SOX9 is colocalized with paraspeckle protein NONO in cultured murine sertoli cells and features structural characteristics of intrinsically disordered proteins.

This study provides supporting evidence for the association between SOX9 and liquid-liquid phase separation. We show that SOX9 colocalized with a paraspeckle protein NONO in many, but not all, of the immortalized and primary murine Sertoli cells examined. In addition, we confirmed that SOX9 has structural characteristics of intrinsically disordered proteins.

Characterization of the stem cell niche components within the seminiferous tubules in testicular biopsies of Klinefelter patients.

OBJECTIVE: To characterize the tubular environment in testicular biopsy tissues from patients with Klinefelter syndrome (KS). DESIGN: Observational immunohistochemical study. SETTING: Academic research unit. PATIENT(S): Males with KS and controls at different developmental time points: fetal, prepubertal, peripubertal, and adult. INTERVENTION(S): Immunohistochemical analysis of testicular biopsies samples to characterize maturation of Sertoli cells and tubular wall components-peritubular myoid cells (PTMC) and extracellular matrix (ECM) proteins. MAIN OUTCOME MEASURE(S): Intensity of antimüllerian hormone staining; proportion of Sertoli cells expressing androgen receptor (AR); and expression of tubular wall markers as characterized by identifying abnormal staining patterns. RESULT(S): Decreased expression for alpha smooth muscle actin 2 (ACTA2) was observed in peripubertal and adult KS as well as in Sertoli cell only (SCO) patients. Altered expression patterns for all ECM proteins were observed in SCO and KS biopsy tissues compared with controls. Only for collagen I and IV were altered expression patterns observed between KS and SCO patients. In peripubertal samples, no statistically significant differences were observed in the maturation markers, but altered ECM patterns were already present in some samples. CONCLUSION(S): The role of loss of ACTA2 expression in PTMC in the disintegration of tubules in KS patients should be further investigated. Future research is necessary to identify the causes of testicular fibrosis in KS patients. If the mechanism behind this fibrotic process could be identified, this process might be altered toward increasing the chances of fertility in KS patients.

Exploration of small RNA biomarkers for testicular injury in the serum exosomes of rats.

Testicular injury is often observed in drug development. Serum hormones are usually used as noninvasive biomarkers for testicular injury; however, their sensitivities are low. Therefore, it is difficult to monitor testicular injury in drug development. In recent years, molecules in body fluid exosomes have attracted attention as biomarkers for diseases. In this study, small RNAs in serum exosomes were analyzed to identify noninvasive biomarkers of testicular injury in rats, which are often used in preclinical drug development. The rat models of testicular injury were prepared by a single oral administration of 2000 mg/kg ethylene glycol monomethyl ether, in which spermatocyte degeneration and Sertoli cell vacuolation were observed, or 400 mg/kg carbendazim, in which Sertoli cell vacuolation and seminiferous tubule dilation were observed. Serum exosomal small RNA-seq analysis of these models was performed. The analysis identified 3 small RNAs that fluctuated in common between the models, and miR-423-5p and miR-128-3p were selected as candidate markers. For evaluating these candidate markers in other testicular injury models, the models were prepared by a single oral administration of 60 mg/kg 1,3-dinitrobenzene or 500 mg/kg nitrofurazone, and spermatocyte degeneration and Sertoli cell vacuolation were observed. In qPCR analysis, these exosomal miRNAs were upregulated in all models except for the 1,3-dinitrobenzene model, in which severe hemolysis was observed. By contrast, these miRNAs in whole serum extracts did not significantly change in any of the models. In conclusion, we identified miR-423-5p and miR-128-3p in serum exosomes as noninvasive biomarkers for testicular injury in rats.

A Novel Dual Immunostain to Characterize Sloughed Cells in Testicular Biopsies for Infertility.

