in aged hamsters: an ultrastructural, immunohistochemical and quantitative morphological study

In this study, we examined the age-related changes on morphometric parameters and ultrastructure of seminiferous tubules, and on the expression of extracellular matrix proteins in lamina propria of Syrian hamsters. A significant decrease in the percentage of normal tubules and an increase in the percentage of hypospermatogenic and arrested maturation tubules was observed with aging. Aged animals showed a decrease in tubular diameter, tubular lumen, seminiferous epithelium volume and total tubular volume. However, the total length of seminiferous tubules was significantly increased with aging. The most important ultrastructural changes with aging were the thickening of the lamina propria, the presence of diverse abnormalities in the spermiogenesis process, degeneration of germ cells, and vacuolization and flattening of Sertoli cells showing abundant lipofucsin droplets and residual bodies. Laminin immunoreactivity was found along the lamina propria of seminiferous tubules both in young and aged animals. Fibronectin immunoreactivity was found along the lamina propria and blood vessels. Both laminin and fibronectin total volume of immunostaining per testis was increased in aged hamsters. In conclusion, the age-related changes in seminiferous tubules of hamster include: a decrease in tubular width and an increase in tubular length; widening of the lamina propria caused by a more extensive connective matrix between the peritubular cells and the basal membrane; and a strong disarrangement of the seminiferous epithelium, including germ cell degeneration and important alterations in both spermiogenesis and Sertoli cell structure.     

Concretions in the human testis are derived from the basal lamina of seminiferous cords

Summary We provide evidence that testicular concretions form exclusively in association with seminiferous cords. Testicular material obtained from 6 patients with various disorders of spermatogenesis was studied by means of light-and electron microscopy. Seminiferous cords in the testes examined form so-called hypoplastic zones. They contain immature Sertoli cells and are surrounded by an unusually thick basal lamina and one or two layers of myofibroblasts. They display extensive coiling and form ring-like structures. Concretions are located at the periphery or in the center of the cords and consist of a hyaline material with an occasional granular core. They are enveloped by Sertoli cells or myofibroblasts. Data presented indicate that concretions may develop within the above-mentioned ring-like structures or in deep indentations of the surrounding connective tissue. Computer-assisted 3-D reconstructions of a seminiferous cord based on 24 serial semithin sections revealec that the majority of concretions communicate with the interstitial tissue through a broad opening comparable to a meso. On the basis of our observations we could demonstrate that concretions are accumulations of basal lamina that occasionally contain remnants of degenerated myofibroblasts. They form outside the cords and belong to the connective tissue compartment. As the components of the basal lamina are thought to be produced by the interaction of Sertoli cells and myofibroblasts we tentatively assume that the development of concretions reflects a disturbance in this complex interaction. We suggest that the presence of concretions refers to a developmental disorder and may be pathognomonic for the occurrence of defective seminiferous cords.Supported by Deutsche Forschungsgemeinschaft (Ho 388/6-1) and Verein zur Förderung der Forschung auf dem Gebiet der Fortpflanzung e.V; Hamburg     

The fine structure of the terminal segment of the bovine seminiferous tubule

Summary The intratesticular excurrent duct system of the bull is composed of rete testis, tubuli recti, and the terminal segment of the seminiferous tubules. Each terminal segment is surrounded by a vascular plexus and may be subdivided into a transitional region, middle portion, and terminal plug. The modified supporting cells of the middle portion and the terminal plug no longer display the typical Sertoli-Sertoli junctions seen in the transitional region and the seminiferous tubule proper. In the region of the terminal plug a distinct central lumen is generally not observed: spermatozoa and tubular fluid must pass through an intricate system of communicating clefts between the apices of the closely attached modified supporting cells. Vacuoles in the supranuclear region of the cells in the middle portion indicate strong transepithelial fluid transport. In analogy to the epithelium of rete testis and tubuli recti, the supporting cells of the terminal segment are capable of phagocytosing spermatozoa. The vascular plexus investing the terminal segment serves a dual purpose: it is a regulatory device for fluid and sperm transport, as well as an area of increased diapedesis for white blood cells.Supported by a grant from the Deutsche Forschungsgemeinschaft     

Testis morphology in rats chronically treated with letrozole, an aromatase inhibitor

The aim of this study was to investigate the influence of the long-term treatment of rats with letrozole on the testis morphology. The pharmacologically induced estrogen deficiency caused statistically significant decreases of both intratesticular and serum levels of estradiol, and morphological changes in the seminiferous epithelium and in the interstitial tissue of the testes. Six months of treatment resulted in the sloughing of premature germ cells of the seminiferous epithelium into the tubular lumen and in intraepithelial vacuolization. Multinucleated giant cells composed of premature germ cells, conglomerates of various cell nuclei and cell debris as well as irregularities and infoldings of the tubular basement membrane were also seen. Moreover, deep invaginations of the lamina propria with myoid cells were observed. Cells in the interstitial tissue showed changes similar to that observed in aging processes. The cytoplasm of LH-R-positive Leydig cells was loaded with lipofuscin granules. The number of lipofuscin-loaded cells was significantly increased in the interstitial tissue of testis in letrozole-treated rats. The results indicate the direct influence of estrogens on seminiferous tubules and the interstitial tissue morphology.     

