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Transitional cells at the junction of seminiferous tubules with the rete testis of the rat: their fine structure, endocytic activity, and basement membrane.

Transitional cells line the intermediate region of rat seminiferous tubules situated between the rete testis and the seminiferous epithelium proper. These tall elongated cells orient themselves in a downstream direction and converge on one another distally in the lumen of the rete testis where they form a distinct papillalike structure through which a narrow patent lumen is apparent. In addition to widely dispersed Golgi apparatus and mitochondria, these cells contain an abundance of microtubules, cisternae of endoplasmic reticulum, and a distinct lobulated nucleus showing clumps of chromatin and a prominent nucleolus. The endocytic activity of these cells was examined by employing adsorptive (cationic ferritin, concanavalin A ferritin) and fluid-phase tracers (native ferritin, horseradish peroxidase-colloidal gold complex, and concanavalin A ferritin in presence of alpha methyl-D-mannoside). Such tracers were injected separately into the lumen of the rete testis, and the animals were killed at 2, 5, 15, and 30 min and 1, 2, and 6 hr after injection. At 2 min, both adsorptive and fluid-phase tracers were found within coated and uncoated pits of the apical plasma membrane of these cells as well as in large, subsurface, uncoated spherical, C-shaped, and tubular membranous elements. At 5 min the tracers were seen in endosomes of different sizes; while at 15 min and 30 min, pale and dense multivesicular bodies of small and large sizes, respectively, were labeled. At 1-hr and longer time intervals secondary lysosomes became labeled. While both fluid-phase and adsorptive tracers followed the same pathway and fate, binding to the apical and lateral plasma membranes of the transitional cells and to the membrane delimiting coated and uncoated pits was observed only with the adsorptive tracers. These results demonstrate that the transitional cells are actively involved in both fluid-phase and adsorptive endocytosis, which may play an important role in modifying the composition of the luminal fluid. The transitional cells of the distal zone of the intermediate region rest on an elaborate basement membrane (BM) complex which includes a thin BM immediately underlying these cells, a thick distal layer of BM, and strands of BM spanning the distance between the two in the form of a loose anastomotic network. Use of antisera against heparan sulfate proteoglycan, laminin, and type IV collagen revealed the presence of all three components within all areas of the BM complex. In the meshes of the anastomotic BM network, extracellular vesicles were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Microvasculature of the human testis and excurrent duct system. Resin-casting and scanning electron-microscopic studies.

The vascular microarchitecture of the human testis and excurrent duct system was examined by use of a casting method combined with scanning electron microscopy. In the testicular parenchyma, the capillaries form a fine plexus around the seminiferous tubules. The commonly described rope-ladder-like pattern of the peritubular capillaries is absent in the human testis. The microvasculature of the rete testis consists of a poorly organized plexus. Throughout the epididymis including the ductuli efferentes, the peritubular capillary network encircles each tubule cylindrically. In the cauda epididymidis, a coarse venous network surrounds the capillary network externally. Small arteries of the epididymis not only penetrate the connective tissue septa, but also run along the surface. In the ductus deferens, three distinct microvascular systems are distinguished: (1) a venous network located in the connective tissue surrounding the muscular coat, (2) a microvascular plexus within the muscular coat, and (3) a peritubular capillary network. The plexus within the muscular coat interconnects the venous and the peritubular capillary networks.

Adult↗

Observations on the blood-testis barrier in a frog and a salamander.

A blood-testis barrier has been demonstrated in a frog, Rana esculenta, and in a salamander, Salamandra salamandra, using lanthanum as an electron-dense marker during fixation. The tracer penetrates the interstices between somatic follicle or Sertoli cells and germ cells in regions of the testis containing spermatogonia and spermatocytes, up to the level of punctate tight junctions. The latter can be localized between the somatic cells that line seminiferous units containing spermatids and mature spermatozoa. The barrier thus appears to be established after meiosis in both species investigated, although spermatids of different developmental stages can be found in open compartments of the testis in S. salamandra.