Evaluation of testicular biopsies from azoospermic men requires recognition of phases of germ cell maturation as organized architecturally within the seminiferous tubule, as well as distinguishing the inability to generate mature spermatozoa (germ cell aplasia or maturation arrest) from normal spermatogenesis, which may be associated with a reversible obstruction. While traditional fixatives (eg, Bouin solution) provide exquisite nuclear detail and preserve the architectural integrity of the seminiferous tubule, formalin fixation yields biopsies with relatively poor nuclear detail and frequent luminal sloughing of cells, making it difficult to assess sperm maturation. One clone of the anti-DOG1 antibody was recently found to be expressed in late (postspermatogonial) germ cells. We developed a dual stain including DOG1 and SF-1 to mark late germ cells and Sertoli cells, respectively, in both sloughed and intact cells. Consecutive testicular biopsies (N=28) from men with azoospermia were classified by hematoxylin and eosin morphology and stained with a dual SF-1 (Perseus)/DOG1 (Cell Marque) immunohistochemical stain. Histologic patterns included normal spermatogenesis (5 cases), hypospermatogenesis (5 cases), late maturation arrest (2 cases), Sertoli cell only pattern (15 cases), and extensive tubular hyalinization (1 case). Architectural disruption of seminiferous tubules with sloughing of cells into the lumens was noted in all biopsies, at least focally. SF-1 (nuclear) was expressed in sloughed Sertoli cells; DOG1 (cytoplasmic) in sloughed postspermatogonial germ cells (spermatocytes and spermatids). This resulted in two distinct immunophenotypes: SF-1(+)/DOG1(-) sloughed cells in cases with the Sertoli cell only pattern and SF-1(+)/DOG1(+) sloughed cells in all other histologic patterns (normal spermatogenesis, hypospermatogenesis, and maturation arrest). Because the rate of sperm retrieval is lower in men with the Sertoli cell only pattern, this immunohistochemical stain may assist pathologists in the proper interpretation of testicular biopsies, allowing better-informed decision making by patients and clinicians regarding the subsequent use of assisted reproductive technologies.

Characterization, recellularization, and transplantation of rat decellularized testis scaffold with bone marrow-derived mesenchymal stem cells.

BACKGROUND: Regenerative medicine potentially offers the opportunity for curing male infertility. Native extracellular matrix (ECM) creates a reconstruction platform to replace the organs. In this study, we aimed to evaluate the efficiency of the testis decellularized scaffold as a proper niche for stem cell differentiation toward testis-specific cell lineages. METHODS: Rats' testes were decellularized by freeze-thaw cycle followed by immersion in deionized distilled water for 2 h, perfused with 1% Triton X-100 through ductus deferens for 4 h, 1% SDS for 48 h and 1% DNase for 2 h. The decellularized samples were prepared for further in vitro and in vivo analyses. RESULT: Histochemical and immunohistochemistry studies revealed that ECM components such as Glycosaminoglycans (GAGs), neutral carbohydrate, elastic fibers, collagen I & IV, laminin, and fibronectin were well preserved, and the cells were completely removed after decellularization. Scanning electron microscopy (SEM) showed that 3D ultrastructure of the testis remained intact. In vivo and in vitro studies point out that decellularized scaffold was non-toxic and performed a good platform for cell division. In vivo implant of the scaffolds with or without mesenchymal stem cells (MSCs) showed that appropriate positions for transplantation were the mesentery and liver and the scaffolds could induce donor-loaded MSCs or host migrating cells to differentiate to the cells with phenotype of the sertoli- and leydig-like cells. The scaffolds also provide a good niche for migrating DAZL-positive cells; however, they could not differentiate into post meiotic-cell lineages. CONCLUSION: The decellularized testis can be considered as a promising vehicle to support cell transplantation and may provide an appropriate niche for testicular cell differentiation.

Internalization of Intact Intercellular Junctions in the Testis by Clathrin/Actin-Mediated Endocytic Structures: Tubulobulbar Complexes.

