Localization of epoxide-metablozing enzymes in rat testis

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much intensely for epoxide hydrolase and glutathione S-transferase B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to expoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.     

The distribution of sex-specific (H-Y) antigens within the seminiferous tubules of the testis: an immunohistochemical study

Summary The cellular distribution of H-Y antigen within the seminiferous tubules of testes from both 20-day-old and adult rats has been examined immunohistochemically. Large amounts of diffuse-staining material surrounding the germ cells were observed within the tubules of 20-day-old rats while the germ cells appeared to have little H-Y positive material on them. In the sexually mature rat, the seminiferous tubules contained cells at various stages of development. Peroxidase staining was evident on many, but not all of these cells. On spermatids and spermatozoa with cytoplasmic droplets attached, peroxidase staining appeared to be present in only a proportion of these cells.     

Inhibition of epoxide hydrolases and glutathione S-transferases by 2-, 3-, and 4-substituted derivatives of 4'-phenylchalcone and its oxide

4'-Phenylchalcones, chalcone oxides, and related compounds were synthesized and tested as inhibitors of cytosolic epoxide hydrolase, microsomal epoxide hydrolase, and glutathione S-transferases from mouse and rat liver. Several compounds were more potent inhibitors of the cytosolic epoxide hydrolase than the parent 4'-phenylchalcone oxide while large substituents in the 4- and especially the 2-position caused a reduction in inhibition. The chalcone oxides showed selectivity as inhibitors of the cytosolic epoxide hydrolase acting on trans-stilbene oxide, while chalcones were inhibitors of cytosolic glutathione S-transferase acting on cis-stilbene oxide. Data are consistent with the hypothesis that much of the inhibition of the glutathione S-transferase is caused by the glutathione conjugate of the chalcone.     

Structural and functional abnormality of ectopic testes in rats

Some males of a mutant strain of King-Holtzman rats exhibit an anomalous heritable defect manifested as either unilateral or bilateral ectopic testes. In the adult, these testes contain seemingly immature Sertoli and Leydig cells, seminiferous tubules greatly reduced in diameter, and exhibit arrested spermatogenesis. Thus, the affected testis is essentially sterile. An inability to produce normal amounts of testosterone and androstenedione by these gonads is probably a reflection of changes that have been effected in their Leydig cells. Thus, this study suggests that abnormal function of the Leydig and Sertoli cells and seminiferous tubule failure in these mutant animals result from the physiologically cryptorchid condition.     

Nuclear testicular 1,25-dihydroxyvitamin D3 receptors in Sertoli cells and seminiferous tubules of adult rodents

1,25(OH)2D3 receptors were studied in whole testes, Sertoli cells, seminiferous tubules, Leydig cells and spermatogonia of adult NMRI mice and SD rats. Specific reversible high affinity binding (KD 1.4 x 10(-10)M; Nmax 72 fmol/mg protein) by a 3.5 S macromolecule was demonstrated in whole testes, Sertoli cells and seminiferous tubules. With identical techniques, no receptors were found in Leydig cells despite previous reports of 1,25(OH)2D3 actions on Leydig cell function.     

Observations on the Leydig cells in the male East African spring hare (Pedetes surdaster larvalis).

The morphology of Leydig cells of the testis of sexually mature and sexually immature spring hares was studied. The cytoplasm of the Leydig of cells the sexually immature spring hares was packed with large lipid droplets leaving little space for the other organelles. Smooth endoplasmic reticulum was poorly developed and occasionally formed concentric layers of fenestrated cisterns around the large lipid droplets. The Leydig of cells the sexually mature spring hares were almost devoid of lipid droplets and their cytoplasm was occupied by abundant tubular smooth endoplasmic reticulum. Cells which shared characteristics with both immature Leydig cells and undifferentiated mesenchymal cells were observed in the limiting membrane of the seminiferous tubulus. These Leydig-like cells may play a role in the differentiation of Leydig cells in the spring hare.     

Location of enzymes of androgen and estrogen biosynthesis in the testis of the ground squirrel (Citellus lateralis)

