Immunolocalization of antioxidant enzymes in testis of rams submitted to long-term heat stress.

Long-term heat stress (HS) induced by testicular insulation generates oxidative stress (OS) on the testicular environment; consequently activating antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx). The aim of this work was to immunolocalize antioxidant enzymes present in different cells within the seminiferous tubule when rams were submitted to HS. Rams were divided into control (n = 6) and treated group (n = 6), comprising rams subjected to testicular insulation for 240 h. After the testicular insulation period, rams were subjected to orchiectomy. Testicular fragments were submitted to immunohistochemistry for staining against SOD, GR and GPx enzymes. We observed immunolocalization of GPx in more cell types of the testis after HS and when compared with other enzymes. In conclusion, GPx is the main antioxidant enzyme identified in testicular cells in an attempt to maintain oxidative balance when HS occurs.

Functional importance of palmitoyl protein thioesterase 1 (PPT1) expression by Sertoli cells in mediating cholesterol metabolism and maintenance of sperm quality.

Sertoli cells are a type of nurse cell in the seminiferous epithelium that are crucial for sustaining spermatogenesis by extending nutritional and energy support to the developing germ cells. Dysfunction of Sertoli cells could cause disordered spermatogenesis and reduced fertility in males. In this study, we focused on the expression and function of palmitoyl protein thioesterase 1 (PPT1), a lysosomal depalmitoylating enzyme, in Sertoli cells. Here, we show that PPT1 expression in Sertoli cells is responsive to cholesterol treatment and that specific knockout of Ppt1 in Sertoli cells causes male subfertility associated with poor sperm quality and a high ratio of sperm deformity. Specifically, Ppt1 deficiency leads to poor cell variably accompanied with abnormal lysosome accumulation and increased cholesterol levels in Sertoli cells. Further, Ppt1 deficiency results in poor adhesion of developing germ cells to Sertoli cells in the seminiferous epithelium, which is likely to be responsible for the reduced male fertility as a consequence of declines in sperm count and motility as well as a high incidence of sperm head deformity. In summary, PPT1 affects sperm quality and male fertility through regulating lysosomal function and cholesterol metabolism in Sertoli cells.

Deletion of DDB1- and CUL4- associated factor-17 (Dcaf17) gene causes spermatogenesis defects and male infertility in mice.

DDB1- and CUL4-associated factor 17 (Dcaf17) is a member of DCAF family genes that encode substrate receptor proteins for Cullin-RING E3 ubiquitin ligases, which play critical roles in many cellular processes. To unravel the function of DCAF17, we performed expression profiling of Dcaf17 in different tissues of wild type mouse by qRT-PCR and generated Dcaf17 knockout mice by gene targeting. Expression profiling of Dcaf17 showed highest expression in testis. Analyses of Dcaf17 transcripts during post-natal development of testis at different ages displayed gradual increase in Dcaf17 mRNA levels with the age. Although Dcaf17 disruption did not have any effect on female fertility, Dcaf17 deletion led to male infertility due to abnormal sperm development. The Dcaf17 -/- mice produced low number of sperm with abnormal shape and significantly low motility. Histological examination of the Dcaf17 -/- testis revealed impaired spermatogenesis with presence of vacuoles and sloughed cells in the seminiferous tubules. Disruption of Dcaf17 caused asymmetric acrosome capping, impaired nuclear compaction and abnormal round spermatid to elongated spermatid transition. For the first time, these data indicate that DCAF17 is essential for spermiogenesis.

Impaired spermatogenesis in the twitcher mouse: A morphological evaluation from the seminiferous tubules to epididymal transit.

Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa. In this developmental process the interactions between different cell types are finely regulated, hence any disruption in these relationships leads to male infertility. The twitcher mouse, the murine model of Krabbe disease, is characterized by deficiency of galactosylceramidase, an enzyme also involved in the metabolism of the galactosyl-alkyl-acyl-glycerol, the precursor of sulfogalactosyl-alkyl-acyl-glycerol, the most abundant glycolipid in spermatozoa. Twitcher mice are sterile due to alterations of spermatogenesis resulting in the production of spermatozoa with abnormally swollen acrosomes and bent flagella, mainly at the midpiece-principal piece junction. The current study employs light, fluorescence, and electron microscopy to examine the defective spermiogenesis leading to the morphological abnormalities of mature sperm. This study reveals that alterations in germ cell development can be initially detected at the stage VIII and IX of spermatogenesis. The disrupted spermatogenetic process leads to a reduced number of elongating spermatids and spermatozoa in these mutant animals. Electron microscopy analysis demonstrates major acrosomal and chromatin condensation defects in the mutants. In addition, in twitcher mice, the epididymal architecture is impaired, with stereocilia of caput and corpus broken, detached and completely spread out into the lumen. These findings indicate that seminolipid expression is crucial for proper development of spermatocytes and spermatids and for their normal differentiation into mature spermatozoa. ABBREVIATIONS: GALC: galactosylceramidase; GalAAG: galactosyl-alkyl-acyl-glycerol; SGalAAG: sulfogalactosylalkylacylglycerol; PND: postnatal day; PAS: periodic acid-Schiff stain; TEM: transmission electron microscopy; SEM: scanning electron microscopy; PFA: paraformaldheyde.

Ouabain interactions with the α4 isoform of the sodium pump trigger non-classical steroid hormone signaling and integrin expression in spermatogenic cells.

In addition to the ubiquitous α1 isoform of the sodium pump, sperm cells also express a male-specific α4 isoform whose function has been associated with sperm motility, fertility, and capacitation. Here we investigate in the murine spermatogenic cell line GC-2 interactions of the α4 isoform with the cardiotonic steroid ouabain in signaling cascades involved in the non-classical action of steroid hormones. Exposure of GC-2 cells to low concentrations of ouabain stimulates the phosphorylation of Erk1/2 and of the transcription factors CREB and ATF-1. As a consequence of this signaling cascade, ouabain stimulates on the mRNA level the expression of integrins αv, ß3 and α5, whose expression is also modulated by the cAMP response element. Increased expression of integrins αv and ß3 is also seen in cultures of seminiferous tubules exposed to 10nM ouabain. At the protein level we observed a significant stimulation of ß3 integrin expression by ouabain. Abrogation of α4 isoform expression by siRNA leads to the complete suppression of all ouabain-induced signaling mentioned above, including its stimulatory effect on the expression of ß3 integrin. The results presented here demonstrate for the first time the induction of signaling cascades through the interaction of ouabain with the α4 isoform in a germ-cell derived cell line. The novel finding that these interactions lead to increased expression of integrins in GC-2 cells and the confirmation of these results in the ex vivo experiments indicate that hormone/receptor-like interactions of ouabain with the α4 isoform might be of significance for male physiology.

Protective effects of grape seed extract on cadmium-induced testicular damage, apoptosis, and endothelial nitric oxide synthases expression in rats.

This study aims to evaluate the protective effect of grape seed proanthocyanidin extract (GSPE) on cadmium (Cd)-induced testicular apoptosis, endothelial nitric oxide synthases (eNOS) expression, and toxicity in rats. A total of 24 male Wistar rats were divided into four groups, namely, control, Cd (2.5 mg/kg), Cd + GSPE (100 mg/kg/day), and GSPE. Spermatogenesis and mean seminiferous tubule diameter were significantly decreased in the Cd groups. Furthermore, the GSPE-treated animals showed an improved histological appearance in the Cd group. The immunoreactivity of eNOS and the number of apoptotic cells were increased in Cd group. Our data indicate a significant reduction of terminal deoxynucleotide transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end-labeling staining and a decrease in the expression of eNOS in the testes tissue of the Cd group treated with GSPE therapy. Therefore, our results suggest that GSPE acts as a potent protective agent against Cd-induced testicular toxicity in rats.

