A molecular rheostat adjusts auxin flux to promote root protophloem differentiation.

Auxin influences plant development through several distinct concentration-dependent effects 1 . In the Arabidopsis root tip, polar auxin transport by PIN-FORMED (PIN) proteins creates a local auxin accumulation that is required for the maintenance of the stem-cell niche2-4. Proximally, stem-cell daughter cells divide repeatedly before they eventually differentiate. This developmental gradient is accompanied by a gradual decrease in auxin levels as cells divide, and subsequently by a gradual increase as the cells differentiate5,6. However, the timing of differentiation is not uniform across cell files. For instance, developing protophloem sieve elements (PPSEs) differentiate as neighbouring cells still divide. Here we show that PPSE differentiation involves local steepening of the post-meristematic auxin gradient. BREVIS RADIX (BRX) and PROTEIN KINASE ASSOCIATED WITH BRX (PAX) are interacting plasma-membrane-associated, polarly localized proteins that co-localize with PIN proteins at the rootward end of developing PPSEs. Both brx and pax mutants display impaired PPSE differentiation. Similar to other AGC-family kinases, PAX activates PIN-mediated auxin efflux, whereas BRX strongly dampens this stimulation. Efficient BRX plasma-membrane localization depends on PAX, but auxin negatively regulates BRX plasma-membrane association and promotes PAX activity. Thus, our data support a model in which BRX and PAX are elements of a molecular rheostat that modulates auxin flux through developing PPSEs, thereby timing PPSE differentiation.

Isolation and gene expression analysis of single potential human spermatogonial stem cells.

STUDY HYPOTHESIS: It is possible to isolate pure populations of single potential human spermatogonial stem cells without somatic contamination for down-stream applications, for example cell culture and gene expression analysis. STUDY FINDING: We isolated pure populations of single potential human spermatogonial stem cells (hSSC) without contaminating somatic cells and analyzed gene expression of these cells via single-cell real-time RT-PCR. WHAT IS KNOWN ALREADY: The isolation of a pure hSSC fraction could enable clinical applications such as fertility preservation for prepubertal boys and in vitro-spermatogenesis. By utilizing largely nonspecific markers for the isolation of spermatogonia (SPG) and hSSC, previously published cell selection methods are not able to deliver pure target cell populations without contamination by testicular somatic cells. However, uniform cell populations free of somatic cells are necessary to guarantee defined growth conditions in cell culture experiments and to prevent unintended stem cell differentiation. Fibroblast growth factor receptor 3 (FGFR3) is a cell surface protein of human undifferentiated A-type SPG and a promising candidate marker for hSSC. It is exclusively expressed in small, non-proliferating subgroups of this spermatogonial cell type together with the pluripotency-associated protein and spermatogonial nuclear marker undifferentiated embryonic cell transcription factor 1 (UTF1). STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We specifically selected the FGFR3-positive spermatogonial subpopulation from two 30 mg biopsies per patient from a total of 37 patients with full spermatogenesis and three patients with meiotic arrest. We then employed cell selection with magnetic beads in combination with a fluorescence-activated cell sorter antibody directed against human FGFR3 to tag and visually identify human FGFR3-positive spermatogonia. Positively selected and bead-labeled cells were subsequently picked with a micromanipulator. Analysis of the isolated cells was carried out by single-cell real-time RT-PCR, real-time RT-PCR, immunocytochemistry and live/dead staining. MAIN RESULTS AND THE ROLE OF CHANCE: Single-cell real-time RT-PCR and real-time RT-PCR of pooled cells indicate that bead-labeled single cells express FGFR3 with high heterogeneity at the mRNA level, while bead-unlabeled cells lack FGFR3 mRNA. Furthermore, isolated cells exhibit strong immunocytochemical staining for the stem cell factor UTF1 and are viable. LIMITATIONS, REASONS FOR CAUTION: The cell population isolated in this study has to be tested for their potential stem cell characteristics via xenotransplantation. Due to the small amount of the isolated cells, propagation by cell culture will be essential. Other potential hSSC without FGFR3 surface expression will not be captured with the provided experimental design. WIDER IMPLICATIONS OF THE FINDINGS: The technical approach as developed in this work could encourage the scientific community to test other established or novel hSSC markers on single SPG that present with potential stem cell-like features. STUDY FUNDING AND COMPETING INTERESTS: The project was funded by the DFG Research Unit FOR1041 Germ cell potential (SCH 587/3-2) and DFG grants to K.v.K. (KO 4769/2-1) and A.-N.S. (SP 721/4-1). The authors declare no competing interests.