Sertoli cells of the mammalian seminiferous epithelium form unique subcellular actin-related structures at intercellular junctions. The appearance of these so called "tubulobulbar complexes" (TBCs) precedes both sperm release at the apex of the epithelium and the movement of early spermatogenic cells out of the spermatogonial stem cell niche at the base of the epithelium. TBCs are considered to be part of the mechanism of junction endocytosis by Sertoli cells. The structures contain junction proteins and morphologically identifiable junctions, and are associated with markers of endocytosis. Here we review the current state of knowledge about the structure and function of TBCs. As the complexes form, they morphologically resemble and have the molecular signature of clathrin-coated pits with extremely long necks. As they mature, the actin filament networks around the "necks" of the structures progressively disassemble and the membrane cores expand or swell into distinct "bulbs". These bulbs acquire extensive membrane contact sites with associated cisternae of endoplasmic reticulum. Eventually the bulbs undergo scission and continue through endosomal compartments of the Sertoli cells. The morphology and composition of TBC indicates to us that the structures likely evolved from the basic clathrin-mediated endocytosis mechanism common to cells generally, and along the way they incorporated unique features to accommodate the cyclic turnover of massive and "intact" intercellular junctions that occurs during spermatogenesis. Anat Rec, 301:2080-2085, 2018. © 2018 Wiley Periodicals, Inc.

NLRP3 in somatic non-immune cells of rodent and primate testes.

NLRP3 is part of the NLRP3 inflammasome and a global sensor of cellular damage. It was recently discovered in rodent Sertoli cells. We investigated NLRP3 in mouse, human and non-human primate (marmoset and rhesus macaque) testes, employing immunohistochemistry. Sertoli cells of all species expressed NLRP3, and the expression preceded puberty. In addition, peritubular cells of the adult human testes expressed NLRP3. NLRP3 and associated genes (PYCARD, CASP1, IL1B) were also found in isolated human testicular peritubular cells and the mouse Sertoli cell line TM4. Male infertility due to impairments of spermatogenesis may be related to sterile inflammatory events. We observed that the expression of NLRP3 was altered in the testes of patients suffering from mixed atrophy syndrome, in which tubules with impairments of spermatogenesis showed prominent NLRP3 staining. In order to explore a possible role of NLRP3 in male infertility, associated with sterile testicular inflammation, we studied a mouse model of male infertility. These human aromatase-expressing transgenic mice (AROM+) develop testicular inflammation and impaired spermatogenesis during aging, and the present data show that this is associated with strikingly elevated Nlrp3 expression in the testes compared to WT controls. Interference by aromatase inhibitor treatment significantly reduced increased Nlrp3 levels. Thus, throughout species NLRP3 is expressed by somatic cells of the testis, which are involved in testicular immune surveillance. We conclude that NLRP3 may be a novel player in testicular immune regulation.

Correlation between testicular mast cell count and spermatogenic epithelium in non-obstructive azoospermia.

Although there is emerging evidence that mast cells are involved in infertility, their exact role has not been elucidated clearly. Here we carried out a retrospective case-control study to find out whether there is a correlation between mast cell (MC) count and proliferation (Ki67 index) of the spermatogenic epithelium as well as of the Sertoli cells (vimentin-positive) in non-obstructive azoospermia (NOA). We assessed MCs, Ki67 and vimentin expression in Sertoli cells in testicular biopsies of germ cell aplasia (GCA, n = 14) and maturation arrest (MA, n = 14) vs. normal spermatogenesis (n = 14) cases. There was a significant decrease in the spermatogonial Ki67 index (1.25 ± 0.91, 4.21 ± 1.81 vs. 39.57 ± 3.92) and Johnsen score (2.48 ± 0.65, 4.89 ± 1.05 vs. 9.75 ± 0.30) as well as a significant increase (P < 0.001) in MC count (29.00 ± 4.11, 7.57 ± 1.95 vs. 3.00 ± 1.30) in seminiferous tubules of infertile cases with GCA and MA vs. controls. On the other hand, the percentage of vimentin-expressing Sertoli cells was significantly decreased (P < 0.001) in biopsies of cases with MA (35.50 ± 15.62) compared to those of cases with GCA and controls (72.64 ± 10.67 and 98.57 ± 1.45 respectively). Additionally, a significant negative correlation was detected between MC count and Ki67 index as well as Johnsen score in the MA group which became more significant in the GCA group. The significant increase in MC count in the GCA group and to a lesser extent in the MA group indicates their possible role in NOA particularly at the spermatogonial proliferation level and this is supported by the significant negative correlation with the Ki67 index.