Up-regulation of heme oxygenase-1 expression modulates reactive oxygen species level during the cryopreservation of human seminiferous tubules.

OBJECTIVE: To study the effect of freezing techniques and to optimize a method for trace amounts of testicular spermatozoa from biopsed seminiferous tubules. The level of reactive oxygen species (ROS) and the gene expression of heme oxygenase-1 was evaluated. DESIGN: Prospective experimental study. SETTING: University-based laboratory. PATIENT(S): Eighteen adults with male fator infertility underwent testicular biopsy surgery. INTERVENTION(S): Seminiferous tubular fragments from each man were evenly allocated to three groups: fresh control, slow freezing, and vitrifiaction groups. The morphology and ROS levels before and after freezing were evaluated for seminiferous tubular fragments. The expression of heme oxygenase-1 (HO-1) at both the transcriptional and protein levels was determined. MAIN OUTCOME MEASURE(S): The morphology was analyzed by light microscopy. The ROS levels were measured with ELISA. The proliferation and differentiation were evaluated by immunohistochemistry, and the expression of HO-1 was evaluated using a real-time polymerase chain reaction (PCR) and Western blotting. RESULT(S): Decreased ROS levels and increased HO-1 expression at the transcriptional and protein levels were observed after thawing the human seminiferous tubules. The ROS level was negatively correlated with HO-1 expression. Slow freezing was more effective than vitrification in terms of HO-1 up-regulation and ROS alteration. CONCLUSION(S): Based on our study, the slow freezing technique was more effective compared with the vitrification method.

Localization of cytochrome P450 and related enzymes in adult rat testis and downregulation by estradiol and bisphenol A.

There is a growing body of evidence that exposure to endocrine disrupting chemicals and to estrogenic compounds in particular can affect the testis and male fertility. In the present study, the constitutive expression of steroidogenic and non-steroidogenic cytochrome P450 (CYP) and related enzymes in adult rat testis, and their regulation by estradiol and bisphenol A, were investigated. CYP1B1, CYP2A1, NADPH-cytochrome P450 oxidoreductase (POR) and microsomal epoxide hydrolase (mEH) proteins, together with CYP17A1 and 3ß-hydroxysteroid dehydrogenase (HSD3B), were detected by immunoblot analysis in testicular microsomes prepared from untreated adult Sprague Dawley rats. In contrast, CYP1A, CYP2B, CYP2E, CYP2D, CYP2C, CYP3A, and CYP4A enzymes were not detected. Immunofluorescence staining of cryosections of perfusion-fixed testes showed that CYP1B1, CYP2A1, CYP17A1, and HSD3B were expressed exclusively or mainly in interstitial cells, whereas mEH and POR protein staining was detected both in interstitial cells and in seminiferous tubules. Testicular CYP1B1 and CYP2A1 protein levels were decreased following treatment of adult rats with estradiol benzoate at 0.004, 0.04, 0.4, or 4 µmol/kg/day or bisphenol A at 400 or 800 µmol/kg/day, for 14 days, whereas expression of HSD3B was unaffected. Testicular CYP17A1, POR, and mEH protein expression was also downregulated at the three highest dosages of estradiol benzoate and at both dosages of bisphenol A. The present study is the first to establish the cellular localization of CYP1B1, mEH, and POR in rat testis and to demonstrate the suppressive effect of bisphenol A on testicular CYP1B1, CYP2A1, mEH, and POR protein levels.

Glutathione peroxidase-3 produced by the kidney binds to a population of basement membranes in the gastrointestinal tract and in other tissues.

