Bismuth uptake in rat testicular macrophages: a follow-up observation suggesting that bismuth alters interactions between testicular macrophages and Leydig cells.

Recent studies suggest that bismuth accumulates in Leydig cells. In addition, a reduced level of serum testosterone and a statistically significant reduction of Leydig cells have been observed. It was therefore hypothesized that Bi has a direct toxic effect on rat Leydig cells. We have now developed a method for double labeling of bismuth and ED-2 (a marker for testicular macrophages). The present data demonstrate that the heavily bismuth-loaded cells in rat testis, originally interpreted as being Leydig cells, are bismuth-loaded macrophages. Consequently, our data suggest a modified hypothesis regarding bismuth-induced interactions between testicular macrophages and Leydig cells.     

Development of cytoplasmic digitations between Leydig cells and testicular macrophages of the rat

Summary Testicular macrophages and Leydig cells from adult animals are known to be functionally coupled. For example, secreted products from macrophages stimulate testosterone secretion by Leydig cells. In adult rat testes, structural coupling also exists between these cells. This coupling consists of cytoplasmic projections from Leydig cells located within cytoplasmic invaginations of macrophages. Although macrophages are known to exist in the testis in immature animals, it is not known when these digitations develop. The purpose of the present study was to determine whether the time of their development coincides with known maturational events that occur in Leydig cells, particularly during the peripubertal period. Testes from rats at 20, 30 and 40-days-of-age as well as testes from mature rats weighing more than 500 gm were prepared for ultrastructural analysis. It was found that digitations form between 20 and 30-days-of-age. These structures varied from simple tubular projections to complicated branched structures, suggesting that digitations are more than simple invaginations of microvilli into coated vesicles as previously described. Subplasmalemmal linear densities were also observed within macrophages juxtaposed to Leydig cells. Collagen was commonly observed between macrophages and Leydig cells in animals 20 days old. These studies demonstrate that although macrophages are present in the testis in maximal numbers at 20 days-of-age, they do not form junctions with Leydig cells until day 30. This is just prior to the major increase in secretory activity of rat Leydig cells that occurs during puberty.     

Omega-3 fatty acids differentially modulate enzymatic anti-oxidant systems in skeletal muscle cells

During physical activity, increased reactive oxygen species production occurs, which can lead to cell damage and in a decline of individual’s performance and health. The use of omega-3 polyunsaturated fatty acids as a supplement to protect the immune system has been increasing; however, their possible benefit to the anti-oxidant system is not well described. Thus, the aim of this study was to evaluate whether the omega-3 fatty acids (docosahexaenoic acid and eicosapentaenoic acid) can be beneficial to the anti-oxidant system in cultured skeletal muscle cells. C2C12 myocytes were differentiated and treated with either eicosapentaenoic acid or docosahexaenoic acid for 24 h. Superoxide content was quantified using the dihydroethidine oxidation method and superoxide dismutase, catalase, and glutathione peroxidase activity, and expression was quantified. We observed that the docosahexaenoic fatty acids caused an increase in superoxide production. Eicosapentaenoic acid induced catalase activity, while docosahexaenoic acid suppressed superoxide dismutase activity. In addition, we found an increased protein expression of the total manganese superoxide dismutase and catalase enzymes when cells were treated with eicosapentaenoic acid. Taken together, these data indicate that the use of eicosapentaenoic acid may present both acute and chronic benefits; however, the treatment with DHA may not be beneficial to muscle cells.     

Control and production of leukotriene B4 in rat tumour and testicular Leydig cells.

Plant mitochondrial ATPase has been chloroform-solubilized and purified by gel filtration from spadices of cuckoo-pint (Arum maculatum). The subunit composition of purified plant and rat liver ATPase were compared by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The delta- and epsilon-subunits of the plant enzyme are larger than their supposed rat liver counterparts and, as such, A. maculatum mitochondrial ATPase shows structural homologies with the enzyme from Escherichia coli [Futai, Sternweis & Heppel (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2725-2729] rather than with the rat liver enzyme.     