PLAG1 deficiency impairs spermatogenesis and sperm motility in mice.

Deficiency in pleomorphic adenoma gene 1 (PLAG1) leads to reduced fertility in male mice, but the mechanism by which PLAG1 contributes to reproduction is unknown. To investigate the involvement of PLAG1 in testicular function, we determined (i) the spatial distribution of PLAG1 in the testis using X-gal staining; (ii) transcriptomic consequences of PLAG1 deficiency in knock-out and heterozygous mice compared to wild-type mice using RNA-seq; and (iii) morphological and functional consequences of PLAG1 deficiency by determining testicular histology, daily sperm production and sperm motility in knock-out and wild-type mice. PLAG1 was sparsely expressed in germ cells and in Sertoli cells. Genes known to be involved in spermatogenesis were downregulated in the testes of knock-out mice, as well as Hsd17b3, which encodes a key enzyme in androgen biosynthesis. In the absence of Plag1, a number of genes involved in immune processes and epididymis-specific genes were upregulated in the testes. Finally, loss of PLAG1 resulted in significantly lowered daily sperm production, in reduced sperm motility, and in several animals, in sloughing of the germinal epithelium. Our results demonstrate that the subfertility seen in male PLAG1-deficient mice is, at least in part, the result of significantly reduced sperm output and sperm motility.

Different expression of leptin and IGF1 in the adult and prepubertal testis in dogs.

Leptin (Lep) and insulin-like growth factor 1 (IGF1) are implicated in the regulation of testicular function, but in dogs, our knowledge is limited to the possible role of the IGF1 system in testicular tumours. In this study, we aimed to describe and compare gene expression and protein localization of Lep, IGF1 and their receptors (LepR and IGF1R, respectively) in the testis of healthy adult and prepubertal dogs. Testes were collected from sexually healthy mature (n = 7) and from 8-week-old dogs (n = 7). Relative gene expression of Lep, LepR, IGF1 and IGF1R was determined by semi-quantitative real-time (TaqMan) PCR and cellular distribution in the testis by immunohistochemistry. Statistical analysis was carried out with Student's t test. Lep and LepR mRNA concentration was similar between the two groups, but IGF1 and IGF1R gene expression was significantly higher in the 8-week-old pups. Protein localization and the intensity of signals differed by age. In adults, Lep and LepR immunoreactivity was detected in spermatocytes and spermatids. Leydig cells showed sporadic, weak Lep staining. In prepubertal animals, intense Lep signals were present in Leydig and Sertoli cells, and LepR was found in Leydig cells. IGF1 and IGF1R protein was expressed in spermatogonia of the mature testis; IGF1 signals in Leydig cells seemed stronger than IGF1R. In the pups, IGF1 and IGF1R staining was detected in Leydig cells and in gonocytes. Sertoli cells showed weak IGF1 and sporadic, weak IGF1R signals. In conclusion, Lep and IGF1 may support spermatogenesis in adult dogs and mediate Leydig cell function. In the immature testis, they may promote development of Sertoli and Leydig cells and gonocytes.

Immunocytochemical localization of kisspeptin and kisspeptin receptor in the primate testis.

BACKGROUND: Hypothalamic kisspeptin-kisspeptin receptor signalling in primates ensures the successful progression into puberty during development and maintenance of reproductive capacity during adulthood. Human testis has been shown to express high-to-moderate levels of kisspeptin and kisspeptin receptor gene expression. In this study, we aimed at characterizing the localization of kisspeptin and kisspeptin receptor in adult primate testis tissue. METHODS: Immunocytochemistry was performed on paraffin-embedded testicular sections from adult rhesus monkeys and from common marmoset monkeys. RESULTS: Kisspeptin receptor was detected in Sertoli cells in the periphery of the seminiferous tubules in adult testes of both species. In contrast, kisspeptin was not localized in the seminiferous epithelium and was detected only in the interstitial compartment of the adult rhesus monkey testis. CONCLUSION: Kisspeptin receptor and kisspeptin are localized in the testis of Old World and New World primates.

