Comparative Proteomics Studies Of Human Follicular Fluid Between The Different Periods Of Follicular Development

BackgroudPrevious research have already proved that oocyte maturation accompany protein synthesization and phosphorylation / dephosphorylation status exchange during the follicular development. StAR(stcroidogenic acute regulatory protein), M PF, MAPK, PKC, and CAMP/PKA have already been proved that involved in oocyte maturation, however, there are still many novel proteins or unknown proteins which might take part in this process. The molecular mechanism of oocyte maturation is being the hot project in the female reproduction field. Proteomic methods detect the functioning units of expressed genes, through biochemical analysis of cellular proteins, to provide a protein fingerprint. The proteomic reflects both the intrinsic genetic programme of the cell and the impact of its immediate environment and is valuable in biomarker discovery therefore. Distinct changes that occur at the protein level during the process of follicular development include unknown protein synthesization, differential protein modification, and changes in specific activity, all of which may affect oocyte maturation. Twodimensional electrophoresis, mass spectrometry and SELDI-TOF (surface-enhanced laser desorption/ionization time of flight) mass spectrometry are the main proteomic technologies, which based on capturing proteins/peptides by chemically modified surface, are specifically powerful for analyzing the complex biological samples. In this research, we want to identify the possible marked proteins which might regulate oocyte maturation / follicular development in the human follicular fluid by using the high resolution two-dimensional electrophoresis and SELDI-TOF-MS.Objectives1. To compare the changes of different proteins between mature follicles and antral follicles then to evaluate the possible marked proteins in the study of follicular development and oocyte maturation by SELDI-TOF mass spectrometry.2. To establish the full-scale protein expression spectrum of human follicular fluid by two-dimensional polyacrylamide gel electrophoresis, to compare those different proteins between mature follicles and antral follicles and to evaluate the possible marked proteins in the study of follicular development and oocyte maturation.3. To study the effect of those proteins, which might regulate oocyte maturation by 2-DE, on maturation in vitro and the kinetics of meiotic maturation in murine cumulus oocyte complex.Methods1. Mature follicular fluids were obtained from forty-eight females after oocyte collection during in vitro fertilization, and antral follicular fluids were obtained from twenty-one females during ascendent follicle pricking. The proteins in follicular fluid were detected by SELDI-TOF-MS and weak cation exchange proteinchip (WCX-2). The data were read with PBS II-C type proteinchip reader and analyzed with Biomarker Wizard and Biomarker Patterns Software of Ciphergen Company.2. Two-dimensional electrophoresis with matrix-assisted laser desorption-mass spectrometry(MALDI-MS) was used to identify the key proteins of oocyte maturation in the same HFF samples.3. Immature oocytes were retrieved from murine ovarian stimulated by human menopausal gonadotropin. GV stage oocytes were matured for 24 hours in human tubal fluid supplemented with 1, 10 and 100ng/ml transferrin or 5, 25 and 125ng/ml apolipoprotein E and human tubal fluid with 75mlU/ml follicle stimulating hormone (control group ). Germinal vesical breakdown and extrusion of the first polar body were observed with invert microscope during the maturation process at 16h, 20h and 24h. When the first polar body was found in the perivitelline space, the oocytes were classified as nuclear maturation.Results1. Through noise filtration conducted by Ciphergen ProteinChip Software 3.1, there were 53 peaks detected for mature follicular fluid and antral follicular fluid. The peaks were between 2kDa and 30kDa. Peaks with a M/Z <2kDa were mainly ion noise form the matrix and therefore excluded. In comparison with the antral follicular fluid, there were significant deviations in the M/Z of the 4 candidate biomarkers, which were 6622.43, 7525.63, 2291.65 and 9131.65, in the mature follicular fluid.2. Using ExPASy Peptldent with Tagldent tool based on molecular mass from SWISS-PROT, the protein, which theoretic molecular mass was 6622.43, was presumed as apolipoprotein C-I precursor. But the three other proteins could not be presumed.3. The expression of different proteins in the mature follicular fluid and the antral follicular fluid were similar. We selected 5 increased protein spots present in mature FF for MS by comparative proteomic analysis. Using ExPASy Peptldent with an error tolerance of 100 ppm, information on the peptide mass fingerprinting data was searched against databases of relevant species. Database searching revealed that the 5 increased proteins in mature FF were apolipoprotein E (ApoE), PRO2044, hypothetical protein, IgG heavy chain and transferrin(TRF).4. The 1ng/ml TRF group had similar percentage of undergoing germinal vesical breakdown and similar percentage of PBI extrusion compared with the control group (P＞0.05); The 10ng/ml TRF group and the 100ng/ml TRF group both had lower percentage of undergoing germinal vesical breakdown and PBⅠextrusion (P＜0.05) compared with control group; TRF groups had higher percentage of the murine matured oocytes' fertilization rate in IVF (P＜0.05) compared with control group, but percentage of fertilization rate were similar in groups treated with different TRF concentration (P＞0. 05).5. The 5ng/ml ApoE group had similar percentage of undergoing germinal vesical breakdown and similar percentage of PBⅠextrusion compared with the control group (P＞0. 05); The 25ng/ml ApoE group and the 125ng/lnl ApoE group both had lower percentage of undergoing germinal vesical breakdown and PBⅠextrusion (P＜0.05) compared with control group; ApoE groups had similar percentage of the murine matured oocytes' fertilization rate in IVF (P＞0.05) compared with control group.Conclusions1. We have established the full-scale protein map for human mature follicular fluid in the different periods of follicular development by 2-DE and SELDI-TOF-MS. The expressions of different proteins in HFF in the different periods of follicular development were similar. The contents were different for the same protein in HFF between the different periods. The differences of those proteins' volume indicate that those proteins maybe the candidates of specific functions during folliculogenesis, hormonesecretion regulation, or oocyte maturation. These results would provide a basis for the further research of oocyte maturation.2. High dosage TRF could inhibit oocyte maturation by inducing the improvement of FSH to oocyte meiosis in vitro. Higher TRF in the follicular fluid at physiological condition may play a significant role in the nature selection of preponderant follicle.3. High dosage ApoE could inhibit the germinal vesical breakdown and the PBⅠextrusion of murine immatured oocytes in vitro. Higher ApoE in the mature follicular fluid at physiological condition could be an important marker of oocyte matured.