Expression of miRNA-371a-3p in seminal plasma and ejaculate is associated with sperm concentration.

BACKGROUND: The microRNAs of the miR-371-3 cluster are novel serum markers for testicular germ cell tumors. Sporadic reports suggested the expression of this miRNA in semen. OBJECTIVES: To verify the expression of miR-371a-3p in seminal plasma and unprocessed ejaculate; to compare seminal plasma miRNA levels in germ cell tumors patients with those of controls; to look for an association of miRNA levels with semen quality. MATERIALS AND METHODS: The miR-371a-3p expression was analyzed with qPCR. The study population consisted of 100 participants: seminal plasma samples from 20 germ cell tumors patients and 30 controls, serum samples from 12 healthy men, ejaculate samples from 38 men undergoing fertility testing. RESULTS: The seminal plasma miR-371a-3p levels of germ cell tumors patients were not different from controls. The miRNA expression was very low in serum but much higher in seminal plasma. In ejaculate samples, the miRNA expression significantly correlated with sperm concentration and the total sperm count. DISCUSSION: miR-371-a-3p is present in sperm-containing fluids. Seminal plasma levels cannot be used to distinguish germ cell tumors from controls. The correlation with sperm concentration in ejaculate samples suggests the spermatozoa as possible source of miR-371a-3p production. CONCLUSION: The miR-371a-3p levels in ejaculate could represent a novel biomarker for the non-invasive evaluation of male infertility.

Multicolor fluorescence in situ hybridization (M-FISH) on cells from urine for the detection of bladder cancer.

Bladder cancer is the fifth most common cancer in adults. Because of the high recurrence rate (up to 70%) new tumor markers for urine are necessary for monitoring patients. In this study, we investigated the value of M-FISH on cells from urine for the detection of bladder cancer. Urine samples from 141 patients suspicious of bladder cancer were analyzed in this study. Cells were isolated from urine before surgical therapy. For FISH analysis, a commercial kit (UroVysion) containing hybridization probes for chromosomes 3, 7, 9p21 and 17, was used. Twenty-five cells were analyzed in each case by two observers. A FISH result was obtained in 121 cases. Overall, sensitivity was 60% and specificity reached 82.6%. Sensitivity and specificity by cytology were 24.1% and 90.5%, respectively. Analyzing results concerning T-category, sensitivity of FISH and cytology was 36.1% and 15% in pTa, 65.2 and 25.7% in pT1, 100% and 66.7% in pT2-3 tumors, respectively. Concerning tumor grade, similar results were obtained: sensitivity was 37% and 14% in G1, 65.4% and 40% in G2, 91.7% and 50% in G3 tumors, for FISH and cytology, respectively. In conclusion, FISH on cells from urine has been shown in all studies to be highly sensitive and specific for detection of bladder cancer. Sensitivity of FISH is higher than conventional cytology and can be used in routine diagnosis additionally to conventional cytology especially in doubtful or negative cases. FISH can detect recurrence earlier than other methods like cytology, cystoscopy or biopsy histological examination.

Ignition of calcium sparks in arterial and cardiac muscle through caveolae.

Ca(2+) sparks are localized intracellular Ca(2+) events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca(2+) spark triggering depends on precise delivery of Ca(2+) ions through dihydropyridine (DHP)-sensitive Ca(2+) channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca(2+) influx channels and RyRs. Because caveolae contain DHP-sensitive Ca(2+) channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca(2+) sparks. Using methyl-ss-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca(2+) sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca(2+) sparks were not significantly affected. We ruled out the possibility that the decreases in Ca(2+) spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca(2+) load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca(2+) sparks in a caveolae microdomain. The transient elevation in [Ca(2+)](i) at the inner mouth of a single caveolemmal Ca(2+) channel induces simultaneous activation and thus opens several RyRs to generate a local Ca(2+) release event, a Ca(2+) spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca(2+) signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease.

Mass isolation of cell surface membrane fragments from pigeon heart.

