[Murine model of busulfan-induced spermatogenesis regeneration: a quantitative evaluation].

OBJECTIVE: To quantitatively evaluate the murine model of spermatogenesis regeneration induced by two-dose busulfan injection. METHODS: Fifty-four male mice were randomly divided into a control and two model groups of equal number, the former treated by two-dose intraperitoneal injection of 50% DMSO solution at 10 ml/kg, and the latter by that of busulfan at 10 mg/kg and 15 mg/kg respectively to establish spermatogenesis regeneration models, both at the interval of 24 days between the two doses. Spermatogenesis in seminiferous epithelia was evaluated by Johnsen score, and the expressions of GATA-4 and GDNF mRNA in Sertoli cells were detected by real time quantitative PCR at 3, 4 and 8 weeks after the treatment. RESULTS: Johnsen score kept stable in the control group at all stages (P > 0.05), but higher than in the model groups at 3 and 4 weeks (P < 0.01). It was lower in the 15 mg/kg than in the 10 mg/kg model group at 4 and 8 weeks (P < 0.01) , and than in the control group at 8 weeks (P < 0.05), but had no significant difference between the 10 mg/kg and the control groups (P > 0.05). Nor did the expression of GATA-4 mRNA in Sertoli cells show any significant difference among the three groups at different stages after the treatment (P > 0.05), and that of GDNF mRNA at different stages in the control group (P > 0.05). Compared with the controls, the level of GDNF mRNA in Sertoli cells was significantly higher at 3 weeks but lower at 4 weeks in the model groups (P < 0.01), and lower in the 15 mg/kg group (P < 0.01) and comparable in the 10 mg/kg group at 8 weeks (P > 0.05); and it was lower in the 15 mg/kg than in the 10 mg/kg group at all stages (P < 0.01). CONCLUSION: Two-dose intraperitoneal injection of 10 mg/kg busulfan at the interval of 24 days is an optimal option for the establishment of a murine model of spermatogenesis regeneration. Higher dose of busulfan may induce deficient expression of GDNF in Sertoli cells and result in incomplete restoration of spermatogenesis.

[Reconstruction of rabbit urethra using urethral extracellular matrix].

OBJECTIVE: To evaluate the feasibility of reconstruction of rabbit urethra using urethral extracellular matrix. METHODS: Extracellular matrix was obtained from the urethra of 20 donor New Zealand rabbits. In experimental group, 20 rabbits underwent segmental urethral resection (about 1.0 to 1.5 cm in length) and the defects were replaced by a tube of extracellular matrix. The serum TNFalpha was detected by ELISA to assess the immunity response preoperatively and 12, 24, 48 h postoperatively. The regenerated urethral segments were taken for histologic and pathologic study 10 days, 3 weeks, 6 weeks and 24 weeks after operation. The urodynamics, urethroscopy and urethrography were also performed. RESULTS: The serum TNFalpha in experiment group slightly rised, with no significant difference when compared with that in control group. 10 days after operation, epithelial cell migrated into the extracellular matrix from two ends, and small vessels were also found. 3 weeks later, several layers of urothelium covered the whole surface of the matrix tube. 6 weeks later, the irregularly arranged smooth muscle fibers were fist observed by Van Gieson staining. 24 weeks after operation, the smooth muscle cells increased, the appearance of the regenerated urethra segments were very similar to normal urethral wall components. The urethrography and urodynamic evaluation revealed no difference between the normal and the regenerated urethral tube. CONCLUSIONS: The urethral extracellular matrix might be an ideal replacement material for urethral defect.

[Long-term culture and identification of spermatogonial stem cells from BALB/c mice in vitro].

