Expressions Of Extracellular Signal-regulated Kinases In The Model Of Rat Testicular Transplantation

BackgroundInfertility is defined by WHO as one year of unprotected intercourse without conception.On evaluation,about 5%～8%couples in developed countries is involved in infertility,while it is 30%high in some area of developing countries.In our country, the morbility is rising.A data from Beijing Xie He hospital showed that male patients increased over 100%during 1980～1990.The cause of infertility is complicate.Testicular transplantation is an important way to cure infertility and subfertility.By now,only allogeneic orthotopic testicular transplantation is applied in the clinical research.But the development was restricted because the results were not satisfied.To find out an effective way of protecting the transplanted testes,many kinds of testicular transplantation models were established to investigate the mechanism of inducing testis injury.In the past,big animals were chosen for testicular transplantation.But big animals always cost too much and the anaesthesia requirement was high.Testicular transplantation model in rat was established in 1971 and promoted the development of testicular transplantation,but it required high microsurgery technology.In 2003 a novel method for testicular transplantation in rat was reported by Tan Fu-Qing which simplified the operation process.However,there were many kind of variations in rats' testicular vessels,one method was not enough for all the variations.In our study,we found four kinds of testicular vessels in 100 rats samples,type 1:the left testicular artery originated from abdominal aorta while the vein joined the left common iliac vein;typeⅡ:the artery originated from left renal artery while the vein joined the left common iliac vein;typeⅢ:the left testicular artery originated from left renal artery while the vein joined the left renal vein;typeⅢ:the left testicular artery originated from abdominal aorta while the vein joined the left renal vein.The percentages of the four types were about 63%, 13%,14%and 10%respectively.It was necessary to design different operation methods for the different four types.Ischemia-reperfusion injury played an important role in transplanted testes injury. Some study revealed that ERK(extracellular signal-regulated kinase)participated in inducing ischemia-reperfusion injury while others suggest opposite results.It is still unclear whether ERK was activated in testicular transplantation and what's the relationship between ERK and testis injury.In our study,the method reported by Tan Fu-Qing was used and modified. ERK1(extracellular signal-regulated kinase 1),ERK2(extracellular signal-regulated kinase 2), pERK1(phosphorylated extracellular signal-regulated kinase 1)and pERK2(phosphorylated extracellular signal-regulated kinase 2)levels of transplanted testes were investigated and compared for revealing the relationship between ERK and testis injury and the protective effect of MEK1/2(mitogen-activated protein kinase kinases 1/2)inhibitor U0126(1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene)to the testes.Part 1Modification of orthotopic testicular transplantation in ratObjective To establish and modify the model of orthotopic rat testicular transplantation which was reported by Tan Fu-Qing in 2003,and create a stable platform for the research of ischemical and reperfusion injury and other basic research of the rat testicular transplantation.Methods The male rats weighted 230～250g were chosen.According to the four different kinds of left testicular vessels anatomic types founded in the study,we designed different ways to cut donors' testicular vessels,and anastomosed the vessels and the deferent ducts.The normal color of donor testis and active bleeding of deferent duct suggest successful transplantation.Compare the average operation time, average cold ischemical time,average anastomosis time and immediate vascular patency rate among the four methods as well as the blood supplies of the transplanted testes and the left lower extremities of the recipients.Results 1.The immediate vascular patency rate of control group and the three modify groups were all 100%,without any difference among the four groups.2.There were some differences on average operation time,average cold ischemia time and average anastomosis time among the four methods.3.The blood supplies of the transplanted testes of the four groups wer all good one week after operation,the colors of the transplanted testes were all normal;,and the blood supplies of left lower extremities of all the four methods were all good.Conclusions1.In this part,we successfully established and modified the model of orthotopic rat testicular transplantation.2.The differences of operation time among the four methods didn't affect the immediate vascular patency rate of the transplantation operation.3.Ligation of receipts' left