Cell surface membrane fragments were isolated and purified by successive rate zonal and isopycnic centrifugation of calcium oxalate-loaded pigeon heart microsomes in sucrose density gradients. The most highly purified cell membrane fraction sediments at a buoyant density of 1.105 g/ml. Some of the membrane pieces are present as open fragments and leaky vesicles, while others form tightly sealed vesicles of both inside-in and inside-out membrane orientation. The pigeon heart cell membrane preparation exhibits high (Na+ + K+ + Mg2+)-ATPase and adenylate cyclase activities. Additional activity of these enzymes is uncovered by sodium dodecyl sulfate and alamethicin, respectively. Electron microscopic inspection of the cell surface membrane preparation revealed (a) a predominance of thick-walled vesicles with smooth surfaces on negative staining and (b) binding of concanavalin A to the bulk of isolated membrane pieces following their incubation with the lectin.

Functional characterization of mitochondrial oxidative phosphorylation in saponin-skinned human muscle fibers.

The conditions of treatment of human skeletal muscle fibers from M. vastus lateralis with saponin were optimized to achieve complete permeabilization of cell membrane at intact mitochondrial oxidative phosphorylation. After 30 min of incubation with saponin all lactate dehydrogenase, 50% of creatine kinase, 30% of adenylate kinase and less than 20% of citrate synthase was released into the permeabilization medium. These skinned fibers behave similar to isolated mitochondria from human skeletal muscle: (i) the respiration with mitochondrial substrates can be stimulated by ADP, (ii) inhibited by carboxyatractyloside and (iii) it is possible to detect fluorescence changes of mitochondrial NAD(P)H on additions of substrates, uncoupler and cyanide. From a comparison of rates of respiration per cytochrome aa3 content of isolated human skeletal muscle mitochondria and saponin-skinned muscle fibers it was possible to calculate that almost 85% of mitochondria in those fibers are accessible for the investigation of oxidative phosphorylation. As shown by the investigation of biopsy samples of two patients with undefined myopathies these fibers are a suitable object for the replacement of isolated mitochondria in the diagnosis of mitochondrial myopathies and encephalomyopathies.

Tachykinin (substance-P) gene expression in Leydig cells of the human and mouse testis.

Specific substance-P immunoreactivity can be detected in the Leydig cells, particularly of human testes, and to a lesser degree in mouse Leydig cells, but not in the rat. Using a modified polymerase chain reaction (PCR) assay, preprotachykinin-A (substance-P) mRNA could be detected in extracts of human, mouse, and bovine testes, but not in rat or boar testes or in bovine thyroid or corpus luteum used as negative controls. This assay is able to discriminate among the alpha, beta, and gamma transcripts of the gene and shows that only the beta and gamma transcripts are present in the testes. Sequencing analysis of the PCR products from bovine hypothalamus, mouse brain, and human testis confirmed the structure of these transcripts, which encode both substance-P and neurokinin-A (substance-K) neuropeptide hormones. Using a variant of this assay it was possible to identify tachykinin transcripts in as few as 500 freshly prepared purified mouse Leydig cells. In parallel studies PCR analysis was also able to confirm the presence of mRNA for both substance-P and neurokinin-A receptors in human testes. Thus, the tachykinins substance-P and neurokinin-A must now be added to the list of potentially paracrine substances regulating intratesticular function.

Impairment of cardiac function and bioenergetics in adult transgenic mice overexpressing the bovine growth hormone gene.

Cardiovascular abnormalities represent the major cause of death in patients with acromegaly. We evaluated cardiac structure, function, and energy status in adult transgenic mice overexpressing bovine GH (bGH) gene. Female transgenic mice expressing bGH gene (n = 11) 8 months old and aged matched controls (n = 11) were used. They were studied with two-dimensional guided M-mode and Doppler echocardiography. The animals (n = 6) for each group were examined with 31P magnetic resonance spectroscopy to determine the cardiac energy status. Transgenic mice had a significantly higher body weight (BW), 53.2+/-2.4 vs. 34.6+/-3.7 g (P < 0.0001) and hypertrophy of left ventricle (LV) compared with normal controls: LV mass/BW 5.6+/-1.6 vs. 2.7+/-0.2 mg/g, P < 0.01. Several indexes of systolic function were depressed in transgenic animals compared with controls mice such as shortening fraction 25+/-3.0% vs. 39.9+/-3.1%; ejection fraction, 57+/-9 vs. 77+/-5; mean velocity of circumferential shortening, 4.5+/-0.8 vs. 7.0+/-1.1 circ/sec, p < 0.01. Creatine phosphate-to-ATP ratio was significantly lower in bGH overexpressing mice (1.3+/-0.08 vs. 2.1+/-0.23 in controls, P < 0.05). Ultrastructural examination of the hearts from transgenic mice revealed substantial changes of mitochondria. This study provides new insight into possible mechanisms behind the deteriorating effects of long exposure to high level of GH on heart function.