OBJECTIVE: To establish a long-term culture system for mouse spermatogonial stem cells (SSCs) and to discuss the key factor that supports mouse SSC self-renewal and proliferation. METHODS: Testis cells from 4-6 days postpartum male transgenic BALB/c mce were collected by a modified two-step enzymatic digestion method and plated on 0. 2% elatin-coated tissue culture plates. The germ cells were enriched by differential adherence selections after respectively incubated for 1, 5 and 24 h and then plated on the mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder layer. The basal culture medium was StemPro-34 SFM supplemented with other 15 nutrient factors. The 20 ng/ml Glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml basic fibroblast growth factor (bFGF) and 200 ng/ml GDNF-family receptor alpha 1 (GFRalpha1) were added to the serum-free medium to promote SSC proliferation. Several important surface markers and special genes were examined by immunocytochemical staining and RT-PCR analysis. RESULTS: After 3-4 days culture on the MEF feeder, SSCs proliferated continuously and formed typical colonies. SSCs from the BALB/c mice could be cultured in a steady state for 3 months. Immunocytochemical staining showed that Oct4 was specifically expressed in the cultured SSC nucleus and GFRalpha1 strongly expressed on the surface of the membrane. RT-PCR confirmed that the cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia. CONCLUSION: SSCs from BALB/c mice could be cultured in the improved culture system for 3 months. This culture system could help further understand the regulating mechanism of SSCs and might provide an opportunity for the treatment of male infertility by SSC transplantation.

Curative effect and histocompatibility evaluation of reconstruction of traumatic defect of rabbit urethra using extracellular matrix.

OBJECTIVE: To investigate the curative effect and histocompatibility of reconstruction of traumatic urethral defect of rabbit using urethral extracellular matrix (ECM). METHODS: Urethral ECM was obtained by excision of the urethra in 20 donor rabbits. In experimental group, 20 rabbits were resected a 1.0 cm-1.5 cm segment of the urethra and artificially made a model of traumatic urethral defect, then reconstructed by the urethral extracellular matrix of the same length. The rabbit immunity response was assessed by lymphocyte transformation test and serum TNF-alpha level. The reconstructed urethral segments were stained with hematoxylin-eosin and Van Gieson stain and observed by histological examination postoperatively. The urethrography, urethroscopy and urodynamic examinations were performed. RESULTS: There was no significant difference in stimulative index of lymphocyte transformation between ECM group and control group. The serum TNF-alpha levels of ECM group slightly rose, but the increase was not significant as compared with control group. On postoperative day 10, epithelial cell had migrated from each side and small vessels were found in the extracellular matrix. In the 3rd week, several layers of urothelium covered the whole surface of the matrix tube. In the 6th week, the disorganized arrangements of smooth muscle fibers were firstly observed by Van Gieson staining. In the 24th week, the smooth muscle cells increased and the matrix tube appeared fairly similar to normal urethral wall components. The urethroscopy and urodynamic evaluation revealed that the surface of reconstructed urethra was smooth and emiction was unobstructed. CONCLUSION: The urethral extracellular matrix might be an ideal and safe biomaterial for the reconstruction of urethral traumatic defect.

[Sorting of spermatogonial stem cells in mice].

OBJECTIVE: To search for an effective method for enriching spermatogonial stem cells in mice. METHODS: Bilateral artificial cryptorchidism was performed on 20 six-week old male Kunming mice. Three months after the operation, the testes were removed and single cell suspension prepared by two-step enzyme digestion. FITC-conjugated anti-alpha6-integrin antibody and PE-conjugated anti-c-kit antibody were added for adequate time on ice. Then the cells with low side scatter light-scattering properties were sorted and positively stained for alpha6-integrin and negative c-kit expression. And the viability of the isolated cells was assessed by trypan blue exclusion. RESULTS: The sorted spermatogonial stem cells constitute 2.8% of the testis cells and over 95% of them were viable. CONCLUSION: FACS can be used to isolate quantities of viable spermatogonial stem cells.

[Treatment of buried penis in children with fixation at the dartos coat of penis through prepubic route].

OBJECTIVE: To observe the long-time results of the treatment of buried penis with fixation at the base of penis through pre-pubic route. METHODS: From Aug 2002 to Dec 2003, the procedure was performed in 34 children without penile skin insufficiency. The major technique involved the release of hided penis shaft by partly shearing suspensory ligament, fixating the bucks fascia and tunica albuginea at 3 and 9 o'clock positions with the subcutaneous penile skin at the base of penis just under the pubic symphysis. RESULTS: Twenty-one patients have been reviewed over one year with satisfactory appearance. Thirteen patients were lost to follow-up. No recurrence happened in our group. CONCLUSION: We compare this technique with major treatment methods for buried penis in children at present, and we conclude this technique is a reasonable, simplified and aesthetical method for the treatment of buried penis in children.

[Modified technique of testis autotransplantation in dogs].

