Sefika C. Mizrak

Propagation of human spermatogonial stem cells in vitro.

CONTEXT Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility. OBJECTIVE To establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation. DESIGN, SETTING, AND PARTICIPANTS Study performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation. MAIN OUTCOME MEASURES Propagation of spermatogonial stem cells over time. RESULTS Testicular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and increased 18,450-fold within 64 days in the germline stem cell subculture. CONCLUSION Long-term culture and propagation of human spermatogonial stem cells in vitro is achievable.

Embryonic stem cell-like cells derived from adult human testis

BACKGROUND Given the significant drawbacks of using human embryonic stem (hES) cells for regenerative medicine, the search for alternative sources of multipotent cells is ongoing. Studies in mice have shown that multipotent ES-like cells can be derived from neonatal and adult testis. Here we report the derivation of ES-like cells from adult human testis. METHODS Testis material was donated for research by four men undergoing bilateral castration as part of prostate cancer treatment. Testicular cells were cultured using StemPro medium. Colonies that appeared sharp edged and compact were collected and subcultured under hES-specific conditions. Molecular characterization of these colonies was performed using RT-PCR and immunohistochemistry. (Epi)genetic stability was tested using bisulphite sequencing and karyotype analysis. Directed differentiation protocols in vitro were performed to investigate the potency of these cells and the cells were injected into immunocompromised mice to investigate their tumorigenicity. RESULTS In testicular cell cultures from all four men, sharp-edged and compact colonies appeared between 3 and 8 weeks. Subcultured cells from these colonies showed alkaline phosphatase activity and expressed hES cell-specific genes (Pou5f1, Sox2, Cripto1, Dnmt3b), proteins and carbohydrate antigens (POU5F1, NANOG, SOX2 and TRA-1-60, TRA-1-81, SSEA4). These ES-like cells were able to differentiate in vitro into derivatives of all three germ layers including neural, epithelial, osteogenic, myogenic, adipocyte and pancreatic lineages. The pancreatic beta cells were able to produce insulin in response to glucose and osteogenic-differentiated cells showed deposition of phosphate and calcium, demonstrating their functional capacity. Although we observed small areas with differentiated cell types of human origin, we never observed extensive teratomas upon injection of testis-derived ES-like cells into immunocompromised mice. CONCLUSIONS Multipotent cells can be established from adult human testis. Their easy accessibility and ethical acceptability as well as their non-tumorigenic and autogenic nature make these cells an attractive alternative to human ES cells for future stem cell therapies.

The effects of different endocrine disruptors defining compound-specific alterations of gene expression profiles in the developing testis

Environmental contaminants considered endocrine disruptors have been shown to affect testis development and function but the mechanisms of action are not clear. We now have analyzed the effects on the transcriptome in testes of mice exposed to mono-(2-ethylhexyl)-phthalate (9.2; 46.3 or 92.7 mg/kg/d), zearalenone (1.3; 3.9 or 6.6 mg/kg/d), lindane (16.6; 32.2 or 64.4 mg/kg/d), bisphenol-A (0.16; 16 or 64 mg/kg/d) or 17β-estradiol (0.006; 0.012 or 0.048 mg/kg/d). The compounds were orally administered in the drinking water during distinct developmental periods: (A) mothers were exposed only during the two weeks before mating; (B) the exposure was continued during pregnancy until birth or (C) exposure was continued for a further four weeks after birth. Testes were studied at four weeks of age. Mono-(2-ethylhexyl)-phthalate and zearalenone, both produced specific alterations of gene signatures. Interestingly, this was irrespective of the concentration of the toxicant or the developmental period during which exposure occurred.

Enrichment of spermatogonial stem cells from long-term cultured human testicular cells

OBJECTIVE To evaluate the degree of enrichment of spermatogonial stem cells (SSCs) from human testicular cell cultures by ITGA6+, HLA-/ITGA6+, GPR125+, and HLA-/GPR125+ magnetic-assisted cell sorting (MACS). DESIGN Experimental basic science study. SETTING Reproductive biology laboratory. PATIENT(S) Multiple samples of cryopreserved human testicular cells from two prostate cancer patients with normal spermatogenesis. INTERVENTION(S) Cultured human testicular cells subjected to four sorting strategies based on MACS and xenotransplanted to the testes of mice to determine the enrichment for SSCs. MAIN OUTCOME MEASURE(S) Enrichment for human spermatogonia and SSCs tested by expression analysis of spermatogonial markers ITGA6, GPR125, ZBTB16, UCHL1, and ID4 using quantitative real-time polymerase chain reaction (qPCR) and by xenotransplantation into the testes of mice, respectively. RESULT(S) Compared with the nonsorted cultured testicular cells, only the ITGA6+ and HLA-/GPR125+ sorted cells showed enrichment for ID4. No difference in expression of ZBTB16 and UCHL1 was observed. Xenotransplantation of the sorted cell fractions showed a 7.1-fold enrichment of SSCs with ITGA6+. CONCLUSION(S) Magnetic-assisted cell sorting of cultured human testicular cells using ITGA6 allows for enrichment of SSCs, which aids in further molecular characterization of cultured human SSCs and enhances testicular colonization upon transplantation in future clinical settings.