Animals↗

Viability of ram testicular spermatozoa following cryopreservation in rete testis fluid.

Ram testicular spermatozoa, collected continuously from the cannulated testis, were frozen in rete testis fluid in straws using the cryoprotective agents egg yolk and glycerol. The effect of cryopreservation on the viability of the spermatozoa was assessed by studying their metabolism, morphology, ultrastructure, and radioiodination patterns. Freeze-thawing significantly depressed the respiration rate and glycolytic activity of testicular spermatozoa. Morphologically, there was little evidence of cryodamage in frozen-thawed testicular spermatozoa. Except for some slightly corrugated acrosomes and a more loosely attached plasma membrane over the sperm head, frozen-thawed testicular spermatozoa were indistinguishable from nonfrozen control spermatozoa. Surface radioiodination of frozen-thawed testicular spermatozoa was highly selective and resulted in a labeling pattern similar to that of the nonfrozen controls. In contrast, the radiolabeling pattern of frozen-thawed electroejaculated spermatozoa was characterized by high background radioactivity and low selectivity. These results confirm previous suggestions that testicular spermatozoa have a greater low-temperature tolerance than do ejaculated spermatozoa and indicate that cryopreservation of immature testicular spermatozoa in rete testis fluid with added egg yolk and glycerol may be a useful approach to extend the availability of these cells.

Animals↗

Testis physiology relevant to immunoregulation.

Intra-testicular transplants are placed in rodents into the large lymphatic sinusoids of the interstitial tissue of the testis. These sinusoids are filled with a protein-rich extracellular fluid that supplies all the requirements of the grafts until vascularization takes place. The testicular microvascular endothelium regulates transport of T lymphocytes and immunoglobulin G to the testis and may thus contribute to regulation of the immune system in this organ. Differences in the organization of the lymphatic drainage exist between species, but in every studied species lymphatic drainage from the testis leads to lymph nodes.

Animals↗

Cytokines and junction restructuring during spermatogenesis--a lesson to learn from the testis.

In the mammalian testis, preleptotene and leptotene spermatocytes residing in the basal compartment of the seminiferous epithelium must traverse the blood-testis barrier (BTB) at late stage VIII through early stage IX of the epithelial cycle during spermatogenesis, entering the adluminal compartment for further development. However, until recently the regulatory mechanisms that regulate BTB dynamics remained largely unknown. We provide a critical review regarding the significance of cytokines in regulating the 'opening' and 'closing' of the BTB. We also discuss how cytokines may be working in concert with adaptors that selectively govern the downstream signaling pathways. This process, in turn, regulates the dynamics of either Sertoli-Sertoli tight junction (TJ), Sertoli-germ cell adherens junction (AJ), or both junction types in the epithelium, thereby permitting TJ opening without compromising AJs, and vice versa. We also discuss how adaptors alter their protein-protein association with the integral membrane proteins at the cell-cell interface via changes in their phosphorylation status, thereby altering adhesion function at AJ. These findings illustrate that the testis is a novel in vivo model to study the biology of junction restructuring. Furthermore, a molecular model is presented regarding how cytokines selectively regulate TJ/AJ restructuring in the epithelium during spermatogenesis.

Blood-Testis Barrier↗

Selected other problematic testicular and paratesticular lesions: rete testis neoplasms and pseudotumors, mesothelial lesions and secondary tumors.

The proximity and, in some instances, communication between several structures in the testis and paratestis (rete testis, epididymis, mesothelium, vestigial epithelium and paratesticular soft tissue) result in a plethora of interesting tumors and tumor-like lesions that together pose a formidable diagnostic challenge both because of their morphologic overlap and rarity. The occasional spread of tumors primarily at other sites to this region adds to the potential problem encountered. This review provides an overview of the pathology of nonmesenchymal paratesticular neoplasms and pseudotumors with a focus on the approach to tubulopapillary neoplasms for which diagnostic considerations may include carcinoma of the rete testis, malignant mesothelioma, ovarian-type epithelial tumors, epididymal carcinoma and metastatic carcinomas. The cornerstone of accurate characterization of these lesions is still a comprehensive, traditional clinicopathologic approach, clinical history (of another primary), gross examination (location) and routine light microscopy, but judicious incorporation of contemporary immunohistochemical markers may aid or in some instances be crucial in resolving the problems encountered.