The intratesticular localization of enzymes of androgen and estrogen biosynthesis was studied in the ground squirrel (Citellus lateralis). In mature animals, interstitium and tubules were isolated by manual dissection. Microsomes were prepared and enzymes assayed by analysis of product formation after incubation with appropriate 3H-labeled substrates. In the immature testis, tubules and interstitium are not readily separable; thus, distribution was inferred after analysis of whole testicular microsomes from control, follicle-stimulating hormone (FSH)-treated, and luteinizing hormone (LH)-treated animals. To verify the cellular composition of tissues and the status of steroidogenic organelles in Leydig and Sertoli cells, samples were also analyzed by light and electron microscopy. In mature squirrels, enzymes of androgen biosynthesis were concentrated in the interstitium; however, levels present in the tubules were sufficient to account for a substantial fraction of whole testicular activity (1/3 to 1/5). By contrast, virtually all of the testicular aromatase was accounted for by that in the seminiferous tubules. The purity of these fractions was checked by light microscopy; they showed little cross-contamination. In whole testicular microsomes of immature squirrels, androgen biosynthetic enzymes had a much lower specific activity than in mature animals; however, the opposite was true for aromatase, its activity being approximately 5-fold higher in prepubertal animals. Luteinizing hormone treatment markedly stimulated hydroxylase and lyase but not aromatase. Luteinizing hormone also induced an increase in Leydig cell size and a dramatic proliferation of smooth endoplasmic reticulum. These changes were correlated with increased serum testosterone. As shown previously in rats, 3 beta-hydroxysteroid dehydrogenase was independent of LH control. Follicle-stimulating hormone had no effect on any of the enzymes studied, but induced some increase of agranular reticulum in Sertoli cells. Results from immature squirrels thus corroborate data from mature animals, showing a predominant interstitial location of androgen biosynthetic enzymes. While we cannot explain the absence of FSH stimulation of aromatase activity, the data do not refute the findings in mature animals showing a predominant tubular location of this enzyme. We conclude that the distribution of steroidogenic enzymes in the testis of squirrels differs in several important respects from rats, although both are members of the order Rodentia.     

Stimulatory effect of follicle-stimulating hormone on rat Leydig cells

Summary The effects of FSH on the testicular interstitial tissue of immature hypophysectomized rats were studied by comparing morphological changes in Leydig cells with quantitative changes in interstitial tissue histology using morphometric analysis. Three groups of rats received subcutaneous injections of 0.5 ml saline vehicle or 10 g rFSH or 20 ng oLH (equivalent to the amount of LH known to contaminate the FSH), twice daily for 7 days. Administration of FSH significantly increased testis weight and stimulated more advanced spermatogenesis compared to saline or LH. Morphometric analysis of testes of LH-treated rats showed a small but significant increase in total interstitial cell volume compared to saline treatment. FSH caused much greater increases in the total volume of interstitial tissue and interstitial cells than either saline or LH and significantly increased the total volume of interstitial fluid by comparison with the other groups. FSH but not saline or LH treatment resulted in a striking hypertrophy of Leydig cells, to produce cells ultrastructurally identical to Leydig cells from adults. Since the target tissue of FSH is the seminiferous epithelium, the observed effects on Leydig cells by FSH treatment suggest that the secretion of factors by the seminiferous tubules may mediate the maturation of Leydig cells.     

Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree).

A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Peroxisomes and intracellular cholesterol trafficking in adult rat Leydig cells following Luteinizing hormone stimulation

The present study was designed to explore the intracellular cholesterol trafficking in Leydig cells of adult rats following Luteinizing hormone (LH) injection. Histochemical techniques were used to demonstrate distribution of free cholesterol in Leydig cells of control and LH-injected rats. Two groups of sexually mature male Sprague Dawley rats (n=4/group) were used. Fifteen min following an injection of 200 microl of either saline (control) or luteinizing hormone (LH, 500 microg in saline) testes of rats were fixed by whole body perfusion using 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer for 20 min. Fixed testes were cut into 3 mm3 and kept immersed in the fixative for further 15 min. Tissue cubes were then incubated at 37 degrees C in a medium containing cholesterol oxidase, 3,3'-diaminobenzidine tetrahydrochloride, horseradish peroxidase and dimethyl sulfoxide to histochemically localize free cholesterol in Leydig cells and processed for electron microscopy. Thin sections of these tissues were stained with aqueous uranyl acetate and lead citrate and examined with a Philips 201C electron microscope. In Leydig cells of control rats, free cholesterol was detected primarily in lipid droplets and plasma membrane. In the majority of Leydig cells, peroxisomes were unstained for free cholesterol, but occasionally few stained ones were present. Staining was not detected in mitochondria and smooth endoplasmic reticulum (SER) in Leydig cells of control rats. In LH-injected rats, lipid droplets, many peroxisomes, inner and outer mitochondrial membranes and some cisternae of SER in Leydig cells showed staining for free cholesterol. Fusion of Leydig cell peroxisomes with lipid droplets and mitochondria was also observed in the LH treated rats. These findings suggested that peroxisomes in adult rat Leydig cells participate in the intracellular cholesterol trafficking and delivery into mitochondria during LH stimulated steroidogenesis. Lipid droplets are used as one source for cholesterol for this process.     