Transient receptor potential vanilloid receptor-1 confers heat resistance to male germ cells

Testicular hyperthermia in mice lacking transient receptor potential vanilloid receptor-1 results in a much more rapid and massive germ cell depletion from the seminiferous tubules than in wild-type animals, indicating that this receptor protects germ cells against heat stress.

Spermatogonial stem cell sensitivity to capsaicin: An in vitro study

BackgroundConflicting reports have been published on the sensitivity of spermatogenesis to capsaicin (CAP), the pungent ingredient of hot chili peppers. Here, the effect of CAP on germ cell survival was investigated by using two testis germ cell lines as a model. As CAP is a potent agonist of the transient receptor potential vanilloid receptor 1 (TRPV1) and no information was available of its expression in germ cells, we also studied the presence of TRPV1 in the cultured cells and in germ cells in situ.MethodsThe rat spermatogonial stem cell lines Gc-5spg and Gc-6spg were used to study the effects of different concentrations of CAP during 24 and 48 h. The response to CAP was first monitored by phase-contrast microscopy. As germ cells appear to undergo apoptosis in the presence of CAP, the activation of caspase 3 was studied using an anti activated caspase 3 antibody or by quantifying the amount of cells with DNA fragmentation using flow cytometry. Immunolocalization was done with an anti-TRPV1 antibody either with the use of confocal microscopy to follow live cell labeling (germ cells) or on Bouin fixed paraffin embedded testicular tissues. The expression of TRPV1 by the cell lines and germ cells was confirmed by Western blots.ResultsInitial morphological observations indicated that CAP at concentrations ranging from 150 uM to 250 uM and after 24 and 48 h of exposure, had deleterious apoptotic-like effects on both cell lines: A large population of the CAP treated cell cultures showed signs of DNA fragmentation and caspase 3 activation. Quantification of the effect demonstrated a significant effect of CAP with doses of 150 uM in the Gc-5spg cell line and 200 uM in the Gc-6spg cell line, after 24 h of exposure. The effect was dose and time dependent in both cell lines. TRPV1, the receptor for CAP, was found to be expressed by the spermatogonial stem cells in vitro and also by premeiotic germ cells in situ.ConclusionCAP adversely affects spermatogonial survival in vitro by inducing apoptosis to those cells and TRPV-1, a CAP receptor, may be involved in this effect as this receptor is expressed by mitotic germ cells.

Phosphoprotein enriched in astrocytes-15 is expressed in mouse testis and protects spermatocytes from apoptosis

Phosphoprotein enriched in astrocytes (PEA-15) is a 15 kDa acidic serine-phosphorylated protein expressed in different cell types, especially in the CN. We initially detected the expression of PEA-15 in primary cultures of Sertoli cells. To assess the presence and localization of PEA-15 in the mouse testis, we studied the expression pattern of the PEA-15 protein by immunohistochemistry and mRNA by in situ hybridization. Both the protein and the mRNA of PEA-15 were localized in the cytoplasm of Sertoli cells, all types of spermatogonia, and spermatocytes up till zygotene phase of the meiotic prophase. Subsequently, with ongoing development of the spermatocytes, the expression decreased and was very low in the cytoplasm of diplotene spermatocytes. To analyze the possible role of PEA-15 in the developing testis, null mutants for PEA-15 were examined. As the PEA-15 C terminus contains residues for ERK binding, we studied possible differences between the localization of the ERK2 protein in wild type (WT) and PEA-15(-/-)mice. In the WT testis, ERK2 was localized in the cytoplasm of Sertoli cells, B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes, whereas in the KO testis, ERK2 was primarily localized in the nuclei of these cells and only little staining remained in the cytoplasm. Moreover, in PEA-15-deficient mice, significantly increased numbers of apoptotic spermatocytes were found, indicating an anti-apoptotic role of PEA-15 during the meiotic prophase. The increased numbers of apoptotic spermatocytes were not found at a specific step in the meiotic prophase.

Correction: Spermatogonial stem cell sensitivity to capsaicin: An in vitro study (vol 6, pg 52, 2008)

Since publication of our article Spermatogonial stem cell sensitivity to capsaicin: an in vitro study. Reprod Biol Endocrinol 2008, 6:52., we have realised that the legend of Figure 1 needs to be corrected and should read as follows: