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To explore a new method of in vitro culture and purification of rat corpus cavernosum endothelial cells (CCECs). Male Sprague-Dawley rats' penile tissue were digested with elastase or collagenase combined with mechanical extrusion to isolate and culture the CCECs. The fixed-point digestion method was used to purify the primary cells. High-purity CCECs were successfully isolated. Following the digestion of the primary CCECs by elastase or collagenase coupled with mechanical extrusion, the cells were paving stone- and cobblestone-shaped over 10 days. The cell purity yielded in the second generation (P2) CCECs after using the fixed-point digestion method was significantly high. Compared with primary CCECs extracted by elastase digestion combined with the mechanical extrusion method, CCECs cultured by collagenase digestion yielded higher purity and a more stable morphology after fixed-point digestion and purification. Immunofluorescence staining of the third generation CCECs and the expression results of endothelial cell-associated marker antibodies CD31 and VWF were positive, and flow cytometry showed the purity of CCECs was 96.9%. Enzymatic digestion combined with mechanical extrusion and fixed-point digestion is a simple, economical method for in vitro culture and purification of CCECs, which is conducive to studying the pathophysiological mechanisms of endothelial dysfunction and erectile dysfunction.

To analyze the embryo development potential after intracytoplasmic injection of sperm from azoospermia patients with different spermatogenic functions.We performed ICSI with sperm retrieved from azoospermia patients with different spermatogenic functions using percutaneous epididymal sperm aspiration (PESA) and testicular sperm aspiration (TESA). Then we recorded and analyzed the rates of normal fertilization, cleavages, excellent embryos and pregnancies.No statistically significant differences were found between the PESA and TESA groups in the rates of normal fertilization ([74.9 +/- 19.6] vs [66.3 +/- 22.7]%, P0.05), cleavages ([96.7 +/- 8.6] vs [92.8 +/- 19.8]%, P0.05), excellent embryos ([43.5 +/- 26.2] vs [35.0 +/- 29.4]%, P0.05) and pregnancies (44.0 vs 52.0%, P0.05). The normal fertilization rates in the patients with normal spermatogenesis, mild spermatogenic dysfunction (SD), moderate SD and severe SD were (77.8 +/- 18.4), (68.4 +/- 18.5), (73.5 +/- 19.8) and (51.4 +/- 27.9)%, respectively, with significant difference between the normal spermatogenesis and mild SD groups (P0.05) as well as between the severe SD and the other groups (P0.05); the cleavage rates were (96.7 +/- 9.2), (96.5 +/- 15.0), (93.9 +/- 12.1) and (93.7 +/- 11.1)%, respectively, with no significant difference among the four groups; the excellent embryo rates were (47.1 +/- 25.8), (40.3 +/- 27.6), (36.2 +/- 23.1) and (15.0 +/- 24.6)%, respectively, with significant difference between the severe SD and the other groups; the pregnancy rates were 54.8, 50.0, 13.6 and 10.0%, respectively, with significant differences among the four groups (P0.001).ICSI by PESA or TESA is an effective approach to azoospermia. There are no significant differences between PESA and TESA in the rates of normal fertilization, cleavages, excellent embryos and pregnancies. The severity of spermatogenic dysfunction affects fertilization and initial development of embryos, which were shown in the rates of normal fertilization, excellent embryos and pregnancies but not that of cleavages.

To study the differentially expressed genes in asthenospermia to gain a deeper insight into the molecular mechanisms of the disease.We analyzed the differentially expressed genes in asthenospermia using GATHER, PANTHER and ToppGene online bioinformatics tools.Our bioinformatics mining and analyses revealed that the differentially expressed genes in asthenospermia played important roles in the cellular protein and macromolecular metabolism, protein modification, cell death, cell apoptosis and apoptosis induction.Asthenospermia patients experience a decline in sperm activity and the basic life activities of sperm simultaneously, and are also prone to cell apoptosis or death. Such differentially expressed genes as KIF3B, MYO15A, KIF6, KIF26B, KIF3A, DNHD2, DMN, DYNC2H1, STARD9, MYOHD1, and TPM1, which are involved in cytoskeletal structure, microtubule movement and cell movement, may be associated with asthenospermia, and therefore deserve further studies.

To investigate the mRNA and protein expression levels of cysteine-rich secretory protein 2 (CRISP2) in the sperm of asthenospermia patients, and explore their relationship with sperm motility and related molecular mechanism.We collected 78 semen samples from adult male patients with asthenospermia and another 70 from healthy volunteers as controls. We extracted total RNA and total protein from the sperm following purification of the sperm by Percoll gradient centrifugation, and detected the relative expressions of CRISP2 mRNA and protein in the two groups by RT-PCR, SYBR Green real-time PCR and Western blot.The expression of CRISP2 mRNA was down-regulated by 4.3 times and that of the CRISP2 protein by 1.71 times in the asthenospermia patients, significantly lower than in the normal control group (P0.05).The down-regulation of CRISP2 mRNA and protein expressions in the sperm of asthenospermia patients may be closely related with decreased sperm motility, which suggests that CRISP2 may serve as a potential molecular target for the research of asthenospermia.