An ultrastructural study on the Leydig and Sertoli cells in the immature lesser mouse deer (Tragulus javanicus)

Leydig and Sertoli cells of the immature lesser mouse deer testes, obtained in East Malaysia, were observed using light and transmission electron microscopy (TEM). The testes were fixed in 5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in ethanol, and embedded in Araldite M. Serial semi-thin sections were cut, stained with toluidine blue and observed using light microscopy. Serial ultra-thin sections were cut, stained with uranyl acetate and lead citrate, and examined using TEM. As a result, ultrastructurally, two types of underdeveloped filament bundles were infrequently recognized in Leydig cells, but not in other testicular cells. One type was the underdeveloped bundles of actin filaments (approximately 5 nm in diameter), which were found in the nucleus of Leydig cells. The other type was the underdeveloped bundles of intermediate filaments (approximately 10 nm in diameter), which were found in the cytoplasm of Leydig cells. A multivesicular nuclear body (MNB)--specifically present in the Sertoli cell nucleus of ruminant testes--was infrequently observed. The MNB is situated in the vicinity of nuclear membrane, still in an underdeveloped stage.     

Immunohistochemical localization of steroidogenic enzymes in the testis of Hokkaido Sika deer (Cervus nippon yesoensis)

The testes from 15 adult male Hokkaido Sika deer (Cervus nippon yesoensis) were collected during the rutting season (October and November). We investigated the localization of 4 kinds of steroidogenic enzymes (P450scc, 3betaHSD, P450c17 and P450arom) immunohistochemically in these testicular samples. The specific immunoreactivities to these enzymes were detected only in the cytoplasm of Leydig cells. This differs to the enzyme distributions reported previously in Japanese black bear, Japanese raccoon dog, Hokkaido brown bear and American black bear, in which the same immunoreactivities were detected in Leydig cells, Sertoli cells and/or spermatogenic cells. The current study suggests that in the testes of the Hokkaido Sika deer, testosterone and estradiol-17beta may be synthesized in the Leydig cells only.     

Vimentin expression in testes of Arabian stallions

Reasons for performing study: Specific patterns of cytoskeletal filaments reflect a functional state of the cell. In testicular cells intermediate filaments (IFs) are of the vimentin type. Since it is known that Sertoli cells regulate the spermatogenic function in the male gonad, it became important to propose a system that could quantify the state of seminiferous tubular quality. To date, a Johnsen score system has never been used to equine testes. Objectives: To demonstrate the expression pattern of vimentin in testes of mature Arabian stallions and correlate its distribution with grade of seminiferous tubule impairment as indicated by a Johnsen score. Methods: For histological examination by the Johnsen method, routine haematoxylin‐eosin staining was used. Vimentin expression and its presence in testicular sections and testicular homogenates were detected by immunohistochemistry and western blot, respectively. Both analyses were performed qualitatively and quantitatively and further validated by ANOVA tests. Results: Distinct morphology of seminiferous tubules was found in testes harvested from 3 stallions. Vimentin in IFs was immunolocalised to the cytoplasm of Sertoli, Leydig and peritubular‐myoid cells. The intensity and pattern of the IFs staining was different in individual seminiferous tubules suggesting a correlation between vimentin expression and the severity of tubule degeneration. Qualitative results by immunohistochemistry and western blot were confirmed by further quantitative analyses. Conclusions: In equine testes, differential expression of vimentin was found to be correlated with the impairment of seminiferous tubules indicated by a decrease in Johnsen score. Potential relevance: The Johnsen score system may be a useful method to facilitate the identification of tubular alterations in the stallion testes. Combined histological and immunohistochemical approach may provide a detailed phenotypic classification of stallions with decreased fertility.     

