| Objective By cyclophosphamide administered to rats of different ages, we try to determine whether the younger ,the less toxicity effect of acute cyclophosphamide on rat spermatogenesis, and find theory base of gonadal protective agents .Methods According to the different development stages of testis, that is period of sperm preproduction, earlier period of sperm production, period of sperm production , period of sexual maturity, we selected 24 1-week old ,3-week old ,5-week old ,9-week old male Wistar rats, respectively. Every age group was randomly divided into 2 groups: earlier observation group and later observation group, then every observation group was randomly divided into control group and experiment group. The rats of earlier observation group were killed 24-hour after cyclophosphamide administered, and the apoptosis rates of spermatogenic cells of testis samples were detected by TUNEL, the expression of Bcl-2 protein by immunohistochemistry and spermatogenic cell cycle by Flow cytometry. The rats of later observation group were killed at 9-weekafter drug administered, the testis samples were observed by light microscope, then we calculated the average diameter and area of seminiferous tubule, count measure spermatogenic epithelium (CMSE), and Johnsen's score.Results: 1 Acute effects of CTX on rat testis of different development phase:.Apoptosis of spermatogenic cellsNormal testis tissue of every age phase has apoptosis. among control groups of every age group , the apoptosis rate of 3-week group is highest, and the one of 1-week group is lowest, whereas the apoptosis rate of every experiment group of every age group is lower significantly (P<0.01), corresponding to the control group respectively, expect for 1-week group . Among experiment group of every age group, the apoptosis rate of 3-week and 5-week group are also higher, but there is no significant differentiation between them(P>0.05)..Expression of Bcl-2 protein Except for 1-week group, expression of Bcl-2 protein of every experiment group descended significantly (P<0.01), corresponding to the control group, respectively. And the age tendency of Bcl-2 expression is similar to the one of spermatogenic cell apoptosis. Bcl-2 protein expression of 3-week group is the highest..Proportion of S period cells:Except for 1-week group, proportion of S period cells of every experiment group descended significantly (P<0.01), corresponding to the control group, respectively. Among experiment groups, the reduction of 3-week and 5-week group are lower than others and they have no significant differentiation.(P>0.05).2 Long-term effect of CTX to testis of different age.Except for 1-week group, every observation index of every experiment group is lower significantly (P<0.01), corresponding to the control group ,respectively. Besides, the age tendency is that the younger of CTX administered, the less of reduction of every observation index. Moreover, there is significant differentiation among them by analysis of variance (P<0.01).Conclusion:1. Spontaneous apoptosis aslo exsit in normal testis tissue. The apoptosis of testis in the phase before initial of spermatogenesis is rare. And testis in the initial and above phase of spermatogenesis have apoptosis, which is highest in testis exsiting in the peak period of proliferation .2. CTX can induce significant cell apoptosis of testis in the initial and above phase of spermatogenesis, the effect is severest on the testis in the most active phase of spermatogenesis, and the effect is little on the testis inthe phase before initial of spermatogenesis3. CTX inducing spermatogenic cell apoptosis may be by down-regulation of Bcl-2 gene.4. CTX can inhibit the proliferation of spermatogonium and proleptotene spermatocyte, especially to the testis in the peak period of proliferation.5. By histology observation to long-term testis tissue, we can conclude the younger of CTX administered, the less toxicity effect of acute cyclophospham...
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