Objective Gonadotropin-releasing hormone(GnRH) could regulate the release of glucagon.The present study aimed to investigate the effect of GnRH analog on glucagon release in In-R1-G9 cells.Methods The expression and localization of the GnRH receptor(GnRHR) in In-R1-G9 cells were detected by Western blot and immunocytochemistry.Various concentrations of GnRH analog(GnRHa) or GnRH antagonist(GnRHant)(0,0.1,10 and 1000 nmol/L)were respectively added to the KRB buffer followed by incubation of the cells for 2 hours.At the end of the incubation period,the medium was collected,and glucagon secretion in the control and experimental groups was measured by radioimmunoassay.Results GnRHR was expressed and located on the surface of In-R1-G9 cells.Compared with the control group,higher concentrations(10 and 1000 nmol/L) of GnRHa markedly stimulated glucagon release(P0.05),while the lower concentration(0.1 nmol/L) exhibited no significant increasing effect on it(P0.05).Higher concentrations of GnRHant remarkably inhibited glucagon release(P0.05),while the lower concentration showed no significant inhibitory effect on it(P0.05).Conclusion GnRH analog regulates glucagon release in a concentration-dependent manner in In-R1-G9 cells,in which GnRHR seems to be involved.

Objective To investigate whether the effect of GnRH agonist(GnRHa) on glucagon release in In-R1-G9 cells is relevant to mitogen-activated protein kinases(MAPK) signaling pathway.Methods The In-R1-G9 cells were serum-starved for 24 h before GnRHa treatment,and then 100 nmol/L GnRHa stimulated the cells for 0,5,10,20,30,60,90 min,respectively.Phosphorylated extracellular signal-regulated kinase(p-ERK),total ERK(T-ERK),phosphorylated p38,and β-actin were detected by Western blotting.After In-R1-G9 cells were treated with ERK inhibitor PD98059(50 μmol/L),glucagon was measured by radioimmunoassay.Results The level of p-ERK increased significantly at 20 min(P0.05) after In-R1-G9 cells were stimulated by GnRHa and the peak of p-ERK level occurred at 30 min(P0.01).p-ERK in the cells treated by GnRHa increased by 169% compared with that in the control group.Then it decreased to normal level at 90 min.However,the level of P-p38 had no significant change at each time point compared with that of the control group.There was significant disparity when PD98059 group was compared with DMSO group(P0.05) and GnRHa+PD98059 group was compared with GnRHa group(P0.01).Conclusions GnRHa regulates glucagon release in In-R1-G9 cells through ERK1/2 MAPK pathway rather than p38 MAPK pathway.

Objective To study the effect of NO donor(sodium nitroprusside,SNP) on testosterone secretion in rat Leydig cells.Methods The primary rat Leydig cells were cultured in vitro for 12 h,and then treated with different concentrations of SNP for various time courses(4 h,8 h,and 12 h).The level of testosterone was detected by chemiluminescence,and the concentrations of 3β-hydroxysteroid dehydrogenase enzyme(3β-HSD) and annexin 5 were analyzed by Western blotting.Results After treatment with 10-4 mol/L SNP for 8 h,with 10-6 mol/L SNP for 12 h,or with 10-4 mol/L SNP for 12 h,the level of testosterone was increased by 23%,28%,and 37% respectively,compared with that of the control(P0.05).Western blot analysis showed that,when Leydig cells were stimulated by SNP,3β-HSD expression had no significant difference(P0.05),compared with that in the control.After Leydig cells were treated with different concentrations of SNP for 4 h or 8 h,the annexin 5 level also had no difference compared with that in the control(P0.05).However,after Leydig cells were stimulated by 10-6 mol/L SNP for 12 h,the annexin 5 was increased by 55% compared with that of the control(P0.05),and the expression of annexin 5 also increased by 48% in the cells treated with 10-4 mol/L SNP(P0.05).Conclusions The results suggest the low concentration of SNP induces testosterone secretion in rat Leydig cells in a dose-and time-dependent manner.3β-HSD is not involved in the process of testosterone secretion induced by SNP,but the process may be related to the expression of annexin 5.