Features of impaired seminiferous tubule differentiation are associated with germ cell neoplasia in adult men surgically treated in childhood because of cryptorchidism

Seminiferous tubule differentiation was related to the occurrence of germ cell neoplasia in 38 men, aged 17-47, treated surgically in childhood for cryptorchidism. Tissues from 46 testes obtained from biopsies taken as a neoplastic preventive procedure or whole testes removed because of GCT were evaluated quantitatively. Paraffin sections were treated with antibodies against placental like alkaline phosphatase (PLAP), a marker of germ cell neoplasia, and cytokeratin 18 (CK-18), a marker of immature Sertoli cells. Quality of spermatogenesis and number Leydig cells were assessed with a score count. Seminiferous tubules diameter, thickness of basal membrane and size of intertubular spaces were measured with image analysis software. In 17.4% of testes spermatogenesis was normal (9.9 points) (N) and neoplasia was not found there. In the other 38 specimens (83%) spermatogenesis was abnormal (A). When spermatogenesis was arrested or when germ cells were absent (3.7+/-1.8 points), neoplastic lesions were found in 13.1% of the specimens. In A group 5.1+/-7.1% of tubules contained immature Sertoli cells, while in N they were not found. Tubular diameter was significantly lower in A (161.5+/-31.8 microm) than in N (184.6+/-24.3 microm) and the percentage of seminiferous tubules with the thickening of tubular basal membrane was also greater in A. Intertubular spaces were significantly larger in A (49.9+/-18.6%) in comparison to N group (32.6+/-12.5%). Mean number of Leydig cells was similar in both groups. To conclude, in most of the formerly cryptorchid testes, despite surgical treatment, impaired seminiferous tubules differentiation is predominant. Germ cell neoplasia is present in testes with retarded seminiferous tubules differentiation. Retardation of seminiferous tubule differentiation consists of inhibited spermatogenesis, presence of tubules with immature Sertoli cells, decreased tubular diameter, increased thickness of basal membrane and enlarged intertubular spaces. Examination of testicular biopsy with respect to the state of seminiferous tubule differentiation may be helpful to predict the appearance of germ cell neoplasia in adult men with cryptorchidism in anamnesis. Orchiopexy of cryptorchid testes may not prevent the occurrence of features of testicular dysgenesis and the associated germ cell neoplasia.     

Stimulation of testicular ornithine decarboxylase activity by arginine vasopressin

Intratesticular injection with arginine vasopressin caused stimulation of ornithine decarboxylase activity in the testes of immature rats. The increase in ornithine decarboxylase activity in response to arginine vasopressin was dose and time dependent. Maximal stimulation of ornithine decarboxylase activity occurred at 2 h after injection with 0.1 micrograms of arginine vasopressin. It was observed that stimulation of ornithine decarboxylase activity occurred in seminiferous tubules and in Leydig cells of the testis in response to arginine vasopressin.     

The effect of prenatal treatment with busulfan on in vitro androgen production by testes from rats of various ages

Treatment of rats with busulfan in utero severely depletes the germ cell population of the seminiferous tubules. These studies have examined the in vitro capacity of testicular tissue and Leydig cells from such testes to secrete androgens. Leydig cells were identified by staining for 3 beta-hydroxy steroid dehydrogenase. Rats were studied at several ages to identify any developmental changes in the androgen-secreting capacity of control and treated gonads. At 30 days of age, no effect of treatment on serum androgen was found. At 60 and 90 days of age, treatment caused decreased androgen and increased LH content of the serum. At 12, 30, 60, and 90 days of age, the amount of androgen secreted per milligram of testicular tissue in response to LH was higher in busulfan-treated rats. Leydig cells from 60- and 90-day-old rats which had received busulfan were also hyperresponsive to LH. It was concluded that Leydig cells from testes essentially devoid of germ cells were hyperresponsive to LH. Serum androgen levels were decreased yet androgen production per Leydig cell was increased. A possible explanation of this apparent paradox is that busulfan treatment resulted in decreased numbers of Leydig cells in the gonads.     

Effect of nutrition, temperature and photoperiod on the rate of sexual maturation of the field vole (Microtus agrestis)

Weanling male and female field voles were placed in long or short photoperiods, kept at 18 degrees C or 4 degrees C, and fed (ad libitum) diets containing 24%, 16%, 8% and 4% protein, for 6 weeks. Animals in the long photoperiod were more sexually mature than were animals in the short photoperiod. Temperatures had no effect on females, but did affect males: those kept at 4 degrees C had heavier testes and wider seminiferous tubules than those kept at 18 degrees C. There was little difference between the animals on 24%, 16% and 8% protein diets. Animals on 4% protein diets had retarded growth rates and were significantly less sexually mature than those on the other 3 diets, males having smaller testes and seminal vesicles and narrower seminiferous tubules and females having smaller ovaries and uteri.     

