[Decreased expression of CatSper1 in the sperm of varicocele rats and protective effect of L-carnitine].

OBJECTIVE: To investigate the expression of the sperm-specific cation channel (CatSper1) in the epididymal sperm of varicocele (VC) rats and the effect of L-carnitine (LC) on the CatSper1 level. METHODS: Seventy male rats were equally randomized into groups A (normal control), B (VC model control), C (VC treated with normal saline), D (VC treated with low-dose LC), E (VC treated with medium-dose LC), F (VC treated with high-dose LC), and G (VC treated by prolonged medication of high-dose LC). The VC model was established by partial ligation of the left renal vein. At 12 weeks after modeling, the model rats in group C were treated intragastrically with normal saline at 1 ml/kg/d, those in groups D, E and F with LC at 0.05, 0.1 and 0.2 g/kg/d respectively, all for 5 consecutive weeks, and those in group G with LC at 0.2 g/kg/d for 7 successive weeks. Then, all the animals were sacrificed and their epididymides harvested for obtainment of the semen parameters by computer-assisted semen analysis (CASA) and determination of the mRNA and protein expressions of CatSper1 in the sperm by RT-PCR and Western blot. RESULTS: Compared with the rats in group A, those in group B showed significantly decreased percentage of grade a+b sperm (P < 0.01), sperm viability (P < 0.01), sperm concentration (P < 0.01) and expressions of CatSper1 mRNA (1.44 ± 0.67 vs 0.71 ± 0.38, P < 0.01) and protein (1.87 ± 0.67 vs 0.84 ± 0.42, P < 0.01). In comparison with the animals in group C, those in the four LC intervention groups exhibited a markedly increased percentage of grade a+b sperm, sperm viability and mRNA and protein expressions of CatSper1, even more remarkably in groups F and G (P < 0.01). No statistically significant difference, however, was observed in sperm concentration between group C and the LC intervention groups (P > 0.05), nor in the mRNA and protein expressions of CatSper1 between groups F and G. CONCLUSIONS: The expression of CatSper1 is decreased in the epididymal sperm of varicocele rats, and L-carnitine can increase the sperm viability, percentage of grade a+b sperm and CatSper1 expression of the rats.

Inhibiting transforming growth factor-ß signaling regulates in vitro maintenance and differentiation of bovine bone marrow mesenchymal stem cells.

Bovine bone marrow mesenchymal stem cells (bBMSC) are potential stem cell source which can be used for multipurpose. However, their application is limited because the in vitro maintenance of these cells is usually accompanied by aging and multipotency losing. Considering transforming growth factor-ß (TGF-ß) pathway inhibitor Repsox is beneficial for cell reprogramming, here we investigated its impacts on the maintenance and differentiation of bBMSC. The bBMSC were enriched and characterized by morphology, immunofluorescent staining, flow cytometry, and multilineage differentiation. The impacts of Repsox on their proliferation, apoptosis, cell cycle, multipotency, and differentiation were examined by Cell Counting Kit-8 (CCK-8), real-time polymerase chain reaction, induced differentiation and specific staining. The results showed that highly purified cluster of diffrentiation 73+ (CD73 + )/CD90 + /CD105 + /CD34 - /CD45 - bBMSC with adipogenic, osteogenic, and chondrogenic differentiation capacities were enriched. Repsox treatments (5 µM, 48 hr) enhanced the messenger RNA mRNA levels of the proliferation gene (telomerase reverse transcriptase [ TERT]; basic fibroblast growth factor [ bFGF]), apoptosis-related gene ( bax and Bcl2), antiapoptosis ratio ( Bcl2/bax), and pluripotency marker gene ( Oct4, Sox2, and Nanog), instead of changing the cell cycle, in bBMSC. Repsox treatments also enhanced the osteogenic differentiation but attenuated the chondrogenic differentiation of bBMSC, concomitant with decreased Smad2 and increased Smad3/4 expressions in TGF-ß pathway. Collectively, inhibiting TGF-ß/Smad signaling by Repsox regulates the in vitro maintenance and differentiation of bBMSC.