Glutathione peroxidase-3 (Gpx3), the extracellular glutathione peroxidase synthesized largely in the kidney, binds to basement membranes of renal cortical epithelial cells. The present study assessed extrarenal expression of Gpx3 using RT-PCR and presence of Gpx3 protein using immunocytochemistry. Gpx3 expression was higher in kidney and epididymis than in other tissues. Gpx3 bound to basement membranes of epithelial cells in the gastrointestinal tract, the efferent ducts connecting the seminiferous tubules with the epididymis, the bronchi, and type II pneumocytes. It was not detected on the basement membrane of type I pneumocytes. Gpx3 was also present in the lumen of the epididymis. Transplantation of Gpx3(+/+) kidneys into Gpx3(-/-) mice led to Gpx3 binding to the same basement membranes to which it bound in Gpx3(+/+) mice but not to its presence in the epididymal lumen. These results show that Gpx3 from the blood binds to basement membranes of specific epithelial cells and indicate that the cells modify their basement membranes to cause the binding. They further indicate that at least two Gpx3 compartments exist in the organism. In one compartment, kidney supplies Gpx3 through the blood to specific basement membranes in a number of tissues. In the other compartment, the epididymis provides Gpx3 to its own lumen. Tissues other than kidney and epididymis express Gpx3 at lower levels and may supply Gpx3 to other compartments.

Expression of steroidogenic enzymes during equine testicular development.

In the mammalian testis, Leydig cells are primarily responsible for steroidogenesis. In adult stallions, the major endocrine products of Leydig cells include testosterone and estrogens. 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase (3ßHSD) and 17α-hydroxylase/17,20-lyase (P450c17) are two key steroidogenic enzymes that regulate testosterone synthesis. Androgens produced by P450c17 serve as substrate for estrogen synthesis. The aim of this study was to investigate localization of the steroidogenic enzymes P450c17, 3ßHSD, and P450arom and to determine changes in expression during development in the prepubertal, postpubertal, and adult equine testis based upon immunohistochemistry (IHC) and real-time quantitative PCR. Based on IHC, 3ßHSD immunolabeling was observed within seminiferous tubules of prepubertal testes and decreased after puberty. On the other hand, immunolabeling of 3ßHSD was very weak or absent in immature Leydig cells of prepubertal testes and increased after puberty. HSD3B1 (3ßHSD gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal testes (P=0.0041). P450c17 immunolabeling was observed in small clusters of immature Leydig cells in prepubertal testes and increased after puberty. CYP17 (P450c17 gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.030) and postpubertal testes (P=0.0318). A weak P450arom immunolabel was observed in immature Leydig cells of prepubertal testes and increased after puberty. Similarly, CYP19 (P450arom gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal (P=0.0001) testes. In conclusion, Leydig cells are the primary cell type responsible for androgen and estrogen production in the equine testis.

Immunohistochemical localization of steroidogenic enzymes in the testis of the sika deer (Cervus nippon) during developmental and seasonal changes.

Testicular steroidogenesis and spermatogenesis during developmental and seasonal changes were investigated in male sika deer (Cervus nippon), a short-day seasonal breeder, to clarify the physiological mechanisms for reproductive function. The immunohistochemical localization of steroidogenic enzymes (P450scc, P450c17, 3betaHSD and P450arom), spermatogenesis and cell proliferation were analyzed in the testes of fetal (164 to 218 days of fetal age), fawn (0 years old), yearling (1 year old) and adult (more than 2 years old) male sika deer. Three kinds of steroidogenic enzymes, P450scc, P450c17 and 3betaHSD, essential for the synthesis of testosterone were located only in the Leydig cells of the testes from the fetal period, and these localizations did not change during developmental or seasonal stages. Immunoreactivity for P450arom, a key enzyme converting testosterone to estradiol, was also localized only in the Leydig cells of testes but was also further limited to the testes of yearlings and adults. Seminiferous tubules had already formed in the fetal testes examined in the present study. Spermatogenesis started in yearlings and was more active in the breeding season. In the adult sika deer testes, the Leydig cells, which displayed immunoreactivities for steroidogenic enzymes, changed to have more cytoplasm in the breeding season than in the non-breeding season. Cell proliferation of Leydig cells was hardly observed in adult testes during seasonal changes. The present results suggested that sika deer testes start to synthesize testosterone from the fetal period, that seasonal changes in testosterone and estradiol syntheses are dependent on the quantitative variation of steroidogenic enzymes synchronized with the size of Leydig cells and that estradiol synthesized in yearling and adult testes makes a contribution to the initiation and recrudescence of spermatogenesis and spermiogenesis in the sika deer.