Significance of mutations in the androgen receptor gene in males with idiopathic infertility.

Abnormal human spermatogenesis is caused by a variety of genetic and acquired conditions. Because spermatogenesis is dependent on androgens, some males may have a minimal form of androgen insensitivity that does not inhibit virilization but impairs fertility. This has lead us to investigate the possibility of abnormalities in the androgen receptor (AR) gene in a large cohort of males suffering from infertility of unknown cause. We studied 180 males with variable impairment of spermatogenesis. In all patients, serum levels of testosterone and gonadotropins were analyzed to define an androgen sensitivity index (ASI). Single-strand conformation analysis and direct DNA sequencing of PCR-amplified blood leukocyte DNA were used to identify mutations within the whole coding region of the AR-gene. Endocrine and molecular investigations were compared with 53 normal males with proven fertility. In three infertile males, mutations in the AR were identified. Two unrelated males had the same variation within the first exon encoding for the transactivation domain of the receptor (Pro390Ser), whereas, in the third, a mutation in the hormone-binding region was characterized (Gln798Glu). All identified mutation carriers had a significantly elevated ASI. A proportion of males with idiopathic infertility carry relevant variations within the AR-gene. These males may be distinguished on the basis of hormone levels, calculating the ASI, although this index lacks specificity.

Function of the cytosolic N-terminus of sucrose transporter AtSUT2 in substrate affinity.

AtSUT2 was found to be a low-affinity sucrose transporter (K(M)=11.7 mM at pH 4). Chimeric proteins between AtSUT2 and the high-affinity StSUT1 were constructed in which the extended N-terminus and central loop of AtSUT2 were exchanged with those domains of StSUT1 and vice versa. Chimeras containing the N-terminus of AtSUT2 showed significantly lower affinity for sucrose compared to chimeras containing the N-terminus of StSUT1. The results indicate a significant function of the N-terminus but not the central cytoplasmic loop in determining substrate affinity. Expression of AtSUT2 in major veins of source leaves and in flowers is compatible with a role as a second low-affinity sucrose transporter or as a sucrose sensor.

Autoantibodies against the angiotensin receptor (AT1) in patients with hypertension.

Sera from patients with malignant essential hypertension (n = 14), malignant secondary hypertension mainly attributable to renovascular diseases (n = 12) and renovascular diseases without malignant hypertension (n = 11) and from normotensive healthy blood donors (n = 35) were studied for the presence of autoantibodies against G-protein-coupled cardiovascular receptors. Autoantibodies against the angiotensin II receptor (AT1) were detected in 14, 33, 18 and 14% of patients with malignant essential hypertension, malignant secondary hypertension, renovascular diseases and control patients, respectively. Sensitivity of the enzyme immunoassay was assessed as 5 microg/ml IgG. Patients did not show antibodies against bradykinin (B2) or angiotensin II subtype 2 (AT2) receptors. Autoantibodies affinity-purified from positive patients localized AT receptors in Chinese hamster ovary transfected cells, and displayed a positive chronotropic effect on cultured neonatal rat cardiomyocytes. These results demonstrate the existence of autoantibodies against a functional extracellular domain of human AT1 receptors in patients with malignant hypertension, and suggest that these autoantibodies might be involved in the pathogenesis of malignant hypertension.

Immunocytochemical localization of M2 muscarinic receptors in rat ventricles with anti-peptide antibodies.

