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

Spermatogonial stem cells (SSCs) are essential for spermatogenesis throughout the lifespan of the male. However, the rarity of SSCs has raised the need for an efficient selection method, but little is known about culture conditions that stimulate monkey SSC proliferation in vitro. In this study, we report the development of effective enrichment techniques and in vitro culturing of germ cells from pre-pubertal monkey testes. Testis cells were analyzed by fluorescence-activated cell sorting techniques and were transplanted into the testes of nude mice to characterize SSCs. Thy-1-positive cells showed a higher number of colonies than the unselected control after xenotransplantation. Extensive colonization of monkey cells in the mouse testes indicated the presence of highly enriched populations of SSCs in the Thy-1-positive sorted cells. Furthermore, monkey testis cells were enriched by differential plating using extracellular matrix, laminin, and gelatin, and then cultured under various conditions. Isolation of monkey testicular germ cells by differential plating increased germ cell purity by 2.7-fold, following the combinational isolation method using gelatin and laminin. These enriched germ cells actively proliferated under culture conditions involving StemPro medium supplemented with bFGF, GDNF, LIF, and EGF at 37 ℃. These results suggest that the enrichment and in vitro culture method proposed in the present study for harvesting a large number of functionally active monkey SSCs can be applied as the basis for efficient in vitro expansion of human SSCs.
Animals ; Colon ; Epidermal Growth Factor ; Extracellular Matrix ; Flow Cytometry ; Gelatin ; Germ Cells ; Glial Cell Line-Derived Neurotrophic Factor ; Haplorhini* ; Humans ; In Vitro Techniques* ; Laminin ; Male ; Methods ; Mice ; Mice, Nude ; Spermatogenesis ; Stem Cells* ; Testis* ; Transplantation, Heterologous

BACKGROUND: The normal cells derived from human embryonic stem cells (hESCs) are regarded as substitutes for damaged or dysfunctional adult cells. However, tumorigenicity of hESCs remains a major challenge in clinical application of hESC-derived cell transplantation. Previously, we generated monoclonal antibody (MAb) 57-C11 specific to the surface molecule on undifferentiated hESCs. The aim of this study is to prove whether 57-C11-positive hESCs are pluripotent and tumorigenic in immunodeficient mice. METHODS: Undifferentiated hESCs were mixed with retinoic acid (RA)-differentiated hESCs at different ratios prior to 57-C11-mediated separation. To isolate 57-C11-positive hESCs from the mixture, biotinylated 57-C11 and streptavidin-coated magnetic beads were added to the mixture. Unbound 57-C11-negative hESCs were first isolated after applying magnet to the cell mixture, and 57-C11-bound hESCs were then released from the magnetic beads. In order to measure the efficiency of separation, 57-C11-positive or -negative hESCs were counted after isolation. To evaluate the efficiency of teratoma formation in vivo, 57-C11-positive or negative cells were further injected into left and right, respectively, testes of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. RESULTS: Approximately 77~100% of undifferentiated hESCs were isolated after applying 57-C11-coated magnetic beads to the mixed cell populations. Importantly, teratomas were not observed in NOD/SCID mice after the injection of isolated 57-C11-negative hESCs, whereas teratomas were observed with 57-C11-positive hESCs. CONCLUSION: 57-C11-positive hESCs are pluripotent and tumorigenic. The combination of 57-C11 and magnetic beads will be useful to eliminate remaining undifferentiated hESCs for the safe cell transplantation.
Adult ; Animals ; Cell Transplantation ; Human Embryonic Stem Cells* ; Humans* ; Mice ; Teratoma ; Testis ; Transplants ; Tretinoin

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

ObjectiveTo assess the effect of corporoplasty with autologous tunica vaginalis graft in the treatment of Peyronie's disease.

METHODSTen patients with Peyronie's disease underwent plaque excision and corporoplasty with autologous tunica vaginalis graft. We obtained and compared IIEF-5 scores of the patients before and at 1 and 5 years after operation.

RESULTSAfter surgery, penile curvature was obviously relieved and all the patients achieved normal penile erection and satisfactory sexual intercourse without erection-related pain or recurrent erectile dysfunction. The mean IIEF-5 score was significantly improved at 1 year (22.40±1.08) and 5 years postoperatively (23.00±1.14) as compared with the baseline, (19.20±2.28) (P<0.05 or 0.01).

