[Differentially expressed proteins in the process of annexin 5 stimulating testosterone secretion from rat Leydig cells].

OBJECTIVE: To study the differentially expressed proteins in the process of annexin 5 stimulating testosterone secretion in cultured rat Leydig cells. METHODS: Primary rat Leydig cells were cultured in vitro and treated with annexin 5 at the concentration of 1 nmol/L for 24 hours, and the cell proteins were extracted to be compared by two-dimensional gel electrophoresis (2-DE). The differentially expressed protein spots were selected to be analyzed by mass spectrometry. RESULTS: We obtained electrophoresis profiles with high resolution and reproducibility, found 50 differentially expressed protein spots, and identified 36 by mass spectrometry, of which 23 were overexpressed and 13 underexpressed in the Leydig cells treated with annexin 5. CONCLUSION: Differentially expressed protein profiles were established in the process of annexin 5 stimulating testosterone secretion in cultured rat Leydig cells, and identified the key role of these proteins in testosterone secretion. Our findings might be helpful to illuminate the mechanism of annexin 5 regulating testosterone secretion in rat Leydig cells.

[Effect of annexin 5 on StAR and testosterone synthesis related enzyme in rat Leydig cells].

OBJECTIVE: To investigate the effect of annexin 5 on the expressions at mRNA levels and protein levels of StAR, P450scc, 3ß-HSD, 17α-hydroxylase and 17ß-HSD in rat Leydig cells. METHODS: The primary rat Leydig cells were cultured for 24 h and then stimulated with 10(-9) mol/L annexin 5 for 12 h and 24 h respectively. Cellular total RNA and total protein were extracted respectively. The expressions of StAR, P450scc, 3ß-HSD, and 17α-hydroxylase and 17ß-HSD(10) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR)and the protein levels were detected by Western blotting. RESULTS: Compared with the control group, at the mRNA level, after being treated with annexin 5 for 12 h, only 17ß-HSD(10) expression had a 26% increase (P<0.05) while the others had no significant difference. The expressions of StAR, P450scc and 3ß-HSD elevated 55%, 69% and 59%(P<0.05) respectively, and 17ß-HSD(10) increased 104%(P<0.01) while 17α-hydroxylase had no significant difference after being treated with annexin 5 for 24 h. At the protein level, after being treated with annexin 5 for 12 h, 17ß-HSD expression had a 39% increase (P<0.05). After 24 h, P450scc, 3ß-HSD and 17ß-HSD elevated 35%, 88% (P<0.05) and 47% (P<0.01) respectively while StAR had no significant difference. CONCLUSION: Annexin 5 regulates testosterone synthesis by affecting the expressions of P450scc, 3ß-HSD and 17ß-HSD at gene and protein levels.

[Protective effect of Annexin 5 on human sperm membrane and DNA integrity].

OBJECTIVE: To investigate the role of Annexin 5 in protecting human sperm membrane and DNA integrity. METHODS: We collected 53 semen samples based on the criteria of sperm density > 20 x 10(6)/ml and motility > 60%, and divided them into an experimental group (2.5 microl 10(-6) mol/L Annexin 5 added to 47.5 microl semen), a negative control group (2.5 microl 1 mol/L Tris-HCl [pH 8.0, 25 degrees C] added to 47.5 microl semen), and a blank control group (2.5 microl 0.01 mol/L PBS [pH 7.4] added to 47.5 microl semen). After 20 minutes of incubation, we evaluated the sperm membrane integrity using the hypoosmotic swelling test and, after another 60 minutes of treatment with H2O2 at 2.5 microl 10.02 mol/L, measured the sperm nuclear DNA integrity by acridine orange fluorescent staining. RESULTS: After 20 minutes of treatment with Annexin 5, the experimental group showed extremely significant difference in the percentage of hypoosmotic swelling sperm ([66.17 +/- 12.02] %) from the blank control ([58.13 +/- 13.08]%, P < 0.01) and the negative control group ([59.94 +/- 11.91]%, P < 0.01), but there was no significant difference between the latter two. Treatment with H2O2 remarkably increased DFI in the experimental group (6.39 +/- 1.07) as compared with the blank control (11.16 +/- 1.16) and the negative control group (10.86 +/- 1.05, P < 0.01), but no significant difference was observed between the latter two. CONCLUSION: Annexin 5 can increase the percentage of hypoosmotic swelling sperm in vitro and protect sperm membrane integrity, and it can also protect sperm DNA from H2O2 damage.