Adenocarcinoma↗

Functional relationships of the mammalian testis and epididymis.

The development of knowledge in four areas of research in male reproductive physiology of particular interest is reviewed. The concept of the blood-testis barrier (BTB), which arose following dye exclusion from the seminiferous tubules, has now been established as the differential transfer from interstitial fluid to tubular and rete testis fluids of molecules of physiological importance. The composition of fluid collected mostly from the rete testis of several species, not only reflects the nature of the barrier, but also the secretory capacity of the Sertoli cell. The functional significance of the transfer of molecules into testicular fluid and the composition of the fluid flowing into the epididymis are discussed. Sertoli cells establish the structural basis of the BTB during puberty and divide the seminiferous epitheliuym into basal and adluminal compartments. The Sertoli cell is the prime target for follicle stimulating hormone (FSH). The responses evoked by FSH are discussed, including special mention of androgen binding protein (ABP) and the protein hormone, 'inhibin', with FSH-suppressing properties. The control of FSH in the lamb is mentioned including new evidence to support a tubular source of a feedback agent with significance during the impuberal stage. Finally, some of the biochemical properties of the epididymis and its fluid contents are reviewed and the epididymal sperm are identified as the site of the antifertility action of the 6-chloro-6-deoxy sugars.

Animals↗

Localization of tritiated 1-(2, 4-dichlorobenzyl)-1H-indazole-3-carboxylic acid ([3H] AF 1890) in rat testis using freeze-drying autoradiography.

Tritiated 1-(2, 4-dichlorobenzyl)-1H-indazole-3-carboxylic acid ([3H]AF 1890) was localized within rat testis using freeze-drying autoradiographic techniques. Ligated seminiferous tubule preparations were exposed in vitro to 80 microCi of [3H]AF 1890 in the absence or presence of excess unlabeled AF 1890 for 30 min at 31 degrees C. Autoradiographs showed a generalized distribution of silver grains over the seminiferous epithelium, in the tubular lumen, and in the interstitial spaces. In addition, clusters of grains were concentrated over the seminiferous epithelium in regions of spermatid heads and tails and in the basal portion of the Sertoli cell cytoplasm, and over Leydig cells in the interstitial space. The generalized distribution was reduced and the grain clusters eliminated by incubation in an excess of unlabeled AF 1890. [3H]inulin was used to assess the effectiveness of the tubular ligation because inulin does not normally penetrate the blood-testis barrier. This extracellular marker was not localized in the tubular lumen or in the seminiferous epithelium. Therefore, the labeling observed with [3H]AF 1890 indicates that this compound crosses the blood-testis barrier.

Animals↗

Estrogen receptor alpha has a functional role in the mouse rete testis and efferent ductules.

Previous studies of the estrogen receptor-alpha knockout (alpha ERKO) in the male mouse demonstrate that the rete testis and efferent ductules are targets of estrogen. Because the alpha ERKO mouse lacks a functional estrogen receptor alpha (ER alpha) throughout development, it was not known whether the morphological and physiological abnormalities observed in the alpha ERKO male were due to developmental defects or to dysfunctions concurrent with the lack of ER alpha in the tissue. This study was designed to determine if treatment of normal wild-type (WT) mice with the pure antiestrogen, ICI 182,780, (ICI) could reproduce the morphological characteristics seen in alpha ERKO mice. Thirty-day-old male mice were treated for 35 days with either castor oil or ICI. Age-equivalent alpha ERKO mice were used for comparison. Light microscopic examinations of the reproductive tracts revealed dramatic changes in the efferent ductules of treated mice: a 1.7-fold increase in luminal diameter, a 56% reduction in epithelial cell height, a 60% reduction in brush boarder height of nonciliated cells, and an apparent reduction of the number of observable lysosomes and endocytotic vesicles. Testes of ICI-treated mice showed swollen rete testes area (6.5 times larger than control) and a 65% reduction in rete testis epithelium height. However, there were no significant changes in body and testis weights. These results indicate that ER blockage with ICI in WT mice results in morphological changes of the efferent ductules resembling those seen in alpha ERKO siblings of the same age. Based on this study, we conclude that ER alpha has a functional role in the mouse reproductive tract and the aberrant morphology observed in the efferent ductules of the alpha ERKO mouse is likely the result of a concurrent response to the lack of functional ER alpha, and not solely due to the lack of ER alpha during early developmental times.