Immunoexpression of androgen receptors and aromatase in testes of patient with Klinefelter's syndrome

Klinefelter's syndrome (47, XXY) is the most common chromosome aneuploidy in men and is usually characterized by underdeveloped testes and sterility. The aim of the present study was to detect cellular distribution of androgen receptors (AR) and aromatase in testes of patient with KS. The tissue sections were processed for morphological and immunohistochemical staining. Additionally, levels of FSH, LH, PRL, estradiol, and testosterone were measured in the plasma. Morphological analysis revealed a complete absence of spermatogenesis. No germ cells were present in seminiferous tubules. In some tubules, nests of apparently degenerating Sertoli cells were found. In the interstitium, Leydig cell hyperplasia was observed. Using immunohistochemistry, nuclear AR staining was detected in Sertoli cells and peritubular cells, whereas in Leydig cells the staining was exclusively cytoplasmic. The immunostaining of aromatase was detected in the cytoplasm of Sertoli cells and Leydig cells. Increased levels of gonadotropins and decreased level of testosterone concomitantly with the cytoplasmic localization of AR in Leydig cells might contribute to the impaired testicular function in patient with KS.     

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

Immunocytochemical localization of cytochrome P450 aromatase in the testis of prepubertal, pubertal, and postpubertal horses

The large amount of testicular estrogens produced by the stallion is unique compared to the amounts found in other domestic species. Although the cellular locale of the cytochrome P450 aromatase (P450arom) enzyme that converts C19 androgens to C18 estrogens has been identified in the Leydig cell of adult equine testis, the location in the immature equine testis is not known. The goal of this work was to localize the enzyme in colts and stallions during sexual development. Testes were obtained from prepubertal (n=7), pubertal (n=6), and postpubertal (n=8) colts and stallions during both the breeding and non-breeding seasons. Tissue was fixed and prepared for immunocytochemistry (ICC), carried out with an antiserum against human placental P450arom. In prepubertal colts, there was distinct immunopositive staining of a similar degree within both the Leydig cell and the seminiferous tubule. Horses in the pubertal group had strong Leydig cell immunopositive staining and a slight degree of positive staining within the seminiferous tubules. Postpubertal stallions exhibited definitive immunopositive staining within Leydig cells but not within the seminiferous tubules. Therefore, P450arom is present within the Leydig cell throughout sexual development. In contrast, the presence of P450arom within the seminiferous tubule based upon ICC appeared to be gone by adulthood, suggesting that an age-dependent shift in the locale of this enzyme as the stallion matures.     

双峰驼睾丸和隐睾细胞外基质相关蛋白的组织化学特征及超微结构比较

为探索细胞外基质相关蛋白在隐睾双峰驼的分布情况及其组织化学特征,应用电镜技术和多种组织化学方法比较了隐睾和正常睾丸的超微结构,组织化学特点及层粘连蛋白（LN）、Ⅳ型胶原（Col Ⅳ）和硫酸乙酰肝素糖蛋白（HSPG）的分布特征。结果显示：(1)与正常睾丸间质结构相比,光镜下隐睾生精小管发育不全,间质内胶原纤维稀疏,网状纤维分布明显,间质血管及生精小管固有膜PAS及AB-PAS阳性反应较弱。电镜下,隐睾生精上皮基膜明显增生,外围I型胶原纤维较少,管周肌样细胞不典型;间质毛细血管及Leydig细胞周围纤维细胞多见,而正常睾丸在间质毛细血管及Leydig细胞周围多分布有成纤维细胞。(2) 免疫组织化学染色显示,正常睾丸组织的Col Ⅳ、LN及HSPG在Leydig细胞内均为强阳性表达,Col Ⅳ和LN在毛细血管内皮细胞强阳性表达,后者在Sertoli细胞的表达尤为明显,HSPG在精原细胞无表达;隐睾时Col Ⅳ、LN及HSPG在Leydig细胞内阳性表达均明显减弱,Col Ⅳ、LN在管周肌样细胞及毛细血管内皮细胞阳性表达也减弱明显,HSPG在精原细胞较强阳性表达,且在精子细胞呈强阳性表达。免疫组织化学图像分析结果显示,双峰驼正常睾丸组织中Col Ⅳ和LN的分布显著高于隐睾组织（P<0.05）,HSPG检测结果在正常睾丸与隐睾之间无统计学差异（P>0.01）。该研究表明,双峰驼隐睾生精小管发育异常,间质组织中合成胶原纤维的能力下降,睾丸细胞外基质的重要成分Col Ⅳ,LN与正常组差异显著与生精小管及Leydig细胞异常发育有关,而HSPG在隐睾生精上皮的强阳性表达与精原细胞发育不成熟密切相关。