[Construction of a mouse model of spermatogonial stem cell transplant recipient by high-temperature heat stress].

OBJECTIVE: To investigate the feasibility of constructing a mouse model of spermatogonial stem cell (SSC) transplant recipient by high-temperature heat stress. METHODS: Four-week-old C57BL/6 male mice and B6(Cg)-Tyrc-2J/J coat color gene homozygous mutant male mice were heat-treated at 43 ℃ for an hour in the incubator. The best transplantation time was determined by HE staining, immunohistochemistry and TUNEL and the SSCs were transplanted into the seminiferous tubules of the mice followed by regular observation of the proliferation, differentiation and spermiogenesis of the SSCs in the testis of the recipient mice. Then the recipients were mated with age-matched normal female mice and the epigenetic features of their offspring were observed. RESULTS: After 3－5 days of high-temperature heat stress, the spermatogenic cells in the testicular seminiferous tubules of the recipient mice showed obviously decreased layers, disordered and loose arrangement, massive deletion, significant apoptosis, reduced mesenchymal cells and increased autophagy, which were basically recovered in about 12 days. At 8 weeks after transplantation, the isolated and purified SSCs were differentiated into spermatogenic cells and sperm with genetic function in the testicular seminiferous tubules of the recipient mice, and normal offspring were reproduced after natural mating. CONCLUSIONS: High-temperature heat stress can be used as an efficient method for rapid construction of the mouse model of spermatogonial stem cell transplantation recipient.

[Rho/ROCK signaling pathway and anti-cryodamage ability of human sperm].

OBJECTIVE: To investigate the influence of the Rho/ROCK signaling pathway on the anti-cryodamage ability of human sperm and provide some theoretical evidence for the development of high-efficiency semen cryoprotectants. METHODS: We collected semen samples from 25 healthy males, each divided into a fresh, a normal cryopreservation control and an Rho-inhibition group. Before and after freezing, we detected sperm motility, viability, membrane integrity, morphology, DNA fragmentation index (DFI), acrosomal enzyme activity (AEA) and mitochondrial membrane potential (MMP) and determined the expressions of RhoA and ROCK proteins in the sperm by immunofluorescence staining. RESULTS: Compared with the normal cryopreservation control, the frozen-thawed sperm of the Rho-inhibition group showed significantly increased sperm motility （ ï¼»51.20 ± 7.70ï¼½% vs ï¼»57.50 ± 6.83ï¼½%, P = 0.002), survival rate ( ï¼»52.87 ± 5.07ï¼½% vs ï¼»60.24 ± 5.53ï¼½%, P = 0.001), membrane integrity (ï¼»59.78±5.56ï¼½% vs ï¼»67.10 ± 4.43ï¼½%, P = 0.001), percentage of morphologically normal sperm (ï¼»4.83 ± 1.11ï¼½% vs ï¼»7.46 ± 1.28ï¼½, P = 0.001) and MMP (56.30 ± 4.28 vs 63.11 ± 2.97, P = 0.001), but decreased DFI (ï¼»27.64 ± 6.64ï¼½% vs ï¼»18.87 ± 4.07ï¼½%, P = 0.001). There was no statistically significant difference in the AEA of the frozen-thawed sperm between the control and Rho-inhibition groups (97.65 ± 9.31 vs 98.30 ± 11.33, P > 0.05). Immunofluorescence staining revealed extensive expressions of RhoA and ROCK proteins in the head and neck of the sperm. CONCLUSIONS: The Rho/ROCK signaling pathway plays a role in the cryodamage to human sperm, and inhibiting the activity of Rho/ROCK can significantly improve the ability of sperm to resist cryodamage.

Evaluation of vitrification protocol of mouse ovarian tissue by effect of DNA methyltransferase-1 and paternal imprinted growth factor receptor-binding protein 10 on signaling pathways.

