| Objective:This study was performed to research the reproductive toxicity on BALB/c male mice infected with the virulent RH strain of toxoplasma gondii (T. gondii). To construct the reproductive toxicity model of T. gondii infection, male mice were infected with toxoplasma by intraperitoneal injection with RH tachyzoites, RNA in situ hybridization was used to detect the T. gondii in testes and epididymidis of infection mice. Transmission election microscopy was used to observe changes on ultrastructure of the testicular tissues caused by T. gondii. The testicular cells were applied onto single cell gel electrophoresis for detection of DNA damage. Observation the changes of oxygen free radials in testes and blood serum.Methods:1. 20 BALB/c male mice were randomly divided into two groups: infected group and negative control group. The negative control group was injected intraperitoneally with PBS and the experimental groups were inoculated intraperitoneally with 2×103 tachyzoites of T. gondii. Four mice in infected group and one in negative control group were sacrificed at 1 day, 3 days, 5 days, and 7 days after infection. The testes and epididymidis were collected and the paraffin sections were prepared to perform the RNA in situ hybridization.2. 10 BALB/c male mice were randomized into 2 groups, 5 mice per group. The negative control group was injected intraperitoneally with PBS and the experimental group was inoculated intraperitoneally with 2.5×103 T. gondii tachyzoites. All mice were sacrificed 7 days after infection, 1 mm3 testicular tissues was used to observe changes on ultrastructure caused by toxoplama gondii.3. 20 BALB/c male mice were randomized into 4 groups, 5 mice per group: one negative control group(C), three experimental groups (G1: 2.5×103, G2: 5×103, and G3: 1×104 tachyzoites). The negative control group was injected intraperitoneally with PBS and the experimental groups were inoculated intraperitoneally with T. gondii tachyzoites. All mice were sacrificed 7 days after infection and the testicular cells were applied onto single cell gel electrophoresis for detection of DNA damage.4. 25 BALB/c male mice were randomized into 5 groups, 5 mice per group: one negative control group, four experimental groups. The control group was injected intraperitoneally with PBS and the experimental groups were inoculated intraperitoneally with 2×103 tachyzoites of T. gondii. Mice were sacrificed at 1 day, 3 days, 5 days, and 7 days after infection. The testes and blood were collected to observe the changes of oxygen free radials. Results:1. Positive results were observed on sections of testes and epididymidis 3 days after infection. The number of tachyzoites in the testis and epididymidis increased with the infection time. The toxoplasma tachyzoites could invade into the different developmental stage of spermatogenic cells, such as spermatogenous cells, first spermatocyte, prespermatid and spermatid. The tachyzoites could either invade into the intracytoplasm or nucleolus of spermatogenic cells. Leydig cell, Sertoli cells and epididymidis could also be invaded into. Significant difference in the number of tachyzoites was seen among different infection time, and no statistically significant difference was observed between the testes and epididymidis at the same infection time.2. Changes on ultrastructure of the testicular tissues: Compared with negative control group, the infection group generally displayed decreases in the spermatogenous cell layers and the quantity of sperm. Mitochondrion vacuolization and the crista mitochondriales decreased, endoplasm reticulum vacuolization, lysosome and other organelles also decreased. Vacuolated cytoplasm was observed in some Sertoli cells, spermatogonia and Leydig cells. Some spermatogenic cells showed the nuclear pyknosis. The tight junction among Sertoli cells showed the interspace widening.3. The coment tail can be abserved in testicular cells of infection mice by single cell gel electrophoresis. The analysis of the comet image showed that significant difference in tail moment and Olive tail moment were seen between the experimental groups and the negative control group. The result indicated that T. gondii infection could cause DNA damage in mice testicular cells, and the level of DNA damage might increase depending on the severity of infection.4. Infection with toxoplasma gondii resulted in an increase of oxygen free radials (NO,·OH, O2- ) in serum and testes, and the increasing tendency seemed at equal pace. The concentration of SOD in serum and testes increased at the first and the third day after infection, subsequently reduced at the fifth day and the seventh day.Conclusions:1. It was considered that T. gondii could invade into the testicular and epididymidis.2. T. gondii could significantly lead to pathologic changes of ultrastructure of Leydig cell, Sertoli cell, spermatogonium, and sperm in testis.3. T. gondii infection could cause DNA damage in mice testicular cells, and the level of DNA damage might increase depending on the severity of infection.4. Infection with T. gondii resulted in an increase of oxygen free radials in serum and testes, and the increasing tendency seemed at equal pace.
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