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.

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.

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.