Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation.

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.

Suppression of transforming growth factor-beta signaling enhances spermatogonial proliferation and spermatogenesis recovery following chemotherapy.

STUDY QUESTION: Could small molecules (SM) which target (or modify) signaling pathways lead to increased proliferation of undifferentiated spermatogonia following chemotherapy? SUMMARY ANSWER: Inhibition of transforming growth factor-beta (TGFb) signaling by SM can enhance the proliferation of undifferentiated spermatogonia and spermatogenesis recovery following chemotherapy. WHAT IS KNOWN ALREADY: Spermatogonial stem cells (SSCs) hold great promise for fertility preservation in prepubertal boys diagnosed with cancer. However, the low number of SSCs limits their clinical applications. SM are chemically synthesized molecules that diffuse across the cell membrane to specifically target proteins involved in signaling pathways, and studies have reported their ability to increase the proliferation or differentiation of germ cells. STUDY DESIGN, SIZE, DURATION: In our experimental study, spermatogonia were collected from four brain-dead individuals and used for SM screening in vitro. For in vivo assessments, busulfan-treated mice were treated with the selected SM (or vehicle, the control) and assayed after 2 (three mice per group) and 5 weeks (two mice per group). PARTICIPANTS/MATERIALS, SETTING, METHODS: We investigated the effect of six SM on the proliferation of human undifferentiated spermatogonia in vitro using a top-bottom approach for screening. We used histological, hormonal and gene-expression analyses to assess the effect of selected SM on mouse spermatogenesis. All experiments were performed at least in triplicate and were statistically evaluated by Student's t-test and/or one-way ANOVA followed by Scheffe's or Tukey's post-hoc. MAIN RESULTS AND THE ROLE OF CHANCE: We found that administration of SB431542, as a specific inhibitor of the TGFb1 receptor (TGFbR1), leads to a two-fold increase in mouse and human undifferentiated spermatogonia proliferation. Furthermore, injection of SB to busulfan-treated mice accelerated spermatogenesis recovery as revealed by increased testicular size, weight and serum level of inhibin B. Moreover, SB administration accelerated both the onset and completion of spermatogenesis. We demonstrated that SB promotes proliferation in testicular tissue by regulating the cyclin-dependent kinase (CDK) inhibitors 4Ebp1 and P57 (proliferation inhibitor genes) and up-regulating Cdc25a and Cdk4 (cell cycle promoting genes). LIMITATIONS, REASONS FOR CAUTION: The availability of human testis was the main limitation in this study. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study to report acceleration of spermatogenesis recovery following chemotherapy by administration of a single SM. Our findings suggest that SB is a promising SM and should be assessed in future clinical trials for preservation of fertility in men diagnosed with cancer or in certain infertility cases (e.g. oligospermia). STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Royan Institute and National Institute for Medical Research Development (NIMAD, grant no 963337) granted to H.B. The authors have no conflict of interest to report.

Inhibition of glycogen synthase kinase-3 promotes efficient derivation of pluripotent stem cells from neonatal mouse testis.

BACKGROUND: Several studies have demonstrated the derivation of multi- or pluripotent stem cells from testicular cells of both newborn and adult mice by a spontaneous conversion process, when these cells are cultured in vitro for an extended time. To obtain a better and robust derivation, we have attempted to identify small molecules (SMs) that induce reprogramming of testicular cells in culture into germline-derived pluripotent stem cells (gPSCs). METHODS: We tested several SMs based on previous reports that have shown enhancement of establishment of induced pluripotent stem cells or embryonic stem cells (ESCs) on mouse NMRI (outbred strain) and C57BL/6 (inbred strain) testicular cells. After appearance of ESC-like colonies at Day 6, they were passaged on mitotically arrested mouse embryonic fibroblasts in mouse ESC medium in the absence or presence of SMs up to Day 14. The generated cells were characterized using a variety of experimental approaches. RESULTS: The application of several SMs involved in pluripotent reprogramming led to the discovery that CHIR99021 (CHIR), a glycogen synthase kinase-3 (GSK-3) inhibitor, promotes efficient derivation of gPSCs from neonatal mouse NMRI and C57BL/6 testes. The pluripotency of the generated cell lines has been confirmed by in vitro spontaneous and direct differentiation toward cardiac and neural lineages, and formation of chimeras after injection of gPSCs into blastocysts. We have shown that the generated gPSCs could be maintained and expanded under chemically defined serum and feeder-free conditions by inhibition of both the extracellular signal-regulated kinases (Erk1/2) and GSK-3. CONCLUSIONS: To our knowledge, this is the first report of a simple and efficient protocol to reprogram gPSCs from testicular cells solely by inhibition of GSK-3 in two strains of mice with different genetic backgrounds. Additionally, this brings us closer to eliminating the need for genetic modification in pluripotent reprogramming. Future studies will determine whether the inhibition of GSK-3 could affect the generation of naïve gPSCs lines in other mammals.

In vitro investigation of zinc oxide nanoparticle toxic effects in spermatogonial cells at the molecular level.

Because spermatogonia transmit genetic information across generations, their DNA must be protected from environmental damages, including exposure to zinc oxide nanoparticles (ZnO NPs), which are frequently used in modern technology. Here, we used an in vitro system enriched for spermatogonia and exposed them to 10 and 20 µg/ml ZnO NPs for one/seven days. We did not detect any significant cell death, chromosomal instability, or DNA fragmentation in the spermatogonia treated with the ZnO NPs following one-day treatment with 10 or 20 µg/ml ZnO NPs. However, ZnO NPs (both 10 and 20 µg/ml) induced chromosomal instability in the spermatogonia after seven days of treatment. Moreover, one-day exposure to these NPs induced reactive oxygen species (ROS) generation and upregulation of apoptotic pathway-related genes p53, Caspase3 and Il6, as an inflammatory factor. Taken together, our study provides preliminary evidence for possible damages induced by low concentrations of ZnO NPs in spermatogonia. We should pay increased attention when using these NPs because of the silent damages in spermatogonia that can be transmitted to the next generation and cause severe effects. However, more data and validation of these results are required to determine the extent of this concern.

Short time exposure to low concentration of zinc oxide nanoparticles up-regulates self-renewal and spermatogenesis-related gene expression.

Extensive application of zinc oxide (ZnO) nanoparticles (NPs) in everyday life results in increased exposure to these NPs. Spermatogonial stem cells (SSCs) guarantee sperm production throughout the male reproductive life by providing a balance between self-renewal and differentiation. We used an in vitro platform to investigate the ZnO NPs effects on SSCs. We successfully synthesized ZnO NPs. In order to investigate these NPs, we isolated SSCs from mouse testes and cultured them in vitro. Our results confirmed the uptake of ZnO NPs by the cultured SSCs. We observed a dose- and time-dependent decrease in SSC viability. Both spherical and nanosheet ZnO NPs had the same cytotoxic effects on the SSCs, irrespective of their shapes. Moreover, we have shown that short time (one day) exposure of SSCs to a low concentration of ZnO NPs (10 µg/mL) promoted expressions of specific genes (Plzf, Gfr α1 and Bcl6b) for SSC self-renewal and differentiation genes (Vasa, Dazl, C-kit and Sycp3) expressed by spermatogonia during spermatogenesis. Our study provides the first insight into ZnO NPs function in SSCs and suggests a new function for ZnO NPs in the male reproductive system. We demonstrated that ZnO NPs might promote spermatogenesis via upregulation of gene expression related to SSC self-renewal and differentiation at low concentrations. Additional research should clarify the possible effect of ZnO NPs on the SSC genome and its effects on human SSCs.