Normal Men And White Blood Cells In Patients With Azoospermia Sperm Mitochondrial Gene Deletion And Content

The objectives of the study are to evaluate the spermatozoa MtDNA content and MtDNA^4977bp deletion in normal and leukocytospermia men, and to investigate the relationship between the leukocyte and ROS with the changes of the sperm MtDNA.According to classic theory, the energy of spermatozoa is only from the mitochondial shell. The construction and function of the spermatozoa mitochondria influence the sperm motility, which is one of the important indexes to evaluate the male fertility. The reactive oxygen species (ROS) is mainly from the electron transfer chain in sperm mitochondria, under the physiological conditions the anti-oxidation reaction system can clean the ROS; but under the pathohogical conditions (e.g. leukocytospermia) spermatozoa will be attacked by large amount of ROS. Because sperm mitochondia is uncovered DNA molecular and there is no repair system in mitochondria, the spermatozoa MtDNA is easily injuried.The fluorescence of the spermatozoa was detected through the flow cytometry. This technique is rapid, high flux, accurate. When Dichloroflqorescin diacetate (DCFH-DA) enters the sperm, the group-"diester" will be shucked off. The production of this reaction is 2′,7′-dichlorofluorescin (DCFH). It can be oxidized to form 2′,7′-dichlorofluorescein (DCF). Because the DCF can shine the fluorescence and the fluorescence density of the probe positively correlated with the ROS content, the ROS content can be detected depending on the fluorescence value.Using the flow cytometry, the levels of ROS in control group and the laukocytospermia group were determined according to this theory. We found that the ROS of seminal plasma in the leukocytospermia group was significant higher than in the control group (p＜0.001) . This result was consistent with Saleh' s result.As a popular technique, Real-time PCR has been widely applied in cytobiology and molecular biology field. This technique is rapid, high flux, accurate. The sperm MtDNA content and gene deletion can be detected with this technique. In this study, all the samples were divided into three parts: supernatant fluid,30% and 80% layers by Percoll gradient centrifugation. By the Real-time PCR, the sperm MtDNA content and MtDNA^4977bp deletion of all the samples were quantitively determined.We found that the average MtDNA content was from 1.05 to 9.78 copies in supernatant fluid, 30%and 80% parts of leukocytospermia group and control group. Compared with the control group, the sperm MtDNA contents of three parts of the leukocytospermia group were all significantly higher (p＜0.001). In supernatant fluid and 80% parts, the difference of MtDNA^4977bp deletion between the control group and leukocytospermia group was not significant, but 30% part was significantly higher in leukocytospermia group (p＜0.0001). Significantly positive correlation was observed between the ROS and MtDNA content in 30% part (r=0.347, p＜0.0001) and in 80% part (r=0.456, p＜0.0001) but no correlation in supernatant part. The conclusion is that the leukocyte and ROS may be one of the causes of the increase of the MtDNA content of spermatozoa, and there is no clear conclusion of the effect on the MtDNA^4977bp deletion, which needs further investigation.