An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg^-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg^-1) on day 36 or a dose of 40 mg kg^-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg^-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg^-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.
Adult Germline Stem Cells/transplantation* ; Animals ; Azoospermia/chemically induced* ; Busulfan/toxicity* ; Disease Models, Animal ; Infertility, Male/chemically induced* ; Injections, Intraperitoneal ; Male ; Mice ; Spermatogenesis/drug effects* ; Spermatogonia/drug effects* ; Stem Cell Transplantation/methods*

Animals ; Antineoplastic Agents, Alkylating ; Busulfan ; Cell Transplantation ; Chimera ; Coturnix/physiology* ; Female ; Male ; Spermatogonia/transplantation* ; Testicular Diseases/therapy* ; Testis/transplantation*

Successful male germ cell transplantation requires depletion of the host germ cells to allow efficient colonization of the donor spermatogonial stem cells. Although a sterilizing drug, busulfan, is commonly used for the preparation of recipient models before transplantation, the optimal dose of this drug has not yet been defined in dogs. In this study, 1-year-old mongrel dogs were intravenously injected with three different concentrations of busulfan (10, 15, or 17.5 mg/kg). Four weeks after busulfan treatment, no fully matured spermatozoa were detected in any of the busulfan-treated groups. However, small numbers of PGP9.5-positive spermatogonia were detected in all treatment groups, although no synaptonemal complex protein-3-positive spermatocytes were detected. Of note, acrosin-positive spermatids were not detected in the dogs treated with 15 or 17.5 mg/kg busulfan, but were detected in the other group. Eight weeks after busulfan treatment, the dogs treated with 10 mg/kg busulfan fully recovered, but those in the other groups did not. PGP9.5-positive spermatogonia were detected in the 10 mg/kg group, and at a similar level as in the control group, but these cells were rarely detected in the 15 and 17.5 mg/kg groups. These results suggest that a dose of 15-17.5 mg/kg is optimal for ablative treatment with busulfan to prepare the recipient dogs for male germ cell transplantation. At least eight weeks should be allowed for recovery. The results of this study might facilitate the production of recipient dogs for male germ cell transplantation and can also contribute to studies on chemotherapy.
Animals ; Busulfan* ; Colon ; Dogs* ; Drug Therapy ; Germ Cells ; Humans ; Male ; Spermatids ; Spermatocytes ; Spermatogonia ; Spermatozoa ; Stem Cell Transplantation* ; Stem Cells* ; Synaptonemal Complex ; Testis* ; Tissue Donors

BACKGROUND: Mesenchymal stem cells (MSCs), have been suggested as a potential choice for treatment of male infertility. Yet, the effects of MSCs on regeneration of germinal epithelium of seminiferous tubules and recovery of spermatogenesis have remained controversial. In this research, we have evaluated and compared the fate of autologous bone marrow (BM)-MSCs during three different periods of time- 4, 6 and 8 weeks after transplantation into the testes of busulfan-induced infertile male rats. METHODS: Rats BM samples were collected from tibia bone under anesthesia. The samples were directly cultured in culture medium. Isolated, characterized and purified BM-MSCs were labeled with PKH26, and transplanted into the testes of infertile rats. After 4, 6 and 8 weeks, the testes were removed and underwent histological evaluations. RESULTS: Immunohistochemical analysis showed that transplanted BM-MSCs survived in all three groups. Some of the cells homed at the germinal epithelium and expressed spermatogonia markers (Dazl and Stella). The number of homed spermatogonia-like cells in 4-week testes, was more than the 6-week testes. The 8-week testes had the least numbers of homed cells (p<0.05). Immunostaining for vimentin showed that BM-MSCs did not differentiate into the sertoli cells in the testes. CONCLUSIONS: From our results, it could be concluded that, autologous BM-MSCs could survive in the testis, migrate onto the seminiferous tubules basement membrane and differentiate into spermatogonia. Although, no more differentiation was observed in the produced spermatogonia, generation of such endogenous GCs would be a really promising achievement for treatment of male infertility using autologous stem cells.
Anesthesia ; Animals ; Basement Membrane ; Bone Marrow* ; Epithelium ; Germ Cells* ; Humans ; Infertility, Male ; Male* ; Mesenchymal Stromal Cells* ; Rats* ; Regeneration ; Seminiferous Tubules ; Sertoli Cells ; Spermatogenesis ; Spermatogonia ; Stem Cells ; Testis* ; Tibia ; Transplantation ; Vimentin

Transplantation of spermatogonial stem cells (SSCs) in experimental animal models has been used to study germ line stem cell biology and to produce transgenic animals. The species-specific recipient model preparation is important for the characterization of SSCs and the production of offspring. Here, we investigated the effects of surgically induced cryptorchidism in dog as a new recipient model for spermatogonial stem cell transplantation. Artificially unilateral or bilateral cryptorchidism was induced in ten mature male dogs by surgically returning the testis and epididymis to the abdominal cavity. The testes and epididymides were collected every week after the induction of artificial cryptorchidism (surgery) for one month. To determine the effect of surgical cryptorchidism, the seminiferous tubule diameter was measured and immunohistochemistry using PGP9.5 and GATA4 antibodies was analyzed. The diameters of the seminiferous tubules of abdominal testes were significantly reduced compared to those of the scrotal testes. Immunohistochemistry results showed that PGP9.5 positive undifferentiated spermatogonia were significantly reduced after surgical cryptorchidism induction, but there were no significant changes in GATA-4 positive sertoli cells. To evaluate the testis function recovery rate, orchiopexy was performed on two dogs after 30 days of bilateral cryptorchidism. In the orchiopexy group, SCP3 positive spermatocytes were detected, and spermatogenesis was recovered 8 weeks after orchiopexy. In this study, we provided optimum experimental conditions and time for surgical preparation of a recipient canine model for SSC transplantation. Additionally, our data will contribute to recipient preparation by using surgically induced cryptorchidism in non-rodent species.
Abdominal Cavity ; Animals ; Animals, Genetically Modified ; Antibodies ; Biology ; Cryptorchidism* ; Dogs ; Epididymis ; Germ Cells ; Humans ; Immunohistochemistry ; Male ; Models, Animal ; Orchiopexy ; Recovery of Function ; Seminiferous Tubules ; Sertoli Cells ; Spermatocytes ; Spermatogenesis ; Spermatogonia ; Stem Cell Transplantation* ; Stem Cells* ; Testis

