[Lycium barbarum miR2911-loaded exosomes promote spermatogenic function recovery in rats with non-obstructive azoospermia by regulating Wnt/ß-catenin signaling pathways].

OBJECTIVE: To investigate the effect of exosomes loaded with Lycium barbarum miRNA (Lb-miR2911) on spermatogenic function recovery in non-obstructive azoospermia (NOA) rats through cross-regulation of the Wnt/ß-catenin signaling pathways. METHODS: We established an NOA model in 30 four-week-old male SD rats by intraperitoneal injection of busulfan. At 5 weeks after modeling, we equally randomized the rats into a model control group (MC，untreated), an Lb-miR2911EXO group (Lb-miR2911EXO ，treated by intratesticular injection of Lb-miR2911-loaded exosomes), and a sham group (Shame，treated by intratesticular injection of exosomes-empty drug), with another 10 male SD rats taken as normal controls（NC）. We observed the uptake and metabolic changes of Lb-miR2911 in the testis tissue of the rats by RNA FISH at 2 and 6 weeks after treatment, detected cell proliferation, spermatogenesis and gene expressions of the Wnt/ß-catenin signaling pathways in the testis tissue by Transcriptome sequencing analysis combined with Western blot and RT-PCR at 12 weeks, evaluated the recovery of the spermatogenic function based on the testis tissue morphology and sperm quality, and assessed the organ toxicity of Lb-miR2911 in the tissue and organs of the rats based on histomorphological analysis and the levels of serum TNF-α, IL-1ß, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and other relevant indicators. RESULTS: After 12 weeks of treatment, histomorphological analysis showed regular arrangement of spermatogenic cells at all levels in the testis tissue, with a large number of mature sperm in the tubular lumen, and with significantly higher Johnsen scores, testis weight, testicular index, sperm concentration and sperm motility in the Lb-miR2911EXO than in the sham group (all P< 0.05). Compared with the model controls, the Lb-miR2911EXO group exhibited remarkably down-regulated gene expression of DACT3 (P< 0.05), up-regulated expressions of DVL2 and ß-catenin (P< 0.05), elevated levels of p-DVL2 and ß-catenin (nucleus) proteins (P< 0.05), increased expressions of cell proliferation-related genes CCND1, CCNE1 and CCNE2 (P< 0.05) and spermatogenesis-related genes DMC1, CCR6, JAM2 and KLC3 (P< 0.05). No pathological changes were observed in the lung, liver and kidney tissues of the rats, or in the levels of serum TNF-α, IL-1ß, AST, ALT, creatinine and urea nitrogen in the rats treated with Lb-miR2911EXO compared with the normal controls (P > 0.05). CONCLUSION: Lb-miR2911-loaded exosomes promote spermatogenic function recovery in NOA rats through cross-regulation of the DACT3, Wnt and ß-catenin signaling pathways.

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.

[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.

[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.