| ObjectiveTo research intraperitoneal injection of ethanol to affect the growth anddevelopment, testicular weight and spermatogenesis of mice, to master thepreparation of paraffin sections of mouse and HE staining and the other technology,combining with TdT-mediated dUTP nick end labeling (TUNEL) to assay theapoptosis of germ cell of testis, using real-time quantitative-PCR to detect theexpression of Caspase-3mRNA, and using Western-Blot to detect the expression ofCaspase-3protein. To discuss the mechanisms of ethanol-induced testicular injury,providing new ideas for the patients with testicular damage who have accepted thetreatment of ethanol long-term, and exploring new directions for the clinical treatmentof male infertility.Object and Methods1. Selected24male Kunming mice which were similar weight, randomly dividedinto control (n=12) and experimental (n=12) groups recording the initial weightof each mouse. The mice in the experimental-group were given intra-peritonealinjection (IP) of ethanol at a dose of3g (15%，v/v) per kg body weight per dayduring the period of14days. The mice in control group were injected withphysiological saline for14consecutive days, the successful establishment ofethanol-induced testicular damage model, and then record the weight of each mouse.2. All mice were sacrificed by cervical and testicular specimens rapidly collected,weighed, calculated organ index. All the left testicles were saved into Bouin’sfixative, and all the right testicles were saved into liquid nitrogen tank.3. To complete the preparation of paraffin-embedded specimens testicles fixed byBouin’s fluid and slicing, The morphological changes of testicular seminiferoustubules and spermatogenic cell were observed by using HE staining. Theapoptosis of testicular germ cell were detected by using TUNEL. Using real-timequantitative polymerase chain reaction (PCR) and Western-Blot to measure theexpression of Caspase-3mRNA and protein.4. Using SPSS l7.0to do statistical data processing, Two independent samples ofquantitative data, while meeting the use of two independent samples t-test whennormality and homogeneity of variance; meet normality but heterogeneity ofvariance, choose to use the corrected t test (t ’test); do not follow a normaldistribution, choose to use the rank sum test. Select test level α=0.05.ResultsUsing15%ethanol3g/kg to mice by intraperitoneal injection for14days in thisexperiment, then successfully established the model of ethanol-induced testicularinjury. During the experiment, the control group of mice have normal diet andactivities, and the experimental group showed some phenomena like drunkennesssuch as excitement, grasp and bite and so on. We consider from the change of weight,the gained weight of experimental group significantly less than the control group (P=0.03), while detect the index of mice testis (relative to the weight of the testes ofmice before the experiment) and variation of seminiferous tubule diameter by HEstaining in optical microscopy, we has also been consistent results. The ratio oftesticular weight and body-weight in the experimental group significantly lower thanthe control group (P <0.05); HE staining show that seminiferous tubule diameter,spermatogenic cells and sperm counts are abnormal. The mean apoptotic index of thespermatogenesis was detected by TUNEL in the two growps mice, the mean apoptotic index of spermatogenic cells in the control group was (0.1219±0.0184), significantlylower than the experimental group (0.5732±0.0223)(P <0.05); The results ofReal-time quantitative PCR showed that the expression of Caspase-3mRNA inethanol group （2-△△Ct=3.35±0.13）was significantly higher than control group（2-△△Ct=1）(P <0.05); The results of Western-Blot showed Caspase-3protein expression（IntDen=44.51±8.94）was significantly higher than control group（IntDen=140.66±6.25）(P <0.05).ConclusionThe growth and development of mammals, the function of testis andspermatogenesis was significantly inhibited by ethanol. Caspase-3played animportant role in the the process of the ethanol-induced mammalian testicular injuryand closely related to the apoptosis of spermatogenic cells, and the mechanism ofaction may be connected with ethanol itself or its metabolites, but still are not surethat need more experiments to further confirm. This experiment confirms the negativeimpact of ethanol on mammals, that may be able to change the undertanding ofethanol by people, providing new ideas for the patients with testicular damage whohave accepted the treatment of ethanol long-term, and exploring new directions forthe clinical treatment of male infertility. |
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