Transplantation of cryopreserved ovarian tissue has been considered as a promising way of fertility preservation for women. however, this cryopreservation method is prone to post-resuscitation follicle proliferation and oocyte development stagnation, affecting late transplant survival. To evaluate current vitrification works, we investigated the critical pathway alternations in vitrified-warmed juvenile 10-day-old mouse ovary. We showed a significant decrease of protein kinase B (Akt) and Mitogen-activated protein kinase (Mapk) phosphorylation, during which serine/threonine kinases play central roles in coordinating follicle and oocyte development and stress response. Inhibition of Akt and Mapk activity were associated with one of the imprinted insulin pathway negative regulatory genes, Growth factor receptor-binding protein 10 (Grb10) which remarkably increased in vitrified-warmed juvenile mouse ovary than that of fresh group (p < 0.05). RNAi-induced Grb10 down-regulation reversed the decrease in Akt and Mapk phosphorylation. The increase of Grb10 expression was partially caused by the hyper-methylation of the promoter region, associated with the decrease of follicular DNA methyltransferase (Dnmt) 1 protein in different stages of vitrified-warmed group, compared to fresh group (p < 0.05). The mRNA and protein expression of Dnmt1 in ovary of vitrified-warmed juvenile mouse were remarkably lower than those in fresh group (p < 0.05). Dnmt1 overexpression dramatically reversed Grb10 up-regulation and Akt and Mapk phosphorylation reduction. Taken together, our findings suggest that Grb10 expression might be helpful in evaluation of effectiveness of vitrification, and considered as a potential target for further vitrification protocols improvement in the future.

[Dynamic changes of pH2AX expression in the reversibility of mouse testicular reproductive function impaired by single heat stress].

OBJECTIVE: To investigate the role of pH2AX in the reversibility of mouse testicular reproductive function impaired by single heat stress. METHODS: Twenty-four C57 male mice were randomly divided into heat stress and control groups and immersed in water at 43℃ and 25℃, respectively, for 15 minutes. At 1, 7, and 14 days of heat exposure, all the mice were sacrificed and their testis tissues collected for determining the apoptosis of the germ cells by TUNEL and measuring the expression level of the pH2AX protein by immunohistochemistry and Western blot. RESULTS: The highest percentage of apoptotic cells were found in the seminiferous tubules of the mice in the heat stress group on the 1st day of the exposure and almost no apoptosis was observed at 7 and 14 days. The pH2AX protein was expressed in the nuclei of the basement membrane of adjacent seminiferous tubules. Compared with the control group, the expression of pH2AX was significantly increased on the 1st day of exposure (0.47 ± 0.02 vs 1.61 ± 0.04, P <0.01), then decreased at 7 days (0.85 ± 0.03) in comparison with that on the 1st day (P <0.01), and again elevated at 14 days (1.72 ± 0.02) as compared with either those at 1 and 7 days (P <0.01) or that of the control (P <0.01). CONCLUSIONS: Heat stress causes dynamic changes of the pH2AX expression in the testis of the mouse, which are associated with heat stress-induced proliferation and division of the testicular spermatogenic cells.

[Effects of single heat stress treatment on spermatogenic cells in mice].

OBJECTIVE: To investigate the effects of single heat stress treatment on spermatogenic cells in mice. METHODS: We randomly divided 36 C57 male mice into a control and a heat stress treatment group and submerged the lower part of the torso in water at 25 °C and 43 °C, respectively, both for 15 minutes. At 1, 7, and 14 days after treatment, we obtained the testicular organ indexes, observed the changes in testicular morphology by HE staining, and determined the location and expression levels of the promyelocytic leukemia zinc finger (PLZF) and synaptonemal comlex protein-3 (SCP-3) in the testis tissue by immunohistochemistry and Western blot. RESULTS: The testicular organ index was significantly lower in the heat stress treatment than in the control group (P < 0.05). Compared with the controls, the heat shock-treated mice showed loosely arranged spermatogenic cells scattered in the seminiferous tubules at 1 day after heat stress treatment, atrophied, loosely arranged and obviously reduced number of spermatogenic cells at 7 days, and relatively closely arranged seminiferous tubules and increased number and layers of spermatogenic cells at 14 days. The number of SCP-3 labelled spermatocytes obviously decreased in the heat stress-treated animals at 1 and 7 days and began to increase at 14 days. The PLZF protein expression was significantly reduced in the heat stress treatment group at 1 day as compared with that in the control (0.19 ± 0.12 vs 0.64 ± 0.03, P < 0.01), but elevated to 0.77 ± 0.02 at 7 and 14 days, even remarkably higher than in the control animals (P < 0.01). CONCLUSION: Heat stress treatment can induce short-term dyszoospermia in mice, which can be recovered with the prolonged time after treatment.

[Dynamic study and screening of new markers of spermatogonial stem cells by iTRAQ protein mass spectrometry].

OBJECTIVE: To study the dynamic changes in the protein marker expression in the spermatogonial stem cells (SSCs) of mice at different ages by iTRAQ protein mass spectrometry and to screen new markers using the bioinformatic proteome database. METHODS: Based on the postnatal weeks, we divided 80 healthy male C57BL/6 mice into eight age groups of equal number, harvested their testicular tissues, extracted proteins following purification of the SSCs by compound enzyme digestion and magnetic-activated cell sorting. Then we analyzed and identified proteins using two-dimensional electrophoresis, protein mass spectrometry, and protein database information. RESULTS: Totally, 248,510 mass spectra were obtained from the MS experiment and 1132 proteins were identified. By the criteria of >1.2-fold for protein abundance difference and P value <0.05, we identified 298 differentially expressed proteins and 9 currently known makers of SSCs (PCNA, GFRalpha1, CDH1, Annexin A7, UCHL1, VASA, CD49f, CD29, and PLZf). Compara- tive analysis showed different expressions of the proteins in the SSCs of the mice of different ages, and the differences in the expressions of GFRalpha1, CD49f, and CD29 were consistent with the findings in other published literature. Ten proteins (P63, CD71, CD98, K19, ACE, K18, K15, K17, SH2, and SH3) were selected as SSC markers to be further studied. CONCLUSION: The proteins in SSCs are differentially expressed in mice of different ages. The technology of iTRAQ protein mass spectrometry can be used to analyze and compare the proteome information of mouse SSCs, obtain differentially expressed proteins in mice of different ages, and thus offers a new ap- proach to further analysis and study of the function and roles of these differential proteins.

[Yimusake Tablet: safe and efficacious for premature ejaculation].

OBJECTIVE: To objectively evaluate the efficacy and safety of Yimusake Tablet in the treatment of premature ejaculation (PE) through a multi-centered large-sample trial. METHODS: We conducted a multi-centered, open, fixed-dose, and self-compared clinical trial among 300 patients with diagnosed PE. The trial lasted 12 weeks, including 4 weeks without any medication and 8 weeks of treatment with Yimusake Tablet, 2 pills (1 g) per night. We observed the intravaginal ejaculation latency time (IELT) before and after treatment, evaluated the safety of medication, and performed a questionnaire investigation on the patients' satisfaction. RESULTS: Of the 300 PE patients, 288 accomplished the clinical trial. The patients ranged in age from 22 to 60 years, averaging at 31.6 years. The mean IELT of the patient was 62.5 seconds at baseline, 168.9 seconds after 4 weeks of treatment with Yimusake Tablet, and 222.2 seconds after 8 weeks of medication. Among the 157 patients with normal erectile function (IIEF >21), the mean IELT was 71.4 seconds before treatment, 147.4 seconds after 4 weeks of medication, and 172.5 seconds after 8 weeks of medication. The patients' satisfaction was significantly increased after treatment. Those complicated by mild to moderate erectile dysfunction achieved different degrees of improvement in the IIEF-5 score, with a mean increase of 3.8. Only a few patients experienced mild adverse events, including constipation, dry mouth, nose bleeding, abdominal pain, and lumbosacral pain, which were all relieved without drug withdrawal. CONCLUSION: Yimusake Tablet is a safe and effective medicine for the treatment of PE.

A prospective, randomized clinical trial comparing plasmakinetic resection of the prostate with holmium laser enucleation of the prostate based on a 2-year followup.

PURPOSE: We compared plasmakinetic resection with holmium laser enucleation of the prostate for the treatment of benign prostatic hyperplasia by analyzing 2-year followup data from a prospective randomized clinical trial. MATERIALS AND METHODS: A total of 280 patients were randomly treated with plasmakinetic resection or holmium laser enucleation of the prostate. Perioperative and postoperative outcome data were obtained during a 2-year followup. RESULTS: No significant differences between the 2 surgical groups were observed in the preoperative data. Both groups displayed significant improvements after surgery. However, we identified no significant differences between the 2 groups in the 2-year followup data for I-PSS (International Prostate Symptom Score), quality of life scores or maximum flow rate values. Patients in the holmium laser enucleation group displayed a lower risk of hemorrhage, shorter bladder irrigation and catheter times, and shorter hospital stays. A larger amount of prostate tissue was retrieved in the holmium laser enucleation group, but the operation time was longer for this group than for the plasmakinetic resection group. CONCLUSIONS: Plasmakinetic resection and holmium laser enucleation of the prostate are effective and safe treatments for benign prostatic hyperplasia. Holmium laser enucleation of the prostate can be applied to prostates of all sizes, and involves less risk of hemorrhage, decreased bladder irrigation and catheter times, as well as reduced hospital stay. Thus, we believe holmium laser enucleation of the prostate should be proposed as a potential new gold standard surgical therapy instead of transurethral resection of the prostate for patients with benign prostatic hyperplasia.

Relative safety of various spermatogenic stem cell purification methods for application in spermatogenic stem cell transplantation.

BACKGROUND: Spermatogonial stem cell (SSC) transplantation technology as a promising option for male fertility preservation has received increasing attention, along with efficient SSC purification technology as a necessary technical support; however, the safety of such application in patients with tumors remains controversial. METHODS: In this study, we used a green fluorescent protein mouse xenograft model of B cell acute lymphocytic leukemia. We isolated and purified SSCs from the testicular tissue of model mice using density gradient centrifugation, immune cell magnetic bead separation, and flow cytometry. The purified SSCs were transplanted into convoluted seminiferous tubules of the nude mice and C57BL/6 male mice subjected to busulfan. The development and proliferation of SSCs in the recipient testis were periodically tested, along with whether B cell acute lymphocytic leukemia was induced following SSC implantation. The genetic characteristics of the offspring obtained from natural mating were also observed. RESULTS: In testicular leukemia model mice, a large number of BALL cells infiltrated into the seminiferous tubule, spermatogenic cells, and sperm cells in the testis tissue decreased. After spermatogonial stem cell transplantation, the transplanted SSCs purified by immunomagnetic beads and flow cytometry methods colonized and proliferated extensively in the basement of the seminiferous tubules of mice; a large number of spermatogenic cells and sperm were found in recipient testicular tissue after 12 weeks of SSC transplantation. In leukemia detection in nude mice after transplantation in the three SSC purification groups, a large number of BALL cells could be detected in the blood of recipient mice 2-3 weeks after transplantation in the density gradient centrifugation group, but not in the blood of the flow cytometry sorting group and the immunomagnetic bead group after 16 weeks of observation. CONCLUSIONS: In this study, we confirmed that immunomagnetic beads and flow cytometry methods of purifying SSCs from the testicular tissue of the testicular leukemia mouse model could be safely applied to the SSC transplantation technology without concomitant tumor implantation. The results thus provide a theoretical basis for the application of tumor SSC cryopreservation for fertility preservation in patients with tumors.

[Isolation, purification and immunochemical characteristics of spermatogonia of rat].

OBJECTIVE: To explore the approach of isolation and purification of spermatogonia and its immunochemical characteristics. METHODS: Compound enzymatic digestions were used to prepare germ cell suspensions of Sprague-Dawley rats aged 10 days, and velocity sedimentation and discontinuous Percoll density gradient centrifugation were used to isolate and purify the spermatogonia. Using c-kit and alpha(6)-integrin multiclone antibodies as markers respectively, the immunochemical characteristics of the spermatogonia in the testicular tissue were observed and the c-kit and alpha(6)-integrin expression rates of the purified cells were detected by flow cytometry. RESULTS: The spermatogonia uniquely expressed c-kit and alpha(6)-Integrin in the testicular tissue. C-kit and alpha(6)-integrin were positively expressed in the purified cell suspensions. Using c-kit as the cell marker, the positive rate was 1.59% +/- 0.04% in the unpurified group, significantly lower than that of the purified group (68.33% +/- 2.45%, P < 0.01). Using alpha(6)-integrin as the cell marker, the positive rate of the unpurified group was 2.38% +/- 0.60%, significantly lower than that of the purified group (72.04% +/- 3.65%, P < 0.01). Trypan blue staining showed that the cell viability of the purified cell suspensions was more than 95%. CONCLUSION: c-kit and alpha(6)-integrin can be used as the molecular markers of spermatogonium at special stage. Spermatogonia with high purity and viability can be obtained via the steps including digestions with enzymes, velocity sedimentation and discontinuous percoll density gradient centrifugation.

[Advances in xenogeneic transplantation of spermatogonial stem cell and its bewilderment in clinical application].

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.