The study on stem cells is a hot field in biomedical science in recent years, and has furthered from laboratory to clinical application. Stem cells, according to their developmental stage and differential properties, can be divided into embryonic stem cells, induced PS cells and adult stem cells, among which, adult stem cells have already been applied to the clinical treatment of many systemic diseases. Currently, the study of spermatogonial stem cells and adult stem cells is in the front of the basic researches on the treatment of male infertility, but the time has not yet arrived for their clinical application. This paper outlines the application prospect of adult stem cells in male infertility.
Adult Stem Cells ; cytology ; Humans ; Infertility, Male ; therapy ; Male ; Mesenchymal Stromal Cells ; cytology ; Spermatogonia ; cytology ; Stem Cell Transplantation

Spermatogonial stem cells (SSCs) play an important role in spermatogenesis and have a unique mode of replication. A single SSC can produce two differentiating cells, or one stem cell and one differentiating cell. The self-renewal and differentiation of SSCs are precisely regulated as relating the niche of SSCs, glial cell line-derived neurotrophic factor, and several signaling pathways. This article reviews the self-renewal and differentiation of SSCs and their regulation mechanisms, which may offer a deeper insight into spermatogenesis and male infertility and pave a theoretical ground for studying testicular tumorigenesis and searching for new potential approaches to the treatment of testicular cancer and other related diseases.
Adult Stem Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Male ; Spermatogenesis ; Spermatogonia ; cytology ; Stem Cell Transplantation

Spermatogonial stem cells are male germ line stem cells, whose potency of proliferation is indispensable for the permanent production of male germ cells. With the advances in the technology of cryopreservation, in vitro culture and intracytoplasmic sperm injection (ICSI), the transplantation of spermatogonial stem cells is showing a splendid vista of application in basic medical research and clinical practice. This review briefly introduces the genesis and differentiation of spermatogonial stem cells, the current situation of their transplantation and the prospects of its application in medical science.
Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Humans ; Infertility, Male ; surgery ; Male ; Spermatogonia ; cytology ; Stem Cell Transplantation

Results from the transplantation of donor spermatogonia into xenogeneic recipient seminiferous tubules indicate that donor germ cells are capable of differentiating to form spermatozoa with morphological character of the donor species. With the advances in freezing, culturing in vitro and enriching germ cell populations, germ cell transplantation procedures have applications of paramount values in medicine, basic science and animal reproduction. Additionally, these techniques can serve as an alternative approach for gonadal protection and fertility preservation especially in patients accepting large dose of chemotherapy or radiotherapy. In this article we reviewed the recent advances in xenogeneic transplantation of spermatogonial stem cell and also analyzed the potential problems existing in its clinical application.
Animals ; Cell Transplantation ; methods ; Humans ; Male ; Mice ; Rats ; Seminiferous Tubules ; Spermatogonia ; transplantation ; Stem Cell Transplantation ; Transplantation, Homologous

OBJECTIVEThe culture of human spermatogonial stem cells (SSC) has not been studied in detail yet. Here we tried to explore the optimized culture method of human SSC by using several different co-culture systems.

METHODSThe alpha6 +Thy-1 +c-kit- cells acquired by the immunomagnetic beads sorting technique were cultured in different co-culture systems. Their morphological, biological characteristics and survival rates were intensively observed by microscopic or immunocytochemical assay. The long-term survival rate of human SSC during culture period was evaluated by germ cell transplantation technique.

RESULTSThe alpha6 +Thy-1 +c-kit- cells could stably survive in the DMEM and DMEM/F12 mediums with fetal bovine serum (FBS) on feeder layer. The survival rates within 1 week were more than 90%. The long-time culture showed the cells were gradually attached on the surface of Sertoli cells by the manner of scattered single cell or accumulated masses. Part of the SSC became more tightly attachment with Sertoli cells or mounted among the Sertoli cells. They could survive or even proliferate for more than 3 months in vitro. Germ cells transplantation study showed that some alpha6 +Thy-1 +c-kit- cells labeled by PKH26 could resided on the basal membrane of seminiferous tubule of nude mice, appearing as single or coupled cells 2 months later after transplantation. The function evaluation of the cultured cells by counting the fluorescent cells in the seminiferous tubule showed 54.9% and 9.2% of SSC in the alpha6 +Thy-1 +c-kit- cells were still remained after cultured for 2 and 4 weeks, respectively.

CONCLUSIONHuman SSC could maintain survival in vitro for more than 3 months, but it was still need to seek for a more optimized and successful culture system for its efficient expansion and proliferation. Thus it will open up a wide prospect for the understanding of the biology of human SSC and the treatment of male sterility.

Adult ; Cell Culture Techniques ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Humans ; Male ; Sertoli Cells ; cytology ; Spermatogonia ; cytology ; physiology ; Stem Cell Transplantation ; Stem Cells ; cytology