Inhibin-α immunohistochemical expression in mature and immature canine Sertoli and Leydig cells

Formalin-fixed, paraffin-embedded sections from 32 canine pairs of testes were immunohistochemically examined for Inhibin-α (INHα). Samples were subdivided into two groups (group 1, neonates; group 2, puppies and adults) and results statistically compared. Inhibin-α was significantly expressed only in Sertoli cells of neonatal testes, while in Leydig cells it was expressed without significant difference between groups. These results suggest that, in dogs, INHα expression switches from Sertoli to Leydig cells during testicular maturation and that, in adult, Leydig cells represent the main source of INHα.     

Mouse hepatoblastomas: a histologic, ultrastructural, and immunohistochemical study

Hepatoblastomas from B6C3F1 and BALB/c mice were examined by light and electron microscopy and by immunohistochemical reactions for alpha-fetoprotein, keratin, and vimentin. Tumors occurred in one group of a chronic bioassay for the interaction of diet, genetic strain, and the carcinogen, 2-acetylaminofluorene. Tumors had several populations (including epithelial and mesenchymal cells) in various stages of differentiation. Neoplastic epithelial cells had features of embryonal hepatocytes, such as sparse cytoplasmic organelles, absence of glycogen, abundant free ribosomes, occasional bile canaliculi, and peroxisome-like dense bodies. Embryonal fibroblast-like cells had pleomorphic and folded nuclei with prominent perinuclear chromatin and dispersed cytoplasmic organelles. Fibroblast-like cells were surrounded by bundles of collagen fibrils. Intermediate or transitional types of cells were seen. No tumor cells were immunoreactive for mouse alpha-fetoprotein (AFP) antibody, unlike those in hepatocellular adenomas or carcinomas. Epithelial and mesenchymal tumor cells contained intermediate filaments throughout the cytoplasm; some of these cells stained for keratin but not for vimentin. These findings suggest that mouse hepatoblastomas are derived from bipotential liver blastema cells and are composed of a mixture of several cell populations.     

Actin in Stratified Squamous Keratinized Epithelium*

Actin filament distribution patterns were revealed in a stratified squamous keratinized epithelium using phalloidin-fluorescent and immunogold labeling techniques applied on bovine luminal pilar as a model tissue. In non-keratinized cell types, actin concentrates on the microfilament-rich cellular cortex as well as on cytoplasmic processes and protrusions. In cornified cells labeling is distributed diffusely over the amorphous cytoplasm. A constant feature in all cell types is plasmalem-mal labeling. Desmosomes exhibit deposition on their plasmalemmal leaflets, the dense central stratum and plaques. Desmosomal as well as cytoplasmic keratinfilament bundles also label for actin, the latter often in a cross-banded manner. These cellular distribution patterns of actin filaments are discussed with respect to the singificance of the microfilaments in the process of cell shape determination, stratification, and cell adhesion.     

Seasonal changes in spermatogenesis and immunolocalization of inhibin/activin subunits in the wild male ground squirrel (Citellus dauricus Brandt)

The objective of this study was to investigate the seasonal changes in spermatogenesis and the immunolocalization of the inhibin alpha and inhibin/activin (betaA and betaB) subunits during the breeding and non-breeding seasons in the wild male ground squirrel. The testicular weight and size and seminiferous tubule diameter were measured, and histological observations of testes were performed. The sections of the testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA and inhibin/activin betaB during the breeding and non-breeding seasons. There were marked variations in testicular weight and size and seminiferous tubule diameter between the breeding and non-breeding seasons, and all types of spermatogenic cells, including spermatozoa, were found in the breeding season. In addition, immunoreactivity was also detected for the inhibin alpha, betaA and betaB subunits in Sertoli and Leydig cells during the breeding season, but immunostaining was only present for the inhibin alpha and inhibin/activin betaB subunits in Sertoli cells during the non-breeding season. These results suggest that seasonal changes in testicular weight and size and seminiferous tubule diameter of wild ground squirrels are correlated with changes in spermatogenesis, and the cellular localization of the inhibin/activin subunits showed season related changes in the breeding and non-breeding seasons.     

