Phosphatidylinositol 3-Kinase δ-Specific Inhibitor-Induced Changes in the Ovary and Testis in the Sprague Dawley Rat and Cynomolgus Monkey.

Phosphatidylinositol 3-kinase (PI3K) δ is a lipid kinase primarily found in leukocytes, which regulates important cell functions. AMG2519493 was a PI3K δ-specific inhibitor in development for treatment of various inflammatory diseases. AMG2519493-related changes in the male and/or female reproductive organs were observed in the 1- and 3-month oral repeat dose toxicology studies in the rat and cynomolgus monkey. Hemorrhagic corpora lutea cysts and increased incidence of corpora lutea cysts without hemorrhage were observed in the ovaries at supra pharmacological doses in the rat. A decrease in seminiferous germ cells in the testis, indicative of spermatogenesis maturation arrest, was observed in both the rat and cynomolgus monkey. Although the characteristics were comparable, the drug systemic exposures associated with the testicular changes were very different between the 2 species. In the rat, the testicular change was only observed at supra pharmacologic exposure. Isotype assessment of PI3K signaling in rat spermatogonia in vitro indicated a role for PI3K ß, but not δ, in the c Kit/PI3K/protein kinase B signaling pathway. Therefore, changes in both the ovary and testis of the rat were considered due to off target effect as they only occurred at suprapharmacologic exposure. In contrast, the testicular changes in the cynomolgus monkey (decrease in seminiferous germ cells) occurred at very low doses associated with PI3K δ-specific inhibition, indicating that the PI3K δ isoform may be important in spermatogenesis maturation in the cynomolgus monkey. Our results suggest species-related differences in PI3K isoform-specific control on reproductive organs.

Germ cell depletion in recipient testis has adverse effects on spermatogenesis in orthotopically transplanted testis pieces via retinoic acid insufficiency.

Germ cell depletion in recipient testes is indispensable for successful transplantation of spermatogonial stem cells. However, we found that such treatment had an adverse effect on spermatogenesis of orthotopically transplanted donor testis tissues. In the donor tissue, the frequency of stimulated by retinoic acid (RA) 8 (STRA8) expression was reduced in germ cells, suggesting that RA signalling indispensable for spermatogenesis was attenuated in germ cell-depleted recipient testes. In this context, germ cell depletion diminished expression of testicular Aldh1a2, which is responsible for testicular RA synthesis, while Cyp26b1, which is responsible for testicular RA metabolism, was still expressed even after germ cell depletion, suggesting an alteration of the RA synthesis/metabolism ratio. These observations suggested that RA insufficiency was one of the causes of the defective donor spermatogenesis. Indeed, repetitive RA administrations significantly improved donor spermatogenesis to produce fertile offspring without any side effects. These findings may contribute to improving fertility preservation techniques for males, especially to prevent iatrogenic infertility induced by chemotherapy in prepubertal cancer patients.

Toxic effects of arsenic trioxide on spermatogonia are associated with oxidative stress, mitochondrial dysfunction, autophagy and metabolomic alterations.

Arsenic is a toxic metalloid that can cause male reproductive malfunctions and is widely distributed in the environment. The aim of this study was to investigate the cytotoxicity of arsenic trioxide (ATO) induced GC-1 spermatogonial (spg) cells. Our results found that ATO increased the levels of catalase (CAT) and malonaldehyde (MDA) and reactive oxygen species (ROS), while decreasing glutathione (GSH) and the total antioxidant capacity (T-AOC). Therefore, ATO triggered oxidative stress in GC-1 spg cells. In addition, ATO also caused severe mitochondrial dysfunction that included an increase in residual oxygen consumption (ROX), and decreased the routine respiration, maximal and ATP-linked respiration (ATP-L-R), as well as spare respiratory capacity (SRC), and respiratory control rate (RCR); ATO also damaged the mitochondrial structure, including mitochondrial cristae disordered and dissolved, mitochondrial vacuolar degeneration. Moreover, degradation of p62, LC3 conversion, increasing the number of acidic vesicle organelles (AVOs) and autophagosomes and autolysosomes are demonstrated that the cytotoxicity of ATO may be associated with autophagy. Meanwhile, the metabolomics analysis results showed that 20 metabolites (10 increased and 10 decreased) were significantly altered with the ATO exposure, suggesting that maybe there are the perturbations in amino acid metabolism, lipid metabolism, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins. We concluded that ATO was toxic to GC-1 spg cells via inducing oxidative stress, mitochondrial dysfunction and autophagy as well as the disruption of normal metabolism. This study will aid our understanding of the mechanisms behind ATO-induced spermatogenic toxicity.