OBJECTIVE: To establish a dog model of testis autotransplantation with a modified technique. METHODS: Testis autotransplantations were performed in 30 dogs. After detachment of the spermatic artery with a cuff of the abdominal aorta and the spermatic vein with a cuff of inferior vena cava, the testis was perfused and kept at ice temperature. An end-to-side anastomosis of the spermatic vessels to the external iliac vessels was conducted. RESULTS: The success rate of the testis autotransplantations was 90% (27/30) and the time for heat ischemia, cold ischemia, anastomosis of spermatic vessels and the whole operation were (4.5 +/- 0.9) minutes, (50.0 +/- 5.0) minutes, (35.5 +/- 5.5) minutes and (3.5 +/- 0.5) hours respectively. CONCLUSION: A stable and feasible model of testis autotransplantation was established, which provides a reliable experimental base for testis autotransplantation.

[Advances in testis transplantation].

Testis transplantation is an important and effective way to treat abdominal impalpable cryptorchidism, male hypogonadism and male infertility. Since 1990s a lot of advances have been made in microsurgical autotransplantation, homotransplantation, testicular tissue transplantation and Leydig cell transplantation. The main achievements include the application of laparoscopy in autotransplantation, researches on the influential factors in spermatogenesis after homotransplantation, explorations of new treatment methods such as fetal testis transplantation, spermatogonial stem cell transplantation and so on. The advances in testis transplantation are summarized in this paper based on the related literature of recent years.

Reconstruction of rabbit urethra using urethral extracellular matrix.

BACKGROUND: Urethral reconstruction for both congenital and acquired etiologies remains a challenge for most urologic surgeons. Tissue engineering has been proposed as a strategy for urethral reconstruction. The purpose of this study was to determine whether a naturally derived extracellular matrix substitute developed for urethral reconstruction would be suitable for urethral repair in an animal model. METHODS: A urethral segmental defect was created in 20 male rabbits. The urethral extracellular matrix, obtained and processed from rabbit urethral tissue, was trimmed and transplanted to repair the urethral defect. Then, the regenerated segment was studied histologically by haematoxylin-eosin staining and Van Gieson staining at 10 days, 3 weeks, 6 weeks, and 24 weeks postoperation. Retrograde urethrography was used to evaluate the function of the regenerated urethras of 4 rabbits 10 and 24 weeks after the operation. The urodynamics of 4 rabbits from the experimental group and control group I were assessed and compared. In addition, 4 experimental group rabbits were examined by a urethroscope 24 weeks after the operation. RESULTS: At 10 days after operation, epithelial cells had migrated from each side, and small vessels were observed in the extracellular matrix. The matrix and adjacent areas of the host tissue were infiltrated with inflammatory cells. The epithelium covered the extracellular matrix fully at 3 weeks postoperation. Well-formed smooth-muscle cells were first confirmed after 6 weeks, at which point the inflammatory cells had disappeared. At 24 weeks postoperation, the regenerated tissue was equivalent to the normal urethra. Urethrography and urodynamic evaluations showed that there was no difference between normal tissue and regenerated tissue. CONCLUSIONS: Urethral extracellular matrix appears to be a useful material for urethral repair in rabbits. The matrix can be processed easily and has good characteristics for tissue handling and urethral function.

[Effects of icariin on the erectile function and expression of nitrogen oxide synthase isoforms in corpus cavernosum of arterigenic erectile dysfunction rat model].

OBJECTIVE: The study the effects of oral administered icariin on intracavernosal pressure (ICP) and on expression of the nitrogen oxide synthase (NOS) isoforms in corpus cavernosum (CC) of arteriogenic erectile dysfunction (A-ED) rat model. METHODS: Forty adult male Wistar rats were randomly divided into 4 groups of 10 rats: shame operated group (group A) and three A-ED model groups (group B, C and D). The internal pudendal arteries were isolated and ligated with 7-O nylon thread at both the main trunk and the penile branches to establish the A-ED model. ICP were tested after the operation to make sure the successful model establishment. The groups A and B were treated with saline: and the groups C and D were treated with icariin (5 mg/kg/day and 10mg/kg/day respectively) orally for 30 days. Then the ICP was measured again. The tissues of corpus cavernosum were taken and RT-PCR was used to detect the mRNA expression of nNOS, iNOS and eNOS in CC, and Western-blot was used to detect the protein expression of these NOS isoforms. RESULTS: The ICP in the group B was significantly decreased compared to the group A (P < 0.01), but the ICP values in the groups C and D were both increased compared to those in the group B (both P < 0.01). The expressions of the mRNA and protein of nNOS, iNOS, and eNOS were all decreased in the group B, however, the mRNA and protein expressions of eNOS were increased a in the groups C and D. In the group C, iNOS also increased. The expression of nNOS showed no obvious changes in the group C and group D. CONCLUSION: Chronic oral treatment with Icariin increases the erectile function (ICP) and restores the eNOS expression in CC of A-ED rats. Icariin may have a long-term therapeutic effect on ischemia/hypoxia induced ED.

[Effects of icariin on intracavernosal pressure and systematic arterial blood pressure of rat].

OBJECTIVE: To realize the effect of icariin on erectile function of penis. METHODS: After the cavernous nerve (CN) of rat was isolated unilaterally, the corpora cavernosa (CC) and right carotid artery were exposed. A 26G needle catheter was inserted into the right CC to monitor the intracavernous pressure (ICP), and another 26G needle catheter was inserted into the left CC for drug administration. Another catheter was placed into the carotid artery to monitor mean systematic arterial blood press (MBp). Icariin of different concentrations was administrated intracavernosally, and the ICP and MBp were recorded during electric stimulation on CN. Sildenafil and papaveine were used as controls. The effects of nitric oxide syntheses (NOS) inhibitor N(omega)-nitro-L-arginine (LNNA), and soluble guanylate cyclase inhibitor H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (ODQ) on icariin (10(-4)mol/L) induced ICP changes were investigated also. RESULTS: Icariin, sidenafil and papaverine increased the ICP in a dose-depended manner (P < 0.01). Icariin and sildenafil did not influence the MBp (P > 0.05), however, papaverine significant influenced MBp (P < 0.01). EC(50) of Icariin, sildenafil and papaveine on ICP/MBp were 2.23 micro mol/L, 0.24 micro mol/L and 9.73 micro mol/L respectively. ODQ and LNNA significantly decreased ICP induced by icariin (10(-4)mol/L). CONCLUSION: Icariin increases ICP without influence on MBp and such effect is inhibited by LNNA and ODQ significantly. Icariin regulates the activity of NO-cGMP signal pathway on CC to enhance erectile function by oral therapy.

[Allotransplantation of spermatogonial stem cells in KM mice].

OBJECTIVE: To study the feasibility of spermatogonial stem cell allotransplantation. METHODS: The spermatogonial stem cell allotransplantation was performed, without the use of immune inhibitor, in KM mice of similar gene types, and the spermatogenesis in recipients' testes was evaluated. The right testes were pierced for transplantation while the left ones were taken as control. RESULTS: Allotransplant germ cells in KM mice can recover normal function of spermatogenesis in the transplanted testis without any immune suppression. CONCLUSION: Allospermatogonial stem cells can be transplanted successfully among KM mice.

[Expression and significance of GFR alpha 1 gene in the recovery spermatogenesis of mice].

OBJECTIVE: To discuss the expression and significance of glial cell-derived neurotrophic factor (GDFN) receptor alpha 1 gene (GFR alpha 1) in the recovery spermatogenesis of mice. METHODS: Adult Kunming mice were injected intraperitoneally with 2 doses of busulfan (10 mg/kg) 24 days apart so as to establish the recovery spermatogenesis model. Testes were harvested 1 w, 2 w, 3 w, 4 w, 6 w, 8 w and 10 w after the second injection, and normal testes were used as control. The recovery spermatogenesis was observed by light and electron microscopy, and the GFR alpha 1 mRNA was measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: The expression of GFR alpha 1 mRNA increased significantly at 1 w and reached its peak at 2 w after the second injection [(104.72 +/- 24.4)% vs normal control, P < 0.01]; its expression reduced significantly at 3 w and reached its valley at 4 w [(20.77 +/- 4.25)% vs normal control, P < 0.01], and then increased gradually and restored to the normal level at 10 w. GFR alpha 1 mRNA was mainly expressed by undifferentiated spermatogonia. CONCLUSIONS: In the course of recovery spermatogenesis, the expression of GFR alpha 1 plays a key role in turning the spermatogonial stem cell reactivity to GDNF, which promotes self-renewal at a high level, or results in differentiation at a low level.