Aggressive chemotherapy may lead to permanent male infertility. Prepubertal males do not generate sperm, but their testes do contain spermatogonial cells (SPGCs) that could be used for fertility preservation. In the present study, we examined the effect of busulfan (BU) on the SPGCs of immature mice, and the possible induction of the survivor SPGCs to develop spermatogenesis in 3D in-vitro culture. Immature mice were injected with BU, and after 0.5–12 weeks, their testes were weighed and evaluated histologically compared to the control mice. The spermatogonial cells [Sal-like protein 4 (SALL4) and VASA (a member of the DEAD box protein family) in the testicular tissue were counted/seminiferous tubule (ST). The cells from the STs were enzymatically isolated and cultured in vitro. Our results showed a significant decrease in the testicular weight of the BU-treated mice compared to the control. This was in parallel to a significant increase in the number of severely damaged STs, and a decrease in the number of SALL4 and VASA/STs compared to the control. The cultures of the isolated cells from the STs of the BU-treated mice showed a development of colonies and meiotic and post-meiotic cells after four weeks of culture. The addition of homogenates from adult GFP mice to those cultures induced the development of sperm-like cells after four weeks of culture. This is the first study demonstrating the presence of biologically active spermatogonial cells in the testicular tissue of BU-treated immature mice, and their capacity to develop sperm-like cells in vitro.

Sertoli cell-only syndrome (SCOS) affects about 26.3–57.8% of azoospermic men, with their seminiferous tubules containing only Sertoli cells. Recently, it was reported that testicular biopsies from nonobstructive azoospermic (NOA) patients contained germ cells, and that sperm could be found in the tubules of 20% of SCOS patients using testicular sperm extraction technology. Since the patients without sperm in their testicular biopsies do not have therapy to help them to father a biological child, in vitro maturation of spermatogonial stem cells (SSCs) isolated from their testis is a new approach for possible future infertility treatment. Recently, the induction of human and mice SSCs proliferation and differentiation was demonstrated using different culture systems. Our group reported the induction of spermatogonial cell proliferation and differentiation to meiotic and postmeiotic stages in mice, rhesus monkeys, and prepubertal boys with cancer using 3D agar and methylcellulose (MCS) culture systems. The aim of the study was to identify the type of spermatogenic cells present in biopsies without sperm from SCOS patients, and to examine the possibility of inducing spermatogenesis from isolated spermatogonial cells of these biopsies in vitro using 3D MCS. We used nine biopsies without sperm from SCOS patients, and the presence of spermatogenic markers was evaluated by PCR and specific immunofluorescence staining analyses. Isolated testicular cells were cultured in MCS in the presence of StemPro enriched media with different growth factors and the development of colonies/clusters was examined microscopically. We examined the presence of cells from the different stages of spermatogenesis before and after culture in MCS for 3–7 weeks. Our results indicated that these biopsies showed the presence of premeiotic markers (two to seven markers/biopsy), meiotic markers (of nine biopsies, cAMP responsive element modulator-1 (CREM-1) was detected in five, lactate dehydrogenase (LDH) in five, and BOULE in three) and postmeiotic markers (protamine was detected in six biopsies and acrosin in three). In addition, we were able to induce the development of meiotic and/or postmeiotic stages from spermatogonial cells isolated from three biopsies. Thus, our study shows for the first time the presence of meiotic and/or postmeiotic cells in biopsies without the sperm of SCOS patients. Isolated cells from some of these biopsies could be induced to meiotic and/or postmeiotic stages under in vitro culture conditions.

Male fertility preservation is required when treatment with an aggressive chemo-/-radiotherapy, which may lead to irreversible sterility. Due to new and efficient protocols of cancer treatments, surviving rates are more than 80%. Thus, these patients are looking forward to family life and fathering their own biological children after treatments. Whereas adult men can cryopreserve their sperm for future use in assistance reproductive technologies (ART), this is not an option in prepubertal boys who cannot produce sperm at this age. In this review, we summarize the different technologies for male fertility preservation with emphasize on prepubertal, which have already been examined and/or demonstrated in vivo and/or in vitro using animal models and, in some cases, using human tissues. We discuss the limitation of these technologies for use in human fertility preservation. This update review can assist physicians and patients who are scheduled for aggressive chemo-/radiotherapy, specifically prepubertal males and their parents who need to know about the risks of the treatment on their future fertility and the possible present option of fertility preservation.