Novel localization of Aurora A kinase in mouse testis suggests multiple roles in spermatogenesis.

Male germ cells are transformed from undifferentiated stem cells into spermatozoa through a series of highly regulated steps together termed spermatogenesis. Spermatogonial stem cells undergo mitosis and differentiation followed by two rounds of meiotic division and then proceed through a series of dramatic cell shape changes to form highly differentiated spermatozoa. Using indirect immunofluorescence, we investigated a role for the mitotic kinase, Aurora A (AURKA), in these events through localization of this protein in mouse testis and spermatozoa. AURKA is expressed in several cell types in the testis. Spermatogonia and spermatocytes express AURKA as expected based on the known role of this kinase in cell division. Surprisingly, we also found AURKA localized to spermatids and the flagellum of spermatozoa. Total AURKA and activated AURKA are expressed in different compartments of the sperm flagellum with total AURKA found in the principal piece and its phosphorylated and activated form found in the sperm midpiece. In addition, active AURKA is enriched in the flagellum of motile sperm isolated from cauda epididymis. These results provide evidence for a unique role for AURKA in spermatogenesis and sperm motility. Defining the signaling mechanisms that govern spermatogenesis and sperm cell function is crucial to understanding and treating male infertility as well as for development of new contraceptive strategies.

VEGFC/VEGFR3 Signaling Regulates Mouse Spermatogonial Cell Proliferation via the Activation of AKT/MAPK and Cyclin D1 Pathway and Mediates the Apoptosis by affecting Caspase 3/9 and Bcl-2.

We have previously shown that the transcript levels of Vegfc and its receptor Vegfr3 were high in spermatogonia and extremely low in spermatocytes and spermatids. However, it remains unknown about the functions and the mechanisms of VEGFC/VEGFR3 signaling in regulating the fate determinations of spermatogonia. To this end, here we explored the role and signaling pathways of VEGFC/VEGFR3 by using a cell line derived from immortalized mouse spermatogonia retaining markers of mitotic germ cells, namely GC-1 cells. VEGFR3 was expressed in mouse primary spermatogonia and GC-1 cells. VEGFC stimulated the proliferation and DNA synthesis of GC-1 cells and enhanced the phosphorylation of PI3K-AKT and MAPK, whereas LY294002 (an inhibitor for AKT) and CI-1040 (an inhibitor for MAPK) blocked the effect of VEGFC on GC-1 cell proliferation. Furthermore, VEGFC increased the transcripts of c-fos and Egr1 and protein levels of cyclin D1, PCNA and Bcl-2. Conversely, the blocking of VEGFC/VEGFR3 signaling by VEGFR3 knockdown reduced the phosphorylation of AKT/MAPK and decreased the levels of cyclin D1 and PCNA. Additionally, VEGFR3 knockdown not only resulted in more apoptosis of GC-1 cells but also led to a decrease of Bcl-2 and promoted the cleavage of Caspase-3/9 and PARP. Collectively, these data suggested that VEGFC/VEGFR3 signaling promotes the proliferation of GC-1 cells via the AKT /MAPK and cyclin D1 pathway and it inhibits the cell apoptosis through Caspase-3/9, PARP and Bcl-2. Thus, this study sheds a novel insight to the molecular mechanisms underlying the fate decisions of mammalian spermatogonia.

High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells.

Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans.

Comparison of enzymatic digestion and mechanical dissociation of human testicular tissues.