Animals↗

Effect of triolein emulsion on the blood-testis barrier in cats.

PURPOSE: Although the blood-testis barrier (BTB) is beneficial physiologically, it acts as an obstacle to chemotherapy. We wished to determine whether a triolein emulsion could alter the permeability of the BTB in cats. MATERIALS AND METHODS: An emulsion of 0.05-mL triolein in 20 mL of saline was infused into the abdominal aorta just proximal to the branching of the renal arteries of each of 15 cats (embolic group). As controls, 12 cats were each similarly infused with 20 mL of normal saline (control group). Pre and postcontrast MR images were obtained 30 minutes and 2 hours after embolization. The images were analyzed for the presence and degree of contrast enhancement (qualitative analysis), and the contrast enhancement ratios (CER) derived from one testis of each animal were compared and statistically analyzed by the mixed linear model (quantitative analysis). RESULTS: Contrast enhancement of the testis was observed in both groups at both time points, but it was more prominent in the embolic group. The CERs in the embolic group (0.63 at 30 minutes, 0.42 at 2 hours) were significantly higher than in the control group (0.36 at 30 minutes, 0.28 at 2 hours; P = 0.0001). In each group, the CERs at 2 hours were significantly lower than those at 30 minutes (P = 0.006). CONCLUSION: Intra-arterial infusion of triolein emulsion increased the permeability of the BTB. This result may be useful in future studies of BTB disruptions caused by fat emulsions.

Animals↗

The connection between the seminiferous tubules and the rete testis in the domestic fowl (Gallus domesticus). Morphological study.

The tubules connecting the seminiferous tubules proper to the rete testis in the fowl were studied with the aid of light and electron microscopy. The material examined ultrastructurally was fixed by vascular perfusion through the thoracic aorta. The seminiferous tubules were joined to the rete testis in three different ways; they were either linked by a terminal segment and a tubulus rectus, by a terminal segment only, or opened directly into the rete cavities. The terminal segment of the seminiferous tubules was lined with columnar cells (modified Sertoli cells). These cells were characterized by having an indented nucleus with a prominent nucleolus, many mitochondria, a sizable Golgi apparatus, electron dense bodies and many cytoplasmic protrusions into the lumen. Intraepithelial lymphocytes as well as macrophages in the lumina of the terminal segment, the tubuli recti and the rete testis were also observed. Myoid cells were found in the boundary tissue of the terminal segment.

Animals↗

Ultrasonography revealing the accumulation of rete testis fluid in bull testicles.

Ultrasonography was used for diagnosing accumulation of rete testis fluid in live bulls. When 447 bulls of Ayrshire breed selected for artificial insemination were studied by ultrasonography, eleven were detected in which the ultrasonograms visualized accumulation of rete testis fluid in the testicles. In 7 bulls the accumulation (spermiostasis) was bilateral and in 4 unilateral. In only four out of 18 testicles, macroscopically epididymal cysts were seen after slaughter. At the age of 16 months four bulls with bilateral spermiostasis were azoospermic. When studied by ultrasonography, the rete testis of normal testicles was echogenic and narrow but in affected testicles anechogenic and wide. Nine of the affected bulls were sons of three fathers. We suggest that spermiostasis is hereditary in Ayrshire bulls and, as in the goat, is progressive by nature.

Animals↗

Regulation of blood-testis barrier dynamics: an in vivo study.