common iliac veins wouldn't affect the blood supplies of left lower extremities.4.Excision of left kidneys did not affect the survival rate of recipients.Part 2The effects of testicular transplantation to the expressions of ERK1, ERK2,pERK1 and pERK2 of the donor testesObjective To determine the expressions of ERK1,ERK2,pERK1 and pERK2 of the donor testes,and the Relationship between the expression levels of ERK1,ERK2, pERK1 and pERK2 and the ischemia-reperfusion injury.Methods The rats were divided into four groups:group1:normal control group, group 2:cold ischemia group,group3:sham operation group,group 4:transplantation group.Determine the expression levels of ERK1,ERK2,pERK1 and pERK2 of all the four groups by western blot.Examine the histological change of the three groups under microscope and electroscope and figure out the MTBS.The ratio of ERK1's gradation toβ-actin's gradation and of ERK2's gradation toβ-actin's gradation represented the ERK1 and ERK2's expression levels respectively.The same way was used for representing the levels of pERK1 and pERK2.Then figure out the ratios of pERK1 to ERK1 and of pERK2 to ERK2.Compare the results of the four groups respectively.Results1.There were no significant differences in the level of ERK1,ERK2,pERK1 and pERK2 among group 1,group 2 and group 3.2.The histological changes of donor testes under microscope and electroscope were similar among group1,group2 and group3,and there were no difference in MTBS among the three groups.3.The levels of ERK1,ERK2,pERK1 and pERK2 were sharply higher in group 4 than in group 1,but the ratios of pERK1 to ERK1 and of pERK2 to ERK2 had not significant difference between group 4 and group 1.4.The histological changes were more severe in group 4 than in group 1,and MTBS was lower in group 4 than in group 1.Conclusions1.Common operation process and cold ischemia did not affect the expressions of ERK1,ERK2,pERK1 and pERK2,and did not cause significant histological injury to the donor testes.2.The ischemia-reperfusion process could activate the expressions of ERK1,ERK2, pERK1 and pERK2.In addition,the expression levels of ERK1 and pERK1 as well as of ERK2 and pERK2 increased in a invariable ratio.3.The histological injury caused by ischemia-reperfusion process is positive correlated with the expression levels of ERK1,ERK2,pERK1 and pERK2.Part 3The protective effect of MEK1/2 inhibitor U0126 to the spermatogenesis of transplanted testes in a short periodObjective To investigate the protective effect of U0126 to the transplanted testes during a short period.Methods Establish two groups:transplantation group,U0126 pretreatment group. Every group was divided into three subgroups according to the different times when the testes were harvested:30min,one week and one month after operation.The serum T,FSH and LH levels of recipients were determined with ELISA kit.The histological changes of the grafts were observed under light microscope and electron microscope. Compare the hormone levels and histological changes among all the groups.Results1.Compared to transplantation group,the levels of ERK1 and ERK2 of U0126 pretreatment group were similar to it;but the levels of pERK1 and pERK2 were decreased obviously and the level of pERK2 decreased more significantly;the ratios of pERK1 to ERK1 and of pERK2 to ERK2 of were also decreased sharply.2.As the time passed after operation,the histological changes of donor testes became more and more server,and the MTBS as well as hormones decreased continuous.3.30min after operation,the histological changes of donor testes under microscope and electroscope were lighter in U0126 pretreatment group than in transplantation group.4.One week after operation,the levels of hormones in U0126 pretreatment group were similar to normal control group,but were higher than transplantation group; the histological changes in U0126 pretreatment group were slighter than transplantation group;MTBS in U0126 pretreatment group were higher than transplantation group;The atrophy percentages of the transplanted testes in U0126 pretreatment group were lower than transplantation group.5.One month after operation,only atrophy percentages of the transplanted testes in U0126 pretreatment group were lower than transplantation group,and there were no differences in other results between U0126 pretreatment group and transplantation group.Conclusions1.As the time passed after operation,the damage to the spermatogenesis became more and more server.2.U0126 did not affect the expression of ERK1 and ERK2,but can inhibit the phosphorylation of ERK1 and ERK2,especially the phosphorylation of ERK2.3.Pretreatment with U0126 could reduce the histological injury of donor testes and maintain the hormones levels of recipients in one week after operation.4.One month later,Pretreatment with U0126 could only reduce the atrophy percentage of the transplanted testes without other protective ettects.