OBJECTIVE: To compare mechanical dissociation, employing the Medimachine system, and enzymatic digestion of human testicular tissues with respect to the proportion of spermatogonia and somatic cells, with the long-term objective of establishing human spermatogonial cultures. DESIGN: Experimental basic science study. SETTING: Reproductive biology laboratory. PATIENT(S): Testicular tissues were obtained from patients with gender dysphoria on the day of sex reassignment surgery. On the basis of the histological evaluation, tissue samples with complete spermatogenesis (fresh, n = 6; cryopreserved, n = 7) and with meiotic arrest (cryopreserved, n = 4) were selected. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The composition of testicular cell suspensions was assessed performing quantitative real-time polymerase chain reaction (qPCR) analyses for germ cell-specific (FGFR3, SALL4, UTF1, MAGE-A4) and somatic marker genes (ACTA2 and VIM). Additionally, flow-cytometric analyses were used to evaluate the percentage of SALL4-and vimentin-positive cells. RESULT(S): While Medimachine dissociation yielded higher cell numbers in all patient groups, viability of cells was highly variable and correlated with the histological status of the tissue. Interestingly, qPCR analysis revealed a significantly decreased expression of the somatic marker genes ACTA2 and VIM and an increased expression of the spermatogonial marker genes FGFR3 and SALL4 after Medimachine dissociation. These findings were corroborated by flow-cytometric analyses that demonstrated that the proportion of SALL4-positive cells was up to 4 times higher after mechanical dissociation. CONCLUSION(S): Medimachine dissociation of human testicular tissues is comparably fast and leads to an enrichment of SALL4-positive spermatogonia. The use of this method may therefore constitute an advantage for the establishment of human spermatogonial cell cultures.

CDK14 expression is down-regulated by cigarette smoke in vivo and in vitro.

In this study, DNA arrays have been employed to monitor gene expression patterns in testis of mice exposed to tobacco smoke for 24 weeks and compared to control animals. The results of the analysis revealed significant changes in expression of several genes that may have a role in spermatogenesis. Cdk14 was chosen for further characterization because of a suggested role in the testis and in regulation of Wnt signaling. RT-PCR analysis confirmed down regulation of Cdk14 in mice exposed to cigarette smoke (CS). Cdk14 is expressed in all testicular cells; spermatogonia- and Sertoli-derived cell lines treated with cigarette smoke extract (CSE) in vitro showed down-regulation of CDK14 mRNA and protein levels as well as down-regulation of ß-catenin levels. CS-induced down-regulation of CDK14 mRNA and protein levels was also observed in several lung epithelium-derived cell lines including primary normal human bronchial epithelial cells (NHBE), suggesting that the effect is not restricted to the testis. Similar to testicular cells, CS-induced down-regulation of CDK14 in lung cells correlated with decreased levels of ß-catenin, a finding suggesting impaired Wnt signaling. In the lungs, CDK14 was localized to the alveolar and bronchial epithelium.

A G-quadruplex DNA structure resolvase, RHAU, is essential for spermatogonia differentiation.

G-quadruplex (G4) DNA and G4 DNA resolvase are involved in a variety of biological processes. To understand the biological function of G4 DNA structures and their resolvases in spermatogenesis, we investigated the distribution of G4 structures in mouse testis and identified their alterations during spermatogenesis. Meanwhile, we studied the function of RNA helicase associated with AU-rich element (RHAU), a G4 DNA resolvase, in spermatogenesis with a germ-cell-specific knockout mouse model. The results showed that the ablation of RHAU in germ cells caused the increase of G4 structures and thus resulted in the decrease of spermatogonial differentiation. c-kit, a spermatogonia differentiation-related gene, contains two G4 DNA motifs on its promoter. We found its expression was significantly downregulated in RHAU conditional knockout testis. A further analysis demonstrated that RHAU directly bound to the G4 structures to activate c-kit expression. We concluded that RHAU regulates spermatogonia differentiation by promoting c-kit expression via directly binding to the G4 DNA motifs c-kit promoter.

The caspase-dependent apoptosis gradient in the testis of the blue shark, Prionace glauca.