We produced antibodies against a synthetic peptide corresponding to amino acids 168-192 of the second extracellular loop of the M2 human muscarinic receptor in rabbits. In immunoblot, affinity-purified antibodies specifically recognized a major band of rat ventricular muscarinic receptor protein with a molecular weight of about 80 KD. This recognition could be blocked by pre-incubation with peptide. Moreover, with both light (LM) and electron microscopic (EM) immunocytochemistry techniques, muscarinic receptors were detected on sarcolemma and T-tubules of rat cardiomyocytes. In addition, immunoreactions were localized in membranes of capillaries. Likewise, these reactivities were abolished by pre-incubation with peptide. These results suggest that the antibodies against the second extracellular loop of human M2 muscarinic receptor could specifically recognize rat ventricular muscarinic receptor protein and could be a powerful tool to study the fate of this receptor under different pathological or physiological conditions.

Synergistic cytotoxic effect of human recombinant tumor necrosis factor alpha combined with dihydroambazone.

Dihydroambazone (DHA) is a water-soluble derivative of the experimental anticancer drug ambazone. In vitro, a combination of DHA and human recombinant tumor necrosis factor alpha (TNF) exerted a strong synergism of cytotoxicity against both mouse melanoma B16K cells and the TNF-sensitive mouse fibroblast line L-M (S). Furthermore, in a colony-forming assay with B16K cells a combination of TNF and DHA inhibited colony-formation much more severely than either drug alone. An increased antiproliferative efficiency was also confirmed in vivo against established subcutaneous melanoma B16 tumors of C57BL/6 mice.

Human seminal fluid contains significant quantities of prorenin: its correlation with the sperm density.

The relevance of the tissue prorenin-renin-angiotensin system (PRAS) to male reproduction has been suggested by several investigators in the past. Although the presence of angiotensin converting enzyme in semen has been demonstrated, unequivocal evidence for the presence of prorenin and renin in the semen is not yet available. We have used a specific immunoradiometric assay based on an antibody directed against the pro-segment of the prorenin molecule to demonstrate that significant quantities of prorenin are present in human semen samples. Although semen is a rich source of proteases and protease inhibitors, the assay used by us, unlike the usual enzymatic renin assay, is not affected by such proteases, and their inhibitors. Furthermore, Western blotting data clearly demonstrated that prorenin is present in semen as a 48 kDa protein. In a majority of semen samples, the prorenin content was found to be several fold greater than that measured in EDTA-plasma samples. Interestingly, the level of prorenin was found to be directly proportional to the sperm density in semen samples. Our results suggest that seminal prorenin is produced locally within the male reproductive system, although its exact origin is yet to be defined, that a complete prorenin-renin-angiotensin system exists in human semen and that this system may be relevant to sperm function.

Induction of cardiomyopathy in severe combined immunodeficiency mice by transfer of lymphocytes from patients with idiopathic dilated cardiomyopathy.

Growing evidence suggests that autoimmune mechanisms play an important role in the pathogenesis of idiopathic dilated cardiomyopathy (DCM). The aim of the study was to evaluate the effects of transfer of lymphocytes from patients with DCM into severe combined immunodeficiency (SCID) mice on the heart structure and function. Thirty CB-17 SCID (6-8 weeks old) mice were used and divided into 3 groups (n = 10). Mice were injected intraperitoneally with up to 25 x 10(6) peripheral blood lymphocytes (PBL) from either patients with DCM which contain human autoantibodies against cardiac beta1-adrenergic receptors and M2-muscarinic receptors (DCM group) or PBL from healthy controls (control-H group). Ten mice did not receive any injections and were used as baseline controls (control-N group). Echocardiography and morphological studies were performed seventy five days after the transfer. Results showed that in DCM group, left ventricle dimensions (LVD) in diastole were increased (4.2 +/- 0.1mm) as compared to both control-H group (3.8 +/- 0.1mm) and control-N group (3.6 +/- 0.1 mm) (p < 0.01). Further, there was a trend for increased LVD in systole. Fractional shortening was not different between groups. Histological evaluation revealed accumulation of human lymphocytes in the capillaries and scarce infiltration of the lymphocytes in the hearts from DCM group. Diffuse fibrosis was significant increased in DCM mice as compared to mice receiving PBL from normal subjects (2.2 +/- 0.3% vs. 0.8 +/- 0.1%, p < 0.01). In conclusion, transfer of the PBL from the patients with DCM was able to induce early stage of heart dilatation in SCID mice. These data provide for the first time the direct evidence supporting that the autoimmune mechanism is important in the pathogenesis of human DCM.

Synergistic cytotoxicity of human recombinant tumour necrosis factor alpha combined with microtubule effectors.

