| It is estimated that10%-20%of couples in worldwide are diagnosed with infertility, among which half of them are male infertility. With the development of economy, the sperm quality has been declined because of the increasing harmful environmental factors and unhealthy habits. Human sperm bank is a department which aims to cure the infertility and prevent the genetic diseases, and collect, test, provide sperm by ultralow cryopreservation. The human sperm bank has provided the patients of azoospermatism and oligoasthenoterazoospermia with option. However, the motility, viability, capacitation and reactive action will damage. It is believed that the changes in osmotic pressure is the main reason of damage in ultra-structure, however, the changes in ultra-structure can not explain the function loss. As the proteins are the important executor on behalf of cell function. Knowing the protein changes after thawing plays a very important role in understanding the mechanism of human sperm cryoinjury. As a result, we previously made a differencial proteomic research in fresh and thawing sperm specimen of9healthy volunteers in sperm bank of our center, finding that Aconitate hydratase, mitochondrial (ACO2) in tricarboxylic acid cycle pathway might play important role in human sperm cryoinjury. The western blot experiment verified the same trend with that of the proteomic research. And immunofluorescence test located at the midpiece of sperm as the main expression region, also called the mitochondrial sheath. By reviewing the articles and protein location indicates that ACO2may be involved in the process of tricarboxylic acid cycle in mitochondrial, and the degradation of AC02after thawing decreasing the ATP product might be the reason of motility loss after thawing. Furthermore, we investigated the changes in ATP content, mitochondrial membrane potiential, and the downstream product of ACO2(Isocitrate). Our findings suggested that the ATP content, Isocitrate content and the mitochondrial membrane potiential significantly decreased after thawing. In summary, we generated a hypothesis that ACO2as mitochandial protein has degraded after thawing, which lowered the content of its downstream product Isocitrate and caused the ATP content decreased, the obvious phenotype was motility loss. This clarification for the target lay a theoretical foundation for mechanism of human sperm cryoinjury and provide a systematic and precise method for selecting, isolating and identifying proteins in using proteomics. Meanwhile, this may also provide with an idea and target to improve the cryoprotectants in increasing the thawing sperm motility. |
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