The severe degenerative phenomena that characterises spermatogenesis in mating blue sharks involves spatially separated germ cell and Sertoli cell apoptosis. Unlike that observed in multilayered type B spermatogonial and spermatocyte cysts caspase-3-depend entapoptosis of single and multinucleate type B spermatogonia in one to three spermatogonial layered cysts resulted in their complete fragmentation, delayed phagocytic removal and displacement of the apoptotic bodies towards the perilumenar Sertoli nuclei. Changes were observed in the immunostaining patterns of proliferating cell nuclear antigen (PCNA), including subtle changes in cytoplasmic and overall intense immunostaining, labelled single and multinucleate cell (MNC) apoptotic spermatogonial masses in premeiotic cysts indifferent stages of the protracted death process. Initial massive MNC formation at the mitosis­meiosis transition eventually left its imprint in the spermatogenic sequence in the form of vacuolated areas in the affected and subsequent stages. Some of the latter attempted further developmental advance but eventually degenerated. The observed higher PCNA index of spermatogonia in vacuolated testes compared to testes with the MNC type of degeneration indicated that the former testicular morphology represented, in essence, the recovery phase from the pronounced MNC death earlier. Events culminating in the eventual apoptotic demise of the Sertoli cells themselves included the abortion of further development (presumably due to a suboptimal Sertoli: germ cell ratio) of those germ cells left over from the wave of MNC death that swept the cysts. Eventually the Sertoli-cell-only cysts became apoptotic as they were engulfed by the infiltrating lymphomyeloid cells from the epigonal organ associated with the mature pole of the testis.

Effects of basic fibroblast growth factor and leukaemia inhibitory factor on proliferation and short-term culture of human spermatogonial stem cells.

In this study, isolated spermatogonial stem cells (SSCs) and Sertoli cells using enzymatic digestion from patients with maturation arrest of spermatogenesis were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum in three different groups: (1) SSCs cultured without Sertoli cells (2) SSCs co-cultured with Sertoli cells (as control group), (3) SSCs co-cultured with Sertoli cells and adding different concentrations of basic fibroblast growth factor (0.1, 1, 10 ng ml(-1)) and human leukaemia inhibitory factor (1000, 1200, 1500 unit ml(-1)) as experimental groups. The assessment of colonies every 10 days during 5-week cultures showed that in the first group, the average number and diameter of the colonies were significantly lower than in the other groups (P < 0.05). The largest number of colonies was observed in control condition (32.29 ± 9.15) in day 30. The largest diameter of colonies was formed in combination dosages of 1 ng ml(-1) basic fibroblast growth factor (bFGF) + 1500 unit ml(-1) leukaemia inhibitory factor (LIF) (302.93 ± 37.68) and 10 ng ml(-1) bFGF and 1200 unit ml(-1) LIF (262.87 ± 35.54) in day 30 respectively. Isolated SSCs were positive for spermatogonial cell markers such as Oct4, Stra8, Piwil2 and Vasa but negative for Nanog. Transplantation technique indicated that hSSCs have good efficiency for colonisation of mouse seminiferous tubules after proliferation in culture system.

Imatinib has deleterious effects on differentiating spermatogonia while sparing spermatogonial stem cell self renewal.

Imatinib mesylate is among a growing number of effective cancer drugs that provide molecularly targeted therapy; however, imatinib causes reproductive defects in rodents. The availability of an in vitro system for screening the effect of drugs on spermatogenesis would be beneficial. The imatinib targets, KIT and platelet derived growth factor receptor beta (PDGFRB), were shown here to be expressed in "germline stem" (GS) cell cultures that contain spermatogonia, including spermatogonial stem cells (SSCs). GS cell cultures were utilized to determine whether imatinib affects SSC self renewal or differentiation. GS cells grown in imatinib retained self renewal based on multiple assays, including transplantation. However, growth in imatinib led to decreased numbers of differentiated spermatogonia and reduced culture growth consistent with the known requirement for KIT in survival and proliferation of spermatogonia. These results build upon the in vivo studies and support the possibility of utilizing GS cell cultures for preclinical drug tests.

Stage-specific gene expression during spermatogenesis in the dogfish (Scyliorhinus canicula).

In the dogfish testis, the cystic arrangement and polarization of germ cell stages make it possible to observe all stages of spermatogenesis in a single transverse section. By taking advantage of the zonation of this organ, we have used suppressive subtractive libraries construction, real-time PCR, and in situ hybridization to identify 32 dogfish genes showing differential expressions during spermatogenesis. These include homologs of genes already known to be expressed in the vertebrate testis, but found here to be specifically expressed either in pre-meiotic and/or meiotic zones (ribosomal protein S8, high-mobility group box 3, ubiquitin carboxyl-terminal esterase L3, 20beta-hydroxysteroid dehydrogenase, or cyclophilin B) or in post-meiotic zone (speriolin, Soggy, zinc finger protein 474, calreticulin, or phospholipase c-zeta). We also report, for the first time, testis-specific expression patterns for dogfish genes coding for A-kinase anchor protein 5, ring finger protein 152, or F-box only protein 7. Finally, the study highlights the differential expression of new sequences whose identity remains to be assessed. This study provides the first molecular characterization of spermatogenesis in a chondrichthyan, a key species to gain insight into the evolution of this process in gnathostomes.