Changes in the morphology and protein expression of germ cells and Sertoli cells in plateau pikas testes during non-breeding season.

Plateau pikas are seasonally breeding small herbivores that inhabit the meadow ecosystem of the Qinghai-Tibetan Plateau. Testis regression in plateau pikas begins in early June, and the male pikas are completely infertile, with a dramatically reduced testis size, in late July. In this study, a decreased germ cell number in the testes was first noted in early June. By late June, only Sertoli cells and a small number of spermatogonia remained. Interestingly, large gonocyte-like germ cells were observed in early July. In late July, the number of gonocyte-like cells per tubule increased significantly, and most of the Sertoli cell nuclei moved to and clustered in the center of the seminiferous tubules. The gonocyte-like germ cells and Sertoli cells began to express AP-2γ and anti-Mullerian hormone (AMH) proteins, which were detected in the germ cells and Sertoli cells of juvenile pikas but not in adult testes. Simultaneously, LC3 puncta dramatically increased in the seminiferous tubules of the pikas' testes during the non-breeding season. Our study found that spermatogonia and Sertoli cells in non-breeding adult pikas morphologically resembled those in juvenile pikas and expressed specific markers, indicating that de-differentiation-like transitions may occur during this process.

Histologic effect of a postnatal slow-release GnRH agonist on feline gonads.

In postnatal domestic cats, GnRH agonists suppressed fecal concentrations of sexual steroids and delayed puberty. The aim of this study was to describe the gross and microscopic morphometric effects of a single administration of the GnRH agonist, deslorelin acetate, on the gonads of postnatally treated cats. Twenty-seven postnatal male (n = 14) and female (n = 13) kittens were randomly assigned to one of the following treatment groups within the first 24 hours of birth: deslorelin acetate (1.6 mg, subcutaneous; DA, n = 16) or control that remained untreated (CO, n = 11) and spayed or castrated immediately after the onset of puberty. After surgical removal, the gonads were gross and histologically assessed. Sertoli cells also were examined immunohistochemically. Comparisons between the treatments were carried out by the Student t test. Gross gonadal wet weight and volume as well as gonadosomatic index were significantly lower in the DA than those in the CO males; these same parameters were not different in females. Primordial (461.4 + 3.0 vs. 1074.3 + 117.5; P < 0.01), primary (59.1 + 13.5 vs. 165.4 + 24.6; P < 0.01), and secondary (17.5 + 2.6 vs. 31.17 + 8.1; P < 0.05) follicles per mm(2) were decreased in DA than in CO gonads. Epididymal sperm motility and morphology were normal in all but two DA cats that had too few sperm to be evaluated. Germinal epithelial height (µm; 39.68 + 0.92 vs. 72.7 + 1.2; P < 0.01) and most of their cellular components as well as the Sertoli (cm(3); 0.1 + 0.02 vs. 0.24 + 0.05; P < 0.01) cells were diminished in the DA cats. Gonadotropin-releasing hormone agonist endocrine disruption during the neonatal critical reproductive time window may have a potential as a contraceptive agent in domestic felids.

Immunohistochemical Characterization of Spontaneous Sertoli Cell Clusters in the Seminiferous Tubules of C57BL/6J Mice.

Cell clusters were observed in the seminiferous tubules of C57BL/6J mice as a spontaneous lesion in a 2-week toxicity study, and they were demonstrated to be basically composed of Sertoli cells by immunohistochemistry for claudin-11 and GATA-4 (GATA-binding protein 4), which are both Sertoli cell markers. The clusters were composed of about 5 to 50 cells, which had eosinophilic and occasionally vacuolated cytoplasm with an unclear cell boundary. The cell clusters involved some sperm. No mitotic figures were observed and no immunoreactivity for proliferating cell nuclear antigen (PCNA) was detected in the clusters. In most cases, the cell clusters were observed in seminiferous tubules that also showed degenerative changes. In rare instances, cell aggregates immunohistochemically positive for claudin-11 were observed in the lumen of the epididymis, suggesting that some of the Sertoli cell clusters were sloughed off from the seminiferous epithelium into the epididymal ducts. To our knowledge, this is the first report of Sertoli cell clusters in any animal species except for transgenic or surgically altered animals.