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.

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.

Objective To observe the expression of annexin 5 in testis of SD rats under constant light stress.MethodsSD rats were exposed to constant light to establish short(2d,3d,4d,7d)and long-term (14d,21d,28d) constant-light-stressed models.The testes were then obtained from the model rats and their corresponding controls respectively.Immunolocalization and relative expression of annexin 5 in rats' testes were analyzed by immunohistochemistry and Western blotting.ResultsWestern blotting showed that,compared with the controls,the expression of annexin 5 in the rats' testes of 14d and 21d group was upregulated significantly by about 34.5%±11% and 26%±16% (P<0.01,P<0.05),respectively,and no significant changes were observed in short (2d,3d,4d,7d) constant-light-stress groups and 28d group (by 18%±5%,P>0.05).Immunohistochemistry showed no changes in annexin 5 localization in rats' testes in stress groups compared with that in control groups.However,a stronger coloration of annexin 5 was observed by immunohistochemistry in 14d,21d and 28d constant-light-stress groups,implying a higher expression of annexin 5.ConclusionThe long-term (14d,21d) constant-light-stress may increase the expression of annexin 5 in rat's testis.Along with the prolongation of stress time (28d),with the development of compensatory mechanism,the expression of annexin 5 in rat's testis approaches the normal level,implying that annexin 5 might participate in the stress processes,as well as in the regulation of some important physiological functions.

Objective To investigate the relationship between seminal plasma thymidine kinase 1 (TK1) and sperm parameters.Methods The level of TK1 in seminal plasma of sterile males and healthy males was detected by enhanced chemiluminescence assay.The density and motility of sperm were analyzed by computer-assisted analysis system.The relationship between seminal plasma thymidine kinase 1 (TK1) and sperm parameters was analyzed respectively.Results The level of seminal plasma TK1 in asthenozoospermia group (2.41±0.21)and azoospermia group (2.72±1.16)was significantly higher than that in normal group (1.53±0.22) (P<0.01).The level of seminal plasma TK1 in oligozoospermia group (1.60±0.13) and oligoasthenozoospermia group (1.68±0.24)had no difference compared with that in normal group (P>0.05).There was no significant correlation between seminal plasma TK1 and other biochemical indexes (P>0.05).Conclusion The level of TK1 might be associated with the quality of sperm motility, The high level of TK1 might be one etiology of asthenospermia.There was no close relationship between the level of TK1 and the changes of sperm number.The reason for high level of TK1 in patients with azoospermia needs to be further investigated.

Objective Previous research discovered that after stimulation of GnRH on Leydig cells, the expression of annexin 5 and the level of testosterone were increased, then we guess that there may be interaction between annexin 5 and testosterone secretion. 3β-HSD is a marker enzyme in Leydig cells.This paper is to study the effect of annexin 5 on testosterone secretion and 3β-HSD expression in cultured rat Leydig cells. Methods The primary rat Leydig cells were cultured in vitro and treated with different concentration of annexin 5 (0,10-10,10-9,10-8mol/L) and with 10-9mol/L concentration of annexin 5 at varied time courses (6, 12, 24,36 h).The culture media were collected for testosterone measurement by chemiluminescence assay. The expression of 3β-HSD protein were also analyzed by Western blot. Results The level of testosterone was significantly increased by 18%(14.38±0.30 vs 12.12±0.74)and 26%(15.25±0.47 vs 12.12±0.74)after treatment for 24 h with 10-10mol/L and 10-9mol/L concentration of annexin 5 (over control, P0.01) respectively. There is an increasing trend with treatment of annexin 5 (10-8mol/L)but no significant change(P0.05); Western blotting results showed the 3β-HSD expression was significant increased by 63%(2.19±1.00 vs 1.34±0.19)(P0.01) at 10-9mol/L concentration but no significant change at 10-10and 10-8mol/L concentration. There was a correlation between the level of testosterone and 3β-HSD expression(r=0.430). Compared with control group, after treatment with 10-9mol/L concentrations of annexin 5 for 24 h, the level of testosterone was significantly increased by 18%(12.59±0.41 vs 10.70±0.09)(P0.01), and decreased by 21%(14.44±1.65 vs 17.46±0.31)(P0.01)for 36 h after stimulation. But there were no significant changes for 6h and 12 h. Conclusion In rat Leydig cells, annexin 5 regulates testosterone secretion in a dose-and time-dependent manner. 3β-HSD is one of key enzymes in the process of estosterone secretion.

