| Objective To investigate the effects of testicular cells after acrylamide exposure of mice, the male mice were treated with acrylamide by three ways:intraperitoneal injection, oral exposure and skin exposure. The mouse model of AA exposure were established, we Studied AA-induced apoptosis in testicular cells of male mice and its effects on vimentin expression, and researched the relationship between vimentin expression and spermatogenic cell apoptosis. Preliminary discussion on target cells of male reproductive toxicity induced by AA, to provide laboratory basis for researching reproductive toxicity mechanism of AA.MethodPart one Forty-eight, five-week-old male KM mice, were randomly divided into 4 groups, including the blank control group, intraperitoneal injection group, oral exposure group, and skin exposure group,12 in each. According to exposure dose 25 mg-kg-1·d-1 for 5 successive days, once a day, changes in body weight and testis coefficient were observed before and after exposure to AA. After the first exposure, mice were used for single cell gel electrophoresis experiment on the sixth day, and early testicular sperm cell micronucleus test on nineteenth day.Part two Forty male mice (seven or eight weeks old) were divided into 4 groups randomly,10 in each. All mouse were administrated with Acrylamide for five successive weeks (6 days a week) at the diferent doses (0,10,20,40mg/kg), the mouse were sacrified on the 36th day following the first administration. The normal control group was given an equal volume of distilled water. After five weeks treatment, histo-pathological techniques (HE staining), flow cytometry, TUNEL, immunohistochemistry techniques and RT-PCR were used to research the relationship between the apoptosis and the expression of vimentin in mice, respectively on the tissue, cell, DNA, protein and mRNA level.Result1. In single-cell gel electrophoresis experiments (six days after first exposure to AA), The body weight of intraperitoneal injection group and oral exposure group decreased significantly (P<0.01). Compared with the blank control group, the testis weight and testis coefficient of intraperitoneal injection group, oral exposure group, and skin exposure group are all decreased significantly (P<0.05 or P<0.01). The indexes of testicular cells DNA damage of the experimental groups were higher than the blank control group significantly (P<0.05or P <0.01).2. In early spermatid micronucleus test(nineteen days after first exposure to AA), the body weight and testis weight of the three AA exposure groups are all decreased significantly (P< 0.01), however, compare with the blank control group, the testis coefficient was not significantly decreased (P>0.05). The early spermatid micronucleus rate of oral exposure group were significantly higher than the control group (P<0.05), while the intraperitoneal injection group and the skin exposure group were higher than the blank control group, but the difference was no statistical significance (P>0.05).3. Compared with the normal control group,the HE staining showed that.10 mg/kg group mice testis seminiferous tubes are in the basic rules, and no significant changes in spermatogenic cells at all levels.20 mg/kg group and 40 mg/kg seminiferous tubes are not in the basic rules, seminiferous epithelium level and all levels of spermatogenic cells decreased, mature sperm within the lumen also decreased, and there are vacuoles in tubes.4. Compared with the control group, the FCM test results showed that Haploid cells decreased significantly, Diploid cells increased significantly (P<0.01), and no significant difference in Haploid and Diploid cells between 10 mg/kg and 20 mg/kg group. The proportion of Tetraploid cells, both in 10 mg/kg and 20 mg/kg group are higher than the control group, and which in 40 mg/kg group is lower than the other two groups, but still higher than control group significantly (P<0.01).The proportion of Haploid cells/Tetraploid cells (1C:4C) and Haploid cells/Diploid cells (1C:2C) in each AA exposure group were lower than the control group significantly (P<0.01), but there is no significant difference in each AA exposure group (P >0.05). Compared with the control group, Only in 40 mg/kg dose group, the proportion of Tetraploid cells/Diploid cells (4C:2C) lower significantly (P<0.01).5. The TUNEL test results showed that the apoptotic index of each dose group was significantly higher than the normal control group (P<0.05).6. Immunohistochemistry staining results showed that the average optical density value of vimentin in 20mg/kg group and 40mg/kg group decreased, compared with normal control group (P<0.05), but no statistical difference in 1Omg/kg (P>0.05).7. RT-PCR results showed that vimentin mRNA expression of each dose group in mouse testis were significantly lower compared with the normal control group (P<0.05).Conclusion 1. In this experimental condition, the DNA damage of testicular cells was existence,, and early stage testicular germ cell micronucleus incidence was elevated after exposure to acrylamide in male mice. The toxic effects of oral exposure were the most sensitive way in the three ways.2. Exposure to Acrylamide can induce seminiferous epithelium level and all levels of spermatogenic cells decreased, mature sperm within the lumen also decreased.3. When mice were exposed to Acrylamide, the efficiency from spermatogonia transform into sperm were descended, the number of spermatid and sperm were decreased, and the process of spermatogenic were disordered. The target cell is likely to the spermatogonia.4. The index of apoptosis were increased, and it was dose dependent.5. The expression of vimentin and vimentin mRNA were decreased, and the vimentin decreased principally in the Leydig cells. Presume that the possible mechanism of reproduction toxic effect of AA is to decrease the expression of vimentin mRNA, then the vimentin decreased.6. There are certain relevance between apoptosis of spermatogenic cell and the lower expression of vimentin. |