Immunohistochemical localization of mitochondrial fatty acid ß-oxidation enzymes in rat testis.

The testis consists of two types of tissues, the interstitial tissue and the seminiferous tubule, which have different functions and are assumed to have different nutritional metabolism. The localization of enzymes of the mitochondrial fatty acid ß-oxidation system in the testis was investigated to obtain a better understanding of nutrient metabolism in the testis. Adult rat testis tissues were subjected to immunoblot analysis for quantitation of the amounts of enzyme proteins, to DNA microarray analysis for gene expression, and to immunofluorescence and immunoelectron microscopy for localization. Quantitative analysis by immunoblot and DNA microarray revealed that enzymes occur abundantly in Leydig cells in the interstitial tissue but much less so in the seminiferous tubules. Immunohistochemistry revealed that Leydig cells in the interstitial tissue and Sertoli cells in the seminiferous tubules contain a full set of mitochondrial fatty acid ß-oxidation enzymes in relatively plentiful amounts among the cells in the testis, but that this is not so in spermatogenic cells. This characteristic localization of the mitochondrial fatty acid ß-oxidation system in the testis needs further elucidation in terms of a possible role for it in the nutritional metabolism of spermatogenesis.

Development and application of a system for seminolipid metabolism using mouse seminiferous tubules.

A convenient tool for studying metabolism of seminolipid in testis was developed by using mouse isolated seminiferous tubules prepared by collagenase treatment. Because more than 99% of [(35)S]sulfate-incorporation was distributed in seminolipid, its metabolism in seminiferous tubules can be analyzed without disturbance of the other sulfolipids in this assay system. Furthermore, the contents of seminolipid and its precursor, galactosylalkylacylglycerol, which were determined by liquid chromatography-electrospray ionization mass spectrometry, did not change within a few hours, indicating that the incorporations of [(35)S]sulfate into seminolipid solely reflects the turnover rate of this sulfolipid. As an initial application of this system, we characterized heat-susceptibility of the seminolipid turnover rate in mouse seminiferous tubules. Severe heating (44 degrees C for 10 min) of the isolated seminiferous tubules suppressed the (35)S-incorporation into seminolipid to 47% of heating at scrotal temperature (32 degrees C for 70 min). In contrast, pretreatment of the testis in vivo under the same condition (44 degrees C for 10 min) did not decrease the seminolipid turnover rate in the isolated seminiferous tubules. In addition, the activity of galactocerebroside sulfotransferase decreased in the temperature-dependent manner in seminiferous tubules as well as crude tubular homogenates, where the activity is significantly more stable in the former than the latter. The newly developed system could provide useful basic data for further analyses of seminolipid metabolism in the testis.

Attenuation of cyclosporine A-induced testicular and spermatozoal damages associated with oxidative stress by ellagic acid.