Leydig cell involvement in the paracrine regulation of mast cells in the testicular interstitium of the rat

The accumulation of mast cells in the rat testicular interstitium was studied under different experimental conditions in order to correlate this accumulation with the alterations of specific testicular tissue compartments or cell types. Estrogen treatment was effective in inducing mast cell proliferation when administered on Day 1 or at higher doses at 10 days of age. Estrogens were ineffective beyond 20 days of age. Postnatal treatment of neonatal-estrogen-treated rats with FSH and LH prevented the appearance of mast cells. In contrast, treatment with the Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) was effective in inducing mast cell accumulation only when administered to adult rats, inducing small numbers of mast cells at 45 days of age; it was ineffective on 30-day-old rats. Hypophysectomy alone did not determine the appearance of mast cells. However, when atrophic Leydig cells were destroyed with EDS, high numbers of mast cells accumulated in the testis. These results support the existence of Leydig cell-related inhibitory factors for mast cells in the rat testicular interstitium.     

Aging but not dietary restriction alters the activation-induced apoptosis in rat T cells

The aim of this study was to determine if aging or dietary restriction (DR) alters activation-induced cell death, which is known to regulate cell proliferation and eliminate the high number of activated cells during an immune response. Splenic T cells were isolated from young (4-6 months) and old (25-26 months) Fischer 344 rats that had free access to food, ad libitum (AL), and from dietary-restricted (DR) old (25-26 months) rats that beginning at 6 weeks of age were fed 60% (40% food-restricted) of the diet consume by the AL rats. T cells were incubated with anti-CD3 antibody, or staphylococcal enterotoxin B (primary stimulus) for 72-96 h, followed by restimulation with anti-CD3 (secondary stimulus) for 72 h. Activation-induced apoptosis was assessed by DNA fragmentation and the expression of Fas/CD95 receptor and Fas ligand (Fas-L) was measured by flow cytometry. We found that the amount of DNA fragmentation was significantly (P<0.05) higher in the stimulated and restimulated T cells from AL old rats and DR old rats compared to young rats. The increase in DNA fragmentation with age was paralleled by an increase in the proportion of the cells expressing Fas and Fas-L. However, DR had no significant effect on the age-related increase in DNA fragmentation or the expression of Fas or Fas-L. We also measured the levels of Bcl-2 and Bax protein and found that the level of Bcl-2 decreased and Bax increased with age and that DR had no effect on the age-related changes in the level of Bcl-2 or Bax protein. These results demonstrate that aging but not DR alters activation-induced apoptosis in rat T cells.     

Fate of Leydig cells in testicular organ culture]

In organ culture experiments, the Leydig cells of mouse testes can still be recognized after 12 days at 31 degrees C. HCG binding sites are effectively demonstrated. Meanwhile, when HCG is added at the initiation of the cultures, it is selectively stored in the cytoplasm of the Leydig cells. These findings point to a fairly good survival of Leydig cells in our tissue culture system.     

Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adjudin-mediated germ cell depletion alters the anti-oxidant status of adult rat testis

Successful spermatogenesis is dependent on the proper attachment of developing germ cells to Sertoli cells. Manipulation of these interactions by drugs like Adjudin can hamper the development of germ cells and lead to conditions of temporary infertility. Although studies have shown the contraceptive potential of Adjudin, much is not known about its action in the testis. In this study, we sought to investigate the effect of Adjudin on the oxidative status of mammalian testis. Adult male rats were administered with a single dose of Adjudin (50 mg/kg body weight) by oral gavage and were killed at 1, 2, 4, 7, 15, or 30 days of treatment. Adjudin caused a significant increase in the production of hydrogen peroxide and lipid peroxidation from 4 to 7 days after treatment. There was a significant decrease in the activities of anti-oxidant enzymes superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase from 4 to 7 days after treatment with Adjudin. However, the state of oxidative stress was less pronounced from 15 to 30 days after Adjudin treatment. The level of androgen binding protein (ABP) remained unchanged following Adjudin treatment. These results show that there is an induction of oxidative stress accompanying adherens junction restructuring which suggests a role for reactive oxygen species in the regulation of these testicular junctions. However, transient elevation in reactive oxygen species levels did not affect androgen transport.     