Generation of multipotent cell lines from a distinct population of male germ line stem cells.

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

Localizations of intracellular calcium and Ca2+-ATPase in hamster spermatogenic cells and spermatozoa.

Calcium plays a predominant role regulating many functional processes of spermatogenesis and fertilization. The purpose of the present study is to define the exact location of calcium as well as examine the role it plays during spermatogenesis and sperm capacitation. Testes and epididymides were obtained from adult healthy male hamsters. Spermatozoa were incubated with modified Tyrode's medium up to 4 h at 37 degrees Celsius for sperm capacitation in vitro. Samples of the testes and sperm cells were analyzed by cytochemical techniques to determine the location of calcium and Ca(2+)-ATPase and the percentage of acrosome reactions under light and electron microscopy. The data showed that (1) Sertoli cells exhibited numerous calcium precipitates as large, round, electron-dense bodies distributed throughout the cytoplasm and the mitochondrial matrix. Fine calcium precipitates existed in fewer numbers in the intracellular storage sites of spermatogonia and primary spermatocytes, in sharp distinction to secondary spermatocyte and spermatids, which showed an abundance of large and round calcium precipitates, especially in the mitochondrial matrix of spermatids. More calcium deposits were distributed in the plasma membrane (PM), acrosome membrane, and matrices of the acrosome and mitochondria following capacitation; (2) Ca(2+)-ATPase was found in the endoplasmic reticulum system and PM of noncapacitated spermatozoa as well as Sertoli cells. Capacitated spermatozoa showed a weak signal. These results suggest that the presence of calcium in spermatogenic cells might play a role in cell growth and differentiation during spermatogenesis. The Ca(2+)-ATPase function may be inhibited during capacitation, leading to an increase in acrosomal calcium level and triggering of acrosomal exocytosis.

The telomerase activity of adult mouse testis resides in the spermatogonial alpha6-integrin-positive side population enriched in germinal stem cells.

Testis is one of the organs with the most telomerase activity in the adult. This activity protects chromosomes from telomere attrition and ensures the transmission of full-length chromosomes to progeny. Little is known about telomerase activity during adult germ cell differentiation, however. We demonstrate here that the telomerase activity of adult mouse testis resides in the alpha6-integrin-positive Side Population containing spermatogonia and enriched in spermatogonial stem cells. The telomerase activity of these cells fell upon entry into meiosis and during the subsequent spermiogenesis. In addition, the telomerase activity of cells in various stages of differentiation was unaffected by aging and, notably, remained high in the alpha6-integrin-positive Side Population.

Expression of Dnmt3b in mouse hematopoietic progenitor cells and spermatogonia at specific stages.

Two de novo-type DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. Dnmt3b is specifically expressed in the totipotent cells of mouse early embryos and Dnmt3a, a longer form of the two isoforms, is ubiquitously expressed in mesenchyme cells after the 10 day embryo stage [Mech. Dev. 118 (2002) 187]. In the present study, we demonstrated that Dnmt3b was expressed in the nuclei of specific cells in certain tissues after the 10 day embryo stage. In fetal liver, dorsal aorta and portal vein, Dnmt3b was expressed in cells expressing CD34, indicating that the cells were hematopoietic progenitor cells. However, Dnmt3b was not expressed in the hematopoietic progenitor cells in yolk sac blood islands at 8 day embryo stage and in adult bone marrow cells. Dnmt3b was also expressed in type-A spermatogonia after birth. Dnmt3b was expressed not only in the totipotent stem cells but also in the progenitor cells the direction of differentiation of which had been already determined. On the other hand, the long form of Dnmt3a was not expressed in these hematopoietic progenitor cells in fetal liver or type-A spermatogonia, but was expressed in hepatocytes in fetal liver and type-B spermatogonia. While Dnmt3b was distributed in both the heterochromatin and euchromatin regions, Dnmt3a was specifically localized to the euchromatin region.

Aurora B expression in normal testis and seminomas.