Objective: To study the changes of reproductive related parameters during different term chronic pain stress, including the weight of testis and epididymis, sperm amount in cauda epididymidis and the expression of 3 beta-hydroysteroid dehydrogenase (3β-HSD) in testis. Methods: The established rat model with Spare nerve injury was stimulated with different chronic pain stress. On 14th day, 21th day and 28th day, the rats were sacrificed respectively. The testis and epididymis were isolated and weighted, and the number of epididymal sperm was calculated. The structure of testis was analyzed by HE stains. The expresssion of 3 beta-hydroysteroid dehydrogenase expression in testis was detected by Western blot. Results: (1) The body weights on day 14 and 21 were decreased in stress group compared with that in normal and sham group. However no statistical significance in body weights was found among these groups. No obvious changes in testicular and epididymal coefficients among these groups were found during different time points. (3) The number of spermatogenic cell in seminiferous epithelium and number of sperm in seminiferous lumens was all decreased. (4) the expression of 3 β-HSD on 28 d in stress group was significantly lower than that in normal and sham group. Conclusion: Chronic pain stress might reduce the weight of testis and epididymis, the unmber of sperm in cauda epididymidis and the expression of 3 β-HSD in testis. In conclusion, chronic pain stress might weaken the reproduction function of rat.

Objective: Many kinds of stress can suppress reproduction.The aim of this study was to observe the influence of acute pain stress on the sperm count,reproductive organ weight and expression of 3 beta-hydroysteroid dehydrogenase(3β-HSD) in the testis of male SD rats.Methods: The rat model of acute pain stress was made by subcutaneously injecting 0.2ml of 5% formalin into the plantar surface of the left hind paw.The weights of the body,testis and epididymis and the count of epididymal sperm were obtained.The testis structure was observed by HE staining,and the intensity of 3β-HSD was analyzed with the Imageproplus 6.0 software following immunohistochemistry.Results: Compared with the control group,the acute pain stress group showed no significant changes in the weights of the body,testis and epididymis,the count of epididymal sperm and the testis structure(P 0.05);the epididymal indexes were significantly increased 6,24 and 72 hours after formalin injection(P 0.05);the 3β-HSD expression was markedly reduced 1 hour after formalin injection and then gradually increased at 6,24 and 72 hours,with statistical differences between 1 and 72 hours(P 0.05).Conclusion: Formalin-induced acute pain stress has no obvious effect on the testis and epididymis weight and spermatogenesis,but affects testosterone secretion by decreasing the expression of 3β-HSD.However,it can be restored to normal by adjustment of homeostasis.

AIM: To investigate the effects of GnRHa on the expression of annexin5 and 3β-HSD in rat Leydig cells.METHODS: The primary Leydig cells were isolated,purified and cultured in vitro.The cells were identified by 3β-HSD immunocytochemistry method.Different concentrations of GnRHa(1×10-5,1×10-6,1×10-7 mol/L) were administrated on the Leydig cells for 24 hour.The changes of annexin5 and 3β-HSD expression were investigated by Western blot.RESULTS: GnRHa stimulated the annexin5 and 3β-HSD expression.Treatment with 10-5mol/L of GnRHa increased annexin5 expression by 69.2%(P<0.01) and 3β-HSD expression by 25.2%(P<0.05).Treatment with 10-6mol/L and 10-7mol/L of GnRHa increased annexin5 expression by 61.5% and 16.64%(P>0.05),and exerted no significant effects on 3β-HSD expression(increased by 7.7% and 2.22% respectively,P>0.05).CONCLUSION: GnRHa can regulate the annexin5 and 3β-HSD expression in primary cultured Leydig cells.