CONCLUSIONSCorporoplasty with autologous tunica vaginalis graft is a safe, simple and effective option for the treatment of Peyronie's disease, though its definite efficiency is to be further supported by large-sample clinical studies.

Erectile Dysfunction ; Humans ; Male ; Penile Erection ; Penile Induration ; surgery ; Penis ; surgery ; Postoperative Period ; Testis ; transplantation

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

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) have potential of differentiation and they secrete anti-inflammatory cytokines and growth factors which make them appropriate for cell therapy. AIM OF THE WORK: Were to evaluate the healing effect of BM-MSCs transplantation on germinal cells of busulfan-induced azoospermic hamsters. MATERIAL AND METHODS: In the present experimental case control study, BM-MSCs were isolated from bone marrow of donor albino hamsters. Five mature male recipient hamsters received two doses of 10 mg/kg of busulfan with 21 days interval to stop endogenous spermatogenesis. After induction of azoospermia, right testis of hamsters was injected with 106 BM-MSCs via efferent duct and the left one remained as azoospermia control testis. Five normal mature hamsters were selected as normal intact control. After 35 days, testes and epididymis of three groups were removed for histological evaluation. RESULTS: Histomorphological analyses of BM-MSCs treated testes and epididymis showed the epithelial tissue of seminiferous tubules had normal morphology and spermatozoa were present in epididymis tubes. Spermatogenesis was observed in most cell-treated seminiferous tubules. The untreated seminiferous tubules were empty. CONCLUSION: Transplanted BM-MSCs could successfully induce spermatogenesis in seminiferous tubules of azoospermic hamster. Therefore, BM-MSCs can be an attractive candidate in cell transplantation of azoospermia.
Animals ; Azoospermia* ; Bone Marrow ; Busulfan ; Case-Control Studies ; Cell Transplantation ; Cell- and Tissue-Based Therapy ; Cricetinae* ; Cytokines ; Epididymis ; Humans ; Intercellular Signaling Peptides and Proteins ; Male ; Mesenchymal Stromal Cells* ; Seminiferous Tubules ; Spermatogenesis* ; Spermatozoa ; Testis ; Tissue Donors ; Transplants

OBJECTIVETo investigate the clinical effect of modified Koyanagi technique with coverage by tunica vaginalis of testis in severe hypospadias.

METHODS49 cases with severe hypospadias treated from Jan. 2009 to Sep. 2011 were retrospectively studied. 25 patients underwent Koyanagi technique with coverage by tunica vaginalis of testis. 24 cases underwent one-stage Duplay + Duckett technique in the same term. The patients were followed up for 7-24 months.

RESULTSAmong the 25 children treated with Koyanagi procedure, 20 cases were cured, 5 patients had postoperative complications, including urethral fistula in 3 cases,urethral stenosis in 2 cases. At the same time, in the Duplay + Duckett group, 17 cases were cured, 7 children had postoperative complications, including urethral fistula in 4 cases, and urethral stenosis in 3 cases. All the patients with urethral fistula were repaired successfully 6 months after the first surgery; The urethral stenosis were cured by dilatation within 1 to 3 months. The successful rate in the 2 groups had no significant difference(P >0.05).

CONCLUSIONSKoyanagi technique with coverage by tunica vaginalis of testis is relatively simple with similar effect as Duplay + Duckett technique for severe hypospadias.

Child ; Child, Preschool ; Humans ; Hypospadias ; surgery ; Male ; Postoperative Complications ; etiology ; therapy ; Retrospective Studies ; Surgical Flaps ; transplantation ; Testis ; surgery ; Urethral Diseases ; etiology ; therapy ; Urethral Stricture ; etiology ; therapy ; Urinary Fistula ; etiology ; surgery

OBJECTIVETo explore the treatment of urethral stricture.

METHODSWe retrospectively studied the clinical data of 1 case of long anterior urethral stricture treated by urethroplasty with pedunculated preputial flap and testicular tunica vaginalis, and summarized the treatment of the disease with review of the relevant literature.

RESULTSThe operation was smooth and successful, and no such complications as fistula and urethral stricture were found during the follow-up.

CONCLUSIONUrethroplasty with pedunculated preputial flap and testicular tunica vaginalis as a substitute is feasible for the treatment of urethral stricture. The key to a successful operation is the proper choice of a urethral substitute.

Adult ; Foreskin ; transplantation ; Humans ; Male ; Skin Transplantation ; Surgical Flaps ; Testis ; Urethral Stricture ; surgery