An in vivo model was used to investigate the regulation of tight junction (TJ) dynamics in the testis when adult rats were treated with CdCl(2). It was shown that the CdCl(2)-induced disruption of the blood-testis barrier (BTB) associated with a transient induction in testicular TGF-beta2 and TGF-beta3 (but not TGF-beta1) and the phosphorylated p38 mitogen activated protein (MAP) kinase, concomitant with a loss of occludin and zonula occludens-1 (ZO-1) from the BTB site in the seminiferous epithelium. These results suggest that BTB dynamics in vivo are regulated by TGF-beta2/-beta3 via the p38 MAP kinase pathway. Indeed, SB202190, a specific p38 MAP kinase inhibitor, blocked the CdCl(2)-induced occludin and ZO-1 loss from the BTB. This result clearly illustrates that CdCl(2) mediates its BTB disruptive effects via the TGF-beta3/p38 MAP kinase signaling pathway. Besides, this CdCl(2)-induced occludin and ZO-1 loss from the BTB also associated with a significant loss of the cadherin/catenin and the nectin/afadin protein complexes at the site of cell-cell actin-based adherens junctions (AJs). An induction of alpha(2)-macroglobulin (a non-specific protease inhibitor) was also observed during BTB damage and when the seminiferous epithelium was being depleted of germ cells. These data illustrate that a primary disruption of the BTB can lead to a secondary loss of cell adhesion function at the site of AJs, concomitant with an induction in protease inhibitor, which apparently is used to protect the epithelium from unwanted proteolysis. alpha(2)-Macroglobulin was also shown to associate physically with TGF-beta3, afadin and nectin 3, but not occludin, E-cadherin or N-cadherin, indicating its possible role in junction restructuring in vivo. Additionally, the use of SB202190 to block the TGF-beta3/p-38 MAP kinase pathway also prevented the CdCl(2)-induced loss of cadherin/catenin and nectin/afadin protein complexes from the AJ sites, yet it had no apparent effect on alpha(2)-macroglobulin. These results demonstrate for the first time that the TGF-beta3/p38 MAP kinase signaling pathway is being used to regulate both TJ and AJ dynamics in the testis, mediated by the effects of TGF-beta3 on TJ- and AJ-integral membrane proteins and adaptors, but not protease inhibitors.

Adherens Junctions↗

The effect of rete testis fluid on the metabolism of testicular spermatozoa.

The O2 uptake of spermatozoa collected from the rete testis of conscious rams and boars has been measured polarographically either in the rete testis fluid (RTF) in which they were collected or in a synthetic saline medium (S-RTF) made to resemble the ionic composition of RTF as closely as possible. In both species, O2 uptake of the testicular spermatozoa was higher in RTF than in S-RTF. The addition of some of the organic constituents of RTF was without effect on O2 uptake. Pyruvate, lactate, acetate or alanine increased the O2 consumption of ram spermatozoa in S-RTF. Lactate had no effect on but acetate increased the O2 consumption of pig testicular spermatozoa. Phospholipid phosphorus and fatty acids decreased during incubation of ram spermatozoa in RTF and S-RTF. The addition of substances which increased the O2 consumption of the spermatozoa in S-RTF was without effect on the lipid composition. The fatty acid composition of the phospholipids and the proportions of the various phospholipids were unaffected by incubation. These results indicate that rete testis fluid has an appreciable effect on the metabolism of the spermatozoa it contains, and that this effect cannot be mimicked by any of the known organic constituents of the fluid.

Animals↗

Seasonal variations in rete testis fluid secretion and sperm production in different breeds of ram.

Production of spermatozoa and secretion of rete testis fluid (RTF) in rams was assessed by a rete testis cannulating technique. Four breeds (Ile-de-France, Romanov, Préalpes du Sud and cross-breed Romanov) were studied throughout the year. Inhibitory effects of the cannulation process on spermatogenesis were observed for some animals. Between-breed differences were found in sperm concentration and flow rate of the RTF. The seasonal variations in the daily sperm production of the testis were more pronounced for Ile-de-France rams than for the other breeds. There was a seasonal variation in the flow rate of RTF in Ile-de-France rams, the minimum flow being in February (winter) and the maximum in August-September (autumn).