AIM:To investigate the cellular distribution of annexin 5 in rat's digestive organs using immunohistochemistry and immunofluorescence. METHODS: Digestive organs from healthy rats were fixed with Bouin's solution. Annexin 5 was examined for its specific distribution in rat digestive organs by immunohistochemistry and immunofluorescence with specific antiserum to rat annexin 5. RESULTS: Annexin 5 was localized in digestive organs of rats in specific cell types. Annexin 5 mainly existed in epithelial cells in stomach, partial cells in fundus glands, goblet cells in small intestine, endothelial cells in bile duct and endothelial cells in blood vessel of liver. Annexin 5 was also observed in mucous membrane and muscular layer of stomach and small intestine, lamina propria cells in small intestine, endothelial cells in liver and endothelial cells in blood vessel in pancreas. CONCLUSION: The existence of annexin 5 localized in many cell types with specific function in rat's digestive organs indicates that annexin 5 might be involved in these physiologic functions.

Objective:To study regulation mechanism of GnRH agonist(GnRHa) on testosterone synthesis in rat Leydig cells.Methods:The primary rat Leydig cells were cultured 24 h in vitro,and then treated with GnRHa(10-7 mol/L) at varied time courses(6,12,24,36,48 h);the expression of 3β-HSD protein was analyzed by Western blot.After stimulation with GnRHa and PD98059(50 μmol/L),the specific inhibitor of ERK1/2,in Leydig cells,the expression of 3β-HSD and the level of testosterone were also investigated.Results:Treatment with GnRHa resulted in 3β-HSD expression in a time-dependent manner.3β-HSD expression was significantly increased 45% over control after 12 h of treatment with GnRHa(P 0.05),maximally elevated 1.0-fold over control at 24 h(P 0.05),and then returned to basic level after 48 h.We also found the activation of 3β-HSD induced by GnRHa was markedly reversed in the presence of PD98059(61% compared with GnRHa group,P 0.05),following the significant decrease of testosterone(45% compared with GnRHa group,P 0.05).Conclusion:The results suggest that GnRHa regulates the expression of 3β-HSD through ERK1/2,and further induces testosterone synthesis.

Objective To study the expression of annexin V in the stomach and submandibular gland of SD rat after acute pain stress induced by formalin.Methods The acute pain stress model was established by subcutaneously injecting rats with 2 ml 5% formalin at plantar surface of the left hindpaw and the control group was injected with isochoric saline.The expression of annexin V in the stomachs and submandibular glands of the stress and control groups was analyzed by Western blotting.Results In the stomach,compared with those of annexin V in the controls,the expressions at 1 h and 72 h after injection of formalin had no significant difference(P0.05),whereas those at 6 h and 24 h significantly increased(P0.01).In the submandibular gland,the expressions at 1 h and 24 h showed no difference between two groups(P0.05),whereas the expressions at 6 h and 72 h in the experimental group enhanced markedly compared to those in the control group(P0.01).Conclusions Annexin V participates in the response of stomach and submandibular gland to acute pain stress induced by formalin and the acute pain may influence on them through annexin V.