Aging alters the skeletal response to disuse in the rat

Disuse has been shown to cause a rapid and dramatic loss of skeletal mass and strength in the load-bearing bones of young and mature animals and humans. However, little is known about the skeletal effects of disuse in aged mammals. The present study was designed to determine whether the skeletal effects of disuse are maintained with extreme age. Fischer 344/Brown Norway male rats (6 and 32 mo old) were hindlimb suspended (HS) or housed individually for 2 wk. Trabecular volume and microarchitecture in the proximal tibia were significantly decreased by HS only in young rats. HS significantly reduced cortical bone mineral density and increased cortical porosity only in old rats by inducing new pore formation. Cortical pore diameter was also increased in old rats, regardless of loading condition. Ex vivo osteogenic and adipogenic cultures established from each group demonstrated that age and HS decreased osteoblastogenesis. Age, but not HS, decreased sensitivity to endogenous bone morphogenetic protein stimulation, as measured by treatment with exogenous Noggin. Adipocyte development increased with age, whereas HS suppressed sensitivity to peroxisome proliferator-activated receptor-gamma-induced differentiation. Serum insulin-like growth factor I levels were reduced with HS in young rats and with age in control and HS rats. These results suggest that the site of bone loss due to disuse is altered with age and that the loss of osteogenic potential with disuse in the old rats may be due to the combined effects of decreased insulin-like growth factor I levels and sensitivity, as well as diminished bone morphogenetic protein production.     

An enzymatic method for the isolation of mouse Leydig cells

Mouse Leydig cells were obtained by dispersion of testes of adult animals (aged 6-15 months) with a neutral protease from B. polymxa (dispase; EC 3.4.24.4). The crude Leydig cell suspension was purified by centrifugation on a discontinuous Percoll gradient using a special centrifugation procedure similar to elutriation. The crude cell suspension obtained from 50 testes could be processed in one run. The combination of these two methods yielded 320000 +/- 53000 Leydig cells/testis (n = 554 testes). The purity of the Leydig cell fraction was greater than or equal to 95% (nucleated cells) based on morphological and histochemical (staining for naphthyl esterase) identification. The purified Leydig cells showed an excellent ultrastructural appearance. More than 98% excluded trypan blue. In the presence of NADPH, testosterone biosynthesis was increased only 1.15 +/- 0.1-fold yielding a "quality factor" of 34.8. Maximal hCG doses induced 10(6) purified Leydig cells to produce 5 nmol testosterone/hr. (40-fold stimulation in comparison to basal values). The Leydig cells showed 43100 +/- 2500 LH/hCG receptors and an association constant of Ka = 1.95 x 10(9) M-1. Due to the reproducibility of the method, to the yield as well as to the morphological and functional state of the purified Leydig cells at least 25% of laboratory animals could be saved.     

Characterization of mouse glandular kallikrein 24 expressed in testicular Leydig cells

Mouse kallikrein 24 is thought to encode a functional serine protease belonging to the mouse glandular kallikrein gene family. Preliminary results suggest that this kallikrein may play a role in testis function in adult mice. In order to obtain insights into its physiological functions, we undertook molecular and biochemical analyses of this enzyme. We cloned a cDNA for kallikrein 24 from the adult mouse testis cDNA library. Kallikrein 24 was expressed in the kidney, submandibular glands, ovary, epididymis, and testis of the mouse. In the testis, kallikrein 24 mRNA was detectable at 4 weeks of postnatal development, and became more prominent thereafter. The kallikrein 24 gene was expressed exclusively in the Leydig cells of adult mice. When Leydig cells isolated from a 2-week-old mouse testis were cultured in the presence of testosterone, kallikrein 24 expression was induced. Active recombinant enzyme showed trypsin-like specificity, favorably cleaving Arg-X bonds of synthetic peptide substrates. The enzymatic activity was strongly inhibited by typical serine protease inhibitors. Mouse kallikrein 24 degraded casein, gelatin, fibronectin and laminin. These results suggest that the enzyme may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis. The present findings should contribute to future physiological studies of this mouse testis protease.     