Aurora/Ipl1-related kinases are a conserved family of proteins that have multiple functions during mitotic progression. High levels of Aurora kinases are characteristic of rapidly dividing cells and tumours. Aurora B encodes a protein that associates with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. In this study the expression and the localisation of Aurora B throughout germinal epithelial progression in normal testis and its neoplastic counterpart were analysed. Immunocytochemistry and RT-PCR analysis of mouse germinal epithelium cells showed the presence of Aurora B in spermatogonia and occasionally in spermatocytes. Western blot analysis revealed the typical Aurora B isoform ( approximately 41 kDa) in the same cellular types. A similar distribution was observed in human testis by immunohistochemistry. Moreover, the distribution and the expression of Aurora B were investigated in neoplasms derived from germ cells. Surgical samples of seminomas were analysed, and a high percentage of Aurora B positive cells (51%) was detected; the expression of Aurora B was significantly related to the MIB-1 proliferation marker (R=0.816). The data presented here demonstrate that Aurora B expression occurs in spermatogonial division. Furthermore, our results indicate that the expression of Aurora B is a consistent feature of human seminomas.

Immunohistochemical localization of hTERT protein in human tissues.

Telomerase is a ribonucleoprotein complex mainly composed of a reverse transcriptase catalytic subunit (telomerase reverse transcriptase gene, hTERT) that copies a template region of its RNA subunit to the end of the telomere. For detecting telomerase activity in a tissue specimen the TRAP assay is a relatively sensitive and specific method, but it can be used only on fresh tissue extracts and offers no information at the single cell level. Immunohistochemistry (IHC) allows to detect hTERT protein expression at an individual cell level in human tissues. We have tested commercially available anti-hTERT antibodies in formalin-fixed and paraffin-embedded human tissues by IHC. Only one monoclonal antibody (NCL-hTERT; Novacastra) was sufficiently specific and this was applied to human tissues in which telomerase activity had been shown by TRAP assay and hTERT mRNA expression by RT-PCR. hTERT protein localized diffusely in the nucleoplasm and more intensely in the nucleoli of cancer cells and proliferating normal cells. Mitotic cells showed diffuse staining of the entire cell. Granular cytoplasmic staining was occasionally found in some tumor cells. In telomerase-positive tumors not all the tumor cells showed hTERT immunoreactivity. A significantly heterogeneous hTERT protein expression was observed in human tumor tissues. The hTERT immunostaining in fixed tissues was concordant with telomerase activity and hTERT mRNA expression in corresponding non-fixed samples. Quantitative RT-PCR of microdissected sections showed that hTERT mRNA expression was higher in cells with nuclear expression than in those with cytoplasmic expression. Double staining with the M30 antibody showed that a subpopulation of hTERT-negative cells is apoptotic. We conclude that: (1) hTERT protein can be detected by IHC in fixed human tissues, but the choice of the antibody, tissue processing, and reaction conditions are critical, (2) hTERT protein localizes in the nucleoplasm, more strongly in the nucleolus, and occasionally in the cytoplasm, (3) telomerase-positive tumors show significant heterogeneity of hTERT protein expression, and (4) a subpopulation of hTERT protein negative tumor cells is identified as apoptotic cells.

Testicular activity of Mos in the frog, Rana esculenta: a new role in spermatogonial proliferation.

Mos is a MAPK kinase kinase with an expression that is highly restricted to the gonads. Its function is mainly associated to the meiotic metaphase II arrest occurring during female gametogenesis, whereas to our knowledge, its role during spermatogenesis has not yet clarified. In the present paper, we report the isolation of c-mos cDNA and the identification of a 60-kDa Mos protein from the testis of the anuran amphibian, Rana esculenta. Both the transcript and the protein are always present at low levels in the testis during the frog annual sexual cycle, with single significant peaks of expression in March and May, respectively. Mos is mainly localized in the cytoplasm of primary and secondary spermatogonia (SPG). Therefore, we have used treatments with ethane-dimethane sulphonate (EDS), which blocks spermatogonial mitosis in frogs. Four days after a single EDS injection, Mos expression in SPG highly increases concomitantly with the temporary arrest of mitosis. From 8 to 28 days after the injection, the normal proliferative activity of SPG is restored, and Mos expression gradually decreases to control levels. These results strongly indicate that the c-mos proto-oncogene exerts a new role associated to the regulation of spermatogonial proliferation.