Objective:Gonadotropin-releasing hormone(GnRH) is a hypothalamic neuronal decapeptide.It induces the synthesis and release of gonadotropins and regulates the synthesis of gonadal hormone.It also has a direct stimulatory effect on Leydig cells.This study investigated the effect of GnRH agonist on ERK MAPK signaling pathway and 3β-HSD mRNA expression in rat Leydig cells.Methods: Primary Leydig cells were cultured for 48 hours,followed by 2 hours of serum-starvation.After the Leydig cells were treated with GnRHa(10-7 mol/L) and the ERK inhibitor PD98059(50 μmol/L),phosphorylated ERK(p-ERK) and total ERK(t-ERK) were detected by Western blot,and 3β-HSD mRNA was analyzed by RT-PCR.Results:After GnRHa stimulation,the p-ERK expression reached the peak at 5 min(3.5 times that of the control,P 0.05),began to decrease at 10 min,though still 2.2 times higher than that of the control(P 0.05),and returned to the basal level at 90 min.When PD98059 was added,the p-ERK expression was decreased by 50%(P 0.05).Another 5 min GnRHa stimulation of the Leydig cells failed to restore the p-ERK expression to the basal level.When the Leydig cells were stimulated by GnRHa after pretreated with PD98059,3β-HSD mRNA was significantly decreased(3.0 times that of the GnRHa group,P 0.05).Conclusion: GnRH agonist regulates the expression of 3β-HSD mRNA and hence the secretion of testosterone via the ERK MAPK signaling pathway.

Objective To investigate the changes of the weight of body,testis and epididymis,the spermtozoon numbers,the expression of 3β-HSD in rat testis under constant-light stress.Methods Rat models with stress induced by constant-light were first established,and then they were divide into GroupA with short constant-light stress(2d,3d, 4 d,7 d) and GroupB with chronic constant-light stress(14 d,21 d,28 d).The testis and epididymis of rat in stress group and control groups were collected respectively.The weight of testis and epididymis was measured and the number of spermtozoon from the caudal epididymis was countered.HE staining,immunohistochemistry and western blot was applied to observe the structure of testis and the change of 3β-HSD expression in rattestis.Results Comparing with those of the control group,body weight,weight of testis and epididymis,number of spermatozoon of the rats in short constant-light stress group(2 d,3 d,4 d,7 d) did not change significantly(P>0.05),the structure of testicle and the expression of 3β-HSD in testis did not change too.Comparing with that of the control group,body weight,weight of testis and epididym and number of spermatozoon of the rats in chronic constant-light stress group(14 d,21 d,28 d) were remarkable increased(P<0.05,P<0.01),and thick somniferous tubule in testicle and many,spermatogenesis cells in lumina were observed by histochemistry analysis.The expressions of 3β-HSD in rat testis in the 14,21 and 28 day group were increased by 20.3%,30.4%and 43.3%fold respectively(P<0.05,P<0.01,P<0.01).Conclusion No significant changes in the reproduction of rats were found under short constant-light stress,which was related to the adjustment of homeostasis.But the chronic constant-light stress not only affected the weight of body,testis and epididym but also affected sperm numbers and testicular structure.It indicated that chronic constant-light stress was an important factor for reproductive function changes of male rats.

Objective To study on the expression of annexin 5 expression in rat testis under acute pain stress. Methods SD rat models with acute pain stress were established by injection of formalin, and their testis were collected respectively at 1 h ,6 h ,24 h and 72 h after injection. The distribution and expression of annexin 3 protein were detected by Immunohistochemistry and western blot. The level of annexin 5 mRNA was measured by quantitative RT-PCR. Results Immunohistochemistry staining showed that annexin 5 was mainly distributed in Sertoli cell and Leydig cells of rat testis. Analysis of western blot exhibited that the expression of annexin 5 decreased about 16.9 % and 12. 8% respectively at lh and 6h after injection of formalin, whereas increased 33.7% and 25% at 24 h and 72 h after injecting formalin. Compared with that of control group, statistical difference were found in the expression of annexin 5 at lh and 24 h. The mRNA level of annexin 5 had a similar expression pattern with its protein expression at different time points. However, no distinct difference in mRNA level of annexin 5 was found among groups. Conclusion Acute pain stress might mainly affect the protein level of annexin 5 in testis, but no mRNA level of annexin 5, which indicated that annexin S might mediated the process of stress inhibiting reproduction, as a stress response gene.

