Proteome Analysis Of Round-headed Sperm By Two-dimensional Difference Gel Electrophoresis And Mass Spectrometry

Introduction: In this study, we performed a comparative analysis of sperm proteins found in normal and round-headed sperm using the novel technique two-dimensional differential in-gel electrophoresis (2-D DIGE) .Thus it is of great research interest to identify a group of consistently changing proteins whose function may reveal insight into critical events in disease progression and which may hold value as potential therapeutic targets.Material & methods: Normal sperm samples were obtained from 12 fertile donors and by masturbation after 4-6 days abstinence, these donors were testified that they all have the normal fertility by the AID, the donors all donated 3 times. And twenty globozoospermia samples were included in this study , samples all from a patient who with exclusively round-headed acrosomeless sperm cells as determined by light and electron microscope. All samples were obtained under informed consent using forms approved by the National Human Reproduction Ethic Investigation Committee.In this work, two-dimensional difference gel electrophoresis(2-D DIGE) coupled with mass spectrometry(MS) was used to investigate changes in the proteome of human round-headed sperm and normal sperm. For one gel, Cy5-labeled proteins from the round-headed sperm were combined with Cy3-labeled proteins isolated from the normal human sperm and separated on the same 2-D gel along with a Cy2-labeled mixture of all samples as an internal standard. For the other gel, we labeled the sample reversed.Results: In this study, Over 61 protein spots were analyzed in each paired normal/abnormal comparison, and using DIGE technology with the mixed-sample internal standard, 36 protein spots were identified by ms/ms as showing significant change(paired t-test, p<0.05) in the level of expression between normal and round-headed. 9 proteins were up-regulated and 27 proteins were down-regulated in round-headed. They seem to play important roles in a variety of pathways including spermatogenesis, cell signaling, cell skeleton and metabolism.