This study was conducted to investigate the possible protective effect of ellagic acid (EA) on cyclosporine A (CsA)-induced testicular and spermatozoal damages associated with oxidative stress in male rats. Forty adult male Sprague-Dawley rats were divided into 4 groups of 10 animals each. Control group was used as placebo. Cyclosporine group received CsA at the dose of 15 mg/kg/day. Ellagic acid group was treated with EA (10 mg/kg/day). Cyclosporine plus ellagic acid group received CsA+EA. Reproductive organs were weighed and epididymal sperm characteristics and histopathological structure of testes were examined along with malondialdehyde (MDA) and glutathione (GSH) levels, glutathione-peroxidase (GSH-Px) and catalase (CAT) activities in testicular tissue. CsA significantly decreased the weights of testes and ventral prostate, epididymal sperm concentration, motility, testicular tissue glutathione (GSH), glutathione-peroxidase (GSH-Px) and catalase (CAT), diameters of seminiferous tubules and germinal cell layer thickness, and it significantly increased malondialdehyde (MDA) level and abnormal sperm rates along with degeneration, necrosis, immature germ cells, congestion and atrophy in testicular tissue. However, the CsA plus EA treatment attenuated all the CsA-induced negative changes observed in the testicular tissue, sperm and oxidant/antioxidant parameters. In conclusion, CsA-induced oxidative stress leads to the structural and functional damages in the testicular tissue and sperm quality of rats, and also EA has a protective effect on these damages.

Two distinct localization patterns of testis-specific serine protease 1 (TESSP1) in the seminiferous tubules of the mouse testis.

Mouse Tesspl has been shown to be a testis-specific gene that may contribute to spermatogenesis. In this report, we raised a specific antibody against TESSP1 to assess its biological role. Western blotting detected testicular TESSP1 in all postnatal developmental stages of the mouse. Experiments using the testes of W/W(V) mice, which lack germ cells, indicated TESSP1 expression in Sertoli cells and Leydig cells. In immunofluorescence staining of the wild-type mouse testis, dot-like signals for TESSP1 were observed in the adluminal compartment of the seminiferous tubules, while diffused signals were found in the basal compartment. Generally, the dot-like and diffused signals overlapped with the trans-Golgi network marker RAB6 and the transmembrane protein CADHERIN 2, respectively. Some TESSP1 staining was also observed in association with interstitial Leydig cells of the testis. The results of this study suggest that TESSP1 is predominantly localized in the plasma membrane of spermatogonia and Sertoli cells in the basal compartment, but exhibits an intracellular localization, presumably in the Golgi apparatus, of spermatocytes and spermatids in the adluminal compartment of the seminiferous tubules. The expression of TESSP1 in both germ cells and somatic cells and alteration in its cellular localization in the germ cells during spermatogenesis indicate that it may have a unique role in the testis.

Morphological and morphometric study on the effect of simulated microgravity on rat testis.

The present study was undertaken to determine the effect of simulated microgravity on the testis of the rats and to evaluate the possibility of spermatogenesis failure in space environment. Fifty-four adult male albino rats were used in this study. They were divided equally into intact control, stress control and experimental groups. The rats of the intact control group (Group Ia) were kept without intervention. The rats of both the stress control and experimental groups were subjected to inguinal canal ligation and tail-suspension. In the stress control group (Group Ib) the hindlimbs were not elevated above the floor of the housing units whereas in the experimental groups the hindlimbs were elevated for one week (Group II) and six weeks (Group III), respectively. In a third experimental group (Group IV) the rats were hindlimb-suspended for six weeks followed by another six weeks without suspension to allow recovery. Prior to sacrifice, the animals were weighed and anesthetized, and the testes were excised and weighed. Testicular specimens were processed for histological, histochemical and morphometric studies. The results of the present study revealed that only after six weeks of hindlimb-suspension, the rats showed a significant decline in testicular weight compared with the control groups. Histologically, few abnormalities were observed in some seminiferous tubules in one-week hindlimb-suspended group. Spermatogenesis was significantly reduced by six-week of hindlimb-suspension marked by atrophy of the testes and loss of all germ cells, except a few spermatogonia. Spermatogenesis was partially restored in the recovery group. In all groups the appearance of Sertoli cells remained the same. Proliferation of Leydig cells was observed in the experimental groups. It is concluded that spermatogenesis is severely inhibited by six weeks of hindlimb-suspension and that it is partially restored following six weeks of recovery. This study provides further insights regarding the serious effects of long-term exposure to microgravity on the testes of mammals, including human beings.