[Improving effect of Shengjingsan on spermatogenic function following testicular torsion/detorsion in rats and its mechanism].

OBJECTIVE: To study the effect of Shengjingsan on spermatogenic function following testicular torsion/detorsion in rats and its action mechanism. METHODS: Forty SD male rats were equally randomized to groups A (sham operation), B (control), C (low-dose Shengjingsan), D (medium-dose Shengjingsan) and E (high-dose Shengjingsan). The model of testicular torsion was established by 720 degrees clockwise torsion of the left testis for 4 hours. An hour before operation, the rats of group B received daily gavage of normal saline at 1 ml per kg per d, while those in groups C, D and E that of Shengjingsan at 0.01, 0.02 and 0.03 g per kg per d, all for 35 days. Then all the rats were sacrificed for measuring the semen parameters by CASA and detecting the expression of the CatSper1 gene in the sperm by RT-PCR. RESULTS: Compared with group A, Sperm concentration, the percentage of grade a + b sperm, sperm vitality and CatSper1 expression were significantly lower in group B ([15.30 +/- 6.30] %, [44.42 +/- 6.36] %, [21.00 +/- 6.14] x 10(6)/ml and 1.12 +/- 0.50) than in A ([51.30 +/- 6.60]%, [69.01 +/- 7.20]%, [40.53 +/- 7.01] x 10(6)/ml and 2.04 +/- 0.77) (P < 0.01). Compared with group B, the four parameters were increased remarkably in groups D ([51.63 +/- 3.20] %, [72.09 +/- 2.20]%, [55.30 +/- 5.90] x10(6)/ml and 2.11 +/- 0.20) andE ([55.93 +/- 3.17]%, [73.01 +/- 2.11]%, [58.33 + 4.90] x 10(6)/ml and 2.31 +/- 0.17) (P < 0.01), but not significantly in C ([18.02 +/- 0.23]%, [48.04 +/- 7.01]%, [22.87 +/- 2.10] x 10(6)/ml and 1.19 +/- 0.51) (P > 0.05). CONCLUSION: Shengjingsan can improve sperm parameters following testicular torsion/ detorsion in male rats by regulating their spermatogenic function and improving the expression of CatSper1 in the sperm.

Transcriptome profile of bovine iPSCs derived from Sertoli Cells.

Although induced pluripotent stem cells (iPSCs) had been generated from several somatic cell types in cattle, their pluripotency and differentiation capacities after freezing/thawing, and the dysregulated transcripts involved in pathways critical for reprogramming were not investigated. Additionally, selection of proper source cells is critical for iPSC derivation because the residual influence of the somatic origin may variegate their differentiation propensity. Sertoli cells (SCs) have special properties suitable for iPSCs derivation. Herein bovine SCs were enriched from the cryopreserved testicular tissues and reprogrammed into iPSCs using lentivirus carrying yamanaka factors (OSKM). These iPSCs have typical morphology resembling human iPSCs and remain normal karyotypes. They can express alkaline phosphatase activity and common pluripotency markers with a low methylation in the promoter region of Nanog. They can also form embryoid bodies and teratomas that give rise to cells/tissues from three embryonic germ layers. Transcriptome profiling showed that the exogenous OSKM were silenced and 8009 dysregulated mRNAs were identified. The pluripotency, methyldioxygenase and anti-apoptosis genes were all upregulated but the apoptotic gene downregulated in these iPSCs. Bunch of pathways related to the reprogramming, inflammation and viral infection pathways were upregulated, while pathways associated with the differentiation, senescence, metabolism and apoptosis were downregulated in these cells. After cryopreservation/thawing, the recovered iPSCs remain strong pluripotency and differentiation capabilities. Together, iPSCs were derived from the bovine SCs isolated from the cryopreserved neonatal bull testis, pluripotency and differentiation capacities verified, iPSCs cryopreserved, cultured and again reverified for pluripotency and differentiation capacities.