Immunolocalization of steroidogenic enzymes and their expression during the breeding season in the testes of wild raccoon dogs (Nyctereutes procyonoides)

The objective of this study was to investigate immunolocalization of steroidogenic enzymes 3βHSD, P450c17 and P450arom and their expression during the breeding season in wild male raccoon dogs. The testicular weight, size and seminiferous tubule diameters were measured, and histological and immunohistochemical observations of testes were performed. The messenger RNA expression (mRNA) of 3βHSD, P450c17 and P450arom was measured in the testes during the breeding season. 3βHSD was found in Leydig cells during the breeding and non‐breeding seasons with more intense staining in the breeding season. P450c17 was identified in Leydig cells and spermatids in the breeding season, whereas it was present only in Leydig cells in the non‐breeding season. The localization of P450arom changed seasonally: no immunostaining in the non‐breeding season; more extensive immunostaining in Leydig cells, Sertoli cells and elongating spermatids in the breeding season. In addition, 3βHSD, P450c17 and P450arom mRNA were also expressed in the testes during the breeding season. These results suggested that seasonal changes in testicular weight, size and seminiferous tubule diameter in the wild raccoon dog were correlated with spermatogenesis and immunoreactivity of steroidogenic enzymes and that steroidogenic enzymes may play an important role in the spermatogenesis and testicular recrudescence and regression process.     

A Stereological Study of the Different Cell Populations in Chicken Testes Treated with Follicle-Stimulating Hormone during Embryonic Development

Quantitative morphological methods were used to analyse the histomorphometric changes and variations in the number and size of cells from diverse cellular populations in testes of newly hatched chicks treated with follicle stimulating hormone (FSH) during embryonic development. The tissue was fixed and embedded in Epon and sections were morphometrically measured under light microscopy, using point counting for volume densities and the Floderus equation to determine numerical density. The average volume of the individual cell was determined by dividing the volume density by the numerical density. Results indicate that FSH administration causes an increase in the number of Sertoli cells and spermatogonia, as well as enlargement of the individual Sertoli cells leading consequently to an increase in the diameter and volume density of the testicular seminiferous tubules. Results also reveal an increase in the volume density of the interstitial cords of the Leydig cells, this expansion is due to the enlargement of the individual Leydig cells and not to an increase in their number, which remains constant. We conclude that testes of chick embryos are able to respond to FSH treatment, as revealed by the changes in the number and size of the cells conforming the diverse cellular populations of the testis. FSH treatment during embryonic development induces histomorphometric changes in both the interstitial tissue and seminiferous tubules, accelerating their growth and differentiation.     

Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue-green alga, Microcystis aeruginosa

The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes. Male Sprague Dawley rats were used for all studies. For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation. Cell suspensions and and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 micrograms/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 micrograms/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg. Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin. In cell suspensions, MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes. In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments. These alterations were dose and time dependent. Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR. In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing. These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments.     

Effects of pre-natal glucocorticoids on testicular development in sheep

We tested the hypothesis that acute pre-natal exposure to high levels of synthetic glucocorticoid (betamethasone) would alter fetal testicular development through actions on gonadal glucocorticoid receptors (GRs). Pregnant Merino ewes bearing singleton male fetuses (n = 24) were allocated randomly among four equal groups to be injected intramuscularly with saline or betamethasone (0.5 mg/kg) either on day 109 of gestation or on both day 109 and day 116 of gestation. Fetal testes were collected at post-mortem, 5 days after each treatment. The volume of interstitial tissue and the volume, length and diameter of the sex cords were measured, and Sertoli cells and gonocytes were counted. For cord volume and interstitial tissue volume, control testes demonstrated maturational changes as fetal age advanced from 109 to 116 days of gestation. For that period, the single injection of betamethasone significantly reduced Leydig cell proliferation (P < 0.05), but had no effect on Sertoli cell numbers. Immunohistochemistry was used to localize GR and proliferating cell nuclear antigen in testicular cells. GR immunoexpression in Leydig cells was higher in fetuses exposed to betamethasone at 109 days of gestation than in control fetuses. Sertoli cells showed low levels of GR. It was concluded that, during mid-gestation, a brief period of glucocorticoid treatment could affect testicular development in male sheep fetuses. The mechanism probably involves direct effects on Leydig cells, as these cells express extra-GR in response to the treatment. Sertoli cells seem to produce less GR than Leydig cells, perhaps explaining their lack of response to betamethasone. These outcomes may have important implications for future fertility in male offspring.     