Age-related morphological and functional changes in the Leydig cells of the horse

Two ultrastructurally distinct types of Leydig cells were observed in the equine testis. Whereas the adult testis exhibited both postpubertal and adult Leydig cells, the testis of the pubertal horse contained only the postpubertal type, and that of the aged horse contained only the adult type. However, Percoll-purified testicular preparations from pubertal, adult, and aged horses all exhibited two distinct Leydig cell populations. The quantitative distribution and the functional characteristics of these Leydig cell populations (ability to bind human chorionic gonadotropin [hCG] and increase of testosterone production after hCG stimulation) evolved with the age of the horse. It is concluded that equine Leydig cells derive from two redundant successive postnatal generations and that there is no strict correlation between the functional properties and the morphological characteristics of these cells.     

Ca-dependent-chloride and potassium currents in rat Leydig cells

Voltage-clamped membrane currents have been investigated from whole-cell patch-clamp recordings performed on single Leydig cells isolated from the adult rat testis. Two outward membrane currents were evoked by depolarizing voltage steps. A potassium current was recorded in cells dialyzed with low (10(-9)-10(-8) M) calcium media. This current was decreased by TEA (10 mM). A chloride current was recorded in cells dialyzed with high (10(-7)-10(-6) M) calcium media. This current was decreased by an external exposure to glutamate. Comparison of the currents at low and high internal calcium concentrations suggests that an increase of the intracellular calcium activates a chloride current.     

Inter-relationship between testicular dysgenesis and Leydig cell function in the masculinization programming window in the rat

The testicular dysgenesis syndrome (TDS) hypothesis proposes that maldevelopment of the testis, irrespective of cause, leads to malfunction of the somatic (Leydig, Sertoli) cells and consequent downstream TDS disorders. Studies in rats exposed in utero to di(n-butyl) phthalate (DBP) have strongly supported the TDS concept, but so far no direct evidence has been produced that links dysgenesis per se to somatic cell dysfunction, in particular to androgen production/action during the ‘masculinization programming window’ (MPW; e15.5–e18.5). Normal reproductive tract development and anogenital distance (AGD) are programmed within the MPW, and TDS disorders arise because of deficiencies in this programming. However, DBP-induced focal testicular dysgenesis (Leydig cell aggregation, ectopic Sertoli cells, malformed seminiferous cords) is not evident until after the MPW. Therefore, we used AGD as a read-out of androgen exposure in the MPW, and investigated if this measure was related to objectively quantified dysgenesis (Leydig cell aggregation) at e21.5 in male fetuses exposed to vehicle, DBP (500 or 750 mg/kg/day) or the synthetic glucocorticoid dexamethasone (Dex; alone or plus DBP-500) from e15.5–e18.5 (MPW), e13.5–e20.5 or e19.5–e20.5 (late window). Dysgenesis was found only in animals exposed to DBP during the MPW, and was negatively correlated (R² = −0.5) with AGD at e21.5 and at postnatal day 8, irrespective of treatment period. Dysgenesis was also negatively correlated (R² = –0.5) with intratesticular testosterone (ITT) at e21.5, but only when treatments in short windows (MPW, late window) were excluded; the same was true for correlation between AGD and ITT. We conclude that AGD, reflecting Leydig cell function solely within the MPW, is strongly related to focal dysgenesis. Our results point to this occurring because of a common early mechanism, targeted by DBP that determines both dysgenesis and early (during the MPW) fetal Leydig cell dysfunction. The findings provide strong validation of the TDS hypothesis.