Objective To study the effect of GnRH agonist (GnRHa) on the expression of glucagon in In-R1-G9 cells. Methods GnRHa was added to the media of the cells at final concentrations of 0.1, 10, 1000 nmol/Lol/L, and normal saline added as control. At the end of the incubation, expressions of protein and mRNA of glucagon in In-R1-G9 cells were measured by Western blot and real time fluorescence PCR respectively. Results As compared with control, high concentrations (10 nmol/L and 1000 nmol/L) of the GnRHa showed a significantly increased expressions of glucagon (all P＜0.05) while low concentration (0.1 nmol/L) did not show any significant change (all P＜0.05). Conclusion GnRHa at 10-1000 nmol/L may increase the expressions of glucagon in In-R1-G9 cells.

AIM: To investigate the effects of formalin-induced acute pain on the expression of annexin 5 mRNA in the stomach and submandibular gland of Sprague-Dawley (SD) rats. METHODS: Acute pain was induced in SD rats by subcutaneous injection of 2 mL of 50 g/L formalin into the plantar surface of the left hind-paw. Control rats were injected with equal volume of normal saline solution. The samples of the stomach and submandibular gland were collected under terminal anaesthesia with ketamine (40 mg/kg) at 1, 6, 24 and 72 h after formalin injection. The expression of annexin 5 mRNA in the stomach and submandibular gland was analyzed by fluorescent quantitative polymerase chain reaction (PCR). RESULTS: Fluorescent quantitative PCR showed that the expression of annexin 5 mRNA in the stomach significantly increased at 1 h after formalin injection compared to that in control mice (7.43±2.67 vs 1.00±0.00, P＜0.01), and then decreased to basic level at 6 h (P＞0.05). In the submandibular gland, the expression of annexin 5 mRNA also increased at 1 h after formalin injection (4.33±1.50 vs 1.00±0.00, P＜0.01), and returned to normal level at 72h (P＞0.05). CONCLUSION: Formalin injection-induced acute pain upregulats the expression of annexin 5 mRNA in the stomach and submandibular gland of rats. Annexin 5 may be an acute stress response protein in the digestive system.

Objective: To investigate the effects of the GnRH analogue on the expressions of annexin 5 and 3β-HSD mRNA in rat Leydig cells.Methods: Rat Leydig cells,after primarily cultured for 24 hours,were treated with different concentrations of GnRH agonist(10-5mol/L,10-6 mol/L,10-7 mol/L) and GnRH antagonist(10-6 mol/L,10-7 mol/L,10-8 mol/L).The expression levels of annexin 5 and 3β-HSD mRNA in the Leydig cells were measured by fluorescence quantitative RT-PCR.Results: Compared with the unexposed controls,the rats treated with GnRH agonist at the concentrations of 10-5mol/L and 10-6mol/L showed an increase in the expression of annexin 5 mRNA by 38.89%(P< 0.05) and 35.29%(P< 0.05),and those treated with GnRH agonist at 10-5mol/L had an increase of 46.43%(P< 0.01) in the expression of 3β-HSD mRNA.While GnRH antagonist significantly decreased the expression of annexin 5 mRNA by 45.45% at 10-6mol/L(P< 0.05),and reduced the expression of 3β-HSD mRNA by 63.64%(P<0.01) and 50%(P<0.05) at 10-7mol/L and 10-8mol/L.There were no statistically significant differences between the control and the other experimental groups.Conclusion: The GnRH analogue can regulate the expressions of annexin 5 and 3β-HSD in primarily cultured Leydig cells,probably by initiating a series of cell transductions after combined with the GnRH receptor.However,the underlying mechanism needs to be further studied.