Relationships of testicular iron and ferritin concentrations with testicular weight and sperm production in boars

The inverse relationship of testicular size and circulating follicle-stimulating hormone (FSH) concentrations has been documented, and accompanying this relationship is the change in color of the parenchymal tissue of the testes. Large testes (300 to 400 g) are pink to light red and small testes (100 g) are dark maroon with color gradations for weights in between. It was hypothesized that this color most likely represented an iron protein. Chromatographic analysis of testicular tissue indicated that the Fe was associated primarily with ferritin, and immunohistochemistry showed that Leydig cells were the primary location of ferritin storage within the testes. Concentrations of Fe and ferritin were higher in small testes and decreased as testes weight increased (P < 0.05). As testicular Fe concentrations increased, daily sperm production (DSP) and total DSP declined (P < 0.05). Genotyping six generations of Meishan x White composite boars (n = 288) for a quantitative trait locus that is indicative of elevated FSH and small testes in boars indicated that the Meishan genotype had elevated testicular iron concentrations and darker color in conjunction with reduced total DSP (P < 0.01). It is not thought the elevated iron concentrations affect testicular weights but are probably a result of elevated FSH and FSH inducement of Fe transport. The storage of Fe in Leydig cells may provide a reservoir of Fe for easy access by Sertoli and germ cells, but still provide a degree of protection to germ cells from ionic iron.     

Morphology and Morphometry of Seminiferous Tubules in Crioulo Horses

This study aimed to assess the biometrics of the testes and the morphology of the seminiferous tubules of Crioulo horses. We studied 10 sexually mature stallions (3–6 years of age). After orchiectomy, testes were perfused with Karnovsky's solution and then embedded in glycol methacrylate. Testis sections (4 μm) were cut and stained with toluidine blue and a solution of 1% sodium borate. The histological images were digitized, and the morphometric analysis was performed using ImageJ software. The average weight of the stallions was 377.5 kg, and the average weight of both testicles was 162.9 g. The percentage of testicular parenchyma occupied by the seminiferous tubules and the intertubular tissue was 77.97% ± 6.34% and 22.03% ± 6.34%, respectively. The average tubular diameter was 205.00 ± 36.91 μm, whereas the average height of the seminiferous epithelium was 70.56 ± 2.82 μm. Average tubular length per testicle and average tubular length per gram of testicle were 4,085.10 ± 1,170.68 m and 26.09 ± 10.63 m/g, respectively. The characteristics of the eight stages of the seminiferous epithelium cycle were similar to those reported in other horse breeds. We conclude that the morphometry of the seminiferous tubules of Crioulo horse resembles what has been reported in other horse breeds. The volumetric proportion of the seminiferous tubules and the Leydig cells of the Crioulo horse is one of the highest ever reported for stallions.     

Immunohistochemical study of intermediate filaments and neuroendocrine marker expression in leydig cells of laboratory rodents

The aims of this study were to detect the expression of intermediate filaments and to verify the existence of marker substances for neuronal and neuroendocrine cells within the interstitial Leydig cells of laboratory rodent's testes, such as it has been described in other species. Adult male rats, mice, gerbils, Syrian hamsters and guinea-pigs were used and the localization of the different markers was achieved by the streptoavidin-peroxidase immunohistochemical method. The present study demonstrates in all rodents studied a similar pattern of localization in Leydig cells of intermediate filaments (vimentin, cytokeratin, neurofilament 200 kD and glial fibrillary acidic protein) and other marker substances (S-100, CgA, substance P and neurone-specific enolase), which are typical of neuroendocrine (APUD cells or paraneurones) and glial cells. The expression of these substances, related to neurotransmitters or neurohomones and other proteins characteristic of neuroendocrine cells, could suggest that it is a neural crest derived cell. Although this study provides more evidences about the immunoexpression of neuronal and glial markers in Leydig cells, this fact cannot be related directly to their embryological origin, because the current data support the hypothesis of a mesenchymal origin of the Leydig cells.     

