Q2 (United States)

STUDY QUESTIONDo the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells?SUMMARY ANSWERWe demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids.WHAT IS KNOWN ALREADYFull spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported.STUDY DESIGN, SAMPLES/MATERIALS, METHODSOrgan culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis.MAIN RESULTS AND THE ROLE OF CHANCEMale germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed.LIMITATIONS, REASONS FOR CAUTIONThe human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans.WIDER IMPLICATIONS OF THE FINDINGSThe successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility.LARGE SCALE DATANone.STUDY FUNDING AND COMPETING INTEREST(S)This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests.

Testicular germ cell transplantation is a novel strategy for preservation of fertility in prepubertal cancer patients, but the risk of reseeding tumor cells into cured patients presently limits clinical application of this approach. To date, no systematic evaluation of the limitations of surface marker-based decontamination of testicular samples with acute lymphoblastic leukemia has been performed. Here, surface markers for leukemic (CD4 and major histocompatibility complex class I) and germ cells (epithelia cell adhesion molecule) in testicular samples infiltrated with Roser's T-cell leukemia were identified. These markers were then used to delete leukemic cells and/or select for germ cells by flow cytometry (FACS). The resulting cell populations were analyzed by FACS, immunocytochemistry, or evaluation of leukemia transmission in syngeneic piebald variegated rats. Simple positive selection of germ cells or deletion of leukemic cells using specific surface markers was unable to effectively decontaminate testicular samples. The poor specificity of spermatogonial surface markers and aggregation of germ and leukemic cells limited the positive selection of germ cells, while immunophenotypic variation among lymphoblastic leukemia cells prevented adequate deletion of leukemic cells. Enzymatic treatment to disperse the testicular cells and feature of the intratesticular environment contributed to this immunophenotypic variation. Only germ cell selection in combination with leukemic cell deletion prevented leukemia transmission in association with intratesticular injection of the sorted cells. However, with such combined sorting, only 0.23% of the original testicular cells were recovered. With presently available techniques, flow cytometric purification of germ cells from a leukemic donor is not sufficiently effective or safe for clinical use.