Analysis Of The Differential Proteomics Between Fresh And Frozen-thawed Ram Spermatozoa

Spermatozoa cryopreservation offers potential for long-term storage of genetic resources. Many aspects of spermatozoa physiology and morphology are affected by cryopreservation. Many factors contribute to spermatozoa damage, including temperature and osmotic effects during freezing and thawing, accompanied with the lose of proteins, the change of expression and function. These injuries affect spermatozoa function, including plasma membrane ?uidity, permeability, lipid composition, and mitochondrial activity.The proteomics technique platform of ram frozen spermatozoa was established by exploring the different sample preparation methods and the different sample quality of two-dimensional gel electrophoresis(2-DE). The same batch of fresh and frozen-thawed spermatozoa were also explored, the protein expression patterns of fresh and frozen-thawed spermatozoa were constructed by using 2-DE. Differentially expressed proteins were detected by PDQuest 8.0 software.The results showed: (1) Compared with three sample preparation methods using 1-DE,the hot Trizol method was much more suitable for extracting the ram spermatozoa proteins. (2) The 2-DE gels were stained with Coomassie Brilliant Blue, the suitable sample quality was 0.45mg using 17cm(pH4～7)IPG strips. (3) The fourteen differentially protein sopts were found that five spots were increased, three spots decreased, two newly expressed and four absent significantly in frozen-thawed spermatozoa 2-DE gels. Valid spots were analyzed through liquid chromatography tandem ion trap mass spectrometry (LC-MS/MS). A search against protein sequences in the National Center for Biotechnology Information databases indicated that the differentially expressed proteins correspond to five proteins including putative heat shock protein 70.1, transcription factor AP-2-alpha, enolase, enolase1 and serum albumin precursor that heat shock protein 70.1, enolase and enolase1 were down-regulated, transcription factor AP-2-alpha was up-regulated in frozen-thawed spermatozoa,serum albumin precursor was seminal plasma protein. The putative heat shock protein 70.1 is needed to be analyzed sequence homology with heat shock protein 70.1 and predicted function by multi-stage mass spectrometry, spermatozoa motility may be related with enolase and enolase1, transcription factor AP-2-alpha can be combined with the specific DNA sequence and activated gene transcription, the serum albumin precursor can be combined with spermatozoa surface and protected spermatozoa in cryopreservation.It was concluded there was a significant difference at protein level in ram spermatozoa during cryopreservation. Differential proteome expression analysis may be useful to clarify the physiology state of ram spermatozoa during cryopreservation for further studies on ram spermatozoa proteomic, searching for the biomarker of cryodamage,,and,,revealing,,the,,cryodamage,,mechanism,,of,,spermatozoa,,cryopreserv-ation. ,...