[Expression of rat annexin 5 and its effect on human sperm motility in vitro].

OBJECTIVE: Gonadotropin releasing hormones (GnRH) regulate the expression of annexin 5 in Leydig cells, and annexin 5 is supposed to be a signal molecule in regulating testosterone secretion. This study aimed to investigate the function of annexin 5 in male reproduction by observing its effect on human sperm motility in vitro. METHODS: The encoding sequence of rat annexin 5 was chemically synthesized and inserted into the HIS fusion expression vector pET28a. The expression of the fusion protein HIS-annexin 5 was induced by isopropyl-beta-D-thiogalactoside (IPTG) under the control of the T7 promoter, and the products were purified by affinity chromatography. The anticoagulant activity of annexin 5 was determined by the modified activated partial thromboplastin time (APTT) test. Semen samples from 15 donors were assigned to a control and an annexin 5 group, the latter treated with recombinant annexin 5 at the concentration of 10(-8) mol/L. Sperm motility and the percentage of grade a + b sperm were measured by computer-assisted semen analysis (CASA) after 20 and 60 min exposure, and the sperm ascending experiment was done after 20 min treatment. RESULTS: The product of the synthesized target gene was 947 bp in length, and the inserted sequence corresponded to the published encoding sequence of rat annexin 5. The plasmid pET28a-annexin 5 was transformed into E. coli BL21(DE3) and IPTG induced a fusion protein with a relative molecular weight of about 36,000, a purity of 95% and a high anticoagulant activity. Compared with the control group, sperm motility and the percentage of grade a + b sperm were increased by 40% (P < 0.01) and 21% (P < 0.01), respectively, after 20 min treatment with annexin 5, but neither showed any significant improvement after 60 min. The sperm ascending altitude was remarkably elevated after annexin 5 treatment, with extremely significant difference from the control group (37.84 +/- 6.35 vs. 49.5 +/- 12.27, P < 0.01). CONCLUSION: An annexin 5 recombinant expression vector was successfully constructed. The protein annexin 5 can be efficiently expressed in E. coli and effectively improve human sperm motility in vitro.

[Effects of GnRH analogues on MAPK pathway in rat Leydig cells].

OBJECTIVE: To investigate the effects of GnRH analogues GnRHa and GnRHant on the MAPK pathway in rat Leydig cells. METHODS: Rat Leydig cells were primarily cultured for 24 hours in vitro and serum-starved for 2 hours, followed by treatment with GnRHa (10(-7) mol/L) or GnRHant (10(-6) mol/L) for 0, 5, 15, 30, 60 and 90 minutes, with the 0 min group as the control. Then the protein levels of phosphorylated ERK (p-ERK) and phosphorylated p38 (p-p38) were detected by Western blot, and that of p-ERK determined by the same means after co-incubation of GnRHa or GnRHant with the PKC inhibitor GF109203X at 1, 5, 10 and 20 micromol/L. RESULTS: After stimulation of the Leydig cells with GnRHa or GnRHant for different times, the protein level of p-p38 showed no significant difference from that of the control group (P > 0.05). Then the Leydig cells were treated with GF109203X at different concentrations for 20 minutes and with addition of GnRHa for another 10 minutes. The level of p-ERK was significantly decreased (P < 0.05) by GF109203X at 10 and 20 micromol/L. Compared with the control, the p-ERK expression was increased by 65% at 15 minutes (P < 0.05) in the GnRHant stimulation group, by 81% (to the peak) at 30 minutes (P < 0.05), began to fall at 60 minutes, and returned to the base level at 90 minutes. The p-ERK level exhibited no significant difference from that of the control (P > 0.05) after treatment of the Leydig cells with different concentrations of GF109203X for 20 minutes and then with GnRHant for 30 minutes. CONCLUSION: The ERK MAPK activation induced by GnRHa depends on the PKC pathway, but not that induced by GnRHant. The p-38 MAPK pathway may not be involved in the effect of GnRH analogues on rat Leydig cells.