Expression of steroid hormone receptors in the genital structures of a true hermaphrodite pug dog.

Hermaphroditism is a rare and a not well-understood disordered sexual development (DSD) in dogs. The objective of the study was to analyse the sex steroid hormone receptor (STHR) expression patterns in the internal genital structures, because the responsiveness of the different tissue types to the steroid hormones may have a key role in pathological alterations based on DSDs. Furthermore, the adhesion molecule ß-catenin was investigated by means of immunohistochemistry because of its important role in development, tissue integrity and disease. Molecular sexing was performed via PCR targeting DBX/DBY genes to identify the pug dog as a true XX hermaphrodite. The portions of uterine tissue revealed comparable expression patterns for STHRs as investigated in normal female reproductive tissue. In the male parts, ß-catenin showed strong expression in the Sertoli cells of the seminiferous tubules; this was in contrast to normal testicular tissue. Likewise, the layers of smooth muscle actin-positive cells surrounding the seminiferous tubules were reduced in the hermaphrodite. The results of this study deepen the knowledge of tissue characteristics in a hermaphrodite dog and highlight the importance of early diagnosis because the STH responsiveness in maldeveloped reproductive tissue might lead to serious problems for the dog.

The role of Pten/Akt signaling pathway involved in BPA-induced apoptosis of rat Sertoli cells.

Bisphenol-A (BPA), one of endocrine-disrupting chemicals, is a male reproductive toxicant. Previous studies have revealed the direct cytotoxicity of BPA in many cultured cells, such as mitotic aneuploidy in embryonic cells and somatic cells, and apoptosis in neurons and testicular Sertoli cells. To understand the action of BPA and assess its risk, the Pten/Akt pathway was investigated in cultured Sertoli cells to elucidate the mechanism of the reproductive effects of BPA. The results showed that over 50 µM BPA treatment could decrease the viability of Sertoli cells and cause more apoptosis. In addition, BPA could induce the increase in mRNA levels of Pten and Akt. The protein level of Pten was increased; however, the protein levels of phospho-Akt and procaspase-3 were decreased after BPA exposure. Taken together, observed results suggested that the Pten/Akt pathway might be involved in the apoptotic effects of BPA on Sertoli cells.

Germ cell migration across Sertoli cell tight junctions.

The blood-testis barrier includes strands of tight junctions between somatic Sertoli cells that restricts solutes from crossing the paracellular space, creating a microenvironment within seminiferous tubules and providing immune privilege to meiotic and postmeiotic cells. Large cysts of germ cells transit the Sertoli cell tight junctions (SCTJs) without compromising their integrity. We used confocal microscopy to visualize SCTJ components during germ cell cyst migration across the SCTJs. Cysts become enclosed within a network of transient compartments fully bounded by old and new tight junctions. Dissolution of the old tight junctions releases the germ cells into the adluminal compartment, thus completing transit across the blood-testis barrier. Claudin 3, a tight junction protein, is transiently incorporated into new tight junctions and then replaced by claudin 11.

Inhibin B and anti-Müllerian hormone/Müllerian-inhibiting substance may contribute to the male bias in autism.