Combinations of human recombinant tumour necrosis factor alpha (rhTNF alpha) with each of four different agents disturbing the microtubule system of the cellular cytoskeleton were tested for synergistic cytotoxic action against murine melanoma B16K and L-M(S) cells. In addition to the known microtubule effectors colchicine, vincristine, and taxol, the influence of the fluorenone-azo-methine derivative alpha-diphenylene-N-(p-[bis-(beta-hydroxyethyl-amino]-phenyl)- nitrone (DHPN) on the rhTNF alpha cytotoxicity was studied. Applying a novel computer-based isobole method [Suehnel J (1990) Antiviral Res 13:23-40] concentration ranges of synergistic, zero, and antagonistic interaction were found after in vitro combination of rhTNF alpha with each of the drugs tested in a 72-h cytotoxicity assay. In contrast, a 24-h exposure of B16K cells to these combinations still did not inhibit in vitro colony formation to a greater extent than either drug alone. A preliminary in vivo experiment revealed an increased antitumour effect after treatment of established subcutaneous melanoma B16 tumours with a combination of rhTNF alpha and DHPN.

Adenylyl cyclase in the heart: an enzymocytochemical and immunocytochemical approach.

This review provides a discussion of the localization of adenylyl cyclase (AC) in normal mammalian heart tissue employing enzymocytochemistry (detection of the catalytic activity of AC by a metal precipitation technique) and immunocytochemistry (immunolabeling of the enzyme protein with antibodies against AC subtypes). By the metal precipitation technique, AC activity was localized in adult guinea pig cardiomyocytes along the sarcolemma and the T-tubule membranes. This reaction can be enhanced by hormones and guanylyl imidodiphosphate, fluoride, and forskolin. With this technique, no precipitates were detected at the sarcoplasmic reticulum. However, under ischemic conditions, AC activity was also found in the junctional sarcoplasmic reticulum of rat cardiomyocytes. Immunocytochemistry revealed AC in the plasma membrane of rat cardiomyocytes. Detection of AC in the perinuclear space of cardiomyocytes might reflect initiation of synthesis and processing of the enzyme protein. Colocalization of AC with cytoskeleton fibers of non-cardiomyocytes emerging in the cell culture of neonatal rat cardiocytes imply a direct cytoskeletal-AC interaction. Finally, it can be stated that the immunolabeling pattern of AC in cryosections of adult and new-born rat hearts reveals a good correspondence with the localization of AC activity in cardiomyocytes demonstrated by enzymocytochemistry.

Growth of rodlike silver nanoparticles by vapor deposition of small clusters.

A beam of silver clusters of 3 nm average diameter is produced by the gas-aggregation technique and deposited onto the NaCl (100) surface. Subsequent agglomeration leads in part to the growth of rodlike particles with the structure of truncated decahedra of typical dimensions 70 nm long and 10 nm diameter and a fivefold symmetry, as shown in the picture. A growth mechanism is inferred from a detailed analysis of electron microscopy data.

Telomerase activity and expression of telomerase subunits in the testicular tissue of infertile patients.

OBJECTIVE: Determination of telomerase activity and the expression of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in the testicular tissue of patients with infertility arising from various causes. DESIGN: Prospective observational study. SETTING: A university hospital. PATIENT(S): Thirty-three patients with azoospermia arising from various causes. There were 12 testicular biopsy specimens from patients with Sertoli cell-only syndrome, 9 from patients with maturation arrest, and 12 from patients with obstructive azoospermia and normal histologic findings. INTERVENTION(S): Thirty-three testicular biopsies. MAIN OUTCOME MEASURE(S): Correlation of histologic findings at testicular biopsy with telomerase activity, hTERT, and hTR. RESULT(S): All 12 biopsy specimens from patients with obstructive azoospermia were positive for telomerase activity, hTR, and hTERT. Biopsy specimens from the 9 patients with maturation arrest were positive for telomerase activity in 8 cases, hTR in 9 cases, and hTERT in 5 cases. None of the patients with Sertoli cell-only syndrome showed either telomerase activity or hTERT, but all of them showed hTR. CONCLUSION(S): Telomerase activity and evidence of hTERT in testicular tissue are highly sensitive and highly specific markers of gametogenesis, which could gain in importance as part of the fertility workup before microinjection procedures.