Temporary disturbance of actin stress fibers in swine kidney cells during pseudorabies virus infection

Rounding and loosening of cells is a consequence of infection with pseudorabies virus (PrV), both in vitro and in vivo. These changes in the normal structure of the cell may be the result of cytoskeletal changes. Immunofluorescence staining of actin filaments and microtubule bundles was performed to examine whether PrV induces a reorganization of these cytoskeletal components in infected swine kidney (SK) cells. Every 2h until 12h post-inoculation (p.i.), cells were washed in cytoskeleton stabilizing buffer (CSB), fixed with paraformaldehyde and washed again with CSB. Cells were permeabilized with a 1/1000 dilution of Triton X-100 and actin filaments were stained by incubating cells with phalloidin-Texas Red. Staining of microtubules was done by incubating the cells subsequently with mouse monoclonal anti-alpha-tubulin and goat anti-mouse IgG-FITC. During the course of infection, actin fibers of SK cells were rearranged in the following sequence: (1) disappearance of thick actin stress fibers between 4 and 6h p.i., (2) complete loss of stress fibers between 6 and 8h p.i., and (3) reappearance of thin stress fibers starting from 10h p.i. In contrast to herpes simplex virus 1 (HSV1) or equine herpesvirus 1 (EHV1), PrV infection did not induce changes in the cellular microtubule network. PrV infection induces a temporary disassembly of actin stress fibers.     

Seasonal Changes in the Immunolocalization of Cytoskeletal Proteins and Laminin in the Testis of the Black‐Backed Jackal (Canis mesomelas)

Manipulation of the reproductive activity of jackals is dependent on a thorough understanding of the reproductive biology of this species. This study describes seasonal morphological changes in the adult testis of the black‐backed jackal in relation to the immunoexpression of the basement membrane marker, laminin and the cytoskeletal proteins, cytokeratin, smooth muscle actin and vimentin. Laminin was immunolocalized in basement membranes surrounding seminiferous tubules, as well as in basement membranes associated with Leydig, peritubular myoid and vascular smooth muscle cells. Scalloped basement membranes enclosed seminiferous tubules in regressing testes. The seminiferous epithelium and interstitial tissue in all animals studied were cytokeratin immunonegative. Smooth muscle actin was demonstrated in vascular smooth muscle cells, as well as in peritubular myoid cells encircling seminiferous tubules. Vimentin immunoreactivity was exhibited in the cytoplasm of Sertoli cells, Leydig cells, vascular endothelial cells, vascular smooth muscle cells and fibrocytes. Vimentin immunostaining in Sertoli, Leydig and peritubular myoid cells varied depending on the functional state of the testis. The results of the study have shown that dramatic seasonal histological changes occur in the testes of the jackal. In addition, the use of immunohistochemistry accentuates these morphological changes.     

Seasonal changes in immunolocalization of inhibin/activin subunits and testicular activity in wild male raccoon dogs (Nyctereutes procyonoides)

Thirty-four pairs of testes from wild adult raccoon dogs (Nyctereutes procyonoides) were obtained between September 2000 and May 2003. The cellular localization of the inhibin alpha and inhibin/activin (betaA and betaB) subunits in wild raccoon dog testes was investigated. The testicular weight and size and seminiferous tubule diameters were measured. There were marked seasonal variations in testicular weight and size and seminiferous tubule diameters, with values relatively low in September and high in March. Spermatogonia and primary spermatocytes were observed in September, and spermatogonia, spermatocytes, and round spermatids were present in January. All types of spermatogenic cells, including mature spermatozoa, were found in March, indicating that the breeding season is around March in Japan. Thereafter, spermatogonia and degenerating spermatocytes were observed in April. The sections of testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA and inhibin/activin betaB. The inhibin alpha and inhibin/activin (betaA and betaB) subunits were only expressed in Leydig cells in September. On the other hand, the inhibin alpha, betaA, and betaB subunits were observed in Leydig cells and Sertoli cells, but not in germ cells, in March. These results suggest that the testes of wild raccoon dogs have the ability to synthesize inhibins, and the cellular localization of inhibin/activin subunits showed season-related changes in the breeding and non-breeding seasons.     