The autistic spectrum disorders have a significant male bias in incidence, which is unexplained. The Sertoli cells of the immature testes secrete supra-adult levels of Müllerian-inhibiting substance/anti-Müllerian hormone (AMH) and inhibin B (InhB), with both hormones being putative regulators of brain development. We report here, that 82 boys with an autism spectrum disorder have normal levels of InhB and AMH. However, the boys' level of InhB correlated with their autism diagnostic interview-revised (ADI-R) scores for the social interaction (R=0.29, P=0.009, N=82) and communication domains (R=0.29, P=0.022, N=63), and with the number of autistic traits the boys exhibited (R=0.34 and 0.27, respectively). The strengths of the abovementioned correlates were stronger in the boys with milder autism (R=0.42 and 0.50, respectively), with AMH exhibiting a significant negative correlation to the ADI-R score in these boys (R=-0.44 and R=-0.39, respectively). Neither hormone correlated to the incidence of stereotyped and repetitive behaviours. This suggests that the male bias in the autistic spectrum has multiple determinants, which modulate the effects of an otherwise non-dimorphic pathology. Furthermore, AMH and InhB have opposing effects on the SMAD1/5/8 pathway, and opposing correlates to autistic traits, implicating the SMAD pathways as a putative point of molecular convergence for the autistic spectrum.

Presence, localization, and origin of clusterin in normal human spermatozoa.

PURPOSE: Clusterin in mammalian semen is a secretory form of clusterin (sCLU) with the heterodimeric structure. It is secreted by the epididymis and seminal vesicle. It is generally agreed that clusterin mainly exists on the surface of abnormal spermatozoa and is implicated in decreased sperm motility, sperm aggregation and infertility. However, few studies observe clusterin in normal spermatozoa, which is presumed to be a novel form. Up to now, the systematical information about the presence, localization, origin and function of clusterin in normal human spermatozoa has yet not been established. The aim of our current study is to systematically research clusterin in normal human spermatozoa. METHODS: We detected the presence of clusterin via western blot, explored the localization of clusterin using immunofluorescence, and investigated the origin and distribution of clusterin in human testis by western blot and immunohistochemistry. RESULTS: We found native clusterin in the inner plasma membrane of normal human spermatozoa. It was derived from the testis and showed similar molecular weight and heterodimeric structure compared with sCLU in semen and on the surface of abnormal spermatozoa. CONCLUSION: Clusterin in normal spermatozoa should be self-synthesized during the later stage of spermatogenesis. The different localization and origin suggested that the clusterin observed by us may be a novel form compared with conventional sCLU on the surface of abnormal spermatozoa.

Reduced numbers of Sertoli, germ, and spermatogonial stem cells in impaired spermatogenesis.

A key step in the investigation of male infertility is the appropriate classification of impaired spermatogenesis. In this study, we precisely identified Sertoli and distinct germ-cell types in the rat, the mouse, and in the human testis. As a proof of principle, we studied testis biopsy samples from azoospermic patients with defined spermatogenic defects. Remarkably, we found that already the numbers of Sertoli cells, spermatogonia and a subset of spermatogonia including stem cells are significantly reduced in patients with maturation arrest at the level of primary spermatocytes (n=33) compared with patients with histologically normal spermatogenesis (n=33). In patients with hypospermatogenesis (n=44) a significant reduction of spermatogonial cell numbers was observed. The numbers of primary and diplotene spermatocytes were reduced by 84%. However, the strongest reduction (96%) was revealed in the numbers of spermatids in patients with maturation arrest. In contrast, patients with hypospermatogenesis showed only modestly reduced numbers of spermatocytes and spermatids compared with normal spermatogenesis. No correlation was found with age or obstruction. For a detailed analysis of the patients, we distinguished between 'pool of founder cells'-related deficiencies (reduced numbers of Sertoli cells, spermatogonia, and spermatogonial stem cells) and 'meiotic' deficiencies (reduced numbers of spermatocytes, meiotic divisions, and spermatids). Interestingly, patients with maturation arrest showed meiotic deficiencies (36%), while the majority additionally demonstrated deficiencies in the founder pool (58%). In contrast, patients with normal spermatogenesis most often had no deficiencies at all (45%) or founder pool-related deficiencies (33%) but an apparently normal meiosis. This is the first report showing that many infertile patients face besides meiotic defects the problem of reduced numbers of Sertoli cells, spermatogonia, and spermatogonial stem cells.