Effect of monensin, a Na+-specific carboxylic ionophore on the oxidative defense system in rat testis.

The effect of monensin, a Na+-specific ionophore on the oxidative defense system in rat testis was studied. Monensin mixed in the animal diet was administered at the dose levels of 2.5, 5 and 10 mg/kg b.w. to Wistar rats for a period of 67 days. A marked inhibition in the activities of different oxidative defense enzymes such as catalase, glutathione peroxidase, glutathione-S-transferase, superoxide dismutase and glutathione reductase was noticed, which indicates the possible involvement of free radicals in the antispermatogenic effects of monensin in rat testis. This was further substantiated by a significant increase in the generation of lipid peroxides along with the depletion of reduced glutathione. The drug treatment resulted in a significant change in apoptotic cell death as seen by an elevated fragmentation in the testicular genomic DNA. Monensin treatment also resulted in marked degenerative changes in the histoarchitecture of testis, such as depletion of different germ cell populations, vacuole formation and disorganization of seminiferous tubules. The results are indicative of the potential antispermatogenic effects of monensin in the rat.

The association between intratubular seminoma and invasive germ cell tumors.

Intratubular seminoma (ITS) has been defined as the complete filling of the seminiferous tubules with seminoma cells with no Sertoli cells present. This contrasts with intratubular germ cell neoplasia, unclassified (IGCNU), where the malignant germ cells are interspersed by Sertoli cells. We aimed to determine the relationship between these 2 entities and the association between ITS and invasive classic seminomas. We therefore examined the morphology and immunochemistry of ITS and IGCNU adjacent to germ cell tumors to differentiate the patterns, frequency, and distribution of these lesions. We found that ITS was seen in equal frequency adjacent to seminomas as it was to nonseminomas. The presence of ITS in non-seminomatous germ cell tumors suggests that it is a true in situ lesion rather than representative of intratubular spread of an existing seminoma. However, because it is not specifically associated with seminoma, we suggest that it is not useful to discriminate this lesion from IGCNU and that it merely represents an advanced form of IGCNU on the way to invasive malignancy.

Expression of cathepsin L in human testis under diverse infertility conditions.

The cathepsin family of proteolytic enzymes play an important role in the remodeling seminiferous epithelia in rodent testis. In an effort to uncover the cathepsin L expression in diverse pathological conditions in human testis, the immunohistochemical localization of cathepsin L was conducted in human testis under diverse male infertility condition including spermatogenic hypoplasia and testis cancer. In seminiferous tubule of normal, non-obstructive azoospermic, decreased spermatogenesis, and maturation arrest conditions, cathepsin L was found in both germ cells and Sertoli cells. In contrast, there was no visible expression of cathepsin L in seminiferous tubule tissues from Sertoli cell-only syndrome, spermatogenic hypoplasia, and testicular cancer. Our result suggests that the cross-talk between germ cells and Sertoli cells is crucial for the control of cathepsin L expression in human testis. The absence of expression of cathepsin L in germ cell cancer emphasizes that cathepsin L expression in Sertoli cells is regulated by functional germ cells in human testis.

Evidence for the presence of angiogenin in human testis.

We have reported the expression and possible roles of angiogenin, a potent angiogenic factor, in human female reproductive organs. In this study, we investigated the expression of angiogenin in the human testis, a male reproductive organ. Western blot analysis showed the presence of angiogenin in the human testis, with a single band of the same size as recombinant human angiogenin. Immunohistochemical study and in situ hybridization showed that the angiogenin protein and messenger RNA (mRNA) localized in peritubular myoid cells (PTMCs) and vascular endothelial and smooth muscle cells. PTMCs are known to play various roles in the testes concerned with spermatogenesis, transport of spermatozoa, structural support to the seminiferous tubules, and mediation of Sertoli cell function. The specific localization of angiogenin in PTMCs suggests that angiogenin plays physiologic roles in the human testis.