Animals↗

Testosterone concentration in the fluids of seminiferous tubules, the interstitium and the rete testis of the rat.

Testosterone was measured by radioimmunoassay in interstitial fluid, 'free-flow' seminiferous tubular fluid, obtained by micropuncture, and rete testis fluid from intact adult anaesthetized rats. Under non-stimulated conditions the concentration of testosterone in interstitial fluid was below the limit of detection in two rats and achieved a mean level of 150 +/- 27 (S.E.M.) ng/ml in the remaining 17 determinations. The testosterone concentration of the seminiferous tubular fluid was below the limit of detection in two rats, and had a mean level in the remaining 15 determinations of 91 +/- 14 ng/ml, which is significantly lower (P less than 0-02) than that in interstitial fluid. The mean ratio of seminiferous tubular:interstitial fluid testosterone concentration calculated in 14 rats was 0-94 +/- 0.24. This ratio was less than unity whenever the interstitial fluid testosterone concentration was more than 50 ng/ml, whereas in all animals with interstitial fluid testosterone of 50 ng/ml, or less, the ratio was greater than or equal to one. The mean testosterone concentration of rete testis fluid in 32 samples was 33 +/- 3 ng/ml. After HCG stimulation in 12 rats, testosterone concentration in interstitial fluid increased to a mean value of 660 +/- 83 ng/ml, and in seminiferous tubular fluid to 460 +/- 44 ng/ml; the difference between the two was significant (P less than 0-05). These results are discussed in relation to the presumed dilution of seminiferous tubular fluid in rete testis fluid and the role of androgen-binding proteins in the transport of steroids.

Animals↗

Dynamin II interacts with the cadherin- and occludin-based protein complexes at the blood-testis barrier in adult rat testes.

In adult rat testes, blood-testis barrier (BTB) restructuring facilitates the migration of preleptotene spermatocytes from the basal to the adluminal compartment that occurs at stage VIII of the epithelial cycle. Structural proteins at the BTB must utilize an efficient mechanism (e.g. endocytosis) to facilitate its transient 'opening'. Dynamin II, a large GTPase known to be involved in endocytosis, was shown to be a product of Sertoli and germ cells in the testis. It was also localized to the BTB, as well as the apical ectoplasmic specialization (apical ES), during virtually all stages of the epithelial cycle. By co-immunoprecipitation, dynamin II was shown to associate with occludin, N-cadherin, zonula occludens-1 (ZO-1), beta-catenin, junctional adhesion molecule-A, and p130Cas, but not nectin-3. An in vivo model in rats previously characterized for studying adherens junction (AJ) dynamics in the testes by adjudin (formerly called AF-2364, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide) treatment was used in our studies. At the time of germ cell loss from the seminiferous epithelium as a result of adjudin-induced AJ restructuring without disrupting the BTB integrity, a significant decline in the steady-state dynamin II protein level was detected. This change was associated with a concomitant increase in the levels of two protein complexes at the BTB, namely occludin/ZO-1 and N-cadherin/beta-catenin. Interestingly, these changes were also accompanied by a significant increase in the structural interaction of dynamin II with beta-catenin and ZO-1. Beta-catenin and ZO-1 are adaptors that structurally link the cadherin- and occludin-based protein complexes together at the BTB in an 'engaged'state to reinforce the barrier function in normal testes. However, beta-catenin and ZO-1 were 'disengaged' from each other but bound to dynamin II during adjudin-induced AJ restructuring in the testis. The data reported herein suggest that dynamin II may assist the 'disengagement' of beta-catenin from ZO-1 during BTB restructuring. Thus, this may permit the occludin/ZO-1 complexes to maintain the BTB integrity when the cadherin/catenin complexes are dissociated to facilitate germ cell movement.

Adherens Junctions↗