Morphological features of the spermatic cord in the musk shrew (Suncus murinus) with special reference to extratesticular Leydig cells

Morphological features of the testicular artery and vein in the spermatic cord of the musk shrew (Suncus murinus) were evaluated by light microscopy, transmission electron microscopy, corrosion cast technique combined with scanning electron microscopy and immunohistochemistry. The vascular architecture in the spermatic cord of the musk shrew was simple. The testicular artery in the musk shrew was straight and accompanied by 1 to 3 branches of testicular vein. The testicular vein was also straight and anastomosed with each other in some points along its length, but it did not form a delicate pampiniform plexus. In the middle and distal portions of the spermatic cord, the tunica adventitia of the artery and vein was joined together to form a single connective tissue septum. Clusters of cells were found in this connective tissue septum in the middle portion of the cord. These cells were located close to the arterial wall and nerve endings, but they did not appear inside of neurium. They showed several typical characteristics similar to Leydig cells, and they were positive for 3beta hydroxysteroid dehydrogenase (HSD) antibody. Ultrastructural and immunohistochemical studies also indicated that the cells in cluster found in the vascular wall of the musk shrew spermatic cord may be equivalent to Leydig cells in testes. These extratesticular Leydig cells had characteristics of the active steroid-producing cell and seemed to be another source of testosterone.     

Distribution of Lectin‐Bindings in the Testis of the Lesser Mouse Deer,Tragulus javanicus

The distribution of lectin bindings in the testis of the smallest ruminant, lesser mouse deer (Tragulus javanicus), was studied using 12 biotinylated lectins specific for d ‐galactose (peanut agglutinin PNA, Ricinus communis agglutinin RCA I), N‐acetyl‐d ‐galactosamine (Dolichos biflorus agglutinin DBA, Vicia villosa agglutinin VVA, Soybean agglutinin SBA), N‐acetyl‐d ‐glucosamine and sialic acid (wheat germ agglutinin WGA, s‐WGA), d ‐mannose and d ‐glucose (Lens culinaris agglutinin LCA, Pisum sativum agglutinin PSA, Concanavalin A Con A), l ‐fucose (Ulex europaeus agglutinin UEA I), and oligosaccharide (Phaseolus vulgaris agglutinin PHA‐E) sugar residues. In Golgi‐, cap‐, and acrosome‐phase spermatids, lectin‐bindings were found in the acrosome (PNA, RCA I, VVA, SBA, WGA and s‐WGA), and in the cytoplasm (PNA, RCA I, VVA, SBA, WGA, LCA, PSA, Con A and PHA‐E). s‐WGA binding was confined to the spermatid acrosome, but other lectins were also observed in spermatocytes. In spermatogonia, VVA, WGA, Con A, and PHA‐E bindings were observed. Sertoli cells were intensely stained with DBA and Con A, and weakly with PHA‐E. In interstitial Leydig cells, RCA I, DBA, VVA, Con A, PSA, LCA, WGA and PHA‐E were positive. UEA I was negative in all cell types including spermatogenic cells. Unusual distribution of lectin‐bindings noted in the testis of lesser mouse deer included the limited distribution of s‐WGA only in the spermatid acrosome, the distribution of DBA in Sertoli cells, Leydig cells and lamina propria, and the absence of UEA I in all type cells. The present results were discussed in comparison with those of other animals and their possible functional implications.     

Leydig cell hyperplasia in an ENU-induced mutant mouse with germ cell depletion

Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3beta-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.