Ultrastructural study of the Sertoli cell and the limiting membrane in the seminiferous tubule of the adult cryptorchid rat

Cryptorchidism was simulated in 13-15-day-old rats by severing the gubernaculum testis and fixing the testis to the abdominal wall. Ultrastructural examination of the testis was made 100 days after birth when a number of modifications to the seminiferous tubules were noted. Germ cells were scanty, with only occasional spermatogonia and primary spermatocytes persisting. The nuclei of Sertoli cells were regular and oval or indented in shape. Their cytoplasm was characterized by a rich smooth endoplasmic reticulum, lipid inclusions and mitochondria with tubulo-vesicular cristae indicative of stero?dogenic activity. The decrease in the number of the germ cells induced a membrane rearrangement with numerous tight junctions and interdigitations between the Sertoli cells. Sertoli cell-specific junctional complexes were very extensive. The lamina propria of the seminiferous tubule appeared thickened and folded and the multilayered basal lamina had complex folds. After fixation with glutaraldehyde containing lanthanum, the latter substance was identified in the basal intercellular spaces of the seminiferous tubules indicating that the blood-testis barrier remains functional in the intra-abdominal testis.     

Testicular involution following optic enucleation

Summary The testes of adult male Syrian hamsters underwent involution within six weeks after optic enucleation. The diameter of the seminiferous tubules was 39% less than controls. Sertoli cells, spermatogonia, and primary spermatocytes were still present, but all steps of spermatids were completely absent from the involuted testes. Lipid droplets filled the Sertoli cell cytoplasm and often encroached upon the nucleus. Sertoli cells had sparse mitochondria and smooth endoplasmic reticulum, but Golgi cisternae were abundant. Typical SertoliSertoli junctions attached contiguous Sertoli cells. With lanthanum tracers it was demonstrated that these junctions were impenetrable; therefore, the bloodtestis barrier was deemed intact. Irregularly shaped protrusions often arose from the peritubular tissue and extended inward toward the seminiferous epithelium, often displacing the cytoplasm of the Sertoli cells and spermatogonia. The core of these protrusions consisted of irregular extensions of myoid cell cytoplasm surrounded by the myoid cells' basal lamina. External to the myoid cell basal lamina were bundles of collagen filaments with the basal lamina of the seminiferous epithelium forming the outermost layer of these protrusions. The apices of the Sertoli cells gave rise to numerous leaf-like processes that extended into and obliterated the lumen of the tubules. The Sertoli cell basal cytoplasm often contained phagocytized degenerating germ cells that appeared to give rise to the lipid droplets that filled the Sertoli cell cytoplasm. Acid phosphatase rich lysosome-like organelles were seen fusing with the degenerating germ cells and lipid droplets. The degenerating germ cells also were shown to contain acid phosphatase activity.     

The hypoxic testis and post-meiotic expression of PAS domain proteins

Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption. Because of the lack of blood vessels, the oxygen partial pressure in the lumen of the tubuli is very low. However, the consequences of these environmental conditions on spermatogenesis are unknown. The PAS domain is found in environmental protein sensors involved in the perception of oxygen partial pressure, light intensity, redox potentials, voltage and certain ligands. We previously identified two PAS proteins highly expressed in the testis: a novel isoform of the hypoxia-inducible factor (HIF)-1alpha and PASKIN, a PAS-Ser/Thr kinase related to bacterial oxygen sensing PAS-domain proteins.     

The epididymal region of the Japanese quail (Coturnix coturnix japonica).

The epididymal region of the Japanese quail was studied histologically. The organ consists of the extratesticular portion of the rete testis, the ductuli efferentes proximales and distales, the ducti conjugentes and ductus epididymidis. Distinct tubuli recti link the seminiferous tubules with the rete testis. The non-ciliated cells of the ductuli efferentes proximales and distales show, between them, certain internal structural differences which were highlighted. In 40% of the birds, the ductus deferens showed dark-grey pigments, regarded as melanin. The epididymal region was generally similar in structure to that of the domestic fowl, turkey and duck.     

Characterization of nuclear pore distribution in freeze-fracture replicas of seminiferous tubules isolated by transillumination.

Transilluminated seminiferous tubules were staged and utilized to determine the distribution of nuclear pore complexes in seminiferous tubules of the rat. Segments of seminiferous tubules of adult albino rats were separated and identified (in stages VII-VIII, IX-XI, XII-XIV, and V-VI), and then processed by freeze-fracture. Type A spermatogonia, the only spermatogonia located in seminiferous segments possessing stages IX-XI and XII-XIV, are oval cells in contact with the basal lamina. They either exhibit a random distribution of nuclear pores or a slight degree of clumping. Type B spermatogonia, found in segments possessing stages V-VI, exhibit, instead, a noticeable pore clustering. The identification of intermediate spermatogonia was not undertaken in this study. Preleptotene spermatocytes are easily identified in freeze-fracture by their location in segments with stages VII-VIII, by their arrangement in numerous groups between the basal lamina and the pachytene spermatocytes, and by their comparatively small size. They exhibit noticeable pore clustering. Leptotene (segments containing stages IX-XI) and zygotene (XII-XIV) spermatocytes show a more homogeneous distribution of nuclear pores. Pachytene spermatocytes are identified by their large size, by consistent detachment from the basal lamina and by being rather numerous and found in all the stages explored. Diplotene spermatocytes have the largest nuclei of all germ cells. They are always detached from the basal lamina and found only in seminiferous segments containing stage XIII. Pachytenes display a regular geometric array of pore aggregation with striking clustering, whereas diplotene nuclear pores takes on a random distribution. Secondary spermatocytes, only present in stage XIV intermingled with metaphase-anaphase profiles, are characterized in replicas by a paucity of evenly distributed nuclear pores.     

Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization

The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules.